Honokiol possesses potential anti-inflammatory effects on rheumatoid arthritis and GM-CSF can be a target for its treatment

Objective: To observe the anti-inflammatory effects of honokiol in primary cultures of peripheral blood mononuclear cells of rheumatoid arthritis patients, the pro-inflammatory cytokines and potential targets were investigated. Methods: The levels of GM-CSF, IL-1β, TNF-α and IL-8 were determined by ELISA assay. The genes and proteins expression were analyzed by real-time PCR and Western blotting respectively. Results: The serum IL-1β, TNF-α and GM-CSF levels were 1.76-, 2.16- and 3.57-fold increased in patients with RA as compared to those of control group. Honokiol inhibited the expression levels of IL-1β, TNF-α, GM-CSF and IL-8 in PBMCs with a dose-dependent manner. Measurements obtained from supernatants were positively correlated between TNF-α and IL-1β, moreover, similar results found TNF-α levels positively correlated with GM-CSF and IL-8 activity in the supernatants of PBMCs isolated from RA patients. Furthermore, the mRNA and protein expression of IL-1β, GM-CSF and IL-8 were up-regulated when the PBMCs exposure to TNF-α, however, honokiol treatment significantly reversed the expression of IL-1β, TNF-α and GM-CSF in response to TNF-α with a dose-dependent manner. Conclusions: This study demonstrates that honokiol could possess potential anti-inflammatory effects and inhibits TNF-α-induced IL-1β, GM-CSF and IL-8 production in PBMCs from rheumatoid arthritis patients.     

Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels

Gamma globulin complexes were demonstrable in certain joint fluids from patients with rheumatoid arthritis by analytic and density gradient centrifugation. They form a continuum of high molecular weight components ranging from 7S to 30S and were dissociable primarily to 7S γG globulin. The larger complexes were also detectable by precipitation reactions with C1q and with γM rheumatoid factor. This permitted the isolation and partial characterization of the complexes. Non-immunoglobulin constituents were not detectable. Evidence was obtained that 7S γG globulin rheumatoid factors represented an important constituent of the complexes.

A relationship was encountered between the amount of γ globulin complex present in the joint fluids and diminution in total haemolytic complement activity. All fluids with abundant γ globulin complexes contained markedly lowered complement levels. A decrease in levels of C1q and β_1A was found to correlate with the amount of γG globulin complexes. Although patients who had diminished complement levels in their joint fluid have serum γM rheumatoid factor, its titre does not correlate well with the extent of complement depression.

Joint fluids with abundant γ globulin complexes manifested an anticomplementary effect. This activity was most apparent at 37°C when fresh rheumatoid serum was used as a source of complement. Evidence indicating the participation of γM rheumatoid factor in this anticomplementary effect was obtained. All fluids with significant anticomplementary activity formed precipitin bands with C1q on agarose plates. γM rheumatoid factor was not necessary for this reaction.

     

Measurement of Inflammatory Biomarkers in Synovial Tissue Extracts by Enzyme-Linked Immunosorbent Assay

We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1β (IL-1β), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of ≤25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1β, IL-6, IL-8, and TNF-α levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy.     

Interaction between chronically HIV-infected promonocytic cells and human umbilical vein endothelial cells: role of proinflammatory cytokines and chemokines in viral expression modulation

HIV type 1 expression was significantly up-regulated in chronically infected promonocytic cell line (U1) co-cultured with human umbilical vein endothelial cells (HUVEC). Virus replication, evaluated as supernatant p24 release, was higher when U1 were co-cultured with IL-1β-activated HUVEC than with unstimulated HUVEC. When non-adherent U1 were removed from co-cultures, the remaining U1 cells adherent to the endothelial monolayer still showed enhanced HIV replication in comparison with an equal number of U1 cultured alone. While addition of adhesion molecule blocking antibodies (anti-intercellular adhesion molecule-1 (ICAM-1), -vascular cell adhesion molecule-1 (VCAM-1), -CD18 and -very late antigen-4 (VLA-4)) strongly inhibited adherence of U1 cells to endothelial monolayers, such treatment resulted in only a partial reduction in p24 release. Furthermore, HIV replication in U1 cells was enhanced on culture in HUVEC-conditioned media. Such data suggest that soluble mediators secreted by endothelial monolayers may modulate HIV-1 expression. Indeed, addition of cytokine and chemokine antagonists to both U1/HUVEC co-cultures and to U1 cultured in HUVEC-conditioned media clearly down-regulated p24 release. Anti-IL-6, anti-tumour necrosis factor-alpha (TNF-α) and, particularly, anti-MCP-1 MoAbs reduced p24 release, while anti-IL-8 polyclonal antiserum and IL-1 receptor antagonist (IL-1Ra) had no significant effect. Thus, the interaction between HUVEC and infected monocytic cells up-regulates HIV-1 replication predominantly through production of endothelium-derived soluble factors including MCP-1, TNF-α and IL-6. This phenomenon may influence the passage of HIV-1 from latency to productive replication and enhance virus spreading during physiological and/or pathological contact of monocytes with endothelium.     

The functional CD40 antigen of fibroblasts may contribute to the proliferation of rheumatoid synovium

This paper demonstrates that CD40 is expressed on rheumatoid synovial pannus and primary fibroblast cell lines established from rheumatoid and osteoarthritic synovium as well as normal skin. Among various tested cytokines, interferon-gamma (IFN-γ) and to a lower extent, tumour necrosis factor-alpha (TNF-α) were found to upregulate CD40 expression on fibroblasts. Synovial and skin fibroblasts cultured over CD40 Ligand transfected L cells (L-CD40 L) demonstrate a CD40 specific increase of DNA synthesis as measured by tritiated thymidine incorporation. Cell-cycle analysis and enumeration of viable cells further show that CD40 induced fibroblast proliferation. Costimulation with L-CD40 L and IFN-γ resulted in maximal proliferation. Engagement of fibroblasts CD40 increased the IL-1-induced production of granulocyte macrophage-colony stimulating factor and macrophage inflammatory protein-1α MIP-1α. CD40 L activated fibroblasts showed decreased levels of CD40, but only marginal alterations of other cell-surface antigens. Taken together, the present results indicate that fibroblasts express functional CD40 and suggest a possible role of CD40 L expressing cells, such as activated T cells and mast cells, in the development of synovium hyperplasia.     

Identification of human IgG autoantibodies specific for IL-10

Since autoantibodies to IL-1α, interferon-alpha (IFN-α) and IL-6 have been described, this study concentrated on the search for autoantibodies to hIL-10 using an assay based on the precipitation of ¹²⁵I-hIL-10 anti-IL-10 autoantibody complexes using Protein G-Sepharose. Among 1860 tested sera, only seven were found to specifically precipitate IL-10, thus indicating the rare occurrence of such autoantibodies. Four of those seven anti-IL-10 autoantibody sera were specific for hIL-10, two recognized both human and viral IL-10, while the last one recognized human, viral and murine IL-10, thus suggesting the existence of at least three different epitopic specificities. The purification of anti-IL-10 autoantibody from one serum demonstrated the existence of a single (IgG1, λ) autoantibody that neutralized IL-10 biological activity. Thus, autoantibodies to IL-10 may represent natural antagonists to IL-10.     

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective

Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin (IL) -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells (HMCs).

Methods

The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay (RPA). Activity of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) was examined by electrophoretic mobility shift assay (EMSA). NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase (JNK) activity were assayed by immunoblot.

Results

Recombinant IL-13 inhibited tumor necrosis factor-α (TNF-α), IL-1α, IL-1β, monocyte chemoattractant protein-1 (MCP-1), IL-8, and transforming growth factor-β1 (TGF-β1) mRNA expressions in a dose-dependent manner. Lipopolysacchorides (LPS) dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner.

Conclusion

IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.     

The bacterial superantigen Staphylococcal enterotoxin B stimulates lymphocyte locomotor capacity during culture in vitro.

The bacterial superantigen Staphylococcal enterotoxin B (SEB) was investigated for its effects on lymphocyte locomotion in vitro. Culture of peripheral blood mononuclear cells (PBMC) for 24-72 hr in SEB (1-100 micrograms/ml) increased the proportion of lymphocytes in locomotor (polarized) morphology and capable of invading collagen gels, to the same extent as the established locomotor activator, anti-CD3 (alpha-CD3), though the conventional antigen, tetanus toxoid was ineffective. The cells responding to SEB were predominantly T cells. SEB had no effect on lymphocyte locomotion in short-term (45 min) assays, thus its effect is to stimulate growth-related locomotor capacity and it does not act as a chemoattractant. During culture of PBMC in SEB, the chemokines interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1) were released into the culture medium. The presence of anti-IL-8, but not of anti-MCP-1, either during culture or added to SEB culture supernatants and tested in short-term assays, inhibited the development of polarization suggesting that IL-8, which is a lymphocyte chemoattractant, also plays a key role in SEB-induced locomotor activation. Among SEB-activated lymphocytes, CD45RO+CD45RA- lymphocytes showed enhanced locomotor responses, but a relation was not found between locomotor activity and the presence of cell surface CD69.     

Proteolytic Activation of the Interleukin-1β Precursor by Candida albicans

Chronic inflammation rather than invasion is characteristic of some forms of superficial candidiasis such as denture stomatitis. We hypothesized that Candida albicans may play a critical role in the pathogenesis of inflammatory lesions observed in chronic candidiasis by activating the proinflammatory cytokine interleukin-1β (IL-1β) from epithelial stores of the precursor. The aim of this study was therefore to demonstrate the proteolytic cleavage and activation of the inactive precursor of IL-1β (pro-IL-1β) by C. albicans. After incubation of either blastospores or hyphae with the inactive precursor, proteolytic cleavage was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western immunoblotting analysis, and the biological activity of the cleavage products was tested in a bioassay. We report here that late-stationary-growth-phase blastospores as well as hyphae of C. albicans, but not exponentially growing cells, can efficiently cleave pro-IL-1β to yield fragments of molecular masses compatible with mature biologically active IL-1β (17 to 19 kDa). Assays conducted in the presence of selected proteinase inhibitors suggest that the cleavage of pro-IL-1β involves the participation of one or more aspartyl proteinases. Cleavage products showed a dose-dependent IL-1β-like activity in a thymocyte proliferation bioassay, which was inhibited by anti-IL-1β neutralizing antibodies. The present data thus suggest a role for C. albicans proteinases in the activation and maintenance of the inflammatory response at epithelial surfaces.     

Immunofluorescent demonstration of an IgG–β1C complex in synovial lining cells of rheumatoid synovial membrane

Synovial tissues obtained at arthrotomy or by needle biopsy were digested with trypsin, and suspensions of synovial cells isolated. Immunofluorescent staining of these cells has shown the simultaneous presence of immunoglobulin G (IgG) and the β_1C component of complement in the cytoplasm of most of the phagocytic lining cells of fifteen of seventeen synovia obtained from both seropositive and seronegative patients with rheumatoid arthritis. This staining pattern was not found in synovial cells obtained from patients with other inflammatory or non-inflammatory arthritides. Discrete cytoplasmic inclusions composed of: (1) IgG and α_1C, and (2) IgG and rheumatoid factor were also observed, but only in synovial cells from seropositive rheumatoid patients. The significance of the presence of an apparent IgG–β_1C complex in the synovial lining cells of most patients with rheumatoid arthritis is discussed.     

Caspase-1 Level in Synovial Fluid Is High in Patients with Spondyloarthropathy but Not in Patients with Gout

Activation of caspase-1 by NALP3 inflammasomes has been shown to be important in initiating acute gouty arthritis. The objectives of this study were to measure the levels of caspase-1 in synovial fluid in gout and various arthritides, and to elucidate the clinical significance of caspase-1 levels in synovial fluid. Caspase-1, IL-1β, IL-18, and uric acid were measured in synovial fluid from 112 patients with gout and other arthritides, such as rheumatoid arthritis, osteoarthritis, and spondyloarthropathy. Caspase-1 in synovial fluid from patients with crystal-induced arthritis, inflammatory arthritis, osteoarthritis, and spondyloarthropathy was 35.9 ± 86.7, 49.7 ± 107.7, 2.1 ± 7.0, and 152.6 ± 155.7 pg/mL, respectively. The mean level and the frequency of high levels (≥125 pg/mL) of caspase-1 in spondyloarthropathy were significantly higher than those in the other arthritides including gout. Caspase-1 was detectible in the synovial fluid of patients with the various arthritides. Contrary to our hypothesis, the caspase-1 level in the synovial fluid of patients with gout was not higher than in that of other arthritides. High levels of caspase-1 may be helpful in differentiating spondyloarthropathy from other arthritides.     

Immunomodulatory Effects of CpG Oligodeoxynucleotides on Established Th2 Responses

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-γ) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-γ, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8⁺ T lymphocytes. Although IFN-γ plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-γ was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-γ, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-γ was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-γ and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.     

Mechanisms of binding of cutaneous lymphocyte-associated antigen-positive and αeβ7-positive lymphocytes to oral and skin keratinocytes

Intraepithelial lymphocytes (IEL) utilize the integrin αeβ7 on their surface to bind to E-cadherin on epithelial cells in the gut and breast. In oral mucosa and skin IEL express αeβ7 and the cutaneous lymphocyte-associated antigen (CLA) but the mechanisms of adhesion of these subsets to keratinocytes are unknown. Levels of αeβ7 and CLA were up-regulated on peripheral blood lymphocytes (PBL) by transforming growth factor-β (TGF-β) and interleukin-12 (IL-12), respectively, and both groups of lymphocytes adhered onto oral and skin keratinocytes. Adhesion of IL-12-activated PBL was totally abolished by anti-lymphocyte-associated function antigen type 1 (anti-LFA-1) antibodies but was unaffected by anti-αeβ7 antibodies indicating that adhesion of the CLA-positive subset is mediated via LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Adhesion of TGF-β-activated PBL to E-cadherin-positive oral and skin keratinocytes was partially inhibited by anti-αeβ7 antibodies but was unaffected by the blocking antibody E4.6 against E-cadherin which detects the binding site for αeβ7-positive lymphocytes in breast and gut epithelium. TGF-β-activated PBL also bound to an E-cadherin-negative oral keratinocyte cell line and adhesion was inhibited by anti-αeβ7 antibodies. These results strongly suggest that in oral epithelium and epidermis αeβ7-positive lymphocytes do not bind to E-cadherin and there may be a novel second ligand for the αeβ7 integrin.     

IRAK4 inhibition: a promising strategy for treating RA joint inflammation and bone erosion

Flares of joint inflammation and resistance to currently available biologic therapeutics in rheumatoid arthritis (RA) patients could reflect activation of innate immune mechanisms. Herein, we show that a TLR7 GU-rich endogenous ligand, miR-Let7b, potentiates synovitis by amplifying RA monocyte and fibroblast (FLS) trafficking. miR-Let7b ligation to TLR7 in macrophages (MΦs) and FLSs expanded the synovial inflammatory response. Moreover, secretion of M1 monokines triggered by miR-Let7b enhanced Th1/Th17 cell differentiation. We showed that IRAK4 inhibitor (i) therapy attenuated RA disease activity by blocking TLR7-induced M1 MΦ or FLS activation, as well as monokine-modulated Th1/Th17 cell polarization. IRAK4i therapy also disrupted RA osteoclastogenesis, which was amplified by miR-Let7b ligation to joint myeloid TLR7. Hence, the effectiveness of IRAK4i was compared with that of a TNF inhibitor (i) or anti-IL-6R treatment in collagen-induced arthritis (CIA) and miR-Let7b-mediated arthritis. We found that TNF or IL-6R blocking therapies mitigated CIA by reducing the infiltration of joint F480⁺iNOS⁺ MΦs, the expression of certain monokines, and Th1 cell differentiation. Unexpectedly, these biologic therapies were unable to alleviate miR-Let7b-induced arthritis. The superior efficacy of IRAK4i over anti-TNF or anti-IL-6R therapy in miR-Let7b-induced arthritis or CIA was due to the ability of IRAK4i therapy to restrain the migration of joint F480⁺iNOS⁺ MΦs, vimentin⁺ fibroblasts, and CD3⁺ T cells, in addition to negating the expression of a wide range of monokines, including IL-12, MIP2, and IRF5 and Th1/Th17 lymphokines. In conclusion, IRAK4i therapy may provide a promising strategy for RA therapy by disconnecting critical links between inflammatory joint cells.     

Mechanism of T-Cell Activation by Mycobacterial Antigens in Inflammatory Synovitis

Earlier studies demonstrated enhanced proliferative responses to an acetone precipitable Mycobacterium tuberculosis (AP-MT) antigenic complex by T lymphocytes from the synovial fluid, compared with the peripheral blood, of patients with inflammatory synovitis, including rheumatoid arthritis. In contrast, decreased proliferation and interleukin 2 (IL-2) production in response to mitogens by synovial fluid lymphocytes from patients with rheumatoid arthritis has been demonstrated. In order to determine if IL-2 was produced in response to AP-MT, the peripheral blood and synovial fluid of patients with inflammatory arthritis were analysed by measuring proliferation and IL-2 production in response to AP-MT and tetanus toxoid. A reduction of IL-2 production relative to proliferation was observed in some, but not all, synovial fluids of patients who responded to the AP-MT. Nevertheless, antibodies to IL-2 as well as interleukin 4 (IL-4), significantly inhibited proliferation of synovial fluid lymphocytes by AP-MT. There was no inhibition by antibodies to interleukin 6 (IL-6). We conclude that AP-MT induced proliferation by synovial fluid lymphocytes is mediated by both IL-2 and IL-4.     

Production of IL-6 by T cells from the femoral head of patients with rapidly destructive coxopathy (RDC)

RDC is a syndrome with unknown etiology that causes rapid destruction of a hip joint. We have investigated the production of osteoclast-activating cytokines (IL-6, IL-1α and tumour necrosis factor-alpha (TNF-α)), interferon-gamma (IFN-γ) and IL-8 by T cells in the affected joint. The level of IL-6 produced by the T cell lines (TCL) established from the femoral head was significantly higher than that from patients' or healthy donors' peripheral blood mononuclear cells (PBMC). IL-6 production by the TCL from synovial membrane or from patients' PBMC was also significantly higher than that from healthy donors' PBMC. IL-1α production by the TCL from the femoral head was significantly higher than any of the other groups when all the TCL were used for the analysis. TNF-α production was highest in the TCL from patients' PBMC. The levels of IFN-γ or IL-8 were not significantly different among these four groups. The plasma levels of all these cytokines except for IFN-γ, that was rather lower, in RDC patients were not significantly different from those in osteoarthrosis or trauma patients, or healthy donors. These results suggest that T cells at the affected femoral head, and also synovial membrane to some extent, are involved in bone resorption through the production of IL-6 and probably IL-1α in patients with RDC.     

Involvement of macrophage chemotactic protein-1 and interleukin-1beta during inflammatory but not basic fibroblast growth factor-dependent neovascularization in the mouse cornea

Corneal neovascularization develops in several pathologic conditions, but its underlying mechanisms remain elusive. We used a mouse inflammatory corneal model (corneas cauterized with silver nitrate) and assessed the role of monocyte/macrophage-attracting factors, macrophage chemotactic protein-1 (MCP-1), and a proinflammatory cytokine, IL-1beta, on macrophage recruitment and neovascularization. Both MCP-1, IL-1beta protein, and mRNA levels increased markedly 12 hours after the chemical cauterization. In situ hybridization showed that MCP-1 was located in corneal epithelial cells, and IL-1beta was located in corneal epithelial cells and infiltrating inflammatory cells. In addition, double staining of corneas with antibodies specific for monocytes/macrophages and IL-1beta revealed that IL-1beta was found in infiltrating monocytes/macrophages at Day 2 after cauterization. Both IL-1beta and MCP-1 induced neovascularization in a rat cornea model, and the cauterization-induced corneal neovascularization was partially inhibited by subconjunctival injection of anti-IL-1beta or anti-MCP-1. Coadministration of two antibodies inhibited corneal neovascularization slightly more than that by the administration of each. In contrast, administration of the anti-MCP-1 or anti-IL-1beta showed minimal inhibition of basic fibroblast growth factor-driven corneal neovascularization by mouse cornea assay. Cauterized corneas treated with anti-MCP-1 antibody had significantly fewer monocytes/macrophages than control. These results indicate the existence of distinct monocyte/macrophage-involved angiogenic pathways in mouse cornea, in which MCP-1 released from corneal epithelial cells attracts monocytes/macrophages into the cornea, where they release IL-1beta leading to inflammatory neovascularization. In addition, the IL-1beta and MCP-1 released from the corneal epithelial cells may directly induce corneal neovascularization.     

Inhibitory effect of clarithromycin on costimulatory molecule expression and cytokine production by synovial fibroblast-like cells

This study was undertaken to investigate the immunomodulatory effect of clarithromycin against synovial fibroblast-like cells (synoviocytes). Synovial tissue obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients was enzymatically digested to separate synoviocytes. The synoviocytes were cultured with or without cytokines in the presence of various concentrations of clarithromycin. The expression of costimulatory molecules was examined on the surface of the synoviocytes, using specific MoAbs and flow cytometry. The production of cytokines by synoviocytes was also measured using an immunoenzymatic assay. Finally, autologous T cells were stimulated by interferon-gamma (IFN-γ)-treated synoviocytes in response to purified protein derivative (PPD). In some experiments, MoAbs specific for costimulatory molecules or clarithromycin were added and ³H-thymidine incorporation was counted. Intercellular adhesion molecule-1 (ICAM-1), LFA-3 and vascular cell adhesion molecule-1 (VCAM-1) were detected on the surface of both RA and OA synoviocytes. However, ICAM-2, B7–1 and B7–2 were not detected, and cytokines failed to induce these molecules. Both spontaneous and up-regulated expression of ICAM-1, LFA-3 and VCAM-1 by IFN-γ, IL-1β or 12-o-tetradecanoyl phorbol 13-acetate (TPA) were markedly suppressed by clarithromycin in a dose-dependent manner at concentrations between 0.1 and 10 μg/ml. The production of IL-1β, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-1α and tumour necrosis factor-alpha (TNF-α) by synoviocytes was detected. Clarithromycin significantly suppressed the production of these cytokines, but did not enhance IL-10 production. Finally, autologous T cells were stimulated by IFN-γ-treated synoviocytes in response to PPD. As clarithromycin suppressed HLA-DR and costimulatory molecule expression was enhanced by IFN-γ, autologous T cell proliferation was markedly inhibited by clarithromycin. Clarithromycin has a considerable immunosuppressive effect on synoviocytes by inhibiting costimulatory molecule expression, cytokine production and antigen-specific T cell proliferation induced by synoviocytes.