Fooyew Liew

The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis.

Interleukin 15 (IL–15) is a novel cytokine with interleukin–2–like activity. It is also a potent T–lymphocyte chemoattractant. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the presence of activated T lymphocytes, macrophages and synoviocytes in the synovial membrane. The mechanisms of T–cell activation in RA are currently unclear. We report the presence of high concentrations of IL–15 in rheumatoid arthritis (RA) synovial fluid and have demonstrated its expression in the synovial membrane lining layer by immunohistochemistry. RA synovial fluids were found to contain chemotactic activity, which was attributable in part to the presence of IL–15. Moreover, in a murine model, injection of recombinant IL–15 was found to induce a local tissue inflammatory infiltrate consisting predominantly of T lymphocytes. Synovial fluid T lymphocytes proliferate in response to IL–15, demonstrating that continued responsiveness to IL–15 is a feature of T cells after entry into the synovial compartment. These data suggest that IL–15 can recruit and activate T lymphocytes into the synovial membrane, thereby contributing to RA pathogenesis.

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2‐deficient mice infected with Leishmania major in vivo or stimulated with IFN‐γ or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL‐12 than those from heterozygous or wild‐type mice. A macrophage cell line, J774, produced significant amounts of IL‐12 following activation with LPS, or LPS plus IFN‐γ. This could be markedly enhanced by the NOS inhibitor L‐NG monomethyl arginine (L‐NMMA), but profoundly inhibited by the NO‐generating compound S‐nitroso‐N‐acetyl‐penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L‐NMMA decreased TNF‐α synthesis by LPS‐activated J774 cells. The differential effect of NO on IL‐12 and TNF‐α is at the transcriptional level and is activation dependent. Since IL‐12 is a major inducer of Th1 cells which produce IFN‐γ that can activate macrophages to produce IL‐12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

Mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced Th1 cell responses.

Mice deficient in the inducible nitric-oxide synthase (iNOS), constructed by gene-targeting, were significantly more susceptible to herpes simplex virus (HSV)-1 infection, displayed a delayed clearance of virus from the dorsal root ganglia (DRG) and exhibited an increase in the frequency of virus reactivation in DRG compared with similarly infected heterozygous mice. The infected iNOS-deficient mice developed enhanced Th1-type immune responses and their spleen cells produced higher concentrations of IL-12 than similarly infected heterozygous mice. This finding suggests that iNOS plays an important role in resistance against HSV-1 infection. Furthermore, nitric oxide (NO) may block the development of Th1 cells via inhibition of IL-12 synthesis and thereby play a role in immune regulation.

Immunological mechanisms and clinical implications of regulatory T cell deficiency in a systemic autoimmune disorder: roles of IL-2 versus IL-15.

Regulatory T cell deficiency is evident in patients with lupus, but the casual relationship and underlying mechanism leading to Treg deficiency are unclear. We analyzed the Treg profile, induction and functions of Treg in a lupus mouse model. A characteristic age‐dependent biphasic change of Treg frequency was observed in the MRL/lpr mice, which developed a spontaneous lupus‐like disease. After an early increase, Treg frequency in the peripheral lymphoid organs rapidly declined with age. Functionally, Treg from both young and old MRL/lpr mice were fully competent in suppressing the wild‐type MRL/+ T effector cell (Teff) responses. Adoptive transfer of MRL/+ Treg markedly suppressed clinical disease in the MRL/lpr mice. We demonstrated that the reduced Treg frequency was a result of insufficient peripheral Treg expansion due to defective MRL/lpr Teff in IL‐2 production, and the associated defects in dendritic cells, which could be fully restored by exogenous IL‐2. In the absence of IL‐2, MRL/lpr Teff but not MRL/lpr Treg were highly responsive to IL‐15 and could expand rapidly due to enhanced IL‐15R expression and IL‐15 synthesis. These findings thus provide a clear causal relationship and immunological mechanism underlying Treg deficiency and systemic autoimmunity.

Skin allograft rejection in mice lacking inducible nitric oxide synthase.

BACKGROUND During allograft rejection, up-regulation of cytokine-inducible nitric oxide synthase (iNOS) leads to the production of large amounts of nitric oxide (NO). The net effect of NO on the alloimmune response is, however, difficult to predict because of its diverse biological effects, which include potentially opposing roles as an effector and immunoregulatory molecule. METHODS In this study, the role of iNOS on the in vitro and in vivo alloimmune response was defined using mutant mice that lack a functional iNOS gene. The ability of spleen cells obtained from iNOS-deficient mutants to proliferate and to produce cytokines in response to irradiated BALB/c stimulator cells was determined, and the rate at which iNOS-deficient mice were able to reject BALB/c skin allografts was observed. RESULTS Spleen cells from homozygous iNOS-deficient (129xMF1)F1 mice, when compared with cells from heterozygous control mice, showed an increased in vitro proliferative response and produced substantially higher levels of interferon-gamma, and also more interleukin-2 and interleukin-12, in response to allogeneic stimulation. The kinetics of BALB/c skin graft rejection were comparable in heterozygous control animals and iNOS-deficient mice. Moreover, no net effect of iNOS on skin allograft rejection was apparent in mice treated with depleting monoclonal antibodies (mAb) to CD4 or CD8 T cells, either alone or in combination, or in mice treated with both anti-CD8 mAb and a neutralizing anti-tumor necrosis factor mAb. CONCLUSIONS These results show that iNOS has an immunomodulatory effect on the in vitro alloimmune response but lack of iNOS has no net influence on the kinetics of murine skin allograft rejection in either unmodified recipients or recipients in which the early contribution of T-cell subsets and tumor necrosis factor-alpha to graft rejection has been abrogated.

Regulating innate immune response cytokines in synovitis

Several studies have provided powerful proof of concept for the notion that single cytokine targeting can manifest effective immune suppression even in the context of complex networks of cytokine activities. We have studied the biology and functional expression of the innate response cytokine IL-15 and, more recently, members of the IL-1/IL-1 receptor superfamily in inflammatory synovitis. These cytokines are characterised by early upregulation during inflammatory responses and characteristic functional synergy and cross-regulation in the intact immune response. IL-15 is an IL-2-like cytokine of 15 kDa that possesses broad proinflammatory activities via its heterotrimeric receptor (IL-15Rα/IL-15/2Rβ/common γ), including promoting T-cell maturation, activation, and cytokine production, natural killer cell maturation and effector function, neutrophil activation, dendritic cell activation, endothelial cell rescue from apoptosis, macrophage activation and B-cell activation and isotype switching. IL-15 is expressed at mRNA and protein levels in several inflammatory arthritides including rheumatoid arthritis. When targeted in rodent models of inflammation using either soluble IL-15Rα or modified IL-15 mutant fusion proteins, amelioration of onset and of existing arthritis is observed. A recent phase I study in which a fully human IgG1 monoclonal antibody (HuMax-IL15; Genmab, Copenhagen, Denmark) was administered to 30 patients with active rheumatoid arthritis indicated that IL-15 blockade was well tolerated up to 8 weeks, with early indications of efficacy detected. We have now observed close interactions between IL-15 and IL-1 receptor superfamily signalling in promoting T-cell activation. Specifically, IL-15 synergises with the TLR2 ligand BLP to promote memory T-cell activation (co-stimulatory) and also via a cell membrane-dependent pathway to promote adjacent macrophage activation and tumour necrosis factor release. We have also observed that a further member of the IL-1 receptor superfamily, namely ST2, is expressed in synovial fibroblasts. Membrane-bound ST2 negatively regulates type I IL-1 receptor and TLR4 signalling by sequestrating the adaptors MyD88 and Mal. Intriguingly, soluble ST2 suppresses development of collagen-induced arthritis in DBA/1 mice if given prophylactically, and also established collagen-induced arthritis if administered after disease onset. Together these data suggest that complex interactions between the common γ-receptor chain and IL-1 receptor superfamily signalling cytokines are of importance in developing synovial inflammatory responses and that elucidation of these pathways offers therapeutic utility.

A novel therapeutic approach to cytokine modulation in articular inflammation using filarial nematode derived ES-62

Meeting abstract on a novel therapeutic approach to cytokine modulation in articular inflammation. Discovering safe, novel immunomodulators that are effective in RA is currently a major therapeutic objective. Long-term immune system deviation is most striking in the host-parasite relationship, in which microbes may coexist with a human host. ES-62 exhibited powerful immunomodulation of CIA, preventing initiation of inflammatory arthritis. Crucially, ES-62 suppressed even established disease. These effects were due to inhibition of cytokine release, specifically TNF-α, and reversal of collagen specific Th1 responses associated with reduced expression of IFN-γ. The physiologic relevance of these observations was confirmed, as ES-62 down-regulated the release of proinflammatory cytokines (TNF-α and IL-6) from patient-derived samples.

Suppression of in vitro and in vivo parameters of inflammatory synovitis by simvastatin

We have explored in vitro and in vivo the immunomodulatory activities of simvastatin, an HMG Co-A reductase (HMGR) inhibitor, in the context of inflammatory arthritis.