VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase.

Adhesion molecules such as VLA-4 are important not only for monocyte adhesion to extracellular matrix proteins, but also for subsequent cell activation. Monocyte adherence to fibronectin or engagement of VLA-4 has been demonstrated to stimulate production of potent inflammatory mediators such as tumor necrosis factor-alpha, interleukin-1, and the procoagulant tissue factor protein. However, the intracellular signaling cascades leading to gene expression have not been elucidated. Using the human monocytic THP-1 cell line, VLA-4 cross-linking by monoclonal antibodies directed against its alpha4 and beta1 subunits produced a time-dependent increase in tyrosine phosphorylation of a broad range of cellular proteins. Using Western blot analysis directed against the phosphorylated form of the extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase proteins, as well as immunoprecipitation and in vitro kinase assays, we found that VLA-4 cross-linking increased ERK1/ERK2 tyrosine phosphorylation and activity. In conjunction, integrin cross-linking also increased NF-kappaB nuclear translocation and 4-h expression of tissue factor. Inhibition of tyrosine kinase activity with genistein (10 microg/ml) as well as selective MAP kinase inhibition with the MEK-1 inhibitor PD98059 abolished the VLA-4-dependent ERK tyrosine phosphorylation, inhibited NF kappaB nuclear binding, and abrogated tissue factor expression induced by both VLA-4 cross-linking and adhesion to fibronectin in THP-1 cells and human peripheral blood monocytes. These studies point to the involvement of the MAP kinase pathway in the activation of monocytic cells during transmigration to inflammatory sites.

N-acetyl cysteine attenuates acute lung injury in the rat.

The development of the adult respiratory distress syndrome (ARDS) in the critically ill patient is associated with a significant morbidity and mortality. The pulmonary dysfunction in ARDS is largely secondary to neutrophil-mediated oxidant injury. The purpose of these studies is to examine the effect of the antioxidant N-acetyl cysteine (NAC) on a rodent model of lung injury. We postulated that NAC might attenuate lung injury following intratracheal challenge with endotoxin (lipopolysaccharide; LPS). Male Sprague-Dawley rats were administered NAC systemically either before or after intratracheal administration of LPS. Lung injury was assessed by measuring the transpulmonary leakage of 125I-labeled albumin, pulmonary myeloperoxidase content, bronchoalveolar lavage fluid cell counts, pulmonary lipid peroxidation and histology. NAC administration significantly attenuated the LPS-induced increases in lung permeability (LPS: .24 +/- .08 vs. LPS + NAC: .12 +/- .03, p < .05) and reduced the LPS-dependent increase in lipid peroxidation. However, total and differential bronchoalveolar lavage cell counts and myeloperoxidase content were not affected by NAC pretreatment. Although neutrophil influx was unaffected, neutrophil activation as assessed by surface CD11b expression and chemiluminescence was significantly down-regulated by NAC. Importantly, NAC administration up to 2 h after endotoxin challenge was still able to significantly ameliorate LPS-induced lung injury. Our data suggests that the attenuation of acute lung injury by NAC in our rodent model is related to free radical scavenging and inhibition of the neutrophil oxidative burst, rather than by an effect on inflammatory cell migration. These results suggest novel approaches for therapeutic interventions in acute lung injury.

Improved tracheal allograft viability in immunosuppressed rats.

Airway ischemia has been a common cause of morbidity and mortality in clinical lung transplantation. The present study examined the effects of cyclosporin A (CsA) and methylprednisolone (MP) on the viability of the devascularized trachea after heterotopic transplantation and omentopexy. Thirty-six tracheal segments were harvested from 18 donor Lewis rats, wrapped in omentum, and heterotopically implanted into the abdomen of recipient rats. Tracheal segments were randomly allocated into one of six recipient groups (n = 6): Lewis syngeneic controls, and five groups of Brown Norway recipients, receiving either no treatment, CsA alone (5 mg.kg-1.day-1 or 15 mg.kg-1.day-1), or CsA in combination with MP (5 mg CsA + 1 mg MP per kg/day or 15 mg CsA + 2 mg MP per kg/day, respectively). After 14 days, the tracheal segments were histologically evaluated. Epithelial thickness and the degree of epithelial regeneration were significantly different (p < 0.05) between the syngeneic control group and the untreated Brown Norway group. Without immunosuppression there was virtually no epithelium, whereas low-dose immunosuppression yielded intermediate viability, and with high dose CsA and MP we observed improved tracheal viability. In this high-dose group, the epithelium was thicker than in even the syngeneic control group. These results indicate that, in heterotopic tracheal allografts, viability may be improved with an optimum combination of CsA and MP.