Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

Tadayuki Oshima1, Kevin P. Pavlick1, F. Stephen Laroux1, S. Kris Verma2, Paul Jordan2, Matthew B. Grisham1, Larry Williams3, and J. Steven Alexander1

Departments of 1 Molecular and Cellular Physiology and 2 Gastroenterology, Louisianna State University Health Sciences Center, Shreveport, Louisiana, 71130-3932; and 3 Guilford Pharmaceuticals, Baltimore, Maryland 21224


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-alpha using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-alpha induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappa B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-alpha within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-alpha activated NF-kappa B through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-alpha stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-kappa B/PARP. MAdCAM-1 expression requires NF-kappa B translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

mucosal addressin cell adhesion molecule-1


    INTRODUCTION
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LYMPHOCYTE TRAFFICKING is mediated by interactions between homing receptors on the lymphocyte and vascular addressins expressed on specific endothelia that are controlled by a variety of inflammatory mediators (10, 42, 51). In particular, lymphocyte homing to mucosal-associated lymphoid tissues, such as Peyer's patches and the lamina propria of the intestine, is critically dependent on a single-chain 60-kDa glycoprotein adhesion molecule, the mucosal vascular addressin or mucosal addressin cell adhesion molecule (MAdCAM-1; see Refs. 52 and 35). MAdCAM-1 is also expressed on endothelial cells within the mesenteric lymph nodes, the lamina propria of both the small and large intestine, and in the mammary gland during lactation. In addition to its normal role in lymphocyte trafficking to mucosal lymphoid tissue, MAdCAM-1 expression is dramatically increased in chronic inflammatory and disease states (39), e.g., inflammatory bowel disease (IBD; see Refs. 9 and 15) and in diabetes (20, 21, 60), and may play important roles in these conditions.

MAdCAM-1 is believed to play a central role in the etiology of colitis through its ability to direct circulating lymphocytes to enter gut-associated lymphoid tissues and the gut interstitium tissues (17, 56). The adhesion between the alpha 4beta 7-integrin, expressed on a subset of T cells, and MAdCAM-1 presented on cytokine-activated endothelial cells, facilitates the emigration of lymphocytes to inflamed sites in the gut in several models of colitis (9). Furthermore, although MAdCAM-1 is constitutively expressed in some regions of the colon and mesenteric lymph nodes, the expression of MAdCAM-1 in these tissues increases at least 10- to 11-fold during periods of active colitis (14, 26, 27). In the interleukin (IL)-10-deficient murine model of spontaneous colitis, the colon expresses very high basal levels of MAdCAM-1 and during active colitis shows robust increases in MAdCAM-1 expression (26). MAdCAM-1 has also been detected at high levels in colon lamina propria venules from mice with hapten-induced colitis (57) and in the colon of the IL-2-deficient mouse (32). In another colitis model, the severe combined immunodeficient (SCID) mouse, reconstituted with CD4+ CD45RBhigh T cells, develops a severe form of colitis (27). This injury can be blocked by antibodies directed against either MAdCAM-1 or its ligand alpha 4beta 7 (41). These data demonstrate that these two adhesive determinants mediate cell trafficking in the chronically inflamed gut. Interestingly, MAdCAM-1 may sustain chronic inflammation by stimulating proliferation of extravasated lymphocytes via MAdCAM/beta 7-mediated signaling (30).

Tumor necrosis factor (TNF)-alpha appears to be one of the most potent cytokines stimulating MAdCAM-1 expression both in vivo and in vitro. Levels of TNF-alpha and other proinflammatory cytokines are dramatically elevated in CD45RBhigh/SCID and IL-10-deficient (-/-) mice (26, 27). Expression of MAdCAM-1 on endothelial cells has been reported after TNF-alpha stimulation (50) and in the colons of mice injected with TNF-alpha (15). Besides MAdCAM-1, TNF-alpha also induces the expression of several other endothelial cell adhesion molecules (ECAMs), including E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 (44), by activating signals leading to activation of the transcription factor nuclear factor (NF)-kappa B. TNF-alpha -induced expression of ECAMs like E-selectin, VCAM-1, and ICAM-1 (25, 43) is almost completely blocked by peptide-aldehyde inhibitors like MG-132 and lactacystin, which block the 26S proteasome, a central regulator of NF-kappa B translocation. The genes for these ECAMs share specific DNA binding motifs in their promoters that allow for NF-kappa B-driven transcriptional initiation of ECAM messages (12, 13). However, although the pathways governing E-selectin, VCAM-1, and ICAM-1 have been studied intensively, the specific cell regulatory mechanisms through which MAdCAM-1 expression is controlled have not been as closely examined. Although the MAdCAM-1 promoter is known to contain NF-kappa B binding sites and may enhance MAdCAM-1 transcripts via NF-kappa B based on luciferase assays (54), NF-kappa B-dependent MAdCAM-1 protein expression has not been demonstrated.

Since the original report on MAdCAM-1 (hereafter MAdCAM) regulation by Sikorski et al. (50), few other studies have examined the regulation of MAdCAM expression, especially within high endothelial venule cells, which are relevant to chronic gut inflammation and IBD. Because entry of lymphocytes into specialized venules bearing MAdCAM in lymphoid tissues appears to regulate chronic gut inflammation, an improved understanding of the signals that promote the expression of this molecule may provide an important therapeutic means for limiting MAdCAM expression, beta 7-lymphocyte binding, and activation and inflammation in these tissues. To date, most experimental studies on MAdCAM expression have been obtained using an endothelioma cell line (bEnd.3) derived from brain microvessels. Although bEnd.3-based models are clearly useful, cells derived from brain capillaries may not accurately model high endothelial venules. Here we have compared MAdCAM expression in murine high endothelial venule cells (SVEC4-10) with brain-derived cells (bEnd.3). We determined how several second-message systems, e.g., NF-kB, poly-ADP ribose polymerase (PARP), p38, p42/44 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and protein kinase G (PKG), control MAdCAM expression in response to TNF-alpha . We measured lymphocyte binding to MAdCAM expressed in our cells to document the functional significance of these expression studies. Last, we have also localized the MAdCAM expressed in stimulated endothelial cells and in the intestine and found that MAdCAM is enriched at cell-cell junctions. These data show several novel paths for MAdCAM regulation and suggest potential pathways for limiting MAdCAM-dependent gut inflammation in IBD.


    MATERIALS AND METHODS
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Reagents. MG-132 (46) was purchased from Biomol (Plymouth Meeting, PA). GPI-6150, the PARP inhibitor, was obtained from Guilford Pharmaceuticals (Baltimore, MD). Genistein (1) was purchased from Sigma (St. Louis, MO). PD-98059 (2), SB-202190 (29), chelerythrin chloride, and KT-5823 were all purchased from Calbiochem (La Jolla, CA).

Cell culture. SVEC4-10 (SVEC) is a cell line derived by Simian virus-40 transformation of murine high endothelial cells (37). The mouse brain endothelial cell line bEnd.3 was obtained from Dr. Eugene Butcher (50). LEII cells are an endothelial cell line derived from murine lung capillary and were supplied by Dr. Thomas Maciag (55). These cell types were all maintained in DMEM with 10% FCS with 1% antibiotic/antimycotic. Mouse CD8+ T cell lymphoma tyrosine kinase (TK)-1 cells that constitutively express alpha 4beta 7 (3) were also donated by Dr. Butcher. These cells were cultured in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol.

Quantification of mRNA levels by RT-PCR. SVEC were pretreated for 1 h with or without inhibitors and then were incubated for 12 h with either vehicle or 20 ng/ml TNF-alpha (Endogen, Stoughton, MA). MAdCAM message was measured by RT-PCR in response to treatments. Total RNA was extracted from SVEC cells using the RNeasy Kit (QIAGEN, Valencia, CA). First-strand cDNAs were prepared from 4 µg of total RNA using a mixture of oligo(dT)12-18 and random hexamer primers with Superscript RT (Promega, Madison, WI). The following oligonucleotides were synthesized and used as primers: P1, 5'-CCTAGTACCCTACCAGCTCA-3'; P2, 5'-ATCTCCTCTTCTTGCTCTGG-3' (P1-P2; 474 bp); P3, 5'-AGAAGAGGAGATACAAGAGG-3'; and P4, 5'-TAGGTGTGTACATGAGCTAGTGTCTGGGCG-3' (P3-P4; 717 and 286 bp).

As controls, a 307-bp (sense, 5'-CGGTGTGAACGGATTTGGCCGTAT-3'; antisense, 5'-GGCCTTCTCCATGGTGGTGAAGAC-3') fragment of murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified in the same tube that contained primers 1 and 2. Primer sequences for GAPDH were separated by introns to control for potential sample contamination by genomic DNA. PCR amplification was performed at 95°C for 3 min, followed by 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, and then at 72°C for the final 5 min. The PCR products were then separated on 1.2% agarose gels. To normalize mRNA levels, the density of the MAdCAM and GAPDH bands from the same lane were scanned, and the data were calculated as the ratios of the optical density values of MAdCAM relative to those of GAPDH and are presented as a percentage of the TNF-alpha -stimulated density ratio. Experiments shown were repeated three times using independent extracts.

Western analysis of cell lysates. Each protein sample (75 µg each) was separated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with 1° anti-mouse MAdCAM monoclonal antibody (mAb; 10 µg/ml MECA-367; Pharmingen, San Diego, CA; see Ref. 52) or 0.05 µg/ml anti-activated MAPK polyclonal antibody (Promega, Pittsburgh, PA). Goat anti-rat or anti-rabbit horseradish peroxidase-conjugated 2° antibody (Sigma) was added at a 1:2,000 dilution. Last, membranes were developed by enhanced chemiluminescence (Amersham, La Jolla, CA). MAdCAM staining density was measured by scanning the 58- to 60-kDa band and densitometry using Image Pro Plus (Media Cybernetics, Bethesda, MD). The data are expressed as the percentage of the level of density induced by TNF-alpha (set at 100%). All experiments were performed at least in triplicate.

Trypsin protection assay. Cell surface and cytoplasmic MAdCAM were compared by trypsinizing TNF-alpha -stimulated endothelial monolayers using the technique described by Takeichi (53). After treatment, cells were trypsinized with 0.1% trypsin (type I; Sigma) in Hanks' balanced salt solution (HBSS) for 5 min. Trypsinized monolayers were then neutralized with 5 mg/ml soybean trypsin inhibitor for 5 min, and samples were solubilized in electrophoresis sample buffer. These samples were Western blotted as described and were compared with monolayers of equivalent surface area that were not exposed to trypsin.

TK-1 lymphocyte adhesion assay. Lymphocyte adhesion assays were performed as previously described (50). Briefly, TK-1 cells were radiolabeled with 30 µCi Na51CrO4/ml (New England Nuclear, Natick, MA) at 37°C for 60 min. The cells were then washed two times with ice-cold HBSS and resuspended in HBSS. Labeled TK-1 cells were then added to the endothelium at a lymphocyte-to-endothelial cell ratio of 5:1 (28) and were allowed to bind for 30 min. After incubation, monolayers were washed two times with HBSS, and the adherent TK-1 cells were solubilized with 1 N NaOH. The 51Cr activity of the labeled cells was measured by gamma -counting. The percentage of adhered TK-1 cells was quantified as follows: %adhesion = {[counts/min (cpm) in adhered cells]/(cpm of total added cells)} × 100. All data represent the averages of four identically treated monolayers.

Electrophoretic mobility shift assay. SVEC were pretreated for 1 h with or without inhibitors and then were incubated for 1 h with either vehicle or TNF-alpha (20 ng/ml). Nuclear extracts were prepared as described previously (48). The NF-kappa B consensus oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC was end labeled with [gamma -32P]ATP using T4 polynucleotide kinase according to the manufacturer's instructions (Promega). Labeled oligonucleotide (35 fmol) was incubated with 20 µg nuclear extracts for 10 min on ice in binding buffer [1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris · HCl (pH 7.5), 0.05 µg/µl poly(dI-dC) · poly(dI-dC), and 4% glycerol] in a total volume of 30 µl. A 50-fold molar excess of nonlabeled consensus or mutated NF-kappa B consensus oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) was included in respective reactions. Samples were subsequently incubated for 30 min at 25°C. For supershift studies, antibody specific for either the p50 (Santa Cruz Biotechnologies) or p65 (Rockland, Gilbertsville, PA) subunits was added after the initial incubation on ice. Protein-DNA complexes were resolved on 4% nondenaturing polyacrylamide gels by electrophoresis in 0.5× Tris-borate-EDTA. Gels were dried and visualized by a PhosphorImager (Molecular Dynamics, Hercules, CA). Activation of NF-kappa B (relative to non-TNF-treated controls) was determined by performing densitometric analysis (ImageQuant software; Molecular Dynamics, Sunnyvale, CA) on shifted bands from scanned autoradiograms. Experiments shown were repeated three times using independent extracts.

Immunofluorescence staining of MAdCAM. IL-10(-/-) mice (n = 4) were anesthetized, and 10 µg of MAdCAM mAb (Pharmingen) was injected in the jugular vein. After 5 min, the vasculature was completely flushed with bicarbonate-buffered saline, and colons were excised as previously described (26). Frozen samples were sectioned in a cryostat at 10 µm. The sections were blocked with 10% normal donkey serum and then incubated with a cytomegalovirus (Cy3) anti-rat secondary antibody (Jackson Laboratories, Westgrove, PA) for 1 h. Photographs were taken with a Sen Sys digital camera. SVEC were grown to confluency on coverslips, stimulated with TNF-alpha (20 ng/ml, 24 h), and stained for MAdCAM without fixation. Rat anti-mouse MAdCAM mAb was used at a concentration of 10 µg/ml. Cy3 goat anti-rat secondary antibody was used at a 1:100 dilution. The specificity of the reaction was tested by incubation with HBSS or rat IgG1.

Statistical Analysis. All values are expressed as means ± SE. Data were analyzed using one-way ANOVA with Bonferroni's correction for multiple comparisons. Significance was accepted at P < 0.05.


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Analysis of MAdCAM expression on bEnd.3 cells. The expression of MAdCAM protein was measured by Western blotting using the MECA-367 mAb. MAdCAM was not constitutively expressed on unstimulated bEnd.3 cells but was induced by TNF-alpha (20 ng/ml; Fig. 1A). Expression of MAdCAM in bEnd.3 cells was dose- and time-dependently increased by TNF-alpha , with maximal levels at 24 h with 20 ng/ml TNF-alpha (data not shown). These data are similar to previous reports (50).


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Fig. 1.   A: effect of signal transduction blockers on mucosal addressin cell adhesion molecule (MAdCAM) in bEnd.3 cells. Cells were pretreated with MG-132 (MG, 5 µM), GPI-6150 (GPI, 60 µM), genistein (gen, 30 µM), PD-98059 (PD, 20 µM), SB-202190 (SB, 10 µM), chelerythrin chloride (chel Cl, 60 µM), or KT-5823 (KT, 0.5 µM) and then exposed to tumor necrosis factor (TNF)-alpha (20 ng/ml) stimulation in the continued presence of inhibitors. MAdCAM expression is significantly increased after TNF-alpha . MG-132, GPI-6150, genistein, PD-98059, or SB-20219 blocked the increase of MAdCAM induced by TNF-alpha . Values represent means ± SE; n = 4 experiments in each group. *P < 0.001 vs. untreated controls; #P < 0.001 vs. TNF-alpha treatment. B: RT-PCR analysis of MAdCAM and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in bEnd.3 (a) or LEII (b) cells. a: Control conditions, no MAdCAM transcript is detected, whereas a 474-bp MAdCAM band is detected after stimulation with TNF-alpha . b: No band is detected as MAdCAM transcript on LEII cells after stimulation with TNF-alpha . GAPDH was run in the same PCR reaction as MAdCAM as an internal control. In a and b, 307-bp bands indicated GAPDH transcripts evaluating quality and amount of RNA. Similar results were obtained in 4 separate experiments.

To examine the second messages involved in the induction of MAdCAM expression by TNF-alpha , bEnd.3 were pretreated with either a proteasome inhibitor (MG-132), a poly-ADP ribose polymerase inhibitor (GPI-6150), the TK inhibitor genistein, a mitogen/extracellular signal-regulated kinase (MEK)-1 inhibitor (PD-98059), a p38 MAPK inhibitor (SB-202190), the nonselective PKC antagonist chelerythrin chloride, or a PKG inhibitor (KT-5823). Preincubation of monolayers with 5 µM MG-132 significantly decreased TNF-alpha -induced MAdCAM protein expression. TNF-alpha -induced MAdCAM protein induction was also blocked by 60 µM GPI-6150, 30 µM genistein, 20 µM PD-98058, or 10 µM SB-202190 but not by either 0.5 µM KT-5823 or 60 µM chelerythrin chloride. None of the tested inhibitors induced expression of MAdCAM in the absence of TNF-alpha (data not shown).

Analysis of the bEnd.3 cell MAdCAM message is shown in Fig. 1B. To confirm the existence of alternative MAdCAM messages (47), we amplified total RNA from bEnd.3 cells with or without 12 h of TNF-alpha stimulation using primers (see MATERIALS AND METHODS). MAdCAM message (with primers 1 and 2) is not expressed on unstimulated bEnd.3 cells but is induced after treatment with mouse TNF-alpha (20 ng/ml) for 12 h (Fig. 1B). MAdCAM message was not detected on LEII lung endothelial cells, even after 12 h of TNF-alpha treatment (Fig. 1B). There was also a dramatic fall in TNF-alpha -induced transcription levels of MAdCAM seen by RT-PCR analysis of RNA from inhibitor-treated endothelial cells. GAPDH was run in the same PCR reaction as MAdCAM as an internal control.

Changes in expression of MAdCAM message after TNF-alpha stimulation. To confirm the effects of these second message inhibitors on expression, we next examined MAdCAM expression in the mouse high endothelial cell line SVEC4-10. MAdCAM message on SVEC was first measured by RT-PCR. Figure 2A shows the PCR products obtained with specific primers (primers 3 and 4) that distinguish the longer and shorter isoforms of the mouse MAdCAM message. MAdCAM message is not transcribed in unstimulated SVEC but is induced in a time-dependent manner after treated with mouse TNF-alpha (20 ng/ml). The kinetics observed for mRNA accumulation here are consistent with those observed for expression of MAdCAM protein. With respect to these two MAdCAM isoforms (detected as 286 and 717 bp with RT-PCR), the longer type was dominant after stimulation with TNF-alpha .


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Fig. 2.   MAdCAM expression in SVEC cells. A: RT-PCR was carried out using TNF-alpha -stimulated and nonstimulated SVEC RNA with primers P3 and P4. In controls, no transcript was detected. Two PCR products (286 and 717 bp) were detected after TNF-alpha stimulation. MAdCAM transcripts were increased time dependently after TNF-alpha stimulation. Experiments were performed 3 times with similar results. B: surface expression of MAdCAM. MAdCAM was localized to the cell surface after TNF-alpha stimulation by trypsin protection. SVEC were treated with medium alone or medium containing 0.1% trypsin. After trypsin, the amount of MAdCAM protein was reduced by 80%. Each value represents the mean ± SE; n = 3 for each group. *P < 0.001 vs. untreated control; #P < 0.001 vs. TNF-alpha treatment.

Effect of TNF-alpha on MAdCAM expression. Expression of MAdCAM in SVEC cells was dose- and time-dependently increased by TNF-alpha , with maximal levels observed after 24 h of incubation with 20 ng/ml TNF-alpha (data not shown). To ensure that the MAdCAM determined by Western blotting was actually expressed on the cell surface, the percentage of the total MAdCAM pool on the surface was determined by 0.1% trypsin treatment (5 min) and immunoblotting (Fig. 2B). Trypsin-treated cells showed significantly lower levels of MAdCAM compared with non-trypsin-treated cells (only 15.6 ± 2.5% of controls without trypsin, P < 0.001). According to densitometry, at least 80% of the MAdCAM was susceptible to trypsin and was therefore present on the cell surface. Because TK-1 cell adhesion to TNF-alpha -treated cells was also significantly reduced by anti-MAdCAM antibody, these data strongly argue that MAdCAM is indeed present on the surface and is functionally active.

Effect of signal transduction blockers on SVEC. Although the intracellular signals that mediate MAdCAM induction have not been completely elucidated in this model, TK, MEK-1, p38 MAPK, and NF-kappa B have been implicated in the cytokine-induced ECAM expression in other cell types. To investigate the signal transduction pathways for MAdCAM, SVEC were pretreated with either the proteasome inhibitor MG-132, the PARP inhibitor GPI-6150, the TK inhibitor genistein, the MEK-1 inhibitor PD-98059, the p38 MAPK inhibitor SB-202190, the nonselective PKC antagonist chelerythrin chloride, or the PKG inhibitor KT-5823. Figure 3A shows the PCR products obtained with primers 3 and 4 (see MATERIAL AND METHODS), which amplify the first and second IgG-like domain of MAdCAM (47). A strong MAdCAM transcript band is detected after stimulation with TNF-alpha (20 ng/ml) for 12 h. Pretreatment with 5 µM MG-132, 60 µM GPI-6150, 30 µM genistein, 20 µM PD-98058, or 10 µM SB-202190 blocked the TNF-alpha (20 ng/ml, 12 h)-induced MAdCAM transcript (to 11.68, 11.9, 23.85, 27.0, and 13.4% of TNF-alpha -treated levels, respectively).


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Fig. 3.   Effect of signal transduction blockers on SVEC. A: RT-PCR analysis of MAdCAM and GAPDH in SVEC exposed to TNF-alpha  ± blockers. In controls, only a faint MAdCAM transcript is detected (474 bp, primers 3 and 4). MAdCAM band is easily detected after TNF-alpha stimulation (20 ng/ml). MG-132 (5 µM), GPI-6150 (60 µM), genistein (30 µM), PD-98059 (20 µM), or SB-202190 (10 µM) blocked TNF-alpha -induced MAdCAM transcript. In A and B, 307-bp bands indicate GAPDH transcripts used to evaluate the amount and quality of RNA. Similar results were obtained in 3 separate experiments. B: cells were pretreated with MG-132 (5 µM), GPI-6150 (60 µM), genistein (30 µM), PD-98059 (20 µM), SB-202190 (10 µM), chelerythrine chloride (60 µM), or KT-5823 (0.5 µM) followed by TNF-alpha (20 ng/ml). MG-132, GPI-6150, genistein, PD-98059, or SB-20219 blocked the increase of MAdCAM on SVEC induced by TNF-alpha . Each value represents the mean ± SE; n = 4 experiments for each group. *P < 0.001 vs. untreated control; #P < 0.001 vs. TNF-alpha treatment.

To determine the effect of second message blockers on TNF-alpha -induced MAdCAM protein expression, immunoblotting was performed. Preincubation of monolayers with 5 µM MG-132 prevented TNF-alpha -induced MAdCAM translation. TNF-alpha -induced MAdCAM translation was also blocked by 60 µM GPI-6150, 30 µM genistein, 20 µM PD-98058, or 10 µM SB-202190 but not by 0.5 µM KT-5823 or 60 µM chelerythrin chloride (Fig. 3B). These inhibitors alone had no effect on MAdCAM translation in SVEC (data not shown).

To determine if the effect of genistein was related to MAPK pathway activation, we performed immunoblotting with anti-active p42/44 MAPK antibody. Pretreatment of monolayers with genistein (30 µM) or PD-98059 (20 µM) prevented the TNF-alpha -induced increase in MAPK phosphorylation (Fig. 4A), suggesting that tyrosine phosphorylation and MEK-1 are important in the activation of extracellular signal-regulated kinase (ERK) 1/2 in response to TNF-alpha .


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Fig. 4.   Effect of signal transduction blockers on p42/44 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B activation. A: activated MAPK was detected using an anti-active MAPK polyclonal antibody. Pretreatment of monolayers with genistein (30 µM) or PD-98059 (20 µM) prevented the TNF-alpha -induced increase in MAPK phosphorylation. Data shown are representative of 3 independent experiments. ERK, extracellular signal-regulated kinase. B: inhibition of TNF-alpha activation of NF-kappa B in SVEC. After 1 h of TNF-alpha (20 ng/ml) treatment, the quantity of protein bound to the NF-kappa B element increased significantly. Pretreatment of the cells with MG-132 (5 µM), GPI-6150 (60 µM), or PD-98059 (20 µM) reduced the TNF-alpha -induced binding of NF-kappa B but not to basal levels. Pretreatment of genistein (30 µM) slightly reduced the binding of NF-kappa B, especially in the band on top. Pretreatment with SB-202190 (10 µM) did not block the NF-kappa B binding. Specificity of the NF-kappa B bands is confirmed by competition with 50× molar excess of cold oligonucleotide and supershift assays performed with alpha -p50 or alpha -p65 antibody. Cont, control; ns, nonspecific complexes.

Inhibition of NF-kB activation. We evaluated the effect of these inhibitors on NF-kappa B activation using the electrophoretic mobility shift assay using an NF-kappa B-radiolabeled double-stranded oligonucleotide probe. As shown in Fig. 4B, nuclear protein from untreated (control) cells produced multiple protein-DNA complexes, of which one complex was dramatically enhanced in extracts from TNF-alpha -treated cells (compare lanes 1 and 2). The increased complex was specifically competed with a 50-fold molar excess of cold competitor (lane 3) but did not compete with the same amount of a mutated probe (lane 4). This suggests that the binding activity is dependent on the NF-kappa B consensus site. Inclusion of either alpha -p50 or alpha -p65 (lanes 5 and 6, respectively) demonstrates that this complex contains the activating form of NF-kappa B (4). Interestingly, pretreatment with PD-98059, MG-132, or GPI-6150 reduced the TNF-alpha -induced activation of NF-kappa B by 29.9, 43.6, and 25.0%, respectively (lanes 7-9). Pretreatment with SB-202190 had no affect on NF-kappa B activation (lane 10), whereas pretreatment with genistein had a slight effect (lane 11).

MAdCAM expressed in high endothelial cells is functional. Having established a role for several signals in the regulation of MAdCAM expression by endothelial cells, we next examined the effects of inhibitors on adhesion of the mouse lymphocyte cell line TK-1 to TNF-alpha -treated SVEC. Figure 5 shows that control adhesion of TK-1 cells was 13.1 ± 1.21%. TNF-alpha induced maximal adhesion (37.3 ± 3.25%, P < 0.001 vs. untreated control) by 24 h. This high level of adhesion was inhibited by preincubation of SVEC with anti-MAdCAM antibody. This antibody reduced TK-1 adhesion to 21.5 ± 1.5% (P < 0.001 vs. TNF-alpha treatment), showing that 65.5% of the TNF-alpha -induced TK-1 adhesion was the result of MAdCAM. Treatment of SVEC with MG-132, GPI-6150, genistein, PD-98058, or SB-202190 significantly reduced adhesion to 15.6 ± 0.96, 13.8 ± 0.66, 16.7 ± 0.60, 18.6 ± 1.01, and 12.6 ± 0.49%, respectively, of that induced by TNF-alpha alone (P < 0.001). None of the inhibitors affected adhesion to untreated SVEC (data not shown).


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Fig. 5.   Adhesion of tyrosine kinase (TK)-1 cells on SVEC. TNF-alpha induced significant increase of TK-1 cell adhesion. This increased adhesion was significantly inhibited by pretreatment with MAdCAM antibody (Ab). Pretreatment of MG-132 (5 µM), GPI-6150 (60 µM), genistein (30 µM), PD-98059 (20 µM), or SB-202190 (10 µM) also significantly inhibited TNF-alpha (20 ng/ml, 24 h)-induced TK-1 adhesion on SVEC. Each value represents the mean ± SE; n = 4 experiments in each group. *P < 0.001 vs. untreated control; #P < 0.001 vs. TNF-alpha treatment.

Immunofluorescent staining of MAdCAM in vitro. Immunolocalization of colonic MAdCAM in IL-10(-/-) colitic mice shows vascular staining at large mucosal and submucosal vessels. The staining appeared to be junctional (Fig. 6, A and B). MAdCAM expression on SVEC was assessed by indirect immunofluorescence and was analyzed by a fluorescence microscope (×200). Control untreated cells exhibited no staining (Fig. 6C). TNF-alpha (20 ng/ml, 24 h) induced a marked increase in the immunofluorescent staining of MAdCAM on the surface and junction to some extent (Fig. 6D). The images shown are representative of at least 20 different fields observed in each experiment.


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Fig. 6.   Immunofluorescent staining of MAdCAM on SVEC. Sectioned colons of interleukin-10-deficient mice and control and TNF-alpha -treated (20 ng/ml, 24 h) SVEC on glass coverslips were stained for MAdCAM localization with cytomegalovirus 3-conjugated anti-rat IgG secondary Ab. A: MAdCAM stains blood vessels in vivo in the mucosa, which appear junctional at higher power (B). Control SVEC cells exhibited no staining (C); however, after TNF-alpha , there was a marked increase in MAdCAM immunostaining, especially at cell junctions (D). Images are representative of at least 20 different fields observed in each experiment and of 3 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAdCAM, a member of the immunoglobulin-like cell adhesion protein superfamily, contains several structural motifs that are homologous with ICAM-1, VCAM-1, and IgA1 and contains a unique mucin-like domain not shared by these molecules (8, 47). MAdCAM is expressed on mucosal venules and directs lymphocytes to traffic into Peyer's patches and the intestinal lamina propria (10). MAdCAM expression in vitro has been previously reported in a mouse brain endothelial cell line (50). MAdCAM appears to play critical roles in several chronic immune and inflammatory conditions, especially IBD, through its ability to direct beta 7-lymphocytes to the gut, where these lymphocytes promote several inflammatory processes leading to IBD (17, 56).

In this study, we have shown that MAdCAM is increased on mouse "high" endothelial cells in vitro after TNF-alpha stimulation, which might represent a more relevant model for evaluating potential mechanisms of human IBD than capillary endothelium from the brain (Fig. 2). On the basis of the described role of MAdCAM in forms of chronic gut inflammation, it is highly likely that TNF-alpha -induced MAdCAM expression would be controlled through TKs, PKC, and perhaps PKG, MAPKs, and NF-kappa B, as has been reported for other endothelial adhesion molecules in colitis and IBD. Furthermore, antagonists to these signaling cascades might provide important therapeutic targets to limit forms of MAdCAM-dependent inflammation.

Recent studies on TNF-alpha /NF-kappa B signals have revealed that receptor-mediated stimuli that mobilize NF-kappa B activate several levels of protein kinases (11, 58), which result in the release and proteasomal degradation of inhibitor protein (I) kappa B from the inactive NF-kappa B-Ikappa B complex. The release of Ikappa B permits the translocation of the activated p50/p65 NF-kappa B complex to the nucleus (18). Read et al. (44) reported that MG-132, an inhibitor of the proteasome, prevented Ikappa Balpha degradation induced by TNF-alpha and nuclear accumulation of NF-kappa B, efficiently inhibiting NF-kappa B function.

Oliver et al. (38) and Hassa and Hottiger (22) reported that PARP is also required for the NF-kappa B-dependent transcription using both PARP gene-deficient mice and cells. Interestingly, Hassa and Hottiger reported that PARP is actually a novel binding partner for NF-kappa B and, apparently, must be activated to activate NF-kB activation. Taken together, our data with MG-132 and GPI-6150 strongly suggest that proteasome activity and PARP are necessary for maximal NF-kappa B activation in the TNF-alpha -mediated induction of MAdCAM in this model.

We also demonstrated that MAdCAM transcription and translation are regulated through the TK MEK-1, p38, p42/44 MAPK, and NF-kappa B signal transduction cascades (Figs. 1-4). PKC and PKG inhibitors did not influence MAdCAM expression. Our data on the regulation of MAdCAM expression in SVEC cells parallel studies of ICAM-1 and VCAM-1 regulation in human umbilical vein endothelial cells. As shown for SVEC, ICAM-1, and VCAM-1, expression is upregulated by TNF-alpha through TK and NF-kappa B signal transduction cascades (58) involving TNF-alpha -induced ERK1 activation (which is inhibited by genistein; see Ref. 31). The effects of the second message inhibitors on MAdCAM mRNA induction are also the same in both bEnd.3 and SVEC cells. However, the effects of genistein and PD-98059 differ slightly in bEnd.3 cells and SVEC, suggesting that the signal transduction pathways in TNF-alpha -induced MAdCAM expression in these two cell types may differ somewhat in their sensitivity to TK/MEK-1 inhibition.

Interestingly, in previous reports, ERK1 inhibition did not block the upregulation of ICAM-1 in IL-1beta -treated cardiac myocytes and fibroblasts (24). In that study, ERK1 inhibition prevented both TNF-alpha -induced NF-kappa B DNA binding and MAdCAM synthesis. Therefore, these data suggest that ERK1 activation precedes NF-kappa B activation in MAdCAM mobilization.

It has recently been reported that p38 MAPK may be required for NF-kappa B-dependent gene expression (11), and inhibition of the p38 MAPK pathway prevented IL-1beta -induced ICAM and VCAM translation (24). Here we have shown that, in addition to NF-kappa B, p38 MAPK was necessary for MAdCAM expression. Our data also indicate that p38 MAPK inhibition can prevent MAdCAM expression induced by TNF-alpha at the level of transcription but did not interfere with NF-kappa B binding to DNA. The effect of the p38 blocker SB-202190 was restricted to the inhibition of trans-activating activity, since activation of NF-kappa B DNA binding (as measured in gel assays) was unaffected. These data support the model proposed by Beyaert et al. (7), which indicated that p38 MAPK was not involved in the release of Ikappa B into the cytoplasm, and the work by Wesselborg et al. (59) and Schulze-Osthoff et al. (49), which both showed that p38 MAPK could activate MAPKAPK, which may function as an NF-kappa B transactivation factor at the nuclear level. Taken together, our observations suggest that several forms of MAPK activation are important in regulating NF-kappa B activity and MAdCAM expression. Apparently, p38 and NF-kappa B involve separate pathways, and p38 may govern other coactivating transcription factors, e.g., p300 (40) or A20 (23), which govern NF-kappa B-mediated transcription but not DNA binding.

With respect to PKC, although previous studies show that direct PKC activation (with phorbol esters) increases E-selectin, VCAM-1, and ICAM-1 expression in endothelial cells (31), TNF-alpha - and IL-1alpha -induced expression of E-selectin and ICAM-1 are, however, independent of PKC activation (34, 45). Our data agree with those findings and fail to show an important role for PKC in MAdCAM regulation, as previously reported in brain endothelial cells (50).

Although our Western blotting data indicate an increase in absolute MAdCAM expression, it is possible that this MAdCAM might not be present on the cell surface or be functional. To address this concern, both total and cell surface MAdCAM pools were determined by trypsin treatment. Trypsin treatment removed at least 80% of the total MAdCAM present on the cell surface induced by TNF-alpha (Fig. 2B). These observations are further supported by our observations that TK-1 cell adhesion (an alpha 4beta 7-expressing lymphocyte line) was significantly reduced by addition of an anti-MAdCAM antibody (Fig. 5). Similarly, immunofluorescence staining of MAdCAM was detected without prior cell permeabilization (Fig. 6B). We also found that MAdCAM antibody reduced maximal TK-1 cell adhesion to SVEC induced by TNF-alpha but that basal adhesion was unaffected by blocking antibody. With the use of MAdCAM antibody to block adhesion, >60% of the TNF-alpha -stimulated adhesion of TK-1 cells was prevented by anti-MAdCAM, as reported by Sikorski et al. (50) in bEnd.3 cells.

Another interesting finding in this study was the distribution of MAdCAM on the cell surface. We showed that TNF-alpha -induced MAdCAM expression often appeared to be concentrated at cell-cell junctions (Fig. 6). This result is supported by the study of Kawachi et al. (26), which first showed MAdCAM expressed on dilated mucosal and submucosal vessels in IL-10(-/-) mouse colon in what appears to be cell-cell junctions. This is the first report to confirm this junctional localization of MAdCAM in vitro.

It has been demonstrated that MAdCAM serves as the main binding partner for alpha 4beta 7-integrin on lymphocytes and plays a role in L-selectin-mediated lymphocyte rolling (5, 6, 16, 19, 57). However, our results show that this junctional MAdCAM appearance is not uniform on all cells. Explanations for the lack of homogeneous junctional staining could be cell alterations induced by transformation or the degree of culture confluency. The possible roles of MAdCAM in endothelial junctions still remain to be elucidated.

In conclusion, the results of our studies show that MAdCAM expression is increased on high endothelial cells by TNF-alpha and that this TNF-alpha -induced MAdCAM expression is mediated through TK, p38, p42/44 MAP, and NF-kappa B signal transduction cascades. The increased MAdCAM expression on these endothelial cells may model the chronic inflammation caused by lymphocyte immune response seen in IBD. Recent studies show that NF-kappa B antagonist NF-kappa B antisense phosphorothioate oligonucleotide and 26S proteosome inhibitor can successfully attenuate inflammation in experimental colitis in vivo (14, 33, 36). In our studies, although we have not considered the expression of VCAM-1 in our cells, these data suggest that MAdCAM might be yet another target of NF-kappa B inhibition. Although we may speculate that additional as-yet-undefined signals may be required for the maximal induction of these genes during exposure to cytokines, it is likely that inhibitors of TKs, MAPKs, and NF-kappa B could be effective in limiting the inflammation associated with human IBD, which is mediated by upregulation of MAdCAM.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HL-47615, DK-43785, and DK-47663.


    FOOTNOTES

Address for reprint requests and other correspondence: J. S. Alexander, Molecular and Cellular Physiology, Louisiana State Univ. Health Sciences Center, 1501 Kings Hwy., Shreveport, LA, 71130-3932 (E-mail: jalexa{at}lsuhsc.edu).

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.

Received 28 March 2001; accepted in final form 14 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akiyama, T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, and Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262: 5592-5595, 1987[Abstract/Free Full Text].

2.   Alessi, DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen- activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489-27494, 1995[Abstract/Free Full Text].

3.   Andrew, DP, Berlin C, Honda S, Yoshino T, Hamann A, Holzmann B, Kilshaw PJ, and Butcher EC. Distinct but overlapping epitopes are involved in alpha 4 beta 7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J Immunol 153: 3847-3861, 1994[Abstract/Free Full Text].

4.   Baeuerle, PA, and Baltimore D. NF-kappa B: ten years after. Cell 87: 13-20, 1996[ISI][Medline].

5.   Berg, EL, McEvoy LM, Berlin C, Bargatze RF, and Butcher EC. L-selectin-mediated lymphocyte rolling on MAdCAM-1. Nature 366: 695-698, 1993[ISI][Medline].

6.   Berlin, C, Berg EL, Briskin MJ, Andrew DP, Kilshaw PJ, Holzmann B, Weissman IL, Hamann A, and Butcher EC. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1 (Abstract). Cell 74: 185, 1993[ISI][Medline].

7.   Beyaert, R, Cuenda A, Vanden Berghe W, Plaisance S, Lee JC, Haegeman G, Cohen P, and Fiers W. The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor. EMBO J 15: 1914-1923, 1996[Abstract].

8.   Briskin, MJ, McEvoy LM, and Butcher EC. MAdCAM-1 has homology to immunoglobulin and mucin-like adhesion receptors and to IgA1. Nature 363: 461-464, 1993[ISI][Medline].

9.   Briskin, M, Winsor-Hines D, Shyjan A, Cochran N, Bloom S, Wilson J, McEvoy LM, Butcher EC, Kassam N, Mackay CR, Newman W, and Ringler DJ. Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue. Am J Pathol 151: 97-110, 1997[Abstract].

10.   Butcher, EC, and Picker LJ. Lymphocyte homing and homeostasis. Science 272: 60-66, 1996[Abstract].

11.   Carter, AB, Knudtson KL, Monick MM, and Hunninghake GW. The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP). J Biol Chem 274: 30858-30863, 1999[Abstract/Free Full Text].

12.   Collins, T. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion. Lab Invest 68: 499-508, 1993[ISI][Medline].

13.   Collins, T, Read MA, Neish AS, Whitley MZ, Thanos D, and Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J 9: 899-909, 1995[Abstract/Free Full Text].

14.   Conner, EM, Brand S, Davis JM, Laroux FS, Palombella VJ, Fuseler JW, Kang DY, Wolf RE, and Grisham MB. Proteasome inhibition attenuates nitric oxide synthase expression, VCAM-1 transcription and the development of chronic colitis. J Pharmacol Exp Ther 282: 1615-1622, 1997[Abstract/Free Full Text].

15.   Connor, EM, Eppihimer MJ, Morise Z, Granger DN, and Grisham MB. Expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in acute and chronic inflammation. J Leukoc Biol 65: 349-355, 1999[Abstract].

16.   Erle, DJ, Briskin MJ, Butcher EC, Garcia-Pardo A, Lazarovits AI, and Tidswell M. Expression and function of the MAdCAM-1 receptor, integrin alpha 4 beta 7, on human leukocytes. J Immunol 153: 517-528, 1994[Abstract/Free Full Text].

17.   Fong, S, Jones S, Renz ME, Chiu HH, Ryan AM, Presta LG, and Jackson D. Mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Its binding motif for alpha 4 beta 7 and role in experimental colitis. Immunol Res 16: 299-311, 1997[ISI][Medline].

18.   Ghosh, S, May MJ, and Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16: 225-260, 1998[ISI][Medline].

19.   Hamann, A, Andrew DP, Jablonski-Westrich D, Holzmann B, and Butcher EC. Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo. J Immunol 152: 3282-3293, 1994[Abstract/Free Full Text].

20.   Hanninen, A, Jaakkola I, and Jalkanen S. Mucosal addressin is required for the development of diabetes in nonobese diabetic mice. J Immunol 160: 6018-6025, 1998[Abstract/Free Full Text].

21.   Hanninen, A, Taylor C, Streeter PR, Stark LS, Sarte JM, Shizuru JA, Simell O, and Michie SA. Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium. J Clin Invest 92: 2509-2515, 1993[ISI][Medline].

22.   Hassa, PO, and Hottiger MO. A role of poly (ADP-ribose) polymerase in NF-kappaB transcriptional activation. Biol Chem 380: 953-959, 1999.

23.   Heyninck, K, De Valck D, Vanden Berghe W, Van Criekinge W, Contreras R, Fiers W, Haegeman G, and Beyaert R. The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB- inhibiting protein ABIN. J Cell Biol 145: 1471-1482, 1999[Abstract/Free Full Text].

24.   Kacimi, R, Karliner JS, Koudssi F, and Long CS. Expression and regulation of adhesion molecules in cardiac cells by cytokines: response to acute hypoxia. Circ Res 82: 576-586, 1998[Abstract/Free Full Text].

25.   Kalogeris, TJ, Laroux FS, Cockrell A, Ichikawa H, Okayama N, Phifer TJ, Alexander JS, and Grisham MB. Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells. Am J Physiol Cell Physiol 276: C856-C864, 1999[Abstract/Free Full Text].

26.   Kawachi, S, Jennings S, Panes J, Cockrell A, Laroux FS, Gray L, Perry M, van der Heyde H, Balish E, Granger DN, Specian RA, and Grisham MB. Cytokine and endothelial cell adhesion molecule expression in interleukin-10-deficient mice. Am J Physiol Gastrointest Liver Physiol 278: G734-G743, 2000[Abstract/Free Full Text].

27.   Kawachi, S, Morise Z, Jennings SR, Conner E, Cockrell A, Laroux FS, Chervenak RP, Wolcott M, van der Heyde H, Gray L, Feng L, Granger DN, Specian RA, and Grisham MB. Cytokine and adhesion molecule expression in SCID mice reconstituted with CD4+ T cells. Inflamm Bowel Dis 6: 171-180, 2000[ISI][Medline].

28.   Kokura, S, Wolf RE, Yoshikawa T, Ichikawa H, Granger DN, and Aw TY. Endothelial cells exposed to anoxia/reoxygenation are hyperadhesive to T-lymphocytes: kinetics and molecular mechanisms. Microcirculation 7: 13-23, 2000[ISI][Medline].

29.   Lee, JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, and Landvatter SW. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739-746, 1994[ISI][Medline].

30.   Lehnert, K, Print CG, Yang Y, and Krissansen GW. MAdCAM-1 costimulates T cell proliferation exclusively through integrin alpha4beta7, whereas VCAM-1 and CS-1 peptide use alpha4beta1: evidence for "remote" costimulation and induction of hyperresponsiveness to B7 molecules. Eur J Immunol 28: 3605-3615, 1998[ISI][Medline].

31.   May, MJ, Wheeler-Jones CP, Houliston RA, and Pearson JD. Activation of p42mapk in human umbilical vein endothelial cells by interleukin-1 alpha and tumor necrosis factor-alpha. Am J Physiol Cell Physiol 274: C789-C798, 1998[Abstract/Free Full Text].

32.   McDonald, SA, Palmen MJ, Van Rees EP, and MacDonald TT. Characterization of the mucosal cell-mediated immune response in IL-2 knockout mice before and after the onset of colitis. Immunology 91: 73-80, 1997[ISI][Medline].

33.   Murano, M, Maemura K, Hirata I, Toshina K, Nishikawa T, Hamamoto N, Sasaki S, Saitoh O, and Katsu K. Therapeutic effect of intracolonically administered nuclear factor kappa B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin Exp Immunol 120: 51-58, 2000[ISI][Medline].

34.   Myers, CL, Desai SN, Schembri-King J, Letts GL, and Wallace RW. Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction. Am J Physiol Cell Physiol 262: C365-C373, 1992[Abstract/Free Full Text].

35.   Nakache, M, Berg EL, Streeter PR, and Butcher EC. The mucosal vascular addressin is a tissue-specific endothelial cell adhesion molecule for circulating lymphocytes. Nature 337: 179-181, 1989[ISI][Medline].

36.   Neurath, MF, and Pettersson S. Predominant role of NF-kappa B p65 in the pathogenesis of chronic intestinal inflammation. Immunobiology 198: 91-98, 1997[ISI][Medline].

37.   O'Connell, KA, and Edidin M. A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells. J Immunol 144: 521-525, 1990[Abstract/Free Full Text].

38.   Oliver, FJ, Menissier-de Murcia J, Nacci C, Decker P, Andriantsitohaina R, Muller S, de la Rubia G, Stoclet JC, and de Murcia G. Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice. EMBO J 18: 4446-4454, 1999[Abstract/Free Full Text].

39.   O'Neill, JK, Butter C, Baker D, Gschmeissner SE, Kraal G, Butcher EC, and Turk JL. Expression of vascular addressins and ICAM-1 by endothelial cells in the spinal cord during chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse. Immunology 72: 520-525, 1991[ISI][Medline].

40.   Perkins, ND, Felzien LK, Betts JC, Leung K, Beach DH, and Nabel GJ. Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Science 275: 523-527, 1997[Abstract/Free Full Text].

41.   Picarella, D, Hurlbut P, Rottman J, Shi X, Butcher E, and Ringler DJ. Monoclonal antibodies specific for beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells. J Immunol 158: 2099-2106, 1997[Abstract].

42.   Picker, LJ, and Butcher EC. Physiological and molecular mechanisms of lymphocyte homing. Annu Rev Immunol 10: 561-591, 1992[ISI][Medline].

43.   Read, MA, Neish AS, Luscinskas FW, Palombella VJ, Maniatis T, and Collins T. The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression. Immunity 2: 493-506, 1995[ISI][Medline].

44.   Read, MA, Whitley MZ, Williams AJ, and Collins T. NF-kappa B and I kappa B alpha: an inducible regulatory system in endothelial activation. J Exp Med 179: 503-512, 1994[Abstract].

45.   Ritchie, AJ, Johnson DR, Ewenstein BM, and Pober JS. Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation. Studies with phorbol myristate acetate and staurosporine. J Immunol 146: 3056-3062, 1991[Abstract/Free Full Text].

46.   Rock, KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, and Goldberg AL. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761-771, 1994[ISI][Medline].

47.   Sampaio, SO, Li X, Takeuchi M, Mei C, Francke U, Butcher EC, and Briskin MJ. Organization, regulatory sequences, and alternatively spliced transcripts of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) gene. J Immunol 155: 2477-2486, 1995[Abstract].

48.   Schreiber, E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells (Abstract). Nucleic Acids Res 17: 6419, 1989[ISI][Medline].

49.   Schulze-Osthoff, K, Ferrari D, Riehemann K, and Wesselborg S. Regulation of NF-kappa B activation by MAP kinase cascades. Immunobiology 198: 35-49, 1997[ISI][Medline].

50.   Sikorski, EE, Hallmann R, Berg EL, and Butcher EC. The Peyer's patch high endothelial receptor for lymphocytes, the mucosal vascular addressin, is induced on a murine endothelial cell line by tumor necrosis factor-alpha and IL-1. J Immunol 151: 5239-5250, 1993[Abstract/Free Full Text].

51.   Springer, TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301-314, 1994[ISI][Medline].

52.   Streeter, PR, Berg EL, Rouse BT, Bargatze RF, and Butcher EC. A tissue-specific endothelial cell molecule involved in lymphocyte homing. Nature 331: 41-46, 1988[ISI][Medline].

53.   Takeichi, M. Functional correlation between cell adhesive properties and some cell surface proteins. J Cell Biol 75: 464-474, 1977[Abstract].

54.   Takeuchi, M, and Baichwal VR. Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins. Proc Natl Acad Sci USA 92: 3561-3565, 1995[Abstract].

55.   Taraboletti, G, Roberts D, Liotta LA, and Giavazzi R. Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J Cell Biol 111: 765-772, 1990[Abstract].

56.   Vainer, B, and Nielsen OH. The influence of adhesion molecules in inflammatory bowel diseases. Ugeskr Laeger 159: 3767-3771, 1997[Medline].

57.   Viney, JL, Jones S, Chiu HH, Lagrimas B, Renz ME, Presta LG, Jackson D, Hillan KJ, Lew S, and Fong S. Mucosal addressin cell adhesion molecule-1: a structural and functional analysis demarcates the integrin binding motif. J Immunol 157: 2488-2497, 1996[Abstract].

58.   Weber, C, Negrescu E, Erl W, Pietsch A, Frankenberger M, Ziegler-Heitbrock HW, Siess W, and Weber PC. Inhibitors of protein tyrosine kinase suppress TNF-stimulated induction of endothelial cell adhesion molecules. J Immunol 155: 445-451, 1995[Abstract].

59.   Wesselborg, S, Bauer MKA, Vogt M, Schmitz ML, and Schulze-Osthoff K. Activation of transcription factor NF-kappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J Biol Chem 272: 12422-12429, 1997[Abstract/Free Full Text].

60.   Yang, XD, Sytwu HK, McDevitt HO, and Michie SA. Involvement of beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the development of diabetes in obese diabetic mice. Diabetes 46: 1542-1547, 1997[Abstract].


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