Inflammatory cytokine response in sulfur mustard-exposed mouse skin.

Assessment of anti-inflammatory therapies against sulfur-mustard (bis(2-chloroethyl)sulfide, HD)-induced skin injury has mainly relied on qualitative histopathological evaluation. Development of quantifiable inflammatory biomarkers using fast and reliable molecular methods is needed for screening anti-inflammatory drugs against HD injury. In this study, we used two different HD exposure models to determine the in vivo cutaneous response of the inflammatory cytokines interleukin-6 (IL-6), IL-1alpha, IL-1beta and tumor necrosis factor alpha (TNF-alpha), in order to identify a suitable inflammatory biomarker common to both models. In the first model, the backs of hairless mice were exposed to HD vapor (1.4 g m(-3)) or sham controls for 6 min using an occluded vapor cup technique. In the second model, right ears of CD1 mice were exposed to a solution (5.0 microl of 195 mM) of HD (0.16 mg) in dichloromethane (CH2Cl2) whereas left ears received only CH2Cl2 (vehicle control). Sulfur-mustard-induced skin inflammation was assessed in skin punch specimens collected at time points up to 24 h post-exposure. Edema was determined by measuring tissue weight, and cytokine content was measured by enzyme immunosorbent assay. Characterized by an increase in edema and IL-6, HD provoked a cutaneous inflammatory response in both models beginning at 6 h post-exposure and continuing to 24 h. An increase in IL-1alpha was observed only in the hairless mouse model, also beginning at 6 h post-exposure and continuing to 24 h. No IL-1beta or TNF-alpha response was observed at any time point in either exposure model. These data document the in vivo production of cutaneous IL-6, a distinct inflammatory biomarker, in two different HD exposure models. We conclude that IL-6 should be a useful in vivo biomarker for evaluating anti-inflammatory drugs against HD-induced skin injury.

Transcriptional regulation of the human polymeric immunoglobulin receptor gene by interferon-gamma.

IgA is transported into external secretions by the polymeric Ig receptor (pIgR). Interferon-gamma (IFN-gamma), a major regulator of pIgR expression, has been shown to increase pIgR mRNA levels in HT-29 human colon carcinoma cells. To determine the molecular mechanisms of pIgR regulation, genomic DNA containing the 5'-flanking region of the human pIgR gene was isolated and a single start site of transcription in human intestinal epithelial cells was identified. Using chimeric reporter plasmids containing flanking regions of the pIgR gene, a segment of the pIgR promoter which is necessary and sufficient for induction of transcription by IFN-gamma in HT-29 cells was identified. Significantly, the pIgR promoter contains three motifs homologous to the interferon-stimulated response element (ISRE), two in the 5'-flanking region and one in exon 1 of the pIgR gene. The upstream ISREs bind nuclear protein(s) which are constitutively expressed by HT-29 cells, while the exon 1 ISRE binds interferon regulatory factor-1 (IRF-1), following stimulation with IFN-gamma. Furthermore, induction of the IRF-1 promoter by IFN-gamma correlates with induction of the pIgR promoter by IFN-gamma. It has previously been demonstrated that induction of pIgR mRNA by IFN-gamma, requires de novo protein synthesis. It is now shown that IRF-1 is not detected in nuclear extracts from HT-29 cells stimulated with IFN-gamma in the presence of cycloheximide, suggesting that de novo synthesis of IRF-1 is required for induction of pIgR transcription by IFN-gamma.

Molecular cloning of the mouse polymeric Ig receptor. Functional regions of the molecule are conserved among five mammalian species.

Transcytosis of polymeric Ig (pIg) by mucosal epithelial cells is mediated by the polymeric Ig receptor (pIgR). Here we describe the characterization of a 3095-bp mouse pIgR cDNA, which encodes a protein of 771 amino acids. Northern blot analysis detected a single mouse pIgR transcript of 3.9 kb, expressed at high levels in small intestine and liver, and at low levels in lung. Alignment of the amino acid sequences of mouse, rat, human, bovine, and rabbit pIgR revealed that functional regions of the molecule are conserved across species. In the extracellular region, conserved motifs include: a 23-amino acid pIg-binding site, 11 intradomain disulfide bonds, consensus sites for N-glycosylation, and a putative cleavage site at which the extracellular region of pIgR (secretory component) is released from the plasma membrane. A 10-amino acid sequence within the transmembrane region is highly conserved, possibly reflecting a mechanism for transmitting signals from the extracellular region to the cytoplasmic tail. Conservation within the cytoplasmic tail of pIgR is clustered in motifs that mediate polarized sorting, endocytosis, and transcytosis.

Interferon-gamma induces polymeric immunoglobulin receptor mRNA in human intestinal epithelial cells by a protein synthesis dependent mechanism.

Transport of secretory IgA into external fluids is mediated by the polymeric immunoglobulin receptor (pIgR) on the surface of mucosal epithelial cells. We studied the mechanism by which interferon-gamma (IFN-gamma) induces pIgR expression in HT-29.74 cells, a subclone of the HT-29 cell line selected for high concns of pIgR. Here we report the isolation of genomic DNA and cDNA clones encoding human pIgR and development of a sensitive ribonuclease protection assay for pIgR mRNA. This assay was used to determine if induction of pIgR by IFN-gamma is mediated by accumulation of pIgR mRNA. After an initial lag of 12 hr, pIgR mRNA increased seven-fold in response to IFN-gamma, reaching a plateau at 24 hr. Concentrations of pIgR protein also increased seven-fold, but the increase was delayed until 48 hr following stimulation with IFN-gamma. Cycloheximide treatment abolished the IFN-gamma induced increase in pIgR mRNA, indicating that induction of pIgR mRNA by IFN-gamma requires de novo protein synthesis. These results suggest that induction of pIgR expression by IFN-gamma involves an increase in steady-state concns of pIgR mRNA via a protein synthesis dependent mechanism.