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.

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.

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.

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.