IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood: II. Application to rheumatoid arthritis and osteoarthritis.

Rheumatoid arthritis (RA) is an immune disease in which the pathological immune reaction is thought to be initiated by the presentation of an (auto) antigen or superantigen by MHC class II positive cells to CD4 T cells. These successive immunological events can be studied by the cytokines produced at the different stages. Cytokine secretion by stimulated cells in autologous diluted whole blood has allowed the study of the immune profile characteristic of rheumatoid arthritis. The pattern of RA patient whole blood cells cultured in autologous blood is characterized by hyperactivity of the mononuclear cells with high secretion of IL-1 beta, TNF-alpha and IL-6 and low production of IFN-gamma, in comparison with the normal (N) and osteoarthrosis (OA) populations. The IL-2 secretion pattern is unique, arising from production followed by consumption. This production-consumption turnover is the most elevated in the RA group. The T cells are indeed activated in rheumatoid arthritis but regulatory events suppress some of their functions. A correlation was found between the inflammatory proteins and mediators of cellular immunity and macrophagic function: IL-1 beta and the sedimentation rate; IL-6 and fibrinogen; TNF-alpha and the number of blood monocytes. The secretion of OA-stimulated whole blood cells was similar to RA for two monokines (overproduction of TNF-alpha and IL-6) and different for IL-1 beta, not different from normal in OA. Stimulated whole blood cell cytokine secretion profile from RA and OA groups, was the same as previously observed in synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of cytokine gene expression in rheumatoid arthritis

Previous studies of the cytokine profile of rheumatoid arthritis (RA) have been primarily limited to the assessment of the levels of these mediators in synovial fluid (SF) or synovial tissues (ST) by biologic or immunologic assays. We have studied cytokine gene expression in RA by in situ hybridization of SF cells, enzymatically dispersed ST cells, and frozen sections of ST. RA ST cells (n = 7) were studied and a high percentage of cells hybridized to the following anti-sense probes: IL-6 = 19 +/- 3.3%; IL-1 beta = 9.9 +/- 1.7%; TNF-alpha = 5.8 +/- 1.4%; granulocyte-macrophage-CSF = 2.2 +/- 0.8%; transforming growth factor-beta 1 = 1.3 +/- 0.2% (p less than 0.05 for each compared to sense probes). Similar results were found using osteoarthritis ST cells, although the percentage of cells expressing the IL-6 gene (7.1 +/- 2.5%) was significantly less in osteoarthritis compared to RA. RA ST cells did not significantly bind the IFN-gamma probe (0.2 +/- 0.1% positive), although they were capable of expressing the IFN-gamma gene if stimulated with PHA. The OKM1+ population of ST cells (i.e., macrophage lineage cells) was greatly enriched for IL-1 beta and TNF-alpha, whereas the OKM1- population (lymphocytes, fibroblasts, and type B synoviocytes) was enriched for IL-6. The vast majority of cells expressing the IL-6 gene were non-T cells. Furthermore, hybridization to RA ST frozen sections localized IL-6 mRNA to the synovial lining layer, which is comprised of type A and type B synoviocytes. In contrast to the high level of cytokine gene expression observed in ST, SF cells did not hybridize significantly to any of the cytokine probes. If stimulated with LPS or PHA, SF cells expressed IL-1 beta or IFN-gamma genes, respectively.     

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.     

The role of T-cell interleukin-17 in conducting destructive arthritis: lessons from animal models

Interleukin-17 (IL-17) is a T cell cytokine spontaneously produced by cultures of rheumatoid arthritis (RA) synovial membranes. High levels have been detected in the synovial fluid of patients with RA. The trigger for IL-17 is not fully identified; however, IL-23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 is a potent inducer of various cytokines such as tumor necrosis factor (TNF)-alpha, IL-1, and receptor activator of NF-kappaB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-alpha in inducing cytokine expression and joint damage have been shown in vitro and in vivo. This review describes the role of IL-17 in the pathogenesis of destructive arthritis with a major focus on studies in vivo in arthritis models. From these studies in vivo it can be concluded that IL-17 becomes significant when T cells are a major element of the arthritis process. Moreover, IL-17 has the capacity to induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis. Anti-IL-17 cytokine therapy is of interest as an additional new anti-rheumatic strategy for RA, in particular in situations in which elevated IL-17 might attenuate the response to anti-TNF/anti-IL-1 therapy.     

Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines

Bone resorption in the joints is the characteristic finding in patients with rheumatoid arthritis (RA). Osteoclast-like cells are present in the synovial tissues and invade the bone of patients with RA. The characteristics of these cells are not completely known. In the work reported here, we generated these cells from peripheral-blood monocytes from healthy individuals. The monocytes were co-cultured with nurse-like cells from synovial tissues of patients with RA (RA-NLCs). Within 5 weeks of culture, the monocytes were activated and differentiated into mononuclear cells positive for CD14 and tartrate-resistant acid phosphatase (TRAP). These mononuclear cells then differentiated into multinucleated giant bone-resorbing cells after stimulation with IL-3, IL-5, IL-7, and/or granulocyte-macrophage-colony-stimulating factor. TRAP-positive cells with similar characteristics were found in synovial fluid from patients with RA. These results indicate that multinucleated giant bone-resorbing cells are generated from monocytes in two steps: first, RA-NLCs induce monocytes to differentiate into TRAP-positive mononuclear cells, which are then induced by cytokines to differentiate into multinucleated giant bone-resorbing cells.     

The role of T cell interleukin-17 in conducting destructive arthritis: lessons from animal models

Interleukin-17 (IL-17) is a T cell cytokine spontaneously produced by cultures of rheumatoid arthritis (RA) synovial membranes. High levels have been detected in the synovial fluid of patients with RA. The trigger for IL-17 is not fully identified; however, IL-23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 is a potent inducer of various cytokines such as tumor necrosis factor (TNF)-α, IL-1, and receptor activator of NF-κB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-α in inducing cytokine expression and joint damage have been shown in vitro and in vivo. This review describes the role of IL-17 in the pathogenesis of destructive arthritis with a major focus on studies in vivo in arthritis models. From these studies in vivo it can be concluded that IL-17 becomes significant when T cells are a major element of the arthritis process. Moreover, IL-17 has the capacity to induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis. Anti-IL-17 cytokine therapy is of interest as an additional new anti-rheumatic strategy for RA, in particular in situations in which elevated IL-17 might attenuate the response to anti-TNF/anti-IL-1 therapy.     

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

Introduction

The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.

Methods

RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.

Results

Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.

Conclusions

These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.     

Urokinase,a constitutive component of the inflamed synovial fluid,induces arthritis

Urokinase plasminogen activator (uPA) is an important regulator of fibrinolysis in synovial fluid. An increase of uPA activity and expression of its receptor have been reported in joints of patients with rheumatoid arthritis (RA). The aim of the present study was to assess the arthritogenic capacity of uPA and the mechanisms by which this effect is mediated. uPA was injected into the knee joints of healthy mice, and morphological signs of arthritis were assessed 4 days after the injection. The prerequisite of different leukocyte populations for the development of uPA-triggered arthritis was assessed by selective cell depletion. The inflammatory capacity of uPA was assessed in vitro. Finally, levels of uPA were measured in 67 paired blood and synovial fluid samples from RA patients. The synovial fluid from RA patients displayed higher levels of uPA compared with blood samples. Morphological signs of arthritis were found in 72% of uPA-injected joints compared with in only 18% of joints injected with PBS (P < 0.05). Synovitis was characterised by infiltration of CD4-Mac-1+ mononuclear cells, by the formation of pannus and by occasional cartilage destruction. The absence of monocytes and lymphocytes diminished the frequency of synovitis (P < 0.01), indicating an arthritogenic role of both these leukocyte populations. Synthetic uPA inhibitor downregulated the incidence of uPA-triggered arthritis by 50%. uPA induced arthritis, stimulating the release of proinflammatory cytokines IL-6, IL-1beta and tumour necrosis factor alpha. Accumulation of uPA locally in the joint cavity is a typical finding in erosive RA. uPA exerts potent arthritogenic properties and thus may be viewed as one of the essential mediators of joint inflammation.     

The influence of synovial fluid on lymphocyte functions in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic recurrent and systemic inflammatory disease affecting around 1% of the population, that primarily involves the joints. In this study, we determined the Th1/Th2 lymphocytes ratio at the site of rheumatoid inflammation and the influence of the synovial fluid (SF) on the secretory and proliferative function in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC), obtained from patients with RA. Our results showed significant differences concerning the mononuclear cells and the CD4/CD8 ratio in synovial fluid and peripheral blood of patients. In SF prevailed Th1 cells, while in peripheral blood we found another cytokine profile of T lymphocytes. Also synovial fluid lymphocytes had a low PHA-stimulated blastogenic response. Patients plasma and synovial fluid showed an inhibitory effect on prolipheration indexes.     

Implication of cytosolic phospholipase A2 (cPLA2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A(2) (cPLA(2)) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA(2) inhibitors prior to IL-1beta or TNF-alpha treatment. Total cellular AA concentrations and PLA(2) activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA(2) inhibitors inhibited both IL-1beta and TNF-alpha-induced superoxide production in RA and OA cells. Basal PLA(2) activity was significantly more important in RA cells than in OA cells; PLA(2) activity was increased in IL-1beta and TNF-alpha pre-treated RA cells, and cPLA(2) inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1beta and TNF-alpha, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H(+) channel.     

α-Enolase expressed on the surfaces of monocytes and macrophages induces robust synovial inflammation in rheumatoid arthritis

α-Enolase (ENO1) is a multifunctional glycolytic enzyme expressed abundantly in the cytosol. It has been implicated in autoimmune and inflammatory diseases. Serum Abs against ENO1 were reported in rheumatoid arthritis (RA). Cell-surface expression of ENO1 has been found to be increased rapidly in response to inflammatory stimuli, but its expression and function has not been reported in RA. In this study, we show that cell-surface expression of ENO1 is increased on monocytes and macrophages isolated from RA patients but not on those from osteoarthritis patients, and Ab against ENO1 can stimulate these cells to produce higher amounts of proinflammatory mediators, such as TNF-α, IL-1 α/β, IFN-γ, and PGE(2) via p38 MAPK and NF-κB pathway. The frequency of ENO1-positive cells in synovial fluid mononuclear cells was higher than PBMCs. ENO1-positive cells were also found in the inflamed synovium from RA patients and arthritic ankle tissues of mice with collagen-induced arthritis. Taken together, these findings suggest that Abs against ENO1 present in RA sera may stimulate monocytes and macrophages expressing cell-surface ENO1 and contribute to production of proinflammatory mediators during the effector phase of synovial inflammation.     

siRNA targeting PLK-1 induces apoptosis of synoviocytes in rheumatoid arthritis

Polo-like kinase-1 (PLK-1) is a member of the PLK family and participates in the control of cell mitosis. Here, we show that immunoreactive PLK-1 is strongly expressed in synoviocytes and some infiltrative mononuclear cells in synovial tissues from patients with rheumatoid arthritis (RA), while patients with osteoarthritis and injury show little or no expression of PLK-1 in synovial tissues. Western blot analysis shows that PLK is expressed and its expression is enhanced by IL-1beta in RA synoviocytes. IL-1beta also enhanced the cell growth of RA synoviocytes. Moreover, siRNA targeted against PLK-1 significantly decreases the expression of PLK-1 of RA synoviocytes stimulated by IL-1beta and suppresses the proliferation of these synoviocytes through apoptosis. These findings suggest that PLK-1 plays a critical role in the proliferation of RA synoviocytes leading to bone destruction, and siRNA against PLK-1 is potentially useful for the treatment of RA.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin

Pathological processes involved in the initiation of rheumatoid synovitis remain unclear. We undertook the present study to identify immune and stromal processes that are present soon after the clinical onset of rheumatoid arthritis (RA) by assessing a panel of T cell, macrophage, and stromal cell related cytokines and chemokines in the synovial fluid of patients with early synovitis. Synovial fluid was aspirated from inflamed joints of patients with inflammatory arthritis of duration 3 months or less, whose outcomes were subsequently determined by follow up. For comparison, synovial fluid was aspirated from patients with acute crystal arthritis, established RA and osteoarthritis. Rheumatoid factor activity was blocked in the synovial fluid samples, and a panel of 23 cytokines and chemokines measured using a multiplex based system. Patients with early inflammatory arthritis who subsequently developed RA had a distinct but transient synovial fluid cytokine profile. The levels of a range of T cell, macrophage and stromal cell related cytokines (e.g. IL-2, IL-4, IL-13, IL-17, IL-15, basic fibroblast growth factor and epidermal growth factor) were significantly elevated in these patients within 3 months after symptom onset, as compared with early arthritis patients who did not develop RA. In addition, this profile was no longer present in established RA. In contrast, patients with non-rheumatoid persistent synovitis exhibited elevated levels of interferon-γ at initiation. Early synovitis destined to develop into RA is thus characterized by a distinct and transient synovial fluid cytokine profile. The cytokines present in the early rheumatoid lesion suggest that this response is likely to influence the microenvironment required for persistent RA.     

Arginine-rich anti-vascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collagen-induced arthritis and VEGF-stimulated productions of TNF-alpha and IL-6 by human monocytes

Vascular endothelial growth factor (VEGF) has been suggested to play a critical role in the pathogenesis of rheumatoid arthritis (RA). We previously identified a novel RRKRRR hexapeptide that blocked the interaction between VEGF and its receptor through the screening of peptide libraries. In this study, we investigated whether anti-VEGF peptide RRKRRR (dRK6) could suppress collagen-induced arthritis (CIA) and regulate the activation of mononuclear cells of RA patients. A s.c. injection of dRK6 resulted in a dose-dependent decrease in the severity and incidence of CIA and suppressed synovial infiltration of inflammatory cells in DBA/1 mice. In these mice, the T cell responses to type II collagen (CII) in lymph node cells and circulating IgG Abs to CII were also dose-dependently inhibited by the peptides. In addition, VEGF directly increased the production of TNF-alpha and IL-6 from human PBMC. Synovial fluid mononuclear cells of RA patients showed a greater response to VEGF stimulation than the PBMC of healthy controls. The major cell types responding to VEGF were monocytes. Moreover, anti-VEGF dRK6 inhibited the VEGF-induced production of TNF-alpha and IL-6 from synovial fluid mononuclear cells of RA patients and decreased serum IL-6 levels in CIA mice. In summary, we observed first that dRK6 suppressed the ongoing paw inflammation in mice and blocked the VEGF-induced production of proinflammatory cytokines. These data suggest that dRK6 may be an effective strategy in the treatment of RA, and could be applied to modulate various chronic VEGF-dependent inflammatory diseases.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFalpha). We examined the in vivo relationship between IL-32 and TNFalpha, and the pathologic role of IL-32 in the TNFalpha-related diseases - arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFalpha reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFalpha, we prepared an overexpression model mouse of human IL-32beta (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFalpha, IL-1beta, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFalpha concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFalpha was increased in resting F4/80+ macrophages, and the expression of TNFalpha, IL-1beta and IL-6 was increased in lipopolysaccharide-stimulated F4/80+ macrophages and CD11c+ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32beta-producing CD4+ T cells significantly exacerbated collagen-induced arthritis, and a TNFalpha blockade cancelled the exacerbating effects of hIL-32beta. We therefore conclude that IL-32 is closely associated with TNFalpha, and contributes to the exacerbation of TNFalpha-related inflammatory arthritis and colitis.