From the
Northwestern University Feinberg School of Medicine, Department of Medicine, Chicago, Illinois 60611,
Veterans Affairs Chicago Health Care System, Lakeside Division, Chicago, Illinois 60611
Received for publication, December 20, 2002
, and in revised form, March 26, 2003.
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ABSTRACT |
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INTRODUCTION |
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The importance of glycoconjugates in inflammation has recently been described by Maly and co-workers (3), who found that mice deficient in the enzymes that generate sialyl-Lex have a partial defect in neutrophil accumulation in response to an acute inflammatory stimulus. If H-2g does exert an effect on cell adhesion, we hypothesized that the 4A11 antigen itself may function as an adhesion molecule or in soluble form induce cell adhesion molecule expression.
Intercellular adhesion molecule (ICAM)-1 belongs to the immunoglobulin superfamily and is constitutively expressed on ECs and other cells. It is up-regulated transcriptionally by proinflammatory cytokines such as TNF- and IL-1 (4). Increased EC expression of ICAM-1 in tissues promotes the recruitment of inflammatory cells expressing its ligand, leukocyte function antigen-1 and CD11a/CD18 (5). Up-regulation of ICAM-1 has been shown in joints and serum of RA patients (6, 7). We have demonstrated previously that ICAM-1 expression on RA synovial tissue ECs is significantly higher compared with normal synovial ECs, suggesting that ICAM-1 promotes leukocyte adhesion and invasion into the RA joint (8, 9).
Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathways have been reported to be involved in many signaling events such as the immune response-induced cytokines and in the actions of growth factors and hormones (10). Specific subtypes of JAK (JAK1, JAK2, JAK3, and Tyk2) are activated by different signals, directing the specificity of response (11). The binding of ligand to its receptor induces assembly of an active receptor complex and consequent phosphorylation of the receptor-associated JAKs. Phosphorylated JAKs lead to the activation of neighboring JAK receptor subunits and several other substrates. JAK-triggered receptor phosphorylation potentially functions via activation of the STAT signaling pathway, Ras-mitogen-activated protein kinase pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway after the interaction of cytokine/interferon receptors with their ligands (12). Aberrations in JAK kinase activity that may lead to derailment of one or more of the above-mentioned pathways disrupt normal cellular responses and result in disease states. Optimal JAK activity is a critical determinant of normal transmission of cytokine and growth factor signals.
Here we show a novel function for Ley/H as a mediator of leukocyte-EC adhesion via induction of EC ICAM-1. Moreover, we demonstrate that the mechanisms underlying Ley/H-induced ICAM-1 expression and cell adhesion include triggering of the JAK-STAT signaling pathway. We propose these pathways are correlated with the development of inflammation in RA.
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EXPERIMENTAL PROCEDURES |
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Cell CultureImmortalized human HMEC-1 cells (HMVECs transformed with SV40 virus) were developed by Dr. Edmund W. Ades of the Centers for Disease Control and Dr. Thomas Lawley of Emory University, Atlanta, GA. Cells were cultured in growth factor complete endothelial basic medium (EBM) supplemented with 5% (v/v) fetal bovine serum (FBS). HMVECs were purchased from BioWhittaker (Walkersville, MD) and maintained in growth factor complete EBM supplemented with 10% (v/v) FBS. HMVECs used in this study were between passage 3 and 12 and did not display discernable phenotypic changes when observed before each assay. HL-60 cells were cultured in RPMI 1640 supplemented with 10% FBS. All cells were maintained at 37 °C, 5% CO2.
Cell Surface Enzyme-linked Immunosorbent Assay for Adhesion Molecule ExpressionHMVECs (1.5 x 104/well) were seeded in 96-well plates in complete EBM with 10% FBS. When 70% confluent, cells were LipofectAMINE-transfected with antisense or sense ODNs to JAK2, PI3K, or Erk1/2 for 4 h. Cells were washed with pre-warmed PBS once, fed with fresh media, and cultured overnight. Transfected or nontransfected cells were allowed to achieve quiescence by incubation in EBM containing 0.1% FBS for 2 h and were pretreated with 50 µM AG-490, 20 µM PD098059, or 20 µM LY294002 for another 2 h. Chemical signaling inhibitors were maintained through the culture period. The pre-treated HMVECs were then exposed to TNF- (1.15 nM) and glycolipids Ley-6 (100 nM), H-5-2 (100 nM), or H-2g (1 nM, 10 nM, 100 nM, or 1000 nM) for the specified times. Media was removed, and cells were fixed in 100 µl of 3.7% formalin, PBS at 37 °C for 15 min. The plates were carefully washed with PBS plus 0.5% Tween 20. Plates were blocked with 1% bovine serum albumin and 30% goat serum. Mouse anti-human ICAM-1, VCAM-1, E-selectin, or P-selectin diluted in blocking buffer were added to the wells, and the plates were incubated at 37 °C for 2 h. Cells were washed twice with PBS plus 0.5% Tween 20, polyclonal goat anti-mouse IgG peroxidase conjugate diluted (1:1000) in blocking buffer was applied, and plates were incubated at 37 °C for 1 h. Cells were then washed twice, and 200 µl/well 3,3',5,5'-tetramethylbenzidine was added. H2SO4 (1 N) was used to stop the reaction after 15 min.
Immunoprecipitation and Western BlottingQuiescent HMEC-1 cells were treated with 100 nM H-2g in the presence and absence of AG-490 for the specified times at 37 °C. An IL-4 (10 ng/ml)/TNF- (1.15 nM) combination was use as the positive control. Alternatively, glycolipids Ley-6 (100 nM) or H-5-2 (100 nM) were used as stimulants. The cells were then lysed in a buffer containing 20 nM HEPES, pH 7.4, 2 nM EDTA, 1 nM
-dithiothreitol, 50 nM glycerophosphate, 1% Triton X-100, 20 µg/ml aprotinin, 1 µg/ml leupeptin, 1 nM sodium orthovanadate, and 400 µM phenylmethylsulfonyl fluoride. For immunoprecipitation, cell lysates containing equal amounts of protein were incubated with appropriate antibodies at 4 °C for 2 h. The antibody-protein complexes were incubated with protein A-Sepharose CL-4B/protein A/G plus agarose beads overnight at 4 °C, and antibody-protein complexes bound to the beads were pelleted at 2000 x g for 2 min. The beads were washed three times with lysis buffer and once with phosphate-buffered saline and resuspended in Laemmli sample buffer. The samples were resolved on 10% SDS-polyacrylamide gels. Western blots were performed as described previously (14).
Pathway Profiling Luciferase Reporter AssayThe JAK/STAT signaling profile system from Clontech was employed for this study. The plasmid containing a STAT3 element tagged with a luciferase expression gene, pSTAT3-TA-Luc, an interferon- activation sequence (GAS) tagged with a luciferase expression gene, pGAS-TA-Luc, or an interferon-stimulated response element (ISRE) tagged with a luciferase expression gene, pISRE-TA-Luc, were transfected into HMEC-1s. HMEC-1s were seeded at 2.50 x 105 cells/well in 35-mm 6-well plates and cultured overnight to 75% confluence. Thirty minutes before transfection, the media was changed to fresh complete EBM media. One µg of pSTAT3-TA-Luc, pGAS-TA-Luc, or pISRE-TA-Luc or pTA-Luc combined with 0.2 µg of green fluorescent protein (GFP) expression plasmid p-EGFP-N1 was added to Eppendorf tubes with 14.5 µl of 2 M calcium chloride and double-distilled H2O to a total volume of 100 µl (DNA-Ca2+ mixture). Hepes-buffered saline solution (2x, 100 µl) in Eppendorf tubes was carefully and slowly vortexed while adding 100 µl of DNA-Ca2+ mixture. The resultant mixture (200 µl) was placed at room temperature for 20 min and then added dropwise onto the cells. The plate was incubated at 37 °C for 4 h in an incubator gassed with 5% CO2. The cells were then washed with pre-warmed PBS once and fed with fresh medium. Fourteen h later the cells were stimulated with 100 nM H-2g or 1 ng/ml recombinant human IL-6 (positive control) for 6 h. After stimulation, the cells were washed twice with PBS, 200 µl of cell lysis buffer was added to cells, and the cells were shaken at 4 °C for 20 min. The cells was scraped into 1.5-ml Eppendorf tubes and spun at 14,000 rpm at room temperature for 1 min to remove cell debris. Forty µl of cell extract was added to the wells, and 50 µl of substrate A and 50 µl of substrate B were added to the plate, and the luciferase activities were measured in a luminometer. GFP expression was measured by fluorescence-activated cell sorting, and efficiency was calculated by the percentage of positive expression.
Preparation of Cytoplasmic and Nuclear Extracts for Western BlottingQuiescent HMEC-1 cells treated with 100 nM H-2g for 5, 10, and 30 min were scraped off the plate, washed with PBS twice at 4 °C, resuspended in 200 µl of buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol, 1.5 µg/ml leupeptin, 20 mM benzamidine, 1.5 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride), and put on ice for 10 min. Two µlof20% Nonidet P-40 was added, the mixture was vortexed for 10 s and pelleted for 30 s, the supernatant (cytoplasmic extract) was collected and stored at70 °C for Western blotting. The nuclear extracts were resuspended in 50 µl of buffer C (20 mM Hepes, pH7.9, 1.5 mM MgCl2, 420 mM NaCl, and 0.5 mM dithiothreitol, 25% glycerol, 0.2 mM EDTA, 1.5 µg/ml leupeptin, 20 mM benzamidine, 1.5 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride), rocked for 15 min in 4 °C, and pelleted at 14,000 rpm for 5 min. The supernatants containing the nuclear extract were stored at70 °C for Western blotting.
Immunofluorescence AssayHMEC-1 cells were seeded on sterile slide coverslips in 6-well plates overnight and allowed to achieve quiescence for 6 h in EBM with 0.1% FBS. Quiescent HMEC-1 cells were stimulated for the indicated times with H-2g (100 nM) or with PBS as a control. Cells were then washed once with PBS and fixed in20 °C cold methanol for 10 min. Rabbit anti-phospho-STAT3 (1:200 in PBS) as primary antibody was added dropwise on the coverslips. The plates were kept at 37 °C for 45 min and washed with 3x PBS. Goat anti-rabbit IgG-fluorescein isothiocyanate antibody (1:200 in PBS) as the secondary antibody was added dropwise on the coverslips. The plates were kept in 37 °C for another 45 min, washed with 3x PBS, and mounted with fluorescence mounting medium on slides. The slides were kept in20 °C in the dark for fluorescence detection. A Zeiss LSM510 laser-scanning confocal microscope was used to detect the translocation of phospho-STAT3.
Cell Adhesion Assay96-well plates (Dynex Technologies, Billingshurst, UK) were placed under UV light for 30 min and coated with 0.02% sterile gelatin. HMEC-1s were plated into the 96-well plates at a concentration of 5 x 104 cells/well and incubated overnight at 37 °C. Cells were stimulated with or without 100 nM H-2g or 1.15 nM TNF- (positive control) for 6 h at 37 °C, 5% CO2. HL-60 cells were washed twice with PBS and adjusted to 5 x 106 cells/ml in RPMI 1640 without FBS. HL-60 cells were incubated with 5 µM calcAM for 30 min at 37 °C (calcAM is a fluorescent dye that is incorporated into living cells). HL-60 cells were then washed twice with pre-warmed RPMI 1640, 1% penicillin/streptomycin to remove unincorporated dye and then adjusted to 2.5 x 106 cells/ml. Labeled HL-60 cells (2.5 x 105/100 µl) were added to each well. Cells were incubated for 1 h at 37 °C and then washed very carefully four times with pre-warmed PBS. Fluorescence was determined using a fluorescent plate reader (SpectraMAX Gemini, Molecular Devices, Sunnyvale, CA) set to 495 nm for excitation and 517 nm for emission. Adhesion was automatically expressed in relative fluorescence units. For better comparisons of the differentially treated groups and to avoid the use of relative fluorescence units, the adhesion of HL-60 cells to non-stimulated HMEC-1 cells was chosen as a reference. The adhesion index was, therefore, defined as the ratio of adhesion of HL-60 to stimulated HMEC-1 (in relative fluorescence units) to adhesion of HL-60 to unstimulated HMEC-1 (in relative fluorescence units). For experiments in which the adhesion to HMEC-1 cells was blocked by antibodies, cells were treated with mAbs to ICAM-1, VCAM-1, P-selectin, E-selectin, or isotype mouse-matched control (2.5 µg/ml) for 1 h at 37 °C and 5% CO2. HL-60 cells were labeled with calcAM and added to the HMEC-1 cells as described above (14).
Statistical AnalysisFor statistical evaluation of all experiments, Student's t tests were performed. Asterisks indicate significantly different values (*, p < 0.05).
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RESULTS |
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H-2g Stimulates JAK2 Kinase Activity in HMEC-1sThe JAK2 inhibitor AG-490 inhibited HMVEC ICAM-1 expression induced by H-2g, suggesting that JAK kinases might play a critical role in the signaling triggered by H-2g. To examine the contributions of the different JAK kinases to H-2g-induced EC ICAM-1 expression, we immunoprecipitated JAKs from HMEC-1s with antibodies directed against JAK1, JAK2, Tyk2, or JAK3 after H-2g stimulation. To assure that all JAKs were activated, an IL-4 (10 ng/ml)/TNF- (1.15 nM) combination was used as the positive control. We probed for tyrosine-phosphorylated proteins with 4G10 (anti-phosphotyrosine mAb). The blots were stripped and re-probed for nonphosphorylated protein. We observed that unlike other JAKs and Tyk2, JAK2 was tyrosine-phosphorylated 1 min after treatment with H-2g (Fig. 2A). H-2g-activated JAK2 was confirmed by Western blot analysis of H-2g-stimulated cell lysates probed with a phospho-specific anti-JAK2 antibody, which demonstrated maximal phosphorylation of JAK2 at 1 min (8.3 ± 1.6-fold increase) (Fig. 2B). Purified native Ley/H antigen up-regulated JAK2 in the same manner as H-2g did (Fig. 2C).
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Phospho-JAK2 Kinase Up-regulates Phospho-STAT3, Which Translocates Quickly into the NucleusWe next examined which STATs were downstream of the H-2g-induced endothelial JAK2 pathway. We employed a signaling pathway profiling system. As shown in Fig. 3A, STAT3, but not STAT1 or STAT2, are involved in the phospho-JAK2 pathway. To determine whether tyrosine phosphorylation of STAT proteins in response to treatment with H-2g was accompanied by translocation into the nucleus, Western blot analyses of nuclear and cytoplasmic fractions of H-2g-treated HMEC-1 cells was performed. H-2g induced nuclear translocation of STAT3 in 5 min, an increase that was sustained for 30 min, whereas no discernible change was observed in the protein levels in the cytoplasmic fraction, suggesting that all STAT3 activated by H-2g was translocated to the nucleus quickly and had no effect on the phospho-STAT3 level in the cytoplasmic fraction. The total cell lysates with non-fractionated cells were also blotted with phospho-STAT3 antibody. The results are shown in Fig. 3B. STAT3 activated by H-2g translocated to nucleus in 5 min, and the effect was still present but diminished at 30 min (Fig. 3B). To confirm this result, we employed an immunofluorescence assay. We labeled the H-2g stimulated or non-stimulated HMEC-1s with anti-phospho-STAT3 (1:200 in PBS) as primary antibody and the goat anti-rabbit antibody conjugated with fluorescein isothiocyanate (1:200 in PBS) as secondary antibody. A Zeiss LSM510 laser-scanning confocal microscope was used to detect the translocation of phospho-STAT3. Phospho-STAT3 was activated and translocated to the nucleus in 5 min, which is consistent with our previous Western blot results. Activated STAT3 entered the nucleus and persisted for 30 min (Fig 3C). STAT3 activation is dependent on phosphorylation of JAK2. Results indicate that the period of STAT3 activation and transient translocation is shorter than JAK2 activation. Similar results were shown in several other studies (16, 17). Gangliosides induce brain microglial cell nitric-oxide synthase, ICAM-1, and monocyte chemotactic protein-1 expression through JAK-STAT pathways; JAK2 activation persisted for 60 min, whereas STAT3 activation diminished after 30 min. Thrombin regulates vascular smooth muscle cell proliferation and heat shock protein expression through the JAK-STAT pathway in the same manner. It is likely that STAT3 activation needs a certain level of phospho-JAK2. Although JAK2 activation was sustained for an hour, that activation level after 30 min may not reach the requirement for STAT3 activation. Thus, the STAT3 activation diminished in 30 min.
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Phospho-JAK2 Kinase Is Upstream of the Erk1/2 and PI3K-Akt PathwaysJAK2 has been suggested to be upstream of the mitogen-activated protein kinase and PI3K-Akt cascades (12, 18, 19), and our data showed that inhibitors of Erk1/2 and PI3K-Akt partially block HMVEC ICAM-1 expression. To examine whether JAK2, Erk1/2, and PI3K-Akt were three independent pathways for H-2g signaling or whether they were contingent on one another, we performed Western blots to define these three pathways. Phosphorylated proteins were measured in Western blots using phospho-specific antibodies. H-2g activated Erk1/2 with peak expression at 5 min. We detected phospho-Akt after H-2g stimulation and found Akt was activated in the same manner as Erk1/2 (Fig. 4A). We examined whether phospho-Erk1/2 and phospho-Akt induced by H-2g were JAK2-related. AG-490 (50 µM) markedly inhibited H-2g-induced activation of Erk1/2 and Akt in Western blots. However, no detectable change was observed in the steady-state Erk1/2 and Akt protein levels after treatment with H-2g either in the presence or absence of AG-490, PD98059, or LY294002 (Fig. 4B).
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H-2g Induces the Adhesion of HL-60 Cells to HMEC-1sThe functional significance of the adhesion molecules induced by H-2g was evaluated with an adhesion assay in which HMEC-1s cultured in 96-well plates were stimulated with H-2g or positive control TNF- and co-incubated with myeloid HL-60 cells labeled with calcAM. HL-60s are known to express high amounts of very late antigen-4 and lymphocyte function-associated antigen-1, two known ligands for ICAM-1 (20, 21). To allow comparison of the different experimental conditions at each incubation time point, we used an adhesion index. After stimulation with H-2g (100 nM) for 6 h, the adhesion index was increased 1.7-fold as compared with nonstimulated HMEC-1s. HL-60 cell adhesion to HMEC-1s was induced by as little as 10 nM H-2g (Fig. 5A). We found that this effect completely declined after 24 h of stimulation with 100 nM H-2g. Interestingly, we observed similar kinetics when HMEC-1s were stimulated with 1.15 nM TNF-
(Fig. 5B). The JAK2 inhibitor (AG-490), the Erk1/2 inhibitor (PD98059), or the PI3K inhibitor (LY294002) inhibited HL-60 cell adhesion to HMEC-1s (Fig. 5C). Antibody to ICAM-1, but not to VCAM-1, E-selectin, or P-selectin, inhibited H-2g (100 nM)-induced HL-60 cell adhesion to HMEC-1s thoroughly, which implies that H-2g-induced HL-60-HMEC-1 adhesion is mediated by ICAM-1 (Fig. 5D).
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Antisense ODNs of Erk1/2 and PI3K Block HMVEC ICAM-1 Expression and HL-60 Cell Adhesion to HMEC-1sWe used inhibitors to the signaling proteins JAK2, Erk1/2, and PI3K to define their pathways. However, these inhibitors may not be completely specific. To confirm the involvement of these pathways in H-2g signaling, antisense ODNs targeting JAK2, Erk1/2, and PI3K and corresponding control sense ODNs were used to LipofectAMINE-transfect HMEC-1s, with subsequent H-2g (100 nM) stimulation. Levels of pan-JAK2/phospho-JAK2, pan-Erk1/2/phospho-Erk1/2, and pan-Akt/phospho-Akt expression were determined by Western blots of total cell lysates. All of the pan-JAK2/phospho-JAK2, Erk1/2/phospho-Erk1/2, and pan-Akt/phospho-Akt expression was blocked by transfection of antisense ODNs to JAK2, Erk1/2, or PI3K (Fig. 6A). Furthermore, we examined ICAM-1 expression and HL-60 adhesion after ODN transfection. ICAM-1 expression was inhibited by antisense ODNs directed against JAK2, PI3K, and Erk1/2 (Fig. 6B). Consistent with the inhibition of ICAM-1 expression, JAK2, Erk1/2, and PI3K antisense ODNs inhibited HL-60 cell adhesion to HMEC-1s (Fig. 6C).
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DISCUSSION |
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In the current study we found that H-2g induced HMVEC ICAM-1 expression in a concentration-dependent manner. The functional association between ICAM-1 expression and adhesion prompted us to examine H-2g-induced leukocyte adhesion. Consistent with a connection between H-2g and induction of ICAM-1 expression, we found that H-2g treatment of HMEC-1s increased HL-60 cell adhesion to HMEC-1s. In addition, this effect was blocked by the JAK2 inhibitor AG-490. These results provide further evidence for involvement of JAK-STAT inflammatory signaling in H-2g-induced HL-60 cell adhesion. Taken together, these results suggest that ICAM-1 may be induced in response to H-2g through JAK-STAT signaling. We next examined other signaling events that may be associated with JAK2-STAT3 activation in H-2g-treated ECs. Having previously observed that Erk1/2 and PI3K were activated by H-2g, we investigated whether H-2g-stimulated JAK2-STAT3 signaling was linked to activation of Erk1/2 and PI3K, key pathways in inflammation. We found that H-2g-induced activation of JAK2 resulted in phosphorylation of Erk1/2 and PI3K-Akt, suggesting cross-talk between JAK2 and Erk1/2 or PI3K-Akt pathways. Interestingly, inhibition of JAK2 by AG-490 resulted in complete inhibition of Erk1/2 and Akt activation with H-2g stimulation, indicating Erk1/2 and PI3K-Akt pathways are downstream of JAK2-STAT3. Because PD98059 and LY294002 and also the antisense ODNs directly against Erk1/2 and PI3K only partially reduce HMVEC ICAM-1 expression induced by H-2g, but AG490 (-cyano-(3,4-dihydroxy)-N-benzylcinnamide) completely inhibits these effects, it is likely that activation of Erk1/2 and PI3K-Akt is downstream of JAK2 activation (Fig. 7). As shown in Fig. 7, the JAK2 antisense ODN did not completely inhibit HMVEC ICAM-1 expression induced by H-2g. Because that the ODN is more specific, we suggest that other pathways, such as G-protein coupled receptors, may be involved in H-2g signaling.
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ICAM-1 is expressed in human ECs, monocytes, and lymphocytes and mediates leukocyte binding to ECs (2426). It is broadly expressed in synovium from RA patients (9, 27, 28). These findings strongly suggest that ICAM-1 plays an important role in inflammation such as RA, particularly in leukocyte-EC interactions. Cytokines such as TNF-, IL-1
, and interferon-
increase ICAM-1 expression in a variety of cell lines, and many of the induced signals are through JAK2 and STAT1. Activated STAT1 dimers are associated with the promoter binding domain of transcription factors such as activator protein-1 and nuclear factor
B to regulate ICAM-1 expression (2931). Ley/H-induced ICAM-1 expression is different in that STAT3 is involved in the pathway instead of STAT1. Kinetics of H-2g-induced ICAM-1 are similar to those observed with TNF-
treatment.
Ilo Jou and co-workers (17) also found that ICAM-1 was induced by activation of the JAK2-STAT3 pathway. They employed a class of anionic glycosphingolipids-gangliosides, a rich component on neuronal cell membranes, in their studies. The gangliosides acted as microglial activators and were, thus, likely to participate in neuronal inflammation. JAK2-STAT3 is the pathway for this signaling. Gangliosides stimulated nuclear factor binding to specific DNA sequences, which are known to be STAT-binding sites. Similar to our study, gangliosides rapidly activated JAK2, induced phosphorylation of STAT3, and increased transcription of inflammation-associated genes such as ICAM-1 and monocyte chemotactic protein-1, which are reported to contain STAT binding elements in their promoter regions. Furthermore, AG490 (-cyano-(3,4-dihydroxy)-N-benzylcinnamide) markedly reduced activation of Erk1/2, indicating that Erks act downstream of JAK-STAT signaling during microglial activation. Other evidence that ICAM-1 could be induced by oligosaccharides was obtained by M. Innocenti and co-workers (32). They found lipopolysaccharides of Helicobacter pylori bacteria up-regulated the adhesion molecules ICAM-1 and VCAM-1 on ECs, thereby potentially contributing to neutrophil recruitment. In addition, one or several unidentified proteins that act via nuclear factor
B activation seem to induce EC cell adhesion molecule expression and activation. Interestingly, the oligosaccharide of lipopolysaccharide from H. pylori is structurally very similar to Ley/H.
In conclusion, our findings suggest that H-2g rapidly triggers JAK-STAT inflammatory signaling, resulting in HMEC-1/HMVEC activation. Our studies indicate that H-2g-stimulated JAK-STAT signaling results in transcription of the inflammatory mediator ICAM-1. We have previously shown that the Ley/H antigen plays a important role in angiogenesis and inflammation in RA. Our current data provide significant new information regarding the molecular mechanisms underlying Ley/H-induced inflammation, and such knowledge will assist in the better understanding the pathogenesis of RA.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: 303 E. Chicago Ave., Ward Bldg. 3-315, Chicago, IL 60611. Tel.: 312-503-1963; Fax: 312-503-0994; E-mail: ae-koch{at}northwestern.edu.
1 The abbreviations used are: mAb, monoclonal antibody; H-2g, 2-fucosyllactose; TNF, tumor necrosis factor; IL, interleukin; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; EC, endothelial cell; HMVEC, human dermal microvascular endothelial cell; HMEC-1, human microvascular endothelial cell line-1; JAK, Janus kinase; STATs, signal transducers and activators of transcription; Erk, extracellular signal-regulated kinase; EBM, endothelial basal medium; ODN, oligodeoxynucleotide; Lex, Lewisx; Ley, Lewisy; FBS, fetal bovine serum; ISRE, interferon-stimulated response element; RA, rheumatoid arthritis; PI3K, phosphatidylinositol 3-kinase; calcAM, calcein-acetoxymethyl ester; PBS, phosphate-buffered saline; GFP, green fluorescent protein.
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REFERENCES |
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