Chemokines play important roles in the development of the inflammatory response by recruiting different types of leukocyte population. Previous in vivo studies indicated that monocyte chemoattractant protein-1 (MCP-1) produced by infiltrating neutrophils was important for the development of delayed-type hypersensitivity (DTH). However, the mechanisms regulating MCP-1 mRNA expression by neutrophils remained unclear. This year we investigated a possible role of cytokines in the regulation of MCP-1 mRNA expression by Neutrophils by using a cytokine- rich crude culture supernatant of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells with (PHA-sup).The PHA-sup dose-dependently induced MCP-1 mRNA expression in neutrophils. A significant amount of MCP-1 mRNA was first detected by Northern analysis at 8 h. The peak MCP-1 mRNA level was detected at 16 h and sustained until 72 h. Cycloheximide and genistein, but not pertussis toxin, inhibited the expression of MCP-1 mRNA. Addition of anti-tumor necrosis factor (TNF)-alpha IgG blocked 30 to 70% of MCP-1 mRNA expression induced with the PHA-sup. PHA-sup-stimulated neutrophils synthesized and secreted 3.1 ? 1.3 ng/5 x 106 neutrophils of MCP-1 within the first 24 h. Hybridization of 32P-labeled cDNA preparations to an array of human cytokine cDNAs further indicated that MCP-1 mRNA was highly expressed in neutrophils along with other chemokines such as interleukin-8 (IL-8), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, and selectively upregulated in the late phase after stimulation with the PHA-sup. Since MCP-1 is produced by infiltrating neutrophils in vivo, it is likely that the molecular events described here occur in vivo. Understanding the mechanisms regulating MCP-1 expression in neutrophils may help treating patients suffering from chronic inflammation including DTH.IL-8 and GRO are two major CXC chemokines that play an important role in the recruitment of neutrophils in the human. Although the functional orthologues, CINC/KC and MIP-2, are present in the rat and mouse, the lack of IL-8 made these animals less useful to study the role of IL-8 and GRO. We previously showed evidence that IL-8 existed in the guinea pig. This year, we cloned the guinea pig orthologues of GRO and IL-1beta, and the expression of gpIL-8, gpGRO, gpTNF-alpha, and gpIL-1beta, was investigated by Northern analysis and/or by in-situ hybridization in different tissues of lipopolysaccharide (LPS)-injected guinea pigs. gpGRO and gpIL-8 mRNA were detected in the lung, heart, liver, kidney, and spleen 1 h after intraperitoneal injection of LPS. Expression of these genes were further studied in the lung. gpGRO, gpIL-8, gpTNF- alpha, and gpIL-1beta expression peaked at 3 h after LPS-injection. Both gpGRO and gpIL-8 mRNA were detected in the cells in alveolar space and bronchial epithelial cells. However, gpGRO mRNA, but not gpIL-8, was also expressed in endothelial cells and vascular smooth muscle cells. Expression of gpIL-8 and gpGRO mRNA appeared to be independent from TNF-alpha- or IL-1beta-stimulation in this model. A high level expression of gpGRO in vascular cells suggest an important role of GRO in the sequestration of neutrophils and multi-organ injuries induced by LPS. Cloning of gpGRO will enable us to identify two chemokine receptors, CXCR1 and CXCR2, in the guinea pig for our future study on the role of IL-8 and GRO, important inflammatory mediators in the human.This project has been transferred from LIB to LMI. - Chemokines, Cytokines, Inflammation, Monocytes/Macrophages, Neutrophils, - Human Subjects
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