Siglec-9 and SHP-1 are differentially expressed in neonatal and adult neutrophils.

Neonatal PMN (polymorphonuclear neutrophils) exhibit altered inflammatory responsiveness and greater longevity compared with adult PMN; however, the involved mechanisms are incompletely defined. Receptors containing immunoreceptor tyrosine-based inhibitory motif (ITIM) domains promote apoptosis by activating inhibitory phosphatases, such as Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1), that block survival signals. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9, an immune inhibitory receptor with an ITIM domain, has been shown to induce cell death in adult PMN in association with SHP-1. To test our hypothesis that neonatal PMN inflammatory function may be modulated by unique Siglec-9 and SHP-1 interactions, we compared expression of these proteins in adult and neonatal PMN. Neonatal PMN exhibited diminished cellular expression of Siglec-9, which was phosphorylated in the basal state. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment decreased Siglec-9 phosphorylation levels in neonatal PMN but promoted its phosphorylation in adult PMN, observations associated with altered survival signaling. Although SHP-1 expression was also diminished in neonatal PMN, GM-CSF treatment had minimal effect on phosphorylation status. Further analysis revealed that Siglec-9 and SHP-1 physically interact, as has been observed in other immune cells. Our data suggest that age-specific interactions between Siglec-9 and SHP-1 may influence the altered inflammatory responsiveness and longevity of neonatal PMN.

Adenovirus E1A proteins are closely associated with chromatin in productively infected and transformed cells.

The adenovirus E1A 243R oncoprotein encodes a potent transcription-repression function within the N-terminal 80 amino acids. Our proposed model of E1A repression predicts that E1A interacts with important cellular proteins on chromatin. Consistent with this idea, we report here that E1A proteins from in vivo formaldehyde cross-linked 293 cells are closely associated with chromatin even after several stringent purification steps including double isopycnic CsCl density gradient centrifugation and size exclusion chromatography. Likewise, E1A proteins expressed from virus during productive infection of HeLa cells are closely associated with chromatin starting at early times after infection. No other adenoviral proteins are necessary for E1A 243R protein to associate with chromatin. Analyses of chromatin from HeLa cells infected with adenovirus vectors expressing E1A 243R protein with deletions in different E1A functional domains indicate that sequences within the E1A N-terminal repression domain are needed for the majority of E1A's interactions with chromatin.

Human intestinal epithelial and smooth muscle cells are potent producers of IL-6.

BACKGROUND: Interleukin-6 (IL-6), a pluripotent cytokine, has traditionally been considered the product of proinflammatory cells. However, many other cell types have been shown to produce IL-6. Since intestinal inflammation is commonly associated with a vigorous systemic inflammatory response, we hypothesized that intestinal epithelial and smooth muscle cells might contribute to that response by producing IL-6. We therefore studied the capacity of differentiated human intestinal epithelial and smooth muscle cell lines to produce IL-6 in response to various proinflammatory stimuli. MATERIALS AND METHODS: CCL-241, a human intestinal epithelial cell line, and HISM, a human intestinal muscle cell line, were grown to confluency and then treated for 24 h with various concentrations of lipopolysaccharide, Clostridium difficile culture extract containing both toxin A and toxin B, recombinant human tumor necrosis factor-alpha (TNF-alpha), or recombinant human interleukin-1 beta (IL-1beta). Supernatants were then collected for IL-6 determination using an enzyme-linked immunosorbent assay. Cell numbers were determined using a Coulter counter. For comparison, parallel studies were performed using phorbol ester-primed U-937 and THP-1 human macrophage cell lines. RESULTS: Both human intestinal epithelial and smooth muscle cells produced IL-6 under basal conditions. In HISM cells, but not in CCL-241 cells, IL-6 release was increased slightly by treatment with C. difficile culture extract containing both toxin A and toxin B and with lipopolysaccharide. In both cell lines, IL-6 production was profoundly stimulated by treatment with IL-1beta and less so with TNF-alpha. Combinations of high-dose TNF-alpha and IL-1beta may have a slightly additive, but not synergistic, effect on IL-6 release. The amount of IL-6 produced by IL-1-stimulated intestinal cell lines was 70-fold higher than that produced by stimulated macrophage cell lines. CONCLUSIONS; Both intestinal epithelial and smooth muscle cells demonstrate the ability to release significant amounts of IL-6. The profound response to IL-1beta and TNF-alpha stimulation by both cell lines suggests that human intestinal parenchymal cells, influenced by paracrine mediators liberated from proinflammatory cells, might significantly contribute to the overall systemic inflammatory response by producing IL-6.