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NEUROREGULATION AND MOTILITY
Division of Gastroenterology, Departments of 1Internal Medicine and 2Neuroscience and Cell Biology, Enteric Neuromuscular Disorders and Visceral Pain Center, The University of Texas Medical Branch at Galveston, Galveston, Texas
Submitted 12 November 2004 ; accepted in final form 10 March 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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. Gene chip array analysis indicated that HCCSMC express a specific panel of 11 cytokines, chemokines, and cell adhesion molecules in a time-dependent manner in response to TNF-. The chip array data were supported by quantitative analysis of mRNA and protein expressions of interleukin (IL)-6, IL-8, intercellular adhesion molecule (ICAM)-1 and IL-11. The proinflammatory mediators were expressed early, whereas the anti-inflammatory cytokine IL-11 was expressed late after TNF- treatment. The expression of ICAM-1 on HCCSMC increased lymphocyte adhesion to these cells, which was blocked by pretreatment with antibody to ICAM-1. TNF- acted on both R1 and R2 receptors to induce the expression of ICAM-1. Pretreatment of HCCSMC with antisense oligonucleotides to p65 nuclear factor-
B (NF-B) blocked the expression of ICAM-1, whereas pretreatment with antisense oligonucleotides to p50 NF-B had little effect. The overexpression of p65 NF-B enhanced the constitutive expression of ICAM-1, and TNF- treatment had no further effect. The delayed expression of endogenous IL-11 limited the expression of ICAM-1, and pretreatment of HCCSMC with antisense oligonucleotides to IL-11 enhanced it. We conclude that TNF- induces gene expression in HCCSMC for programmed synthesis and release of pro- and anti-inflammatory mediators.
nuclear factor- B; myo-immune interactions; motility; cytokines
. However, it is not known whether circular smooth muscle cells that account for the bulk of muscularis externa also can secrete inflammatory mediators, and, if so, whether this is a programmed function regulated at the transcriptional level.
The aim of this study, therefore, was to investigate whether primary cultures of human colonic circular smooth muscle cells (HCCSMC) and intact circular muscle tissue secrete pro- and anti-inflammatory proteins in response to tumor necrosis factor (TNF)-. We used TNF- as the stimulus because its level in the muscularis externa is increased very early on after the onset of mucosal inflammation and remains elevated for a prolonged period (21). The gene expression of inflammatory mediators was examined initially by cDNA microarray analysis. This was followed by more quantitative determinations of mRNA and protein expressions of representative cytokines, chemokines, and adhesion molecules by colorimetric quantification of mRNA, RT-PCR, ELISA, and Western blot analysis. Finally, we determined whether the synthesis of one of these molecules, intercellular adhesion molecule-1 (ICAM-1), is regulated transcriptionally by the activation of p50 and p65 subunits of transcription factor nuclear factor-B (NF-B). We show here that primary cultures of HCCSMC and intact circular muscle strips synthesize and secrete a specific panel of inflammatory proteins in response to TNF-. The proinflammatory mediators are synthesized first, followed a few hours later by the synthesis of an anti-inflammatory mediator IL-11. The delayed synthesis of IL-11 plays a critical role in protecting smooth muscle cells during the inflammatory response by limiting the production of proinflammatory molecule ICAM-1. The p65 subunit of NF-B is a key transcription factor in this programmed response.
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METHODS |
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Smooth muscle cells were dispersed by two consecutive digestions with papain and collagenase, as described previously by Shi et al. (44) and Pazdrak et al. (33). In brief, circular muscle tissue pieces (0.5 x 0.5 cm2) were warmed to 37°C in 20 ml HEPES for 15 min and incubated with 0.4 mg/ml papain and 0.3 mg/ml 1,4-dithiothreitol (DTT) until the tissue appeared loose and sticky (in 
15 min). The tissue was then washed and further digested at 31°C with 0.5 mg/ml collagenase (type II, 319 U/mg) and 0.1 mg/ml soybean trypsin inhibitor for 40 min. The digested tissue was washed three times with enzyme-free HEPES buffer, and the muscle cells were allowed to disperse spontaneously under gentle to-and-fro motion. The dispersed cells were harvested by filtration through a 500-µm Nitex mesh and collected by centrifugation at 350 g for 5 min.
Cell cultures.
The freshly dispersed HCCSMC were washed two times in Hanks' solution before plating. Cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS in the presence of 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphoterecin B. Media were changed every 3 days. Cells in passages 3–5 were used in all experiments. To eliminate the possible effect of growth factors in the serum, all cells were cultured in serum-free media for at least 15 h before treatment with TNF- or vehicle control. Immunofluorescence imaging showed that >95% of the cells stained positive for smooth muscle-specific -actin.
Jurket E6 T-lymphocytes were purchased from American Type Culture Collection (Manassas, VA) for the coculture and adhesion assay. This cell line expresses T cell characteristics and complement receptors and possesses adhesive function (23, 38).
Extraction of cytoplasmic and nuclear proteins. Cytoplasmic and nuclear proteins were prepared as described previously (44). Cells were lysed with 0.25% Nonidet P-40 at 4°C for 10 min in solution A (in mM: 10 HEPES, 1.5 MgCl2, 10 KCl, 0.5 DTT, 0.5 phenylmethylsulfonyl fluoride, and 10 ng/ml each leupeptin and aprotinin). After centrifugation for 10 min at 12,000 g, the supernatant was collected as cytoplasmic protein. The nuclear pellet was suspended in solution C (in mM: 20 HEPES, 420 NaCl, 1.5 MgCl2, 0.2 EDTA, 0.5 DTT, and 25% glycerol, plus protease inhibitors, pH 7.9) and incubated on ice for 15 min while mixing samples frequently by vortexing. The samples were then centrifuged at 14,000 g for 10 min, and the supernatant (nuclear protein) was diluted with five volumes of buffer D (in mM: 20 HEPES, 50 KCl, 0.2 EDTA, 0.5 DTT, and 20% glycerol plus protease inhibitors, pH 7.9).
cDNA microarray. Total RNA was isolated using the Atlas Pure Total RNA Labeling system (Clontech, Palo Alto, CA). Cells were lysed in denaturing solution. After two phenol/chloroform extractions and isopropranalol precipitations, RNA samples were washed with 80% ethanol and dissolved in RNase-free water. Atlas Human Hematology/Immunology Array was purchased from Clontech Laboratories. The manufacturer's array procedure was followed. Briefly, total RNA was treated with DNase I and enriched for poly(A)+ RNA using biotinylated oligo(dT) magnetic beads. The cDNA probes were synthesized on beads using Moloney murine leukemia virus RT and purified using a NucleoSpin column. The arrays were then hybridized overnight with the cDNA probes at 68°C. After gentle washes, the array membranes were exposed to a phosphoimaging screen for 2–4 h. This array contains 406 specific cDNAs, 9 housekeeping cDNAs, and negative controls immobilized in duplicate dots in each array membrane. The cDNAs are organized in four blocks (A, B, C, and D). The block B-containing cDNAs of cytokines, chemokines, and adhesion molecules were analyzed primarily with Clontech AtlasImage 1.5 software, except for vascular cell adhesion molecule (VCAM)-1 and CD44, which were located in block C.
Colorimetric quantification of cytokine/chemokine mRNAs. The IL-6 and IL-8 mRNA expressions were quantitated using the Colorimetric Quantikine mRNA kit purchased from R&D Systems (Minneapolis, MN). This assay allows RNA samples to be hybridized with gene-specific biotin-labeled capture oligonucleotide probes and digoxigenin-labeled detection probes in a microplate. The hybridization solution was then transferred to a streptavidin-coated microplate, and the RNA/probe hybrid was captured. After anti-digoxigenin conjugate and substrate were added, color developed in proportion to the amount of gene-specific mRNA in the original sample. The optical density was read by a Labsystems Multiskan (Helsinki, Finland) microplate reader.
PCR analysis of IL-11 and ICAM-I gene expression.
Quantitative real-time PCR was employed to determine IL-11 gene expression with the help of the University of Texas Medical Branch Real-Time PCR Core Facility using Taqman technology on an Applied Biosystems 7000 sequence detection system. The applied Biosystem's reference numbers for the human IL-11 primers and probe are NM-000641 and Hs00174148-m1, respectively. The expression of ICAM-1 gene was analyzed by RT-PCR. The first-strand cDNA was synthesized using SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). Platinum PCR Supermix (Invitrogen) containing 22 U/ml Taq DNA polymerase was used for PCR. The sense and antisense primers for human ICAM-1 were 5'-CTTCTCCTGCTCTGCAACCC-3' and 5'-GGGAGAGCACATTCACGGTC-3' respectively (2). The PCR was programmed for 30 cycles, each consisting of 95°C for 60 s, 60°C for 60 s, and 72°C for 2 min. The -actin primers for PCR analysis were 5'-ATGGATGATGATATCGCCGC-3' (sense) and 5'-TTAATGTCACGCACGATTTC-3' (antisense; see Ref. 4).
ELISA method for protein determination. The IL-6, IL-8, and IL-11 proteins released in the culture media were quantitated using the R&D Systems colorimetric ELISA kits as per the manufacturer's instructions. These assays employ the quantitative enzyme immunoassay technique. The optical density was determined using a Labsystems Multiskan microplate reader set to 450 nm and corrected at 570 nm.
Electrophoretic mobility shift assay of NF-B nuclear binding.
Specific oligonucleotides containing NF-B common consensus (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) and B sequences in ICAM-1 promoter (5'-GCCCGGGGAGGATTCCTGGG-3' at –504/–485 and 5'-TAGCTTGGAAATTCCGGAGC-3' at –192/–172) were used for electrophoretic mobility shift assay (EMSA). The underlined DNA sequences are NF-B binding motifs. The oligonucleotides were labeled with [
-32P]ATP by T4 polynucleotide kinase (Amersham, Arlington Heights, IL). The EMSA reactions were carried out in 20 µl EMSA binding buffer (in mM: 10 Tris·HCl, 40 NaCl, 1 EDTA, and 1 DTT, pH 7.5) with 6 µg nuclear extract, 0.5 ng 32P-labeled oligo, and 2 µg poly(dI-dC/dI-dC) (Pharmacia, Kalamazoo, MI). After incubation for 20 min at 25°C, the reactions were stopped, and the products were electrophoresed on nondenaturing 4% polyacrylamide gel in 0.5x Tris-borate EDTA at 150 volts. The specific DNA-protein binding was identified by competition with a 50-fold excess of unlabeled oligonucleotides. Gels were dried and analyzed by autoradiography and densitometry.
Western blot analysis. Conventional Western blot analysis was used to examine ICAM-1 protein expression in HCCSMC. Cytoplasmic proteins were extracted as described above, and 10 µg of protein samples were loaded and run on 10% SDS-PAGE. The membrane was incubated with 0.1 µg/ml sheep anti-human ICAM-1 antibody (R&D Systems) at 4°C overnight and 1:10,000 rabbit anti-sheep IgG horseradish peroxidase for 1 h at room temperature.
Transient transfection of HCCSMC.
For transient transfection with plasmids and oligonucleotides, 5 x 104 cells in 1 ml medium were seeded in each well of 12-well culture plates 1 day before transfection. pCMV-p65 or pCMV-p50 (0.5 µg; a gift from Dr. John Hiscott, Montreal, Canada; see Ref. 26) or 4 or 10 µM sense and anti-sense oligonucleotides were used for transfection in the presence of 1.5 µl FuGENE-6 (Roche, Mannheim, Germany) for 24 h. Next, TNF- or vehicle control was added to the cells for another 24 h before the cells were harvested.
The phosphorothioate sense and anti-sense oligonucleotides were synthesized commercially and cartridge purified by Biosource International (Foster City, CA). The sequences of the oligonucleotides (26, 30) were as follows: p65 sense, 5'-GCCATG GACGAACTGTTCCCC-3'; p65 antisense, 5'-GGGGAACAGTTCGTCCATGGC-3'; p50 sense, 5'-AGA ATGGCAGAAGATGATCCA-3'; p50 antisense, 5'-TGGATCATCTTC TGCCATTCT-3'; IL-11 sense, 5'-ATGAACTGTGTTTGC-3'; and IL-11 antisense, 5'-GCAAACACAGTTCAT-3'.
Quantitative cell adhesion assay.
The assay for lymphocyte adherence to HCCSMC was carried out according to the method described by Amrani et al. (4) and Rahman et al. (35). Lymphocytes were activated with phorbol-12,13-dibutyrate (5 ng/ml; Sigma, St. Louis, MO) and ionomycin (250 nM; Sigma) for 42 h at 37°C and labeled with 2 µCi·106 cells·ml–1 [3H]thymidine (Perkin-Elmer, Boston, MA) during the final 16 h of incubation. Lymphocytes (5 x 105/well) were then added to resting or TNF--treated HCCSMC in 24-well culture plates. After 1 h at 37°C, nonadherent lymphocytes were removed by two washings with PBS. The adherent lymphocytes were lysed in 1% Triton X-100 in PBS and counted with a beta counter. Each expression was performed in triplicate, and data are expressed as means ± SE percent bound cells. For neutralizing antibody studies, HCCSMC were pretreated with 10 µg mouse anti-human ICAM-1 antibody (R&D Systems) for 45 min before coculture with lymphocytes.
Immunofluorescence staining of TNF- receptor subtypes.
HCCSMC were fixed in 1% paraformaldehyde in PBS for 30 min and permeabilized with 0.5% Triton X-100 for another 30 min (44). The cells were incubated with anti-TNF- receptor R1 and R2 antibodies (1:200; R&D Systems) for 1 h at 25°C. Cy3-conjugated donkey anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA) was used at 1:1,000 dilution for 1 h at 25°C. The cells were visualized under a Nikon fluorescence microscope.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-induced expression of inflammatory mediators.
Gene expression of cytokines, chemokines, and adhesion molecules by HCCSMC in response to TNF- was examined using the Clontech Atlas Human Hematology/Immunology array. The array data were analyzed at 3 and 24 h after treatment of HCCSMC with 20 ng/ml TNF- or vehicle control alone. Gene expression of a protein was considered to be altered by TNF- if its mRNA in the array was increased by at least 100% or decreased by at least 50% compared with that in vehicle-treated cells. TNF- treatment of HCCSMC enhanced the expression of 11 genes, each with a time-dependent response (Table 1). IL-6, leukemia inhibitory factor (LIF), IL-8, monocyte chemoattractant protein (MCP)-1, eotaxin, MCP-3, CD44, ICAM-1, and VCAM-1 exhibited an early increase of their respective mRNAs at 3 h (Fig. 1). Of these, the expressions of IL-6, LIF, IL-8, MCP-1, MCP-3, and ICAM-1 decreased at 24 h, whereas those of eotaxin and CD44 increased further at 24 h of TNF- treatment. The expression of VCAM-1 at 24 h of TNF- treatment was about the same as that at 3 h. By contrast, IL-11 and RANTES (regulated on activation, normal T cell expressed and secreted) genes were not induced at 3 h, but their expressions were increased severalfold at 24 h. The expression of traditional housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase and -actin, were not altered by TNF- treatment. -Actin was used as a reference for the expression of all genes.
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All inflammatory mediators were expressed at very low levels in untreated cells (Fig. 2). There was little change in their expression in vehicle-treated cells over 24 h (Fig. 2B). The IL-6 and IL-8 mRNAs peaked at 3 h after TNF- treatment, and then they plateaued at a lower level (Fig. 2A). The increase of IL-11 mRNA peaked at 12 h after exposure of the cells to TNF-. The increase in ICAM-1 mRNA was maximal between 3 and 6 h (Fig. 3A). In each case, mRNA expression was followed by an increase in protein expression (Figs. 2B and 3B).
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for 24 h. We found that TNF- treatment significantly increased the expression of IL-6 and IL-8 mRNAs in these tissues 46- and 68-fold, respectively, at 24 h (n = 3).
Role of HCCSMC-derived ICAM-1 in T lymphocyte adhesion.
The induced expression of ICAM-1 increases lymphocyte function-associated (LFA)-dependent adhesion of T lymphocytes in other nonhematopoietic cells (4). To determine whether the expression of ICAM-1 on HCCSMC has a similar function, we cocultured HCCSMC with activated T lymphocytes (23, 38). In vitro adhesion assay showed sparse T cell adherence in untreated cells (Fig. 4A). The adhesion increased to 211 ± 18% of control when HCCSMC were treated with TNF- for 24 h (Fig. 4B), and it was blunted to 136 ± 13% when the cells were preincubated with neutralizing antibody against ICAM-1 (Fig. 4B).
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receptor subtypes and NF-B activation in the expression of ICAM-1 on HCCSMC.
We chose ICAM-1 as a representative molecule to investigate whether its expression is regulated transcriptionally by the activation of NF-B through TNF- receptors. Immunofluorescence staining with antibodies against TNF receptor 1 (TNFR1) (80 kDa) and TNFR2 (60 kDa) demonstrated that both receptor subtypes were present on HCCSMC (Fig. 5). Western blotting, using the same antibodies, confirmed the presence of both receptor subtypes on HCCSMC (data not shown). To investigate the regulation of ICAM-1 expression by these receptors, HCCSMC were incubated with neutralizing antibodies to each receptor subtype 1 h before TNF- treatment. The TNF--induced ICAM-1 protein and mRNA expressions were reduced by 32 ± 2 and 36 ± 2%, respectively, in the presence of neutralizing antibody to TNF- R1 and by 58 ± 3 and 52 ± 4%, respectively, in the presence of TNF- R2 antibody. The combination of both antibodies reduced ICAM-1 protein expression by 66 ± 4%.
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enhancer sequence (5'-GATCCAGAGGGGACTTTCCCAGAG-3') as probe for EMSA, we found that TNF- treatment induced time-dependent activation of NF-B (Fig. 6). Supershift assays using p50 and p65 antibodies indicated that both subunits of NF-B were present in the DNA-binding complex. Pharmacological blockade of NF-B activation by MG-132, an S20 protease inhibitor, or transient transfection of the cells with the dominant-negative mutant of IB blocked the expression of ICAM-1 in response to TNF- (Fig. 7).
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B binding motifs have been identified on the human ICAM-1 promoter located at –499/–490 and –187/–178 from the transcription start site (25, 33, 35). With the use of 32P-labeled probes containing the upstream and downstream B motifs, EMSA showed that specific DNA binding to the probe containing the downstream motif was increased significantly in TNF--treated cells, whereas there was little binding to the probe containing the upstream B motif (Fig. 8). Although both p50 and p65 antibodies decreased DNA binding with the probe containing the consenses B sequence (Fig. 6), only p65 antibody decreased it significantly with the probe containing the downstream B motif of ICAM-1 promoter (Fig. 8).
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. Sense oligonucleotides to p50 and p65 had no effect on ICAM-1 expression in response to TNF- (Fig. 9). Antisense oligonucleotides to p65, but not to p50, blocked the expression of ICAM-1 in response to TNF-. The overexpression of p65 in HCCSMC enhanced the constitutive expression of ICAM-1, and TNF- had little additional effect (Fig. 10). The overexpression of p50, on the other hand, had no significant effect on the constitutive expression of ICAM-1 or on the effect of TNF-.
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(Fig. 11, A and B). On the contrary, the addition of IL-11 neutralizing antibody before TNF- treatment enhanced the expression of ICAM-1 at later time points (Fig. 11, A and C). These data were confirmed by transiently transfecting the cells with sense and antisense oligonucleotides for IL-11. Sense oligonucleotides had little effect on the expression of ICAM-1 at 24 and 48 h after treatment with TNF-. Antisense oligonucleotides, on the other hand, enhanced the expression of ICAM-1 at both time points (Fig. 11D). Further studies using EMSA showed that IL-11 treatment of the cells reduced NF-B/DNA binding (Fig. 11E).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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in the longitudinal muscle-myenteric plexus within 24 h of Trichinella spiralis infection. More recently, Salinthone et al. (36) also found that human colonic smooth muscle cells generate IL-1, IL-6, IL-8, cyclooxygenase-2, and RANTES in response to a cocktail stimulus containing TNF-, IL-1, and interferon-. They used quantitative PCR to determine the expression of specific cytokines and chemokines. Our findings, with the use of an immunology gene chip array, show that these cells synthesize a broad but specific panel of cytokines, chemokines, and cell adhesion molecules with a defined time course in response to TNF-.
The synthesis of inflammatory mediators in HCCSMC in response to TNF- is programmed in two ways. The first is that the genes of different inflammatory mediators in the panel are expressed at different times after exposure of cells to TNF-. The mRNAs of some mediators are expressed as early as 3 h but the increase in their expression is over before 24 h; others are expressed also by 3 h but their levels remain elevated for at least 24 h; those in the third group are expressed only after 3 h and their levels remain elevated for at least 24 h. The initial expression of proinflammatory mediators may boost the inflammatory response in the neuromuscular layer. These inflammatory proteins secreted by smooth muscle cells may also contribute to hyperplasia, hypertrophy, and stricture formation (5, 31, 37). The second programmed sequence is the initial expression of only the proinflammatory proteins and the later expression of anti-inflammatory proteins. The secretion of IL-11, an anti-inflammatory mediator, peaked as late as 12 h after TNF- treatment. Further experiments showed that the inhibition of endogenous IL-11 by the addition of its antibody to the culture medium or the inhibition of its synthesis by transient transfection of HCCSMC with antisense oligonucleotides enhanced the expression of ICAM-1, a proinflammatory mediator. The addition of sense oligonucleotides had no effect. Furthermore, the addition of exogenous IL-11 to the culture medium, before the addition of TNF-, suppressed the expression of ICAM-1. In a recent study, Pazdrak et. al. (33) reported that exposure of human colonic smooth muscle strips to TNF- for 24 h suppresses their contractile response to ACh. This suppression is mediated through the expression of ICAM-1 because it is inhibited if the expression of ICAM-1 is blocked by transient transfection of the muscle strips with antisense oligonucleotides to ICAM-1. Our studies, therefore, suggest that IL-11 may act as an endogenous anti-inflammatory mediator in HCCSMC. Its delayed synthesis limits or blocks the expression of proinflammatory ICAM-1 that suppresses cell contractility by reducing NF-B/DNA binding. It is noteworthy that IL-10 that has been reported to be an anti-inflammatory cytokine in immune cells was not induced by TNF- treatment in HCCSMC (48). IL-10 is usually made by regulatory T cells. The role of ICAM-1 in HCCSMC adhesion to activated lymphocytes is similar to that seen in the adhesion of endothelial cells to lymphocytes (4, 35).
The inflammatory response in the muscularis externa begins soon after the onset of mucosal inflammation (12, 19, 39, 45). The precise nature of stimulus from the mucosa that triggers the inflammatory response in the muscularis externa is not known. There are indications, however, that this trigger may come from enteric neural reflexes and from elevated circulating levels of proinflammatory cytokines as a result of mucosal inflammation. For example, substance P levels are increased in the muscularis externa of ulcerative colitis patients (11) and of rat jejunum when mucosal inflammation is induced by T. spiralis infection (47). Substance P is released from the intrinsic sensory neurons that have their sensory nerve endings in the mucosa (10). Substance P is known to activate resident macrophages to release proinflammatory cytokines, such as TNF-, which may then recruit the surrounding circular muscle cells to amplify the secretion of specific inflammatory mediators. Thus neuronal reflexes and increased circulating levels of proinflammatory cytokines (24) may induce a full-blown inflammation-like response in the muscularis externa, without the infiltration and activation of lymphocytes seen in patients with ulcerative colitis (32). The circular smooth muscle contractility in these patients is suppressed without any apparent presence and activation of immune cells in the muscle layers (45). Of course, the infiltration and activation of immunocytes in the outer muscle layers and myenteric plexus in Crohn's disease (32) and in experimental models of inflammation may further alter the time course and amplitude of the inflammatory response in muscularis propria.
The transcriptional regulation of ICAM-1 expression in HCCSMC in response to TNF- appears to be similar to that reported in human endothelial cells (25) and a melanoma cell line (17). This transcription is mediated primarily by the binding of p65 homodimers to the variant B motif at –187/–178 of ICAM-1 promoter. In support of this finding, we detected only a single complex of p65 in TNF--induced NF-B binding to the ICAM-1 promoter. Other investigators (17, 25) found two complexes, one containing homodimers of p65 and the other containing heterodimers of p65 and p50. In support of our finding, CMV-driven overexpression of p50 had little effect on the constitutive expression of ICAM-1, and antisense oligonucleotides to p50 did not block the expression of ICAM-1 in response to TNF-. Thus ICAM-1 gene may not be a target of p50 NF-B in HCCSMC. However, in the same cells, p50 NF-B has been reported to negatively regulate the expression of the 1C-subunit of L-type calcium channels (41). Our findings show that TNF- activates NF-B through both of its receptor subtypes, but predominantly through TNFR2. By contrast, in tracheal smooth muscle cells, TNF- induces the synthesis of cytokines predominantly through TNFR1 (3).
Our data show that ICAM-1 expression on HCCSMC promotes leukocyte adhesion, which is similar to that seen on its expression on endothelial and epithelial cells (16, 35). In addition, the 10 other cytokines, chemokines, and adhesion molecules secreted by these cells have diverse biological functions associated with inflammation. Therefore, the smooth muscle cells, once considered to be passive contractile cells, may play an active role in the manifestation of the inflammatory response and healing from it. The relatively large volume of smooth muscle cells in the neuromuscular layer compared with that of immune cells may be critical to obtaining an optimal inflammatory response in the muscularis externa. In addition, the secretion of inflammatory mediators may continue even after the infiltration and activation of immunocytes has ceased because of the autocrine effects of inflammatory mediators secreted by smooth muscle cells. In this regard, the measurements of the expression of specific inflammatory mediators in the tissue may be a more inclusive and broader measure of the inflammatory response than immunostaining of the tissue for specific immune cells alone.
In conclusion, TNF- induces gene expression in HCCSMC to initiate programmed synthesis and release of pro- and anti-inflammatory mediators. The secretion of these inflammatory mediators would initially enhance the inflammatory response in the neuromuscular layer, even in the absence of classic immune cell infiltrate, leading to smooth muscle and enteric neural dysfunction in acute and chronic gut inflammation. The secretion of inflammatory mediators by circular smooth muscle cells may be a major source of motility dysfunction in ulcerative colitis in which there is no apparent infiltration of immune cells in the outer muscle layers and myenteric plexus. The delayed expression of anti-inflammatory cytokine IL-11 may limit or terminate the expression of proinflammatory cytokines to initiate the recovery of smooth muscle function from inflammation-induced injury. The expression of ICAM-1 in HCCSMC by TNF- is regulated transcriptionally by the activation of the p65 subunit of NF-B and its binding to the downstream B motif of ICAM-1 promoter.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. Clin Chem 47: 1297–1301, 2001. suppresses human colonic circular smooth muscle cell contractility by SP1 and NF-kB mediated induction of ICAM-1. Gastroenterology 127: 1096–1109, 2004.[CrossRef][Web of Science][Medline]
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B activation by oxidative stress and inflammation suppresses contractility in colonic circular smooth muscle cells. Gastroenterology 124: 1369–1380, 2003.[CrossRef][Web of Science][Medline]
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, Vehicle-treated controls; 
, TNF-
