CCL3/MIP-1alpha is pro-inflammatory in murine T cell-mediated hepatitis by recruiting CCR1-expressing CD4(+) T cells to the liver

T cell-mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C-C chemokine ligand 3/macrophage inflammatory protein-1alpha (CCL3/MIP-1alpha) are elevated in the serum of patients with T cell-mediated liver diseases, but its role is not fully understood. Con A-induced hepatitis is a murine liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of IFN-gamma. In this study, we have used CCL3/MIP-1alpha gene-deficient mice to examine the role of CCL3/MIP-1alpha in the pathogenesis of Con A-induced hepatitis. We demonstrate a novel pro-inflammatory role for CCL3/MIP-1alpha since CCL3/MIP-1alpha deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1-expressing CD4(+) T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP-1alpha deficiency. Correspondingly, hepatic IFN-gamma produced by the recruited CD4(+) T cells was significantly reduced by CCL3/MIP-1alpha deficiency during Con A-induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4(+) T cells within the liver producing IFN-gamma during Con A-induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP-1alpha-CCR1 pathway may represent a novel therapeutic target for treating T cell-mediated liver diseases.     

Regulation of local and metastatic host-mediated anti-tumour mechanisms by L-selectin and intercellular adhesion molecule-1

Malignant melanoma is often accompanied by a host response of inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. To assess the role of adhesion molecules, including L-selectin and intercellular adhesion molecule-1 (ICAM-1), in this process, subcutaneous primary growth and metastasis to the lung of B16 melanoma cells not expressing L-selectin, ICAM-1 or their ligands were examined in mice lacking L-selectin, ICAM-1 or both. Primary subcutaneous growth of B16 melanoma was augmented by loss of L-selectin, ICAM-1 or both, while pulmonary metastasis was enhanced by the loss of L-selectin or combined loss of L-selectin and ICAM-1. In both situations, the combined loss of L-selectin and ICAM-1 exhibited the greatest effect. This enhancement was associated generally with a reduced accumulation of natural killer (NK) cells, CD4+ T cells and CD8+ T cells and also with a diminished release of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha but not interleukin (IL)-6. Cytotoxicity against melanoma was not defective by the absence of ICAM-1, L-selectin or both, suggesting that the enhancement of tumour growth and metastasis caused by the loss of adhesion molecules results from an impaired migration of effector cells into the tissue rather than from a suppression of the cytotoxic response. The results indicate that L-selectin and ICAM-1 contribute co-operatively to the anti-tumour reaction by regulating lymphocyte infiltration to the tumour.     

NC-1500 prevents concanavalin A-induced mice hepatitis without affecting cytokine gene expression.

NC-1500 is a dihydropyridine type calcium channel blocker. The effect of NC-1500 on mice concanavalin A-induced hepatitis was examined. Treatment of mice with concanavalin A (Con A) caused elevation of plasma transaminase. Pretreatment of mice with NC-1500 (3, 10 and 30 mg/kg, p.o.) prevented this Con A-induced elevation of plasma transaminase. Treatment of mice with Con A induced tumor necrosis factor-alpha (TNF-alpha) mRNA expression in the liver. However, NC-1500 (30 mg/kg, p.o.) did not affect this Con A-induced TNF-alpha mRNA expression in the liver. The present results showed that NC-1500 inhibited Con A-induced hepatitis without affecting TNF-alpha mRNA expression in the liver.     

Leptin deficiency, not obesity, protects mice from Con A-induced hepatitis

Leptin-deficient ob/ob mice are protected from Con A-induced hepatitis. However, it is unclear whether leptin deficiency or obesity itself is responsible for this protection. To address this question, wild-type (WT) obese mice with high serum leptin levels were generated by injection of gold thioglucose (WT GTG). Both Con A-injected WT and WT GTG mice developed hepatitis, whereas no hepatic damage was observed in ob/ob mice. Moreover, TNF-alpha and IFN-gamma levels as well as expression of the activation marker CD69 were elevated in liver mononuclear cells of WT and WT GTG mice, but not in ob/ob mice following administration of Con A. The liver of WT and WT GTG mice had the same percentage of NK T cells, a lymphocyte population involved in Con A-induced hepatitis. This population decreased equally in both WT and WT GTG mice after Con A injection. In contrast, the liver of ob/ob mice contained 50% less NK T cells compared to WT and WT GTG mice. Furthermore, no decrease in NK T cells was observed in Con A-injected ob/ob mice. We conclude that leptin-deficiency, not obesity, is responsible for protection from Con A-induced hepatitis.     

Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression

Inflammatory cells play a crucial role in wound healing, but the role of adhesion molecules including L-selectin and intercellular adhesion molecule-1 (ICAM-1) is not known in this process. We examined skin wound repair of excisional wounds in mice lacking L-selectin, ICAM-1, or both. The loss of ICAM-1 inhibited wound healing, keratinocyte migration from the edges of the wound toward the center, and granulation tissue formation. By contrast, L-selectin deficiency alone did not affect any of these parameters. However, the loss of both L-selectin and ICAM-1 resulted in inhibition of keratinocyte migration and granulation tissue formation beyond those caused by loss of ICAM-1 alone. Treatment of platelet-derived growth factor to the wounds normalized delayed wound healing in ICAM-1(-/-) mice, but not in L-selectin/ICAM-1(-/-) mice. Therefore, although ICAM-1 contributes to wound repair to a greater extent than L-selectin, a role for L-selectin was revealed in the absence of ICAM-1. The impaired wound repair was associated with reduced infiltration of neutrophils and macrophages in ICAM-1(-/-) and L-selectin/ICAM-1(-/-) mice. These results demonstrate a distinct role of ICAM-1 and L-selectin in wound healing and that the delayed wound healing in the absence of these molecules is likely because of decreased leukocyte accumulation into the wound site.     

Adhesion molecules on intermediate TCR cells. I. Unique expression of adhesion molecules, CD44+ L-selectin-, on intermediate TCR cells in the liver and the modulation of their adhesion by hyaluronic acid.

In addition to thymus-derived T cells, it was demonstrated recently that extrathymically differentiated T cells exist in the liver and other immune organs of mice. Since such extrathymic T cells have T-cell receptors (TCR) of intermediate intensity (i.e. intermediate TCR cells) and constitutively express IL-2 receptor beta-chain (IL-2R beta) similar to natural killer (NK) cells, they are easily distinguished from thymus-derived T cells with a TCR-bright+ IL-2R beta- phenotype (i.e. bright TCR cells). In the present study, the expression of adhesion molecules CD44 and L-selectin was compared between these T-cell subsets. Intermediate TCR cells in the liver and other organs were found to be CD44+ L-selectin- and, inversely, bright TCR cells were CD44- L-selectin+. CD3- IL-2R beta+ NK cells were also estimated to be CD44+ L-selectin-. Hyaluronic acid, which is known to adhere to a CD44 ligand, bound to intermediate TCR cells, but not to bright TCR cells. Among many extracellular matrices, hyaluronic acid induced a prominent decrease in the numbers and proportions of intermediate TCR cells and NK cells in the liver from 6 to 24 hr after in vivo administration. The half-life span of injected hyaluronic acid was approximately 7 hr in the plasma. These results suggest that the CD44 molecule, which is uniquely expressed on intermediate TCR cells and NK cells, is eventually associated with their adhesion to the sinusoidal walls in the liver.     

L-selectin expression differentiates T cells isolated from different lymphoid tissues in cattle but does not correlate with memory.

L-selectin (LECAM-1, LAM-1) was expressed by a high proportion of CD4+ and CD8+ T cells, as well as almost all of the gamma delta T-cell receptor (TcR)+ (WC1+) T cells, isolated from blood, lymph nodes or tonsils. CD4+ T cells in the lamina propria of the gut villi and CD8+ T cells in the villous epithelium as well as the majority of WC1+ T cells in the gut mucosa were L-selectin-. The proportion of T cells from Peyer's patches that synthesized the molecule was intermediate between the value for blood and gut mucosa. Expression of L-selectin therefore marks T cells in cattle with a distinct tissue distribution that correlates with its function as the peripheral node homing receptor. The proportion of CD4+ and CD8+ T cells in the circulation that were L-selectin+ decreased with age. Unlike CD45R, expression of L-selectin was not related to CD4 T-cell memory as judged by proliferation in transformation assays to soluble antigen. Three-colour immunofluorescent staining demonstrated four subpopulations of CD4 and CD8 T lymphocytes in peripheral blood mononuclear cells (PBMC) that were CD45R+, L-selectin+; CD45R+, L-selectin-; CD45R-, L-selectin+; CD45R-, L-selectin-. CD4(4) memory cells were CD45R- and L-selectin+ or L-selectin-. Taken with earlier studies the reported observations demonstrated that only one of the four phenotypes of the CD4+ T cells in blood is present in the lamina propria of the gut villi and these are CD45R-, L-selectin-. Two of the four phenotypes of CD8+ T cells were present in the gut epithelium; these were CD45R+, L-selectin- or CD45R-, L-selectin-. Expression of the bovine molecule was not rapidly down-regulated on T cells following activation by exposure to phorbol myristate acetate.     

Protection from concanavalin A (Con A)-induced T cell-dependent hepatic lesions and modulation of cytokine release in mice by sodium fusidate

The immunomodulatory effects of the antibiotic sodium fusidate (SF) were tested in a model of T cell-dependent hepatic injury that can be induced in normal mice by a single i.v. injection of Con A. Signs of hepatitis with elevated transaminase activities in plasma, severe infiltration of the liver by neutrophil granulocytes, lymphocytes and monocytes, and necrotic areas were observed in control mice treated intraperitoneally with PBS 24 h and 1 h before Con A challenge. T cell- and macrophage-derived cytokines (IL-2, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), IL-1β, IL-6) were released with different kinetics in the circulation of these mice. SF, 20, 40 or 80 mg/kg, administered 24 h and 1 h before Con A challenge, protected the mice against the hepatitic effects of Con A. The protective effects of SF were dose-dependent and accompanied by profound modifications of blood levels of cytokines induced by Con A, so that, relative to control mice, SF (80 mg/kg)-treated animals showed markedly diminished plasma levels of IL-2, IFN-γ and TNF-α, along with augmented levels of IL-6. These results suggest that SF might be useful in the treatment of immunoinflammatory liver diseases in humans.     

Induction of thymocyte apoptosis by systemic administration of concanavalin A in mice: role of TNF-alpha, IFN-gamma and glucocorticoids

Administration of concanavalin A (Con A) is a well-established model of acute immune-mediated hepatitis. Here, we demonstrate that intravenous injection of Con A in mice induces profound thymic atrophy. Compared to liver damage, the kinetics of Con A-induced thymic atrophy is slower and more prolonged; the nadir in thymocyte number is reached 4 days after Con A injection, whereas peak transaminase levels are observed at 12-24 h. Marked alterations in the ratio of CD4+ and CD8+cells in the thymus and spleen and significantly increased rates of thymocyte and splenocyte apoptosis are observed. Neutralization of the cytokines TNF-alpha or IFN-gamma, which protects mice from Con A-induced hepatitis, prevents thymic atrophy as well as alterations in CD4+ and CD8+ cell numbers and apoptosis rates. However, neither TNF-alpha nor IFN-gamma are detectable in thymocyte lysates after Con A injection, whereas both cytokines are present in liver, spleen and serum. Administration of the glucocorticoid receptor antagonist mifepristone does not prevent thymic atrophy, thus ruling out a possible contribution of endogenous glucocorticoids. Con A-induced thymic atrophy is accompanied by down-regulation of Bcl-2 expression in the thymus, which is prevented by neutralization of TNF-alpha or IFN-gamma. These data demonstrate that the thymus is a critical target organ of Con A-induced inflammation; the effects of Con A on the thymus are mediated by extrathymic production of TNF-alpha and IFN-gamma, but not by glucocorticoids.     

Glycyrrhizin protects mice from concanavalin A-induced hepatitis without affecting cytokine expression.

The administration of concanavalin A (Con A) to mice induces cytokine-dependent hepatitis. In the present study, the effect of glycyrrhizin on Con A-induced hepatitis was examined. Treatment of mice with Con A (0.2 mg/mouse, i.v.) induced elevation of the plasma transaminase activities at 24 h. Mice were treated with glycyrrhizin (100, 200 and 400 mg/kg, i.p.), and glycyrrhizin at the doses of 200 and 400 mg/kg inhibited the Con A-induced elevation of the plasma transaminase activities. At 1 h after Con A treatment, interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-6 proteins were released into the plasma. Although treatment with glycyrrhizin at 200 mg/kg inhibited Con A-induced hepatitis, it did not affect the release of any of these Con A-induced cytokines into the plasma. The present results clearly show that glycyrrhizin inhibited Con A-induced hepatitis without affecting cytokine expression.     

Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and exacerbated by endogenous IL-10 deficiency

One single intra-venous (i.v.) injection of Concanavalin A (Con A) into mice provokes a cell-mediated immunoinflammatory hepatitis. We have presently evaluated the immunopharmacological effects of exogenous interleukin (IL)-10 and the role of endogenous IL-10 in this model by using exogenous IL-10, anti-IL-10 monoclonal antibody (mAb) and mice with disrupted IL-10 gene (IL-10 KO mice). Whilst exogenous IL-10 administered in a prophylactic (1 h prior to Con A) and even "early" therapeutic fashion (30 min after Con A) reduced the elevation of transaminase activities in plasma in a dose-dependent manner, observed in control mice, these biochemical markers of liver injury were significantly increased both in IL-10 KO mice as well as in those receiving anti-IL-10 mAb. Interestingly, doses of Con A lower than 20 mg/kg that were only capable of inducing slight serological signs of hepatitis in mice, exerted marked hepatitic effects when administered to either anti-IL-10 mAb-treated mice or to IL-10 KO mice. The disease modulating effects of exogenous IL-10 and either genetical or pharmacologically-induced IL-10 deficiency were associated with profound and opposite modifications of the Con A-induced increase in the circulating levels of IFN-gamma and TNF-alpha. Relative to control animals, the blood levels of these cytokines were diminished in IL-10-treated mice and augmented in both IL-10 KO mice and anti-IL-10 mAb-treated mice. These results prove the physiological antiinflammatory role of endogenous IL-10 in Con A induced hepatitis and the beneficial effects of IL-10 treatment to prevent this condition.     

Relative contributions of selectins and intercellular adhesion molecule-1 to tissue injury induced by immune complex deposition

Immune complex-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. To assess the relative contribution of adhesion molecules, including selectins and ICAM-1, in this pathogenetic process, the cutaneous passive Arthus reaction was examined in mice lacking E-selectin, P-selectin, or both L-selectin and ICAM-1 with anti-P- or E-selectin mAbs. Edema and hemorrhage were significantly reduced in P-selectin(-/-) mice compared with wild-type mice while they were not inhibited in E-selectin(-/-) mice. Combined E- and P-selectin blockade resulted in more significant reduction relative to L-selectin/ICAM-1(-/-) as well as P-selectin(-/-) mice. Remarkably, both E- and P-selectin blockade in L-selectin/ICAM-1(-/-) mice completely abrogated edema and hemorrhage. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells that expressed significant levels of P-selectin glycoprotein ligand-1. Similarly reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated partly with the decreased production of tumor necrosis factor-alpha and interleukin-6. The results of this study indicate that both endothelial selectins contribute predominantly to the Arthus reaction by regulating mast cell and neutrophil infiltration and that the full development of the Arthus reaction is mediated cooperatively by all selectins and ICAM-1.     

CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis

Natural killer (NK) cells are innate immune cells that are enriched in the liver, but the processes underlying NK cell trafficking to the liver and cellular activation within the liver of patients with T cell-mediated liver diseases remain poorly defined. Concanavalin A (Con A) hepatitis is a murine model mimicking many aspects of human T cell-mediated liver diseases. Here we demonstrate that severe hepatitis in CCR5-deficient (KO) mice is associated with increased hepatic NK cell recruitment driven by enhanced hepatic production of CCL5 acting via CCR1 and by enhanced hepatic NK cell activation relative to that observed in wild-type mice after Con A administration. Furthermore, NK cell depletion ameliorated severe hepatitis in CCR5 KO mice but did not alter hepatitis in wild-type mice after Con A treatment. We propose that in the setting of CCR5 deficiency NK cells assume a profound effector role in Con A hepatitis via enhanced CCL5-CCR1 driven hepatic recruitment in addition to augmented cytokine-driven NK cell activation to produce interferon-gamma. These results highlight the potential profound impact of altered chemokine receptor expression on the innate immune response in the setting of T cell-mediated hepatitis.     

High-mobility group box 1 exacerbates concanavalin A-induced hepatic injury in mice

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as an early endogenous alarmin of inflammation following injury and as a late mediator of lethality in sepsis. Although HMGB1 has been implicated in acute lung injury, rheumatoid arthritis, and allograft rejection, its role in T-cell mediated hepatitis remains obscure. Here, we investigated the role and the underlying mechanisms of HMGB1 in concanavalin A (Con A) induced hepatic injury. We demonstrate that high levels of HMGB1 were detected in the necrotic area and in the cytoplasm of hepatocytes after Con A treatment. Administration of exogenous recombinant HMGB1 enhanced Con A-induced hepatitis, while blockade of HMGB1 protected animals from T cell-mediated hepatitis as evidenced by decreased serum transaminase, associated with reduced hepatic necrosis and mortality. Blockade of HMGB1 by a neutralizing antibody inhibited proinflammatory cytokine production, NFκB activity, and the late stage of T/NKT cell activation. These finding thus suggest a pivotal factor of HMGB1 in Con A-induced hepatitis. Blockage of extracellular HMGB1 may represent a novel therapeutic strategy to prevent hepatic injury in T cell-mediated hepatitis.     

T cells expressing both L-selectin and CD44 molecules increase in number in peritoneal exudate cells and in vitro-stimulated spleen cells from mice immunized intraperitoneally with Listeria monocytogenes.

L-selectin, which was first reported as MEL-14 antigen in mice, is a type of animal lectin and expressed on lymphocytes, neutrophils and macrophages. L-selectin has been reported to be a homing receptor of lymphocytes to peripheral lymph nodes and to have an important role in initial adhesion of lymphocytes and neutrophils to endothelial cells activated by inflammatory cytokines. On the other hand, it has been reported that naive T cells express L-selectin while memory T cells and in vitro antigen-stimulated T cells lose its expression. If all memory T cells lack L-selectin, trafficking of memory T cells into inflammatory sites would be difficult. To know whether all memory T cells lack L-selectin expression, kinetics of expression of L-selectin was analysed on memory T-cell subsets, which are detected by expression of CD44, in mice after intraperitoneal immunization with a sublethal dose of viable Listeria monocytogenes. T cells expressing both L-selectin and CD44 were detected in splenocytes and peritoneal exudate cells (PEC) from untreated mice, though at low levels. L-selectin+ CD44+ T cells increased in PEC, which are known to be highly enriched in antigen-primed T cells, and reached maximum level on day 14 after immunization. Furthermore, we found increases not only of L-selectin- CD44+ but also of L-selectin+ CD44+ T cells by in vitro Listeria antigen stimulation of Listeria-immune spleen cells on day 14. These results showed that T cells expressing both L-selectin and CD44 increase after antigen stimulation in vivo and in vitro. The L-selectin+ CD44+ T cells may be a subset of memory T cells which retain their capacity of trafficking to inflammatory sites.     

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.     

Comparison of thymocyte development and cytokine production in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice

CD7 and CD28 are Ig superfamily molecules expressed on thymocytes and mature T cells that share common signaling 0mechanisms and are co-mitogens for T cell activation. CD7-deficient mice are resistant to lipopolysaccharide (LPS)-induced shock syndrome, and have diminished in vivo LPS-triggered IFN-gamma and tumor necrosis factor (TNF)-alpha production. CD28-deficient mice have decreased serum Ig levels, defective IgG isotype switching, decreased T cell IL-2 production and are resistant to Staphylococcus aureus enterotoxin-induced shock. To determine synergistic roles CD7 and CD28 might play in thymocyte development and function, we have generated and characterized CD7/CD28 double-deficient mice. CD7/CD28-deficient mice were healthy, reproduced normally, had normal numbers of thymocyte subsets and had normal thymus histology. Anti-CD3 mAb induced similar levels of apoptosis in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytes as in control C57BL/6 mice (P = NS). Similarly, thymocyte viability, apoptosis and necrosis following ionomycin or dexamethasone treatment were the same in control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. CD28-deficient and CD7/CD28-deficient thymocytes had decreased [3H]thymidine incorporation responses to concanavalin A (Con A) stimulation compared to control mice (P < or = 0.01 and P < or = 0.05 respectively). CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 double-positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. Con A-stimulated CD4/CD8 double-negative (DN) thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). Anti-CD3-triggered CD7/CD28-deficient thymocytes also had decreased IFN-gamma and TNF-alpha production compared to C57BL/6 control, CD7-deficient and CD28-deficient mice (P < or = 0.05). Thus, CD7 and CD28 deficiencies combined to produce abnormalities in the absolute number of B7-1/B7-2-expressing cells in the thymus, thymocyte IL-2 receptor expression and CD3-triggered cytokine production.     

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.     

CD4+ T-cell dysfunctions through the impaired lipid rafts ameliorate concanavalin A-induced hepatitis in sphingomyelin synthase 1-knockout mice

Membrane microdomains consisting of sphingomyelin (SM) and cholesterol appear to be important for signal transduction in T-cell activation. The present study was designed to elucidate the role of membrane SM in vivo and in vitro using sphingomyelin synthase 1 (SMS1) knock out (SMS1(-/-)) mice and Concanavalin A (ConA)-induced hepatitis. After establishing SMS1(-/-) mice, we investigated CD4+ T-cell functions including proliferation, cytokine production and signal transduction in vivo. We also examined severity of hepatitis, cytokine production in serum and liver after ConA injection at a dose of 20 mg kg(-1). CD4+ T cells from SMS1(-/-) mice showed severe deficiency of membrane SM and several profound defects compared with wild-type controls as follows: (i) cellular proliferation and production of IL-2 and IFN-γ by co-cross-linking of CD3 and CD4; (ii) tyrosine phosphorylation of LAT and its association with ZAP-70; (iii) clustering and co-localization of TCR with lipid rafts. Consistent with these impaired CD4+ T-cell functions in vitro, SMS1(-/-) mice showed decreased serum levels of IL-6 and IFN-γ by ConA injection, which renders SMS1(-/-) mice less sensitive to ConA-induced hepatitis. These results indicated that the deficiency of membrane SM caused the CD4+ T-cell dysfunction through impaired lipid raft function contributed to protection of ConA-induced liver injury, suggesting that the membrane SM is critical for full T-cell activation both in vitro and in vivo.