β2-Glycoprotein I-specific T Cells Are Associated with Epitope Spread to Lupus-related Autoantibodies

Systemic lupus erythematosus (SLE) is a prototypic model for B cell epitope spread in autoimmunity. Autoantibodies to numerous and molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting the apoptotic cell-binding protein β₂-glycoprotein I (β₂GPI). Notably, mice immunized with β₂GPI and LPS display a remarkably similar pattern of autoantibody emergence to that seen in human SLE. Here, we used this model to investigate whether epitope spread to SLE-related autoantibodies is associated with a unique or limited β₂GPI-specific T cell response. We ask whether MHC class II haplotype and its associated T cell epitope restriction impact epitope spread to SLE-related autoantibodies. We found that β₂GPI/LPS-immunized mice produced similar SLE-related autoantibody profiles regardless of their β₂GPI T cell epitope specificity or MHC class II haplotype. Although β₂GPI T cell epitope specificity was clearly determined by MHC class II haplotype, a number of different β₂GPI T cell epitopes were associated with epitope spread to SLE-related autoantibodies. Notably, one β₂GPI T cell epitope (peptide 23, NTGFYLNGADSAKCT) was also recognized by T cells from an HLA-DRB1*0403⁺ autoimmune patient. These data suggest that the generation of a β₂GPI-reactive T cell response is associated with epitope spread to SLE-related autoantibodies, independent of epitope specificity or MHC class II restriction. On the basis of these findings, we propose that factors enabling a β₂GPI-reactive T cell response may predispose individuals to the development of SLE-related autoantibodies independent of their MHC class II haplotype.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of macrophages from nonobese diabetic and systemic lupus erythematosus-prone mice

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.     

Prothrombin binds to the surface of apoptotic,but not viable,cells and serves as a target of lupus anticoagulant autoantibodies

Anti-phospholipid Ab (aPL) are a heterogeneous group of autoantibodies directed against various combinations of phospholipids (PL) and PL-binding proteins. Lupus anticoagulant (LA) Ab, a subset of aPL, exhibit anticoagulant properties in vitro, but are procoagulant in vivo. Most LA Ab are specific for either beta(2)-glycoprotein I (beta(2)GPI) or prothrombin (PT), two PL-binding proteins. We have previously shown that beta(2)GPI and beta(2)GPI-dependent aPL bind specifically to apoptotic, but not viable, thymocytes. In this study, we demonstrate that PT, like beta(2)GPI, binds selectively to the surface of apoptotic, but not viable, Jurkat cells. Furthermore, PT supports the binding of systemic lupus erythematosus-derived polyclonal and murine monoclonal LA Ab to apoptotic cells. Two LA mAb, which differed dramatically in their relative affinities for PT, were studied. Although one mAb (29J3-62) had a high affinity for PT alone, the other (29I4-24) showed minimal reactivity with PT alone and required PL for elevated binding. Monovalent fragments of 29I4-24 reacted with PL-bound PT with high affinity, suggesting that this mAb recognizes a PL-dependent epitope. Despite these differences, PT-dependent binding of both mAb to apoptotic cells was 30-fold greater than that to viable cells. Moreover, binding of PT to apoptotic cells was, itself, increased in the presence of bivalent, but not monovalent, forms of either mAb. In summary, our data demonstrate the following: 1) specific binding of PT to apoptotic cells, an effect enhanced by PT-dependent LA Ab; 2) heterogeneity of PT-dependent LA Ab; and 3) potential pathogenicity of Ab of either low or high affinity for PT.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of murine lupus

Of the multiple murine models of autoimmunity, the three most closely resembling human systemic lupus erythematosus (SLE) are the MRL/lpr, New Zealand Black/White F(1), and male BXSB. Although these strains share many disease characteristics, no common cellular defect has previously been found in prediseased mice from all these strains. We show in this study that macrophages from prediseased mice of all three SLE-prone strains, as well as macrophages from mice whose genomes contribute to the development of SLE (MRL/+, New Zealand White, New Zealand Black, female BXSB, and LG/J), have an identical and profound defect in cytokine expression that is triggered by apoptotic cells. Strikingly, none of 13 nonautoimmune strains tested exhibited this defect. Given that apoptotic Ags have been increasingly recognized as the target of autoantibodies, a defect in cytokine expression that is triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity.     

Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells

Apoptotic cells are considered an important auto-antigenic source in diseases such as systemic lupus erythematosus (SLE). A human monoclonal antibody demonstrating exquisite specificity towards late-stage apoptotic cells was generated from an SLE patient. Polyreactive recognition of ribonucleoproteins Ro52 and Ro60 was observed. The antibody significantly diminished the phagocytosis of apoptotic cells and a concomitant decrease in transforming growth factor-beta (TGF-beta) secretion was observed. Light and heavy chain sequencing revealed the antibody to be in essentially germline configuration. Elicited anti-idiotypic antibodies bound distinct self-antigens and showed augmented reactivity towards apoptotic cells as well. Thus, near-germline encoded antibodies recognizing antigens externalized during the process of apoptosis can mediate a variety of potentially pathogenic effects; decreases in the phagocytic uptake of dying cells would constitute a disease-perpetuating event and stimulation of the idiotypic network could lead to intermolecular epitope spreading, increasing the range of molecular targets..     

Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor.

Anticardiolipin antibodies (aCL) found in sera from patients with SLE react with cardiolipin (CL) in the presence of a 50-kDa serum cofactor. The cofactor, which was identified to be beta 2-glycoprotein I by sequencing the N-terminal amino acids, not only enhances CL binding by antibodies in SLE but also depresses it by antibodies associated with syphilis. Cofactor-dependent binding of aCL in SLE to solid phase CL was competitively inhibited by the simultaneous addition of fluid phase CL but was unaffected by either prior or simultaneous addition of a high excess of the cofactor. Binding of aCL in syphilis to solid phase CL was competitively inhibited by either addition of the cofactor or fluid phase CL. aCL in SLE reacted with CL, PS, and PA in the presence of cofactor. In contrast, biotinyl-cofactor bound directly to these anionic phospholipids (PL) and also to PG. These results show that the cofactor-CL complex bears an epitope that confers recognition specificity for aCL in SLE, in contrast with direct CL recognition by syphilitic aCL. The direct binding of the cofactor to PL suggests that the cofactor dependence of aCL binding to PL is due to recognition by aCL of a unique epitope generated upon the formation of the cofactor-CL complex.     

DNA on membrane receptors: a target for monoclonal anti-DNA antibody induced by a nucleoprotein shed in systemic lupus erythematosus

Antibodies to double stranded (ds) DNA correlate with clinical evolution in systemic lupus erythematosus (SLE) although little is known about the immunogen and target for these antibodies, since ds DNA is poorly immunogenic. We now show that monoclonal anti DNA antibodies similar to those detected in human SLE can be produced by immunization of genetically non-autoimmune mice with a human circulating DNA-protein complex increased in the circulation of SLE patients. One such monoclonal antibody showed antinuclear reactivity, interacting with a 74 kd DNA-binding membrane protein, in reactions prevented by absorption with ds DNA cellulose. Our data suggests that anti ds DNA antibody reactions in SLE may be triggered by circulating nucleoproteins and directed toward membrane receptors capable of interacting with extracellular DNA.     

Antiphospholipid reactivity against cardiolipin metabolites occurring during endothelial cell apoptosis

We have recently shown that cardiolipin (CL) and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. In this study, we investigate the immunoreactivity to CL derivatives occurring during endothelial apoptosis in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). We compared the serum immunoreactivity to CL with that of its derivatives monolysocardiolipin (MCL), dilysocardiolipin (DCL), and hydrocardiolipin (HCL) by means of both enzyme-linked immunosorbent assay and thin-layer chromatography (TLC) immunostaining. In addition, we investigated the composition of phospholipid extracts from the plasma membrane of apoptotic endothelial cells and the binding of patients' sera to the surface of the same cells by using high-performance TLC and immunofluorescence analysis. The average reactivity to MCL was comparable with that of CL and significantly higher than that for DCL and HCL in patients studied, both in the presence or in the absence of beta2-glycoprotein I. Of relevance for the pathogenic role of these autoantibodies, immunoglobulin G from patients' sera showed an increased focal reactivity with the plasma membrane of endothelial cells undergoing apoptosis. Interestingly, the phospholipid analysis of these light membrane fractions showed an accumulation of both CL and MCL. Our results demonstrated that a critical number of acyl chains in CL derivatives is important for the binding of antiphospholipid antibodies and that MCL is an antigenic target with immunoreactivity comparable with CL in APS and SLE. Our finding also suggests a link between apoptotic perturbation of CL metabolism and the production of these antibodies.     

Use of single point mutations in domain I of beta 2-glycoprotein I to determine fine antigenic specificity of antiphospholipid autoantibodies

Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in beta(2)GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole beta(2)GPI containing single point mutations in the first domain. The binding to beta(2)GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40-43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-beta(2)GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-beta(2)GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40-43 contributes to a dominant target epitope.     

IL-1 beta-deficient mice are resistant to induction of experimental SLE

IL-1 is one of the most pleiotropic pro-inflammatory and immunostimulatory cytokines. Overproduction of IL-1 has been shown to be involved in the pathogenicity of various autoimmune inflammatory diseases, including systemic lupus erythematosus (SLE). However, the different contributions that the IL-1 agonistic molecules make in their in vivo native milieu, IL-1beta which is mainly secreted against IL-1alpha which is mainly cell-associated, have not been established. Experimental SLE can be induced in mice by injection with monoclonal anti-DNA antibodies bearing a major idiotype designated, 16/6Id. In the present study, experimental SLE was induced in mice deficient in specific IL-1 molecules, i.e. IL-1alpha(-/-), IL-1beta(-/-), IL-1alpha/beta(-/-) (double KO) and in control BALB/c mice. Mice deficient in IL-1beta , i.e. IL-1beta(-/-) and IL-1alpha/beta(-/-) mice, developed lower levels of anti-dsDNA antibodies after immunization with 16/6Id, as compared to IL-1alpha(-/-) or control BALB/c mice. Disease manifestations were milder in mice deficient in IL-1beta expression. The representative cytokine cascade that is characteristic of overt experimental SLE was also shown to be reduced in groups of mice that lacked IL-1beta as compared to mice deficient in IL-1alpha, which is mainly cell-associated. Altogether, our results point to the importance of secretable IL-1beta, rather than cell-associated IL-1alpha, in the immunostimulatory and inflammatory phenomena that mediate the pathogenesis of experimental SLE.     

A major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein autoantigen

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180-205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher's exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease.     

Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry

The origins of autoimmunity in systemic lupus erythematosus (SLE) are thought to involve both genetic and environmental factors. To identify environmental agents that could potentially incite autoimmunity, we have traced the autoantibody response in human SLE back in time, prior to clinical disease onset, and identified the initial autoantigenic epitope for some lupus patients positive for antibodies to 60 kDa Ro. This initial epitope directly cross-reacts with a peptide from the latent viral protein Epstein-Barr virus nuclear antigen-1 (EBNA-1). Animals immunized with either the first epitope of 60 kDa Ro or the cross-reactive EBNA-1 epitope progressively develop autoantibodies binding multiple epitopes of Ro and spliceosomal autoantigens. They eventually acquire clinical symptoms of lupus such as leukopenia, thrombocytopenia and renal dysfunction. These data support the hypothesis that some humoral autoimmunity in human lupus arises through molecular mimicry between EBNA-1 and lupus autoantigens and provide further evidence to suspect an etiologic role for Epstein-Barr virus in SLE.     

Anti-beta2-glycoprotein I antibodies recognizing platelet factor 4-heparin complex in antiphospholipid syndrome in patient substantiated with mouse model

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies associated with arterial and/or venous thrombosis, and recurrent abortion accompanied often by thrombocytopenia. These antibodies are heterogeneous and react against phospholipid-binding proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin. The recognition of anti-beta2-glycoprotein I (anti-beta2GPI) by platelet factor 4-heparin complex (PF4-Hc) has been previously evoked and partially confirmed by the present inhibition studies. Further, the anti-beta2-glycoprotein I antibodies were purified from a patient with primary antiphospholipid syndrome using Affi-gel-10-beta2GPI immunoaffinity chromatography. The purified anti-beta2GPI IgM as well as patient serum equally recognized PF4-Hc in ELISA mode. In order to substantiate this data and to better understand we studied an animal model using mouse active immunization with the purified human anti-beta2GPI. The mice showed a significant decrease in their platelet count. In addition the ELISA responses of the immunized mice sera were positive against both beta2GPI and PF4-Hc, substantiating the double recognition. Despite many previous reported animal model studies, this is the first time we have shown the specific recognition of anti-beta2GPI antibodies by PF4-Hc, the results in the induced mice correlating the data observed with some patients.     

Evidence for multiple shared antigenic determinants within Ro60 and other lupus-related ribonucleoprotein autoantigens in human autoimmune responses

Ab responses directed against several ribonucleoprotein (RNP) Ags are a characteristic feature of systemic lupus erythematosus (SLE). Previous work in our laboratory using mouse model systems had revealed that both epitope spreading and inherent cross-reactivity between ribonucleoproteins contributes to the observed multiple specificities in autoimmune sera. We have now extended these studies to human autoimmune responses. Using purified polyclonal and mAbs derived from SLE patients, cross-reactivity between Ro60 and SmD was demonstrated. The cross-reactive epitope was mapped to nonhomologous regions on Ro60(481-505) and SmD(88-102). Five mAbs specifically recognized apoptotic cells, demonstrated variable levels of cross-reactivity toward other nonhomologous ribonucleoprotein targets and bound multiple, nonoverlapping and nonhomologous epitopes on Ro60. Our study demonstrates that cross-reactivity between frequently targeted autoantigens is an important aspect of human systemic autoimmune responses. The presence of multiple cross-reactive epitopes on Ro60 might be important for the generation of anti-Ro60 Ab in SLE patients and in normal individuals displaying no evidence of clinical disease.     

Adhesion mechanism of human beta(2)-glycoprotein I to phospholipids based on its crystal structure.

Human beta(2)-glycoprotein I is a heavily glycosylated five-domain plasma membrane-adhesion protein, which has been implicated in blood coagulation and clearance of apoptotic bodies from the circulation. It is also the key antigen in the autoimmune disease anti-phospholipid syndrome. The crystal structure of beta(2)-glycoprotein I isolated from human plasma reveals an elongated fish-hook-like arrangement of the globular short consensus repeat domains. Half of the C-terminal fifth domain deviates strongly from the standard fold, as observed in domains one to four. This aberrant half forms a specific phospholipid-binding site. A large patch of 14 positively charged residues provides electrostatic interactions with anionic phospholipid headgroups and an exposed membrane-insertion loop yields specificity for lipid layers. The observed spatial arrangement of the five domains suggests a functional partitioning of protein adhesion and membrane adhesion over the N- and C-terminal domains, respectively, separated by glycosylated bridging domains. Coordinates are in the Protein Data Bank (accession No. 1QUB).     

Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.     

Anticardiolipin antibodies in NZW x BXSB F1 mice. A model of antiphospholipid syndrome.

NZW x BXSB F1 (W/B F1) male mice develop systemic lupus-like disease, and several autoantibodies, circulating immune complexes, and lupus nephritis become apparent. The abnormally high incidence of degenerative coronary vascular disease with myocardial infarction and thrombocytopenia due to the presence of both platelet-associated antibodies and circulating antiplatelet antibodies in this animal has been reported. We found that W/B F1 male mice produced autoantibodies against cardiolipin (aCL) and that the titer of aCL increases with age. aCL from W/B F1 male mice were mainly IgG and binding activity to cardiolipin was aCL-cofactor (beta 2-glycoprotein I (beta 2-GPI)) dependent. We developed monoclonal aCL from these animals and examined specificity of the autoantibodies. All the mAb used reacted with the negatively charged phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, and some reacted with platelets and DNA. The addition of human or mouse beta 2-GPI enhanced the titer for monoclonal aCL from the W/B F1 mice. From the results of competitive inhibition enzyme immunoassay with monoclonal aCL and purified beta 2-GPI, aCL from the W/B F1 mice recognized the complex of CL and beta 2-GPI. The W/B F1 male mouse may be an appropriate model for use in studies on the pathologic significance of aCL in patients with antiphospholipid syndrome.     

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.     

Cutting edge: lupus susceptibility interval Sle3/5 confers responsiveness to prolactin in C57BL/6 mice

Prolactin is of interest in the pathogenesis of systemic lupus erythematosus (SLE) because almost 25% of SLE patients display hyperprolactinemia, and serum prolactin correlates with disease activity in some patients. Furthermore, hyperprolactinemia causes early mortality in lupus-prone mice and induces a lupus-like phenotype in nonspontaneously autoimmune mice. We show here that the immunomodulatory effects of prolactin are genetically determined; hyperprolactinemia breaks B cell tolerance and causes a lupus-like serology in BALB/c mice expressing a transgene encoding the H chain of an anti-DNA Ab but not in C57BL/6 transgenic mice. In C57BL/6 mice that express both the H chain transgene and the lupus susceptibility interval Sle3/5, prolactin induces increased serum titers of anti-DNA Ab and glomerular Ig depositions. The increase in costimulation due to prolactin-mediated up-regulation of both CD40 on B cells and CD40L on T cells would appear to play a central role in lupus induction in this model.     

Roquinimex-mediated protection effect on the development of chronic graft-versus-host disease in mice is associated with induction of Th1 cytokine production and inhibition of proinflammatory cytokine production

Roquinimex is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models, but the mechanism is still unknown. In this study, we investigated the effect of roquinimex on chronic graft-versus-host disease (GVHD) in mice, a well-established model for human systemic lupus erythematosus (SLE). Oral administration of roquinimex significantly suppressed the development of proteinuria and ameliorated nephritis symptoms in chronic GVHD mice. In addition, renal histopathology and immunohistochemistry studies revealed reduced glomerulonephritis and decreased IgG deposition in chronic GVHD mice treated with roquinimex. Chronic GVHD is characterized by a predominance of Th2 cytokines, and proinflammatory cytokines that also play an important role in the pathology of tissue damage. Therefore, we focused on the effect of roquinimex on cytokine production. Chronic GVHD mouse splenocytes exhibited severely reduced interferon (IFN)-gamma production in response to Concanavalin (Con A) stimulation and an overt Th2 skewness. Roquinimex treatment, however, induced IFN-gamma production and restored the Th1/Th2 cytokine balance, although only a minimal effect of roquinimex on interleukin (IL)-4 secretion was observed. The production of the proinflammatory cytokines TNF-alpha and IL-1 beta by peritoneal macrophages from lipopolysaccharide (LPS)-treated GVHD mice was significantly inhibited by roquinimex treatment. These data suggested that the beneficial effect of roquinimex on lupus might, at least in part, result from a restoration of Th1/Th2 cytokine balance and inhibition of inflammatory cytokine production.