Down-Modulation of Monocyte Transendothelial Migration and Endothelial Adhesion Molecule Expression by Fibroblast Growth Factor
2002; Elsevier BV; Volume: 160; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61169-8
ISSN1525-2191
AutoresHong Zhang, Andrew C. Issekutz,
Tópico(s)Platelet Disorders and Treatments
ResumoBasic and acidic fibroblast growth factor (bFGF and aFGF, respectively) and vascular endothelial growth factor (VEGF) exert angiogenic actions and have a role in wound healing, inflammation, and tumor growth. Monocytes and endothelial cells are involved in these processes, but the effect of FGF and VEGF on monocyte-endothelial cell interactions has not been defined. We observed that monocyte adhesion to resting or cytokine (tumor necrosis factor-α or interleukin-1α)-stimulated human umbilical vein endothelial cells (HUVECs) was markedly inhibited (40 to 65%) by culture (1 to 6 days) of HUVECs with aFGF or bFGF. Monocyte transendothelial migration induced by C5a and chemokines (MCP-1, SDF-1α, RANTES, MIP-1α) was also suppressed (by 50 to 75%) on bFGF-stimulated HUVECs. VEGF did not have these effects at the concentrations used (10 to 20 ng/ml), although like bFGF, it promoted HUVEC proliferation. Culture of HUVECs with bFGF and aFGF significantly down-regulated intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression on resting or tumor necrosis factor-α-stimulated HUVECs, but had no influence on platelet endothelial cell adhesion molecule (PECAM)-1 and VE-cadherin expression. bFGF also inhibited MCP-1 production by HUVECs. The inhibitory effects of bFGF on monocyte transendothelial migration and adhesion molecule expression were reversed by SU6668, an anti-angiogenic agent and bFGF receptor tyrosine kinase inhibitor. Our results suggest that bFGF and aFGF may suppress endothelial-dependent monocyte recruitment and thus have an anti-inflammatory action during angiogenesis in chronic inflammation but inhibit the immunoinflammatory tumor defense mechanism. However, SU6668 is an effective agent to prevent this down-regulatory action of bFGF on monocyte-endothelial cell interactions. Basic and acidic fibroblast growth factor (bFGF and aFGF, respectively) and vascular endothelial growth factor (VEGF) exert angiogenic actions and have a role in wound healing, inflammation, and tumor growth. Monocytes and endothelial cells are involved in these processes, but the effect of FGF and VEGF on monocyte-endothelial cell interactions has not been defined. We observed that monocyte adhesion to resting or cytokine (tumor necrosis factor-α or interleukin-1α)-stimulated human umbilical vein endothelial cells (HUVECs) was markedly inhibited (40 to 65%) by culture (1 to 6 days) of HUVECs with aFGF or bFGF. Monocyte transendothelial migration induced by C5a and chemokines (MCP-1, SDF-1α, RANTES, MIP-1α) was also suppressed (by 50 to 75%) on bFGF-stimulated HUVECs. VEGF did not have these effects at the concentrations used (10 to 20 ng/ml), although like bFGF, it promoted HUVEC proliferation. Culture of HUVECs with bFGF and aFGF significantly down-regulated intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression on resting or tumor necrosis factor-α-stimulated HUVECs, but had no influence on platelet endothelial cell adhesion molecule (PECAM)-1 and VE-cadherin expression. bFGF also inhibited MCP-1 production by HUVECs. The inhibitory effects of bFGF on monocyte transendothelial migration and adhesion molecule expression were reversed by SU6668, an anti-angiogenic agent and bFGF receptor tyrosine kinase inhibitor. Our results suggest that bFGF and aFGF may suppress endothelial-dependent monocyte recruitment and thus have an anti-inflammatory action during angiogenesis in chronic inflammation but inhibit the immunoinflammatory tumor defense mechanism. However, SU6668 is an effective agent to prevent this down-regulatory action of bFGF on monocyte-endothelial cell interactions. A fundamental feature of inflammation involves the adhesion of leukocytes to vascular endothelium and their emigration into inflamed tissues.1Springer TA Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6522) Google Scholar, 2Cines DB Pollak ES Buck CA Loscalzo J Zimmerman GA McEver RP Pober JS Wick TM Konkle BA Schwartz BS Barnathan ES McCrae KR Hug BA Schmidt AM Stern DM Endothelial cells in physiology and in the pathophysiology of vascular disorders.Blood. 1998; 91: 3527-3561PubMed Google Scholar Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is a feature during chronic inflammation, eg, in arthritis, wound healing, and tissue remodeling or tumor growth. Angiogenesis is a complex process regulated by interactions of endothelial cells with growth factors and cytokines as well as with extracellular matrix proteins via adhesion molecules.3Iruela-Arispe ML Dvorak HF Angiogenesis: a dynamic balance of stimulators and inhibitors.Thromb Haemost. 1997; 78: 672-677PubMed Google Scholar Monocytes play an important role in collateral vessel formation (arteriogenesis) by attaching to activated endothelium and by invading the walls of innate collateral vessels, where they produce growth factors. Previous studies have demonstrated that this process can be promoted by several chemokines and growth factors.4Heil M Clauss M Suzuki K Buschmann IR Willuweit A Fischer S Schaper W Vascular endothelial growth factor (VEGF) stimulates monocyte migration through endothelial monolayers via increased integrin expression.Eur J Cell Biol. 2000; 79: 850-857Crossref PubMed Scopus (101) Google Scholar Recruitment of monocytes has been suggested to be important in the angiogenic cascade.5Albini A Florio T Giunciuglio D Masiello L Carlone S Corsaro A Thellung S Cai T Noonan DM Schettini G Somatostatin controls Kaposi's sarcoma tumor growth through inhibition of angiogenesis.FASEB J. 1999; 13: 647-655Crossref PubMed Scopus (116) Google Scholar Furthermore, the cytokines basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) produced by monocytes, as well as other cells, are among the most potent mediators of angiogenesis. These factors are frequently present at sites of physiological and pathological angiogenesis and growing tumors are dependent on this process.6Brown LF Detmar M Claffey K Nagy JA Feng D Dvorak AM Dvorak HF Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine.EXS. 1997; 79: 233-269PubMed Google Scholar, 7Schweigerer L Neufeld G Friedman J Abraham JA Fiddes JC Gospodarowicz D Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth.Nature. 1987; 325: 257-259Crossref PubMed Scopus (664) Google Scholar, 8McCourt M Wang JH Sookhai S Redmond HP Proinflammatory mediators stimulate neutrophil-directed angiogenesis.Arch Surg. 1999; 134: 1325-1332Crossref PubMed Scopus (227) Google Scholar, 9Yeh CH Peng HC Huang TF Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.Exp Cell Res. 1999; 251: 57-66Crossref PubMed Scopus (31) Google Scholar During inflammation, the cytokines tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1α and β) have been shown to induce leukocyte infiltration in part by up-regulating the expression of leukocyte adhesion molecules on vascular endothelial cells.1Springer TA Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6522) Google Scholar, 10van de Stolpe A van der Saag PT Intercellular adhesion molecule-1.J Mol Med. 1996; 74: 13-33Crossref PubMed Scopus (672) Google Scholar It is well established that the recruitment and emigration of circulating leukocytes is dependent on a multistep cascade of events involving leukocyte tethering, rolling, firm adhesion, and emigration and these steps are mediated by distinct adhesion molecules and activation pathways.1Springer TA Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6522) Google Scholar, 11Butcher EC Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2646) Google Scholar The selectins (L-, P-, and E-selectin) and α4-integrins (α4β1 and α4β7)11Butcher EC Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2646) Google Scholar, 12Berlin C Bargatze RF Campbell JJ von Andrian UH Szabo MC Hasslen SR Nelson RD Berg EL Erlandsen SL Butcher EC Alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow.Cell. 1995; 80: 413-422Abstract Full Text PDF PubMed Scopus (920) Google Scholar mediate rolling of leukocytes in postcapillary venules of the systemic circulation, whereas firm adhesion and emigration of rolling leukocytes including monocytes is mostly dependent on two members of the CD18 (β2) integrin family, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) as well as α4β1,13Luscinskas FW Cybulsky MI Kiely JM Peckins CS Davis VM Gimbrone Jr, MA Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1.J Immunol. 1991; 146: 1617-1625PubMed Google Scholar which engage intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 and VCAM-1 on endothelium.14Issekutz AC Rowter D Springer TA Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration.J Leukoc Biol. 1999; 65: 117-126Crossref PubMed Scopus (132) Google Scholar, 15Smith CW Leukocyte-endothelial cell interactions.Semin Hematol. 1993; 30: 45-54PubMed Google Scholar, 16von Andrian UH Chambers JD McEvoy LM Bargatze RF Arfors KE Butcher EC Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo.Proc Natl Acad Sci USA. 1991; 88: 7538-7542Crossref PubMed Scopus (974) Google Scholar These findings suggest that modulation of the adhesion molecule expression on endothelium can influence the trafficking of leukocytes into tissues. In comparison with our understanding of the mechanisms in normal vessels, relatively little is known about factors that regulate monocyte or other leukocyte adhesion and transmigration across angiogenic vessels in inflammation or in tumors. Recent studies in vivo have suggested that rolling, adhesion, and transmigration of leukocytes in angiogenic blood vessels may be impaired.17Stromblad S Cheresh DA Integrins, angiogenesis and vascular cell survival.Chem Biol. 1996; 3: 881-885Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 18Borgstrom P Hughes GK Hansell P Wolitsky BA Sriramarao P Leukocyte adhesion in angiogenic blood vessels. Role of E-selectin, P-selectin, and beta2 integrin in lymphotoxin-mediated leukocyte recruitment in tumor microvessels.J Clin Invest. 1997; 99: 2246-2253Crossref PubMed Scopus (51) Google Scholar Previous studies have shown that many tumor cells produce bFGF and VEGF,19Kolomecki K Stepien H Narebski JM Vascular endothelial growth factor and basic fibroblast growth factor evaluation in blood serum of patients with hormonally active and inactive adrenal gland tumours.Cytobios. 2000; 101: 55-64PubMed Google Scholar, 20Komorowski J Jankewicz J Stepien H Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble interleukin-2 receptor (sIL-2R) concentrations in peripheral blood as markers of pituitary tumours.Cytobios. 2000; 101: 151-159PubMed Google Scholar and freshly isolated tumor endothelium exhibits decreased expression of ICAM-1 as compared to endothelium in normal tissue.21Griffioen AW Tromp SC Hillen HF Angiogenesis modulates the tumour immune response.Int J Exp Pathol. 1998; 79: 363-368Crossref PubMed Scopus (24) Google Scholar Furthermore, endothelial VCAM-1 expression is suppressed in melanomas and carcinomas.22Piali L Fichtel A Terpe HJ Imhof BA Gisler RH Endothelial vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma.J Exp Med. 1995; 181: 811-816Crossref PubMed Scopus (166) Google Scholar High levels of endothelial growth factors are often found in plasma of patients with various tumors.19Kolomecki K Stepien H Narebski JM Vascular endothelial growth factor and basic fibroblast growth factor evaluation in blood serum of patients with hormonally active and inactive adrenal gland tumours.Cytobios. 2000; 101: 55-64PubMed Google Scholar, 20Komorowski J Jankewicz J Stepien H Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble interleukin-2 receptor (sIL-2R) concentrations in peripheral blood as markers of pituitary tumours.Cytobios. 2000; 101: 151-159PubMed Google Scholar, 23Fuhrmann-Benzakein E Ma MN Rubbia-Brandt L Mentha G Ruefenacht D Sappino AP Pepper MS Elevated levels of angiogenic cytokines in the plasma of cancer patients.Int J Cancer. 2000; 85: 40-45Crossref PubMed Scopus (83) Google Scholar The potential impact of these factors on leukocyte-endothelial cell adhesion and emigration has not been fully evaluated. Griffioen and colleagues24Griffioen AW Damen CA Martinotti S Blijham GH Groenewegen G Endothelial intercellular adhesion molecule-1 expression is suppressed in human malignancies: the role of angiogenic factors.Cancer Res. 1996; 56: 1111-1117PubMed Google Scholar has observed a decreased expression of ICAM-1 on bFGF-treated human umbilical vein endothelial cells (HUVECs) in vitro. However these studies did not investigate the effects on monocyte adhesion or transendothelial migration (TEM). Furthermore, most previous studies have used HUVECs grown to confluence with endothelial cell growth supplements that contain a mixture of growth factors and these may influence the basal and stimulated endothelial responses.25Zhang H Issekutz AC Growth factor regulation of neutrophil-endothelial cell interactions.J Leukoc Biol. 2001; 70: 225-232PubMed Google Scholar In this study we examined the effect of bFGF, aFGF, and VEGF on endothelial cell interaction with monocytes resulting in monocyte adhesion and TEM. The effects were further analyzed on basal and inflammatory cytokine-stimulated ICAM-1, VCAM-1, and E-selectin expression and chemokine production by endothelium. The effects of a potent anti-angiogenic agent (SU6668)26Laird AD Vajkoczy P Shawver LK Thurnher A Liang C Mohammadi M Schlessinger J Ullrich A Hubbard SR Blake RA Fong TA Strawn LM Sun L Tang C Hawtin R Tang F Shenoy N Hirth KP McMahon G Cherrington JM SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors.Cancer Res. 2000; 60: 4152-4160PubMed Google Scholar on reversing growth factor-induced changes was also determined. Recombinant human bFGF, VEGF, and IL-8 were purchased from Peprotech Inc. (Rocky Hill, NJ); TNF-α was from Genetech Inc. (San Francisco, CA); interferon-γ was from InterMune Pharmaceutical Inc. (Palo Alto, CA); IL-1α was a gift from Immunex Corp. (Seattle, WA); human serum albumin (HSA) (pyrogen-free) was from Connaught Laboratories (Downsview, Ontario, Canada). Monoclonal antibodies (mAbs) used were mouse mAb R6.5 to ICAM-1 (gift from Dr. TA Springer, Boston, MA); mAb 4B9 to VCAM-1 and BB11 to E-selectin (gifts from R Lobb, Biogen Inc., Cambridge, MA); 3H11B9 to pertussis toxin (gift from Dr. T Issekutz, Halifax, Canada); 5H2 to PECAM-1 (in-house generated). mAb to VE-cadherin was from Chemicon (Temecula, CA); peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were from Bio-Can Scientific (Mississauga, Ontario, Canada); peroxidase conjugate streptavidin was from Amersham (Oakville, Ontario, Canada), fluorescein isothiocyanate-conjugated sheep F(ab)2 anti-mouse IgG was from Sigma Chemical Co. (St. Louis, MO) and Alexa 488-conjugated goat anti-mouse IgG was from Molecular Probes (Eugene, OR). SU6668 (kind gift from Dr. J Cherrington, SUGEN, Inc., South San Francisco, CA) was dissolved in dimethyl sulfoxide at a stock concentration of 50 mmol/L. Recombinant human C5a, a gift from CIBA-Geigy Pharmaceuticals (Summit, NJ) and human monocyte chemotactic protein-1 (MCP-1), RANTES, stromal-derived factor-1α (SDF-1α), bFGF, aFGF, VFGF165, and macrophage inflammatory protein-1α (MIP-1α) were from Preprotech. Human monocytes were purified as described previously27Chuluyan HE Issekutz AC VLA-4 integrin can mediate CD11/CD18-independent transendothelial migration of human monocytes.J Clin Invest. 1993; 92: 2768-2777Crossref PubMed Scopus (144) Google Scholar from ethylenediaminetetraacetic acid (EDTA)/acid citrate dextrose (ACD) anti-coagulated venous blood of healthy donors. Briefly, red cells were sedimented with 6% dextran-saline (Abbott Laboratories, Montreal, Canada), leukocyte-rich plasma was collected and after centrifugation (150 × g for 10 minutes at 22°C), the leukocyte pellet was resuspended in Ca2+, Mg2+-free Tyrode's solution with 5% autologous platelet-poor plasma and labeled with Na251CrO4 (Amersham) for 30 minutes at 37°C. During this incubation, the osmolarity of the medium was gradually increased in three steps from 290 to 360 mOsm by addition of 9% NaCl. This improved the monocyte purity and did not affect cell viability or function, as shown previously.28Boyum A Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium.Scand J Immunol. 1983; 17: 429-436Crossref PubMed Scopus (184) Google Scholar The labeled leukocytes were washed once with Ca2+, Mg2+-free Tyrode's solution-5% platelet-poor plasma (360 mOsm), and resuspended in Ca2+, Mg2+-free Tyrode's solution (360 mOsm) containing 0.2% EDTA, 10% platelet-poor plasma, and 56% Percoll (Pharmacia Fine Chemicals, Dorval, PQ). The leukocytes were separated on a discontinuous Percoll gradient of 73%, 62%, 56% (containing the labeled leukocytes), 50%, 46%, and 40% by centrifugation (400 × g for 25 minutes at 22°C). The purest monocyte faction was recovered at the 46 to 40% Percoll interphase. The monocytes were resuspended for migration studies at 7 × 105/ml in RPMI 1640, 0.5% HSA containing 10 mmol/L Hepes (pH 7.4). This method yielded monocytes of ≥92% purity with no neutrophil contamination based on neutral red and Wright's staining and >98% viability by trypan blue exclusion.27Chuluyan HE Issekutz AC VLA-4 integrin can mediate CD11/CD18-independent transendothelial migration of human monocytes.J Clin Invest. 1993; 92: 2768-2777Crossref PubMed Scopus (144) Google Scholar HUVECs were isolated and cultured in flasks29Jaffe EA Nachman RL Becker CG Minick CR Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.J Clin Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6261) Google Scholar and grown on filters as previously described.30Issekutz AC Chuluyan HE Lopes N CD11/CD18-independent transendothelial migration of human polymorphonuclear leukocytes and monocytes: involvement of distinct and unique mechanisms.J Leukoc Biol. 1995; 57: 553-561PubMed Google Scholar Briefly, endothelial cells were detached from human umbilical veins by treatment with 0.5 mg/ml of collagenase (Cooper Biomedical, Ontario, Canada) in 0.01 mol/L of phosphate-buffered saline (PBS) (pH 7.4) and grown in basal medium composed of RPMI 1640 (Sigma Chemical Co.) containing 2 mmol/L of l-glutamine, 2-mercaptoethanol, sodium pyruvate, penicillin/streptomycin, and supplemented with 20% heat-inactivated human AB serum (ABS). To initially establish the cultures, endothelial cell growth supplement (12.5 μg/ml) (Collaborative Research, Lexington, MA) and 45 μg/ml of heparin (Sigma) were added to the basal medium. Cells were cultured in 2% gelatin (Difco Inc., Detroit, MI)-coated culture flasks (NUNC, Life Technologies, Mississagua, Ontario, Canada). The HUVECs were harvested using 0.025% trypsin and 0.01% EDTA (Sigma) and cultured in gelatin-coated 96-well plates at 2.5 × 104 cells/well without growth factor or 1.5 × 104/well with added growth factors as indicated until confluence at 3 days and used for enzyme-linked immunosorbent assays (ELISA) or adhesion assays. For monocyte TEM, HUVECs were seeded on polyvinyl pyrolidene (PVP)-free polycarbonate filters (5-μm pores) in Transwell culture plate inserts (6.5-mm diameter; Fisher Scientific, Ottawa, Ontario, Canada) up to the third passage. The filters were pretreated with 0.01% gelatin (37°C, 18 hours) followed by 3 μg of human fibronectin (Collaborative Research) in 50 μl of water at 37°C for 2 hours. Fibronectin was then replaced by HUVECs (1.5 × 104 or 2.5 × 104 in basal medium with or without the indicated growth factor, respectively), added above the filter in 0.1 ml and 0.6 ml of basal medium was added to the lower compartment beneath the filter. On filters, the HUVECs required 5 to 6 days to form a tight permeability barrier that was evaluated by 125I-labeled-HSA diffusion as previously described.30Issekutz AC Chuluyan HE Lopes N CD11/CD18-independent transendothelial migration of human polymorphonuclear leukocytes and monocytes: involvement of distinct and unique mechanisms.J Leukoc Biol. 1995; 57: 553-561PubMed Google Scholar Under all conditions, <1.5% labeled HSA diffused across the HUVEC/filter unit in 45 minutes with 1-mm applied positive hydrostatic pressure, whereas bare filters showed ≥30% diffusion of 125I-labeled HSA in this test. The HUVECs were grown in 96-well culture plates under the indicated conditions and 51Cr-labeled monocytes were added at a concentration of 105 cells/well for 45 minutes at 37°C. Afterward, HUVECs were gently washed three times with prewarmed RPMI 1640 medium. Bound 51Cr monocytes were harvested from wells by addition of 1 N of NaOH and quantified with γ-spectrometer (Wallac LKB-1282, Fisher Scientific). The percentage of added monocytes adherent to the HUVECs was calculated. Adhesion was measured with triplicate or quadruplicate replicates. HUVECs were cultured on gelatin/fibronectin-coated polycarbonate filters with (1.5 × 104/well) or without (2.5 × 104/well) an indicated growth factor for 6 days. Migration assays were performed as described previously.30Issekutz AC Chuluyan HE Lopes N CD11/CD18-independent transendothelial migration of human polymorphonuclear leukocytes and monocytes: involvement of distinct and unique mechanisms.J Leukoc Biol. 1995; 57: 553-561PubMed Google Scholar, 31Issekutz AC Lopes N Endotoxin activation of endothelium for polymorphonuclear leucocyte transendothelial migration and modulation by interferon-gamma.Immunology. 1993; 79: 600-607PubMed Google Scholar Briefly, HUVEC monolayers on the filters and the lower compartments were washed with RPMI 1640 and then were transferred to a new, clean well of a 24-well plate (lower compartment). When the HUVECs were prestimulated with cytokines, the well contained RPMI 1640 to 10% AB serum and stimulation was for 4 hours. After washing, 0.6 ml of RPMI 1640, 10 mmol/L HEPES, and 0.5% HSA was added to the well containing as indicated the chemotactic stimulus. Before immersion of the HUVEC filter unit, 0.1 ml of medium containing 1 × 105 51Cr-labeled monocytes were added above the HUVECs. After incubation (75 minutes at 37°C, 5% CO2), migration was stopped by washing the upper compartment twice with 0.1 ml of RPMI 1640 to remove nonadherent monocytes. The undersurface of the filter was wiped with a cotton swab saturated with an ice-cold PBS-0.2% EDTA solution and this was added to the lower compartment. The cells that spontaneously detached from the undersurface of the filter or were removed by the swab were lysed by the addition of 0.5% Triton X-100 and all of the 51Cr released in the lower compartment and from the swab was quantitated with a γ-spectrometer. The results are expressed as the percentage of the total 51Cr monocytes added above the HUVECs that migrated through the HUVEC filter unit. The monocyte adhesion to the HUVECs was quantified by lysis with 0.5 N NaOH of the 51Cr-monocytes that remained on the HUVEC monolayer after three washes of the monolayer/filter unit with warm RPMI 1640. The 51Cr in this NaOH lysate was quantified and is expressed as the percentage of the total 51Cr monocytes added above the HUVECs that adhered on the HUVEC monolayer. All experiments were performed with triplicate replicates. The expression of ICAM-1, VCAM-1, and E-selectin on HUVECs was determined with whole-cell ELISA as described previously with minor modification.31Issekutz AC Lopes N Endotoxin activation of endothelium for polymorphonuclear leucocyte transendothelial migration and modulation by interferon-gamma.Immunology. 1993; 79: 600-607PubMed Google Scholar Briefly, HUVEC monolayers in 96-well plates were incubated with (1.2 × 104/well) or without (2 × 104/well) a specific growth factor at various concentrations for various times. In some experiments the HUVEC monolayers were treated with TNF-α or IL-1α for 4 hours. For SU6668 treatment, varying concentrations of this compound were added 1 hour before bFGF was added to the wells. Before ELISA, medium was removed and HUVECs were washed twice with warm RPMI medium. The HUVEC monolayer was fixed by adding 50 μl of 1% paraformaldehyde for 15 minutes at room temperature and then 50 μl of 0.05 mol/L Tris/0.1 mol/L glycine (pH 7.2) was added for an additional 15 minutes at room temperature. After washing, 100 μl of RPMI 1640 to 5% fetal calf serum (FCS)-0.1% NaN3 containing mAb to ICAM-1, to VCAM-1, E-selectin, PECAM-1, VE-cadherin, or control mAb was added. After 60 minutes (37°C, 5% CO2), the monolayers were washed four times and then 100 μl of peroxidase-conjugated goat anti-mouse IgG (1:4000 in RPMI 1640 to 5% FCS) was added for 60 minutes (37°C, 5% CO2). The monolayers were washed four times and then 100 μl of substrate (o-phenylenediamine, 12.5 mg/ml; 0.1 mol/L citrate-phosphate buffer, pH 5; 0.012% H2O2) was added. The enzyme reaction was stopped by adding 100 μl of 4 N H2SO4, and absorbance at the 490 nm was measured. Results are expressed as OD × 1000. The expression of endothelial adhesion molecules was also determined by immunofluorescence flow cytometry using a standard immunofluorescence protocol.32Issekutz TB Wykretowicz A Effect of a new monoclonal antibody, TA-2, that inhibits lymphocyte adherence to cytokine stimulated endothelium in the rat.J Immunol. 1991; 147: 109-116PubMed Google Scholar Briefly, HUVECs were detached by brief treatment with 0.01% trypsin and 0.02% EDTA. Cell surface expression of ICAM-1, VCAM-1, and E-selectin was assessed using mAb R6.5, 4B9, and BB11 (5 μg/ml each), respectively. Binding was assessed by secondary detection with fluorescein isothiocyanate conjugated to sheep F(ab)2 anti-mouse IgG (Sigma). Nonspecific fluorescence was assessed by substituting the primary mAb with a nonbinding isotype-matched control mAb (3H11B9). Analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA). Results are expressed as fluorescence histograms plotted on a log scale. Immunofluorescence microscopy was performed on HUVECs to localize junctional proteins PECAM-1 and VE-cadherin. The HUVECs were grown on gelatin/fibronectin-coated Perplex culture slide chambers (Labtek, Life Technologies, Mississauga, Ontario, Canada) in the indicated growth media. The monolayers were stained with primary mAb (anti-PECAM-1 or anti-VE-cadherin or isotype control 3H11B9) in RPMI 1640 to 10% FCS, 0.1% NaN3 (45 minutes, 37°C). After washing, HUVECs were fixed with 1% paraformaldehyde (15 minutes, 22°C) followed by sequential blocking with 0.05 mol/L Tris and 0.1 mol/L glycine (pH 7.2) and 10% FCS-PBS (15 minutes each at 22°C). Secondary goat anti-mouse IgG conjugated with Alexa 488 was applied for detection. Slides were examined and photographed with a Nikon fluorescence microscope. MCP-1 and IL-8 concentrations were quantified by sandwich ELISA as previously described.33Chuluyan HE Lang BJ Yoshimura T Kenney JS Issekutz AC Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma.Life Sci. 1998; 63: 1939-1952Crossref PubMed Scopus (18) Google Scholar MCP-1 in the HUVEC culture supernatants was captured with mAb E11 immobilized on ELISA plates. The secondary antibody was polyclonal rabbit IgG anti-human MCP-1 (kind gifts from T Yoshimura, National Cancer Institute, Frederick, MD). This was detected with goat anti-rabbit IgG alkaline-phosphatase conjugate. Sensitivity of this assay was down to 300 pg/ml of MCP-1. For IL-8 capture, mAb 3IL-8-h10 was used and the secondary was biotinylated mAb 2IL-8-HIA (kind gifts from JS Kenney, Antibody Solutions, Palo Alto, CA). This was detected with peroxidase conjugated to streptavidin. The detection limit for this assay was 600 pg/ml. The HUVECs were cultured in 96-well plates at a density of 104 cells/well with or without bFGF or VEGF in RPMI 1640 to 20% ABS for 2 days. In some wells, SU6668 was added 30 minutes before growth factor addition. On the third day, the medium was changed to RPMI 1640 with 5% ABS and the above agents were added again as above. 3H-thymidine (Amersham, Oakville, Ontario, Canada) (1 μCi) was added to each well for an additional 6 hours at 37°C. 3H-thymidine-labeled DNA was then quantitated by harvesting the cells onto Whatman GF/B paper filter mats using a cell harvester. The filter content of 3H was measured with a Beckman liquid scintillation counter. Results are expressed as cpm of 3H-thymidine incorporated into DNA. One-way analysis of variance, Student's t-test, or paired t-test was used for statistical analysis of the data as indicated. P values exceeding 0.05 were not considered significant. Initially, we examined the effect of bFGF, aFGF, and VEGF on the adhesion of monocytes to unstimulated TNF-α
Referência(s)