Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease.

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.

Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.

The human gastrointestinal mucosa is exposed to a diverse normal microflora and dietary Ags and is a common site of entry for pathogens. The mucosal immune system must respond to these diverse signals with either the initiation of immunity or tolerance. APCs are important accessory cells that modulate T cell responses which initiate and maintain adaptive immunity. The ability of APCs to communicate with CD4+ T cells is largely dependent on the expression of class II MHC molecules by the APCs. Using immunohistochemistry, confocal microscopy, and flow cytometry, we demonstrate that alpha-smooth muscle actin(+), CD90+ subepithelial myofibroblasts (stromal cells) constitutively express class II MHC molecules in normal colonic mucosa and that they are distinct from professional APCs such as macrophages and dendritic cells. Primary isolates of human colonic myofibroblasts (CMFs) cultured in vitro were able to stimulate allogeneic CD4+ T cell proliferation. This process was dependent on class II MHC and CD80/86 costimulatory molecule expression by the myofibroblasts. We also demonstrate that CMFs, engineered to express a specific DR4 allele, can process and present human serum albumin to a human serum albumin-specific and DR4 allele-restricted T cell hybridoma. These studies characterize a novel cell phenotype which, due to its strategic location and class II MHC expression, may be involved in capture of Ags that cross the epithelial barrier and present them to lamina propria CD4+ T cells. Thus, human CMFs may be important in regulating local immunity in the colon.

Subepithelial myofibroblasts express cyclooxygenase-2 in colorectal tubular adenomas.

PURPOSE: Recent data support the hypothesis that the inducible isoform of cyclooxygenase (COX-2) plays a role in the early stages of colonic carcinogenesis and that nonsteroidal anti-inflammatory drugs (NSAIDs) retard the development of colon cancer by modulating COX-2. However, the cell types responsible for producing COX-2 in colorectal adenomas remain a subject of controversy. EXPERIMENTAL DESIGN: COX-2 expression in normal colonic mucosa (n = 50), hyperplastic polyps (n = 43), sporadic adenomas (n = 67), and invasive colonic adenocarcinoma (n = 39) was studied in formalin-fixed and paraffin-embedded tissue sections from endoscopy biopsy and colonic resection specimens. Immunohistochemistry (avidin-biotin complex technique with double immunolabeling) was used to identify the phenotypes of COX-2-producing cells. RESULTS: In colorectal adenomas, increased expression of COX-2 was detected and localized to alpha smooth muscle actin ( proportional, variant SMA)-positive subepithelial stromal cells (myofibroblasts) in the periluminal region of the lamina propria in 63 (94%) of 67 cases. In contrast, in normal colonic mucosa and in hyperplastic polyps with intact epithelium, COX-2 expression was found only in macrophages and endothelial cells. In areas in which the surface epithelium was ulcerated in normal mucosa as well as hyperplastic or neoplastic polyps, COX-2 expression was increased in granulation tissue (and present in macrophages, endothelium, and myofibroblasts). In invasive carcinoma, COX-2 expression in myofibroblasts was limited to the adenomatous portion of the tumor and was detected in 62% of cases (n = 39). In addition, focal expression of COX-2 by malignant epithelial cells was observed in 23% of invasive adenocarcinoma. CONCLUSIONS: These results show that increased COX-2 expression in sporadic adenoma of the colon is common and is localized specifically to subepithelial intestinal myofibroblasts. These findings further support the hypothesis that myofibroblasts are important target cells for NSAID-mediated chemoprevention of colorectal cancer.

Aspirin-mediated COX-2 transcript stabilization via sustained p38 activation in human intestinal myofibroblasts.

Acetylsalicylic acid (aspirin) is a cyclooxygenase (COX) inhibitor, yet some of its therapeutic effects are thought to derive from mechanisms unrelated to prostaglandin synthesis inhibition. In human intestinal myofibroblasts, aspirin, at therapeutic doses, had the unexpected effect of inducing prolonged COX-2 expression. This induction was especially pronounced when cells were treated with interleukin-1alpha (IL-1) plus aspirin for 24 h. Sodium salicylate, a poor COX inhibitor, likewise enhanced IL-1-mediated COX-2 gene expression whereas 5-aminosalicylic acid (5-ASA) or indomethacin had no effect. The COX-2 transcriptional rate, measured by nuclear runoff analysis and heterogeneous nuclear RNA reverse transcription-polymerase chain reaction, was only modestly elevated by aspirin treatment. In contrast, aspirin treatment dramatically stabilized the COX-2 message. The COX-2 mRNA half-life in IL-1 treated cells was 1 h and was increased in excess of 5 h in IL-1 + aspirin-treated cells. Phosphorylation of p38 MAPK was enhanced in aspirin-treated cells (but not in cells treated with 5-ASA or indomethacin) for up to 24 h after treatment. Inhibition of p38 activity negated aspirin-mediated COX-2 mRNA stabilization and the resultant increase in COX-2 mRNA and protein levels. The modest transcriptional response seen in aspirin treated cells was also abolished by p38 inhibition. We conclude that aspirin enhances COX-2 expression via sustained activation of p38, which results in prolonged stabilization of the COX-2 message and a slightly elevated transcription rate. Aspirin also enhanced steady-state mRNA levels of other IL-1 modulated genes (IL-1beta, IL-6, groalpha, and TNFalpha) that are likewise regulated at the level of message stability via p38 activation.

IL-1alpha-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCzeta-ROS pathway.

BACKGROUND & AIMS: Intestinal myofibroblasts (IMFs) express cyclooxygenase 2 (COX-2) early on in polyp progression and respond to pro-inflammatory cytokines. Interleukin (IL)-1alpha induces COX-2 expression in IMF via mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and nuclear factor kappa B (NF-kappaB)-dependent pathways. Because NF-kappaB activity can be mediated by PKC activation and reactive oxygen species (ROS) generation, we examined the relationship of these pathways to IL-1alpha-induced COX-2 expression. METHODS: The effects of specific PKC inhibitors and antioxidants on PKC activation, ROS generation, and COX-2 expression were studied. RESULTS: Immunoprecipitation/kinase (IPK) analysis showed that IL-1alpha increased PKC alpha, delta, and zeta activity 4.5-, 3.1-, and 2.6-fold, respectively, within 5 minutes. Single-cell fluorescence microscopy of 2',7'-dichlorofluorescin diacetate (DCF)-loaded cells showed that IL-1alpha increased ROS levels 2-fold within 15 minutes and this increase was inhibited by 10 micromol/L bisindolylymaleimide I (BIS), a pan-specific PKC inhibitor that also inhibits COX-2 expression. Chelerythrine chloride (CC) (0.5 micromol/L) inhibited classic and novel PKC activity, but not PKCzeta, and enhanced IL-1alpha-mediated ROS generation 4.0-fold and COX-2 expression 1.8-fold. The use of a PKCzeta pseudosubstrate prevented IL-1 from increasing ROS greater than control levels and abolished IL-1alpha-induced COX-2 expression. Small inhibitory RNA (siRNA) for PKCzeta confirmed its role in COX-2 expression. Antioxidants inhibited ROS generation and diminished IL-1alpha-induced COX-2 expression by 80%, without affecting PKC activation. Neither the PKC inhibitors nor the antioxidants prevented NF-kappaB-mediated transcription as determined by reporter gene analysis. CONCLUSIONS: PKCzeta and threshold ROS generation are critical for IL-1alpha-induced COX-2 expression and act concomitantly with NF-kappaB translocation in IMF.

Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction.

Elevated mucosal interleukin-1 (IL-1) levels are frequently seen during acute and chronic intestinal inflammation, and IL-1 neutralization lessens the severity of inflammation. One major effect of IL-1 is the increased release of eicosanoid mediators via induction of cyclooxygenase-2 (COX-2). One site of COX-2-derived prostaglandin synthesis during acute and chronic intestinal inflammation is the intestinal myofibroblast. COX-2 expression has also been documented in these cells in colonic neoplasms. Thus an understanding of the regulation of COX-2 expression in human intestinal myofibroblasts is important. As an initial step toward this goal we have characterized IL-1alpha signaling pathways that induce COX-2 expression in cultured human intestinal myofibroblasts. IL-1 treatment resulted in a dramatic transcriptional induction of COX-2 gene expression. Activation of nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated protein kinase (ERK), p38, and protein kinase C (PKC) signaling pathways was each necessary for optimal COX-2 induction. In contrast to what occurs in other cell types, including other myofibroblasts such as renal mesangial cells, PKC inhibition did not prevent IL-1-induced NF-kappaB or mitogen activated protein kinase/ stress-activated protein kinase activation, suggesting a novel role for PKC isoforms during this process. The stimulatory effects of PKC, NF-kappaB, ERK-1/2, and presumably c-Jun NH(2)-terminal kinase activation were exerted at the transcriptional level, whereas p38 activation resulted in increased stability of the COX-2 message. We conclude that, in intestinal myofibroblasts, IL-1-mediated induction of COX-2 expression is a complex process that requires input from multiple signaling pathways. Each parallel pathway acts in relative autonomy, the sum of their actions culminating in a dramatic increase in COX-2 transcription and message stability.