A Novel Function for a Glucose Analog of Blood Group H Antigen as a Mediator of Leukocyte-Endothelial Adhesion via Intracellular Adhesion Molecule 1*

Kui Zhu {ddagger}, M. Asif Amin {ddagger}, Michael J. Kim {ddagger}, Kenneth J. Katschke, Jr. {ddagger}, Christy C. Park {ddagger} and Alisa E. Koch {ddagger} § 

From the {ddagger} 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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 4A11 antigen is a unique cytokine-inducible antigen up-regulated on rheumatoid arthritis synovial endothelium compared with normal endothelium. In soluble form, this antigen, Lewisy-6/H-5-2 (Ley/H), or its glucose analog, 2-fucosyllactose (H-2g), mediates angiogenesis. The Ley/H antigen is structurally related to the soluble E-selectin ligand, sialyl Lewisx, and is selectively expressed in skin, lymphoid organs, thymus, and synovium, suggesting that it may be important in leukocyte homing or adhesion. In the present study, we used H-2g as a functional substitute to demonstrate a novel property for Ley/H antigen in inducing leukocyte-endothelial adhesion. H-2g significantly enhanced the expression of human dermal microvascular endothelial cells (HMVECs) intercellular adhesion molecule-1 (ICAM-1), but not vascular cell adhesion molecule-1, E-selectin, and P-selectin. Immunoprecipitation and Western blotting showed glycolipids Ley-6, H-5-2, or the glucose analog H-2g quickly activated human microvascular endothelial cell line-1 (HMEC-1) Janus kinase 2 (JAK2) and that the JAK2 inhibitor, AG-490, completely inhibited HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Use of a JAK/signal transducer and activator of transcription (STAT) profiling system confirmed that H-2g selectively activated STAT3 but not STAT1 and STAT2. AG-490 inhibited H-2g-induced Erk1/2 and PI3K-Akt activation, suggesting that JAK2 is upstream of the Erk1/2 and PI3K-Akt pathways. Furthermore, the JAK2 inhibitor AG-490, the Erk1/2 inhibitor PD98059, or the phosphatidylinositol 3-kinase inhibitor LY294002 or antisense oligodeoxynucleotides directed against JAK2, Erk1/2, or phosphatidylinositol 3-kinase blocked H-2g-induced HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Hence, H-2g signals through JAK2 and its downstream signal transducers STAT3, Erk1/2, and phosphatidylinositol 3-kinase result in ICAM-1 expression and cell adhesion. Potential treatment strategies through the inhibition of JAK-dependent pathways to target H-2g signals may provide a useful approach in inflammation-driven diseases like rheumatoid arthritis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Monoclonal antibody (mAb)1 4A11 was developed in our laboratory using cells from rheumatoid arthritis (RA) patient joint synovial tissue. Later we identified the 4A11 antigen as an endothelial cell (EC) molecule, Lewisy-6/H-5-2 (Ley/H), which is structurally related to the soluble E-selectin ligand, sialyl Lewisx (Lex) (1, 2). H structures are primarily known for their role as blood group antigens. Ley/H is mainly expressed on some epithelial cells and on the EC membrane. The antigen is selectively expressed on ECs of skin, lymphoid organs, thymus, and synovium. We also found that the Ley/H antigen was up-regulated in the skin of a human poison ivy dermatitis model before the ingress of inflammatory cells (1). Moreover, in human RA synovial tissue, the Ley/H endothelial antigen is up-regulated compared with normal synovial tissue. In soluble form, Ley/H is increased in inflammatory RA compared with non-inflammatory osteoarthritis synovial fluids. In vitro cytokines such as interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), which are important in inflammatory states, up-regulate the expression of Ley/H on ECs. We have identified 2-fucosyllactose, a readily available glucose analog of H-5-2, termed H-2g, as a potent mediator of angiogenesis, thus potentially contributing to the growth and proliferation of the RA synovial pannus. Taken together, our results suggest that Ley/H may contribute to cellular ingress or adhesion in inflammatory states like RA.

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-{alpha} 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—H-2g, the Src inhibitor PP2, the PI3K inhibitor LY294002, and the Erk1/2 inhibitor PD98059 were purchased from Calbiochem. The JAK2 inhibitor AG-490 was purchased from LC Laboratories, Woburn, MA. Calcein-acetoxymethyl ester (calcAM) was from Molecular Probes, Eugene, OR. Antibodies used were as follows. Anti-JAK1, JAK2, and Tyk2 antibodies were from Upstate Biotechnology, Inc., Lake Placid, NY. Anti-phospho-STAT1, phospho-JAK2 and anti-phosphotyrosine (4G10, IgG 2b) mAbs were from BIOSOURCE International, Camarillo, CA. Anti-phospho-STAT2, phospho-STAT3, and anti-JAK3 were from Cell Signaling, Beverly, MA. Anti-phosphospecific and total Akt and Erk1/2 were from New England Biolabs, Beverly, MA. Anti-ICAM-1 (mAb 720), -E-selectin (BBA16), -P-selectin (BBA30), and -VCAM-1 (BBA5) were from R&D Systems, Minneapolis, MN. Goat anti-rabbit IgG-fluorescein isothiocyanate antibody was from Sigma-Aldrich. Protein A-Sepharose CL-4B/protein A/G plus agarose were purchased from Amersham Biosciences. The Mercury JAK/STAT pathway profiling system was from Clontech Laboratory, Palo Alto, CA. The sequences of the oligodeoxynucleotide (ODNs) employed in this study were as follows: JAK2 antisense, AAG GCA GGC CAT TCC CAT; JAK2 sense, ATG GGA ATG GCC TGC CTT; PI3K antisense, GTA CTG GTA CCC CTC AGC ACT CAT; PI3K sense, ATG AGT GCT GAG GGG TAC CAG TAC; Erk1/2 antisense, GCC GCC GCC GCC GCC AT; Erk1/2 sense, ATG GCG GCG GCG GCG GC (13). The corresponding sense ODN was used as a control for each antisense ODN. The ODNs were synthesized and purified by the Northwestern University Biotechnology Laboratory and modified with phosphorothioate. LipofectAMINE was from Invitrogen. H-5-2 and Ley-6 glycolipids, the native blood group antigens, were purified by Annika E. Bäcker, Department of Laboratory Medicine, Sahlgrenska Hospital, Goteborg, Sweden, stock solution 60 mM, dissolved in ethanol. Recombinant human TNF-{alpha} (specific activity 1.3 x107 units/mg) was obtained from Upjohn Co., Kalamazoo, MI and human recombinant IL-4 (specific activity 5 x 106 units/mg) was from Peprotech, Rocky Hill, NJ.

Cell Culture—Immortalized 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 Expression—HMVECs (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-{alpha} (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 Blotting—Quiescent 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-{alpha} (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 {beta}-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 Assay—The 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-{gamma} 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 Blotting—Quiescent 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 at–70 °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 at–70 °C for Western blotting.

Immunofluorescence Assay—HMEC-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 in–20 °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 in–20 °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 Assay—96-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-{alpha} (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 Analysis—For statistical evaluation of all experiments, Student's t tests were performed. Asterisks indicate significantly different values (*, p < 0.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
H-2g Induces HMVEC ICAM-1 Expression—Our previous work has shown that molecules such as VCAM-1 and E-selectin expressed on ECs may be shed. The shed molecules in turn bind adjacent ECs via their respective ligands and exert direct cell effects on ECs or leukocytes mediating inflammation and angiogenesis (15). Ley/H is expressed on the EC surface and also circulates in soluble form. Because of the selective expression of Ley/H in organs often targeted by inflammation, such as the joint synovial tissue in RA, we hypothesized that Ley/H might be involved in inflammatory cell adhesion. To examine the hypothesis, we studied HMVEC cell adhesion molecule expression in response to glycolipids Ley-6 (100 nM), H-5-2 (100 nM), or H-5-2 glucose analog H-2g using a cell based enzyme-linked immunosorbent assay. We examined E-selectin, P-selectin, VCAM-1, and ICAM-1 expression. Ley-6, H-5-2, and H-2g induced ICAM-1 expression. Ley-6 had the most potent effect. Because H-2g acted similarly to the native glycolipid antigens, we used H-2g in subsequent experiments. We examined H-2g-induced HMVEC ICAM-1 expression after 1, 2, 4, 6, 8, 12 and 24 h of stimulation with 1, 10, 100, and 1000 nM H-2g. We found that H-2g induced ICAM-1 expression in a concentration-dependent manner beginning at 10 nM (Fig. 1A). The peak time of H-2g-induced HMVEC ICAM-1 expression was 6 h. In contrast, VCAM-1, E-selectin, and P-selectin were not induced (Fig. 1B). PBS was used as a negative control, and 1.15 nM TNF-{alpha} was used as a positive control. We employed the Erk1/2 inhibitor (PD98059), the PI3K inhibitor (LY294002), the Src inhibitor (PP2), and the JAK2 inhibitor (AG-490) before (6 h) and during the experiment and found that the Erk1/2 inhibitor, the PI3K inhibitor, and the JAK inhibitor blocked ICAM-1 expression induced by H-2g. AG-490 had the most dramatic effect among these three inhibitors and completely inhibited HMVEC ICAM-1 expression induced by H-2g, suggesting that JAK2 was a key signaling intermediate (Fig. 1C).



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FIG. 1.
Ley-6, H-5-2, and H-2g induce HMVEC ICAM-1 expression. A, quiescent HMVECs incubated with 0.1% FBS were stimulated for 6 h with the purified glycolipids Ley-6 (100 nM), H-5-2 (100 nM), the H-5-2 glucose analog H-2g (100 nM), or TNF-{alpha} (1.15 nM); PBS served as a negative control. Both Ley-6 and H-5-2 induced HMVEC ICAM-1 expression. Aside from TNF-{alpha}, Ley-6 was the most potent stimulus. Beginning at 10 nM, H-2g-induced HMVEC ICAM-1 expression. The optimal concentration of H-2g was 100 nM. B, H-2g failed to induce VCAM-1, P-selectin, and E-selectin expression. The peak expression of HMVEC ICAM-1 induction occurred at 6 h. C, the JAK kinase inhibitor AG-490 (50 µM), the Erk1/2 kinase inhibitor PD98059 (PD, 20 nM), or the PI3K inhibitor LY294002 (LY, 20 nM) inhibited HMVEC ICAM-1 expression. AG-490 completely inhibited the effect, whereas PD or LY inhibited the effect by 28 and 39%, respectively (p < 0.05). Data from four separate experiments are presented as the mean ± S.E.; n is the number of experiments.

 

H-2g Stimulates JAK2 Kinase Activity in HMEC-1s—The 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-{alpha} (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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FIG. 2.
HMEC-1 JAK2 kinase is activated by H-2g. A, quiescent HMEC-1s were stimulated with H-2g (100 nM) for 1 min. Equalized (0.75 mg/ml) protein from cell lysates was immunoprecipitated (IP) with anti-JAK1, JAK2, Tyk2, or JAK3 antibodies, and Western blots were performed using phosphotyrosine (4G10) mAb (upper panel). Only JAK2 was activated by H-2g. Blots were stripped and reprobed with anti-JAK1, JAK2, Tyk2, JAK3 antibodies to assure the equal loading of proteins (lower panel). Results are representative of four assays. B, quiescent HMEC-1s were stimulated for the indicated times with H-2g (100 nM). Cell lysates were blotted for phospho-JAK2 (*p-JAK2). Blots were stripped and reprobed with non-phosphorylated JAK2 (pan-JAK2) antibody. Blots were scanned and analyzed for quantification with the UN-SCAN-IT software. Band intensities for phospho-JAK2 were normalized to the corresponding band intensities for pan-JAK2. Data from four separate experiments were presented as the mean ± S.E.; NS, nonstimulated; n, number of experiments. C, quiescent HMEC-1s were stimulated for the indicated times with Ley-6 or H-5-2. Cell lysates were blotted for phospho-JAK2 (*p-JAK2). Blots were stripped and reprobed with non-phosphorylated JAK2 (pan-JAK2) antibody. Results are representative of four assays.

 

Phospho-JAK2 Kinase Up-regulates Phospho-STAT3, Which Translocates Quickly into the Nucleus—We 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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FIG. 3.
Phospho-STAT3 is activated by phospho-JAK2 and translocates to the nucleus quickly. A, pSTAT3-TA-Luc (1 µg), pGAS-TA-Luc (1 µg), pISRE-TA-Luc (1 µg), or pTA-Luc (1 µg) were co-transfected with 0.2 µg of p-EGFP-N1 into HMEC-1 cells as described under "Experimental Procedures," and GFP expression levels were measured by fluorescence-activated cell sorting. The luciferase activities induced by recombinant human IL-6 and H-2g represent the activation of GAS (STAT1/STAT1), IRES (STAT1/STAT2), and STAT3 (STAT3/STAT3), which are three elements downstream of JAK pathways. Results were normalized with the amount of GFP for consistent transfection efficiency. Although recombinant human IL-6 (1 ng/ml) activated all three STATs, H-2g (100 nM) only activated the STAT3 pathway. B, quiescent HMEC-1 cells were stimulated for the indicated times with H-2g (100 nM). Cell lysates were separated into nuclear and cytoplasmic fractions and run on SDS-PAGE gels. Gels were blotted for phospho-STAT3 (*p-STAT3). The cell lysates were also probed with pan-STAT3 antibody. All blots were scanned and analyzed for quantification with UN-SCAN-IT software. Band intensities for phospho-STAT3 were normalized to pan-STAT3. H-2g-induced nuclear translocation of STAT3 at 5 min, an increase that was sustained for 30 min, whereas no discernible change was observed in the protein levels in the cytoplasmic fraction of phospho-STAT3. Data from four separate experiments are presented as the mean ± S.E.; NS, nonstimulated; n, number of experiments. C, HMEC-1 cells were seeded on sterile slide coverslips in 35-mm 6-well plates overnight and allowed to achieve quiescence for 6 h in EBM complete medium with 10% FBS. Quiescent HMEC-1 cells were stimulated for the indicated times with H-2g (100 nM). Cells were fixed and stained with {alpha}-phospho-STAT3 (1:200 in PBS) as the primary antibody and goat anti-rabbit-fluorescein isothiocyanate as the secondary antibody. At time 0, no nuclear phospho-STAT3 was detectable. After 5 min of H-2g stimulation, phospho-STAT3 reached its peak expression, and after 30 min of stimulation, nuclear phospho-STAT3 was still detectable.

 

Phospho-JAK2 Kinase Is Upstream of the Erk1/2 and PI3K-Akt Pathways—JAK2 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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FIG. 4.
Erk1/2 and Akt are activated by H-2g inhibition by AG-490, PD98059, and LY294002. A, quiescent HMEC-1 cells were stimulated for 5, 10, and 30 min with H-2g (100 nM). Cell lysates were blotted for phospho-Erk1/2 (*p-Erk1/2) and phospho-Akt (*p-Akt). The same blots was stripped and reprobed for their steady-state protein separately. Blots were scanned and analyzed for quantification with UN-SCAN-IT software. Band intensities for phospho-Erk1/2 and phospho-Akt were normalized to the corresponding band intensities for their steady-state protein. All their peak expression times were 5 min, and the activation was sustained for more than 30 min. B, the JAK2 inhibitor (AG-490), the Erk1/2 inhibitor (PD98059), and the PI3K inhibitor (LY294003) were added separately to quiescent HMEC-1s 2 h before H-2g (100 nM) stimulation. Cell lysates were blotted for phospho-Erk1/2 and phospho-Akt. The same blots were stripped and reprobed for pan-Erk1/2 or pan-Akt. Blots were scanned and analyzed for quantification with UN-SCAN-IT software. Band intensities for phospho-Erk1/2 and phospho-Akt were normalized to the corresponding band intensities for their total protein. AG-490 inhibited activation of the PI3K effector Akt. PD98059 only blocked Erk1/2 activation, and LY294002 only blocked Akt activation. Data from four separate experiments are presented as the mean ± S.E.; *p, phospho; NS, nonstimulated; n, number of experiments. DMSO, Me2SO.

 

H-2g Induces the Adhesion of HL-60 Cells to HMEC-1s—The 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-{alpha} 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-{alpha} (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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FIG. 5.
H-2g-induced HL-60 adhesion to HMEC-1s is mediated by JAK2-STAT3, Erk1/2, and PI3K-Akt pathways. A, quiescent HMEC-1 cells were treated with H-2g for 6 h. HL-60 cells (2.5x 106 cells/ml, 100 µl) labeled with calcAM were added and incubated for 1 h. Plates were carefully washed four times with PBS, and fluorescence was determined by measurement with a fluorescent plate reader set to 495 nm for excitation and 517 nm for emission. Adhesion was expressed in relative fluorescence units. For better comparisons of the differentially treated groups and to avoid the use of relative fluorescence units, we use an adhesion index, which is 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). Beginning at 10 nM, H-2g induces HL-60 adhesion to HMEC-1s to a maximum of a 1.7-fold increase. TNF-{alpha} was used as a positive control. B, HL-60 cell adhesion to HMEC-1s was diminished at 24 h of H-2g incubation with the same kinetics as TNF-{alpha}. C, the JAK2 inhibitor (AG-490), the Erk1/2 inhibitor (PD98059), and the PI3K (LY294002) were added separately to quiescent HMEC-1s 2 h before H-2g (100 nM) stimulation and were maintained during the experiment. All inhibitors blocked adhesion to different extents. D, anti-ICAM-1 mAb (2.5 µg/ml) inhibited HL-60 cell adhesion to HMEC-1s by 88%. Data from four separate experiments are presented as the mean ± S.E.; {alpha}, antibody; n, number of experiments.

 

Antisense ODNs of Erk1/2 and PI3K Block HMVEC ICAM-1 Expression and HL-60 Cell Adhesion to HMEC-1s—We 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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FIG. 6.
Antisense JAK2, Erk1/2, and PI3K ODNs block pan-JAK2/phospho-JAK2, pan-Erk1/2/phospho-Erk1/2, and pan-Akt/phospho-Akt expression, decrease HMVEC ICAM-1 expression, and diminish HL-60 adhesion to HMEC-1s induced by H-2g stimulation. HMEC-1s or HMVECs were seeded at 2.50 x 105/3 ml in 35-mm 6-well plates and transfected with 4 µg of ODNs using LipofectAMINE. Fourteen hours after transfection, cells were allowed to achieve quiescence for 2 h in EBM with 0.1% FBS. Quiescent HMVECs or HMEC-1s were treated with H-2g (100 nM). A, HMEC-1 lysates were blotted for phospho-JAK2 (*p-JAK2), phospho-Erk1/2 (*p-Erk1/2), and phospho-Akt (*p-Akt). The same blots were stripped and reprobed for their steady-state protein, respectively. The antisense ODNs directed against JAK2, Erk1/2, and PI3K blocked phospho-JAK2 and pan-JAK2, phospho-Erk1/2 and pan-Erk1/2 expression, and also, both phospho-Akt and pan-Akt expression. The same results were obtained with HMVECs (data not shown). B, HMVEC ICAM-1 expression induced by H-2g was down-regulated by LipofectAMINE transfection of antisense ODNs directed against JAK2, Erk1/2, and PI3K. C, HL-60 adhesion to HMEC-1s induced by H-2g was blocked by antisense ODN transfection. Data from four separate experiments are expressed as the mean ± S.E.; n, number of experiments; NS, nonstimulated; AS, antisense; S, sense.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The finding that the human genome encodes no more than 30,000–50,000 proteins has emphasized the importance of post-translational modification in modulating the activities and functions of proteins in health and disease. One excellent example of such a modification is glycosylation, and information is accumulating about this process through oligosaccharide recognition. These carbohydrate expression proteins mediate critical processes such as protein recognition and cell trafficking and play important roles in mechanisms of immunity and cell-cell interactions (22, 23). Our previous studies described the 4A11 antigen as a glycoconjugate containing Ley/H. H-2g, a glucose analog of this antigen with similar functional properties, such as mediating angiogenesis, was used in this study. Our knowledge that 4A11 antigen was cytokine-inducible and was selectively expressed on the ECs of skin, thymus, lymph nodes, and synovium suggested that it may be important in leukocyte homing and infiltration.

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 ({alpha}-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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FIG. 7.
Hypothesized H-2g-induced EC signaling pathways. H-2g binds to an unknown receptor and activates JAK2. Phosphorylation (P) of JAK2 provides the docking site for STAT3, which in turn becomes phosphorylated at tyrosine and serine residues. Phosphorylated STAT3 is released from the receptor complex and form dimers. Phospho-STAT3 dimers translocate to the nucleus, where they directly bind to the promoter region of specific target genes, e.g. the ICAM-1 gene, thus regulating transcription of ICAM-1, which is involved in cell adhesion. Erk1/2 and PI3K-Akt are two parallel pathways that may be involved to lesser extent. Erk1/2 and PI3K may act directly on the transcription factor in the nucleus or through STAT3 dimers.

 

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-{alpha}, IL-1{beta}, and interferon-{gamma} 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 {kappa}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-{alpha} 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 ({alpha}-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 {kappa}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.


    FOOTNOTES
 
* This work was supported by the Gallagher Professorship for Arthritis Research and Veterans Affairs Research Service, National Institutes of Health Grants AI-40987 (to A. E. K.), HL-58695 (to A. E. K.), and AI-49287 (to C. C. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Koch, A. E., Nickoloff, B. J., Holgersson, J., Seed, B., Haines, G. K., Burrows, J. C., and Leibovich, S. J. (1994) Am. J. Pathol. 144, 244–259[Abstract]
  2. Halloran, M. M., Carley, W. W., Polverini, P. J., Haskell, C. J., Phan, S., Anderson, B. J., Woods, J. M., Campbell, P. L., Volin, M. V., Backer, A. E., and Koch, A. E. (2000) J. Immunol. 164, 4868–4877[Abstract/Free Full Text]
  3. Lacha, J., Bushell, A., Smetana, K., Rossmann, P., Pribylova, P., Wood, K., and Maly, P. (2002) J. Leukocyte Biol. 71, 311–318[Abstract/Free Full Text]
  4. Lindsley, H. B., Smith, D. D., Cohick, C. B., Koch, A. E., and Davis, L. S. (1993) Clin. Immunol. Immunopathol. 68, 311–320[CrossRef][Medline] [Order article via Infotrieve]
  5. Porter, J. C., and Hogg, N. (1997) J. Cell Biol. 138, 1437–1447[Abstract/Free Full Text]
  6. Tak, P. P., Thurkow, E. W., Daha, M. R., Kluin, P. M., Smeets, T. J., Meinders, A. E., and Breedveld, F. C. (1995) Clin. Immunol. Immunopathol. 77, 236–242[CrossRef][Medline] [Order article via Infotrieve]
  7. Furuzawa-Carballeda, J., and Alcocer-Varela, J. (1999) Scand. J. Immunol. 50, 215–222[CrossRef][Medline] [Order article via Infotrieve]
  8. Koch, A. E., Shah, M. R., Harlow, L. A., Lovis, R. M., and Pope, R. M. (1994) Clin. Immunol. Immunopathol. 71, 208–215[CrossRef][Medline] [Order article via Infotrieve]
  9. Szekanecz, Z., Haines, G. K., Lin, T. R., Harlow, L. A., Goerdt, S., Rayan, G., and Koch, A. E. (1994) Arthritis Rheum. 37, 221–231[Medline] [Order article via Infotrieve]
  10. Ihle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K., Thierfelder, W. E., Kreider, B., and Silvennoinen, O. (1994) Trends Biochem. Sci. 19, 222–227[CrossRef][Medline] [Order article via Infotrieve]
  11. Kisseleva, T., Bhattacharya, S., Braunstein, J., and Schindler, C. W. (2002) Gene (Amst.) 285, 1–24[CrossRef][Medline] [Order article via Infotrieve]
  12. Rane, S. G., and Reddy, E. P. (2000) Oncogene 19, 5662–5679[CrossRef][Medline] [Order article via Infotrieve]
  13. Morel, J. C., Park, C. C., Zhu, K., Kumar, P., Ruth, J. H., and Koch, A. E. (2002) J. Biol. Chem. 277, 34679–34691[Abstract/Free Full Text]
  14. Morel, J. C., Park, C. C., Woods, J. M., and Koch, A. E. (2001) J. Biol. Chem. 276, 37069–37075[Abstract/Free Full Text]
  15. Koch, A. E., Halloran, M. M., Haskell, C. J., Shah, M. R., and Polverini, P. J. (1995) Nature 376, 517–519[CrossRef][Medline] [Order article via Infotrieve]
  16. Madamanchi, N. R., Li, S., Patterson, C., and Runge, M. S. (2001) J. Biol. Chem. 276, 18915–18924[Abstract/Free Full Text]
  17. Kim, O. S., Park, E. J., Joe, E. H., and Jou, I. (2002) J. Biol. Chem. 277, 40594–40601[Abstract/Free Full Text]
  18. Matsumiya, T., Imaizumi, T., Itaya, H., Shibata, T., Yoshida, H., Sakaki, H., Kimura, H., and Satoh, K. (2002) Life Sci. 70, 3179–3190[CrossRef][Medline] [Order article via Infotrieve]
  19. Uddin, S., Majchrzak, B., Wang, P. C., Modi, S., Khan, M. K., Fish, E. N., and Platanias, L. C. (2000) Biochem. Biophys. Res. Commun. 270, 158–162[CrossRef][Medline] [Order article via Infotrieve]
  20. Katagiri, K., Kinashi, T., Irie, S., and Katagiri, T. (1996) Blood 87, 4276–4285[Abstract/Free Full Text]
  21. Mizoguchi, A., Takasaki, S., Maeda, S., and Kobata, A. (1984) J. Biol. Chem. 259, 11949–11957[Abstract/Free Full Text]
  22. Lee, S. J., Evers, S., Roeder, D., Parlow, A. F., Risteli, J., Risteli, L., Lee, Y. C., Feizi, T., Langen, H., and Nussenzweig, M. C. (2002) Science 295, 1898–1901[Abstract/Free Full Text]
  23. Feizi, T. (2000) Glycoconj. J. 17, 553–565[CrossRef][Medline] [Order article via Infotrieve]
  24. Duperray, A., Languino, L. R., Plescia, J., McDowall, A., Hogg, N., Craig, A. G., Berendt, A. R., and Altieri, D. C. (1997) J. Biol. Chem. 272, 435–441[Abstract/Free Full Text]
  25. Striz, I., Mio, T., Adachi, Y., Heires, P., Robbins, R. A., Spurzem, J. R., Illig, M. J., Romberger, D. J., and Rennard, S. I. (1999) Am. J. Physiol. 277, L58–L64[Medline] [Order article via Infotrieve]
  26. Yusuf-Makagiansar, H., Anderson, M. E., Yakovleva, T. V., Murray, J. S., and Siahaan, T. J. (2002) Med. Res. Rev. 22, 146–167[CrossRef][Medline] [Order article via Infotrieve]
  27. Wilson, J. L., Walker, J. S., Antoon, J. S., and Perry, M. A. (1998) J. Rheumatol. 25, 499–505[Medline] [Order article via Infotrieve]
  28. Lindsley, H. B., Smith, D. D., Davis, L. S., Koch, A. E., and Lipsky, P. E. (1992) Semin. Arthritis Rheum. 21, 330–334[Medline] [Order article via Infotrieve]
  29. Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A., and Springer, T. A. (1986) J. Immunol. 137, 245–254[Abstract/Free Full Text]
  30. Pober, J. S., Bevilacqua, M. P., Mendrick, D. L., Lapierre, L. A., Fiers, W., and Gimbrone, M. A., Jr. (1986) J. Immunol. 136, 1680–1687[Abstract/Free Full Text]
  31. Wu, A. J., Chen, Z. J., Kan, E. C., and Baum, B. J. (1997) J. Cell. Physiol. 173, 110–114[CrossRef][Medline] [Order article via Infotrieve]
  32. Innocenti, M., Thoreson, A. C., Ferrero, R. L., Stromberg, E., Bolin, I., Eriksson, L., Svennerholm, A. M., and Quiding-Jarbrink, M. (2002) Infect. Immun. 70, 4581–4590[Abstract/Free Full Text]