Sherry Thornton

Association of the course of collagen-induced arthritis with distinct patterns of cytokine and chemokine messenger RNA expression.

OBJECTIVE To quantitate changes in cytokine and chemokine messenger RNA (mRNA) levels during the development and progression of collagen-induced arthritis (CIA) in mice. METHODS Mice with CIA were scored for arthritis and killed at weekly intervals. Cytokine and chemokine mRNA levels were determined by RNase protection assays of total paw RNA. RESULTS Arthritic paws exhibited mRNA levels of interleukin-1beta (IL-1beta), IL-2, macrophage inflammatory protein 2 (MIP-2), IL-6, IL-1 receptor antagonist, RANTES, tumor necrosis factor alpha (TNFalpha), TNFbeta, MIP-1alpha, IL-11, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and TGFbeta3 that were increased above mRNA levels in paws of normal, unimmunized mice and that exhibited distinct temporal patterns of mRNA expression. Clinically uninvolved paws also exhibited an increase in mRNA levels of IL-11, RANTES, TNFalpha, TNFbeta, and MIP-1alpha. CONCLUSION The observed differential temporal cytokine and chemokine mRNA expression patterns suggest that specific cytokines and chemokines have defined roles at various times during the course of autoimmune arthritis. Since most of these cytokines and chemokines are found in human rheumatoid arthritis (RA) synovium and synovial fluids, these findings may have relevance to RA.

Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin αMβ2 binding motif

Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib–) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin αMβ2 binding motif (Fibγ390–396A) or the αIIbβ3 platelet integrin-binding motif (FibγΔ5), were challenged with collagen-induced arthritis (CIA). Fib– mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibγ390–396A mice, which retain full clotting function. In contrast, arthritis in FibγΔ5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib– and Fibγ390–396A mice with CIA displayed reduced local expression of TNF-α, IL-1β, and IL-6, which suggests that αMβ2-mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-α expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to αMβ2-mediated inflammatory processes.

Viral IL-10 and soluble TNF receptor act synergistically to inhibit collagen-induced arthritis following adenovirus-mediated gene transfer.

Viral IL-10 (vIL-10) and soluble TNF receptor (sTNFR) are anti-inflammatory proteins that can suppress collagen-induced arthritis (CIA). These and related proteins have shown efficacy in the treatment of human rheumatoid arthritis; however, neither alone is able to completely suppress disease. Furthermore, they have short half-lives, necessitating frequent administration. To determine the ability of these proteins to act synergistically following gene transfer, arthritis was induced in DBA/1 male mice by immunization with type II collagen on days 0 and 21. Mice were injected i.v. either before disease onset (day 20) or after disease onset (day 28) with 1010 particles of adenovirus encoding vIL-10, a soluble TNF receptor-IgG1 fusion protein (sTNFR-Ig), a combination of both vectors, or a control vector lacking a transgene. Significant synergism was observed with the combination of vIL-10 and sTNFR-Ig, with a substantial reduction in both the incidence and severity of disease as well as inhibition of progression of established disease. sTNFR-Ig alone had no effect on CIA. vIL-10 alone inhibited disease when given before disease onset, but had minimal effect on established disease. Both proteins inhibited spleen cell proliferation and IFN-γ secretion in response to stimulation with type II collagen, but only vIL-10 reduced the synovial mRNA levels of the proinflammatory cytokines IL-1β, TNF-α, and IL-6. These findings demonstrate that vIL-10 and sTNFR-Ig act synergistically in suppressing CIA and suggest that gene transfer offers a potential therapeutic modality for the treatment of arthritis.

Altered susceptibility to collagen‐induced arthritis in transgenic mice with aberrant expression of interleukin‐1 receptor antagonist

OBJECTIVE To determine the effect of overexpression or deletion of interleukin-1 receptor antagonist (IL-1Ra) in collagen-induced arthritis (CIA). METHODS Mice overexpressing the IL-1Ra gene under the control of its endogenous promoter, mice lacking IL-1Ra, and normal littermate controls were immunized with bovine type II collagen (CII) and compared in terms of features of CIA. RESULTS Mice overexpressing IL-1Ra had a significant reduction in the incidence and severity of CIA. After CII immunization, IL-1Ra messenger RNA was overexpressed in the spleens, but not in the paws, of transgenic mice. Minimal differences were observed in the humoral or cellular immune responses to CII. Mice lacking IL-1Ra had a significantly earlier onset of CIA, with increased severity. CONCLUSION Endogenous expression of IL-1Ra is a critical determinant of susceptibility to CIA. These findings suggest potential therapeutic interventions for autoimmune arthritis.

Gene expression signatures in polyarticular juvenile idiopathic arthritis demonstrate disease heterogeneity and offer a molecular classification of disease subsets.

OBJECTIVE To determine whether peripheral blood mononuclear cells (PBMCs) from children with recent-onset polyarticular juvenile idiopathic arthritis (JIA) exhibit biologically or clinically informative gene expression signatures. METHODS Peripheral blood samples were obtained from 59 healthy children and 61 children with polyarticular JIA prior to treatment with second-line medications, such as methotrexate or biologic agents. RNA was extracted from isolated mononuclear cells, fluorescence labeled, and hybridized to commercial gene expression microarrays (Affymetrix HG-U133 Plus 2.0). Data were analyzed using analysis of variance at a 5% false discovery rate threshold after robust multichip analysis preprocessing and distance-weighted discrimination normalization. RESULTS Initial analysis revealed 873 probe sets for genes that were differentially expressed between polyarticular JIA patients and healthy controls. Hierarchical clustering of these probe sets distinguished 3 subgroups within the polyarticular JIA group. Prototypical patients within each subgroup were identified and used to define subgroup-specific gene expression signatures. One of these signatures was associated with monocyte markers, another with transforming growth factor beta-inducible genes, and a third with immediate early genes. Correlation of gene expression signatures with clinical and biologic features of JIA subgroups suggested relevance to aspects of disease activity and supported the division of polyarticular JIA into distinct subsets. CONCLUSION Gene expression signatures in PBMCs from patients with recent-onset polyarticular JIA reflect discrete disease processes and offer a molecular classification of disease.

Heterogeneous effects of IL-2 on collagen-induced arthritis.

IL-2 is generally considered a pro-inflammatory cytokine that exacerbates Th1-mediated disease states, such as autoimmune arthritis. Consistent with this role for IL-2, recent studies from our laboratory demonstrate that IL-2 mRNA is markedly increased during the acute stage of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. To further define the role of IL-2 in CIA, the levels of IL-2 protein and its receptor and the effects of IL-2 administration were analyzed during CIA. IL-2 protein and IL-2R were preferentially expressed at disease onset, compared with later stages of disease. Administration of recombinant human IL-2 (rhIL-2) at, or just before, disease onset exacerbated disease; surprisingly, rhIL-2 given before disease onset inhibited CIA, associated with reduced cellular and humoral responses to type II collagen. Determination of in vivo serum levels of Th1 and Th2 cytokines in response to rhIL-2 treatment demonstrated that IFN-γ, but not IL-4, was markedly up-regulated in response to IL-2. In mice treated with anti-IFN-γ Ab, both early and late IL-2 administration exacerbated CIA. Thus, IL-2 can have two opposite effects on autoimmune arthritis, a direct stimulatory effect and an indirect suppressive effect that is mediated by IFN-γ.

Immature cell populations and an erythropoiesis gene-expression signature in systemic juvenile idiopathic arthritis: implications for pathogenesis

IntroductionPrevious observations suggest that active systemic juvenile idiopathic arthritis (sJIA) is associated with a prominent erythropoiesis gene-expression signature. The aim of this study was to determine the association of this signature with peripheral blood mononuclear cell (PBMC) subpopulations and its specificity for sJIA as compared with related conditions.MethodsThe 199 patients with JIA (23 sJIA and 176 non-sJIA) and 38 controls were studied. PBMCs were isolated and analyzed for multiple surface antigens with flow cytometry and for gene-expression profiles. The proportions of different PBMC subpopulations were compared among sJIA, non-sJIA patients, and controls and subsequently correlated with the strength of the erythropoiesis signature. Additional gene-expression data from patients with familial hemophagocytic lymphohistiocytosis (FHLH) and from a published sJIA cohort were analyzed to determine whether the erythropoiesis signature was present.ResultsPatients with sJIA had significantly increased proportions of immature cell populations, including CD34+ cells, correlating highly with the strength of the erythropoiesis signature. The erythropoiesis signature strongly overlapped with the gene-expression pattern in purified immature erythroid precursors. The expansion of immature cells was most prominently seen in patients with sJIA and anemia, even in the absence of reticulocytosis. Patients with non-sJIA and anemia did not exhibit the erythropoiesis signature. The erythropoiesis signature was found to be prominent in patients with FHLH and in a published cohort of patients with active sJIA, but not in patients with inactive sJIA.ConclusionsAn erythropoiesis signature in active sJIA is associated with the expansion of CD34+ cells, also is seen in some patients with FHLH and infection, and may be an indicator of ineffective erythropoiesis and hemophagocytosis due to hypercytokinemia.

The limited role of interferon-γ in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness.

OBJECTIVE Systemic juvenile idiopathic arthritis (JIA) is an autoinflammatory syndrome in which the myelomonocytic lineage appears to play a pivotal role. Inflammatory macrophages are driven by interferon-γ (IFNγ), but studies have failed to demonstrate an IFN- induced gene signature in active systemic JIA. This study sought to characterize the status of an IFN-induced signature within affected tissue and to gauge the integrity of IFN signaling pathways within peripheral monocytes from patients with systemic JIA. METHODS Synovial tissue from 12 patients with active systemic JIA and 9 with active extended oligoarticular JIA was assessed by real-time polymerase chain reaction to quantify IFN-induced chemokine gene expression. Peripheral monocytes from 3 patients with inactive systemic JIA receiving anti-interleukin-1β (anti-IL-1β) therapy, 5 patients with active systemic JIA, and 8 healthy controls were incubated with or without IFNγ to gauge changes in gene expression and to measure phosphorylated STAT-1 (pSTAT-1) levels. RESULTS IFN-induced chemokine gene expression in synovium was constrained in active systemic JIA compared to the known IFN-mediated extended oligoarticular subtype. In unstimulated peripheral monocytes, IFN-induced gene expression was similar between the groups, except that lower levels of STAT1, MIG, and PIAS were observed in patients with active disease, while higher levels of PIAS1 were observed in patients with inactive disease. Basal pSTAT-1 levels in monocytes tended to be higher in systemic JIA patients compared to healthy controls, with the highest levels seen in those with inactive disease. Upon stimulation of monocytes, the fold increase in gene expression was roughly equal between groups, except for a greater increase in STAT1 in patients with inactive systemic JIA compared to controls, and a greater increase in IRF1 in those with active compared to inactive disease. Upon stimulation, the fold increase in pSTAT-1 was highest in monocytes from patients with inactive systemic JIA. CONCLUSION Monocytes in patients with active systemic JIA retain the ability to respond to IFNγ, suggesting that the lack of an IFN-induced gene signature in patients with active disease reflects a limited in vivo exposure to IFNγ. In patients with inactive systemic JIA who received treatment with anti-IL-1β, hyperresponsiveness to IFNγ was observed.

The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A

Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl₃ vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

NK cells secrete high levels of IFN-γ in response toin vivo administration of IL-2

Interleukin‐2 is an immunotherapeutic agent for the treatment of metastatic tumors. Administration of recombinant human IL‐2 (rhIL‐2) in vivo activates lymphocytes and cell‐mediated immune responses. In mice, we have recently observed a dramatic increase of serum IFN‐γ levels in response to in vivo administration of rhIL‐2, which was necessary for the observed protective effects of IL‐2 against the development of collagen‐induced arthritis. To explore further the basis of this phenomenon, the kinetics and source of IFN‐γ in response to IL‐2 was investigated. Highest serum levels of IFN‐γ were observed within 3 h of IL‐2 administration, with levels decreasing over time. Anti‐IL‐2 receptor β antibody blocked this IFN‐γ induction. Multiple doses of rhIL‐2 resulted in corresponding increases in circulating IFN‐γ. IFN‐γ induction was dose‐dependent between doses of 240 to 30,000 U of rhIL‐2. Analysis of the cellular source of IFN‐γ secretion using NK‐ and T cell‐deficient mice demonstrated that NK cells are the likely source of IFN‐γ. Furthermore, IFN‐γ secretion in response to IL‐2 administration was not affected bythe absence of IL‐12, the pivotal cytokine for determination of Th1 responses. These results suggest that effects of IL‐2 on immune responses in vivo may be mediated by IFN‐γ.