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Originally published online as doi:10.1189/jlb.0504304 on September 30, 2004

Published online before print September 30, 2004
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(Journal of Leukocyte Biology. 2005;77:44-51.)
© 2005 by Society for Leukocyte Biology

Transmigrated neutrophils down-regulate the expression of VCAM-1 on endothelial cells and inhibit the adhesion of flowing lymphocytes

Philip C. W. Stone*, Frank Lally*, Mahbub Rahman*, Emily Smith*, Christopher D. Buckley{dagger}, Gerard B. Nash* and G. Ed Rainger*,1

* Departments of Physiology and
{dagger} Rheumatology, The Medical School, The University of Birmingham, United Kingdom

1 Correspondence: Department of Physiology, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK B15 2TT. E-mail: g.e.rainger{at}bham.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As the first leukocytes recruited during inflammation, neutrophils are ideally situated to regulate the subsequent recruitment of mononuclear leukocytes. Here, we found that human neutrophils recruited by endothelial cells (EC), which had been stimulated with tumor necrosis factor {alpha} for 4 h, inhibited the adhesion of flowing, mixed mononuclear cells or purified lymphocytes over the subsequent 20 h but did not affect the adhesion of a secondary bolus of neutrophils. The degree of inhibition of lymphocyte adhesion increased with the duration of neutrophil-EC contact and with the number of recruited neutrophils. Antibody-blocking studies showed that lymphocyte adhesion was mediated predominantly by vascular cell adhesion molecule-1 (VCAM-1). Recruited neutrophils reduced the EC expression of VCAM-1 but not intercellular adhesion molecule-1 (ICAM-1) or E-selectin in a manner that mirrored the time- and number-dependent reduction in lymphocyte adhesion. VCAM-1 was not shed into the culture supernatant, and a panel of protease inhibitors was unable to reverse its down-regulation, indicating that it was not proteolytically degraded by neutrophils. In EC that had been in contact with neutrophils, the mRNA message for VCAM-1 but not ICAM-1 was down-regulated, indicating that alterations in transcriptional activity were responsible for the reduction in VCAM-1. Thus, under some inflammatory milieu, neutrophils may delay the recruitment of mononuclear leukocytes by regulating the expression of EC adhesion receptors.

Key Words: adhesion molecules • lymphocyte trafficking • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During acute inflammation, leukocytes are recruited to the tissues by the endothelial cells (EC) of the post-capillary venules, which have been activated by inflammatory agents such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-1ß [1 , 2 ]. EC activation promotes the expression of specialized adhesion receptors [E- and P-selectin and vascular cell adhesion molecule-1 (VCAM-1)], which have rapid forward kinetics for bond formation essential for binding flowing leukocytes [1 2 3 4 ]. These bonds have high tensile strength but are short-lived, and a repeated cycle of bond formation and disengagement supports leukocyte rolling adhesion in the direction of the shear forces generated by blood flow [3 , 4 ]. Rolling allows slow-moving leukocytes to assimilate signals from the EC-borne chemoattractants [e.g., IL-8, monocyte chemotactic protein-1 (MCP-1), platelet activating factor], which promote immobilization and transendothelial migration via adhesive interactions between leukocyte integrin receptors and members of the immunoglobulin (Ig) superfamily such as intercellular adhesion molecule-1 (ICAM-1) and VCAM-1 on EC [1 , 2 ].

It is now well established that during an evolving, inflammatory reaction, the recruitment of different leukocyte subsets alters in a dynamic manner to meet the specific needs of the inflamed tissue [5 ]. Indeed, there is compelling evidence from some animal models of acute inflammation that an influx of neutrophilic granulocytes (neutrophils) precedes a phase in which the recruitment of mononuclear leukocytes (monocytes and lymphocytes) predominates [6 , 7 ]. These changes in the composition of the leukocytic infiltrate probably reflect alterations in the patterns of adhesion receptor and chemokine expression on the EC surface, which in turn, are thought to be regulated by the patterns of cytokines and other inflammatory mediators released from cells within the tissue stroma [8 ].

As rolling receptors support the initial tethering interactions, which are a prerequisite for leukocyte activation and tissue penetration, they represent a key regulatory point in the process of leukocyte recruitment. We believe that newly recruited leukocytes might be able to influence the progression of a developing inflammatory response by regulating the expression of these receptors and thus, subsequently controlling the cellular content of the inflammatory infiltrate. For example, monocytes and T lymphocytes are able to promote the expression of E-selectin and VCAM-1 on EC, and EC activated by monocyte coculture can support the secondary adhesion and migration of other leukocytes [9 , 10 ]. Neutrophils, however, have long been considered to be terminally differentiated effectors of the innate immune response, which execute their bactericidal and cytotoxic functions aggressively once localized at the inflammatory locus. They are not widely thought of as regulators of the cellular composition of the inflammatory infiltrate and have not been shown to directly regulate the expression of EC rolling receptors. In the present study, we show that neutrophils possess the ability to regulate the expression of VCAM-1 on EC. In consequence, they can inhibit the adhesion of flowing lymphocytes, thus demonstrating that recruited neutrophils have the capacity to delay the switch from an acute to a resolving phase of inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte isolation
Blood was collected from healthy volunteers into EDTA (1.6 mg/ml). Neutrophils and mixed mononuclear cells were separated using two-step density gradients of Histopaque 1119 and 1077 (Sigma, UK) as described previously [11 ]. Cells were washed, counted, and adjusted to concentrations between 1 x 105 and 2 x 107/ml in phosphate-buffered saline (PBS; Aldrich, Milwaukee, WI) containing 0.1% bovine serum albumin (BSA; PBS/Alb; Sigma). Neutrophil preparations were greater than 95% granulocytes with some residual mononuclear leukocyte contamination; mixed mononuclear cells were 29% monocytes and 71% lymphocytes as determined by cell volume distribution using a Coulter Multisizer II. Lymphocytes were purified using Histopaque 1077 density media followed by panning on culture plastic to remove contaminating monocytes [11 ]. Preparations typically contained 93% lymphocytes with some residual monocyte contamination and were adjusted to a concentration of 1 x 106/ml in PBS/Alb prior to adhesion assay.

EC isolation and coculture with neutrophils
Human umbilical vein EC (HUVEC) were isolated and cultured in EC medium, which was Medium 199 (Invitrogen, Paisley, UK) containing 28 µg/ml gentomycin (Sigma), 20% fetal calf serum (Sigma), 1 µg/ml hydrocortisone (Sigma), and 10 ng/ml epidermal growth factor (Sigma) as described previously [11 ]. EC were grown to confluence in 25 cm2 culture flasks (BD Falcon, Oxford, UK) precoated with 1% gelatin solution (Sigma).

Primary cultures of HUVEC were passaged and cultured in microslides (glass capillaries with rectangular cross-section of 3 x 0.3 mm and good optical qualities) for 24 h until confluent as described previously [12 ]. EC monolayers in microslides were activated with 100 U/ml recombinant human TNF-{alpha} (R&D Systems, Abingdon, UK) for 4 h. TNF was washed from the system, and 50 µl purified neutrophils was injected into the microslides at concentrations sufficient to yield between 0.05 and six neutrophils/EC based on a count of 1000 EC/mm2. In microslides, a ratio of six neutrophils/EC equated to neutrophil concentration of 2 x 107/ml. After 1 h, nonadherent cells were washed from the microslides, and neutrophils were counted by phase-contrast microscopy. We found that an average of 74.9 ± 23.2% of injected neutrophils was adherent to the EC monolayer of which 55.0 ± 20.2% transmigrated into the subendothelial space (data are mean±SEM of four experiments). Neutrophils and EC were then cocultured for 4, 8, or 20 h with an automated change of culture medium made hourly using a culture system described previously [12 ].

Alternatively, EC were passaged into 96-well plates and subjected to coculture with neutrophils under the same regimes as in microslides. In some experiments, inhibitors of protease function were added to the coculture medium after neutrophil adhesion had occurred, and nonadherent cells had been washed from the system. Antiproteases were the broad-spectrum serine proteinase inhibitors aprotinin (50 µg/ml, Sigma) and {alpha}1-antitrypsin (2.5 µg/ml, Sigma), the broad-spectrum cysteine protease inhibitor E-64 (Sigma), the neutrophil elastase inhibitor eglin-C 60–63 (1 µM, Sigma), matrix metalloproteinase (MMP) inhibitor 1 (a peptide inhibitor that neutralizes the function of MMP-1, -3, -8, and -9, 50 µM, CalBiochem, Nottingham, UK), and the TNF-{alpha}-converting enzyme inhibitor TNF-{alpha} protease inhibitor-1 (TAPI-1; 40 µM, Peptides International, Louisville, KY).

Mixed mononuclear cell, lymphocyte, and neutrophil adhesion assay under conditions of flow
After 4, 8, or 20 h of coculture, microslides were incorporated into a flow-based adhesion assay and viewed by video-microscopy as described previously [10 , 12 ]. Prior to the perfusion of lymphocytes, 10 microscope fields selected at random were recorded. This allowed determination of the number of neutrophils remaining after coculture but before adherent leukocytes had entered the system. Mixed mononuclear cells or purified lymphocytes or neutrophils at a concentration of 1 x 106/ml were then perfused through microslides for 3 min at a flow rate calculated to give a wall shear stress of 0.1 Pa (1 dyne/cm2). Nonadherent cells were then washed from the microslide using cell-free medium. After washing for 2 min (total perfusion time, 5 min) five randomly selected fields were video-recorded for 30 s each.

Video records of experiments were analyzed off-line. Using phase-contrast microscopy, we were unable to distinguish cocultured leukocytes from the freshly introduced ones. Thus, in most experiments, leukocyte adhesion from flow was quantified by counting all adherent cells and subtracting the number of neutrophils that was present at the end of coculture but before the perfusion of a secondary bolus of leukocytes. The number of leukocytes adherent to the EC was normalized for the number of cells perfused and expressed as adherent cells/mm2/106 cells perfused. In a separate series of experiments, we verified that counting adherent cells by subtracting the number present after coculture from the total population after a secondary bolus of leukocytes was accurate. We found their was no significant difference when we compared the subtractive count attained using phase-contrast microscopy (134±25 cells/mm2/106 perfused) with inflowing lymphocytes labeled with the fluorescent dye Calcein-AM and counted using fluorescent microscopy (131±6 cells/mm2/106 perfused; data are mean±SEM of three experiments).

Measuring the expression of VCAM-1 in EC and the levels of soluble VCAM-1 (s-VCAM-1) in coculture supernatants
VCAM-1 expression was measured using standard enzyme-linked immunosorbent assay (ELISA) techniques. Briefly, neutrophils were established in coculture with EC in 96-well plates at the cellular ratios and for durations that paralleled microslide experiments. Cocultures were fixed for 15 min in 1% gluteraldehyde in PBS, rinsed thoroughly, and blocked with 1% BSA. This strategy of fixation renders the internal pools of protein available for labeling, as it disrupts the cell membrane, resulting in measurement of the total cellular pool of target epitopes [13 ]. VCAM-1 was labeled with a mouse anti-human VCAM-1 monoclonal antibody (mAb; clone 1.4C3; 17.5 µg/ml, Dako, UK) or an isotype-matched, nonimmune control antibody (20 µg/ml, Dako). A horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody was used to label the primary antibody and colorimetric conversion of the substrate O-phenylene diamine used to quantify VCAM-1 expression. Results were expressed as absorbance at 490 nm after subtraction of absorbance in control wells. The levels of s-VCAM-1 were measured using a commercially available ELISA kit from R&D Systems using the manufacturer’s published protocol. Data are expressed as µg/ml s-VCAM-1 released per 105 EC.

Measuring mRNA expression for VCAM-1 and ICAM-1 by real-time polymerase chain reaction (PCR)
RNA for real-time PCR was extracted from EC monolayers using TRizol reagents (Invitrogen) according to the manufacturer’s instructions. Contaminating genomic DNA was removed using a DNA-freeTM kit (Ambion, Huntingdon, UK). The reverse transcription (RT) of mRNA to first-strand cDNA used Geneamp PCR reaction kit reagents (Perkin Elmer, Warrington, UK), i.e., 10 mM Tris HCl, pH 8.3; 50 mM KCl; 5 mM MgCl; 1 mM deoxy-guanosine 5'-triphosphate, deoxy-adenosine 5'-triphosphate, deoxy-cytidine 5'-triphosphate, thymidine 5'-triphosphate; 1 U/µl RNase inhibitor; 2.5 U/µl Mu-MLV reverse transcriptase; 2.5 mM random hexamers. Quantitative real-time PCR was performed using 500 ng cDNA and Assays-on-DemandTM gene expression mix for the target genes ICAM-1 and VCAM-1 (using 6-carboxyfluorescein-labeled probe, Applied Biosystems, Warrington, UK), 18-S endogenous control with forward and reverse primers [using VIC-labeled probe (Eurogentec Ltd., Hampshire, UK)], and Taqman® Universal PCR master mix (Applied Biosystems). cDNA was amplified using a standard protocol of 40 cycles of 2 min at 50°C, 10 min at 95°C, 15 s at 95°C, and 1 min at 60°C. Results were analyzed using the sequence detection system 2.2 software, and results are presented as relative quantity normalized to the expression levels in unstimulated EC.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effects of coculture on EC and neutrophils
EC monolayers were stimulated for 4 h with 100 U/ml TNF (Fig. 1A ). After removal of the TNF with a wash in culture medium, purified neutrophils were injected into microslides and allowed to settle and adhere to the EC surface for 1 h. After washing out nonadherent neutrophils with culture medium, we found that ~70% of infused neutrophils had adhered to the EC of which approximately half had transmigrated into the subendothelial space (phase dark cells in Fig. 1B ). Microslides were incorporated into a system, which perfused culture medium periodically through the glass capillary (at 0.2 Pa for 30 s, hourly, to replenish spent medium), and after 20 h of coculture, we observed that the majority of apically adherent cells (phase bright cells in Fig. 1B ) had been washed from the system (Fig. 1C) . Prolonged coculture with neutrophils did not alter the appearance of the EC monolayer, and there was no difference in the density of EC cultured in the presence or absence of neutrophils (1057±99 and 1010±96, respectively; data are mean±SEM of three experiments). When EC that had been cocultured with neutrophils for 20 h were stained to show their nuclear morphology, there was no indication of nuclear condensation in EC, and surprisingly, little apoptosis was evident in the neutrophils (Fig. 1D ; note that Fig. 1C and 1D , are phase-contrast and fluorescent images of the same microscope field). To verify that apoptotic neutrophils were not responsible for alterations in EC function, we established a static version of the coculture system based in 24-well plates. When apically adherent neutrophils were collected from this system after 20 h of coculture with EC, we found that neutrophil apoptosis had been delayed so that less than 5% showed nuclear condensation compared with greater than 60% in neutrophils cultured in identical medium for 20 h on plastic (Fig. 2A ). Using the proteolytic activity of caspase-3 to determine the levels of neutrophil apoptosis also demonstrated significantly attenuated levels of cell death in neutrophils cocultured with EC (Fig. 2B) . Additionally, we were able to account for all of the neutrophils originally adherent to the EC by counting the collected cells and those remaining in coculture (data not shown), demonstrating that neutrophils had not been lost by necrosis. These data strongly imply that alterations in EC function as a result of coculture with neutrophils are not a result of death of EC or neutrophils.



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  		Figure 1. Photomicrographs showing (A) a phase-contrast image of an EC monolayer treated for 4 h with 100 U/ml TNF prior to the addition of isolated neutrophils; (B) a phase-contrast image of an EC monolayer on which isolated neutrophils have been incubated for 1 h and nonadherent cells removed with wash buffer. Phase dark cells are transmigrated under the monolayer [transmigrated cells; TM]. Phase bright cells are adherent to the apical surface of the monolayer [apically adherent cells, AA]. (C) A phase-contrast image of an EC monolayer cocultured with neutrophils for 24 h. The majority of the surface-adherent neutrophils had been washed from the system during coculture, leaving a population of TM. (D) A fluorescent microscopy image of the same field of view shown in C after staining with acridine orange. EC nuclei show no sign of condensation that would be indicative of apoptosis. It is interesting that the majority of neutrophils also demonstrates a multilobed nuclear morphology, indicating that apoptosis has been delayed in these cells.

 


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  		Figure 2. The affect on neutrophil apoptosis of coculture with HUVEC. Neutrophils were isolated from whole blood and maintained on culture plastic or in coculture with HUVEC for 20 h, after which time, they were collected from culture and levels of apoptosis compared with freshly isolated neutrophils. Apoptosis was assessed by (A) nuclear morphology or (B) caspase-3 cleavage of substrate containing the Asp-Glu-Val-Asp (DEVD) sequence. Data are the mean ± SEM of four experiments. *, P < 0.05; **, P < 0.01, for comparison of levels of apoptosis in freshly isolated and plastic-cultured neutrophils by paired t-test; +, P < 0.05, for comparison of levels of apoptosis in plastic-cultured and EC-cocultured neutrophils by paired t-test.

 
Neutrophils inhibit the adhesion of flowing mixed mononuclear cells and lymphocytes but not neutrophils to TNF-stimulated EC
Coculture of EC with mononuclear leukocytes has been reported to promote the expression of EC adhesion receptors (e.g., ref. [14 ]) and chemokines (e.g., ref. [15 ]) so that in consequence, EC support the adhesion of a secondary bolus of flowing leukocytes [10 ]. We conducted analogous experiments using neutrophils cocultured with TNF-{alpha}-stimulated EC. In our initial experiments, we saw that flowing mixed mononuclear cells bound to TNF-stimulated EC (167±8 cells/mm2/106 perfused), and in the presence of cocultured neutrophils, adhesion was reduced by nearly 90% (21±16 cells/mm2/106 perfused; data are mean±SEM of three experiments). These experiments indicated that adhesion of monocytes and lymphocytes was inhibited by the coculture of neutrophils with EC.

We went on to investigate this phenomena using purified peripheral blood lymphocytes. When confluent monolayers of EC were stimulated with 100 U/ml TNF-{alpha} for 24 h, they could support the adhesion of flowing lymphocytes (Fig. 3A ). In comparison, when the same TNF-{alpha}-stimulated EC were cocultured with neutrophils for 20 h at a ratio of six neutrophils:one EC, the adhesion of flowing lymphocytes was almost abolished (Fig. 3A) . The ability of neutrophils to inhibit lymphocyte adhesion was dependent on the number of neutrophils established in coculture (Fig. 3A) , and significant reductions in lymphocyte adhesion were evident in the presence of as few as 0.3 neutrophil/EC. When EC were cocultured with neutrophils at the highest coculture ration for 8 h, inhibition of lymphocyte adhesion was apparent but to a lesser degree than at 24 h (Fig. 3B) , and after 4 h of coculture, the level of lymphocyte adhesion was unchanged compared with TNF-{alpha}-stimulated control EC (Fig. 3B) .



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  		Figure 3. The effects of coculturing neutrophils with HUVEC on subsequent lymphocyte or neutrophil adhesion. Monolayers of EC that had been cocultured with neutrophils were incorporated into a flow-based adhesion assay, and the levels of leukocyte adhesion were assessed. Data are shown for (A) the effect of coculturing EC with different ratios of neutrophils on the number of adherent lymphocytes. Data are the mean ± SEM of four experiments. Analysis of covariance (ANCOVA) showed a significant effect (P=0.024) of neutrophil coculture ratio on lymphocyte adhesion; (B) the effect of duration of neutrophil (PMN):EC coculture at a ratio of 6:1 on the percentage inhibition of lymphocyte adhesion compared with EC cultured alone. Data are the mean ± SEM of four experiments. ANCOVA showed no significant effect of duration of neutrophil coculture on lymphocyte adhesion. *, P < 0.05, for comparison of lymphocyte adhesion at 0 h and 20 h of coculture by paired t-test; (C) the effect of neutrophil:EC coculture on the adhesion of a secondary bolus of neutrophils. Data are mean ± SEM of three experiments.

 
Upon investigating the effects of neutrophil-EC coculture on the behavior of a secondary bolus of flowing neutrophils, we found that the levels of neutrophil adhesion were not significantly affected after 8 h or 20 h of coculture, although there was a small increase in the levels of neutrophil binding at both time-points (Fig. 3C) . It is interesting that we found that the efficiency with which adherent neutrophils could migrate through the EC monolayer was significantly increased by coculture: 66 ± 4% of adherent cells transmigrated on EC cocultured for 20 h compared with 35 ± 0.6% on TNF-stimulated EC without coculture (data are mean±SEM of three experiments; P=0.02 by paired t-test). This implied that neutrophils might penetrate tissue more efficiently in the presence of previously migrated neutrophils.

Coculture with neutrophils reduces the expression of VCAM-1 but not ICAM-1 or E-selectin on EC
Studies about the molecular basis of the adhesion of peripheral blood lymphocytes to EC activated by TNF-{alpha} indicate that the interaction of lymphocyte {alpha}4ß1-integrin with VCAM-1 on the EC surface makes an important contribution to the capture of flowing cells (e.g., ref. [16 ]). Here, we found that 72% of lymphocyte adhesion could be inhibited by antibody blockade of VCAM-1, confirming the importance of this interaction in our model (Fig. 4 ).



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  		Figure 4. The effect of an anti-VCAM-1 antibody (Ab) on the levels of lymphocyte adhesion to TNF-stimulated EC, which were stimulated with 100 U/ml TNF for 24 h, and the levels of adhesion of flowing lymphocytes were assessed in the presence or absence of a function-neutralizing mAb directed against VCAM-1. Data are mean ± SEM of three experiments. **, P < 0.01, for comparison by paired t-test of lymphocyte adhesion to EC in the presence or absence of anti-VCAM-1.

 
We followed the expression of EC VCAM-1, ICAM-1, and E-selectin by ELISA in our coculture experiments. In these experiments, cocultures were fixed with 1% gluteraldehyde, which permeabilizes the cell membranes and allows labeling of the total cellular pool of receptors. Using this strategy, we found that unstimulated EC barely expressed detectable levels of VCAM-1, but upon treatment with 100 U/ml TNF-{alpha}, absorbance levels were increased over 30-fold (Fig. 5A ). Coculture of TNF-{alpha}-stimulated EC with six neutrophils/EC for 20 h reduced the total expression of VCAM-1 by 65% (Fig. 5A) . This result strongly suggests that upon neutrophil coculture, total VCAM-1 expression is reduced markedly. The degree of inhibition of VCAM-1 expression was also dependent on the ratio of neutrophils to EC in coculture, so that VCAM-1 expression was reduced at concentrations of neutrophil coculture as low as 0.03 neutrophil/EC (Fig. 5A) . When EC were cocultured with the highest ratio of neutrophils for shorter durations, a significant reduction in VCAM-1 expression was evident after periods of coculture as short as 4 h (Fig. 5B) . Unstimulated EC constitutively expressed ICAM-1 (Fig. 5C) , which was increased approximately twofold upon EC activation with TNF-{alpha}. Coculturing the TNF-{alpha}-stimulated EC with six neutrophils/EC did not affect the expression of ICAM-1 (Fig. 5C) . E-selectin was not constitutively expressed on EC, but expression was strongly induced by treatment with 100U/ml TNF (Fig. 5D) . Expression peaked at ~6 h of TNF treatment and thereafter, was down-regulated so that expression could not be demonstrated 24 h after the initiating stimulus (Fig. 5D) . Coculture of EC with neutrophils did not significantly alter the dynamics of the time-course of E-selectin expression or the levels of expression at any given time-point (Fig. 5D) .



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  		Figure 5. The effect of neutrophil coculture with EC on adhesion receptor expression. EC that had been cocultured with neutrophils were fixed in 1% formaldehyde, and the expression of VCAM-1, ICAM-1, and E-selectin was assessed by ELISA. Data are shown for (A) the effect of different coculture ratios on VCAM-1 expression [ANCOVA showed a significant effect (P<0.01) of neutrophil (PMN) coculture ratios on VCAM-1 expression]; (B) the effect on VCAM-1 expression of different durations of coculture at a ratio of six neutrophils/EC [ANCOVA showed a significant effect (P<0.01) of coculture duration on the levels of VCAM-1 expression]; (C) the effect of neutrophil coculture on ICAM-1 expression; and (D) the effect of the duration of coculture on the expression of E-selectin. Data are the mean ± SEM of four experiments.

 
VCAM-1 is not shed into the coculture medium
We speculated that VCAM-1 might be shed from the EC surface proteolytically as a means of down-regulation. When we measured the levels of s-VCAM-1 in the supernatants from EC cultures, we found that it was absent from unstimulated EC but appeared in increasing concentration over a 24-h period in EC stimulated with TNF-{alpha} (Fig. 6 ). However, when EC stimulated with TNF-{alpha} were cocultured with neutrophils, the rate and levels of VCAM-1 release into the culture supernatant did not increase (Fig. 6) . Additionally, down-regulation of VCAM-1 expression could not be inhibited using a panel of antiproteases that inhibited the function of proteolytic pathways, which have been reported previously to mediate VCAM-1 shedding (Table 1 ).



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  		Figure 6. The effect of duration of coculture on the levels of s-VCAM-1 in culture supernatants from EC stimulated with TNF in the presence (open bars) or absence (solid bars) of cocultured neutrophils. ANOVA showed that there was a significant effect (P<0.01) of time on the expression of s-VCAM-1 in the presence or absence of cocultured neutrophils. Data are mean ± SEM of three experiments.

 

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  		Table 1. Effects of Treating Neutrophil (PMN)/EC Cocultures with Antiprotease Reagents on the Levels of Expression of VCAM-1 after 20 h Coculture

 
VCAM-1 but not ICAM-1 is regulated at the transcriptional level in EC cocultured with neutrophils
Using real-time PCR, we investigated whether VCAM-1 and ICAM-1 expression in cocultured EC was regulated at the transcriptional level. Compared with unstimulated EC, TNF-treated EC had significantly increased mRNA expression for VCAM-1 and ICAM-1 (Fig. 7 ). In the presence of cocultured neutrophils, mRNA levels for ICAM-1 were unaltered. However, there was a marked reduction in VCAM-1 mRNA, implying that VCAM-1 expression is regulated by cocultured neutrophils at the transcriptional level.



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  		Figure 7. The effects of neutrophil (PMN):EC coculture at a ratio of 6:1 on levels of adhesion receptor mRNA. EC were cocultured with neutrophils and isolated by trypsin digestion. After purification, mRNA levels were assessed by real-time PCR. Data are shown (A) for VCAM-1 and (B) for ICAM-1 and are mean ± SEM of three experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we have shown that interactions between EC and transmigrated neutrophils can inhibit the adhesion of a secondary bolus of flowing mixed mononuclear cells or lymphocytes but not the adhesion of a secondary bolus of neutrophils. There is good evidence that monocytes and T cells can modulate the reponses of EC during inflammation. Monocytes or T lymphocytes cocultured with EC induced the expression of the adhesion receptors E-selectin and VCAM-1 [9 , 10 , 14 , 17 ], the chemokines MCP-1, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1{alpha}, IL-8, interferon (IFN)-inducible protein 10, and monokine induced by IFN-{gamma} [15 , 17 18 19 20 21 ], as well as the proinflammatory cytokines IL-1-ß, TNF-{alpha}, IL-6, and IFN-{gamma} [17 , 19 , 22 , 23 ]. Furthermore, EC cocultured with monocytes are stimulated in a manner that supports the secondary adhesion, activation and transendothelial migration of leukocytes [9 , 10 , 17 , 24 ]. These data show that recruited mononuclear cells can reinforce the inflammatory process, leading to increased recruitment of leukocytes.

Although we have shown that neutrophils can inhibit the adhesion of mononuclear leukocytes, a proinflammatory role for neutrophils in the regulation of leukocyte recruitment by EC is indicated in some in vitro and animal models. For example, recombinant cationic antimicrobial protein of molecular weight 37 kDa (CAP37), a neutrophil-derived protein, stimulates the expression of VCAM-1 and E-selectin in EC [25 ]. Notably, however, CAP37 derived upon neutrophil coculture with EC has not been demonstrated to support such a route of activation. Peroxynitrite generated by the interaction of superoxide and nitric oxide in EC and neutrophil cocultures is reported to mobilize endothelial nuclear factor-{kappa}B and induce the expression of E-selectin [26 ]. However, in this study, neutrophils were incapable of activating EC without preactivation by the nonphysiological agonists phorbol 12-myristate 13-acetate or calcium ionophore. It is interesting that the same authors reported that incubation of unstimulated neutrophils with TNF-stimulated EC did not result in EC activation. Neutrophils may also be important in up-regulating mononuclear cell adhesion during complement-mediated inflammation by a process dependent on neutrophil-mediated IL-6 signaling [6 , 27 28 29 ]. This mechanism of regulation may, however, be restricted to EC responding to mediators of acute inflammation such as complement, as cytokines (e.g., IL-1, TNF, IFN-{gamma}) and bacterial endotoxin activate EC to support the adhesion of mononuclear leukocytes without a requirement for neutrophil recruitment or IL-6 signaling.

Previous studies indicate that interactions between lymphocyte {alpha}4ß1-integrin and VCAM-1 on the EC surface support the majority of the adhesive contacts made by flowing lymphocytes [16 ]. Here, again, we found that lymphocyte adhesion could be inhibited largely by antibody blockade of VCAM-1, confirming the importance of this interaction in our model. When we cocultured EC with neutrophils, the expression of VCAM-1 was down-regulated, strongly indicating that this was the mechanism underlying the observed reduction in leukocyte adhesion. Our initial hypothesis was that VCAM-1 was cleaved by EC- or neutrophil-derived proteases. In fact, very little is known about the enzymatic processing of surface VCAM-1 after expression. Nevertheless, the presence of increased VCAM-1 levels in the blood plasma during inflammatory episodes indicates that a route for VCAM-1 shedding does exist, especially as there is no direct evidence for a secretable form of the receptor. Recent evidence indicates that the neutrophil serine proteases cathepsin-G or elastase may be able to support VCAM-1 shedding [30 ], as these proteases appear to promote mobilization of hematopoietic progenitor cells from the bone marrow by down-regulating VCAM-1 on bone marrow stromal cells. However, in the current study, the use of inhibitors of serine proteases such as aprotinin or eglin-c had no effect on the neutrophil-induced reduction in surface expression of VCAM-1 on EC. Zinc-dependent MMPs have also been reported to regulate the release of VCAM-1 from the surface of cerebral EC, a process that was abrogated using a broad-spectrum inhibitor of MMP function, marimastat, but not broad-spectrum antiproteases such as aprotinin or leupeptin [31 ]. Again, in the current study, the use of an inhibitor of MMP function had no effect on neutrophil-induced loss of VCAM-1 from HUVEC, although the inhibitor used (MMP inhibitor-1) was limited in its specificity. In addition, upon assessment of the levels of s-VCAM-1 in coculture supernatants, it was clear that proteolytic shedding from the surface of cells was unlikely to have occurred in our model, unless the molecule was internalized and degraded by neutrophils, a process that has not previously been described.

Initially, we thought it unlikely that neutrophils would regulate transcription of the VCAM-1 gene. However, studies using real-time PCR indicated that this was the case. It is interesting that the control of VCAM-1 transcription was quite specific, as message for ICAM-1 was unaltered. This result implies that neutrophils are not only aggressive executioners of the innate immune response, but they also have the potential to participate in a subtle process of cross-talk with other cell types at sites of their recruitment in a manner that might regulate the evolution of the inflammatory response. Indeed, the ability of neutrophils to down-regulate transcriptional activity of the VCAM-1 gene and thus, the adhesion of lymphocytes might act as a negative-feedback mechanism to fine-tune the inflammatory response, for example, delaying the transition of inflammation to a mononuclear phase and maintaining a response of acute character. Indeed, our observation, that neutrophils penetrate the EC monolayer more efficiently when they follow neutrophils recruited up to 20 h previously, adds weight to this hypothesis. Alternatively, there may be instances when a fully developed, inflammatory response is unnecessary, for example, during mild tissue trauma when neutrophils entering the damaged site do not require a secondary infiltrate of mononuclear leukocytes to resolve the inflammatory process and accordingly, bar their entry into the tissue. It is noteworthy that although we have managed to virtually abolish lymphocyte adhesion, this phenomenon was titratable and dependent on the number of neutrophils recruited into coculture by activated EC. Pathophysiologically, this would allow neutrophils to grade the degree of mononuclear leukocyte recruitment depending on the severity and/or stage of development of the inflammatory response. Mononuclear cells, once recruited to tissue, would probably also be able to modify the responses of EC [9 , 10 , 14 , 15 , 17 18 19 20 21 22 23 24 ] so that the nature of the leukocytic infiltrate during progression of inflammation would be dependent on the integration of a diverse array of signals by vascular EC, including those from recruited leukocytes.


   ACKNOWLEDGEMENTS
 
This work was supported by BHF Nonclincal Senior Lectureship BS/97001, BHF Program Grant RG/200011, and ARC Project Grant B0964.

Received May 25, 2004; accepted September 2, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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