No abstract

The molecular stimuli involved in receptor-induced integrin activation are still poorly defined. We have investigated the role of receptors for the Fc portion of immunoglobulin G molecules (Fc gammaR) on activation of integrins in human neutrophils. Cross-linking of Fc gammaRIIA induced an increase in surface expression of beta2 integrins but had no effect on beta1 integrins. In contrast, cross-linking of Fc gammaRIIIB not only increased beta2 integrins on the cell surface but also induced beta1 integrin activation, as indicated by an increase in binding to fibronectin and the appearance of an activation epitope detected by the monoclonal antibody 15/7. The Fc gammaRIIIB-induced increase of beta2 integrins required Src-family tyrosine kinases, Syk kinase, and phosphatidylinositol-3 kinase (PI-3K), as the corresponding, specific inhibitors, PP2, Piceatannol, and LY294002, completely blocked it. Contrary to this, Fc gammaRIIIB-induced beta1 integrin activation was not blocked by PP2 or LY294002. It was, however, enhanced by Piceatannol. After Fc gammaRIIIB cross-linking, colocalization of Fc gammaRIIIB and active beta1 integrins was detected on the neutrophil membrane. These data show, for the first time, that cross-linking of Fc gammaRIIIB induces an inside-out signaling pathway that leads to beta1 integrin activation. This activation is independent of Src-family kinases, and PI-3K and may be induced in part by the interaction of Fc gammaRIIIB with beta1 integrins.

Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (∼0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation.

Allergic asthma is a T cell-dependent inflammatory lung disease that results from complex interactions between genetic predisposition and environmental factors, including exposure to lipopolysaccharide (LPS). In this study, we have shown that airway LPS exposure was sufficient to induce airway hyperreactivity (AHR) and eosinophil recruitment in mice that had previously experienced an acute episode of allergic asthma. LPS-induced disease reactivation depended on the activation of allergen-specific CD4(+) T cells by a subset of lung langerin(+) dendritic cells (DCs) that retained the allergen. Upon LPS exposure, migration of langerin(+) DCs from lungs to draining lymph nodes increased and LPS-exposed langerin(+) DCs instructed CD4(+) T cells toward a T helper (Th) 2 response. Selective depletion of langerin(+) DCs prevented LPS-induced eosinophil recruitment and T-cell activation, further demonstrating a critical role for langerin(+) DCs in disease reactivation. This finding provides a possible explanation for the subclinical worsening of asthmatics following exposure to low-dose LPS.