Connexin 43-dependent tumor-suppressing effect of the Bowman-Birk protease inhibitor on M5076 ovarian sarcoma-bearing mice.

The present study was designed to confirm whether the Bowman-Birk inhibitor (BBI) induces an increase in p27 accumulation without S phase kinase-associated protein 2 (skp2) degradation by means of the expression of connexin (Cx) 43 as a gap junctional intercellular communication (GJIC)-dependent pathway in mice with M5076 ovarian sarcoma. M5076 ovarian sarcomas (1x105 cells/animal) were subcutaneously transplanted onto the backs of BDF1 mice receiving 10, 20 or 40 mg/kg of purified BBI intraperitoneally. Relative tumor weight (p<0.01, r=0.503) was negatively correlated with the dose of BBI. In contrast, the relative density of Cx43 mRNA (p<0.01, r=0.570) and Cx43 (p<0.01, r=0.718) was positively correlated with the dose of BBI, as were p21 (p<0.01, r=0.633), p27 (p<0.01, r=0.561) and skp2 (p<0.01, r=0.733). We therefore suggest that the anti-carcinogenic effects of BBI induce negative growth control by means of an increase in p27 accumulation caused by the expression of Cx43 as a GJIC pathway.

Endothelial selectins regulate skin wound healing in cooperation with L-selectin and ICAM-1.

Skin wound healing is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. Mice lacking intercellular adhesion molecule-1 (ICAM-1) delayed skin wound healing and mice lacking both L-selectin and ICAM-1 (L-selectin/ICAM-1(-/-)) show more delayed wound healing. Deficiency of both endothelial selectins (E-selectin or P-selectin) also delays wound healing. However, the relative contribution and interaction of selectins and ICAM-1 to the wound healing remain unknown. To clarify them, repair of excisional wounds was examined in L-selectin/ICAM-1(-/-) mice, wild-type mice with both E- and P-selectin blockade, and L-selectin/ICAM-1(-/-) mice with both E- and P-selectin blockade. Wild-type mice with both E- and P-selectin blockade showed delayed wound healing that was comparable with that in L-selectin/ICAM-1(-/-) mice. Combined E- and P-selectin blockade in L-selectin/ICAM-1(-/-) mice resulted in more significant delay. Mice lacking or blocked for adhesion molecules also showed suppressed keratinocyte migration, angiogenesis, granulation tissue formation, leukocyte infiltration, and cytokine expression, including transforming growth factor-beta and interleukin-6. Application of basic fibroblast growth factor (bFGF) but not platelet-derived growth factor to the wounds significantly improved wound healing in L-selectin/ICAM-1(-/-) mice with both E- and P-selectin blockade. bFGF significantly increased the leukocyte infiltration and subsequent fibrogenic cytokine production, as well as keratinocyte migration, angiogenesis, and collagen synthesis despite the loss of four kinds of adhesion molecules. These results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM-1 and may provide critical information for the therapy of skin wounds.

Decreased expression levels of CD22 and L-selectin on peripheral blood B lymphocytes from patients with bullous pemphigoid.

Bullous pemphigoid (BP), an autoimmune subepidermal-blistering disease of the elderly, is caused by antibodies against BP antigens at the epidermal basement membrane zone (BMZ). CD22 is a B lymphocyte specific response regulator, which is down-regulated after B-cell activation. Old CD22-deficient mice produce class-switched autoantibodies. To assess the role of CD22 in the pathogenesis of BP, we examined CD22 expression on B cells from BP patients and correlated its expression with clinical parameters. B cell expression of CD22 was 20% lower in BP patients when compared to healthy control subjects. In addition, B cells from BP patients showed decreased expression of L-selectin, which is an indicator of leukocyte activation, and CD22 expression levels were correlated with L-selectin expression. These results suggest that the decreased CD22 expression may be associated with the activation of B cells in BP. CD22 expression levels in BP patients did not correlate with the levels of anti-epidermal BMZ antibodies, and old CD22-deficient mice did not develop the anti-epidermal BMZ antibody. These results suggest that a decrease in CD22 expression may not be associated with BP-specific antibody production.

Serum levels of tumor necrosis factor and interleukin-13 are elevated in patients with localized scleroderma.

BACKGROUND: The enhanced production of some cytokines may be related to the pathogenesis of localized scleroderma (LSc). OBJECTIVE: To determine whether serum levels of tumor necrosis factor (TNF) and interleukin (IL)-13 are elevated, and whether they correlated with clinical or serological features in patients with LSc. METHODS: Serum levels of TNF and IL-13 were examined by ELISA in 45 patients with LSc and in 20 healthy controls. RESULTS: The frequency of serum TNF detection was significantly higher in patients with LSc (24%) compared with that in healthy controls (0%). The frequency of serum IL-13 detection was also significantly higher in patients with LSc (29%) compared with that in controls (0%). In patients with LSc, elevated TNF levels significantly correlated with the presence of IgM antihistone antibodies, anti-single-stranded DNA antibodies, elevated serum IL-6 levels, the number of linear lesions, and muscle involvement. Elevated IL-13 levels were significantly associated with the number of plaque lesions and the total number of lesions. CONCLUSIONS: These results suggest that TNF and IL-13 may be associated with the development of LSc.

Intercellular adhesion molecule-1 and L-selectin regulate bleomycin-induced lung fibrosis.

The development of bleomycin-induced lung injury, a model of pulmonary fibrosis, results from inflammatory cell infiltration, a process highly regulated by the expression of multiple adhesion molecules. At present, the identity and role of the adhesion molecules involved in the fibrotic process are unknown. Therefore, bleomycin-induced pulmonary fibrosis was examined in mice lacking L-selectin (L-selectin(-/-)) expression, intercellular adhesion molecule-1 (ICAM-1) expression, or both. After 16 days of intratracheal bleomycin challenge, collagen deposition was inhibited in both L-selectin(-/-) and ICAM-1(-/-) mice when compared with wild-type littermates. Interestingly, collagen deposition was virtually eliminated in L-selectin/ICAM-1(-/-) mice relative to either the L-selectin(-/-) or ICAM-1(-/-) mice. Decreased pulmonary fibrosis was associated with reduced accumulation of leukocytes, including neutrophils and lymphocytes. Decreased mRNA expression of proinflammatory cytokines and transforming growth factor (TGF)-beta1 paralleled the inhibition of collagen deposition. The present study indicates that L-selectin and ICAM-1 play a critical role in pulmonary fibrosis by mediating the accumulation of leukocytes, which regulate the production of proinflammatory cytokines and TGF-beta1. This suggests that these adhesion molecules are potential therapeutic targets for inhibiting human pulmonary fibrosis.

CD19-dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight-skin mouse.

The tight-skin (TSK/+) mouse, a genetic model for human systemic sclerosis (SSc), develops cutaneous fibrosis and autoantibodies against SSc-specific target autoantigens. Although molecular mechanisms explaining the development of fibrosis and autoimmunity in SSc patients or TSK/+ mice remain unknown, we recently demonstrated that SSc patients overexpress CD19, an important regulatory molecule expressed by B lymphocytes. B cells from CD19-deficient mice are hyporesponsive to transmembrane signals, while B cells overexpressing CD19 are hyperresponsive and generate autoantibodies. In this study, TSK/+ B cells also exhibited a hyperresponsive phenotype with decreased surface IgM expression, enhanced serum Ig production, and spontaneous autoantibody production. Moreover, CD19 tyrosine phosphorylation was constitutively augmented in TSK/+ B cells. CD19-mediated [Ca(2+)](i) responses, Vav phosphorylation, and Lyn kinase activity were similarly enhanced. Studies of TSK/+ mice deficient in CD19 expression demonstrated that CD19 deficiency significantly decreased skin fibrosis in TSK/+ mice. Additionally, CD19 loss in TSK/+ mice upregulated surface IgM expression and completely abrogated hyper-gamma-globulinemia and autoantibody production. CD19 deficiency also inhibited IL-6 production by TSK/+ B cells. Thus, chronic B cell activation resulting from augmented CD19 signaling in TSK/+ mice leads to skin sclerosis possibly through IL-6 overproduction as well as autoimmunity.

The cutaneous reverse Arthus reaction requires intercellular adhesion molecule 1 and L-selectin expression.

The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by expression of multiple adhesion molecules. To assess the role of L-selectin and ICAM-1 in this pathogenetic process, the cutaneous reverse passive Arthus reaction was examined in mice lacking L-selectin (L-selectin(-/-)), ICAM-1 (ICAM-1(-/-)), or both (L-selectin/ICAM-1(-/-)). Edema and hemorrhage, which peaked 4 and 8 h after IC challenge, respectively, were significantly reduced in L-selectin(-/-), ICAM-1(-/-), and L-selectin/ICAM-1(-/-) mice compared with wild-type littermates. In general, edema and hemorrhage were more significantly inhibited in ICAM-1(-/-) mice than in L-selectin(-/-) mice, but were most significantly reduced in L-selectin/ICAM-1(-/-) mice compared with ICAM-1(-/-) or L-selectin(-/-) mice. Decreased edema and hemorrhage correlated with reduced neutrophil and mast cell infiltration in all adhesion molecule-deficient mice, but leukocyte infiltration was most affected in L-selectin/ICAM-1(-/-) mice. Reduced neutrophil and mast cell infiltration was also observed for all mutant mice in the peritoneal Arthus reaction. Furthermore, cutaneous TNF-alpha production was inhibited in each deficient mouse, which paralleled the reductions in cutaneous inflammation. These results indicate that ICAM-1 and L-selectin cooperatively contribute to the cutaneous Arthus reaction by regulating neutrophil and mast cell recruitment and suggest that ICAM-1 and L-selectin are therapeutic targets for human IC-mediated disease.