Hirotsugu Ide

Vascular Endothelial Growth Factor Expression and Regulation of Murine Collagen-Induced Arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.

The role monokines in granuloma formation in mice: The ability of interleukin 1 and tumor necrosis factor-α to induce lung granulomas

Abstract Granulomatous inflammation is associated with many significant human diseases, including tuberculosis, leprosy, sarcoidosis, parasite infection, and berylliosis. Very little is known about the basic mechanism of this type of inflammation. In the present study, we showed that pulmonary granulomas were induced in mice by the intractracheal injection of agarose beads coupled to recombinant interleukin 1 (IL-1) or tumor necrosis factor-α (TNF-α). Histologically, the bulk of granulomas was composed of macrophages and their derivatives. In contrast, the inflammatory reactions induced by beads coupled to either recombinant IL-2 or murine interferon-γ were considerably smaller than those induced by beads coupled to monokines. These results suggest that macrophages and monokines such as IL-1 and TNF-α, but not T cell-derived lymphokines, play an essential role in granuloma formation.

Vascular endothelial growth factor expression by activated synovial leukocytes in rheumatoid arthritis: critical involvement of the interaction with synovial fibroblasts.

OBJECTIVE To examine the expression and regulation of the angiogenic factor, vascular endothelial growth factor (VEGF), by fibroblast-like synoviocytes (FLS), monocytes, and polymorphonuclear neutrophils (PMNs) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS Monocytes or PMNs obtained from RA SF were cocultured with unstimulated, semiconfluent RA FLS. Culture supernatants were assayed for the proliferation and in vitro tube formation of endothelial cells, and for the production of VEGF, by enzyme-linked immunosorbent assay. The expression of VEGF messenger RNA and protein was also determined by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS We found that the interaction of inflammatory, activated leukocytes with FLS resulted in synergistic increases in VEGF expression and secretion, which contributed to the proliferation of endothelial cells and to in vitro endothelial tube formation. The induction of VEGF was mediated via specific adhesion molecules, as indicated by the finding that anti-integrin antibodies significantly inhibited VEGF. Furthermore, the levels of VEGF secretion correlated with the expression of cell surface integrin (CD11b and CD18) on both monocytes and PMNs in the SF. CONCLUSION VEGF expression within inflamed joints thus appears to be regulated not only by inflammatory cytokines, but also by the physical interaction of activated leukocytes and FLS. Once expressed, VEGF likely plays a crucial role in the neovascularization of the pannus and the progressive joint destruction associated with the synovial inflammation of RA.

Production of interleukin 1-like factor from human peripheral blood monocytes and polymorphonuclear leukocytes by superoxide anion: the role of interleukin 1 and reactive oxygen species in inflamed sites.

In the present study, we investigated the possibility that oxidative stress to human peripheral blood monocytes and polymorphonuclear leukocytes (PMNs) can induce interleukin 1 (IL-1)-like activity. Oxidative stress that we used was superoxide anion (O2-) and hydrogen peroxide (H2O2). O2-, but not H2O2, could induce an IL-1-like factor(s) from monocytes and PMNs. IL-1-like activity from monocytes and PMNs induced by O2- was due to de novo synthesis because no IL-1-like activity was found in culture supernatants and in the lysate of unstimulated cells. We next examined the effects of radical scavengers on production of an IL-1-like factor(s). Generation of IL-1-like activity from monocytes was amplified by preincubation with catalase (H2O2 scavenger), although it was suppressed by preincubation with either superoxide dismutase (O2- scavenger) or vitamin E (antioxidant analogs). These results suggest that production of an IL-1-like factor(s) from monocytes and PMNs was due to O2- stimulation. Our data that production of an IL-1-like factor(s) from inflammatory cells by stimulation with O2- imply a model of the up-regulation mechanism of inflammation mediated by enhanced IL-1-like factor production stimulated with reactive oxygen species.

Macrophage inflammatory protein 1 alpha expression by synovial fluid neutrophils in rheumatoid arthritis

OBJECTIVE To determine the contribution made by synovial fluid (SF) neutrophils to the augmented expression of macrophage inflammatory protein 1 α (MIP-1α) in rheumatoid arthritis (RA). METHODS Neutrophils were isolated from samples of SF from RA patients and peripheral blood (PB) samples from RA patients and healthy controls. Cell associated MIP-1α was visualised immunohistochemically, and cell associated MIP-1α as well as MIP-1α secreted into the SF was assayed by ELISA. Steady state expression of MIP-1α mRNA was assessed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS Freshly isolated SF neutrophils contained significantly higher concentrations of both MIP-1α protein and its transcript than PB neutrophils from either RA patients or healthy controls; incubation in the absence or presence of tumour necrosis factor α for 24 hours resulted in a significant increase in MIP-1α secretion by RA SF neutrophils compared with neutrophils obtained from either normal PB or RA PB; and expression of MIP-1α by SF neutrophils was well correlated with both RA disease activity and SF mononuclear cell (MNC) counts. CONCLUSION Expression and secretion of MIP-1α by SF neutrophils may be indicative of local and systemic inflammation in RA. Moreover, this C-C chemokine may contribute to the recruitment of MNCs from the bloodstream into synovial joints and tissues.

Biphasic regulation of the development of murine type ii collagen-induced arthritis by interleukin-12 : Possible involvement of endogenous interleukin-10 and tumor necrosis factor α

OBJECTIVE To examine the dose-specific effects of interleukin-12 (IL-12) on the evolution of murine type II collagen-induced arthritis (CIA). METHODS From day 24 through day 33 following primary immunization, mice received daily intraperitoneal injections of murine recombinant IL-12. Measurements of anticollagen IgG, cytokines, and corticosterone were performed using enzyme-linked immunosorbent assay and radioimmunoassay. RESULTS CIA mice injected with a low dose of IL-12 (5 ng/day) exhibited accelerated onset and increased severity of arthritis. In contrast, administration of a high dose of IL-12 (500 ng/day) attenuated arthritic inflammation. The low dose of IL-12 induced tumor necrosis factor alpha (TNFalpha) production, whereas the high dose induced production of both IL-10 and corticosterone and suppression of anticollagen antibody levels. Administration of neutralizing anti-TNFalpha and anti-IL-10 antibodies reversed the dose-specific effects of IL-12. CONCLUSION IL-12 is an important immunomodulator during the pathogenesis of CIA. It appears to act by regulating humoral and cellular immune responses, as well as by mediating the expression of immunoregulatory cytokines and glucocorticoids.

Follow-up study of lipid peroxides, superoxide dismutase and glutathione peroxidase in the synovial membrane, serum and liver of young and old mice with collagen-induced arthritis

Because reactive oxygen species (ROS) are generally believed to play an important role in tissue injury in rheumatoid arthritis, we examined the levels of lipid peroxides, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the synovial membrane, serum and liver of young (8 wk) and old (12 mo) mice with collagen-induced arthritis. In the synovial membrane, serum and liver, lipid peroxide levels of both young and old mice were increased beginning on the 3rd day after the onset of arthritis. SOD activity, which scavenges O2- and inhibits lipid peroxidation, rose markedly in the synovial membrane of young mice in parallel with the increase in lipid peroxide levels, but not so markedly in old mice. Liver GSH-Px activity, which metabolizes already formed lipid peroxides, also rose in young arthritic mice to a greater degree than in old mice. This study suggests that in inflammatory synovial lesions, lipid peroxides are generated due to an increase in ROS concentration, with resultant cytotoxicity, and that younger animals or humans can prevent this unfavorable reaction more effectively than aged ones by enzyme induction. The hypothesis that lipid peroxides formed in the oxidative lesions of the primary organ are released into the serum, trapped by the liver and metabolized there is further supported by the present study.

Enhanced expression of interferon-inducible protein 10 associated with Th1 profiles of chemokine receptor in autoimmune pulmonary inflammation of MRL/lpr mice

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic lupus erythematosus (SLE)-like disease. The natural history of the pulmonary involvement and the underlying mechanism of leukocyte infiltration into the lungs of MRL/lpr mice and SLE patients remains elusive. We aimed to investigate the expression profiles of chemokines and chemokine receptors in the lung of the SLE-prone mouse. We examined the correlation between lung inflammation and expression of IP-10 (interferon-γ-inducible protein 10), a CXC chemokine, and TARC (thymus- and activation-regulated chemokine), a CC chemokine, in MRL/lpr mice, MRL/Mp-+/+ (MRL/+) mice, and C57BL/6 (B6) control mice. The extent of cell infiltration in the lung was assessed histopathologically. Reverse transcriptase PCR showed up-regulation of IP-10 mRNA expression in the lungs (P < 0.05) of MRL/lpr mice, in comparison with MRL/+ or B6 mice. The increase paralleled increased expression of a specific IP-10 receptor, CXCR3, and correlated with the degree of infiltration of mononuclear lymphocytes. In contrast, lung expression of TARC and its specific receptor, CCR4, were suppressed in MRL/lpr mice. Immunohistology showed that macrophage-like cells were the likely source of IP-10. Flow cytometric analyses revealed that the CXCR3-expressing cells were mainly infiltrating CD4 T cells and macrophages, which correlated with the degree of mononuclear lymphocyte infiltration. Recent data suggest that Th1 cells and Th1-derived cytokines play an important role in the development of SLE-like disease in MRL/lpr mice. Our results suggest that IP-10 expression in the lung is involved, through CXCR3, in the pathogenesis of pulmonary inflammation associated with migration of Th1 cells.

Inflammatory Cytokines and Enzymes in Synovial Fluid of Patients with Rheumatoid Arthritis and Other Arthritides

Cytokines and lysosomal enzymes, which are produced by inflammatory cells, play a role in inflammation. We have found that synovial fluid (SF) in rheumatoid and septic arthritis contained a large number of white blood cells (WBCs) and high levels of cytokines and enzymes, while in contrast the SF of osteoarthritis and traumatic arthritis did not contain significant amounts. Measurements of WBCs, cytokines and enzymes in SF are useful for evaluating clinical disease activity. Assays for WBCs and enzymes are simple and rapid when compared to those for cytokines.

Tacrolimus-induced lung injury in a rheumatoid arthritis patient with interstitial pneumonitis

A 74-year-old woman was experiencing rheumatoid arthritis complicated with interstitial pneumonitis (IP), and tacrolimus treatment was started. She presented with dyspnea. Chest X-ray and computed tomography (CT) showed ground-glass opacity and IP. Although tacrolimus was stopped, she died of respiratory failure. At autopsy, both the upper and lower lung fields showed usual IP and the organizing stage of diffuse alveolar damage. The former is common, but the latter is uncommon, suggesting tacrolimus may cause severe alveolar damage.