We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1β. IL-1β induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1β for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1β. IL-1β primed neutrophils for enhanced release of superoxide (O2−) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1β also induced O2− release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1β-induced O2− release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1β and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1β induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1β and activation of this cascade mediates IL-1β-induced O2− release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.
Abstract-Cytokine activation of vascular endothelial cells renders the hyperadhesiveness for neutrophils. During the processes of inflammation and atherosclerosis, the production of reactive oxygen species by neutrophils contributes to endothelial cell (EC) damage and injury. However, the precise mechanisms for neutrophil activation by ECs remain unknown. Thus, we investigated what kinds of pathophysiological factors synthesized by inflammatory cytokineactivated ECs potentiated the activity of neutrophil functions. The magnitude of O 2 Ϫ release from neutrophils, which is one of pivotal neutrophil functions, was measured as an indicator potentiated by activated ECs. Neutrophils release massive amounts of O 2 Ϫ on coculture with activated ECs. Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (Ab) or specific platelet-activating factor (PAF)-receptor antagonist suppressed the O 2 Ϫ release from neutrophils on coculture with the activated ECs by 50% to 70%. The supernatants from activated ECs also induced O 2 Ϫ release by neutrophils. This stimulatory effect of activated EC supernatants on O 2 Ϫ release by neutrophils was abolished by anti-GM-CSF Ab or by PAF-receptor antagonist. As we previously reported, we demonstrated the expression of GM-CSF mRNA by Northern blotting and protein synthesis of GM-CSF by ELISA on tumor necrosis factor as well as interleukin-1-activated ECs. Although phosphorylation of mitogen-activated protein kinases was observed in ECs stimulated by tumor necrosis factor and interleukin-1, treatment of ECs with PD98059 (MEK1 inhibitor) and SB203580 (p38 mitogen-activated protein kinase inhibitor) in the presence of the cytokine failed to attenuate the stimulatory effect of activated ECs on neutrophil activation. We found that activated ECs regulated neutrophil function on coculture. We show here for the first time, to our knowledge, that the collaboration between GM-CSF and PAF synthesized by activated ECs markedly potentiated neutrophil activation.
A randomized double-blind placebo-controlled clinical trial was conducted to evaluate the chondroprotective action of salmon nasal cartilage proteoglycan on joint health. The effect of oral administration of proteoglycan (10 mg/day) on cartilage metabolism was evaluated in individuals with knee joint discomfort but without diagnosis of knee osteoarthritis. The average age of patients was 52.6±1.1 years old. The effect of proteoglycan was evaluated by analyzing markers for type II collagen degradation (C1,2C) and synthesis (PIICP), and the ratio of type II collagen degradation to synthesis. The results indicated that the change in C1,2C levels significantly differed in the proteoglycan group compared with the placebo group following 16 weeks intervention among subjects with high levels of knee pain and physical dysfunction (total score of Japan Knee Osteoarthritis Measure ≥41) and subjects with constant knee pain (both P<0.05). There was a greater increase in PIICP levels in the proteoglycan group than the placebo group following intervention, although this difference was not significant in both sets of patients. Thus, the C1,2C/PIICP ratios decreased in the proteoglycan group, whereas they slightly increased in the placebo group following the intervention. Furthermore, no test supplement-related adverse events were observed during the intervention. Therefore, oral administration of salmon nasal cartilage proteoglycan at a dose of 10 mg/day may exert a chondroprotective action in subjects with knee joint discomfort. This effect was achieved by improving cartilage metabolism (reducing type II collagen degradation and enhancing type II collagen synthesis), without causing apparent adverse effects.
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