Calpain regulates neutrophil chemotaxis.

Cell polarization is required for directed cell migration. We investigated the role of the calcium-dependent protease calpain during neutrophil chemotaxis and found that calpain inhibition induced neutrophil adhesion, polarization, and rapid chemokinesis in the absence of exogenous activators. Resting neutrophils display constitutive calpain activity with mu-calpain being the predominant active isoform. Our findings suggest that constitutive calpain activity in resting neutrophils may function as a negative regulator of protrusion and migration. Specific inhibition of mu-calpain, but not m-calpain, induced neutrophil polarization and chemokinesis. In contrast to IL-8-induced chemokinesis, the chemokinesis induced by calpain inhibition was not reduced in the presence of pertussis toxin, suggesting that calpain functions downstream of G protein-coupled receptors. Further, both calpain inhibition and stimulation with IL-8 and formyl-Met-Leu-Phe (fMLP) induced an increase in Cdc42 and Rac activation. These findings are consistent with the involvement of calpain in chemotaxis pathways. Accordingly, calpain inhibition decreased neutrophil chemotaxis and directional persistence in a gradient of IL-8 and fMLP. Together, these data reveal a previously uncharacterized function for calpain in neutrophils and suggest that localized modulation of calpain activity may regulate neutrophil chemotaxis downstream of G-protein-coupled receptors.

Identification of an additional isoform of STAT5 expressed in immature macrophages.

We are interested in understanding the molecular basis of macrophage (Mphi) differentiation and activation by cytokines. Recent reports have suggested that the transcription factor STAT5 may play a role in Mphi differentiation. In the experiments described here, we assessed the expression of STAT5-related molecules in three Mphi cell lines, RAW 264.7, WEHI-3, and WEHI-3D+, which represent different stages of Mphi maturation, and also in primary peritoneal and bone marrow Mphi from BALB/c mice. The studies revealed that the previously characterized STAT5a and STAT5b isoforms are detectable at both the mRNA and protein levels in these Mphi populations. Additional STAT5-related proteins were detected by immunoblot analysis and were preferentially expressed in both the immature WEHI-3 cell population and the adherent bone marrow population containing immature Mphi. These results identify new isoforms of STAT5 and demonstrate that distinct patterns of expression of STAT5-related proteins are observed in Mphi at different stages of maturation.

Analysis of the IFN-gamma-signaling pathway in macrophages at different stages of maturation.

We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-gamma. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the gamma-activated sequence DNA regulatory element in response to stimulation with IFN-gamma, while other aspects of IFN-gamma gene induction remain intact. In the work described here, we examined the components of the IFN-gamma signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-gamma receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-gamma stimulation. These results demonstrate that the components of the IFN-gamma signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-gamma does not result in STAT1 activation.

Mechanisms of murine RANTES chemokine gene induction by Newcastle disease virus.

We have previously defined the lipopolysaccharide (LPS)-responsive element (LRE) in the promoters of murine RANTES (regulated on activation normal T-cell expressed) (MuRantes) and murine IP-10/crg-2, chemokines which have potent chemotactic properties for inflammatory cells including monocytes and T lymphocytes. In the present work, we studied the transcriptional mechanism of MuRantes gene induction by virus and compared it with that of LPS in an effort to understand the host responses to virus and bacterial toxins at the molecular level. MuRantes mRNA expression is induced by Newcastle disease virus (NDV) and LPS in the RAW 264.7 macrophage cell line and peritoneal macrophages of LPS-responsive C3HeB/FeJ mice. In LPS-hyporesponsive C3H/HeJ mice, only NDV induces this chemokine gene, indicating that the pathways of transcriptional activation by NDV and LPS are not identical. Using a transient transfection assay, the minimal virus-responsive element (VRE) was localized between nt -175 and -116. The VRE contains previously defined LRE motif 1 (TCAYRCTT) and motif 3 ((T/A)GRTTTCA(G/C)TTT), which were shown to also be important for initiation of transcription by virus. NDV-stimulated nuclear extracts were tested for trans-activating factors able to bind the VRE. The chromosomal protein HMG-I(C) was shown to bind the 3'-A.T-rich domains of the VRE, and the presence of HMG-I(C) was demonstrated in the VRE-protein complex formed with nuclear extracts from NDV-stimulated, but not unstimulated cells. These findings demonstrate the role of HMG-I(C) in activation of MuRantes promoter by NDV.