The effect of the autoimmunity-associated gene, PTPN22, on a BXSB-derived model of lupus.

A single nucleotide polymorphism in PTPN22 is linked to increased disease susceptibility in a range of autoimmune diseases including systemic lupus erythematosus (SLE). PTPN22 encodes the Lyp phosphatase that dampens TCR signaling and is necessary for signaling downstream of toll-like receptors in myeloid cells. To understand these dual functions in disease, we examined the impact of deficiency in PTPN22 on a spontaneous murine model of SLE. Male PTPN22 KO mice carrying BXSB chromosome 1 and the Yaa disease accelerating factor developed disease at a similar rate and severity as PTPN22 WT. In contrast, although female mice showed no differences in survival in the absence of PTPN22, autoantibody production was significantly increased and splenic populations associated with pathogenesis in this model were expanded in the PTPN22 KO group. These findings support the notion that when coupled with other predisposing autoimmunity genes, PTPN22 deficiency contributes to a predisposition to lupus pathogenesis.

Role of nucleic acid-sensing TLRs in diverse autoantibody specificities and anti-nuclear antibody-producing B cells.

Nucleic acid (NA)-sensing TLRs (NA-TLRs) promote the induction of anti-nuclear Abs in systemic lupus erythematosus. However, the extent to which other nonnuclear pathogenic autoantibody specificities that occur in lupus and independently in other autoimmune diseases depend on NA-TLRs, and which immune cells require NA-TLRs in systemic autoimmunity, remains to be determined. Using Unc93b1(3d) lupus-prone mice that lack NA-TLR signaling, we found that all pathogenic nonnuclear autoantibody specificities examined, even anti-RBC, required NA-TLRs. Furthermore, we document that NA-TLRs in B cells were required for the development of antichromatin and rheumatoid factor. These findings support a unifying NA-TLR-mediated mechanism of autoantibody production that has both pathophysiological and therapeutic implications for systemic lupus erythematosus and several other humoral-mediated autoimmune diseases. In particular, our findings suggest that targeting of NA-TLR signaling in B cells alone would be sufficient to specifically block production of a broad diversity of autoantibodies.

The Lbw2 locus promotes autoimmune hemolytic anemia.

The lupus-prone New Zealand Black (NZB) strain uniquely develops a genetically imposed severe spontaneous autoimmune hemolytic anemia (AIHA) that is very similar to the corresponding human disease. Previous studies have mapped anti-erythrocyte Ab (AEA)-promoting NZB loci to several chromosomal locations, including chromosome 4; however, none of these have been analyzed with interval congenics. In this study, we used NZB.NZW-Lbw2 congenic (designated Lbw2 congenic) mice containing an introgressed fragment of New Zealand White (NZW) on chromosome 4 encompassing Lbw2, a locus previously linked to survival, glomerulonephritis, and splenomegaly, to investigate its role in AIHA. Lbw2 congenic mice exhibited marked reductions in AEAs and splenomegaly but not in anti-nuclear Abs. Furthermore, Lbw2 congenics had greater numbers of marginal zone B cells and reduced expansion of peritoneal cells, particularly the B-1a cell subset at early ages, but no reduction in B cell response to LPS. Analysis of a panel of subinterval congenic mice showed that the full effect of Lbw2 on AEA production was dependent on three subloci, with splenomegaly mapping to two of the subloci and expansions of peritoneal cell populations, including B-1a cells to one. These results directly demonstrated the presence of AEA-specific promoting genes on NZB chromosome 4, documented a marked influence of background genes on autoimmune phenotypes related to Lbw2, and further refined the locations of the underlying genetic variants. Delineation of the Lbw2 genes should yield new insights into both the pathogenesis of AIHA and the nature of epistatic interactions of lupus-modifying genetic variants.

Combined loss of proapoptotic genes Bak or Bax with Bim synergizes to cause defects in hematopoiesis and in thymocyte apoptosis.

The proapoptotic members of the Bcl-2 family can be subdivided into members that contain several Bcl-2 homology (BH) domains and those that contain only the BH3 domain. Although it is known that BH3-only proteins and the multi-BH domain proteins, Bak and Bax, are essential for programmed cell death, the overlapping role of these two subgroups has not been examined in vivo. To investigate this, we generated Bak/Bim and Bax/Bim double deficient mice. We found that although Bax-/-Bim-/-, but not Bak-/-Bim-/-, mice display webbed hind and front paws and malocclusion of the incisors, both groups of mice present with dysregulated hematopoiesis. Combined loss of Bak and Bim or Bax and Bim causes defects in myeloid and B-lymphoid development that are more severe than those found in the single knock-out mice. Bak-/-Bim-/- mice have a complement of thymocytes that resembles those in control mice, whereas Bax-/-Bim-/- mice are more similar to Bim-/- mice. However, thymocytes isolated from Bak-/-Bim-/- or Bax-/-Bim-/- mice are markedly more resistant to apoptotic stimuli mediated by the intrinsic pathway as compared with thymocytes from single-knockout mice. These data suggest an essential overlapping role for Bak or Bax and Bim in the intrinsic apoptotic pathway.

The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

IL-4 and IL-10 inhibition of spontaneous monocyte apoptosis is associated with Flip upregulation.

Human peripheral blood monocytes undergo spontaneous apoptosis in culture. Spontaneous monocyte apoptosis is regulated by the death ligand, Fas Ligand (FasL) binding to its receptor Fas. The pro-inflammatory molecules, LPS and IL-1beta, prevent spontaneous monocyte apoptosis. Here, we demonstrate that the anti-inflammatory cytokines IL-4 and IL-10 inhibit spontaneous monocyte apoptosis compared to control-treated cells. IL-4- or IL-10-mediated suppression of spontaneous monocyte apoptosis is associated with the induction of Flip, an essential inhibitor of the Fas-death signal. In contrast, IL-4 and IL-10 inhibit LPS or IL-1beta induced pro-inflammatory cytokine production. These data suggest that in monocytes IL-4 or IL-10 has a dual function, to inhibit pro-inflammatory cytokine production and to suppress spontaneous apoptosis.

Systemic autoimmunity and lymphoproliferation are associated with excess IL-7 and inhibited by IL-7Rα blockade.

Lupus is characterized by disturbances in lymphocyte homeostasis, as demonstrated by the marked accumulation of activated/memory T cells. Here, we provide evidence that proliferation of the CD8+ precursors for the accumulating CD4⁻CD8⁻ T cells in MRL-Fas(lpr) lupus-predisposed mice is, in part, driven by commensal antigens. The ensuing lymphadenopathy is associated with increased production of IL-7 due to expansion of fibroblastic reticular cells, the primary source of this cytokine. The excess IL-7 is not, however, consumed by CD4⁻CD8⁻ T cells due to permanent down-regulation of IL-7Rα (CD127), but instead supports proliferation of autoreactive T cells and progression of autoimmunity. Accordingly, IL-7R blockade reduced T cell activation and autoimmune manifestations even when applied at advanced disease stage. These findings indicate that an imbalance favoring production over consumption of IL-7 may contribute to systemic autoimmunity, and correction of this imbalance may be a novel therapeutic approach in lymphoproliferative and autoimmune syndromes.

Bim-Bcl-2 homology 3 mimetic therapy is effective at suppressing inflammatory arthritis through the activation of myeloid cell apoptosis.

OBJECTIVE: Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies that activate the apoptotic cascade may have potential for use in RA; however, few therapeutic agents fit this category. The purpose of this study was to examine the potential of Bim, an agent that mimics the action of Bcl-2 homology 3 (BH3) domain-only proteins that have shown success in preclinical studies of cancer, in the treatment of autoimmune disease. METHODS: Synovial tissues from RA and osteoarthritis patients were analyzed for the expression of Bim and CD68 using immunohistochemistry. Macrophages from Bim(-/-) mice were examined for their response to lipopolysaccharide (LPS) using flow cytometry, real-time polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and immunoblotting. Bim(-/-) mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum-transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT-BH3) was administered as a prophylactic agent and as a therapeutic agent. Edema of the ankles and histopathologic analysis of ankle tissue sections were used to determine the severity of arthritis, its cellular composition, and the degree of apoptosis. RESULTS: The expression of Bim was reduced in RA synovial tissue as compared with controls, particularly in macrophages. Bim(-/-) macrophages displayed elevated expression of markers of inflammation and secreted more interleukin-1beta following stimulation with LPS or thioglycollate. TAT-BH3 ameliorated arthritis development, reduced the number of myeloid cells in the joint, and enhanced apoptosis without inducing cytotoxicity. CONCLUSION: These data demonstrate that BH3 mimetic therapy may have significant potential for the treatment of RA.

Combined deficiency of proapoptotic regulators Bim and Fas results in the early onset of systemic autoimmunity.

Alterations in the stoichiometric balance between members of Bcl-2 and Fas apoptotic pathway could lead to the pathogenesis of systemic lupus erythematosus (SLE). We showed that patients with SLE displayed increased expression in antiapoptotic members of the Bcl-2 and Fas apoptotic pathways in isolated mononuclear cells. Further, mice (Bcl2l11(-/-)Fas(lpr/lpr)) lacking the Bcl-2 pro-apoptotic member, Bim (Bcl2l11(-/-)) and and with an lpr mutation in the gene encoding Fas (Fas(lpr/lpr)) developed severe SLE-like disease by 16 weeks of age unlike Bcl2l11(-/-) or Fas(lpr/lpr) mice. Bcl2l11(-/-)Fas(lpr/lpr) antigen-presenting cells (APCs) were markedly activated, and their numbers were increased in lymphoid tissues and in kidneys, yet numerous TUNEL-positive cells were observed in glomeruli of Bcl2l11(-/-)Fas(lpr/lpr) mice. These data demonstrate that dysregulation of the Bcl-2 or Fas pathways can alter the function of APCs, thereby leading to SLE pathogenesis.

Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis.

Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1beta in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis.

Bim deficiency leads to exacerbation and prolongation of joint inflammation in experimental arthritis.

OBJECTIVE: : Rheumatoid arthritis (RA) is characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. Since there are only a few detectable cells undergoing apoptosis in the joint, it is possible that a defect in apoptosis may contribute to synovial hyperplasia. This study sought to identify and characterize the direct role of apoptotic regulators in a mouse model of inflammatory arthritis. METHODS: Using a serum transfer model, experimental arthritis was induced in mice lacking the proapoptotic Bcl-2 family genes Bak (Bak-/-), Bax (Bax-/-), or Bim (Bim-/-), as compared with wild-type (WT) control mice. Physical examination for edema of the ankles and histopathologic analysis of ankle sections were used to determine the severity of arthritis. The serum and ankles were examined for production of chemokines and cytokines using enzyme-linked immunosorbent or Luminex-based assays. RESULTS: Bim-/- mice displayed increased severity and prolongation of arthritis. In contrast, Bak-/- and Bax-/- mice showed no difference in the severity of arthritis as compared with WT mice. In addition, Bim-/- mice had elevated levels of proinflammatory chemokines and cytokines, decreased joint and serum production of antiinflammatory cytokines, fewer TUNEL-positive cells, and reduced levels of active caspase 3 as compared with WT mice. CONCLUSION: These studies are the first to demonstrate a role for the proapoptotic Bcl-2 protein Bim in the effector phase of RA. The findings indicate that Bim potentially functions to repress the effector phase of arthritis by regulating the milieu of the joint and serum, and by inducing apoptosis.

p21Cip1 is required for the development of monocytes and their response to serum transfer-induced arthritis.

One of the central functions of cyclin-dependent kinase inhibitors, such as p21, p27, or p16, is to prevent entry into the cell cycle. However, the question remains as to whether they have other functions in the cell. We previously demonstrated that overexpression of p21 in fibroblasts isolated from patients with rheumatoid arthritis decreases the production of pro-inflammatory molecules. Overexpression of p21 has been also shown to reduce the development of experimental arthritis in mice and rats. To explore the role of endogenous p21 in the development of arthritis, we induced arthritis in p21(-/-) mice using the K/BxN serum transfer model of arthritis. Mice deficient in p21 were more resistant to serum transfer-induced arthritis (K/BxN) than wild-type (wt) control mice. Fewer macrophages were detected in p21(-/-) as compared to wt joints following transfer of K/BxN serum. Chemotaxis assays of bone marrow-derived macrophages from p21(-/-) and wt mice revealed no difference in migration. However, there was a substantial decrease in inflammatory monocytes circulating in peripheral blood and in monocyte precursors in bone marrow of p21(-/-) mice as compared to wt mice. Adoptive transfer of wt bone marrow-derived macrophages into p21(-/-) mice restored the sensitivity to serum transfer-induced arthritis. These data suggest a novel role for p21 in regulating the development and/or differentiation of monocytic populations that are crucial for the induction of inflammatory arthritis.

A cell-cycle independent role for p21 in regulating synovial fibroblast migration in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21(-/-)) and were examined with respect to growth and migration. p21(-/-) and wild-type (WT) FLS were compared with respect to migration towards chemoattractants found in RA synovial fluid in the presence and absence of cell cycle inhibitors. Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21(-/-) FLS display a marked increase (3.1-fold; p < 0.05) in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.

Fas death receptor signaling represses monocyte numbers and macrophage activation in vivo.

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1beta and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.

Retinoblastoma suppression of matrix metalloproteinase 1, but not interleukin-6, through a p38-dependent pathway in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by increased synovial lining cellularity, inflammation, and destruction of cartilage and bone. During the pathogenesis of RA, synovial fibroblasts reenter the cell cycle and multiply in number. RA synovial fibroblasts express high levels of the MAP kinase p38, which may contribute to the production of interleukin-6 (IL-6) and matrix metalloproteinases (MMPs). IL-6 and MMP-1 promote inflammation and joint destruction, respectively. Taken together, these findings indicate that in RA the enhanced cell cycle activity and production of IL-6 and MMP-1 may be linked. Therefore, we sought to determine if the tumor suppressor gene product retinoblastoma (Rb), a negative regulator of cell cycle activity, inhibits IL-6, MMP-1, and p38 in RA synovial fibroblasts. METHODS: RA and non-RA synovial fibroblasts were examined by enzyme-linked immunosorbent assay (ELISA) for the relative expression of inactive hyperphosphorylated Rb (inactive Rb/total Rb). Ectopic Rb expression was mediated by infection with a replication-defective adenovirus that expresses Rb (Ad-Rb). A control replication-defective adenovirus that expresses beta-galactosidase (Ad-beta-gal) was used. Cell cycle activity was determined by flow cytometry. IL-6 and MMP-1 expression was examined by real-time polymerase chain reaction and ELISA. Expression and activation of p38 were determined by kinase assays and ELISA. The activity of p38 was enhanced by infecting RA synovial fibroblasts with a replication-defective adenovirus that expresses a constitutively active form of MAPK kinase 3 (Ad-CA-MKK3), an upstream activator of p38. RESULTS: Quiescent RA, compared with non-RA synovial fibroblasts, displayed a 200% (P < 0.02) increase in the inactive Rb isoform. Proliferating RA synovial fibroblasts exhibited a 60% (P < 0.12) increase in the inactive Rb isoform compared with non-RA synovial fibroblasts. Increased levels of the active Rb isoform inhibited cell cycle progression and suppressed IL-6 and MMP-1 secretion in RA synovial fibroblasts, although the steady-state levels of IL-6 and MMP-1 messenger RNA remained unchanged. However, Rb overexpression had no effect on spontaneous or IL-1beta-induced production of IL-6 or MMP-1 in non-RA synovial fibroblasts. Ectopic Rb expression reduced the activity of p38. Ad-CA-MKK3 infection in RA synovial fibroblasts increased p38 phosphorylation, and MMP-1 but not IL-6 secretion. In contrast, Rb overexpression inhibited Ad-CA-MKK3-mediated phosphorylation of p38 and subsequent increase in MMP-1. CONCLUSION: Rb-mediated suppression of IL-6 and MMP-1 occurs at a posttranscriptional level. However, Ad-Rb reduction of MMP-1 but not IL-6 requires inhibition of the p38 pathway. These results suggest that Rb negatively regulates p38 activation, leading to decreased MMP-1 secretion in RA synovial fibroblasts.