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Am J Physiol Heart Circ Physiol 274: H513-H519, 1998;
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Vol. 274, Issue 2, H513-H519, February 1998

Protein tyrosine phosphorylation mediates TNF-induced endothelial-neutrophil adhesion in vitro

Susan A. Kelly1,2, Pascal J. Goldschmidt-Clermont3, Emily E. Milliken4, Toshiyuki Arai1, Elise H. Smith1, and Gregory B. Bulkley1

Departments of 1 Surgery and 4 Medicine (Cardiology), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-4685; 2 Department of Surgery, Medical Center of Delaware, Newark, Delaware 19718; and 3 Heart and Lung Institute, University Medical Center, The Ohio State University, Columbus, Ohio 43210-1214

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Proinflammatory cytokines initiate the vascular inflammatory response via the upregulation of adhesion molecules on the luminal endothelial surface. We investigated directly the role of protein tyrosine phosphorylation in the upregulation of the endothelial adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and E-selectin, and the consequent adhesion of neutrophils, after tumor necrosis factor (TNF)-alpha -stimulation of human aortic endothelial cells in vitro. Time- and dose-dependent TNF-alpha -stimulated ICAM-1 and E-selectin upregulation and neutrophil adhesion each were suppressed by tyrosine kinase inhibitors, including genistein (200 µM), but not genistin, its isoflavone analog without tyrosine kinase inhibitory activity. Tyrphostin AG 126, a synthetic selective tyrosine kinase inhibitor, also suppressed ICAM-1 and E-selectin upregulation and neutrophil adhesion, each in a dose-dependent manner, whereas tyrphostin AG 1288 had no effect. Tyrosine phosphorylation of two proteins (85 and 145 kDa in the cytoskeleton fraction) found minutes after TNF-alpha -stimulation was also inhibited by genistein. These findings suggest that, in endothelial cells, TNF-alpha upregulates ICAM-1 and E-selectin expression and consequent neutrophil adhesion via protein tyrosine phosphorylation.

human; intercellular adhesion molecule 1; E-selectin; tyrphostin; genistein; tumor necrosis factor; polymorphonuclear leukocyte

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE RECRUITMENT OF leukocytes from the circulation by the endothelium is essential for the initiation and targeting of an inflammatory response. An important early event is the activation of the endothelium by a variety of proinflammatory stimuli, such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS), ultimately resulting in the upregulation of relatively low-affinity adhesion molecules, such as P- and E-selectin, which mediate leukocyte rolling along endothelium, and relatively high-affinity adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1, which facilitate firm adhesion (2, 5, 11, 17, 21). The intracellular signaling pathways for this response are not fully understood.

Protein tyrosine kinases (PTKs) and phosphatases play a pivotal role in the signaling reactions that mediate targeting and binding to endothelial cells (ECs). In particular, tyrosine kinases generate signals that modulate the activation of adhesion molecules such as integrins and other downstream signals that result from integrin engagement (7, 10). The systemic toxicity of LPS, which is mediated largely by TNF, has been reported to be prevented in mice by the PTK inhibitor tyrphostin AG 126 (19). This reduction in LPS-mediated lethality in vivo could be due to inhibition at the level of either the circulating leukocytes, the ECs, or both. Indeed, tyrphostin AG 126 has been reported to inhibit LPS-stimulated production of TNF and nitric oxide by macrophages themselves in that study. Nevertheless, we hypothesized that protein tyrosine phosphorylation might also be critical for the response of ECs to TNF produced by macrophages. We therefore evaluated the potential role of protein tyrosine phosphorylation in the TNF-alpha -stimulated upregulation of adhesion molecule expression and of consequent neutrophil adhesion in human aortic ECs in vitro.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Human aortic ECs (Clonetics, San Diego, CA) were cultured in T-75 flasks in endothelial growth media with 2% serum (Clonetics) at 37°C with 5% CO2. Cells from passages 6 to 11 from primary cultures were used. The ECs were grown to confluence in uncoated 96-well plates (Falcon; Becton-Dickinson, Bedford, MA) for the assays of both endothelial-neutrophil adhesion and adhesion molecule expression. Separate preparations of cells were grown to confluence on glass coverslips for immunostaining or in 100-mm petri dishes and then serum deprived (0.1% serum) for 48 h for the analysis of protein tyrosine phosphorylation by Western hybridization.

End Points

Leukocyte adhesion assay. Human blood was collected (in 10 U/ml heparin) from laboratory worker volunteers by a protocol approved by the Johns Hopkins University Joint Committee for Clinical Investigation and was centrifuged at 1,300 g for 10 min. The white blood cell layer was removed and layered over cold Accu-prep gradient (Accurate Chemical and Scientific, Westbury, NY) and centrifuged at 600 g for 30 min at 4°C for leukocyte separation. The red blood cell/polymorphonuclear leukocyte (PMN) layer was resuspended in red blood cell lysing buffer (Sigma, St. Louis, MO). After 20 min at room temperature, the preparation was centrifuged at 1,300 g for 2 min, and this step was repeated until the PMN pellet was free of red blood cells. The PMN pellet was then washed and resuspended in calcium-free phosphate-buffered saline (PBS; GIBCO, Grand Island, NY). This suspension was found to contain >95% PMNs by microscopic morphology after modified Wright-Giemsa staining (Diff-Quik Stain Set; Baxter, Miami, FL). The PMNs were labeled fluorescently by incubation with 5 µM calcein acetoxymethyl ester (Molecular Probes, Eugene, OR) for 20 min at 37°C. After being washed with calcium-free PBS, the PMNs were resuspended in Hanks' buffered saline solution (GIBCO) containing 0.2% bovine serum albumin and were plated on the EC monolayers, which had been grown to confluence in 96-well plates and treated with TNF-alpha , with or without an inhibitor. The PMNs were incubated with the EC monolayers for 20-25 min at 37°C. Nonadherent PMNs were then removed by gentle washing with PBS. The wells were visually inspected to ensure that the adhesion of the PMN was to the ECs themselves and not to the plastic. The adherent PMNs (and ECs) were then lysed using 4 mM Zwittergent (Calbiochem, La Jolla, CA), and the plates were read on a fluorescence plate reader at excitatory frequency (EX) 480 nm/emission frequency (EM) 530 nm. Standard curves obtained from a number of different PMN preparations indicated that the fluorescent emission was proportional to the number of fluorescently labeled PMNs that were adherent to the EC monolayer for any given batch of PMNs (data not shown). Because the calcein uptake by each batch of PMNs varied, each experiment was conducted and reported with simultaneous controls for the same batch of PMNs.

Expression of Adhesion Molecules

Enzyme-linked immunosorbent assay for upregulation of EC adhesion molecules. After treatment of the ECs with TNF-alpha , with or without inhibitor, the monolayers (in 96-well plates) were fixed for 20 min at 4°C in a solution containing 2% paraformaldehyde, 0.1 M L-lysine hydrochloride, and 2.1 mg/ml sodium periodate and then blocked in a solution of 0.1 M glycine and 1% bovine serum albumin. The blocking solution was then removed by washing with 0.1% bovine serum albumin in PBS, and the monolayers were probed with either mouse anti-human ICAM-1 or mouse anti-human E-selectin (Pharmingen, San Diego, CA) for 1 h at 37°C. This was followed by incubation with a peroxidase-conjugated sheep anti-mouse secondary immunoglobulin G (Sigma) for 1 h at 37°C. Developing substrate (0.1 M Na2HPO4, 0.05 M citric acid, 0.2% H2O2, and 0.4 mg/ml o-phenylenediamine) was then added for 5 min, and the reaction was stopped with 2 N H2SO4. The plates were then read on a spectrophotometric plate reader at 520 nm. Absorbance was linearly proportional to the amount of antibody bound, as confirmed by standard curves (data not shown).

Immunofluorescent staining of EC adhesion molecules. ECs, grown to confluency on glass coverslips, were treated with TNF-alpha , with and without an inhibitor, fixed with 3.7% formaldehyde in PBS for 20 min at room temperature, and probed with either the anti-ICAM-1 or anti-E-selectin for 20 min at room temperature. After washing with PBS, a fluorescein isothiocyanate-conjugated goat anti-mouse secondary immunoglobulin G (50 µg/ml; American Qualex, San Clemente, CA) was incubated at room temperature with the ECs for 20 min. The coverslips were then mounted on slides with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL) and allowed to dry. The ECs were then visualized on a Zeiss fluorescence microscope (EX 480 nm/EM 530 nm), and photographs of representative cells were taken using Ektachrome film (400 ASA; Kodak) with 30-s exposures for all frames.

Measurement of protein tyrosine phosphorylation by Western hybridization. Confluent EC monolayers, grown in 100-mm petri dishes, were treated with TNF-alpha , with or without a tyrosine kinase inhibitor, and then lysed with low-salt buffer containing 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.1 mM MgCl2, 10 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM Na3VO4, and the protease inhibitors leupeptin (50 mg/l), chymostatin, antipain, and pepstatin [25 mg/l each (Sigma); buffers used for Western hybridization are described in Ref. 9]. Subcellular fractions were separated as follows: the lysates were sonicated and then centrifuged at 150,000 g at 4°C for 30 min. The pellets were resuspended and sonicated in lysing buffer containing, in addition to the above, 145 mM NaCl and 1% Triton X-100. After centrifugation (150,000 g, 4°C, 30 min), the supernatants (enriched in membrane proteins) were diluted 1:2 in Laemmli sodium dodecyl sulfate-loading buffer, and the pellets (containing the Triton-insoluble cytoskeletal fraction) were resuspended in Laemmli buffer. All samples were boiled for 5 min, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient; Jule), transferred to a nitrocellulose filter, and probed with a mouse anti-human monoclonal antibody for phosphotyrosine residues (1 µg/ml; Upstate Biotechnology). The resulting Western blots were developed with a horseradish peroxidase-conjugated goat anti-mouse secondary immunoglobulin G (5 µg/ml; American Qualex) and enhanced chemiluminescence developing reagents (Amersham, Arlington Heights, IL).

Experimental Protocols

Agonists. TNF-alpha was used as our prototypical proinflammatory cytokine to stimulate the ECs. After incubation for periods ranging from 0.5 to 6 h, adhesion and upregulation of ICAM-1 and E-selectin were measured (see Expression of Adhesion Molecules). In the experiments probing for protein tyrosine phosphorylation by Western blotting, the ECs were treated with TNF-alpha for only 3-5 min and then were washed, lysed, and assayed as described.

Inhibitors. Several inhibitors of PTKs were used to evaluate the potential role for protein tyrosine phosphorylation in this response: genistein (50-200 µM) and tyrphostins AG 126 and AG 1288 (10-100 µM). Genistein was added at the same time as the TNF-alpha . Genistin (Sigma), an analog of genistein without PTK inhibitory activity, was used under identical conditions to control for possible nonspecific effects of genistein. The tyrphostins required pretreatment of the ECs for at least 1 h before the addition of TNF-alpha . All inhibitors and TNF-alpha were removed from the ECs by washing with PBS before the PMNs were added. Thus the PMNs were never exposed directly to the TNF-alpha or to the inhibitors.

Statistical analysis. Values were expressed as means ± SD. Time curves from different treatment groups (TNF-alpha alone vs. TNF-alpha with inhibitor) were compared using multivariate analysis of variance. Treatment groups of single concentrations of the inhibitors were compared using a Student's t-test. Values of P < 0.05 were considered to indicate statistically significant differences.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Time Course of TNF-alpha -Mediated EC-PMN Adhesion and Upregulation of Adhesion Molecules

A submaximal dose of TNF-alpha (100 U/ml) was chosen, based on a dose-response curve to TNF-alpha for PMN adhesion to ECs (Fig. 1). Upregulation of PMN adhesion was seen after a lag phase of 1 h and was nearly maximal at 4-5 h (see Fig. 3B). ICAM-1 and P- and E-selectin are key adhesion molecules on the surface of ECs that mediate high (ICAM-1)- and low (selectin)-affinity binding of PMN to EC. To account for the effect of TNF-alpha on the adhesion of PMN to EC, we studied the EC surface expression of adhesion molecules after addition of TNF-alpha . Expression of ICAM-1 and E-selectin was induced on the surface of ECs by TNF-alpha , following a time course that was similar to that of PMN adhesion (see below). In contrast, P-selectin seemed unlikely to be involved in the PMN adhesion to these ECs under our experimental conditions because measurable P-selectin was not constitutively expressed and was not inducible from these cells by TNF-alpha , thrombin, or histamine (data not shown). The upregulation of EC ICAM-1 surface expression after 4 h of TNF-alpha treatment was clearly visualized by immunostaining (Fig. 2).


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  		Fig. 1.   Adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells (ECs). ECs were treated with varying concentrations of tumor necrosis factor (TNF)-alpha for 4 h, after which fluorescently (calcein) labeled PMNs were added. Based on this dose-response curve, a submaximal dose (100 U/ml) of TNF-alpha was chosen for further experiments. Points on the curve (treatment groups) represent means of 7-14 wells ± SD.


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  		Fig. 2.   Immunofluorescent staining of ECs for intercellular adhesion molecule 1 (ICAM-1). A: untreated ECs were fixed and stained with anti-ICAM-1, followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Staining represents the constitutive expression of ICAM-1. B: ECs, treated for 4.5 h with TNF-alpha , were fixed and stained, and an increase in ICAM-1 expression localized to the membrane can be seen. C and D: ECs were treated with 200 µM genistein alone (C) or were treated with genistein and TNF-alpha (D). Faint fluorescent staining in these cells resembles that of untreated cells. (All images represent equal exposure times.) Note that, although upregulation of ICAM-1 was blocked by genistein, there were no signs of structural damage to the ECs resulting from genistein treatment.

Attenuation of PMN-EC Adhesion by Tyrosine Kinase Inhibitors

Next, we studied the role of EC tyrosine kinase pathways in PMN adhesion to ECs. The effects of several PTK inhibitors on the binding of PMN to human aortic ECs were examined. Genistein, a specific but relatively nonselective PTK inhibitor, attenuated TNF-mediated adhesion in a dose-dependent manner (Fig. 3A). Genistein (200 µM) completely blocked TNF-alpha -mediated adhesion (Fig. 3, A and B). To rule out nonspecific injury by these concentrations of genistein as a cause for its inhibitory effect, a separate, identical batch of ECs was incubated with 200 µM genistein following our standard protocol 1 day before the experiment. The inhibitor was then removed by washing, and, the following day, experiments revealed a complete recovery of the TNF-alpha -mediated adhesion response (Fig. 3C). Additionally, to control for possible nonspecific effects of genistein, its isoflavone analog genistin was also used. Genistin, which possesses no PTK inhibitory activity, had no effect on TNF-alpha -mediated adhesion, even at 200 µM (Fig. 3D).


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  		Fig. 3.   Inhibition of TNF-alpha -induced adhesion of PMNs by genistein or tyrphostin. A: tyrphostins were incubated with the ECs for 1-2.5 h before addition of TNF-alpha , which was then added to the ECs for 4.5 h. Genistein was added at the same time as TNF-alpha . Genistein or tyrphostin AG 126 inhibited the TNF-alpha -induced adhesion response in a dose-dependent manner, but no suppression of this response was found with tyrphostin AG 1288 at any concentration; n = 7-21 wells/treatment. Data are presented as %control (fluorescent emission of the PMN adhesion without inhibitor). * P < 0.001 vs. TNF-alpha alone by analysis of variance (ANOVA). Each experiment was repeated 2-3 times. B and D: number of adhering PMNs, represented by fluorescent emission, was increased after ~2 h and was maximal by 5-6 h of treatment. Inhibition of this adhesion response was seen with 200 µM genistein (B) but not the genistein analog genistin (which has no protein tyrosine kinase inhibitory activity; D). C: when genistein (200 µM), previously incubated with the ECs, was removed from the media for a period of 20 h, TNF-alpha -induced response completely recovered; n = 6 for each treatment group.

The tyrphostins are synthetic, selective PTK inhibitors. We used the tyrphostins AG 126 and AG 1288, which have been found to suppress TNF and nitric oxide production in LPS-stimulated macrophages (19). Tyrphostin AG 126 inhibited TNF-alpha -induced adhesion in a dose-dependent manner, whereas AG 1288 had no significant inhibitory effect on the PMN adhesion to ECs (Fig. 3A).

Attenuation of Upregulation of ICAM-1 and E-selectin

Dose-dependent inhibition of the TNF-alpha -induced upregulation of both ICAM-1 and E-selectin was observed with genistein (Fig. 4, A and B). This was confirmed by the immunofluorescent staining of EC ICAM-1 (Fig. 2, C and D). Inhibition of upregulation of both ICAM-1 and E-selectin was complete at a concentration of 200 µM (Fig. 4, A-D), but no inhibition was seen with genistin (Fig. 4, C and D). Consistent with the observations on PMN adhesion, tyrphostin AG 126 suppressed the TNF-alpha -induced upregulation of both ICAM-1 and E-selectin (Fig. 4, A and B). However, the effect of genistein at 50 µM on E-selectin expression was much stronger than the effect of this inhibitor on ICAM-1 expression. Because genistein at 50 µM had a strong effect on PMN attachment to ECs, we concluded that, under our experimental conditions, E-selectin may well play an important mediator role for PMN binding to ECs, consistent with a previous report (4). Tyrphostin AG 1288 also had an inhibitory effect on both adhesion molecules, and, although statistically significant (P < 0.01), this effect was far less than the inhibition provided by genistein and tyrphostin AG 126 (Fig. 4, A and B).


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  		Fig. 4.   Suppression of TNF-alpha -mediated upregulation of EC ICAM-1 and E-selectin by genistein or tyrphostins. TNF-alpha -mediated upregulation of ICAM-1 or E-selectin was measured by enzyme-linked immunosorbent assay after fixation of the ECs, using anti-ICAM-1 or anti-E-selectin and a peroxidase-conjugated secondary antibody. A and B: ECs were treated with genistein or tyrphostins as described in Fig. 3A. Genistein and tyrphostin AG 126 suppressed the TNF-alpha -induced upregulation of ICAM-1 and E-selectin. Tyrphostin AG 1288 also affected ICAM-1 and E-selectin upregulation, although the inhibition seen was not as marked as that seen with tyrphostin AG 126. * P < 0.001 and ** P < 0.01 compared with TNF alone by ANOVA; n = 3-12 wells/group. Each experiment was repeated 2-3 times. C and D: inhibition of TNF-alpha -induced ICAM-1 or E-selectin upregulation was seen with 200 µM genistein but not with genistin (200 µM); n = 3 wells each. Each experiment was performed 2-3 times.

TNF-alpha -Induced Protein Tyrosine Phosphorylation

TNF-alpha induced changes in tyrosine phosphorylation of specific proteins within subcellular compartments of ECs. After 3-5 min of TNF-alpha stimulation, an increase in tyrosine phosphorylation of two proteins (85 and 145 kDa) could be seen in the fraction enriched in cytoskeletal proteins (Triton X-100-insoluble fraction; Fig. 5). Genistein inhibited the constitutive phosphorylation of both of these proteins and inhibited the upregulation of their phosphorylation by TNF-alpha . In contrast, TNF-alpha had no evident effect on the tyrosine phosphorylation of proteins for the cytosolic fraction (data not shown) nor on proteins belonging to membrane-enriched fractions (Fig. 5).


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  		Fig. 5.   Tyrosine phosphorylation of two EC proteins induced by treatment with TNF-alpha . ECs were treated with TNF-alpha for 3-5 min before lysis and separation of subcellular fractions. An increase in tyrosine phosphorylation of two proteins (85 and 145 kDa) could be seen in the fraction enriched in cytoskeletal protein (P.2). Genistein (200 µM) inhibited both the constitutive phosphorylation and the upregulation of phosphorylation of these proteins by TNF-alpha . Equal protein loading (10 µg) of the lanes was confirmed by Coomassie blue staining of a matching gel (right). No significant changes in protein phosphorylation were observed in membrane protein-enriched fractions (P.1). YP, tyrosine phosphorylation.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The TNF-alpha -induced adhesion of PMNs to ECs seen in vivo could reflect quantitative and/or qualitative changes in adhesion molecule expression on either ECs, leukocytes, or both (2, 5, 6, 21). In our model, we focused on the EC events, which must take place initially for the adhesion of circulating leukocytes to occur. Because the mediators (TNF-alpha and inhibitors) were added only to the ECs in our system and because our ECs were then washed before the addition of PMNs, our results should reflect only changes in the expression of EC adhesion molecules. This is also supported by preliminary studies in which we found that phorbol ester-induced neutrophil adhesion to unstimulated endothelium was not affected by the addition of genistein to the endothelium (data not shown). We reproducibly found that TNF-alpha induced adhesion and upregulation of ICAM-1 and E-selectin. Upregulation of PMN adhesion and adhesion protein expression each followed a similar time course, reaching maxima after 4-5 h of treatment.

The pivotal role of protein tyrosine phosphorylation in these EC processes is becoming increasingly evident (15, 19, 23). We found suppression of adhesion and of ICAM-1 and E-selectin upregulation by several PTK inhibitors. Genistein inhibited these processes, whereas its analog genistin, without PTK inhibitory activity, did not. Moreover, cells were able to recover their response to TNF-alpha after removal of genistein from the media. These findings make it unlikely that the inhibition of these TNF-alpha -stimulated events was due to nonspecific or toxic effects of the genistein, even at the higher doses. Although it seems clear from our data that the genistein inhibited ICAM-1 upregulation by TNF in ECs, an opposite effect of genistein has been reported (22). The different experimental conditions and systems, such as incubation time, could account for these disparate findings. For example, oxidants, which are considered to be upstream of tyrosine phospholyration in these signal transduction pathways (16), are generally reported to mediate ICAM-1 induction by cytokines in ECs (3), whereas dual effects of antioxidants on ICAM-1 induction in the same ECs have also been reported, where longer incubation of ECs with antioxidants enhanced ICAM-1 expression (18). During the completion of this work, we became aware of two studies (15, 23) exploring the role of tyrosine phosphorylation in the upregulation of adhesion of PMNs after TNF stimulation of human umbilical vein ECs. Our results are consistent with the findings of these studies (15, 23) but provide more definitive evidence that protein tyrosine phosphorylation itself is essential for TNF-induced ICAM-1 upregulation and consequent PMN adhesion in response to TNF.

The tyrphostins are synthetic PTK blockers designed to be individually specific for particular PTKs (12). Tyrphostin AG 126 has been reported to prevent LPS-mediated lethality in mice in vivo. This has been correlated with the ability of tyrphostins AG 126 and AG 1288 to suppress TNF and nitric oxide production by macrophages in response to LPS in vitro (19). We found that tyrphostin AG 126 has an inhibitory effect on the TNF-alpha -induced activation of ECs as well and on the upregulation of ICAM-1 and E-selectin and on associated PMN adhesion. On the other hand, tyrphostin AG 1288 had no inhibitory activity on adhesion in our model. The difference in the inhibitory effect between AG 126 and AG 1288 may be ascribed to their differences in structures, as reported previously (20). It is possible that the concurrent inhibition of ECs and macrophage function by tyrphostin AG 126 may well have contributed to the remarkable reduction in mortality that has been seen in LPS-stimulated mice that had been treated with AG 126 (19).

An increase in tyrosine phosphorylation was seen in at least two separate proteins associated with the Triton X-insoluble (cytoskeletal) fractions, after stimulation with TNF-alpha for 3-5 min. This effect, like the adhesion molecule expression, was inhibited by genistein. These proteins were all of a higher molecular weight than the 35-kDa protein reported to be phosphorylated on tyrosine after TNF stimulation of human umbilical vein ECs (23).

The TNF-alpha -induced upregulation of EC adhesion molecules and consequent neutrophil adhesion has been well documented to occur in vivo as well as in vitro (2, 5, 6, 21). In vivo, neutrophil adhesion is usually, but not always (atherosclerosis), observed in postcapillary venules rather than in large vessels (11, 14, 17). The ECs used here were derived from human aorta, and, despite their origin, were able to respond vigorously to TNF-alpha and interact with PMNs. Aortic ECs are considered to represent a less specialized EC of those available for investigations ex vivo. They are, therefore, less likely to "drift away" from their generic phenotype and display highly specialized pathways that would not be relevant to in vivo conditions. Likewise, if signaling pathways are detectable within aortic ECs, they are more likely to be functional in many other ECs of the body.

Another notable difference between our in vitro model and an in vivo situation is the lack of blood flow in our system. The absence of flow conditions for the binding of PMN to EC might explain why E-selectin expression and its inhibition by tyrosine kinase antagonists seem to correlate particularly well with PMN adhesion data. However, it is not unlikely that genistein might also have inhibited the production of inflammatory mediators by the endothelium, thereby inhibiting adhesion (13). Shear forces play an important role in the rolling of neutrophils by affecting both the contact time and disruption forces between endothelium and leukocytes (1, 5, 8, 11). We could only carefully control the intensity and number of buffer washes to render this variable as constant as possible, but our assay cannot address the response to shear stress in vivo, nor can it definitively discriminate PMN rolling from firm adhesion. These experiments do show, however, that the upregulation of known adhesion molecules, which was found to require protein tyrosine phosphorylation, could have functionally relevant effects.

In summary, the triggering of an initial vascular inflammatory response by the proinflammatory cytokine TNF-alpha via the upregulation of EC adhesion molecules appears to require the activation of specific PTKs. Because the adhesion of circulating neutrophils to the venular endothelium is necessarily the initial physiological trigger of the microvascular inflammatory response, this information should help us to better understand, and perhaps modulate, the vascular inflammatory reaction in vivo.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants DK-31764 and HL-52315, an Established Investigator Award of the American Heart Association, and a postdoctoral training grant from the Medical Center of Delaware.

    FOOTNOTES

Address for reprint requests: G. B. Bulkley, Blalock 685, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-4685.

Received 30 May 1997; accepted in final form 7 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(2):H513-H519
0363-6135/98 $5.00 Copyright © 1998 the American Physiological Society


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