Ichiko Ezaki

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.

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.

Recombinant human IL-1β and TNF-α stimulate production of IL-1α and IL-1β by vascular smooth muscle cells and IL-1α by vascular endothelial cells

Vascular endothelial cells (EC) produced IL-1 alpha but not IL-1 beta into extracellular fluids. Vascular smooth muscle cells (SMC), on the other hand, produced both IL-1 alpha and IL-1 beta, and IL-1 beta produced was much higher than IL-1 alpha. The addition of recombinant human IL-1 beta or recombinant human TNF-alpha significantly enhanced IL-1 alpha production in EC, and IL-1 alpha and IL-1 beta production in SMC. IL-1 beta release was not observed even when EC were stimulated with TNF-alpha. These results suggest that the species of released form of IL-1 are different in different cell types and that cytokines enhance IL-1 alpha and IL-1 beta production in SMC and IL-1 alpha production in EC.

Human monoclonal rheumatoid factors augment arthritis in mice by the activation of T cells

In order to investigate the in vivo role of rheumatoid factor (RF), the effects of the administration of human monoclonal (m) IgM‐RF and IgG‐RF on the development of arthritis in mice were examined. The administration of human mRFs into mice immunized with type II collagen (CII) markedly enhanced the clinical score and paw swelling. The severity of arthritic joint disease with a marked infiltration of lymphoid cells, proliferation of synovial membrane, pannus formation and destruction of articular cartilage was significantly enhanced in both groups receiving RF (RF‐enhanced arthritis). Skin ulcers were also observed in some of these RF‐enhanced arthritis mice, whereas no such signs were observed in CII‐immunized mice without mRFs. Both IgM‐RF and IgG‐RF increased CII‐specific IgG antibodies in circulation, and the severity of arthritis correlated with the production of high titres of anti‐CII antibodies. In vivo treatment of RF‐enhanced arthritis mice with an anti‐CD4 MoAb or an anti‐CD8 MoAb inhibited the induction and progression of arthritis in these mice. Administration of RF to severe combined immunodeficient (SCID) mice with arthritis developed by the transfer of spleen cells from CII‐immunized mice, prolonged the arthritis and enhanced the severity. This murine model of RF‐enhanced arthritis may provide a useful tool for analysing the pathogenesis of rheumatoid arthritis in RF‐positive patients.

Superoxide generation by snovial fluid neutrophils enhanced by immune complexes and suppressed by rheumatoid factor in synovial fluid

SummarySynovial fluid (SF) from patients with rheumatoid arthritis (RA) enhanced superoxide generation by neutrophils isolated from RA SF, in contrast to SF from patients with osteoarthritis. These superoxide generation-enhancing substances may be intermediate-sized immune complexes and a complement C5-derived fragment. Rheumatoid factor (RF) isolated from RA SF suppressed superoxide generation-enhancing activity of aggregated IgG. Therefore, biologically active RF may block the interaction of the immune complexes with neutrophils accumulating in RA SF, and protect the joint tissue from the effects of oxygen radicals or proteases.

Characterization of T‐cell receptor β chain mRNA expression in IFN‐α‐responsive chronic myelogenous leukaemia patients

Interferon‐α (IFN‐α) has shown promise in the treatment of chronic phase of chronic myelogenous leukaemia (CML). IFN‐α has also been found to indirectly up‐regulate the expression of major histocompatibility (MHC) antigens, and to directly increase the activity of lymphocytes against tumour cells. To elucidate whether IFN‐α induces anti‐leukaemic activity of the autologous T cells in CML patients, we analysed the accumulation of T‐cell receptor (TCR) in each Vβ family using the reverse transcriptase‐polymerase chain reaction (RT‐PCR) followed by single‐strand conformation (SSCP) analysis. We found the predominant expression of the Vβ 10, 12, and 14 families in the peripheral blood (PB) of CML patients, in contrast to healthy donors. Especially, in IFN‐α‐responsive patients, we observed an enhancement of the accumulation of the Vβ 9 and 20 families, suggesting that T cells enhanced by IFN‐α may react with a discrete set of antigens on the surface of malignant cells. These findings may demonstrate that CML cells possess the heterogenous antigens and the clonal expansion of Vβ 9+ and Vβ 20+ T cells may be a prognostic indicator of the immune responsiveness to IFN‐α.

Rheumatoid factor modulation of neutrophil superoxide generation enhancing activity of preformed immune complexes

SummaryHeat aggregated human (HAG) IgG pretreated with total rheumatoid factors isolated from the serum of rheumatoid arthritis patients showed decreased superoxide generation enhancing activity as compared with HAG pretreated with buffer alone. Similarly, monoclonal IgM rheumatoid factor isolated from the serum of a patient with macroglobulinemia complicated by rheumatoid arthritis inhibited superoxide generation enhancing activity of HAG. On the other hand, superoxide generation enhancing activity of BSA-antiBSA immune complexes was not affected by preincubation with rheumatoid factors isolated from the sera of either rheumatoid arthritits patients or the macroglobulinemia patient. Rheumatoid factors isolated from rheumatoid arthritis serum were fractionated by high performance liquid chromatography and IgM-class and IgG-class rheumatoid factors were obtained. IgG-class rheumatoid factor significantly enhanced the superoxide generation enhancing activity of HAG, whereas IgM rheumatoid factor inhibited it. Rheumatoid arthritis sera showed significantly higher superoxide generation enhancing activity than normal sera. HAG preincubated with rheumatoid arthritis sera showed significantly lower superoxide generation enhancing activity than HAG preincubated with normal sera. These results suggest that factors inhibiting superoxide generation enhancing activity of HAG are present in rheumatoid arthritis sera, and that the responsible is IgM rheumatoid factor, whereas IgG rheumatoid factor enhances it. The factors that express superoxide generation enhancing activity in rheumatoid arthritis sera are suggested to be intermediate size immune complexes.

Leukotriene B4 Production by Neutrophils from Patients with Rheumatoid Arthritis

Leukotriene B4 (LTB4) production by unstimululated and stimulated neutrophils from peripheral bloods of normal persons and rheumatoid arthritis patients and from rheumatoid arthritis synovial fluids were examined. Spontaneous production of LTB4 was observed in neutrophils from rheumatoid arthritis synovial fluids but not in peripheral blood neutrophils from rheumatoid arthritis and normal persons. Synovial fluid neutrophils produced significantly higher amounts of LTB4 than peripheral neutrophils of rheumatoid arthritis patients or normal persons, whether neutrophils were stimulated or not. Recombinant human IL-1B did not stimulate LTB4 production by neutrophils. Tiopronin, a SH group-containing compound, inhibited neutrophil LTB4 production. These results indicate that LTB4 plays an important role in rheumatoid joint inflammation.