Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

PMNs present in inflammed tissues provide a defense against the infiltration of infectious agents and the scavenging of necrotic tissue. However, in addition to their beneficial effects, the fulminating PMN infiltration observed in certain disease states involves damaging interactions with vascular tissue that result in injury to the endothelium. At sites of inflammation, PMN accumulation induces damage through the release of a number of mediators (Bevilaqua et al., 1985; Nathen, 1987; Weiss, 1989). In addition to endothelial tissue injury, direct effects of PMNs on vascular smooth muscle in the affected organs could exacerbate the damage by altering normal hemodynamics. Previous results using isolated vascular tissue have shown that the addition of PMNs influences the contractile tone of the vessel wall. However, uncertainty exists about the exact nature of the PMN response under conditions of inflammation in vascular tissue. In certain instances, PMNs have produced a vasorelaxation that was attributed to the release of NO (Rimele et al., 1988; Gonzales et al., 1992). Other investigators have shown that PMNs induce a vasoconstriction mediated by the interaction of reactive oxygen products with basally produced NO (Ohlstein and Nichols, 1989; Murohara et al., 1993). In each of these studies, variation in PMN activation state and species differences may play a role in the effects observed. It is important to note that the effect of human PMNs on human vascular tissue has not yet been adequately addressed.

Because the amount of vascular injury caused by infiltrating PMNs and the direct effect on smooth muscle may depend on their activation state, we were interested in measuring the activation state of PMNs and relating that to their biological response. Activated PMNs have previously been shown to change the level of expression of specific adhesion glycoproteins on their cell surface in response to inflammatory mediators (Carlos and Harlan, 1990; Springer, 1990; Anderson et al., 1986). These surface glycoproteins have been implicated in the adhesion of PMNs to sites of inflammation, so they represent useful markers for PMN activation (Jutila et al., 1989; Lewinsohn et al., 1987). Therefore, we chose to measure the activation state of PMNs by the expression of these proteins and relate that to their biological response on vascular smooth muscle contractility.

We used freshly harvested HUV for the vascular assay because this tissue is easily accessed for prompt experimentation. In addition, using isolated HUV makes it possible to compare vascular function with the large database generated with HUVEC grown in culture. Therefore, we can compare the cellular adhesion dynamics in this system with information already known in cultured HUVEC, including the release of mediators from the endothelium. HUV also offered the advantage of using human tissue to identify inhibitors that may be specific for human proteins involved in these interactions. Therefore, we measured the effect of human PMNs on isolated HUV, while measuring the activation state of the PMNs by immunofluorescence staining and flow cytometry. In an attempt to mimic an inflammatory state, we also tested PMNs using HUV that were stimulated by cytokines and PMNs that were activated with fMLP.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Preparation of umbilical vein segments. Human umbilical cords were obtained from Danbury Hospital within 12 h of delivery and placed into chilled and oxygenated PBS of the following composition (mmol/L): 118, NaCl; 4.7, KCl; 1.6, CaCl₂; 1.2, KH₂PO₄; 1.2, MgCl₂; 10.0, dextrose; 25.0, NaHCO₃; 0.02, NaEDTA; pH 7.25, and kept on ice before dissection. Sections of HUV or human umbilical artery (HUA) within 10 cm of the placenta were carefully dissected from the cord, cleaned of excess connective tissue and cut into 3 to 4-mm rings in a Petri dish filled with 4°C PBS. Rings were then placed into beakers containing PBS or PBS with a mixture of cytokines, TNF-, 50 U/ml, IL-1, 50 U/ml, and IFN-, 50 ng/ml, (Genzyme, Cambridge, MA), and were kept overnight at 4°C for use in tissue bath experiments the next day. In some of the segments, the endothelium was removed by inserting a length of polyethylene tubing into the lumen and gently twisting the ring around the tubing with thumb and forefinger. The rings were then suspended between two stainless steel wires, of which one was fixed to a solid glass support rod and the other was connected to a force transducer (Kent Scientific, Kent, CT), and placed into 37°C water-jacketed tissue baths containing PBS that was constantly oxygenated with 95% O₂ and 5% CO₂. Isometric force was continually measured and the data collected by a digital acquisition system (Po-Ne-Mah Inc., Simsbury, CT). The rings were placed under a preload of 5 g of force, which was continuously adjusted during the equilibration period to serve as base-line force. This amount of force was chosen from a series of preliminary force-force experiments that showed near maximum contraction with 125 mM KCl.

Immunohistochemical analysis of HUV. Segments of HUV were used for immunohistochemical analysis to determine the physical condition of the endothelium and the extent of endothelium denudation. Frozen sections of HUV segments were obtained by placing them in embedding medium (Tissue-Tek, Miles Inc., Elkhart, IN) and immediately immersing them in liquid nitrogen. These frozen tissues were stored at 70°C until thin sections were cut using a cryostat (Hacker Inst., Fairfield, NJ). Frozen tissues were obtained at different times during the experiments. A control frozen tissue was obtained immediately after dissection of the HUV segment. A second set of samples was obtained either following overnight incubation of the segments in PBS or after cytokine stimulation in PBS. A third group of samples was obtained at the end of the experiment and taken directly from the tissue baths. The tissues were treated with a monoclonal antibody for human PECAM-1 (mAb JC/70A) (BioGenex, San Ramon, CA) or ICAM-1 (BIRR-0001) (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT) and visualized by reaction with hemotoxylin-eosin stain. Stained sections were interpreted by an investigator on the basis of the intensity of staining and given a score for the percentage of cells stained (0 = none, 1 = 25%, 2 = 50%, 3 = 75%, 4 = 100%).

Isolation and preparation of neutrophils. Human PMNs were isolated by a method described previously (Henson et al., 1978). Whole blood (200 ml) was drawn by venous puncture of volunteers and collected into 20-ml sodium-heparin collection tubes. Erythrocytes were sedimented with 6% dextran (avg. mol. wt. 580 K; Sigma Chemical Co., St. Louis, MO). Approximately 25 ml of plasma was layered over 15 ml of ficoll-paque (Pharmacia Biotech, Uppsala, Sweden) in a separate 50-ml falcon tube and centrifuged at 1000 × g for 30 min at 4°C in a Sorval RT6000B centrifuge (Sorval Instruments, Wilmington, DE). The supernatant and peripheral blood mononuclear cell layer were aspirated off, and the pelleted cells were vortexed for 15 s with 10 ml of dH₂O to lyse any remaining erythrocytes. The cells were immediately washed two times in RPMI 1640 (Life Technologies Inc., Grand Island, NY). The cells were then suspended in 5 ml of RPMI and kept on ice while an aliquot was taken for cell counting using a hemocytometer. The cells were routinely stained with Wright-Giemsa stain (Sigma Chemical Co.) for analysis by cell differential. The cell preparations were found to be >95% PMNs using these methods. Dye exclusion assays using trypan blue showed the cells to be >95% viable, and staining for platelet contamination showed minimal number of platelets visualized per 100× field of view.

8

Determination of the activation state of neutrophils. In order to assess the level of PMN activation, we measured the level of adhesion glycoprotein expression on the PMNs both before and after stimulation by fMLP. Fluorescence-labeled antibodies for each protein were allowed to bind to the PMNs, and mean fluorescence was determined by flow cytometry. An aliquot of isolated PMNs (4 × 10⁶ cells) was taken both before and after activation by fMLP. In most cases the cells were taken immediately before addition to the tissue baths, but in some experiments the cells were taken directly from the bath and, after centrifugation at 600 × g, resuspended in phosphate buffer solution (Life Technologies Inc.) for treatment with antibodies. After the PMNs were washed two times, the cell pellet was suspended in 100 µl of phosphate buffer, and a 100-µl solution of mouse anti-human monoclonal antibody (200 µg/ml), phosphate buffer control or IgG isotype control was added, and the mixture was incubated on ice for 30 min. The cells were washed two times and incubated for 30 min with a 1:50 dilution of a phycoerythrin-conjugated goat anti-mouse IgG F(ab')₂ secondary antibody (Tago Inc., Burlingame, CA). The cells were washed two times and fixed in a 2% formalin solution (Sigma Chemical Co.). The fixed cells were kept at 4°C and in the dark before analysis by flow cytometry. Analysis was performed on a FACScan (Becton Dickinson, Mississauga, Ontario, Canada), and the data were analyzed using the PC-Lysys II computer software (Becton Dickenson). Cell population gates were set for PMNs on the basis of light scatter. Mean fluorescence intensity was measured and compared with nonbinding isotype IgG and with secondary antibody alone.

Procedure for testing the effect of PMNs on HUV. After the equilibration of either control HUV segments or cytokine-stimulated HUV, a cumulative contractile dose-response curve with serotonin (1 × 10⁹ to 3 × 10⁷ M) was performed on each tissue. This was done to determine the viability of each tissue, and unresponsive tissues were discarded at a rate of approximately 10% of those tested. The tissues were washed after the last dose of serotonin and allowed to equilibrate for 15 min to reach base-line force before any other test agents were added. Cell suspensions of PMNs in RPMI, either unactivated or activated (10⁸ M fMLP), were then added directly to the tissue baths. Cumulative concentrations of PMNs were added (0.15, 0.3, 0.5, 1, 2, × 10⁶ cells/ml), and the contractile response was allowed to plateau before addition of the next dose. In some experiments the PMNs (1 × 10⁶ cells/ml) were added after plateau of a preexisting serotonin contraction (1 × 10⁷ M). After the last dose of cells, the tissues were washed two times and allowed to equilibrate for 15 min before a second post-PMN dose-response curve was performed with serotonin. The response of serotonin before and after PMNs was compared to assess any effect on smooth muscle function. Washout of the serotonin was followed 15 min later by replacement of the PBS with 125 mM KCl. The maximum KCl contraction was used to normalize all previous responses by quantifying them as a percentage of the force generated to KCl.

5

4

Statistical analyses. The results are reported as the mean ± SEM. Data within each group were compared with the respective control value by a paired analysis based on test for normality. Either a paired t test or a Wilcoxon sign rank test for matched pairs was used to test for significance of the difference from control. An adjustment for multiplicity of measurement was done for all multiple comparisons with control values. Statistical significance was considered at the P < .05 level.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Effect of PMNs on isolated human umbilical vein. Both unactivated and fMLP-activated human PMNs produced a robust and dose-dependent contraction of isolated unstimulated HUV. This response was not significantly different between unactivated and activated PMN preparations (P > .05, unpaired analysis), (fig. 1). The contraction was evident within 5 min and, at higher doses, was sustained for up to 15 min. Similar contractile responses were seen using both unactivated and activated PMNs in preparations of HUA. With a single dose of PMNs, the contraction produced was sometimes phasic, represented by a series of contractions. The mean contraction of activated PMNs (1 × 10⁶ cells/ml) was 30 ± 6.8% (n = 11) of the maximum response to KCl. Furthermore, both unactivated and activated PMNs at 1 × 10⁶ cells/ml produced a vasocontraction of HUV precontracted with a submaximal (10⁷ M) dose of serotonin (data not shown). The mean contraction of HUV to 125 mM KCl was 13.2 ± 0.7 g (n = 27). A dose-response curve to fMLP in RPMI (1.0 × 10¹⁰ to 1.0 × 10⁶ M) indicated that there was no effect on HUV at the concentrations used in this study, even though concentrations of fMLP 100-fold greater did show a contractile effect (data not shown). The contractile response to both unactivated and activated PMNs was also tested in cytokine-stimulated HUV (fig. 2), and there was no significant difference between the vasocontraction induced in control vs. cytokine-stimulated HUV (compare figs. 1 and 2). PMN preparations free of contaminating platelets were tested and were shown to have identical contractile response, which indicates that the vascular contractions observed were not due to contaminating platelets (data not shown). In addition, comparison of the dose-response curve to serotonin before and after treatment of HUV showed no difference in the responsiveness of this contractile agonist with either unactivated or activated PMNs (fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Concentration-response curves for the addition of increasing concentrations of unactivated PMNs (, n = 10) and activated PMNs (, n = 10) to unstimulated HUV. Both unactivated and activated PMNs produced contractions of HUV, but their responses were not significantly different from each other. These responses were normalized to the percentage of the maximum response to 125 mM KCl. The activated PMNs were treated with 108 M fMLP 15 min before their addition to the tissue bath. Values shown are means ± S.E.M.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for the addition of increasing concentrations of unactivated polymorphonuclear leukocytes (PMNs) (, n = 8) and activated PMNs (, n = 8) to HUV stimulated with the cytokines IL-1, 50 U/ml; TNF-, 50 U/ml; and IFN-, 50 ng/ml. Both unactivated and activated PMNs produced contractions of HUV, but their responses were not significantly different from each other. These responses were normalized to the percentage of the maximum response to 125 mM KCl. The activated PMNs were treated with 108 M fMLP 15 min before their addition to the tissue bath. Values shown are means ± S.E.M.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Concentration-response curve to serotonin before (, n = 4) and after (, n = 4) the addition of a single dose (1 × 106 cells/ml) of activated PMNs to HUV. Serotonin produced a concentration-dependent contraction of HUV that was not significantly different after the addition of activated PMNs. The response to serotonin was normalized to the percentage of the maximum response to 125 mM KCl. Values shown are means ± S.E.M.

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Representative recorder tracings illustrating the effect of the supernatant (Sup.) from a low-speed spin of unactivated PMNs (2 × 107 cells, panel A) on the tone of HUV. Both the unactivated cell suspension (Unact. Cells + Sup.) and the supernatant produced a contraction of HUV. The resuspended cells (Cells) showed no response. Panel B illustrates the effect of the supernatant from a low-speed spin of activated PMNs (2 × 107 cells) on isolated HUV. Only the activated cell suspension (Act. Cells + Sup.) produced a response. The resuspended cells were difficult to test because of clumping.

Reproducibility of the PMN-induced contraction. In order to determine the consistency of the contractile response to PMNs so that each tissue could be used as its own control, we tested the response of HUV to repeated additions of a single (1 × 10⁶ cells/ml) dose of PMNs. The vascular responses to both unactivated and activated PMNs were shown to be very consistent between the first and second administrations of cells (fig. 5). The mean variation between the first administration of PMNs and the second was less than 10% testing of 4 to 8 separate PMN preparations in seven different HUV tissues.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Bar graph showing the effect of sequential doses of both unactivated and activated PMNs on HUV. The solid bars represent the first dose (1 × 106 cells/ml) of PMNs, and the hatched bars represent an identical dose of cells given 15 min after washout of the first dose. The responses of the two doses were nearly identical for both the unactivated and the activated PMNs. The responses were normalized to the percentage of the maximum contraction to 125 mM KCl. Values shown are means ± S.E.M. (n = 8, unactivated PMNs; n = 6, for activated PMNs).

Vascular response to PMNs. We attempted to determine the nature of the vascular contraction to PMNs by the addition of agents known to interfere with the release of vasoactive compounds from cells. Table 1 shows the results of experiments designed to affect the contraction of HUV by both unactivated and activated PMNs. Treatment with indomethacin, SOD or L-NMMA had no significant effect on the response of either unactivated or activated PMNs. However, treatment of PMNs with a high concentration of the leukotriene biosynthesis inhibitor BIRM-270 (0.5 µM) significantly decreased the contraction of HUV produced by unactivated PMNs by 61 ± 19% (P < .05), but not that produced by activated PMNs. The inhibition shown by lower concentrations of BIRM-270 was not significant, and administration of BIRM-270 alone at 0.5 µM induced no response.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Bar graph summarizing the contractile response of both unactivated and activated PMNs on HUV with endothelium, (solid bars, n = 10) and endothelium-denuded, (hatched bars, n = 6). The responses of two different concentrations of PMNs are shown and normalized to the percent of the maximum contraction to 125 mM KCl. Values are means ± SEM, *P < .05 vs. endothelium-intact tissues.

Measurement of the activation state of PMNs. Because both unactivated and activated PMN preparations caused a vascular response, we measured the activation state of each preparation before addition to HUV by flow cytometric analysis of adhesion glycoprotein expression. After activation by fMLP, expression of Mac-1 on the PMN surface was significantly increased, and expression of L-selectin was significantly decreased, from values for the control unactivated PMNs. Figure 7 shows the ratio of Mac-1 to L-selectin in both preparations of PMNs and demonstrates the activation state of the cells. There was a significant increase in the ratio after incubation with fMLP. We also isolated PMNs directly from the tissue bath 20 min after addition of unactivated PMNs to HUV and found that these cells had a similar ratio to the unactivated PMNs. Using activated PMNs (1 × 10⁶ cells/ml), we observed a significant correlation of the activation state with the vascular response (r = 0.77, P < .05) (fig. 8). The correlation using unactivated PMNs was not significant, the cells showing a range of contractile responses with a fairly consistent Mac-1/L-selectin ratio near unity (data not shown). However, several PMN preparations were identified in which L-selectin was not decreased after activation by fMLP but remained near control levels. These preparations showed varying levels of responsiveness with a low activation state and were not included in the analysis of activated PMNs.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Bar graph showing the activation state of different preparations of PMNs as measured by their Mac-1/L-selectin ratio. This ratio was obtained by FACS analysis of the mean fluorescence intensity of these two proteins on PMNs from unactivated cells (Unact., open bar, n = 10), PMNs activated for 15 min at 37°C with 108 M fMLP (Act., solid bar, n = 10) and unactivated PMNs isolated from the tissue bath after 15 min (Unact. Bath, hatched bar, n = 4). Values shown are means ± SEM (*P < .05 vs. Unact. and Unact. Bath).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Scatter plot of the relationship between the activation state of activated PMNs and the vasocontraction produced. Activation state was determined as the Mac-1/L-selectin ratio, and vasocontraction was measured as a percentage of the response to 125 mM KCl. The correlation coefficient (r) was equal to 0.77 (P < .05). A best-fit line was drawn from the data using the method of least squares. There was no significant correlation when unactivated PMNs were used.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

We investigated the interaction of human PMNs with human vascular tissue to determine the extent to which cellular activation state was involved in PMN-induced vascular responsiveness. We determined that both unactivated and activated PMNs induced a cell number-dependent vasocontraction in HUV, the nature of which was dependent on the activation state of the cells. The vasoconstrictor response observed for unactivated PMNs was endothelium-independent, was due to soluble factor(s) in the cell supernatant and was partially blocked by an inhibitor of leukotriene biosynthesis. For the activated PMNs, the response was endothelium-dependent, was not due to a soluble factor and was linearly related to their activation state.

One of the most interesting findings of this study was that both unactivated and activated PMNs contracted HUV but that their respective responses were quite different in nature. Treatment of HUV with unactivated PMNs had no significant effect on the response to serotonin, which suggests that there was no sustained modulatory effect on vascular smooth muscle function. Moreover, we showed that the observed vasocontraction was due to a soluble factor or factors released into the cell supernatant. This active contractile agent was not affected by indomethacin, SOD or L-NMMA, which indicates that cyclooxygenase products, oxygen radicals or nitric oxide does not account for the vasocontraction observed. We are presently examining whether these pathways may play a role in other human vascular preparations. Though the PMNs appear unactivated, we found evidence that the vasocontraction is partially blocked by a leukotriene biosynthesis inhibitor (table 1), which indicates that the PMNs may release vasoactive leukotrienes such as LTB₄ that may account for their contractile activity. These results would be consistent with our finding that the response to unactivated PMNs was endothelium-independent.

However, the nature of the vasocontraction obtained by treatment with activated PMNs was quite different from that obtained with unactivated PMNs. Much like unactivated PMNs, activated PMNs had no effect on the response of smooth muscle to serotonin. Furthermore, activated PMNs show no activity in their cell-free supernatants, which indicates that the cells were necessary for vasocontraction and that their activity was not due to release of a stable vasoactive substance into the supernatant. However, it is possible that any potential vasoactive agent released was labile and thus was not detected in our assay system. Second, the response to activated PMNs was endothelium-dependent. The data in figure 6 indicate a significant decrease in PMN-induced vasocontraction in endothelium-denuded HUV, but the response was not abolished.

Our failure to block the response to activated PMNs completely may be a consequence of incomplete endothelium removal. Though our analysis included only HUV segments determined immunohistochemically to be endothelium-denuded, our efforts were by the limited ability of our immunohistochemical technique to resolve completely the integrity of the endothelium on an entire segment. Our endothelium-denuded HUV segments probably represent a combination of HUV denuded of endothelium and HUV incompletely denuded. However, one cannot rule out a partial endothelium-independent response. Activated PMNs were not affected by treatment with BIRM-270, which indicates that leukotriene products are not likely to be responsible for the observed vasocontraction.

We were unable to determine the exact nature of the smooth muscle contraction mediated by activated PMNs. Experiments designed to inhibit cyclooxygenase, reactive oxygen species, leukotriene biosynthesis and nitric oxide synthase had no effect on the response to activated PMNs (table 1). Previous studies (Sessa et al., 1991) demonstrated that human PMNs can produce endothelin-1, but these authors found that endothelin-1 production was more pronounced in unactivated than in activated PMNs, so it is unlikely that endothelin-1 mediates the vasocontraction that we observed. The possibility exists that it is the binding event itself that induces the release of a contractile factor from the endothelial cells. In support of this possibility is a previous study that showed that cross-linking of ICAM-1 on leukocytes leads to a signaling event (respiratory burst) in these cells (Rothlein et al., 1994).

The efficacious vasocontraction that we observed during the interaction of PMNs with vascular tissue indicates that infiltrating PMNs at sites of inflammation can be expected to alter vascular smooth muscle function as well as causing the tissue damage that has been documented. The maximum concentrations of PMNs used in this assay (1-2 × 10⁶/ml) were similar to those found in our isolates from whole blood. Therefore, the alterations in smooth muscle contractility at local inflammatory sites would be expected to modulate the hemodynamic character of the affected or inflamed organs, possibly exacerbating the inflammatory response. In a study of the microvascular vessels of humans and other species, Brain et al. (1989) demonstrated a similar finding by showing that endothelin-1 produced local vasoconstraction during neutrophil accumulation in skin tissue. Furthermore, a review of the differential effects of kinins (Regoli et al., 1993) indicates that kinins can produce both arterial vasodilation and venoconstriction. Though we observed the direct effects of PMNs on vascular contractility, we were unable to determine whether treatment of HUV with PMNs produced any endothelial dysfunction. Immunohistochemical results indicated that there was no clear sign of endothelium damage after treatment with PMNs. Our results are supported by a previous study using isolated canine PMNs with canine coronary arteries, in which no clear signs of endothelium damage were detected (Minamino et al., 1996). However, our results are indicative of the level of endothelium that is present and do not address endothelium function.

Because we were unable to demonstrate an endothelium-dependent relaxation in HUV, we were unable to determine the effect of PMNs on endothelium function. A previous report by Bodelsson and Stjernquist (1994) identified substance P as an endothelium-dependent vasorelaxant in HUV, but we were unable to obtain similar results. A recent report (Izumi et al., 1996) suggests that histamine acts as an endothelium-dependent agonist in HUV, but the response was evident only in vessels obtained at midgestation. Our inability to identify an endothelium-dependent agonist in HUV is in agreement with previous studies (Monuszko et al., 1990; Pomperantz et al., 1978; Tulenko, 1979; Altura et al., 1972). We do not know whether this is a tissue-specific phenomenon; other investigators (Murohara et al., 1994; Lefer et al., 1994) have shown endothelial damage after treatment with PMNs using other tissues. The lack of a pronounced endothelium-dependent response in HUV may be indicative of other vascular beds in which there is a reduced response to endothelium-dependent agonists.

There is considerable discrepancy in the literature on the nature of the vascular response to PMNs in vitro. A previous report by Gonzales et al. (1992), the only study wherein human PMNs and human vascular tissue were used, showed that activated PMNs induced a vasorelaxation in human mammary artery that the authors attributed to release of NO. A second study (Rimele et al., 1988) using rat aorta and isolated rat peritoneal PMNs supports the previous results that used human tissue. However, studies by Lefer et al. (Murohara et al., 1994; Lefer et al., 1994) using isolated cat PMNs, Ohlstein and Nichols (1989) using rat peritoneal PMNs and Murohara et al. (1993) using human PMNs on isolated pig coronary arteries have demonstrated PMN-induced vasocontraction. These studies have concluded that the vasocontraction is due to release of reactive oxygen species that inactivate basally produced NO. Much of the difficulty in comparing these different results is the authors' use of PMNs under different activation conditions and with different species. None of these previous studies have attempted to measure the activation state of the PMNs and relate this to activity.

We constructed a ratio analysis, Mac-1 to L-selectin, for determining the activation state of PMNs. The Mac-1/L-selectin ratio was used to normalize the expression levels from different preparations of PMNs and to compare the biological responses of unactivated and activated PMNs. The Mac-1/L-selectin ratio for activated PMNs was 8-fold higher than for unactivated PMNs and was similar to the activated state of PMNs isolated from in vivo models of inflammation if one applies the same ratio of Mac-1/L-selectin expression (Jutila et al., 1989; Lewinsohn et al., 1987). Furthermore, the results in figure 8 show that after PMN activation by fMLP, we were able to show a significant relationship between the activation state of the cells and the vasocontraction observed. It is clear that once they are activated with fMLP, the PMNs show a linear relationship between increasing activation state and increasing vasocontraction. This relationship was not observed with unactivated PMNs, which suggests that fMLP treatment induced a change in "activation" of PMNs in which the expression of Mac-1 and L-selectin were linearly related to vasoactivity and that we were not observing a simple continuum from unactivated to activated PMNs. Using this ratio could also help to elucidate the differences reported in the literature between various studies that have examined the interaction of PMNs and vascular tissue.

Because our measurement of the activation state of PMNs is based on the ratio of adhesion glycoproteins, it is possible that the PMN-induced vasocontraction we observed with activated PMNs is due to a cellular adhesion-dependent event. The cellular adhesion-dependent nature of PMN-induced vasocontraction is supported by previous studies with isolated feline PMNs (Murohara et al., 1994; Lefer et al., 1994) and canine PMNs (Minamino et al., 1996). We are presently conducting experiments to test the cellular adhesion dependence of the response of activated PMNs.

Even though we were unable to determine the exact nature of the PMN-induced vasoconstriction in HUV, we were able to identify an effective, reproducible response using both unactivated and activated PMNs in human tissue. Furthermore, we were able to show a significant correlation between the vasocontraction produced and the activation state of the PMNs being tested. It may also be possible to tailor the conditions to investigate the release of vasoactive factors by using unactivated PMNs or to study the effects of cellular adhesion events by using activated PMNs. This may be a useful in vitro model to investigate whether the response to PMNs has any implications for understanding vasoconstriction events precipitated by infiltrating inflammatory cells in in vivo models of inflammation.