Requirement for dendritic cells in the establishment of anti-phospholipid antibodies

OBJECTIVE: The cross-presentation of cell-associated autoantigens contributes to systemic autoimmune diseases, including systemic lupus erythematosus (SLE). Little is known about the regulation of the immune response against soluble autoantigens targeted in these diseases. METHODS: We immunized the offspring of New Zealand Black and New Zealand White mice (NZB x NZW F(1)) with syngeneic dendritic cells (DC) that had macropinocytosed beta2-glycoprotein 1 (beta(2)GPI) during propagation in normal mouse serum or that had phagocytosed apoptotic thymocytes with syngeneic (murine) or xenogeneic (bovine) beta(2)GPI, which was associated to plasma membrane of the cells. Mice were in parallel immunized with apoptotic thymocytes that had associated the beta(2)GPI to their membranes in the absence of DC. The development of anti-beta(2)GPI antibodies and clinical features were monitored. RESULTS. Apoptotic cells alone, opsonized with beta(2)GPI, failed to induce anti-beta(2)GPI autoantibodies or clinical disease. In contrast, autoimmunity developed in the presence of DC. Furthermore, the syngeneic beta(2)GPI was a more effective antigen than the xenogeneic protein in re-boosted animals. CONCLUSIONS. DC effectively initiate in NZB x NZW F(1) mice self-sustaining autoimmunity against the beta(2)GPI, either associated to apoptotic cells or macropinocytosed from the serum.     

Antiphospholipid antibodies mediate autoimmunity against dying cells

The Antiphospholipid Syndrome (APS) is characterized by thrombosis and pregnancy loss, clinical events mediated by pathogenic anti-phospholipid autoantibodies (aPL). β2-glycoprotein I (β2GPI) is the major autoantigens recognized by aPL. β2GPI is a cationic protein that binds to negatively charged surfaces such as those of apoptotic cells. This feature may lead to two major events: i) immunization with β2GPI fosters the Fc-receptor-mediated uptake by antigen presenting cells of apoptotic material decorated with β2GPIand the activation ofβ2GPI-specific T cells which in turn provide help to β2GPI-specific B cells for the production of anti-β2GPI; ii) apoptotic bodies decorated with β2GPI can be opsonized by anti-β2GPI and shifted towards a pro-inflammatory clearance by macrophages; epitope spread can occur with the generation of autoimmunity against nuclear autoantigens. In the presence of a predisposing genetic background and of a particular cytokine environment (type I interferons), the sequential emergence of autoantibodies can evolve into overt clinical disease. The spectrum of clinical phenotypes of the patients can be modulated by several factors affecting the pathogenicity of anti-β2GPI (e.g. domain specificity). We conclude that dying cells may play a dual role in APS: (I) as immunogen for the induction of aPL (etiology) and (II) as targets of aPL for the chronification of inflammation and the development of autoimmune diseases (pathology).     

Tolerogenic dendritic cells specific for β2-glycoprotein-I Domain-I,attenuate experimental antiphospholipid syndrome

Tolerogenic dendritic cells (tDCs) have the potential to control the outcome of autoimmunity by modulating the immune response. The aim of this study was to uncover the tolerance efficacy attributed to beta-2-glycoprotein-I (β₂GPI) tDCs or β₂GPI domain-I (D-I) and domain-V (D-V)-tDCs in mice with antiphospholipid syndrome (APS). tDCs were pulsed with β₂GPI or D-I or D-V derivatives. Our results revealed that β₂GPI related tDCs phenotype includes CD80^high, CD86^high CD40^high MHC class II^high. The miRNA profiling encompass miRNA 23b^high, miRNA 142-3p^low and miRNA 221^low. In addition the β₂GPI related tDCs showed reduced secretion of IL-1β, IL-12 and IL-23. D-I tDCs treatment was more efficient than β₂GPI tDCs in inducing of tolerance in APS mice, manifested by lowered titers of anti- β₂GPI antibodies (Abs) and reduced percentage of fetal loss. Tolerance induction was accompanied by poor T cell response to β₂GPI, high numbers of CD4 + CD25 + FOXP3 + T-regulatory cells (Treg), reduced levels of IFNγ, IL-17 and increased expression of IL-10 and TGFβ. Tolerance was successfully transferred by Treg cells from the tolerized mice to β₂GPI immunized mice. We conclude that predominantly D-I-tDCs and β₂GPI tDCs have the potential to attenuate experimental APS by induction of Treg cells, reduction of anti- β₂GPI Abs titers and increased expression of anti-inflammatory cytokines. We suggest that β2-GPI-D-I-tDCs may offer a novel approach for developing therapy for APS patients.     

Anti-β2-glycoprotein I paratopes and β2-glycoprotein I epitopes characterization using random peptide libraries

Abstract

Studies concerning interactions between anti-β2-glycoprotein I antibodies (anti-β2GPI) and β2-glycoprotein I (β2GPI) suggest relevance of charge interactions and hydrogen bonds. However, paratope of diagnostically and clinically relevant anti-β2GPI and epitope characteristics of β2GPI, still remain unclear. The aim of our study was to determine paratope characteristics of various anti-β2GPI antibodies and epitope characteristics of β2GPI using phage display. Monoclonal IgG anti-β2GPI, purified polyclonal high avidity and low avidity IgG anti-β2GPI derived from plasma of APS patients were used to screen phage display libraries. The affinity and competition ability of selected clones were evaluated. Various heptapeptides presenting putative paratopes of anti-β2GPI and specific heptapeptides presenting putative epitopes of β2GPI were determined. Epitope presenting peptides bind to the respective anti-β2GPI and consequently interrupt antibody–antigen interaction. The amino acid composition of selected peptides confirmed the importance of hydrogen bonds and charge interactions in the binding of anti-β2GPI to the antigen. Epitopes recognized by high avidity anti-β2GPI predominately contain hydrogen bond forming side chains, while in low avidity anti-β2GPI epitope the charged side chains prevail. The alignment of selected sequences to three-dimensional antigen structure revealed that polyclonal high avidity anti-β2GPI recognize native epitopes that are accessible regardless of β2GPI's conformation whereas the epitope recognized by low avidity anti-β2GPI is cryptic and cannot be accessed when β2GPI takes the closed plasma conformation.     

Anti-β2GPI/β2GPI induced TF and TNF-α expression in monocytes involving both TLR4/MyD88 and TLR4/TRIF signaling pathways

Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2-glycoprotein I (anti-β₂GPI/β₂GPI)-induced tissue factor (TF) expression in human acute monocytic leukemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MyD88 and TRIF, in anti-β₂GPI/β₂GPI-induced the activation of human blood monocytes and THP-1 cells and the relationship among TLR4, β₂GPI and ANX2 in this process. The results showed that treatment of monocytes or THP-1 cells with anti-β₂GPI/β₂GPI complex could increase TF, MyD88, TRIF as well as TNF-α (tumor necrosis factor alpha) expression. These effects were blocked by addition of TAK-242, a blocker of signaling transduction mediated by the intracellular domain of TLR4. Moreover, TLR4/β₂GPI/ANX2 complex could be detected in THP-1 cell lysates. Overall, our results indicate that anti-β₂GPI/β₂GPI complex induced TF and TNF-α expression involving both TLR4/MyD88 and TLR4/TRIF signaling pathways and TLR4 and its adaptors might be molecular targets for therapy of antiphospholipid syndrome (APS).     

Direct and indirect T cell priming by dendritic cell vaccines

The mechanisms by which dendritic cell (DC) vaccines prime host T cells in vivo was analyzed. Mice were immunized with syngeneic bone marrow-derived DC and as surrogate antigen β-galactosidase (β-gal) was used. DC either pulsed with peptide, loaded with β-gal antigen or gene-modified induced β-gal-specific cytotoxic T lymphocytes (CTL) and moderate rejection of an in vivo challenge with β-gal expressing tumors. In addition, β-gal-specific CTL lysed the syngeneic DC that were used as vaccines. Using SCID mice reconstituted with F1 lymphocytes, direct priming by gene-modified DC vaccines was demonstrated by the presence of β-gal-specific CTL of the haplotype exclusively expressed by DC while indirect priming by host antigen-presenting cells (APC) was shown by the detection of CTL of the haplo type exclusively present on host APC and absent on DC vaccines. Since DC immunization in syngeneic mice was associated with an increase in NK1.1⁺/Ly49C⁻ cells and detectable lysis of DC in vitro by lymphokine-activated killer cells, DC vaccines appear to interact with host natural killer cells as well as with antigen-specific T cells. These effector cells in turn may lyse DC vaccines thereby leading to the release of antigens that can be taken up by host APC.     

Mixed haplotypes and autoimmunity

Why do (NZB × NZW)F₁ mice develop an autoimmune lupus-like syndrome? The second exons of the II genes of NZB and NZW are identical to their counterparts of H-2^d and H-2^u haplotypes. Several lines of evidence suggest that this allows the production of a mixed haplotype molecule, I-Eα^dEβ², and that this molecule plays a key role in the developmental of autoimmunity.     

Strain differences in the immune response of mice: II. Responses by neonatal cells in irradiated adult hosts

Lethally irradiated NZB and C57BL mice were injected with syngeneic thymus and marrow, or thymus and liver cells, and immunized with sheep red blood cells (SRBC). As in intact neonatal mice, NZB plaque-forming cell (PFC) responses were significantly higher than C57BL.

Irradiated (NZB×C57BL)F₁ and (NZB×BALB/c)F₁ hybrid mice were given mixtures of parental and syngeneic cells, and it was shown that the high NZB response to SRBC was characteristic of the newborn liver rather than of the thymus. NZB liver gave rise to more PFC against SRBC than BALB/c liver, but not more against chicken RBC. It is argued that liver cells contain genetic information regarding antibody specificity.

C57BL cells grew poorly in F₁ hosts, but the low SRBC response appeared to be characteristic of both liver and thymus.

The nomenclature of the participating cells, and their role in the development of immunological responsiveness, are discussed.

     

Anti-β2-glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

Autoantibodies characteristic for anti-phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are anti-β₂-glycoprotein I (β₂GPI) antibodies and anti-DNA antibodies, respectively, and almost half of APS cases occur in SLE. Anti-β₂GPI antibodies are recognized to play a pivotal role in inducing a prothrombotic state, but the precise mechanism has not been fully elucidated. In a widely accepted view, binding of anti-β₂GPI antibodies to cell surface β₂GPI in monocytes and endothelial cells triggers the Toll-like receptor 4-myeloid differentiation primary response 88 (TLR)-4-MyD88) signaling pathway which leads to activation of p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinases (MEK-1/ERK) and/or nuclear factor kappa B (NF-κB) and expression of tissue factor (TF). However, resting cells do not express substantial amounts of TLR-4. Previously, we generated a mouse monoclonal anti-β₂GPI antibody WB-6 and showed that it induced a prothrombotic state – including TF expression on circulating monocytes – in normal mice. In the current study, we aimed to clarify the mechanism of interaction between WB-6 and resting monocytes, and found that WB-6 exhibits binding activity to DNA and enters living monocytes or a monocytic cell line and, to a lesser extent, vascular endothelial cells. Treatment of the cells with DNase I reduced the internalization, suggesting the involvement of cell surface DNA in this phenomenon. Monocytes harboring internalized WB-6 expressed TF and tumor necrosis factor (TNF)-α which, in turn, stimulated endothelial cells to express intercellular adhesion molecule 1 (ICAM-I) and vascular cell adhesion molecule 1 (VCAM-I). These results suggest the possibility that a subset of anti-β₂GPI antibodies with dual reactivity to DNA possesses ability to stimulate DNA sensors in the cytoplasm, in addition to the cell surface receptor-mediated pathways, leading to produce proinflammatory and prothrombotic states.     

β2GPI/抗β2GPI抗体复合物激活THP-1细胞内TRIF途径

目的:观察β2GPI/抗β2GPI抗体复合物能否激活单核细胞株THP-1的TRIF途径,以探讨TRIF依赖途径在抗磷脂综合征(APS)发病机制中的作用。方法:采用一定剂量β2GPI/抗β2GPI抗体复合物刺激THP-1细胞一定时间,收集细胞总RNA及总蛋白,荧光定量PCR(RT-PCR)检测细胞TRIF mRNA水平,Western blot检测细胞TRIF蛋白表达情况;进一步观察TLR4途径抑制剂-TAK-242是否干预β2GPI/抗β2GPI抗体复合物对TRIF的诱导表达以及相关细胞因子IL-6、IL-8、TNF-α的表达。结果:β2GPI/抗β2GPI抗体复合物(100 mg/L)能够诱导THP-1细胞表达TRIF(mRNA及蛋白),并显示时间效应,分别于刺激1 h和2 h时TRIF mRNA及蛋白表达至高峰。TAK-242(5μmol/L)能够明显抑制β2GPI/抗β2GPI抗体复合物对THP-1细胞TRIF的诱导表达,同时抑制IL-6、IL-8、TNF-α等炎症因子的表达。结论:TRIF依赖的TLR4途径参与了β2GPI/抗β2GPI抗体复合物对THP-1细胞的激活,提示其在抗磷脂综合征的病理机制中发挥一定作用。     

Relative affinity of anti-protein antibodies in New Zealand mice

New Zealand mice—NZB, NZW and NZB/W F₁ hybrids—were immunized with human serum transferrin in saline. The number of responder animals, levels and relative affinity of the antibody produced to this antigen were determined. Distinct trends in those immunologic parameters were noted with strain, sex and age at immunization. The relative affinity of the antibodies produced by the strains was lower than that previously demonstrated for several other inbred mouse strains. These results are discussed with reference to the spontaneous soluble complex disease developed by the New Zealand mice.     

Immune suppression to nucleosides: differences between NZB and NZW mice

Previous studies (Y. Borel and M. C. Young, Proc. Natl. Acad. Sci. USA 1980. 77: 1593) have shown that one can raise nucleic acid-specific suppressor T cells which diminish either the T-dependent immune response in vivo or the T-independent immune response in vitro. The results presented here confirm and extend these observations in several different strains of mice. Administration of nucleoside-modified spleen cells diminishes antibody-forming cells to nucleoside in mice immunized with nucleoside linked to keyhole limpet hemocyanin (KLH). Immune suppression was obtained in all strains except SJL and NZW, which are known to be high responders to denatured DNA. Both the primary and secondary immune responses were suppressed in C57BL/6 mice. Autologous cells exhibit a different ability to function as carriers. Spleen cells are the most effective, and to a certain extent, thymus cells. In contrast, bone marrow cells and red cells fail to induce immune suppression. A strain difference was found between NZB and NZW mice in their susceptibility to immunosuppression by nucleoside-modified spleen cells. Whereas NZB mice are high responders to nucleoside-KLH, they were easily suppressed by nucleoside coupled to spleen cells. In contrast, NZW mice, although relatively low responders to nucleoside-KLH, were not suppressed by administration of nucleoside coupled to spleen cells. Both male and female (NZB × NZW)F₁ mice appeared to behave like the NZW parental strain and were resistant to immunosuppression by nucleoside-modified spleen cells. The significance of this observation for the pathogenesis of murine systemic lupus erythematosus is discussed.     

Dendritic cells generated with Flt3L and exposed to apoptotic cells lack induction of T cell anergy and Foxp3 regulatory T cell conversion in vitro

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3⁺ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.     

Pathological changes of thymic epithelial cells and autoimmune disease in NZB, NZW and (NZB × NZW)F1 mice

An extensive histological study was carried out of NZB, NZW and (NZB × NZW)F₁, (BWF₁), mice of all ages between birth and 18 months. The thymuses of these mice were compared to those of three normal mouse strains. The study of the NZW mice showed that these mice, although they only occasionally have weakly positive Coombs' tests, may develop a renal disease probably of an autoimmune nature, similar to that of the NZB and the BWF₁ mice. Mice of all the three NZ strains developed lesions of the skin, liver, intestines, lymphatic tissues and kidneys much resembling those occurring in human systemic lupus erythematosus (SLE), neonatally thymectomized mice and, with the exception of the renal changes, the lesions of graft versus host disease.

The comparative study of the thymus in autoimmune and normal strains, revealed that important changes of the large medullary epithelial cells, involved in the formation of Hassall's corpuscles, occur very early in the three autoimmune strains. In the NZB mice the large epithelial cells are severely decreased in number in the first weeks following birth. The depletion of epithelial cells could be ascribed to a secondary degeneration of these cells soon after birth.

In contrast with the NZB mice, an extensive hyperplasia of the large epithelial cells and Hassall's corpuscles was observed in the NZW and BWF₁ mice, and was already apparent in the newborn animal. Many of the epithelial aggregates seemed to have been invaded by lymphoid cells. Both epithelial cells and the lymphoid cells engaged in this process showed a variety of degenerative changes. As in the NZB, a depletion of epithelial cells occurred in a later phase, at the age of 8 months in the BWF₁ and at 1 year in the NZW. In the majority of young mice of the normal strains invasion of islands of epithelial cells by lymphoid cells may also be observed, although this process is far less extensive than in the autoimmune strains and does not result in either epithelial hyperplasia or depletion of epithelial cells.

The described phenomenon of invasion of epithelial structures in the thymus by subsequently disintegrating lymphoid cells seems to support Burnet's concept, that so-called `forbidden clones' of lymphoid cells are eliminated in the thymus.

     

Accelerated Autoimmune Disease in MRL/MpJ-Faslpr but not in MRL/MpJ Following Immunization with High Load of Syngeneic Late Apoptotic Cells

Numerous studies have shown that autoantigens may be clustered in the blebs of apoptotic cells. However, it is not yet clear in what circumstances apoptotic cells could be immunogenic rather than tolerogenic when interacting with macrophages, dendritic cells, and B cells. In order to further study this question we compared immunization of high load of syngeneic late apoptotic cells in two genetically close pro-autoimmune mice strains: MRL/MpJ and MRL/MpJ-Fas^lpr. We show that high apoptotic load could accelerate the generation of anti-dsDNA and anticardiolipin, and the extent of kidney disease, in MRL/MpJ-Fas^lpr but could not generate autoimmunity in MRL/MpJ. Thus, in this model, a high load of apoptotic cells could augment the autoimmune response in established autoimmunity, but did not generate de novo autoimmune response in pro-autoimmune mice. Taken together with previous observations, apoptotic cell load may modify autoimmune disease generating either immune inhibition and down regulation of autoimmunity or immune stimulation and acceleration of an autoimmune disease.     

β2-glycoprotein I,lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction.To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC.Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding.Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals.     

Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice

One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β₂GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β₂GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β₂GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.     

Induction of Pathogenic Anti-dsDNA Antibodies Is Controlled on the Level of B Cells in a Non-Lupus Prone Mouse Strain

The SmD1_83–119 peptide is a main target of autoantibodies and T cells in human and murine lupus, but its role in autoimmunity induction remains elusive. Therefore, female Balb/c mice and (NZW × Balb/c)F1 [CWF1] mice with identical MHC haplotype as lupus prone NZB/W mice were immunized with SmD1₈₃₋₁₁₉. Immunizations of CWF1 mice with SmD1_83–119, but not with the controls (irrelevant peptide, HEL peptide, or saline), induced anti-SmD1_83–119 and anti-dsDNA antibodies and proteinuria not present in Balb/c mice. DsDNA-specific plasma cell induction after SmD1_83–119 immunizations was confirmed by ELISPOT assays showing that the generation of dsDNA-specific antibody forming cells (AFC) was mainly driven by increased T-cell help. T-cell help for the generation of dsDNA-specific AFC was also present in saline-treated CWF1 mice but was controlled on the levels of B cells preventing autoimmunity.     

Accelerated autoimmunity and lupus nephritis in NZB mice with an engineered heterozygous deficiency in tumor necrosis factor

Development of autoimmunity and lupus nephritis in New Zealand (NZB x NZW)F1 mice, a model for human systemic lupus erythematosus (SLE), involves both MHC- and non-MHC-linked contributions. A characteristic reduced responsiveness of the Tnf gene, which derives from the NZW parent, has been considered contributory since replacement therapy modifies the course of disease. It has remained unclear whether imbalances in TNF production operate early at the level of autoimmune induction, or, whether TNF interferes with the development of glomerulonephritis independent of the ensuing autoimmunity. To directly assess if reduced TNF production alone is sufficient to exacerbate the innocuous autoimmune responses present in NZB mice, we crossed NZB mice with Tnf-deficient and normal background control mice. Unlike control groups, (NZB x Tnf(0))F1 hemizygous mice develop enhanced autoimmunity and severe renal disease similar to the (NZB x NZW)F1 mice. Autoimmune responses are associated with an early spontaneous increase in serum levels of anti-nuclear autoantibodies and hyperproliferating B cells which readily express anti-dsDNA specificities in response to polyclonal and T helper stimuli. These findings demonstrate a physiological role for TNF in suppressing the emergence of autoreactive lymphocytes in the NZB model, and indicate that defective TNF function may be causative of the autoimmune and pathological phenomena in lupus.