Masashi Nobunaga

The effects of cytokines on metalloproteinase inhibitors (TIMP) and collagenase production by human chondrocytes and TIMP production by synovial cells and endothelial cells.

It has been suggested that lL‐1 produces cartilage matrix degradation by metalloproteinases such as collagenase, and that such degradation is regulated by metalloproteinase inhibitors (TIMP). Therefore, the balance between collagenase and TIMP is an important factor for tissue destruction in inflammatory joints. In the present study the effects of cytokines on collagenase and TIMP production in chondrocytes as welt as the effects of cytokines on TIMP production in connective tissue cells were studied. IL‐Iβ inhibited TIMP production in endothelial cells while enhancing TIMP production in synovial cells and chondrocytes. In addition, tumour necrosis factor‐alpha (TNF‐α) significantly inhibited and IL‐6 significantly enhanced TIMP production in endothelial cells, synovial cells and chondrocytes. In the chondrocyte supernatant, collagenase activity/TIMP ratio was significantly elevated by the addition of either IL‐lβ or TNF‐α to the cells, whereas the ratio was significantly decreased by IL‐6. These results suggest that the cytokine effects on TIMP production are different among the different cell types, and that either IL‐lβ or TNF‐α induce cartilage matrix degradation by disrupting the collagenase/TIMP balance, while, on the other hand, IL‐6 protects the tissue through an opposite effect.

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide

Abstract5Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle ceils and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.

Activation of complement in normal serum by hydrogen peroxide and hydrogen peroxide‐related oxygen radicals produced by activated neutrophils

Neutrophils activated by soluble partculate stimuli generate superoxide anion and subsequently form hydrogen peroxide and other oxygen radicals. The effect of hydrogen peroxide on the complement system in normal serum was investigated. Treatment of normal serum with hydrogen peroxide resulted in a diminution of the haemolytic activity of the total and alternative complement pathways and the haemolytic titres of C3 and C5 but not of C2, in normal serum. These decreases in complement activity depended on the concentration of hydrogen peroxide added to the serum. Immunoelectrophoretic analysis of hydrogen peroxide‐treated serum showed that C3 and C5 proteins were activated. Complement degradation products C3a and C5a were produced in normal serum treated with hydrogen peroxide, and 20 mm EDTA abolished C3a and C5a production in hydrogen peroxide‐treated serum but 20 mm Mg‐EGTA did not. Catalase completely abolished and dimethylsulphoxide and D‐mannitol, hydroxyl radical scavengers, partially inhibited the hydrogen peroxide‐mediated complement activation. Hypochlorite, incubated with normal serum, significantly inhibited serum haemolytic activity, and sodium thiosulphate, a reducing agent, abolished the effect of hypochlorite. Normal serum incubated with activated neutrophils showed neutrophil chemotactic activity and decreased serum haemolytic activity, and the addition of catalase or methionine (S mm) completely abolished the effects of activated neutrophils. These results suggest that hydrogen peroxide activates complement via an alternative pathway of complement activation and that hydroxyl radicals and other hydrogen peroxide‐related species such as hypochlorite are most likely involved in hydrogen peroxide‐mediated complement activation. Complement activation by oxygen radicals produced by activated neutrophils may be one of the mechanisms by which complement is activated in human immune complex diseases.

Relationship between leukotriene B4 and immunological parameters in rheumatoid synovial fluids

Leukotriene B4 (LTB4) was measured in synovial fluid from 20 patients with rheumatoid arthritis and 15 patients with osteoarthritis. The level of LTB4 was significantly higher in synovial fluid from rheumatoid arthritis patients as compared with synovial fluid from osteoarthritis patients. LTB4 levels also significantly correlated with cell numbers, rheumatoid factor, and immune complexes in synovial fluid from rheumatoid arthritis patients. There was an inverse correlation between LTB4 levels and complement components. The high-pressure liquid chromatography peak of immunoreactivity extracted from the synovial fluid occurred at a retention volume identical to that of authentic LTB4. These results suggest that the increased level of this mediator in synovial fluid may contribute to perpetuation of inflammation and tissue destruction in rheumatoid arthritis.

ROLE OF OXYGEN RADICALS AND IL-6 IN IL-1-DEPENDENT CARTILAGE MATRIX DEGRADATION

It has been suggested that IL-1 produces cartilage matrix degradation by metalloproteinases such as collagenase and that such degradation is regulated by metalloproteinase inhibitors. In the present study, the effects of IL-6 and oxygen radical scavengers on cartilage matrix degradation were studied. Superoxide dismutase, catalase, or methionine all significantly inhibited cartilage matrix degradation both in IL-1β-stimulated and unstimulated experimental conditions. Both 10 mM EDTA and 100 nM tissue inhibitor of metalloproteinase (TIMP) significantly inhibited cartilage matrix degradation. The addition of methionine significantly inhibited collagenase activity produced in the culture supernatants of chondrocytes stimulated with IL-1β. IL-6 significantly suppressed cartilage matrix degradation produced spontaneously or by IL-1β stimulation in chondrocytes. IL-6 inhibited superoxide production by chondrocytes both in IL-1β-stimulated or unstimulated conditions. These results suggest that oxygen radicals are involved in cartilage matrix degradation mediated by both paracrine and autocrine IL-1 mechanisms and that oxygen radicalmediated activation of collagenase in chondrocytes may explain the mechanisms of how oxygen radicals are involved in cartilage matrix degradation. IL-6 inhibited superoxide production in chondrocytes and thus inhibited cartilage matrix degradation.

Effects of recombinant human IL-Iβ on production of prostaglandin E2, leukotriene B4, NAG, and superoxide by human synovial cells and chondrocytes

The effects of recombinant human IL-1β on the production of prostaglandin E2 (PGE2), leukotriene B4 (LTB4),N-acetyl-β-D-glucosaminidase (NAG), and superoxide by synovial cells and chondrocytes derived from osteoarthritis patients were determined. IL-1β markedly enhanced PGE2 production in chondrocytes and, to the lesser extent, in synovial cells. Synovial cells and chondrocytes spontaneously released LTB4 into culture medium and IL-1β significantly inhibited LTB4 production by these cells. IL-1β significantly suppressed the release of NAG and superoxide by synovial cells, whereas it significantly enhanced the production of NAG and superoxide by chondrocytes. Production of intracellular superoxide dismutase by synovial cells was significantly enhanced on incubation with IL-1β, but that of chondrocytes was not altered. IL-6, unlike IL-1β, significantly suppressed the production of NAG and superoxide by synovial cells and chondrocytes.These results suggest that IL-1 has differing effects on the release of mediators by synovial cells and chondrocytes and that these cells also vary in their responses to IL-1β and IL-6.

Production of soluble ICAM-1 from human endothelial cells induced by IL-1 beta and TNF-alpha.

The present study was designed to establish the effects of cytokines on soluble ICAM-1 (sICAM-1) production by human endothelial cells (EC) and ICAM-1 expression on these cells and the effects of purified sICAM-1 on lympho-cyte-EC adhesion. Expression of ICAM-1 and production of sICAM-1 were measured by a specific ELISA method. ICAM-1 expression was enhanced by IL-1β, TNF-α, and most effectively by IFN-γ. IL-4, IL-6, M-CSF, or GM-CSF showed no effects on ICAM-1 expression. IL-4 (100 units/ml) or IL-6(100 units/ml) abolished the enhancing effect of IL-1β, while TNF-α (1, 10, 100 units/ml) synergized with IL-1β to promote ICAM-1 expression in EC. In contrast with the transient increase of cell-associated ICAM-1 expression after activiation by IL-1β, which peaked 40 h poststimulation and declined thereafter, sICAM-1 continued to accumulate in culture supernatants even after 48 h poststimulation in IL-1β-stimulated EC. IL-1β treatment resulted in an increase in adhesion. sICAM-1, purified from cell-free supernatants obtained after a 48-h culture of EC in IL-1β by affinity chromatography using monoclonal ICAM-1 antibody coupled to Sepharose beads, significantly inhibited lymphocyte EC adhesion. Preincubation of lymphocytes with conditioned medium of EC cultured with 100 units/ml IL-1β for 48 h, which contained a considerable amount of sICAM-1, resulted in a significant inhibition of lymphocyte adhesion to IL-1β-stimulated EC. These results suggest that there is a cumulative increase in sICAM-1 concentration in the vicinity of cytokine-stimulated EC and that this sICAM-1 modulates ICAM-1-mediated cell to cell interaction.

Anti-inflammatory effects of recombinant human manganese superoxide dismutase on adjuvant arthritis in rats

The anti-inflammatory effects of recombinant human manganese superoxide dismutase on adjuvant arthritis was investigated. Local application of this manganese superoxide dismutase given every 2 days not only significantly reduced foot swelling but also retarded radiological bone destruction in adjuvant arthritis. Copper zinc superoxide dismutase had little effect on foot swelling.

C1q binding to human vascular smooth muscle cells mediates immune complex deposition and superoxide generation.

Evidence was obtained for the binding of Clq to the membrane of cultured vascular smooth muscle cells derived from human umbilical cord veins. Clq was fixed to the cell membrane at 4°C, whereas it was ingested into the cytoplasm, as a cytoplasmic inclusion, when tested at 37°C. The addition of Clq in advance inhibited the subsequent binding of Clq. Neither fibronectin nor laminin was detected on the cell membrane. Aggregated IgG bound to vascular smooth muscle cells in the case of preincubation with Clq at 4°C, whereas aggregated IgG did not bind to the cells in the absence of Clq. The addition of Clq molecules to the cells in suspension enhanced Superoxide generation by vascular smooth muscle cells. There was no effect of Clq on Superoxide generation by the cells in monolayer. These results suggest that Clq binds on the membrane of vascular smooth muscle cells via its specific receptor that mediates immune complex binding to the cells and Superoxide generation. These properties elucidate the mechanisms by which circulating immune complexes deposit in the vascular wall, and subsequent degradation of tissue components surrounding vascular smooth muscle cells occurs through oxidative burst of the cells.

Effect of cytokine-induced soluble ICAM-1 from human synovial cells on synovial cell-lymphocyte adhesion

The present study was designed to establish (i) the effects of cytokines on soluble ICAM‐1 (sICAM‐1) production by human synovial cells (SC) and ICAM‐1 expression on these cells, and (ii) the effects of sICAM‐1 on lymphocyte‐SC adhesion. sICAM‐1 production was enhanced in parallel with ICAM‐1 expression by IL‐1β, TNF‐α and IFN‐γ. IL‐4 showed no effects on ICAM‐1 expression. In contrast with the transient elevation of cell‐associated ICAM‐1 by IL‐1β, which peaked 36 h after stimulation and declined thereafter, sICAM‐1 continued to accumulate in culture supernatants even after 48 h. Purified sICAM‐1 was obtained from a 48h culture synovial cell supernatant by affinity chromatography using ICAM‐1 monoclonal antibody. The purified sICAM‐1 significantly inhibited adhesion of lymphocytes and monocytes to cytokine‐stimulated synovial cells. These results suggest that sICAM‐1 may modulate chronic synovitis by inhibiting ICAM‐1‐mediated cell‐to‐cell adhesion.