Off course in the era of cloning by nuclear transfer, the idea to clone a pig which will be knock-out for the galactosyl-transferase was logical to envision and this past year at Chrismas, two different companies announced the birth of such Gal-knock out cloned pigs (today there is more than a hundred of such heterozigous pigs living in Scotland) . There is, however, not yet any results even «in vitro» which support the importance of these animals as organ donors for xenografting. A major consequence of the Gal-knock out could nevertheless, be that pig retroviruses which would leave such organs would not express the galactosyl epitopes and therefore would not be recognized by the human immune system. A second problem could come from the fact that there is two galactosyl-transferase genes and that both should be knock out to assure the complete absence of galactosyl expression at the pig endothelial cell surface. Finally, it is already known that whether Galactosyl is not present anymore at the surface of the pig cells, there is probably other glycoproteins which can be recognized by anti-pig preformed antibodies.

Although difficult, the use of swine as organ donors for human beings is still a major hope for physicians and patients, but the research is still ongoing and will need some more years to envision the use of pig organs in human medicine. Whereas all the enthusiasm in that field was mainly for vascularized xenografts such as heart or kidney, it is possible that the first success in the field of xenotransplantation will be in cellular transplantation i.e. pancreatic islets, neural cells...

IL-2 and Chemokines as Vaccine Adjuvants

R. C. Fields and J. J. Mulé

Department of Surgery and the Tumor Immunology and Immunotherapy Program of the Comprehensive Cancer Center, University of Michigan Health System

Ann Arbor, Michigan 48109-0666

Because dendritic cells (DC) are critically involved in initiating primary and boosting secondary host immune responses, attention has focused recently on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of potent and specific MHC class I and class II-restricted antitumor T cell responses following stimulation with tumor lysate-pulsed DC in vitro. The use of whole tumor cell lysates as a source of tumor antigen(s) for presentation by DC circumvents the need for viable fresh tumor cells and for the establishment of tumor cell lines in vitro, as well as avoids the necessity for molecular characterization of the tumor antigen(s) for effective immunization. Since human tumors have been shown to elicit multiple specific immune responses in the autologous patient, our approach of using whole tumor lysates pulsed onto DC offers the potential advantage of augmenting a broader T cell immune response to tumor-associated antigens (TAAs) that would not be obtained by pulsing DC with a single (or several), defined peptide(s). In addition, this approach would likely lessen the potential for tumor escape by the broader elicited immune response, yet increase the potential to trigger T cell reactivity to those particular antigens that would result in actual tumor regression in vivo (namely, "tumor rejection" antigens).

In preclinical models, immunization of syngeneic mice with tumor lysate-pulsed DC has resulted in potent specific priming and antitumor effects on micrometastatic pulmonary nodules in several histologically-distinct tumors, which are mediated by CD8+ and, to a lesser extent, CD4+ host-derived T cells (1). We have found tumor lysates to be equivalent to highly purified apoptotic tumor cells as a source of TAAs for pulsing of DC (2).

We have recently utilized TP-DC in a phase I trial of pediatric patients with solid tumors (3). Children with relapsed solid malignancies who had failed standard therapies, including bone marrow transplant, were eligible. The vaccine utilized immature DC generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. These DC were pulsed overnight with tumor cell lysates and the immunogenic protein keyhole-limpet hemocyanin (KLH). A total of 1x10⁶ to 1x10⁷ DC were administered intradermally every two weeks for a total of three vaccinations. Fifteen patients (ages 3-17 years) were enrolled with 10 patients completing all vaccinations. Leukapheresis yields averaged 2.8 x 10⁸PBMC/kg and DC yields averaged ~11% of starting PBMC. Children with neuroblastoma, sarcoma, and renal malignancies were treated without obvious toxicity. DTH response was detected in 7/10 patients for KLH and 3/6 patients for tumor lysates. Priming of T cells to KLH was seen in 6/10 patients and to tumor in 3/7 patients as demonstrated by specific IFN-g-secreting T cells in unstimulated PBMC. Significant regression of multiple metastatic sites was seen in one patient. Five patients showed stable disease including 3 who had minimal disease at time of vaccine therapy and remain free of tumor with 22-36 months follow up. These results demonstrated that it is feasible to generate large numbers of functional DC from pediatric patients even in those of very young age, highly pretreated, and with a large tumor burden. The DC could be administered in an outpatient setting without any observable toxicity.

We have focused our recent efforts on designing new strategies to improve upon our initial encouraging clinical results with DC-based tumor vaccines. In preclinical studies, we have found that approaches or agents that selectively elicit apoptosis of tumors in vivo may profoundly augment both the therapeutic efficacy and immune stimulatory capacity of locally delivered DC alone (4). These new data serve as rationale for local administration of DC alone with or without the systemic administration of IL-2 for the clinical treatment of accessible, recurrent human cancers, particularly for those with a significant baseline level of apoptosis or following treatment with potent apoptosis-inducing agents. Indeed, we have recently initiated phase I clinical trials in advanced breast cancer and invasive muscle bladder cancer patients based on these new experimental findings.

The systemic administration of recombinant IL-2 in combination with tumor lysate-pulsed DC has resulted in enhanced therapeutic effects against well-established tumors at either subcutaneous or pulmonary sites (5,6). Interleukin-2 administration could also markedly increase the vaccine potency of peptide-pulsed DC vaccines against established colon carcinoma (7). We have also evaluated whether KLH can augment the antitumor efficacy of tumor lysate-pulsed DC immunization in vivo in the setting of systemic IL-2 administration (8). In addition to being used as a "surrogate antigen" in vaccine approaches to measure immunologic response in cancer patients, KLH has also been shown to be a strongly immunogenic carrier protein to elicit T cell help. Indeed, the addition of KLH to tumor lysate-pulsed DC immunization can both profoundly augment IFN-gamma production by tumor-specific T cells and result in enhanced antitumor therapeutic efficacy in vivo, particularly when combined with systemic IL-2 administration (8).

We are also investigating the potential of combining chemokines with DC-based vaccines to both recruit/concentrate relevant immune populations at the vaccination site as well as to activate the recruited T cells by potent presentation of TAAs (9-11). Anti-tumor therapies based on chemokine gene transfer and expression have utilized chemokine-transfected tumor cells and adenoviral gene delivery to tumors. Previously, we reported that tumor cells stably expressing the CXC chemokine RANTES failed to grow in immunocompetent hosts (9). The anti-tumor effect elicited by RANTES secreting tumor cells was dependent upon CD8⁺ T-cells. However, we were unable to detect T cell or DC migration in response to RANTES in vitro. Furthermore, RANTES secreting tumors were ineffective as a treatment against established tumors. We have now shown for the first time that the molecule secondary lymphoid tissue chemokine (SLC) can induce a strong antitumor response that results in significant infiltration of immune effector cells into treated tumors and that genetic modification of DC to express SLC enhances their capacity to elicit tumor rejection in vivo (10). Of importance, we have demonstrated that SLC-secreting DC can effectively prime tumor-reactive T cells at the tumor site in the complete absence of functional lymph nodes (11). Because of these results, we will initiate a phase I clinical trial of a new vaccine comprising SLC gene-modified, tumor-pulsed dendritic cells with IL-2 in adult patients with advanced melanoma and colorectal cancer and in pediatric patients with advanced sarcoma and neuroblastoma (12).

1. Fields, R.C., Shimizu, K. and Mulé, J.J.: Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc. Natl. Acad. Sci. USA 95: 9482-9487, 1998.

2. Kotera, Y., Shimizu, K., and Mulé, J.J.: Comparative analysis of necrotic and apoptotic tumor cells as a source of antigen(s) in dendritic cell-based immunization. Cancer Res. (Advances in Brief): 61: 8105-8109, 2001.

3. Geiger, J., Hutchinson, R., Hohenkirk, L., McKenna, E., Chang, A., and Mulé, J.J.: Vaccination of pediatric tumor patients with tumor lysate-pulsed dendritic cells expands specific T cells and mediates tumor regression. Cancer Res. 61: 8513-8519, 2001.

4. Candido, K.A., McLaughlin, J.C., Shimizu, K., Kunkel, R., Fuller, J.A., Redman, B.G., Thomas, E.K., Nickoloff, B.J., and Mulé, J.J.: Local administration of dendritic cells inhibits established breast tumor growth: Implications for apoptosis-inducing agents. Cancer Res. 61: 228-236, 2001.

5. Shimizu, K., Fields, R., Giedlin, M., and Mulé, J.J.: Systemic administration of interleukin-2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines. Proc. Natl. Acad. Sci. USA 96: 2268-2273, 1999.

6. Shimizu, K. and Mulé, J.J.: Recombinant IL-2 potentiates immunologic responsiveness to tumor vaccines. Cancer J from Scientific American 6: 67-75, 2000.

7. Kershaw, M.H., Hsu, C., Mondshire, W., Parker, L.L., Wang, G., Overwijk, W.W., Lapoint, R., Yang, J.C., Wang, R.-F., Restifo, N.P., and Hwu, P. Immunization against endogenous retroviral tumor-associated antigens. Cancer Res. 61: 7920-7924, 2001.

8. Shimizu, K., Giedlin, M., and Mulé, J.J.: Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein. Cancer Res. 61: 2618-2624, 2001.

9. Mulé, J.J., Custer, M.C., Averbook, B., Yang, J.C., Rosenberg, S.A., and Schall, T.J.: Genetic modification of a murine fibrosarcoma to secrete the chemotactic cytokine RANTES: Loss of tumorigenicity in vivo is dependent upon host immune cells. Human Gene Ther. 7: 1545-1553, 1996.

10. Kirk, C.J., Hartigan-O'Connor, D., Nickoloff, B.J., Chamberlain, J.S., Giedlin, M., Aukerman, L., and Mulé, J.J.: T cell dependent immunity mediated by secondary lymphoid tissue chemokine (SLC): Augmentation of dendritic cell based immunotherapy. Cancer Res. 61:2062-2070, 2001.

11. Kirk, C.J., Hartigan-O'Connor, D., and Mulé, J.J.: The dynamics of the T cell antitumor response: chemokine secreting dendritic cells can prime tumor reactive T cells extranodally. Cancer Res. 61: 8794-8802, 2001.

12. Terando, A., Sharma, M., Roessler, B., Yannie, P.J., and Mulé, J.J.: A new approach to enhance human dendritic cell-based tumor vaccines based on chemokine gene modification. (in preparation).

Leukemic dendritic cells : potential for therapy and new insights towards immune escape by leukemic blasts

Béatrice Gaugler^1,2, Daniel Olive^1,2 and Mohamad Mohty¹

¹Laboratoire d'Immunologie des Tumeurs, Institut Paoli-Calmettes, Université de la Méditerranée, Marseille, France.

² Institut National de la Santé et de la Recherche Médicale (INSERM) U119, Marseille, France.

Antigen presenting cells (APCs) are specialized for initiating primary T cell immune responses. Being the most potent APCs in vitro and in vivo, DCs were shown to play a key role in the induction of antigen-specific immune responses to tumor antigens and serve as essential constituents of the immune system for triggering immune reactions and are considered promising targets for immunotherapy. DCs ultimately derive from hematopoietic precursors, although little is known about their lineage of origin. They can be generated in vitro from CD34 progenitors as well as from peripheral blood monocytes. In vitro-differentiated DCs show functional and phenotypic characteristics of immature DCs, able to capture and process antigens, that can be further differentiated in vitro into mature DCs with microbial agents, inflammatory cytokines or CD40L.

There is now some evidence that malignant cells of many tumor types may be recognized and eliminated by the immune system. The unique properties of DCs have suggested that they may have the potential to reverse the immunological unresponsiveness generally seen in cancer patients. This can occur through a more effective presentation of tumor epitopes that might be more efficient inducers of helper and cytotoxic T cells. At present, there is no doubt that, at least in animal models, regression of established tumors or protection against tumors can be effectively induced by DCs. A number of reports also show the clinical feasibility of DC based immunotherapy in a wide variety of human cancers. The use of DCs for immunotherapy is rapidly growing in malignant diseases. Many undergoing clinical trials provided encouraging results, but the real clinical benefit of this promising approach is still under evaluation.

At present little is known about the relationship between DCs and hematological malignancies, and this field is rapidly growing. Acute myeloid leukemia (AML) is characterized by a clonal proliferation of hematopoietic myeloid progenitor cells that do not differentiate into functional leukocytes. A self-renewal capacity of one or several leukemia-initiating cells is necessary to initiate the leukemic process, and is followed by a commitment of low proliferative blasts into particular lineages characterizing the different subtypes of AML. At present, the relationships between the various DC differentiation pathways and the hematopoietic progenitors remain unclear. Interactions between DCs and AML cells might represent an attractive model where DCs can become a valuable therapeutic tool for the adjuvant treatment of leukemic patients. However, DC subsets in vivo may also be affected by leukemogenesis and may contribute to the escape of leukemia from immune control.

Immunoregulatory potential of leukemia-derived DCs

The potency of DCs for induction of immune responses can be affected by a number of aspects related to their maturation stage and state of activation. DCs in vivo might represent a privileged target for cancer evasion from immune control through a number of different mechanisms, resulting in a lack of efficient immune responses. Unlike solid cancers where accumulating evidence has been described in the last few years, the interference between DCs and hematological disorders, especially AML, is not yet well characterized. DCs are phenotypically and functionally heterogeneous in vivo. In humans, in addition to tissue-specific DCs, two distinct subsets of blood dendritic cells have been characterized, myeloid DCs (MDC) and plasmacytoid DCs (PDCwith distinct functions. Recently, we investigated the status of circulating peripheral blood DCs in a large series of AML patients belonging to different FAB subtypes. We asked first, whether circulating DCs could be detected in the peripheral blood of patients with AML. We next examined the functional properties of these cells. We could show a dramatic quantitative imbalance in blood DC subsets among the majority of 37 patients with AML. Both MDC and PDC subsets were found to exhibit the original leukemic chromosomal abnormality as ascertained by FISH experiments. Leukemic PDC had impaired capacity for maturation after culture in vitro, a decreased allostimulatory activity and are altered in their ability to secrete IFN-a after microbial stimulation. Our results established a clear difference in the function of leukemic circulating DCs compared to their normal counterpart. Stimulation via CD40 pathway failed to enhance the allostimulatory activity of PDC from leukemic patients, which is compatible with the absence or low expression of HLA-DR and costimulatory molecules. This defective function of leukemic PDC would have serious consequences on the induction of anti-leukemic immune responses. The major recognized feature of PDC is their ability to secrete large quantities of IFN-a. Thus, PDC have a critical role for linking innate and adaptive immune responses against tumors. Type I IFN plays an essential role in antiviral immunity and is widely used to treat viral hepatitis, and various types of malignancies, especially chronic myeloid leukemia. Thus, the absence of PDC can exert indirect potent immunosuppressive properties on the induction of leukemia-specific responses.

The role of MDC in AML patients is less clear. Although, immature circulating blood MDC have preserved maturation capacities after culture in vitro, recent data suggest that immature monocyte-derived DCs can induce regulatory T cells both in vitro and in vivo. Thus, it is likely that these expanded immature MDC might induce regulatory suppressive T cells impairing the quality of anti-leukemia immune response.

Our observation from different patients that leukemic PDC, like leukemic MDC, can exhibit the same original chromosomal abnormality of the myeloid leukemic clone, support evidence that leukemia initiating progenitors are located at the very early level of hematopoietic stem cells, but also raise the intriguing possibility of the existence in vivo of an early common DC progenitor (CDCP) capable under specific circumstances to give rise to MDC or PDC, that do not necessarily belong to distinct and completely independent lineages. It can be proposed that an oncogenic event associated with the leukemic transformation, can affect this CDCP and thus, be responsible for the defective functions of leukemic DCs in vivo.

Potential for therapy with leukemia-derived DCs

Allogeneic bone marrow transplantation proved to be an efficient treatment for patients with AML. This is mainly attributed to the so-called graft-versus-leukemia effect mediated by the donor-derived immune system, especially T cells. Thus, modulation of the immune system appears to be an attractive modality for the treatment of AML patients, especially those patients at high risk of relapse and that can not benefit from allogeneic stem cell transplantation. We and other investigators have described the successful differentiation of leukemia- derived DCs from patients with AML. We could show that myeloid blasts may be driven to differentiate ex-vivo into fully functional DCs capable of inducing leukemia-specific CTL. These leukemia-derived DCs could be obtained after a short term culture in the presence of GM-CSF, IL-4 and CD40L. They exhibited a typical DC morphology, had a phenotype of mature DCs especially the expression of co-stimulatory molecules, and could induce a potent proliferative response in naive CD4+ T cells, while still retaining the leukemic chromosomal abnormality of the original blasts as ascertained by FISH experiments. These cells secreted significant amounts of IL-12p70, and in some cases, highly efficient autologous leukemia-specific CTL were obtained. The high number of efficient co-stimulatory, adhesion and MHC molecules that are present on the membrane of these leukemic DCs could allow recruitment and activation of the rare specific anti-tumoral T cells that are supposed to belong to the naive lymphocyte pool. Moreover, to overcome the absence of identified leukemia-associated antigens, in the case of leukemic DCs, tumor themselves are used as immunogens. The latter could allow the induction of a T cell response against a wide variety of peptides, which would avoid their escape from CTL specific for only one peptide. In addition, this approach can allow to bypass the potential obstacle represented by the defective function of DCs demonstrated in vivo.

Unfortunately, in all published studies, including the report from our group, and despite the use of a wide variety of cytokine combinations, leukemia-derived DCs could not be obtained from all AML patients. Furthermore, yields of leukemic DC were very heterogeneous between patients, including among patients having the same leukemia subtype. In all studies, it was not possible to postulate any characteristic of an AML predictive of generation of cells with DC features. Since leukemic blasts are heterogeneous at the level of maturation stages, in our recent pre-clinical work, we raised the question of the nature of the blast compartment that can be induced to acquire DC features in vitro. Our aim was to identify a blast subset capable after a short term culture with a minimalist combination of cytokines of giving rise to fully functional leukemic DCs. Such a rapid and predefined culture protocol, avoiding patient-tailored techniques, long term in vitro manipulations and complex and expensive cytokine combinations would allow the access to this strategy to the highest number of patients. We could demonstrate that in most cases, CD14+ blasts are the cells capable to give rise to fully functional DCs after a short term culture (5 to 7 days) in the presence of GM-CSF and IL-4 or GM-CSF and clinical grade IL-13. For clinical application, these cells could also be grown in clinical grade serum-free medium. Another major feature of DC physiology concerns the expression of chemokine receptors. Chemokines and their receptors play a critical role in the selective attraction of various subsets of leukocytes and DCs. Along with the acquisition of adhesion and costimulatory molecules, the immune response requires a timely interaction among different cell types within distinct microenvironments. The migration of DCs to the secondary lymphoid organs is believed to be one of the critical events. CCR-7 is a very important player in the mechanism by which T lymphocytes and DCs enter secondary lymphoid organs through high endothelial venules. As a way to understand the regulation of leukemia-derived DCs traffic, we examined on their surface the chemokine receptor expression in comparison with normal mature monocyte-derived DCs. Flow-cytometry analysis revealed that after maturation, leukemia-derived DCs do not express CCR-5, a marker of immature DCs, but could acquire the expression of CCR-7. After injection in vivo, it could be therefore hypothesized that these leukemia-derived DCs would be trapped in T cell areas of lymph nodes where the initiation of immune responses take place. In addition, for optimal efficiency, one could assume that leukemia-derived DCs must already retain at least some leukemia-related proteins avoiding the antigen capture step and homing directly into lymph nodes where they would be able to initiate an efficient antitumor immune response. In this respect, a critical parameter is to retain part or integrality of the tumor-associated antigens during in vitro differentiation. All investigators who performed FISH analysis, confirmed that leukemia-derived DCs, still retain the same cytogenetic abnormalities expressed by original leukemic blasts. Therefore, leukemia-derived DCs from AML patients may retain at least some features of the malignant clone like some leukemia-related proteins associated with the cytogenetic abnormality. Although the latter does not necessarily mean that they will be efficiently presented to T-cells, one could assume that leukemia-derived DCs, while directing a Th1 response profile, will help generating antileukemic cytotoxic responses better than fresh tumor cells. We have shown that CD14+ blasts-derived DCs can drive naive T cells towards a Th1 response with secretion of IFN-g. The strategy leading to induce or increase AML cells immunogenicity while acquiring essential chemokine receptors for trafficking into secondary lymphoid organs, may help in vivo to generate anti-leukemia cytotoxic T effectors in an autologous setting, or identify AML tumor-associated antigens that might prove active as a cell-free vaccine. Moreover, this strategy is also ripe for therapeutic manipulations and refinement in the allogeneic setting. Leukemia-derived DCs might be used in vitro to elicit a more active and less toxic responder antileukemic T cells in the pool of allogeneic donor lymphocytes.

Study of leukemic DCs gives new insights towards understanding both leukemogenesis and the physiology of DCs, and illustrate the narrow boundary between tolerance and activation. Investigators engaged in this endeavor need to bear in mind, that in AML and other hematological malignancies, the generation of potent tumor-specific cytotoxic effectors capable of tumor control, may be subsequently counterbalanced by a variety of mechanisms leading to anergy. The availability of leukemia-derived DCs and their capacity to enhance tumor recognition is a promising approach to immunotherapy in AML paving the way for investigations in other hematological malignancies. The design of a clinical DC-based vaccine immunotherapy protocol requires a concise functional characterization of DCs as well as a reflection on the crucial role of route and timing of vaccine delivery to ensure potent specific cytotoxic effectors and helper T-lymphocytes. If DC-based therapy is to be of benefit for patients, it is probable that this will be in the setting of minimal residual disease following or concomitantly to other established therapies. The optimization of DC based vaccines, ranks with the development of sensitive techniques to monitor minimal residual disease and reliable methods of measuring patient responses to DC vaccines.

Use of graft versus host natural killer cell alloreactivity in the therapy of leukemia

Loredana Ruggeri, Marusca Capanni, Elena Urbani, Katia Perruccio, Antonella Tosti, Sabrina Posati, Emanuela Burchielli, Vincenzo Fraticelli, Franco Aversa,

Massimo F. Martelli and Andrea Velardi.

Division of Hematology and Clinical Immunology,

Perugia University School of Medicine, Perugia, Italy

About 60% of patients have matched donors and fewer make it to transplant because of the delays of the donor search and bone marrow harvesting. Virtually every patient has a family member who is identical for one HLA haplotype ("haploidentical") and fully mismatched for the other, and who could immediately serve as a donor. In the past, haploidentical transplants were impossible due to unacceptable GVHD rates if they were T cell replete and very high rejection rates if they were T cell depleted. Studies in murine models showed the MHC barrier can be crossed by transplantation of high numbers of T cell depleted hematopoietic stem cells, the so-called megadose. The clinical application of this concept resulted in high engraftment rates and a low incidence of graft versus host disease (GvHD) in advanced stage acute leukemia patients (1).

Haploidentical transplants, besides requiring a higher intensity conditioning, cannot use donor T cell alloreactivity because T cells would be lethal across the HLA barrier. They rely on the megadose of stem cells for engraftment. Since T cells must be removed from the graft, potential problems of haploidentical transplants are leukemia relapse and rejection, toxicity from high-intensity conditioning and delayed immune recovery.

A further level of alloreactivity, mediated by natural killer (NK) cells, comes into play only in mismatched transplants. NK cell lysis is negatively regulated by receptors for MHC class I. Some receptors (killer cell Ig-like receptors, KIRs) are specific for epitopes shared by certain class I alleles (KIR ligands). NK cells expressing a single KIR are blocked only by a specific class I allele group. Thus NK cells make up a repertoire which has a simplified view of class I polymorphisms and can give rise to alloreactions (2).

So there are two kinds of haplo-mismatched transplants. In one the mismatched recipient class I alleles all belong to the same groups as those in the donor (this is a KIR ligand-matched transplant), and all donor NK cells will be blocked by the recipient class I molecules. There will be no NK alloreactions. In the a second kind the host class I alleles belong to different groups and are not recognized by all the NK cells in the donor repertoire. Some of the donor NK cells will not recognize the host mismatched allele and will be activated to kill the recipient target. Under these mismatch conditions the transplanted stem cells rapidly give rise to an NK cell wave which reproduces the same NK repertoire as originally present in the donor, including high-frequency alloreactive NK clones which kill cryo-preserved host cells, in the absence of GVHD (3). In vitro donor alloreactive NK clones killed myeloid leukemias. Most lymphoblastic leukemias (ALL) were resistant to alloreactive NK killing. This correlated with failure to express a major molecule involved in NK to target binding, LFA-1. These in vitro data suggest alloreactive NK cells mediate GVL effects against myeloid leukemia.

To test this hypothesis NOD-SCID mice were engrafted with human myeloid leukemias. Mice rapidly developed advanced disease and, if left untreated, or given non-alloreactive human NK clones, died over the following three weeks. In contrast, the infusion of few human alloreactive NK cells cleared leukemia. Mice went into molecular remission and 100% of them were rescued from leukemic death (4).

To determine whether donor alloreactive NK cells ablate the host T cells and condition the host for haploidentical transplantation, we developed a murine haploidentical transplant model in which one NK population from the donor mouse does not have the correct inhibitory receptor to recognize the recipient MHC and so it is alloreactive and kills recipient target cells in vitro. We infused this NK population into the recipient mouse after mild immune suppression to see whether engraftment was promoted without GVHD. Alloreactive NK cells reduced T cell and granulocyte counts in the bone marrow and spleen of the mice to what was observed after lethal irradiation. The infusion of alloreactive NK cells allowed reduced-intensity, "mini", transplants across the MHC barrier. After non-lethal irradiation or a reduced-intensity regimen based on fludarabine (as in human matched mini transplants), mice promptly rejected. They also rejected when given non-alloreactive NK cells, but after an infusion of few alloreactive NK cells they became full donor chimeras with no GVHD (4).

We next found conditioning with alloreactive NK cells ablated host dendritic cells (DCs) and prevented GVHD. DCs initiate the GVH reaction because they present host alloantigens to donor T cells which then mount an attack on host tissues. The infusion of alloreactive NK cells drastically reduced the DC number. When mice were conditioned with alloreactive NK cells they safely received up to twenty times the lethal dose of allogeneic T cells without signs of GVHD. So, the mouse models proved alloreactive NK cell ablate leukemia, condition the host to transplantation, and protect form GVHD (4).

What is the impact of donor NK cell alloreactivity on clinical transplantation? Our analysis involved 92 acute leukemia patients with either ALL or AML, 85% of whom at high risk of relapse (≥ 3^rd complete remission), or in relapse at transplant.

Patients were divided according to whether or not they had been transplanted from haploidentical donors able to mount NK alloreactions in the GVH direction, i.e., missing expression in the recipient of the class I allele groups recognized by KIRs which were present in the donors. In 100% of these pairs the donors possessed alloreactive NK clones in their repertoires. Pre-transplant HLA typing identifies donors with the potential for NK alloreactions in the graft versus host direction. 100% of the rejections all occurred in patients who had been transplanted from donors unable to mount NK alloreactions. And no rejections whatsoever occurred in patients transplanted from NK alloreactive donors. All the few cases of GVHD were confined to transplants from donors unable to mount NK alloreactions. Transplantation from NK alloreactive donors totally protected from GVHD. As predicted by the in vitro ALL resistance to alloreactive NK killing, transplantation from NK alloreactive donors had no impact on the ALL relapse rate. Our AML patients were also at high risk of relapse. When transplanted from donors who were unable to mount NK alloreactions, their probability of relapse was 75%. The probability of relapse was 0% in AML patients transplanted from NK alloreactive donors. The expected survival for this risk category of patients when transplanted from matched unrelated donors is 7%. Survival of our patients when transplanted from haploidentical donors who could not mount NK reactions is similar, being 5%. Transplantation from NK alloreactive donors had a dramatic impact on survival, which was 60% (4). The implication is that with pre-transplant HLA typing alone one can select as donor the haploidentical family member who offers such a survival advantage (4, 5).

References

1. Aversa F, Tabilio A, Velardi A, Cunningham I, Terenzi A, Falzetti F, Ruggeri L, Barbabietola G, Aristei C, Latini P, Reisner Y, and Martelli MF: Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype.

THE NEW ENGLAND JOURNAL OF MEDICINE, 339:1186, 1998

2. Sherif S. Farag, Todd A. Fehniger, Loredana Ruggeri, Andrea Velardi, and Michael A. Caligiuri. Natural Killer Cell Receptors: New Biology and Insights into the Graft versus Leukemia Effect.

BLOOD, in press

3. Ruggeri L, M Capanni, M Casucci, I Volpi, A Tosti, K Perruccio, E Urbani, RS Negrin, MF Martelli, and A Velardi. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation.

Blood 94:333-339, 1999

4. Loredana Ruggeri, Marusca Capanni, Elena Urbani, Katia Perruccio, Warren D. Shlomchik, Antonella Tosti, Sabrina Posati, Daniela Rogaia, Francesco Frassoni, Franco Aversa, Massimo F. Martelli and Andrea Velardi. Effectiveness of Donor Natural Killer Cell Alloreactivity in Mismatched Hematopoietic Transplants.

SCIENCE, 295:2097-2100, 2002.

5. Klass Krre. A perfect mismatch. SCIENCE, 295:2029-2021, 2002

Monoclonal antibody therapy of lymphomas

Ph. Solal-Céligny, Centre J. Bernard, Le Mans, France

Two main progresses have allowed to use monoclonal antibodies (MoAbs) in the treatment of lymphomas and to obtain during the last 5 years clinically significant improvements in the prognosis of these diseases.

1 - The engineering of hybrid or humanized MoAbs which are made of a small fraction of murine origin, containing the binding site to the antigen and a predominant fraction of human origin which activates complement system and antibody-dependant cellular toxicity much better than murine Abs.

2 - The choice of surface antigens of lymphoma cells which are ideal targets because of some of their properties e.g. absence of shedding, of modulation after binding of the specific MoAbs, absence of serum free antigens, no deleterious effect of specific Mo Abs on normal cells ...

Alemtuzumab (Campath 1H) is a humanized MoAb directed against CD52, an antigen found on almost all normal or malignant lymphoid cells as well as NK cells and monocytes whether of T or B origin. This antibody is now available for the treatment of chronic lymphocytic leukemia (CLL) and similar disorders (prolymphocytic leukemias). It acts mainly on the blood and marrow component of the disease, but is less active on nodal and other extranodal tumors. Its toxicity profile is mainly a risk of prolonged immunosuppression and opportunistic infections (especially HZV and CMV) due to rapid and prolonged depletion of T and B cells. The risk is greater in patients with previous immunodepression. The future development will include the treatment of residual disease after conventional chemotherapy or autologous stem cell transplantation, and immunosuppression in the context of non-myeloablative allogeneic stem cell transplantation for lymphoid or myeloid malignant diseases.

Rituximab is a hybrid MoAb directed against CD20, an antigen expressed by almost all lymphoid cells of B cell origin and all B-cell NHLs (except lymphoblastic lymphomas and plasmocell dyscrasies). CD20 antigen has all the properties of an ideal target. The mechanisms of action of rituximab are not fully understood. They include :

- an activation of the complement system, which may in part depend upon the expression of inhibitors (i.e. CD55 and/or CD59) by lymphoma cells ;

- an activation of antibody dependant cellular toxicity. It has recently been shown that this activation and consequently the clinical activity of rituximab is influenced by genetically dependant affinity of the Fcg III receptor of cytotoxic cells ;

- direct apoptosis effects on lymphoma cells which may be due to a STAT-3 mediated down-regulation of Bcl-2 protein through a down-regulation of IL-10 expression.

From these experimental studies, several new strategies are tested in order to improve the efficacy of rituximab e.g. combined treated with MoAbs directed against complement inhibition. Its clinical activity has first been demonstrated in chemoresistance low-grade B cell non Hodgkin's lymphomas (NHLs) where its use has been approved. Many new indications and/or strategies have since been developed with promising results :

- as a single treatment of patients with a low-tumor burden follicular NHL ;

- in combination with a CHOP chemotherapy in elderly patients with an aggressive B cell NHL or with advanced follicular NHLs ;

- in combination with other cytotoxic agents such as fludarabine ;

- before stem cell harvest in the context of autologous stem cell transplantation in order to treat the patient and to in vivo purge blood and bone marrow and thus obtain a lymphoma cell free graft ;

- in the treatment of some autoimmune disorders, such as idiopathic thrombocytopenic purpura ;

- in the treatment of several other B cell proliferations : Waldenström's disease, mantle-cell NHLs, marginal zone NHL, mainly in combination with conventional chemotherapy.

Other humanized Mo Abs are currently under development :

- epratuzumab, an anti CD22 MoAb, which may act synergistically with rituximab or in rituximab-refractory indolent NHLs ;

- Hu 1D10, directed against a variant of the HLA-DR B chain antigen, expressed by 60% of NHLs whether indolent or aggressive. This MoAb could be able to induce an autologous antilymphoma response, with delayed and prolonged clinical activity. This MoAb could also have activity in other solid tumors (breast, colon) which also express HLA-DR antigen.

To enhance therapeutic potency of MoAbs, investigators have conjugated them to cytotoxic radioisotopes in order to target radiation specifically to tumor sites. Radioimmunoconjugates kill tumor cells even in cases of defective host immune function, and/or if they do not express the antigen (crossfire effect from neighboring antibody-coated cells), and/or in cases with tumor penetration problems of unconjugated MoAbs. Two radionuclides are currently used : (i) I-131 which is widely accessible and easily used but which is rapidly cleared and presents a potential radiation hazard ; (ii) Ytrium-90 which can be employed on an outpatient basis (b emetter only), has a deeper penetration effect than 131-I but which does not allow imaging.

Tositumomab is a 131-I-anti CD20 murine monoclonal antibody which has been largely used by US centers but is not available in Europe. Kaminsky et al. have reported a 71% response rate (34% complete) in 59 chemoresistant patients treated with a single infusion of tositumomab. The median relapse-free survival was 12 months and 20 months for complete responders. The response rate reached 97% with 63% complete responses in 76 previously untreated patients with low-grade NHL.

Ibritumomab is a murine anti CD20 MoAb conjugated to Y-90 using the tiuxetan chelate. Witzig et al. have reported have reported a phase II trial in 51 chemoresistant patients with a 67% overall response rate and a 26% CR rate. In a randomized trial carried out on 143 patients comparing ibritumomab and rituximab, the overall response rate was higher in ibritumomab-treated patients (80% vs. 56%) as well as the the CR rate (30% vs. 16%). However, there was no significant difference in progression-free survival. Targeted radioimmunotherapy has also been used for replacing external beam total body irradiation before autologous stem cell transplantation. Very high doses of radioconjugated 131-I anti CD20 antibodies have used, and, in the most recent trials, combined with intensive chemotherapy. Using such a combined therapy, Press et al. have obtained a 73% 3-year progression-free survival in 52 patients.

Recent years have witnessed the development of a variety of promising immunotherapies for treating patients with non-Hodgkin's lymphomas. Foremost among these advances is the exciting success of monoclonal antibodies. Further studies will be required to determine the optimal settings for radiolabeled and unlabeled antibodies in the armamentarium of lymphoma therapy.

Monoclonal Antibody Therapies in Solid Tumors

Dominique Bellet

Laboratoire d'Immunologie des Tumeurs ESA 8067 CNRS, Université René Descartes - Paris 5, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, Paris et

Département de Biologie Clinique, Institut Gustave Roussy, Villejuif

Introduction

A century ago, Paul Ehrlich coined the imaginative words "magic bullets" for antibodies that target and neutralize their antigens. Seventy-five years later, Georges Köhler and Cesar Milstein invented a means of cloning individual antibodies and described the first monoclonal antibody (1). In theory, replicating the powerful defense system of antibodies paved the way for tremendous advances in the treatment of numerous deadly diseases including cancers. Indeed, when in the early 1980s, monoclonal antibodies (mAbs) first entered clinical studies, they were heralded with Paul Ehrlich's words "magic bullets" for the treatment of cancers. However, the first trials were performed with murine antibodies and it became clear that these mAbs had limited potential as therapeutics. The human immune system recognizes murine mAbs as foreign materials, producing human anti-mouse antibodies (HAMAs) to clear them from the body, thereby limiting their therapeutic benefits. Furthermore, murine mAbs are inefficient at triggering the function of effector cells (e.g., macrophages, T cells, NK cells) involved in the elimination of antibody-antigen complexes. Thus, researchers needed to design nonimmunogenic mAbs with high binding affinities that could trigger the appropriate effectors. The most obvious strategy was to make mAbs more "human-like" by creating mAbs with human protein sequences. Efforts concentrated on using genetic manipulations led to the production of human or "human-like" monoclonal antibodies (Figure 1). "Chimeric" mAbs, specifically human-murine hybrids, were described in several publications during 1984 and the first chimeric mAb entered clinical trials in 1987. However, the composition of "chimeric" antibodies which contain the original murine variable regions is still one-third mouse and two-thirds human. On the assumption that a greater percentage of native human sequences would provide a more effective therapeutic tool, "humanized" mAbs were produced in 1986 by CDR grafting and the first "humanized" mAb entered clinical studies in 1988. "Humanized" antibodies contain the original CDR murine sequences and retain about five percent of murine protein sequences. Finally, production of fully human mAbs from transgenic mice and phage display became possible in the early1990s and the first human antibody derived from transgenic mouse technologies entered clinical trials in 1999.

Clinical trials of antibodies for the treatment of solid tumors

For the most part, the recent success of mAbs for the treatment of malignancies can be attributed to advances in antibody engineering. Indeed, rituximab (Rituxan) which, in 1997, was the first mAb approved by the US FDA for the treatment of cancer (lymphoma), is a "chimeric" antibody, while trastuzumab (Herceptin), which in 1998 was the first mAb approved by the US FDA for the treatment of solid tumors (breast), is a "humanized" antibody. Since 1998, 25,000 patients have been treated with trastuzumab and this antibody was approved by European agencies in 2000. Trastuzumab is an antibody directed against, the HER-2/neu or Erb B-2 glycoprotein, a putative receptor tyrosine kinase involved in the growth and survival of breast carcinoma cells. This glycoprotein is encoded by the human epidermal growth factor receptor 2 (HER-2) gene. This gene is overexpressed by at least one fourth of human breast cancers and its overexpression correlates with poor clinical outcome. Trastuzumab, when combined with cytotoxic agents, enhances their anti-tumor activity and significantly prolongs survival in metastatic breast carcinoma. Moreover, it was reported that trastuzumab, administered as a single agent, produces durable objective responses and is well tolerated by women with HER-2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Interestingly, trastuzumab is not only the first antibody used for treating solid tumors, but it also provides the "proof of concept" of "tailored" treatments based on the genetic profile of patients. Indeed, the use of trastuzumab is recommended for patients with a strong overexpression of HER-2, as determined by a validated immunochemical technique which determines HER-2 expression or by fluorescence in situ hybridization (FISH) which analyzes HER-2 gene amplification.

A survey of clinical trials using the National Cancer Institute website shows that, in May 2002, antibody therapy is the modality of 162 clinical trials: 72 trials for the treatment of hematologic malignancies and 90 trials for the treatment of solid tumors. Amongst these latter trials, 31 trials includes trastuzumab for the treatment of various malignancies including breast, endometrial, prostate, colorectal, bladder, lung and pancreatic cancer. After trastuzumab, bevacizumab (IMC-C225) is the antibody included in the highest number (15 trials) of clinical trials. Bevacizumab is a "chimeric" human-mouse mAb directed to the epidermal growth factor receptor (EGFR or erbB-1), a cell membrane receptor with tyrosine kinase activity that triggers the intracellular signaling pathway. This antibody is included in clinical trials for the treatment of breast, head and neck, colorectal, prostatic and ovarian cancer, glioblastoma and malignant glioma. In addition, 30 antibodies either "naked" or conjugated to a drug, a toxin or a radioelement (¹¹¹In, ⁹⁰Y or ¹³¹I) are evaluated in clinical trials.

The mechanisms of antibody-induced tumor regression.

The most striking point concerning the mechanisms of antibody-induced tumor regression is, as stated by the tumor immunologists Alan Houghton and David Scheinberg, that, "after more than 15 years of experience with monoclonal antibody therapy of cancer, we have no definite idea of what mechanisms are essential for clinical activity" (2). A number of potential mechanisms have been identified that allow mAbs to operate in vivo. The majority of unconjugated mAbs now in clinical development for cancer treatment are intended to opsonize the malignant cells and allow effectors within the immune system to destroy them. Monoclonal antibody bound to antigen activates complement components, leading to opsonization of cancer cells by phagocytic cells expressing complement receptors, direct lysis of tumor cells and inflammation with recruitment of inflammatory cells. Alternatively, mAbs may bind to activating Fc receptors on effector cells, leading to antibody-dependent cellular cytotoxicity (ADCC) or release of cytokines. A third mechanism involves the direct binding of mAbs to growth factor receptors or other signaling molecules on the cancer cells, leading to cell death. Taken together, in vitro experiments and in vivo observations strongly suggest that monoclonal antibody therapies are likely to involve many mechanisms and that these complex multifunctional molecules work through multiple pathways. Trastuzumad provides a representative example of present knowledge on mechanisms involved in antibody-induced tumor regression. Three mechanisms of action are proposed : (i) Potentiation of chemotherapy, but the mechanism of synergies observed with other chemotherapy agents in vitro is unknown; (ii) inhibition of tumor cell proliferation after HER-2 protein receptor endocytosis and alterations in signaling pathways implicated in cell growth; and (iii) antibody-dependent cell mediated cytotoxicity with, eventually, NK cells as effector cells. Moreover, a fourth mechanism has recently been proposed: internalization and degradation of HER-2 might increase the amount of HER protein available for loading into MHC class I molecules, subsequently leading to enhanced tumor lysis by HER-2-specific cytotoxic T lymphocytes (3).

The future of antibody therapy for solid tumors.

After two decades of skepticism, there is little doubt that antibodies will be more widely used for the treatment of solid tumors. However, several obstacles still have to be overcome for developing successful therapeutic monoclonal antibodies. In particular, it would be adventageous to engineer antibodies for non-immunogenicity, for novel effector functions and for improved pharmacokinetics. Numerous solutions have already been proposed including single chain Fv (scFv), diabodies or triabodies (Figure 1). Another key factor is the identification of novel tumor-specific antigens. In the postgenomic era, it is now becoming possible to identify genes with broad overexpression in cancer. Recently, it has been suggested that we focus on gene products potentially involved in carcinogenesis, oncogenesis and/or tumoregenesis (4). Such genes might code for target antigens recognized by antibodies and mAbs directed against these targets might be suitable therapeutic agents for the treatment of cancer. Despite advances in antibody engineering and target antigen identification, it remains unlikely that a single naked antibody would be capable of eradicating tumors and it is highly likely that a combination of several antibodies will soon become a novel treatment modality in addition to or in parallel with other treatments.

Finally, antibody therapy shows that the pessimistic view that "it will never work" might be erroneous and that the early promise of Ehrlich's "magic bullets" might thus be fulfilled after all.

References

1. J.M. Reichert. Monoclonal antibodies in the clinic. Nature Biotechnol. 19:819-822,2001

2. A.N. Houghton, D.A. Scheinberg. Monoclonal antibody therapies-a' constant' threat to cancer. Nature Med. 6::373-374,2000

3. C. M. zum Büschenfelde, C. Hermann, B. Schmidt, C. Peschel, H. Bernhard. Antihuman epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab enhances cytolytic activity of class I-restricted HER2-specific T lymphocytes against HER2-overexpressing tumor cells. Cancer Res. 62:2244-2247,2002

4. J.L. Schultze, R.H. Vonderheide. From cancer genomics to cancer immunotherapy : toward second-generation tumor antigens. Trends Immunol 22:516-523,2001

Targeting of costimulatory molecules in inflammatory bowel disease

Jan L. Ceuppens, Zhanju Liu, Stefaan Colpaert, Karel Geboes, Paul Rutgeerts

University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Belgium

Idiopathic inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract which lead to an unpredictable clinical course with a succession of exacerbations and remissions of variable intensity. The etiology and pathogenesis of human IBD remain elusive. Dysregulation of the immune system, altered luminal flora, a defective mucosal barrier, and genetically predisposing factors as well as environmental factors have been implicated in its development (Fig.1) (for review see MacDermott, 1994; Fiocchi, 1998; Papadakis and Targan, 1999).

Luminal flora

Fig. 1. Potential pathogenic events in IBD
Histological analysis has revealed that both CD and UC are characterized by an influx of activated leukocytes into inflamed mucosa, i.e., CD25+ T, B cells and CD68+ macrophages. In CD, there is a dense accumulation of activated T cells and macrophages, which in some cases are organized into typical granulomas. The earliest microscopic lesions of CD consist of epithelial patchy necrosis, mucosal microulcerations, aphthous ulcers, a naked surface of the dome (the epithelial area overlying a mucosal lymph follicle), villous abnormalities, and damage of small capillaries. In UC mainly diffuse epithelial necrosis is seen. The cellular infiltrates are more changeable and acute inflammatory events (neutrophils forming crypt abscesses) are prominent. Lymphocytes and macrophages, but not granulomas, are present (Geboes, 1994).

Several research groups have pointed out that ongoing activation of CD4⁺ T cells and macrophages in inflamed lamina propria mucosa is a central event in the immunopathology of IBD. These cells secrete proinflammatory cytokines that cause and/or facilitate tissue damage. Treatment with depleting anti-CD4 antibody induces remission in CD (Stronkhorst et al., 1997). Macrophages producing proinflammatory cytokines (e.g., tumor necrosis factor [TNF]-a, interleukin-12) infiltrate the inflamed mucosa of CD (Breese et al., 1994; Parronchi et al., 1997; Monteleone et al., 1997). Remission of CD can be induced by immune target therapy against TNF-a (Targan et al., 1997). Expression of the costimulatory B7 molecules is also prominent (Peetermans et al, 1995; Rugtveit et al, 1997). These B7 molecules are important for interaction between antigen presenting cells (APC) and T lymphocytes.

Importantly, the luminal flora is also associated with the induction, development and chronicity of IBD, although it is not currently defined as to how these antigens trigger the disease. Mucosal mononuclear cells from inflamed areas of IBD patients proliferate when exposed to autologous intestinal bacteria, whereas cells from uninvolved mucosa of the same patients and from patients in remission fail to proliferate to autologous IBD flora (Pirzer et al. 1991; Duchmann et al., 1995). CD4⁺ T cell clones isolated from inflamed mucosa of IBD patients proliferate to anaerobic Bacteroides and Bifidobacteria species (enterobacteria) as well as the isolates of aerobic intestinal flora (Duchmann et al. 1999). CD is found not to occur if the fecal stream is diverted after surgery (Rutgeerts et al., 1991; D'Haens et al., 1998). Antibiotic therapy, especially metronidazole, is a useful adjunct for distal CD and is of therapeutic benefit in extending remission (Rutgeerts et al., 1995). Experimental colitis in some animal models has also proven that luminal florae are potential triggers of the inflammatory response in the intestine (Colpaert et al, 2001). The hypothetical mechanism by which luminal flora initiates the immunopathology is shown in Fig. 2. Specific but unidentified luminal antigens, together with other predisposing factors such as a defect of the intestinal barrier and genetic susceptibility, may directly affect the host immune system in the intestine. These events lead to an exaggerated immune and inflammatory response to luminal antigens with increased secretion of proinflammatory mediators and enhanced mucosal T cell-mediated cytotoxic activity (Muller et al., 1998).

Once activation of lamina propria-T cells occurs, they produce cytokines, leading to amplification of a proinflammatory cascade and tissue damage. Numerous studies have demonstrated that CD has the immune stigma of a Th1 immune response. In contrast, UC rather represents some kind of a Th2 immune response (Fuss et al, 1996; Desreumaux et al, 1997).

Fig. 2. Hypothetical mechanisms in the immunopathogenesis of IBD
Animal models of IBD

A number of experimental animal models of IBD has been established (for review see Elson et al., 1995; Fiocchi 1998; Blumberg et al., 1999). These models provide distinct opportunities to explore the pathogenesis of human IBD.

The model that we have used is an adoptive transfer model of experimental colitis in severe combined immunodeficiency (SCID) mice. SCID mice (C.B-17 or Balb/c strain; H-2^d) with the autosomal recessive mutation on chromosome 16 are unable to rearrange antigen receptor gene segments. These animals have an intrinsic defect of T and B lymphocytes in peripheral lymphoid organs and suffer from severe combined immunodeficiency (Bosma and Carroll, 1991). An experimental colitis model in SCID mice has been established by reconstitution with syngeneic (Balb/c) CD45RB^highCD4⁺ T cells (Morrissey et al., 1993; Powrie et al., 1994a & 1994b). In this model of colitis, CD45RB^high (naïve) CD4⁺ T cells from the spleen and lymph nodes of normal mice are selected based on high expression of the surface molecule, CD45RB, and adoptively transferred into SCID recipients. SCID mice, reconstituted with syngeneic CD45RB^highCD4⁺ T cells but not CD45RB^lowCD4⁺ T cells or total unseparated CD4⁺ T cells, develop loss of body weight 3-5 weeks after T cell transfer and a wasting disease with severe colitis after 6-10 weeks of T cell reconstitution, characterized by diarrhea, and anal prolapse as well as colon enlargement with a greatly thickened colonic wall. Transmural inflammation is common in the ascending and transverse colon. The cellular inflammatory infiltrate is composed of large numbers of lymphocytes and macrophages mixed with a small population of neutrophils and eosinophils. In the mucosa, the infiltrates show a transmucosal distribution with diffuse basal lymphocytes. Epithelial lesions include ulceration and less severe lesions such as mucin depletion, loss of goblet cells, and crypt abscesses (Liu et al, 2000). All of these clinical, histopathological, and immunological features resemble those observed in human CD. This model system offers opportunities to investigate the role of pro- and anti-inflammatory mediators secreted by mucosal lymphocytes and of lymphocyte-specific molecules involved in mucosal immune responses.

The number of interferon (IFN)-g-producing cells is greatly increased in inflamed colon in colitic SCID mice. Anti-IFN-g treatment abrogates the development of severe colitis (Powrie et al., 1994). Moreover, CD45RB^high T cells from IFN-g^-/- mice fail to induce intestinal inflammation in SCID recipients (Ito et al., 1997). Tumor Necrosis Factor (TNF)-a is the most extensively characterized of the proinflammatory cytokines. It plays an important role in many aspects of local and systemic inflammatory responses. An overexpression of TNF-a is observed in the inflamed colon of colitic SCID mice and in vivo administration of anti-mouse TNF-a effectively inhibits intestinal inflammation with down-regulation of proinflammatory cytokines IFN-g and interleukin (IL)-2 and abrogation of leukocyte infiltration in the colon (Powrie et al., 1994; Mackay et al., 1998). Transfer of CD45RB^high cells from TNF-a^-/- mice (Corazza et al., 1999) further demonstrated that TNF-a is necessary to the development of severe colitis. TNF-a production by CD4⁺ T cells is not essential to the onset of colitis, but non-T cell-derived TNF-a (e.g., from macrophages) is required. We ourselves have demonstrated that interleukin-12 also has an essential role in the development of colitis in this model, as a neutralizing monoclonal antibody to interleukin-12 completely abrogated development of the disease (Liu et al, 2001). B7 costimulatory molecules on APC and/or macrophages seem to have an essential role in the induction of pathology, as blocking antibodies to B7 prevent disease induction, apparently by blocking T cell activation (Liu et al, 2001).

This adoptive transfer model of colitis also offers opportunities to study regulatory T cells (Treg).Cotransfer of the reciprocal CD45RB^lowCD4⁺ T cells with an inoculum of potentially pathogenic CD45RB^highCD4⁺ T subset inhibits disease. This immune suppression is dependent on Transforming-growth-factor (TGF)-b (Powrie et al., 1996) and on IL-10 (Asseman et al., 1999), secreted by CD45RB^low cells. These findings indicate that normal mice contain a population of TGF-b- and IL-10-producing regulatory T cells that control inflammatory responses in the gut. Administration of anti-TGF-b reverses the suppressive function of CD45RB^low cells (Powrie et al., 1996). IL-10 treatment of reconstituted SCID mice also blocks intestinal inflammation (Powrie et al., 1994). Interestingly, CD45RB^highCD4⁺ T cells from IL-10 transgenic mice fail to induce IBD-like syndrome in SCID mice (Hagenbaugh et al., 1997), but CD45RB^lowCD4⁺ T cells from IL-10^-/- mice are unable to suppress mucosal inflammation in colitic SCID mice (Asseman et al., 1999). Similar effects were also observed when CD45RB^low T cells were treated with an anti-IL-10R mAb. Recently, CD4⁺ T cells predominantly secreting TGF-b and IL-10 are designated as Th3 and Tr1 type, respectively (Groux and Powrie, 1999). These subsets of CD4⁺ T cells function as regulatory T cells to play an important role in down-regulating immune responses. Cotransfer of Tr1 cells effectively inhibits colitis in SCID mice induced by pathogenic CD45RB^highCD4⁺ T cells (Groux et al., 1997). These results strongly support the concept that TGF-b and IL-10 play an important role in the function of regulatory T cells that control inflammation responses in the gut. The cell population that regulates mucosal inflammation in this model also expresses CTLA-4 (CD152) as recently documented by Read et al (2000) and by ourselves (Liu et al 2001).

Interaction between CD40L and CD40 in IBD

CD40/CD40L have received much attention over the past years as a pivotal ligand-receptor pair in immune regulation and as being important in the pathogenesis of several diseases (Banchereau et al., 1994; Foy et al., 1996; Grewal and Flavell, 1998).

CD40L (CD154), a 33 kDa type II integral membrane glycoprotein, is a member of the TNF family, which include TNF-a, lymphotoxin, FasL, and others. The gene for human CD40L is located to the X-chromosome, position Xq26.3-Xq27.1. The human CD40L gene spans 12-13 kb of chromosomal DNA and consists of five exons. The first exon codes for the intracellular, transmembrane, and a small portion of the extracellular region, whereas exons II-V code for the rest of the extracellular domain. CD40L is transiently expressed on antigen-activated CD4+ Th cells and a small population of activated CD8+ T cells but not on resting T cells. In addition, CD40L is also expressed on other cell types after activation including mast cells, basophils, B cells, eosinophils, dendritic cells (DC), and platelets. Multiple models of polyclonal T cell activation have been demonstrated to induce CD40L expression in vitro, including phytohemagglutinin, PMA, ionomycin, and anti-CD3. Expression of CD40L on activated T cells is closely regulated by various factors. Cyclosporine A, prostaglandin E2 and glucocorticoids have been shown to inhibit its expression, whereas CD28 engagement significantly synergizes with anti-CD3 to induce and maintain CD40L expression on activated T cells. Moreover, some cytokines such as IL-2, IL-12 and IL-15 have also found to be synergistic with anti-CD3 to enhance CD40L expression.

CD40 is a 50 kDa type I membrane glycoprotein of the TNF receptor/nerve growth factor receptor superfamily, which also includes NGFR, Fas, RANK, and OX40L as examples. The mature molecule is composed of 277 amino acids (AA) with a 193-AA extracellular domain, including a 21-AA leader sequence, a 22-AA transmembrane domain, and a 62-AA intracellular tail. CD40 is mainly expressed on B cells, monocytes/macrophages, and DC. Moreover, CD40 is also expressed on activated endothelial cells (EC), fibroblasts, epithelial cells and keratinocytes (Banchereau et al., 1994; Foy et al., 1996; Grewal and Flavell, 1998).

CD40-CD40L interactions play an important role in B cell activation, APC activation and the initiation of antigen-specific T-cell responses (Fig. 3).

Anti-CD40L mAb used in murine colitis induced by rectal administration of 2,4,6-trinitrobenzene-sulfonic acid (TNBS), can effectively prevent mucosal inflammation and IFN-g production by LP-CD4+ T cells (Stuber et al., 1996). Interestingly, CD40L transgenic mice are also generated and these mice with the highest transgene copy numbers acquire a lethal IBD marked by infiltration of CD40L+ T cells and CD40+ cells into diseased tissues (Clegg et al., 1997). These data suggest that the CD40-CD40L costimulatory pathway plays a role in mucosal inflammation in the intestine. However, involvement of this receptor-ligand pair in human IBD has not been demonstrated yet. We found that CD40L is expressed on freshly isolated lamina propria T cells from patients with CD. Importantly, CD40L on these cells was functional to induce interleukin-12 and TNF production by normal monocytes, especially after interferon-gamma priming. The inclusion of a blocking monoclonal antibody to CD40L or CD40 in such cocultures significantly decreased monocyte IL-12 and TNF production. Moreover, lamina propria and peripheral blood T cells from these patients, after in vivo activation with anti-CD3, showed increased and prolonged expression of CD40L as compared with controls. Immunohistochemical analysis indicated that the number of CD40(+) and CD40L(+) cells was significantly increased in inflamed mucosa, being B cells/macrophages and CD4(+) T cells respectively. These findings thus suggest that CD40L up-regulation is involved in pathogenic cytokine production in IBD, and that blockade of CD40-CD40L interactions may have therapeutic effects for these patients.

Prevention and treatment of experimental colitis in SCID mice reconstituted with CD45RB^highCD4⁺ T cells by blocking CD40-CD40L interactions

In this experimental colitic model in SCID mice reconstituted with syngeneic CD45RB^highCD4⁺ T cells, we investigated the pathogenic role of CD40 signaling. First, expression of CD40 and CD40L in the inflamed colon was significantly increased as measured at both protein and mRNA levels by immunohistochemistry and real time RT-PCR, respectively. Administration of anti-CD40L neutralizing mAb MR1, starting immediately after CD45RB^high T cell reconstitution over an 8-wk period, completely prevented symptoms of wasting disease (Fig. 4). Intestinal mucosal inflammation was

Fig4. Effects of anti-CD40L treatment on the development of colitis. Colitic SCID mice were treated with anti-CD40L MR1 or HIg starting at the beginning of T cell transfer. The change of weight over time is expressed as percent of the original weight. Data represent the mean ± SEM of each group from 3 independent experiments.

effectively prevented, as revealed by abrogated leukocyte infiltration (i.e., CD4⁺ cells, macrophages) and decreased CD54 expression in inflamed mucosa, and strongly diminished mRNA levels of the proinflammatory cytokines IFN-g, TNF-a, and IL-12. When colitic SCID mice were treated with anti-CD40L mAb starting at 5 wk after T cell transfer up to 8 weeks, this delayed treatment still led to significant clinical and histological improvement. Treatment also down-regulated proinflammatory cytokine secretion (i.e., IL-2 and IFN-g) by isolated LP-CD4⁺ T cells when stimulated in vitro with immobilized anti-CD3e mAb and mouse mastocytoma P815 cells transfected with mouse CD80. Interestingly, colitis relapses were observed 6-7 wk post-withdrawal of anti-CD40L treatment. These data show that the CD40-CD40L interactions are essential for the Th1-mediated inflammatory responses in the bowel of colitic SCID mice. Our data, together with earlier reports showing severe intestinal mucosal inflammation in CD40L transgenic mice (Clegg et al., 1997) and less inflammation in the colon in CD40L^-/-CD45RB^high cell-reconstituted mice (de Jong, et al. 2000), also suggest that CD40-CD40L interactions may participate in the pathogenesis of IBD. Moreover, these results also indicate that blockade of CD40-CD40L interactions may have a beneficial therapeutic effect on the inflammatory cascade involved in human IBD.

Conclusions

Based on this work, a hypothetical mechanism that governs the immunopathogenesis of human IBD can now be proposed (Fig. 5). A leaky intestinal barrier with increased intestinal permeability leads to intensified absorption of luminal antigens and bacterial products which then directly activate mucosal monocytes/macrophages to secrete IL-12 and IL-15. IL-12 produced by activated mucosal macrophages induces and/or enhances Th1 immune responses. IL-15 contributes to IBD T cell activation, proliferation and cytokine secretion (Liu et al, 2000) and both IL-12 and IL-15 function as important survival factors for IBD LP-T cells. LP-T cells are activated due to exposure to dietary and microbial products, or possibly by intraluminal antigens presented by APC such as macrophages, dendritic cells and IEC in the intestinal mucosa. T cell activation leads to the expression of several activation markers such as CD40L, CD25, and CD69. Interactions of CD40L expressed on LP-T cells with CD40 on APC and macrophages induces costimulatory molecule expression such as B7 and CD54 and secretion of IL-12, IL-15 and TNF, which together further amplify immune and inflammatory responses and recruit activated leukocytes into the inflamed sites.

Lumen

Fig. 4. Hypothetical mechanisms of pathogenesis of IBD

On the basis of animal models, there is resistance to down-regulation by regulatory T cells such as Tr1 and Th3 cells (Groux and Powrie, 1999). Whether the latter deficiency is operative in humans needs to be further explored.

It is unlikely that all components of the inflammatory cascade of IBD have been identified. To date, many signal-receptor ligand pairs, e.g., B7-CD28/CTLA-4, OX40-OX40L, CD54-LFA, CD58-CD2, a4b7-MAdCAM-1, Fas-FasL interactions, have been suggested to participate in T cell effector functions. An increased expression of these accessory molecules is also present in inflamed mucosa of IBD patients and of some experimental colitis models. Therefore, LP-T cell priming in inflamed mucosa may be dependent on multiple luminal antigen and costimulatory signaling stimulation, in addition to modulation by local cytokines and chemokines. Further studies are required to define the precise mechanisms involved in mucosal T cell activation and effector responses through different accessory molecules (e.g., B7, CD28, CTLA-4, OX40, 4-1BB, RANK) and inflammatory mediators (e.g., IL-12, IL-15, IL-17, IL-18).

Several unexplained aspects merit further exploration, such as the exact mechanisms by which luminal florae induce mucosal CD4⁺ T cell and macrophage activation and expansion; whether such an inflammatory response is also initiated via the bystander activation of mucosal T and/or APC cells; potential mechanisms responsible for dichotomous pattern of Th cell commitment at the early stage of mucosal inflammation in CD versus UC; the initiating events for the cytokine cascade dominating the tissue damage; ° whether costimulatory signals are sufficient to trigger LP-T cell and APC priming and to maintain their effector responses; whether currently used immune therapy is enough to completely block the ongoing inflammatory responses.

Immune target therapy is being used for controlling the ongoing immune pathology, but these approaches are still at an early stage. With the new approaches to understand the role of cytokines, costimulatory and adhesion molecules, the research scientists and clinicians will obtain more insight in the immunopathogenesis of IBD and this may help to choose an optimal immune therapy to control the disease.

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Cytokine inhibitors in rheumatoid arthritis

Pierre Miossec, MD PhD

Department of Immunology and Rheumatology, Hôpital Edouard Herriot

69437 Lyon Cedex 03

Phone: 334-72-11-74-87; Fax: 334-72-11-74-29, E-mail: miossec@univ-lyon1.fr

The example of cytokine inhibitors in the treatment of rheumatoid arthritis (RA) is a good demonstration that acting on non-specific down stream targets may still prove beneficial. In RA, the lack of identification of a causal mechanism was felt as a major limitation for improvement of treatment. Clinical benefit was also observed in Crohn's disease where clinical expression, anatomical distribution, underlying mechanisms are very different. Thus most of the mechanisms responsible for the clinical expression, are in fact non-specific.

Reasons to block cytokines started with TNFa and IL-1. Production of active TNFa by synovium membrane cells was demonstrated in the presence of a blocking anti-TNFa polyclonal antibody. Extension of these results used a monoclonal antibody later in animal models of RA. This mouse monoclonal was modified into a chimeric antibody combining the mouse antibody part to a human constant region. The first clinical results were obtained with the cA2 antibody later renamed infliximab and registered as remicade.

Current inhibitors of TNFa:

As for the natural regulation of cytokine action and production, therapeutic control can be specific of a given cytokine, or act on a group of proinflammatory cytokines. For TNFa specific inhibitors, blocking antibodies to TNFa, which are not natural molecules represent the simplest concept. On the contrary, use of soluble receptors (p55 or p75) represents an amplification of a natural regulation since these molecules control endogenous TNFa action.

Infliximab or remicade is a chimeric anti-TNFa specific antibody, which has obtained FDA and EMEA approval for the treatment of RA and Crohn's disease. Etanercept or Enbrel is a fusion protein made of two p75 soluble TNF receptor bound to a human Fc part. It has obtained FDA approval for the treatment of RA, juvenile idiopathic arthritis and psoriatic arthritis.

Advantage or inconvenience between the exclusive inhibition of TNFa (with specific anti-TNFa antibody) or associated to that of LTa (with soluble receptors) has not been really clarified. In addition, remicade binds to membrane bound TNF leading to cell lysis in the presence of endogenous complement.

Local and systemic effects of TNFa inhibition:

The current use of remicade is based on the ATTRACT study. Methotrexate was combined or not to infliximab 3 or 10 mg/kg either every 4 or 8 weeks. From this study remicade is registered as given at 3 mg/kg every 8 weeks by IV infusions combined with Methotrexate.

In preclinical studies, Etanercept was used alone at 10 or 25 mg twice a week and compared to Methotrexate alone. Accordingly etanercept has been registered with two 25 mg s/c injection a week as RA monotherapy.

Both treatments showed a rapid effect on systemic manifestations and on levels of acute phase proteins confirming the importance of TNFa in systemic inflammation. The rapid feeling of well being is particularly impressive confirming the central effect of TNFa on brain targets, in particular on the hypothalamus.

Local actions are dominated by the anti-inflammatory effect on synovitis. Migration of cells contributes to the initiation and chronicity of the inflammatory process, leading to matrix destruction. Formation of RA synovitis is dependent on new blood vessel formation, critical for inflammatory cell migration. Synovium samples obtained from patients treated with anti-TNFa inhibitors showed a reduction of cell infiltrate and of angiogenesis.

Clinical trials have shown a reduction of destruction as measured by an inhibition of further x-rays damage. This result is the most critical in a situation with a major depression of repair capacities. It is of interest to note that in the ATTRACT study, patients considered as non-clinical responders had a protection from further joint destruction. Further studies are needed to demonstrate that such treatment can completely prevent joint damage in early disease.

Limitations and side effects:

The clinical results in RA with the two types of inhibitors showed that a proportion of about a third of the patients does not respond. Similar results were observed when looking at spontaneous TNFa production by RA synovium samples or in response to etanercept. This heterogeneity may be related to the absence of TNFa contribution in some patients and to a reduced production of TNFa in others. Such differences are partially secondary to cytokine gene polymorphisms affecting TNFa and other regulatory cytokines. Combination of other inhibitors of IL-1 or IL-17 may enhance the response incidence.

Another limitation is the progressive loss of adequate response. To some extend, increasing the dose or reducing the infusion intervals may improve the response. Antibodies to TNFa binding sites may also contribute in some patients.

More importantly, this treatment has a suspensive effect with symptoms resuming when treatment is stopped. These data suggest the contribution of other factors and of the other cell types.

TNFa is a critical molecule to control infections as showed by an increased mortality during treatment of septic shock with TNFa inhibitors. In RA, it is now clear that TNF blockade induces an immune defect characterized by opportunistic infections, tuberculosis being the most common. This appears more common with infliximab. Over 200 cases have been reported to the FDA with a 10 % mortality. Pretreatment screening is now in place.

Induction of anti-nuclear antibodies but rarely of lupus has been observed in some patients. A link between TNFa and lupus has been established in mouse. The anti-inflammatory cytokine IL-10 is directly involved in IgG and auto antibody production with a mutual inhibition between TNFa and IL-10. TNFa inhibition favors IL-10 production and a switch from a Th1 to a Th2 cytokine profile.

A possible effect on cancers and specifically lymphomas remains unclear.

A few cases of neurological manifestations have been described including demyelinating diseases. These are in line with worsening of multiple sclerosis with inhibition of TNFa . The mechanism has not been clarified, but could be related to the anatomical site and to a difference regarding the type of TNFa receptors that are involved.

TNFa and the other monocyte-derived cytokines:

In addition to TNFa, IL-1 produced also by monocyte / macrophage, is one of the major components of the inflammatory and destructive reaction. Control of IL-1 with IL-1Ra will be described elsewhere. TNFa and IL-1 are interconnected. Indeed in RA mouse models, the current concept involves TNFa in systemic and local inflammation while IL-1 rather controls local and destructive effects on bone and cartilage. The soluble type II IL-1 receptor is now ready for clinical trials.

In addition to IL-1 and TNFa, other cytokines produced also by monocytes and mesenchymal cells such as IL-18, IL-12, IL-15 are also considered as targets.

Regarding the contribution of T cells to RA, results with IL-17 have shed some new light on this once highly discussed issue. This proinflammatory cytokine is produced by RA synovial membrane, more particularly by infiltrating Th1 lymphocytes. When in contact with synoviocytes, IL-17 producing T cells have an increased ability to favor destruction and to block repair activity. Furthermore, IL-17 bone marrow contributes to juxta-articular bone destruction. Finally, IL-17 acts in synergy or additivity with TNFa and IL-1, increasing their production and action. Accordingly control of IL-17 may be beneficial in RA.

Future of inhibition of the TNFa and conclusion:

Several ways are already used or planned. As for the treatment of many diseases such as retroviral infections or malignancies, combination therapy is the next logical step. Associations with Methotrexate are already common. Interactions between the other inflammatory cytokines suggest the combined control of TNFa, IL-1 even IL-17. Intracellular targets such as p38, NFkB have been identified and their pharmacological control is the target of active research. Combination of specific and non-specific approaches may lead to increased efficacy with reduced incidence and severity of side effects.

Even though the final goal of prevention implies the discovery of the responsible agents, the benefit observed with TNFa inhibitors provides the proof for the role of cytokines and the possibility of a control of complex inflammatory diseases by acting on their balance. The simplest approach is in principle, the specific inhibition of their action. Stimulation of the endogenous production of regulatory cytokines may represent a more physiological way to modify such balance. The absence of response in some patients to these inhibitors implies to consider the level of production and its regulation of a given cytokine and its genetic control in a given patient. Eventually, it will be possible to adapt in a better way the treatment to the patient.

Large scale transcriptional profiling of MS brain lesions: identification of new targets
Dr. Rosetta Pedotti
Beckman Center for Molecular Medicine, Stanford, USA

Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) characterized by demyelination of the white matter in the brain and spinal cord (Steinman, Cell 1996). Environmental factors and a genetically determined susceptibility are both implicated in a misdirected immune response against myelin antigens. Experimental autoimmune encephalomyelitis (EAE) is the experimental model of MS and it shares with the human disease many features, such as acute, chronic and relapsing neurological dysfunction. EAE can be induced in susceptible animal species by active or adoptive immunity to certain myelin proteins. Named by Rivers in 1933 experimental allergic encephalomyelitis, in the 80s, as more was learned about cellular immunology and the T helper 1-T helper 2 (Th1-Th2) paradigm, EAE became experimental “autoimmune” encephalomyelitis, the prototypic Th1 cell-mediated autoimmune model. In EAE, as well as in MS, Th1 cells are the main orchestrator of the disease while Th2 responses, associated with allergic diseases, seems to protect from EAE. A shift of the immune response from Th1 to Th2 seems a promising therapeutic strategy for EAE and MS.
We have recently shown that the boundary between allergy and autoimmunity can be blurred: It is possible to induce "horror autotoxicus" with anaphylaxis against certain self-antigens, exemplified by myelin peptides (Pedotti et al, Nature Immunology 2001). Anaphylaxis to self appears when mice with EAE are re-exposed to certain myelin peptides during the recovery or the relapsing phase of EAE, when Th2 responses are present, and not during the acute phase of the disease. These allergic reactions to self seem to be mediated by IgG1 antibodies, which in mice, together to IgE, are able to elicit anaphylaxis. Allergic reactions to self myelin seemed to depend on whether or not a self-antigen is expressed in the thymus: We showed anaphylaxis to myelin proteolipid protein (PLP)139-151 and myelin oligodendrocyte glycoprotein (MOG)35-55, which are not expressed in the thymus, but not to PLP178-191 and myelin basic protein (MBP) Ac1-11, which are expressed in the thymus. With peptides expressed in thymus, and therefore subjected to some degree of thymic tolerance, there is resistance to the induction of anaphylaxis.
Allergy is classically defined as an immunological reaction to a foreign antigen, and, at the extreme, allergic reactions to foreign protein can lead to anaphylactic shock and death. In contrast, autoimmune diseases, such as multiple sclerosis, result from the lingering attention of the immune system toward self-proteins. In the paradigm Th1-Th2, EAE and allergy seem to be at the opposite side of the spectrum of the immune responses. Nevertheless, Th2 T cells are capable of inducing EAE with features that include eosinophilic inflammation, sometimes also present in MS. In addition, it is known that mast cells and other elements that can participate in allergic responses are present in MS lesions.
We recently performed large scale sequencing of over 11,000 transcripts from libraries derived from MS lesions, as well as gene microarray analyses of transcripts from MS lesions. We reported in two papers (Lock et al, Nature Medicine 2002; Chabas et al, Science, 2001) increased levels of transcripts in MS lesions for several genes encoding molecules traditionally associated with allergic responses, including prostaglandin D synthase, histamine receptor 1, platelet activating factor receptor (PAFR), immunoglobulin Fc e receptor 1, and tryptase. We validated these data obtained in MS brains, with real time PCR studies performed in the EAE model. These studies revealed that transcripts for tryptase, PAFR and prostaglandine D syntase (PGDS) were elevated in the central nervous system in EAE. Moreover, histamine receptor 1 (H1R) was elevated on Th1 cells reactive to myelin, and immunohistochemical staining revealed H1R and H2R in inflammatory lesions. Pyrilamine, an H1R antagonist, blocked EAE PAF plays a major role in murine anaphylaxis, where the role of the IgG1-FcgRIII-macrophage-PAF axis can be more important than that of the IgE-FceRI-mast cell-histamine axis. EAE was ameliorated in mice with disruptions of the _-chain of FcgRIII (FcgRIII-/-) and of the g-chain common to FcgRIII and FceRI (FcR g chain -/-). Furthermore, the PAFR antagonist CV6209 reduced the severity of EAE. A number of molecules that can play important roles in allergic responses were shown to participate in EAE, a model for Th1 mediated autoimmunity. Even if Th1 lymphocytes represent major contributors to the pathogenesis of EAE and MS, molecules involved in the allergic response can potently modulate the disease.
Besides revealing increased amounts of transcripts for several genes encoding molecules associated with allergic responses, microarray analysis of MS lesions, rapidly obtained at autopsy, revealed increased transcripts of genes encoding inflammatory cytokines, particularly IL-6, IL-17, IFN-g, and associated downstream pathways. Comparison of two poles of MS pathology- acute lesions with inflammation versus silent lesions without inflammation- revealed differentially transcribed genes. Some products of these genes were chosen as targets for therapy of EAE. Granulocyte Colony Stimulating Factor (G-CSF) is upregulated in acute, but not in chronic MS lesions, and the effect on ameliorating EAE is more pronounced in the acute phase, in contrast to knocking out the Ig Fc receptor common gamma chain, where the effect is greatest on chronic disease.
Microarray technology provides an image of gene expression in demyelinating lesions on an unprecedented scale. The use of large scale analysis of gene transcripts from EAE lesions has identified another molecule that seems to play a key role in demyelinating disease: osteopontin (OPN). OPN transcripts were elevated with gene microarrays analysis in spinal cords from rats paralyzed from EAE. Furthermore, large scale sequencing of cDNA libraries derived from plaques dissected from three brains of individuals with MS, indicates that OPN transcripts are among the most abundant in MS lesions. OPN expression was demonstrated in both MS and EAE lesions with immunohistochemistry. Progressive paralysis ensues in normal mice after injection of myelin oligodendrocyte glycoprotein, but in OPN-/- knockout mice injected with MOG, progressive EAE is rare and remissions are common. MOG reactive T cells in OPN-/- mice produce more IL-10 than in +/+ mice and less interferon-gamma. Therefore, OPN plays a decisive role in the development of progressive EAE. OPN may be a critical regulator of Th1 responses, which are strongly influenced by IFN-gamma, in MS and EAE. It may be an appropriate target to block development of progressive MS.
Microarray studies provide a powerful technological innovation for the simultaneous imaging of large ensembles of genes in MS tissue. A gold mine of new therapeutic targets will likely emerge from such studies.

Reference
1. Steinman, L. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell. 85, 299-302. (1996).
2. Steinman, L. Myelin-specific CD8 T cells in the pathogenesis of experimental allergic encephalitis and multiple sclerosis. J Exp Med. 194, F27-30. (2001).
3. Pedotti, R. et al. An unexpected version of horror autotoxicus: anaphylactic shock to a self-peptide. Nat Immunol. 2, 216-22. (2001).
4. Lock, C. et al. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med. 8, 500-8. (2002).
5. Chabas, D. et al. The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease. Science. 294, 1731-5. (2001).

IL-1Ra: From pathophysiology to therapeutics

Jean-Michel Dayer, Division of Immunology & Allergy, University Hospital, Switzerland

There is considerable evidence that IL-1 and TNF are key mediators of inflammation and tissue degradation in rheumatoid arthritis (RA) (1). Both cytokines may influence local and systemic disease manifestations, as well as the mechanisms that result in the destruction of cartilage and bone matrix. In particular, the effects of IL-1 on the induction of destructive enzymes such as matrix metalloproteinases and prostaglandins appear to be critical in the pathophysiology of structural joint damage. IL-1 and TNF are produced primarily by macrophages and, to a lesser extent, by fibroblasts and neutrophils. Evidence suggests that the action of T lymphocytes (TL) on monocyte-macrophages (Mf) in synovial tissue is primordial. However, immune complexes may also play a role in stimulating Mf. Direct contact between TL and Mf is the primary pathway inducing the production of IL-1 and TNF in monocytes (2). By impeding this contact, it will be possible to inhibit the production of both cytokines.

The evidence that IL-1 is associated with osteoclast activation and consequently increases bone resorption is based on the observation that (1) IL-1 induces bone resorption in vitro; (2) elevated IL-1b levels in gingival fluid correlate with the severity of bone resorption; (3) osteoporosis in elderly patients is reduced with the spontaneous expression of high IL-1Ra levels, while it increases in the presence of low IL-1Ra levels; (4) blocking of IL-1 receptors reduces the activity in human myeloma cells.

IL-1 acts on osteoblasts and bone formation by inducing the expression of RANKL (receptor activator of NF-kB ligand), also known as ODF (osteoclast differentiation factor). In turn, RANKL stimulates the differentiation of the osteoclast precursor into mature osteoclasts responsible for bone resorption (3). RANKL and IL-1 also stimulate directly mature osteoclasts. Fortunately, this system is not without an inhibitory molecule: osteoprotegerin (OPG), a RANKL antagonist that inhibits osteoclast differentiation. RANKL also induces the production of pro-inflammatory cytokines such as IL-1 and of others that stimulate and induce differentiation of T cells (e.g. IL-12 and IL-15 produced by APC). IL-1 and TNF act on marrow stromal cells and osteoclasts also to secrete OPG. However, in RA the concentration of RANKL exceeds probably that of OPG.

As far as tissue destruction is concerned, IL-1 appears to be more potent than TNF in that it induces matrix metalloproteinases (MMP) and proteoglycanases or aggrecanases. However, once more, the synergism with TNF is important.The respective roles of IL-1 and TNF in terms of tissue destruction remain to be clearly defined in the human system. For the time being, the relevance of IL-1 as crucial destructive mediator and propagator of joint inflammation is highlighted by the absence of chronic arthritis and joint erosions in IL-1b-deficient mice (4). On chondrocytes IL-1 is clearly a more potent catabolic factor than TNF. This may partly be due to the level of receptor expression on chondrocytes.

In chronic inflammation and joint destruction one of the main differences between IL-1 and TNF is probably related to their effect on the repair process. The disparate effects of IL-1 and TNF are illustrated in chronic relapsing SCW arthritis. Repeat injections of SCW activate macrophages and give rise to destructive arthritis, characterized by loss of proteoglycans, erosion of the surface and pronounced infiltration of the synovial tissue.These characteristic features of arthritis can be induced in normal mice but are severe in a TNF-deficient background, which illustrates that erosion and inflammation occur even in complete absence of TNF. In contrast, proteoglycan depletion is obvious in control litter-mates but not in IL-1b-deficient mice. In various in vitro models of human cartilage culture, explants or cell cultures,

IL-1 strongly decreases the synthesis of proteoglycans, whereas TNF has little effect (5).

In summary, IL-1 is a key mediator of rheumatic diseases because of: (1) its high capacity (greater than TNF) to induce the production of MMPs and PGE2 in synovial cells; (2) its role as mediator in bone resorption and cartilage destruction; (3) its property of decreasing the repair process; (4) its strong synergism with TNF in inducing many inflammatory genes at both local and systemic levels; (5) its genetic linkage to highly erosive RA.

The discovery of IL-1 receptor antagonist

As early as 1983/1984, before the cloning of IL-1, we suspected the presence of a potential inhibitor to IL-1. Indeed, pursuing the goal of isolating large amounts of IL-1 using the bioassay of stimulation of collagenase and prostaglandin on synovial cells, our attention was drawn to diseases associated with large amounts of monocytes, i.e. monocytic leukemia, or diseases associated with a high temperature or chronic debilitating diseases such as RA and juvenile rheumatoid arthritis. To our surprise, since the screening for IL-1 was only possible by bioassay (no immunoassays were available at that time), we failed to detect IL-1 biological activities in serum or urine of seriously ill patients suffering from the above diseases.This prompted the hypothesis that IL-1 might be masked by inhibitory molecules, and after biochemical purification a factor of ~17 kDa was isolated from the urine of patients with monocytic leukemia (6, 7).This factor specifically blocked the biological activities of IL-1, without affecting those of TNF.This was the first identification of IL-1 receptor antagonist in vivo.

In 1985 an independent observation was made by W. Arend of an inhibitor to chondrocyte and thymocyte responsiveness to IL-1 in cultured human monocytes, without identifying the mechanism of action. In 1987 we identified the mechanism of action that justified the nomenclature of 'receptor antagonist' (IL-1Ra) using the ligand-binding assay, revealing that natural purified IL-1Ra was interfering with the binding of IL-1 to lymphocytes (8). The same year, our group in collaboration with A.-M. Prieur made the first clinical observation of the variation in IL-1Ra levels in disease, high IL-1Ra levels being observed during the afebrile phase, and low levels in the febrile phase of patients with systemic juvenile rheumatoid arthritis (9).

Based on the blocking effect of natural IL-1Ra on the binding of IL-1 to the cells, IL-1Ra was cloned at Synergen in 1990 (10, 11) and we found that both IL-1Ra purified from urine, and recombinant IL-1Ra inhibited IL-1-mediated bone resorption and PGE2 production (12). Recombinant IL-1Ra was introduced in clinical trials involving patients with RA in 1991, almost eight years after its initial isolation (13, 14).

IL-1 and IL-1 receptor family

The IL-1 family is growing and consists amongst others of two proinflammatory molecules, IL-1a and IL-1b , and the specific receptor antagonist, IL-1Ra. There are two types of IL-1 receptors (IL-1R): IL-1R type I possesses a long cytoplasmic tail and is responsible for the induction of the intracellular response after binding to IL-1. IL-1a or IL-1b binding to IL-1RI results in the formation of a heterodimer with the IL-1 receptor accessory protein (IL-1AcP). The receptor contains a Toll domain, important for signal transduction.The heterodimer complex recruits IL-1 receptor-activating kinase (IRAK), and a signal is transduced to the cell nucleus through different pathways, including TRAF 6 and P1,3 kinase to IkB kinases, but also through MEKK and p38 MAPK. Thus, IL-1Ra inhibits competitively IL-1 binding to the receptor, preventing the formation of the heterodimer and consequently signal transduction. It must be noted, however, that the binding affinity of IL-1Ra to IL-1RI is as strong as the binding of IL-1a or b. However, between 70 and 80% of the binding of IL-1a or IL-1b has to be blocked for the satisfactory inhibition of their biological activities.

IL-1R type II has a short cytoplasmic domain and functions as 'decoy' receptor, leading to a distinct decrease in IL-1 available for binding to IL-1R type I. IL-1R type II is released from cells in a soluble form and by binding to IL-1 it prevents the cytokine from reaching the target cells. We observed that soluble IL-1R type II in association with IL-1Ra is highly efficacious in blocking IL-1-induced production of collagenase and PGE2, whereas soluble IL-1R type I reverses this beneficial blockade by binding to IL-1Ra (15).

Balance and imbalance between IL-1 and IL-1Ra

The relative production of IL-1 to IL-1Ra is important in biology and disease. It is likely that in patients with RA the concentration of endogenous IL-1Ra is not sufficient for regulating the effects caused by increased IL-1b levels. It has also been shown that in patients with Lyme arthritis, the ratio of IL-1b to IL-1Ra in synovial fluid is indicative of the severity of disease. Patients with the shortest time to resolution of the attack had the lowest levels of IL-1b and the highest levels of IL-1Ra. In contrast, patients with the longest time to resolution of the attack had the highest levels of IL-1b and the lowest levels of IL-1Ra.This ratio also proved crucial to the regulation of the temperature and the systemic manifestations in juvenile rheumatoid arthritis.

Animal models as examples for the role of IL-1 and IL-1Ra

The role of IL-1Ra in the potential prevention and/or treatment of disease has been determined in various animal models of destructive arthritic diseases.

Increased destruction of articular joints consecutive to the administration of IL-1: Intra-articular injections of recombinant IL-1 in rodents induced transient synovial inflammation as well as leucocyte infiltration into the joint cavity and synovial lining. It also resulted in the depletion of proteoglycans.TNF caused less extensive damage in articular cartilage.The administration of soluble IL-1 receptor markedly reduced cartilage degradation and white-blood cell infiltration.The association of both soluble IL-1 and TNF receptors enhanced the inhibition of cartilage deterioration, white blood cell infiltration and synovitis. Similar observations were made in models of murine arthritis and rat adjuvant arthritis. In contrast, in models of streptococcal cell wall (SCW) arthritis the blocking of TNF proved more efficient than the blocking of IL-1 (5).

IL-1Ra knockout animals: In collagen-induced arthritis (CIA), mice with an aberrant expression of IL-1 receptor antagonist exhibited an earlier onset with increased severity (16). In mice of BALB/cA background, but not of C57 BL/6J background, the spontaneous, early development of inflammatory arthritis was observed (17), whereas other strains of IL-1Ra-deficient mice presented first signs of arteritis (18).

IL-1Ra transgenic mice show a reduced incidence and severity of arthritis in CIA models

The genetic factor observed in animal models may be of similar importance in humans: IL-1Ra allelic polymorphism was observed when an association exists between IL-1Ra allele A2 and diseases characterized by a decrease in IL-1Ra levels and an increase in IL-1, as in the case of ulcerative colitis, alopecia reata, psoriasis and susceptibility to severe sepsis.

Cell-cell contact and induction of IL-1 and TNF

The interaction between T cells and monocyte-macrophages is believed to play an important role in the production of IL-1 and TNF in the rheumatoid joint.The subtypes of CD4+ T cells in RA synovial tissue were analyzed using CCR5 and CCR3 receptors as 'markers' of Th1 and Th2, with the caveat usually applied to such markers. Thus, Th1 cells were the predominant T cells found in the synovium,Th2 cells being much less detectable.

This finding is important in terms of cytokine production. Direct contact between Th1 clones and macrophages leads to the production of large amounts of IL-1 and TNF, whereas contact with Th2 produces IL-1Ra and very little IL-1 and TNF. Consequently, at the level of rheumatoid synovial tissue, the production of pro-inflammatory cytokines appears to overrule that of IL-1Ra. The interactions between T cells and macrophages leading to IL-1 and TNF production depend only slightly on cell-associated IL-1 and TNF, but to a great extent on other molecules on the surface of stimulated T cells, including the integrins CDll. Blocking of these cellular adhesion molecules at the systemic level, however, may be detrimental to the normal immune response to infection. CD69, an early activation antigen on T cells, is also involved. Antibodies to CD69 inhibited IL-1 production by 40%. However, the association of CD11b antibody and CD69 antibody did not block more than ~50% of the production of IL-1 or TNF (see review in ref. 2). We found the production of IL-1 and TNF by monocytes during their interaction with stimulated TL to be strongly inhibited by HDL/Apo A-I (19).

In summary, the present consensus is that IL-1 and TNF are the principal pro-inflammatory and 'pro-destructive' cytokines in RA and probably in many other chronic inflammatory diseases as well. In addition to the complex balance between pro-inflammatory and anti-inflammatory cytokines, the imbalance between IL-1 and IL-1Ra or IL-1sRII, or between TNF and TNF-sRI or TNF-sRII may be an important factor leading to chronic inflammation. Other examples of such an imbalance are [Th1]/[Th2]; [MMP]/[TIMP]; [RANK]/[OPG].

IL-1,TNF-a and their inhibitory molecules, such as IL-1Ra and TNF-sR, are naturally occurring molecules, discovered by bioassays in vitro.Their use as therapeutic biologics was based on both rational concept and hypothesis when studying the pathogenesis of human diseases.

Treatment of RA with recombinant IL-1 receptor antagonist

Recombinant human sIL-1Ra, also known as anakinra, is produced by Amgen, Inc. (USA). Anakinra has recently been approved for treatment of RA by the FDA and is now commercially available as Kineret^(r).

Dose-range study

One hundred and seventy-five patients with active RA were enrolled in a randomized, double-blind study of recombinant human IL-1Ra administered by subcutaneous injection (13, 14). The rationale for this study was that the administration of IL-1Ra would restore the normal IL-1/IL-1Ra balance and result in suppression of IL-1-mediated pathogenetic events in patients with active RA. Treatment was well tolerated, the most frequent adverse effects being injection-site reactions.

Due to the small groups of patients and the lack of a placebo control group it was not possible to draw firm conclusions regarding efficacy from this study. However, the results did suggest that daily dosing was more effective than weekly dosing with respect to the number of swollen joints, the investigator and patient assessments of disease activity, pain score and C-reactive protein levels.The findings were considered encouraging and a randomized, placebo-controlled, phase II clinical trial was undertaken.

Randomized placebo-controlled clinical trial

In this study, 472 patients with active and severe RA were recruited into a 24-week, double-blind, placebo-controlled, multi-centre study (20). Patients were randomised into one of four groups: placebo or IL-1Ra 30, 75 or 150 mg/day given by a self-administered subcutaneous injection. The primary therapeutic end point was an ACR 20% response, achieved by 27% of the placebo group compared to 43% of the IL-1Ra 150 mg/day group (p = 0.014). The clinical responses in the 150 mg/day group (p = 0.014) were statistically superior to those observed in the other treatment groups with respect to number of swollen joints (p = 0.009), tender joints (p = 0.0009), Health Assessment Questionnaire (HAQ) (p = 0.0007), ESR (p <0.0001) and CRP (p = 0.0007). The clinical responses were observed after two weeks of therapy and the maximal fall in the acute-phase response occurred during the first week of treatment. Radiologic evaluation of the hands demonstrated a statistically significant slowing in the rate of progressive joint damage following treatment, when compared to placebo.

A small subgroup of patients participating in this trial underwent a synovial biopsy before and after treatment to determine the effects of IL-1Ra on inflamed synovial tissue. There was a notable reduction in intimal layer macrophages and subintimal layer macrophages and lymphocytes following IL-1Ra 150 mg/day. These observations represent the inhibition by IL‑1Ra of biologically relevant IL-1-mediated pathogenetic effects (21).

The 178 patients with a complete set of radiographs that completed 12 months' IL-1Ra treatment demonstrated a significant reduction in the total Sharp score: from 1.82, after the first 6-month treatment period, to 1.18 after the second (p<0.001). Comparing the second 6-month treatment period to the first, a statistically significant difference in the erosion score was observed (p<0.001), suggesting that the continuation of treatment produced an accelerated benefit for the prevention of erosion formation.

Randomized clinical trial of IL-1Ra combined with methotrexate

Methotrexate (MTX) is the most widely used disease-modifying therapeutic compound in the treatment of RA. The efficacy of combining IL-1Ra treatment with MTX was evaluated in a randomized, double-blind, placebo-controlled study over 24 weeks. In patients with persistently active RA, the combination of anakinra and MTX provided signficantly greater clinical benefit than MTX alone, and it was safe and well tolerated (22).

Conclusion

The new therapeutic era, in which the two cytokines that are considered to be crucial in RA pathogenesis can be specifically targeted, permits some optimism, both for clinicians and for their patients. In a disease as heterogeneous as RA, it is possible that patients who demonstrate an inadequate therapeutic response to the targeted inhibition of one pivotal pro-inflammatory cytokine may respond well to inhibition of the other. Moreover, the prospect of developing innovative treatment protocols that simultaneously inhibit more than one key mediator in the pathophysiologic networks that influence inflammation, damage, and repair is intriguing and presents new challenges that will demand rigorous and long-term scrutiny of both efficacy and safety (23 - 26).

REFERENCES

1. Arend WP, Dayer J-M. Inhibition of the production and effects of interleukin- 1 and tumor necrosis factor a in rheumatoid arthritis. Arthritis Rheum 1995; 38: 151-60.

2. Burger D, Dayer J-M. The role of human T-lymphocyte-monocyte contact in inflammation and tissue destruction. Arthritis Res 2002; 4(suppl 3): S169-76.

3. Goldring SR, Gravallese EM. Pathogenesis of bone erosion in rheumatoid arthritis. Curr Opin Rheumatol 2000; 12: 195-199.

4. Saijo S. Asano M, Horai R, Yamamoto H, Iwakura Y. Suppression of autoimmune arthritis in interleukin-1-deficient mice in which T cell activation is impaired due to low levels of CD40 ligand and OX40 expression on T cells. Arthritis Rheum 2002; 46: 533-44.

5. van den Berg WB. Uncoupling of inflammatory and destructive mechanisms in arthritis. Sem Arthritis Rheum 2001; 30 (suppl. 2): 7-16.

6. Balavoine JF, De Rochemonteix B, Cruchaud A, Dayer JM. Collagenase and PGE2 stimulating activity (interleukin-1 like) and inhibitor in urine from a patient with monocytic leukaemia. In: Kluger MJ, Oppenheim JJ, Powanda MC, eds. The Physiological, Metabolic, and Immunologic Actions of Interleukin-1. New York: Alan R. Liss, Inc., 1985; 429-36.

7. Balavoine JF, de Rochemonteix B, Williamson K, Seckinger P, Cruchaud A, Dayer J-M. Prostaglandin E2 and collagenase production by fibroblasts and synovial cells is regulated by urine-derived human interleukin 1 and inhibitor(s). J Clin Invest 1986; 78: 1120-4.

8. Seckinger P, Lowenthal JW,Williamson K, Dayer J-M, MacDonald HR. A urine inhibitor of interleukin 1 activity that blocks ligand binding. J Immunol 1987; 139: 1546-9.

9. Prieur A-M, Kaufmann M-T, Griscelli C, Dayer J-M. Specific interleukin-1 inhibitor in serum and urine of children with systemic juvenile chronic arthritis. Lancet 1987; II: 1240-2.

10. Hannum CH,Wilcox CJ, Arend WP, et al. Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature 1990; 343: 336-40.

11. Eisenberg SP, Evans RJ, Arend WP, et al. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 1990; 343: 341-6.

12. Seckinger P, Klein-Nulend J, Alander C,Thompson RC, Dayer JM, Raisz LG. Natural and recombinant human IL-1 receptor antagonists block the effects of IL-1 on bone resorption and prostaglandin production. J Immunol 1990; 145: 4181-4.

13. Lebsack ME, Paul CC, Bloedow DC, et al. Subcutaneous IL-1 receptor antagonist in patients with rheumatoid arthritis. Arthritis Rheum 1991; 34(Suppl): S67.

14. Campion GV, Lebsack ME, Lookabaugh J, Gordon G, Catalano MA. Dose-range and dose-frequency study of recombinant human interleukin-1 receptor antgonist in patients with rheumatoid arthritis. Arthritis Rheum 1996; 39: 1092-101.

15. Burger D, Chicheportiche R, Giri J, Dayer J-M.The inhibitory activity of human interleukin-1 receptor antagonist is enhanced by type II interleukin-1 soluble receptor and hindered by type I interleukin-1 soluble receptor. J Clin Invest 1995; 96: 38-41.

16. Ma Y, Thornton S, Boivin GP, Hirsh D, Hirsch R, Hirsch E. Altered susceptibility to collagen-induced arthritis in transgenic mice with aberrant expression of IL-1 receptor antagonist. Arthritis Rheum 1998; 41: 1798-1805.

17. Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, et al. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med 2000; 191: 313-20.

18. Nicklin MJ, Hughes DE, Barton JL, Ure JM, Duff GW. Arterial inflammation in mice lacking the interleukin 1 receptor antagonist gene. J Exp Med 2000; 191: 303-12.

19. Hyka N, Dayer J-M, Modoux C et al.Apolipoprotein A-I inhibits the production of IL-1b and tumor necrosis factor-a by blocking contact-mediated activation of monocytes by T lymphocytes. Blood 2001; 97: 2381-9.

20. Bresnihan B, Alvaro-Gracia JM, Cobby M, et al.Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum 1998; 41: 2196-204.

21. Cunnane G, Madigan A, Murphy E, Fitzgerald O, Bresnihan B. The effects of treatment with interleukin-1 receptor antagonist on the inflamed synovial membrane in rheumatoid arthritis. Rheumatology 2001; 40: 62-9.

22. Cohen S, Hurd E, Cush J, Schiff M, Weinblatt ME, Moreland LW, Kremere J, Bear MB, Rich WJ, McCabe D. Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate. Arthritis Rheum 2002; 46: 614-24.

23. Bresnihan B.The prospect of treating rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. BioDrugs 2001; 15: 87-97.

24. Jiang Y, Genant HK, Watt I, et al.A multicenter, double-blind, dose-ranging, randomized and placebo-controlled study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis: radiologic progression and correlation of Genant and Larsen scoring methods. Arthritis Rheum 2000; 43:1001-9.

25. Dayer J-M, Feige U, Edwards CK, III, Burger D. Anti-interleukin-1 therapy in rheumatic diseases. Curr Opin Rheumatol 2001; 13: 170-6.

26. Dayer J-M, Bresnihan B. Targeting interleukin-1 in the treatment of rheumatoid arthritis. Arthritis Rheum 2002; 46: 574-6 (Editorial).

AUTOIMMUNE DIABETES : THE ROLE OF THE ISLET

Christian Boitard
INSERM U342
82 avenue Denfert-Rochereau
75014 Paris

The infiltration of the islets of Langerhans by lymphocytes and monocytes (insulitis) represents the histological hallmark of insulin-dependent diabetes mellitus (IDDM) both in the human and in animals. Evidence for autoimmunity in man is indirect. It relies on the detection, in diabetic and prediabetic subjects, of autoantibodies directed against islet cells, the detection of T cell responses to b cell antigens, the association with a restricted set of class II Major Histocompatibility Complex (MHC) alleles and the transient efficacy of immunotherapy at clinical onset of IDDM. Animal models have been instrumental in contributing to our current understanding of the multifactorial facets of IDDM. The overwhelming progress achieved over the last ten years in the field of IDDM are due to a large extent to studies carried in the biobreeding (BB) rat, the nonobese diabetic (NOD) mouse and in transgenic mice.

The implication of the islet of Langerhans in the pathogenesis of IDDM can be envisioned at different levels. The anatomical and physiological organization of the islet tissue governs lymphocyte homing, determines the presentation of autoantigens, the spreading of the autoimmune reaction and, finally, the clinical evolution of the autoimmune disease. The role of the islets of Langerhans has mostly received indirect experimental attention. The clustering of the autoantigens recognized by autoantibodies and by T cells to the secretory granules and the synaptic-like vesicles of the islet b cell points to a possible relationship between autoimmune development and a key function of the b cell. Treatment with exogenous insulin, diazoxide and glucose injections modulate the development of insulitis and diabetes in the BB rat and in the NOD mouse. A reduction of diabetes incidence has been obtained by prophylactic insulin treatment of BB rats and NOD mice. The transfer of diabetes by spleen cells from diabetic NOD mice or BB rats into naive recipients is delayed as well by prophylactic insulin treatment. In human IDDM, intensive intravenous insulin therapy during 15 days at clinical onset of diabetes improves endogenous insulin secretion during the first 12 months of follow up. In the human, however, prevention of diabetes in normoglycemic subjects at risk for the development of diabetes, treated by insulin, has failed. Several hypotheses can explain the beneficial effect of insulin on the development of diabetes. First, insulin modulates T lymphocytes through the insulin receptor which is expressed early on, following antigen stimulation. Insulin affects cytotoxic and helper T-cell functions through the control of intermediary metabolites and substrate oxidation. The reduction in the synthesis of insulin receptors following hyperinsulinemia during a glucose clamp is associated with a reduction in insulin-driven cytotoxic T-cell functions. According to an alternative hypothesis, insulin therapy could down regulate islet antigen expression by favoring b cell rest. Finally, insulin could act as a specific autoantigen whose administration under a tolerogenic form would desensitize the recipient.

We addressed the question of the role of the islet of Langerhans in the autoimmune process by studying NOD mice that have been deprived of b cells at 3 weeks of age following a single dose of alloxan and maintained on insulin for 6 months. The capacity of spleen cells from such mice to transfer diabetes in young irradiated recipients was lost, despite the fact that T cell mediated alloreactivity was intact. Thus, b cell antigen is necessary for maintaining the pool of autoimmune T cells. Interestingly, the development of sialitis was not affected in these mice. These data bring strong evidence that autoimmunity is determined by both the immune system and the target organ.

The precise characterisation of the b cell autoantigens which are recognized by the immune system and elicit the autoimmune process represents another major topic of interest in the field of diabetes. Surprisingly, several of the candidate antigens which have emerged from clinical and experimental studies are not exclusively expressed by b cells. They are also found in the other endocrine islet cells and in neurons (e.g. glutamic acid decarboxylase or GAD, tyrosine phosphatases etc..) or are even more widely distributed like ICA 69 or the 38 kDa imogen. Proinsulin/insulin is so far the only defined b cell specific autoantigen. Most of these autoantigens are recognized by both B and T cells. Animal models provide unvaluable tools not only to identify antigens potentially recognizable by B and T lymphocytes, but also to evaluate the role of these antigens in the autoimmune process. Evidence that supports a critical role for candidate autoantigens includes preventing or accelerating diabetes in NOD mice either by inducing immune tolerance against autoantigen, by passively transferring autoantigen-specific T cell clones or by immunization with plasmid DNAs encoding autoantigens. Further evidence has been sought by inducing an autoimmune response to islets in normal mice immunized against candidate autoantigen. In addition, targeting autoantigen expression as transgenes in antigen presenting cells (APCs), pituitary cells or pancreatic ß-cells has been shown to prevent NOD mice from developing diabetes. More recent experiments has addressed the role of autoantigens in knock out mice in which a null mutation for autoantigen-encoding genes is introduced in a target tissue.

The long but probably still incomplete list of candidate autoantigens presumably responsible for IDDM leads to postulate that there is not a unique antigen involved. Sequential epitope and antigen spreading has been demonstrated in NOD mice by studying the evolution with time of their T cell reactivities. Splenic NOD T cells seem to react first with a limited set of GAD epitopes and to progressively extend their reactivity to other peptides.This observation will need to be extended to the islet level before more definitive conclusions are drawn. The main challenge is to understand how this increasing antigenic diversity remains compatible with the specificity of the disease, the highly restricted MHC complex background associated with genetic susceptibility and the successful attempts at preventing the disease by peptide induced therapy.

Key issues need to be solved before a comprehensive and unified view of IDDM immunopathogenesis is achieved. The susceptibility genes need to be precisely identified as well the environmental elements which control their expression. Among the several candidate autoantigens which have been identified, all do not probably bear the same responsibility in the initiation and the perpetuation of the autoimmune reaction. More generallly, the natural history of the autoimmune response, its primary activation site, the role of external pathogens cross-reactive with islet autoantigens, the effector agents, the regulatory pathways which may control the autoimmune response, are still open issues. Animal models have only provided so far elements of answer. More work is required to sort out what is really essential and relevant to human IDDM. There is still a long way to go, but the reward, namely the cure or, even the better, the prevention of IDDM is at the end.

Immuno-intervention in the treatment of
acute and chronic hepatitis

Heiner Wedemeyer and Michael P. Manns

Department of Gastroenterology, Hepatology, and Endocrinology

Medizinische Hochschule Hannover

Carl-Neuberg-Str. 1

30625 Hannover

Germany

Wedemeyer.Heiner@mh-hannover.de

Infection with the hepatitis C virus (HCV) is one of the leading causes of end-stage liver disease and hepatocellular carcinoma. Worldwide, an estimated 170 million people are chronically infected with HCV. While already in the seventies there was much evidence that a non-A-non-B hepatitis virus must exist, it took until 1989 when the hepatitis C virus was identified by M. Houghton and co-workers (1). Now, 13 years after the discovery of HCV, we have detailed information on prevalence, transmission, replication, natural history and pathogenesis of the virus. Most importantly, meanwhile the majority of infected patients can be successfully treated. While chronic infection can be prevented if acute HCV is treated early with interferon alpha, pegylated interferons in combination with ribavirin can induce a sustained virological response in 50-60% of patients with chronic hepatitis C. For patients chronically infected with HCV genotypes 2 or 3, HCV infection is nowadays a curable disease. The latest development is the initiation of promising vaccine studies and thus, eradication of HCV seems to be an achievable goal for the future.

Natural History of Hepatitis C infection

Although it is widely accepted that hepatitis C virus infection is a major cause of end-stage liver disease and hepatocellular carcinoma (2), the natural history and hence the prognosis of the infection are still controversial. An accurate determination of the natural history of hepatitis C is hampered by the fact that the initial onset of infection is usually devoid of signs and symptoms, that the disease course during the chronic phase is usually unaccompanied by symptoms and that the duration to development of end-stage liver disease may exceed 30-40 years. Acute HCV-infection takes a chronic course in 50-80% of the cases. Chronic carriers usually have either only minimal or moderate hepatitis. The risk to develop liver cirrhosis may range from 0.4 to 40%. Transfusion-associated hepatitis C seems to be more aggressive leading to cirrhosis in as much as 35% of the cases after 25 years of infection if liver enzymes are persistently elevated (3). By contrast, the risk for progressive liver disease was much less in retrospectively identified cohorts of young adults infected with HCV with rates of cirrhosis ranging from 0.4% to 5% after 20 to 45 years (4;5). The outcome of hepatitis C is heavily influenced by co-existing factors such as alcohol consumption, HBV- coinfection, HIV-coinfection, genetics and other liver diseases like hemochromatosis. Overall, HCV seems to be a rather mild virus not leading to significant disease in the majority of patients as long as these additional risk factors can be avoided.

Role of innate and adaptive immune responses in hepatitis C infection

Since HCV is a non-cytopathic virus in most circumstances, the immune response almost certainly plays a central role not only for the control of the infection but also for the pathogenesis of liver disease. On one hand, symptomatic patients with acute HCV infection are more likely to recover than asymptomatic patients (4;6). Symptoms are caused by the hosts' immune system suggesting that stronger cellular immune responses are associated with viral clearance. On the other hand, patients with more severe hepatitis have a higher chance to develop liver cirrhosis and hepatocellular carcinoma (7). The histological activity of the disease is determined by qualitative and quantitative assessment of the cellular infiltrate in the liver. The immune response against HCV is complex and generated by various cell types and tissues. Both, innate and adaptive immune responses contribute to the control of HCV infection.

Innate immunity

In line with previous findings in HBV infection a recent study in HCV infection supported the importance of innate immune responses for clearance of HCV. The analysis of gene expression in liver biopsies from chimpanzees during early HCV infection showed a very early increase of IFN-response genes preceding expression of T lymphocyte surface markers by several weeks (8). In line with this studies, natural killer cell activity has been shown to be impaired in chronic HCV infection (9). However, the mechanisms of down regulation of NK cell function was unclear until recently, when it was demonstrated by two groups that binding of the HCV E2 protein to CD81 inhibits NK cell activation, cytokine production, cytotoxicity, and proliferation but had no effect on T cell function (10;11). Thus, HCV-E2-mediated inhibition of NK cells represents another strategy for the virus to evade from the host immune response and to establish itself a chronic infection.

Adaptive Immunity - Humoral immune responses

Anti-HCV antibodies against epitopes from all HCV proteins usually develop between month 2 and 8 of acute HCV infection which is quite late as compared to other viral infections. No specific antibody pattern is associated with recovery or a specific level of replication. It seems possible that escape from efficient humoral immunity might occur the longer viremia lasts. Subsequently, a more heterogeneous humoral immunity against the so called hypervariable region 1 is associated with chronicity (12). Recently, it has been demonstrated that HCV may be cleared even in the absence of any humoral immunity against envelope proteins (13). In addition, there seems to be no long-lasting protective humoral immunity against HCV. In contrast, anti-HCV antibodies even do decline after recovery from acute HCV infection to undetectable levels after two decades (14).

Passive immunization with anti-HCV immunoglobulins are currently explored for the prevention of HCV re-infection, which occurs in nearly all patients after liver transplantation. Data of clinical trials should be available within a few months.

Adaptive Immunity - Cellular immune responses

In recent years the use of new techniques like MHC class I tetramer staining and ELISPOT assays has led to the identification of an association between a multispecific, strong and maintained HCV-specific CD4+ and CD8+ T cell response and viral clearance during acute HCV infection (15). The CD4+ response is maintained for several years after recovery. The CD8+ response remains also detectable, but there are conflicting data to what extend the CD8+ response decreases over time after recovery (14;16).

Non-cytolitic inhibition of viral replication by antiviral cytokines seems to be of great importance not only for the control of HBV but also for hepatitis C infection (17). In general, resolution of HCV is associated with an early IFN-gamma response by CD8+ T cells and persistence of functionally impaired CD8+ T cells leads to chronic infection (18;19). Activated CD8+ positive T-cells can be found in 30-time higher frequencies in the liver than in the peripheral blood of chronically infected patients (20) and potentially contribute to liver disease.

Therapy of hepatitis C infection

Therapy of acute hepatitis C infection

To preserve cellular immune responses by early control of viral replication, we decided to initiate a treatment trial of acute hepatitis C infection where therapy was started within 4 months after infection. Acute hepatitis C has become a rare event after screening blood products for HCV and most patients with acute HCV infection are commonly seen first by physicians in private practice. In order to enrol such patients for our trial, we conducted a nationwide, prospective study in Germany with the support of the German Association for the Study of the Liver (21).

A total of 44 patients with a mean age of 36 years were enrolled. The average time from infection to the first signs or symptoms of hepatitis was 54 days, and the average time from infection until the start of therapy was 89 days. At the end of both therapy and follow-up, 43 patients (98 percent) had undetectable levels of HCV RNA in serum and normal serum alanine aminotransferase levels. Levels of HCV RNA became undetectable after an average of 3.2 weeks of treatment (21). Thus, our study demonstrated that chronic HCV can be prevented by early treatment of acute HCV infection with interferon alpha monotherapy for just 6 months. Importantly, no combination with ribavirin was necessary.

Therapy of chronic hepatitis C infection

Interferon alpha has been the only antiviral treatment option for patients with chronic hepatitis C infection for almost a decade, leading to a sustained virological response in less than 20% of patients. In 1998, the introduction of combination therapy of interferon alpha and ribavirin (3x3 MU IFN tiw + 1.000/1.200 mg ribavirin daily) significantly improved sustained virological response rates to more than 40% (22). Ribavirin displays no significant direct antiviral activity against HCV but rather causes an TH2-TH1-shift of cellular immune responses. Recently, pegylated interferons with longer half-lifes and more favourable pharmacokinetics improved sustained response rates to more than 55% with an acceptable safety profile (23). A multicentre trial of 1530 patients with chronic hepatitis C compared two regimes of PEG-IFN alpha-2b in combination with ribavirin to the standard therapy of 3 MU IFN alpha-2b plus 1.000 or 1.200 mg ribavirin (24). A high dose of Peg-IFN alpha-2b/ribavirin combination therapy demonstrated significant better sustained response rates especially in patients with the HCV-genotype 1. However, PEG-IFN alpha-2b was not superior to standard combination therapy in patients infected with HCV-genotype 2/3 where response rates of >80% could be achieved with either treatment regimen.

Vaccine for hepatitis C

Therapeutic vaccinations are currently explored in hepatitis B and in hepatitis C to enhance not only humoral but may be even more importantly cellular immune responses. Peptide vaccines targeting CTL epitopes and DNA vaccines combined with different adjuvants (CpG-DNA, cytokines) as well as recombinant viruses expressing HCV proteins might be promising ways to stimulate HCV-specific immune responses. We recently showed that even oral vaccination for hepatitis C using attenuated salmonella as carriers for HCV plasmids might be possible in future (25). However, for therapeutic vaccines we have to take into account that uncontrolled activation of strong cellular immune responses may also be a double-edged sword because intrahepatic inflammatory activity could potentially be worsened.

Current treatment options with interferons are quite successful but associated with significant side effects and are very costly. Not all patients may be treated and for a sub-population infected with genotype 1 and significant fibrosis, response rates are still far below 50%. Thus, the combination of direct inhibition of viral replication by new enzyme inhibitors and specific induction of immune response against HCV seems to be a promising way for immuno-intervention of hepatitis C in the future.

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13. Bassett SE, Thomas DL, Brasky KM, Lanford RE. Viral persistence, antibody to E1 and E2, and hypervariable region 1 sequence stability in hepatitis C virus-inoculated chimpanzees. J Virol 1999; 73: 1118-26.

14. Takaki A, Wiese M, Maertens G, Depla E, Seifert U, Liebetrau A, Miller JL, Manns MP, Rehermann B. Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C. Nat Med 2000; 6: 578-82.

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20. He XS, Rehermann B, Lopez-Labrador FX, Boisvert J, Cheung R, Mumm J, Wedemeyer H, Berenguer M, Wright TL, Davis MM, Greenberg HB. Quantitative analysis of hepatitis C virus-specific CD8(+) T cells in peripheral blood and liver using peptide-MHC tetramers. Proc Natl Acad Sci U S A 1999; 96: 5692-7.

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25. Wedemeyer H, Gagneten S, Davis A, Bartenschlager R, Feinstone S, Rehermann B. Oral immunization with HCV-NS3-transformed Salmonella: induction of HCV- specific CTL in a transgenic mouse model. Gastroenterology 2001; 121: 1158-66.

IMMUNO-INTERVENTION IN SEPSIS

Didier PAYEN, MD, Ph D, Anne Claire LUKASZEWICZ, MD, Valerie FAIVRE

Department of Anesthesiology & Intensive Care Medicine; Laboratoire de Recherche UFR Lariboisiere-Saint Louis.

Lariboisiere University Hospital

University Paris VII

Institut Federatif de Recherche "Circulation"

dpayen1234@aol.com

Human systemic inflammatory response (SIRS) results from a profound stimulation of the immune system. Although infection is the predominant etiology in intensive care unit, SIRS is also observed after different types of injuries such as trauma 1, ischemia-reperfusion 2, and major surgery including cardiac surgery 3. Activation of the inflammatory network is a complex process with many redundancies that involve the inflammatory mediators, the coagulation/fibrinolysis system, and the modification of the balance between system cell survival and cell death. In the past decade, wall constituents of microorganisms such as lipopolysaccharide (LPS) of cell wall of Gram negative bacteria and cell-fragments of Gram positive bacteria, have been shown to be potent activators of a wide range of cells, including monocytes/macrophages, neutrophils and endothelial cells. Upon stimulation these cells release pro-inflammatory mediators that play a critical role in the host response to invasion by micro-organisms, clear bacteria and stimulate growth factors. Inflammation is an essential feature for the host defenses but also must be kept in check. Although a lack of inflammatory response is detrimental and promotes infection, an excessive inflammatory reaction as observed in septic shock or severe sepsis can also be harmful and lead to early multiple organ failure. There is large amount of evidence derived from animals and human studies that pro-inflammatory mediators such as TNF-a and IL-1 are released after injection of LPS or when bacteria are present in blood stream 4, 12. Multiple attempts have been made to test different molecules to modulate or inhibit this pro-inflammatory response 13, but none have shown any benefit on mortality rate in septic patients despite huge expenditures by the pharmaceutical industry. Although there are many possible reasons to explain these negative results including the redundancies in the system, some investigators have questioned the "pro-inflammatory only theory".

Most biological systems have checks and balances. Similarly, during the course of sepsis, patients also undergo an anti-inflammatory response, designated as the "Compensatory Anti-inflammatory Response Syndrome (CARS) 14, 15. This is manifest that monocytes from septic patients are hyporesponsive for cytokine release 16, 17, 18 when stimulated ex-vivo. This observation supports the concept that an anti-inflammatory response may deactivate monocytes and leads to a state of "immunoparalysis". It is, however, not clear whether monocyte/macrophage hyporesponsiveness is a protective phenomenon related to an active downregulation or simply a "post battle" cellular exhaustion of the cells which may then concern both pro- and anti-inflammatory molecules. Whatever the mechanism, it appears clear that a subset of septic patients develop a prominent systemic immuno-suppressive state that might need treatment by "booster" drug promoting the pro-inflammatory response as interferon-g, IL-12, or gCSF 16. Thus, our previous attempts at inhibiting the immune response in septic patients may have been completely the opposite of what was necessary.

Monocyte activation has been extensively demonstrated as a key determinant of the inflammatory profile 16, 19, 22. It is well documented that monocytes from healthy subjects presensitized with LPS have a markedly reduced capacity to produce pro-inflammatory cytokines (especially TNF-a) in response to a second LPS stimulation 23, 24, 25. Similarly monocytes or whole blood of septic patients release minimal amounts of cytokines after LPS stimulation 16, 18, 26, 27. It is however difficult to use these observations on monocytes of healthy volunteers to explain the downregulation observed in monocytes from septic patients. In this situation, monocytes are influenced by an environment of both pro- and anti-inflammatory cytokines which by themselves may induce downregulation 28. If the early phase is mainly hyper-inflammatory, the late phase remains to be characterized. It is obvious that data derived from the analysis of monocytes from a given patient at a given time point may not necessarily be extrapolated to all patients at different time of their evolution and data is needed at multiple time points.

Taking the number of molecules and cell functions that participate in the innate immune response in sepsis, a strategic choice has to be made to select the pertinent parameters to assess the immune function.

The antigen presentation by the monocytes is recognized as a key function in sepsis mediated inflammatory response 16, 21, 24, 29, 30, 31. It has been shown that such a function as assessed by MHC class II HLA-DR expression, is depressed after sepsis and correlates with outcome and/or infection rate 30, 32, 33 and to persistence of the sepsis. This parameter is sensitive enough to be used to evaluate the impact of Ifg. Ifg given as a therapy for patients or added in vitro, corrects the depressed HLA-DR expression of monocytes from septic patients 16. Based on these observations, HLA-DR was chosen to be included on the panel to assess monocyte for the present project.

Additionally, modifications of the monocyte counterpart of T cell surface co-stimulatory molecules might amplify or account for the induced immune depression. As an example, CD80 and CD54 are essential co-stimulatory stimuli for resting cells 34, 35, 36, 37. Interactions between the CD54 molecules and CD11b/CD18 (LFA-1) synergistically increase the signal 34, 38. It is known that the absence of the full set of signals may lead to incomplete T cell activation and even to anergy. The sole reduction in HLA-DR expression might be not then sufficient to explain all the immune depression. Because of this we have decided to add CD54 to the cell surface expression of the monocytes.

Cytokines levels in plasma or in the supernatant of cultured cells can be measured to evaluate the immuno-inflammatory balance between pro- and anti inflammatory cytokines and their impact in the observed monocytes turned off functions. The choice of cytokines is difficult since all of them cannot be measured and only some appear suitable to evaluate the balance between pro and anti inflammation. Since it is logical to look at one of each pro- and anti-inflammatory cytokines, IL-10, IL-12 and TNF-a could be selected. Among the pro-inflammatory cytokines, TNF-a is selected because of its recognized key role mediating the pro-inflammatory response 5, 6, 39, 40, 41. Plasma TNF-a levels and supernatant concentrations after in vitro stimulation by LPS of whole blood or PBMC are suitable test for pro-inflammatory response of white cells. IL-12, a heterodimeric cytokine is produced by phagocyte, dendritic cells, and B lymphocytes in responses to bacteria or bacterial product 42, 43, 44. In addition, IL-12 is required for the production of Ifg by NK cells and T lymphocytes and supports the development of the TH1 phenotype of CD4+ T cells 45. Ifg enhances in turn IL-12 release by phagocytes, thereby inducing positive feedback interactions that are crucial for the activation of the phagocyte system and T cell differenciation. Recently, it has been shown that monocyte IL-12 production was severely and selectively impaired in patients developing post-operative sepsis 45. Interestingly, such an IL-12 depression preceded the onset of surgery and did not occur as a consequence of major surgery. Thus, reduced IL-12 release does not reflect a general defect in monocyte cytokine production 45. Such molecule could be a part of the development of novel therapeutic strategy to stimulate host defense mechanisms.

Among the anti-inflammatory cytokines, IL-10 seems to be the most important to study. During the last 5 years, several studies indicate that IL-10 plays important yet contrasting role in the sepsis response 46 and the expression of IL-10 may contribute to sepsis-induced leukocyte deactivation. In several animal models of sepsis, neutralization of IL-10 results in exaggerated pro-inflammatory cytokine expression and death, while administration of recombinant IL-10 confers significant therapeutic protection 47, 48. Endogenous IL-10 appears also to inhibit protective innate immune response 49 by suppressing macrophage production of cytokines 50 and inhibiting the neutrophil 51 and macrophage phagocytic and bactericidal activity in vitro 52.

In addition, experimental evidence has been recently provide for the hypothesis that modulation of sepsis responses in LPS-primed macrophages is dictated by a cytokine regulatory mechanism derived through a reciprocal regulation of IL-10 and IL-12 production in the cells 28. Although such a mechanism of control has not been shown yet in sepsis-induced immuno-depression, it seems logical to be tested in human inflammation.

In most of the published sepsis trials, the so-called "immuno-compromized" patients (AIDS; cancer, transplanted patients, patients treated by immuno-suppressive drugs) are excluded, although they are a large part of the most difficult septic patients to treat. It is reasonable to think that it is for these patients that the decision to modulate inflammatory response might the most difficult but yet the most useful. For patients having severe sepsis without being immunocompromized, the study over time of the proposed parameters will provide information on the anti-inflammatory shift of the cells, the duration and the time for recovery. The cytokines profile determine whether the mechanism of this shift of the cells from pro to anti inflammatory.

The recent development of genomic technologies allowing to screen simultaneously a large number of genes, at different time evolution and in different conditions will provide soon crucial informations about the genes induction and regression in sepsis. The pioneer papers have shown the choreography of such genes expression and have shown a "common trunk" of genes expression in response to bacterial fragments, yeast or viruses⁵³. Among gene regression, which occurs quasi simultaneously to genes induction, important genes encoding for proteins of metabolic response and antigen presentation have been found.

In conclusion, the complexity and the multifactorial aspects of the host response to microorganism, explain the difficulty to have an adapted intervention. However the recent positive clinical trials on the use of activated protein c⁵⁴ in addition to the one on steroid therapy⁵⁵ have stimulated a huge interest for drug therapy of severe sepsis. To avoid any further failure of such trials, all investigators agree to develop tools for immuno-monitoring and to have a better understanding of such host response particularly the time evolution.

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REFERENCES

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