© 1996 American Heart Association, Inc.
Articles |
Heterogeneity of Expression of E- and P-Selectins In Vivo
the Department of Physiology and Biophysics (M.J.E., D.N.G.), Louisiana State University Medical Center, Shreveport; the Division of Inflammation/Autoimmune Diseases (B.W., M.A.L.), Hoffmann–La Roche Inc, Nutley, NJ; and Discovery Research (D.C.A.), Pharmacia and Upjohn Laboratories, Kalamazoo, Mich.
Correspondence to Michael J. Eppihimer, PhD, Department of Physiology and Biophysics, LSU Medical Center, 1501 Kings Hwy, Shreveport, LA 71130-3932. E-mail meppih@lsumc.edu.
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A novel technique involving radiolabeled monoclonal antibodies was used to characterize and compare the expression of E- and P-selectin on unstimulated, histamine-challenged, and endotoxin-challenged endothelial cells in various tissues of the mouse. Under unstimulated conditions, E-selectin was absent in all organs, but significant expression of P-selectin was observed in several organs. Histamine induced a rapid time-dependent upregulation of P-selectin, with the largest responses observed in mesentery and lung. Significant fold elevations in P-selectin expression occurred as early as 5 minutes after the histamine injection and remained elevated up to 1 hour. Histamine-induced P-selectin upregulation was inhibited by the H1 receptor antagonist diphenhydramine, whereas the H2 receptor antagonist cimetidine had no effect. Endotoxin (lipopolysaccharide [LPS]) also induced a time-dependent expression of P-selectin that reached a maximum between 4 and 8 hours after endotoxin administration. LPS-induced upregulation of P-selectin was greatest in heart and stomach, which exhibited insignificant constitutive expression of P-selectin. LPS also induced a time-dependent upregulation of E-selectin, with maximal expression occurring 3 to 5 hours after intraperitoneal administration. The lung and small intestine exhibited the largest responses to LPS challenge. Histamine administration did not affect E-selectin expression in any tissue. E- and P-selectin–deficient mice were used to test the specificity of monoclonal antibody binding in unstimulated, histamine-challenged, and LPS-stimulated tissues. Vascular binding of the radiolabeled E-selectin and P-selectin monoclonal antibodies was not observed in the respective deficient mice. These findings suggest that P-selectin is constitutively expressed on vascular endothelium in some tissues of the mouse and that there are significant regional differences in the magnitude and time course of histamine- and endotoxin-induced P-selectin expression. In contrast, E-selectin appears to be absent on unstimulated vascular endothelium but is upregulated within 3 hours after the administration of endotoxin in most tissues.
Key Words: endothelium • adhesion molecule • selectin • leukocyte • inflammation
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The emergence of leukocytes (WBCs) from the vascular to the interstitial compartment is regulated at the contact zone between WBCs and ECs by an array of CAMs. This migration process represents a cascade of events initiated by the selectin family of CAMs, which mediates the rolling of WBCs along the endothelium of postcapillary venules. The selectins represent a family of three structurally similar carbohydrate-binding lectins, consisting of an N-terminal lectin domain, an epidermal growth factor domain, and a series of consensus repeats similar to those in complement proteins.1 L-selectin is constitutively expressed on the surface of all WBCs and is shed by proteolysis after cell activation.2 Both E- and P-selectin are expressed on the surface of stimulated ECs, with the latter also found on the surface of activated platelets. P-selectin is stored in the Weibel-Palade bodies of ECs, where it can be rapidly (within minutes) mobilized to the cell surface after stimulation with inflammatory mediators such as histamine, thrombin, or peroxides.3 4 In contrast, there is no preformed pool of E-selectin, but this CAM can be transcriptionally induced, with maximal presentation of the glycoprotein to the cell surface occurring 4 to 6 hours after EC stimulation by cytokines such as tumor necrosis factor-
and interleukin-1ß. Recent studies suggest that P-selectin is also regulated by transcriptional mechanisms that function parallel with and are independent of the rapidly induced translocation of P-selectin from storage granules in ECs.5 Numerous studies have implicated E- and P-selectin as important mediators of WBC-EC interactions during acute and chronic inflammatory reactions.6 7 8 9 Studies in this area have relied on mAbs directed against functional epitopes of the selectins or gene-targeted mice that are deficient in a specific CAM to delineate the relative contribution of selectins to WBC rolling. For example, it has been shown that exposure of a P-selectin mAb to histamine-activated endothelium in vitro10 or in vivo11 completely blocks WBC rolling. Similarly, WBC rolling was observed to be attenuated in postcapillary venules of P-selectin gene–deficient mice.12 In contrast, a definitive role for E-selectin in the inflammatory process has not been established, with several in vivo and in vitro studies13 14 15 arguing that E-selectin has the capacity to support WBC rolling, but with recent reports demonstrating a lesser role for E-selectin in WBC rolling.8 16 A possible explanation for these findings is the proposal that E- and P-selectin have partially overlapping functions in the inflammatory process, as evidenced by the inability of E-selectin–specific antibodies to add to the blocking effect of anti–P-selectin antibodies.17 This redundancy in selectin function has been further substantiated in E-selectin–deficient mice, in which WBC emigration was unaffected (compared with wild-type mice) in several models of inflammation.16 However, administration of a P-selectin–specific antibody in E-selectin–deficient mice does significantly attenuate WBC emigration. Furthermore, this redundancy in selectin function may exist because of the binding of E- and P-selectin to a related site on PGSL-1, which is located on neutrophils, to which P-selectin binds with a greater efficiency and adhesive strength than does E-selectin.18
It has generally been inferred that WBC rolling during an inflammatory process is coupled to an elevation in E- and P-selectin expression on the surface of ECs in postcapillary venules. This is supported by observations of increases in the tissue levels of selectin mRNA19 and the number of microvessels immunostained for E- and P-selectin20 21 in inflamed tissues. Although these indirect methods have provided qualitative data that is consistent with the view that endotoxin and/or histamine increase the expression of E- and P-selectin on microvascular ECs, no quantitative information is available that allows for an accurate assessment and comparison of the magnitude and kinetics of selectin expression in different tissues exposed to an inflammatory stimulus. Recently, a novel technique, based on the use of radiolabeled mAbs, was developed to quantify the expression of CAMs in vivo.22 23 Given the absence of quantitative information regarding the expression of E- and P-selectin in vivo, we have modified the dual-radiolabeled antibody technique, as described by Panes et al,23 in an effort to characterize the time course and magnitude of E- and P-selectin expression in different tissues of mice after histamine and endotoxin challenge.
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mAbs
The mAbs used for the in vivo assessment of P- and E-selectin were RB40.34, a rat IgG1 against mouse P-selectin18 (Pharmingen Inc), and 10E6, a rat IgG2 against mouse E-selectin,24 respectively. The antibody RB40.34, which is directed against P-selectin, has been shown by immunohistochemical staining to be localized on ECs and platelets in blood vessels of wild-type mice and to be absent in mice deficient in the P-selectin gene.25 P-23, a nonbinding murine IgG1 directed against human P-selectin, was also used in the experimental protocols.26
Radioiodination of mAbs
All binding mAbs (RB40.34 and 10E6) and nonbinding mAbs (P-23) were labeled with 125I and 131I (Du Pont NEN), respectively, using the chloramine T method.27 In brief, a 1-mg sample of mAb in 1.5 mL PBS (pH 7.4) was added to 125I or 131I, with a total activity of 1.0 mCi, and 100 µg of chloramine T. The mixture was incubated at room temperature for 1 minute, and 62.5 µg of sodium metabisulfate was then added. The total volume was brought to 2.5 mL by adding PBS. Thereafter, the coupled mAb was separated from free 125I or 131I by gel filtration on a Sephadex PD-10 column. The column was equilibrated and then eluted with PBS containing 1% bovine serum albumin. Two fractions of 2.5 mL were collected, the second of which contained the radiolabeled antibody. Absence of free 125I or 131I was ensured by extensive dialysis of the protein-containing fraction. Less than 1% of the activity of the protein fraction was recovered from the dialysis fluid. These procedures were previously applied in this laboratory for preparing a rat ICAM-1 mAb for measurements of the expression of this CAM in vivo.23
Animal Procedures
Male C57Bl/6J mice (n=110) weighing 23.8±2.7 (mean±SD) g were used in the radiolabeled antibody experiments. The mice were anesthetized intramuscularly with a mixture of ketamine and xylazine at a dose of 150 and 7.5 mg/kg, respectively. The left jugular vein and descending abdominal aorta were cannulated with polyethylene tubing (PE-10). For assessing ECAM expression, a mixture of 10 µg of either 125I P-selectin mAb (RB40.34) or 125I E-selectin mAb (10E6) and a dose (0.5 to 5.0 µg) of 131I nonbinding mAb (P-23) was injected through the jugular vein catheter. A blood sample was obtained through the abdominal aorta catheter 5 minutes after injection of the mAb mixture. The animals were then heparinized (40 U sodium heparin) and rapidly exsanguinated by perfusion of bicarbonate-buffered saline through the jugular vein catheter with simultaneous blood withdrawal through the abdominal aorta catheter. This was followed by perfusion of 10 mL of bicarbonate-buffered saline through the abdominal aorta catheter after severing the inferior vena cava at the thoracic level. Entire organs were harvested and weighed.
Calculation of E- and P-Selectin Expression
The method for calculating the expression of ECAMs has been described previously.23 In brief, the 125I (binding mAb) and 131I (nonbinding mAb) activities in different tissues and in 50 µL samples of cell-free plasma were counted in a 14800 Wizard 3 gamma counter (Wallac), with automatic correction for background activity and spillover. The total injected activity in each experiment was calculated by counting a 4-µL sample of the radiolabeled mAb mixture. The radioactivities remaining in the tube used to mix the mAbs and the syringe used to inject the mixture were subtracted from the total injected activity and were, on average, <1% of the total injected activity. The accumulated activity of each mAb in an organ was expressed as the percentage of the injected activity per gram of tissue. E- and P-selectin expression was calculated by subtracting the accumulated activity per gram tissue of the nonbinding mAb (131I P-23) from the activity of the binding anti–E-selectin mAb (125I 10E6) or anti–P-selectin mAb (125I RB40.34), respectively. Previous studies have shown that mAbs retain their functional activity after radioiodination, as evidenced by a similar effectiveness of labeled and unlabeled mAbs to block WBC adherence in rat mesenteric venules.23
Experimental Protocols
Initial experiments were performed to assess the total doses of anti–E-selectin and anti–P-selectin mAb that were necessary to saturate all of the adhesion receptors expressed on vascular ECs. An optimal dose of labeled (hot) binding mAb was determined when a concomitant dose of unlabeled (cold) binding mAb resulted in a proportional decrease in the percentage of injected activity per gram of tissue. The dose of anti–E-selectin and anti–P-selectin mAbs that saturated all adhesion receptors was found to be 10 µg for each mAb. In order to assess potential differences in the constitutive expression of E- and P-selectin between tissues and the time course and magnitude of E- and P-selectin in different tissues after systemic histamine or endotoxin challenge, the following protocol was used. Constitutive, histamine-induced, and endotoxin-induced expression of E- and P-selectin were assessed by injecting a mixture of radiolabeled binding and nonbinding mAbs. The mixture consisted of either 10 µg of labeled anti–E-selectin (10E6) or anti–P-selectin mAb (RB40.34) and a dose of labeled nonbinding mAb (P-23) ranging from 0.5 to 5 µg. A variable dose of nonbinding mAb was used to compensate for the decay in activity of the 131I isotope, which has a half-life of 
8 days. The time course and magnitude of histamine- and endotoxin-induced selectin expression in different tissues were determined in animals receiving 0.5 mg of histamine (intravenously) and 50 µg of endotoxin (LPS) (intraperitoneally or intravenously), respectively; thereafter, the organs were harvested at several time points. To assess the specificity of the 125I mAbs to bind to their respective ligands, the accumulation of 125I 10E6 and 125I RB40.34 was measured in E- and P-selectin–deficient mice, respectively, after LPS or histamine challenge. The P-selectin–deficient mice were provided by Upjohn Laboratories (Kalamazoo, Mich),25 whereas the E-selectin–deficient mice and the relevant strain of background mice (129sv) were provided by Hoffman–La Roche Laboratories (Nutley, NJ).17 The ability of histamine receptor antagonists to prevent histamine-induced P-selectin expression was assessed by intravenous administration of 0.75 mg of an H1 receptor antagonist (diphenhydramine) or an H2 receptor antagonist (cimetidine) immediately before the injection of histamine. Tissues were harvested 10 minutes after histamine injection, since maximal expression of P-selectin coincided with this time point.
To assess the contribution of platelet-bound 125I mAb RB40.34 to total tissue 125I mAb RB40.34 activity, an IIbß3 (the protein component of adhesion molecule of GPIIb/IIIa) inhibitor (TP9201) (a gift from James O. Tolley, Telios Pharmaceuticals, San Diego, Calif) was administered to unstimulated, histamine-stimulated, and LPS-stimulated mice. The peptide TP9201 has been previously shown to be a potent inhibitor of platelet aggregation and thrombosis by selectively blocking the binding of IIbß3 to fibrinogen.28 29 30 For LPS-stimulated mice, a 7-mg/kg dose of TP9201 was administered intramuscularly 60 minutes before receiving an intraperitoneal injection of 50 µg LPS. Given the clearance of TP9201 from the circulation, an additional dose (3 mg/kg) of TP9201 was administered 3 hours after the LPS injection, and 1 hour later, the mixture of radiolabeled mAbs was then injected into the mice, and the tissues were harvested. Histamine-stimulated mice received a single 7 mg/kg dose of TP9201 intravenously 15 minutes before an intravenous injection of 0.5 mg histamine. Similarly, unstimulated mice were administered a single 7 mg/kg dose of TP9201 intravenously 15 minutes before the injection of the radiolabeled mAb mixture. After the injection of the radiolabeled mAb mixture, all mice were exsanguinated and their tissues harvested as described previously.
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Constitutive and Histamine-Induced P-Selectin Expression
Significant regional differences were found in the tissue distribution of 125I mAb RB40.34 in the unstimulated state (Table 1
), with several tissues exhibiting an insignificant level of 125I mAb RB40.34 binding. The accumulation of 125I mAb RB40.34 observed in most of the tissues of untreated mice suggests the presence of a basal (constitutive) expression of P-selectin. The presence of constitutive P-selectin is substantiated by the ability of unlabeled mAb RB40.34 to reduce the accumulation of 125I mAb RB40.34. Intravenous administration of histamine induced a time-dependent increase in the accumulation of 125I mAb RB40.34 in all tissues except brain. Fig 1 shows the differences in the time course and magnitude of 125I mAb RB40.34 accumulation in the heart, mesentery, small intestine, and muscle after systemic administration (intravenous) of 0.5 mg histamine. There were significant increases in the accumulation of 125I mAb RB40.34 as early as 5 minutes after the injection of histamine, with a sustained accumulation of 125I mAb RB40.34 up to 1 hour in the heart and muscle. In general, maximal accumulation of 125I mAb RB40.34 occurred between 10 and 30 minutes after the injection of histamine. As shown in Fig 1, histamine-induced accumulation of 125I RB40.34 per gram of mesenteric tissue was approximately an order of magnitude greater than that of heart or muscle tissue. Measurements of 125I mAb RB40.34 accumulation at 4 and 8 hours after histamine injection revealed an invariant increase compared with constitutive values.
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Fig 2 illustrates the accumulation of 125I mAb RB40.34 in the small intestine and muscle of mice that were injected with either an H1 (diphenhydramine) or H2 (cimetidine) receptor antagonist before stimulation with histamine. The accumulation in both small intestine and muscle were completely inhibited with diphenhydramine, whereas cimetidine had no significant effect in reducing the accumulation of 125I mAb RB40.34 compared with histamine (P>.05). A similar pattern of mAb accumulation in response to histamine with or without H1 and H2 receptor antagonists was noted in all other tissues studied.
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LPS-Induced Expression of P-Selectin
Fig 3 illustrates the time course and magnitude of P-selectin expression in the heart, mesentery, small intestine, and muscle after LPS challenge. The remaining data for other organs are summarized in Table 2. Significant differences in the time course and magnitude of 125I mAb RB40.34 accumulation were found between tissues. Maximal accumulation of 125I mAb RB40.34 was found to occur between 4 and 8 hours after LPS injection. In some tissues (eg, heart and muscle), significant accumulation of 125I mAb RB40.34 was noted as early as 10 and 30 minutes after intravenous administration of LPS, suggesting the translocation of P-selectin from storage granules to the cell surface. As shown in Fig 3, at 8 hours after injection of LPS, the magnitude of 125I mAb RB40.34 accumulation was significantly attenuated compared with that at 4 hours (P<.05). In most tissues, the activity of 125I mAb RB40.34 remained significantly above constitutive values at 24 hours after LPS administration. In comparison, LPS-induced P-selectin expression was significantly greater at 4 hours compared with histamine-induced P-selectin expression at 10 minutes for all tissues studied (P>.05). For example, in the heart, LPS-induced expression of P-selectin was 20 times greater than that elicited by histamine, whereas only a twofold difference in expression for the two stimuli was noted in the mesentery.
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In order to assess the effect of platelet retention in the vasculature on P-selectin expression in unstimulated tissues and tissues stimulated with LPS or histamine, a peptide (TP9201) directed against the GPIIa/IIIb adhesion receptor was administered to mice to inhibit platelet-EC adhesion. For control, histamine-stimulated, and LPS-stimulated mice, administration of the GPIIa/IIIb receptor antagonist TP9201 resulted in an insignificant change in P-selectin expression in organs compared with organs in mice not treated with the GPIIa/IIIb antagonist (P>.05) (data not shown).
Constitutive and LPS-Induced Expression of E-Selectin
Intraperitoneal injection of LPS induced a time-dependent increase in the accumulation of 125I mAb 10E6 in all tissues studied. Fig 4 illustrates the time course and magnitude of 125I mAb 10E6 accumulation in the heart, mesentery, small intestine, and muscle. The remaining data for other organs are summarized in Table 3. Significant differences in the time course and magnitude of 125I mAb 10E6 were found between tissues (P<.05). In all the tissues except the heart, mesentery, and small intestine, an insignificant accumulation of 125I mAb 10E6 was observed under baseline conditions (P>.05). The addition of a dose of cold mAb 10E6 resulted in a decrease in the accumulation of 125I mAb 10E6 in the hearts but not in the mesentery and small intestine of untreated mice, suggesting that there is a significant amount of constitutively expressed E-selectin in heart vascular endothelium. A 50 µg dose of cold mAb 10E6 was also found to proportionally decrease the accumulation of 125I mAb 10E6 in tissues of LPS-treated mice, indicating that the accumulation of 125I mAb 10E6 in organs is due to its specific binding to its ligand. In all tissues, the accumulation of 125I mAb 10E6 reached maximal levels between 3 and 5 hours after LPS administration. By 8 hours after LPS injection, the accumulation of 125I mAb 10E6 was significantly attenuated compared with values at 3 and 5 hours (P<.05). In contrast to 125I mAb RB40.34 (P-selectin), the tissue activity of 125I mAb 10E6 returned to constitutive values at 24 hours after LPS injection. Intravenous administration of 0.5 mg histamine resulted in an insignificant (P>.05) change in 125I mAb 10E6 accumulation compared with constitutive values (data not shown).
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E- and P-Selectin Expression in Selectin-Deficient Mice
In order to demonstrate the specificity of the 125I mAb 10E6 and 125I mAb RB40.34 accumulation in tissues, 10 µg of either 125I mAb 10E6 or 125I mAb RB40.34 was injected into mice that were deficient in the E- or P-selectin genes, respectively. Fig 5 illustrates the accumulation of 125I mAb RB40.34 in the heart and small intestine of P-selectin–deficient mice (which was not significantly different from a value of zero) and C57Bl/6 background mice that were treated with either histamine or LPS. Tissues of P-selectin–deficient mice injected with histamine or LPS were harvested at 10 minutes and 4 hours after mediator administration, respectively. Similarly, an accumulation of 125I mAb 10E6 was not observed in E-selectin–deficient mice that were either untreated or treated with LPS. As shown in Fig 6, the activity of 125I mAb 10E6 in the hearts and small intestines of E-selectin–deficient mice was not significantly different from a value of zero for all treatments studied (P>.05). Similar findings of the accumulation of 125I mAb 10E6 and 125I mAb RB40.34 were found in all other tissues of selectin-deficient mice (data not shown). The absence of tissue activity of 125I mAb 10E6 and 125I mAb RB40.34 in LPS-treated E- and P-selectin–deficient mice, respectively, demonstrates that the selective accumulation of 125I mAb is due to specific binding of these mAbs to their ligand.
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Radiolabeled mAbs have proved to be a valuable tool for quantifying the in vivo expression of ECAMs with a precision not previously attained using immunohistochemical methods.22 23 Recently, the dual-radiolabeled mAb technique was used to quantify both constitutive and induced ICAM-1 expression in a rat model of inflammation.23 That study revealed that the radiolabeling procedure did not alter the functionality of the mAb and that the selective accumulation of the 125I mAb was due to the specific binding to its ligand. Moreover, rather profound differences in constitutive and induced ICAM-1 expression were noted to exist between tissues, suggesting that the contribution of ICAM-1 to leukocyte adherence and emigration in postcapillary venules may vary between different tissues. In order to determine whether similar regional heterogeneity of CAM expression is also manifested for the leukocyte-rolling receptors expressed by vascular endothelium, we used the dual-radiolabeled mAb technique to quantify both constitutive and induced expression of E- and P-selectin in several tissues of the mouse.
Relatively little is known about the modulation and kinetics of P-selectin expression in vivo. Prior in vitro3 31 and in vivo studies32 using immunohistochemical procedures have suggested that little or no P-selectin is expressed on the surface of unstimulated ECs. In contrast, the present study offers evidence that a significant amount of P-selectin is expressed under basal conditions in many of the tissues examined. Such a discrepancy between our findings and published immunohistochemical studies may result from a weak staining intensity (by ECAMs that exhibit nominal expression) being masked by background staining of other cell types.3 The presence of constitutive P-selectin in the tissues examined is substantiated by two major lines of evidence: (1) The accumulation of 125I mAb RB40.34 can be virtually eliminated by simultaneously administering a dose of cold (unlabeled) RB40.34. (2) The accumulation of 125I RB40.34 in untreated P-selectin–deficient mice is invariant from zero and significantly less than the accumulation of 125I RB40.34 in wild-type mice. The presence of constitutive P-selectin in tissues is supported by intravital measurements of baseline WBC rolling in the dorsal skin chamber9 and in the dermal venules of mice.33 34 The possibility that WBC rolling is normally present in venules in the absence of stimulation or trauma provides an explanation for the resident population of neutrophils in tissues such as the intestinal mucosa.35 Tissues that are constantly exposed to external inflammatory stimuli such as the small intestine would necessitate that WBC rolling, adherence, and emigration occur continuously to sustain a resident tissue cell population. In general, those tissues that expressed significant constitutive P-selectin in the present study are those organs most likely to be exposed to continuous inflammatory stimuli (Table 1
).
P-selectin is unique among ECAMs in that it can be rapidly mobilized to the EC surface after stimulation. The current data indicate that the translocation of P-selectin from cellular storage granules may differ substantially between tissues. For example, in the heart and muscle, P-selectin is significantly expressed within 5 minutes after histamine administration, reaching a maximal level by 30 minutes and remaining elevated for up to 1 hour, whereas the mesentery reaches a peak value of expression by 10 minutes, followed by a decline in expression toward constitutive values. These data indicate that histamine-induced P-selectin expression is not necessarily the transient event that is suggested by previous in vitro studies.4 It was revealed that P-selectin stored in Weibel-Palade bodies of HUVECs could be expressed within minutes in the presence of histamine but was reinternalized after 20 to 30 minutes. The prolonged expression (>30 minutes) of P-selectin measured in the present study is consistent with intravital observations of elevated and persistent levels of rolling leukocytes in venules exposed to histamine.11 Disparities between the present data and previous in vitro studies may arise from functional and structural differences in microvascular ECs and endothelium of large blood vessels. For example, it has been revealed that microvascular ECs have fewer Weibel-Palade bodies compared with ECs of large blood vessels36 and that HUVECs lose some of their ECAMs in culture at early passages (J. Shen, R.G. Ham, S. Karmiol, unpublished data, 1995).
Given that histamine plays an important role in the recruitment of WBCs to inflammatory foci, the present study attempted to assess the impact of histamine receptor antagonists on the magnitude of P-selectin upregulation. As illustrated in Fig 2, diphenhydramine (H1 receptor antagonist) had a significant effect on inhibiting the upregulation of P-selectin in response to histamine. In contrast, cimetidine (H2 receptor antagonist) had no effect on P-selectin expression when administered before histamine. These data agree favorably with intravital microscopic studies that demonstrate an abolishment of histamine-induced WBC rolling following the administration of diphenhydramine but not cimetidine.11 37 Furthermore, diphenhydramine but not cimetidine was shown to inhibit histamine-induced adhesion of human neutrophils to HUVEC monolayers in vitro.37
In addition to demonstrating the mobilization of a preformed pool of P-selectin after histamine challenge, the present study provides data that describe the kinetics of transcription-dependent expression of P-selectin following LPS administration. In a manner consistent with histamine-induced P-selectin expression, we found notable differences in the responses of tissues to LPS stimulation with respect to both the magnitude and kinetics of P-selectin upregulation. In most tissues, maximal expression of P-selectin occurred at 4 hours after LPS administration, with a decline in the level of expression to a steady state value lasting up to 24 hours. Although the time required to achieve peak surface expression of P-selectin in vivo agrees favorably with in vitro estimates,5 31 38 the significant elevation in P-selectin expression observed at later time points is uncharacteristic of previous in vitro studies.5 Studies of P-selectin expression in cytokine-stimulated mouse ECs have revealed that the expression of P-selectin was near basal levels after 24 hours.5 The differences between the expression of P-selectin observed on cultured microvascular ECs and the expression measured in our in vivo study may arise from LPS-induced generation of cytokines by other cells in vivo, which act to sustain the level of P-selectin expression in postcapillary venules. Our data also suggest that LPS also elicits a transient (<30-minute) upregulation of P-selectin that precedes the de novo synthesis of the CAM. The rapid LPS-induced upregulation of P-selectin that was observed in the present study, coupled with the reported absence of a change in P-selectin mRNA within 30 minutes,19 indicates that the initial LPS-induced P-selectin expression is derived from the preformed pool of this glycoprotein. Unlike histamine, LPS induced a significant transient upregulation of P-selectin in about half of the organs studied, and the magnitude of expression was substantially less than that induced by histamine. The disparity between the ability of these two stimuli to induce equivalent P-selectin expression may arise from differences in the tissue densities of LPS and histamine receptors. Despite these differences, the rapid LPS-induced expression of P-selectin is consistent with in vivo observations that an anti–P-selectin mAb inhibits the transient neutropenia induced by LPS.39
An interesting aspect of our findings is the differences (both within and between tissues) in the magnitude of P-selectin expression that were elicited by histamine (preformed pool) and LPS (transcription-dependent pool). For example, in the heart, LPS-induced expression of P-selectin was 20 times greater than that elicited by histamine, whereas only a twofold difference in expression for the two stimuli was noted in the mesentery. Since the endothelia of different types of microvessels express different levels of P-selectin,3 it is possible that tissues with low levels of stored P-selectin may preferentially upregulate the CAM via de novo synthesis, whereas tissues with high levels of stored P-selectin may rely preferentially on mobilization of the preformed pool to sustain leukocyte rolling. Although a definitive explanation for the differential responses of tissues to histamine and LPS is not readily available, differences in the density of Weibel-Palade bodies and/or the relative densities of LPS and histamine receptors may contribute to the observed responses.
Although platelets have been shown to express P-selectin in response to different stimuli, the accumulation of 125I-labeled P-selectin mAb (RB40.34) in tissues observed in the present study does not appear to be associated with platelet-bound 125I RB40.34. Intravital microscopic observations of unstimulated and activated platelets suggest that platelets roll but do not adhere to venular endothelium unless the integrity of the vessel wall is disrupted.40 Based on the rolling velocities of platelets, it would appear that the density of P-selectin on activated platelets and/or the strength of platelet-associated P-selectin binding to its ligands is lower than that exhibited by other cells expressing the CAM, either of which would preclude platelet entrapment within the microvasculature. Our assessment of platelet retention in the vasculature on P-selectin expression in unstimulated tissues and tissues stimulated with LPS or histamine revealed an insignificant contribution of platelets to P-selectin expression, as evidenced by an invariance in P-selectin expression in tissues that were either pretreated with a GPIIa/IIIb antagonist (to inhibit platelet-EC adhesion) and those tissues not receiving the GPIIa/IIIb antagonist. These measurements agree favorably with previous studies, which used markers for platelet-specific mRNA and found an absence of platelet sequestration in tissues of untreated and LPS-treated rats.41 Furthermore, measurements of 125I mAb RB40.34 activity in pelleted blood samples revealed a minimal amount of 125I mAb RB40.34 bound to platelets and was invariant between untreated mice and mice treated with histamine or LPS, indicating that the accumulation of platelets in organs following exsanguination is nominal.
Numerous in vitro42 and in vivo20 21 studies have attempted to assess the kinetics of E-selectin expression under different pathological conditions. The present study provides the first quantitative assessment of the kinetics of E-selectin expression in the vasculature of different organs. Although our in vivo data, which demonstrate an absence of constitutive E-selectin in different tissues, agree with the observations of previous studies,20 42 we found a different time course of LPS-induced E-selectin expression relative to that previously reported. Our study demonstrates that significant upregulation of E-selectin occurs as early as 2 hours after LPS administration, with maximal expression occurring at 3 hours. Bevilacqua et al42 revealed that peak upregulation of E-selectin occurs between 4 and 6 hours in cytokine-stimulated HUVECs. Furthermore, examination (using immunohistochemical methods) of LPS-induced E-selectin in the dermal vasculature of monkeys suggests that maximal expression of E-selectin occurred 8 hours after LPS injection.43 The disparities between our E-selectin expression data and previously published kinetics may represent differences in EC type (microvascular versus HUVECs) or species (mice versus humans). Nonetheless, our findings are similar to the in vitro studies demonstrating that the surface expression of E-selectin returns to constitutive values by 24 hours after EC activation.
A novel feature of the present study was the ability to compare the relative magnitude and kinetics of E- and P-selectin expression in several tissues. Our data indicate that the early histamine-induced expression (<1 hour) of selectins on ECs is due to mobilization of P-selectin from cellular storage granules. However, histamine administration did not result in an upregulation of E-selectin expression in any tissue. It is widely accepted that the mobilization of P-selectin from storage granules accounts for WBC rolling during the early stages of inflammation, whereas E-selectin contributes to a later phase of recruitment of rolling WBCs. Although our data support the first assumption, it is not entirely consistent with the latter. Indeed, our data suggest that LPS-induced (transcription-dependent) upregulation of E-selectin is likely to contribute to WBC rolling at intermediate time points (2 to 4 hours), with transcriptionally controlled P-selectin assuming a greater role at several hours after LPS (or presumably cytokine) challenge. The sustained elevation of P-selectin expression at 24 hours after LPS stimulation, when E-selectin expression has already returned to basal levels, supports our contention that P-selectin may make a more important contribution to the modulation of leukocyte trafficking at several hours after cytokine stimulation.
In contrast to the published reports that invoke a minimal role for E-selectin in mediating WBC rolling, our data on selectin expression indicate that E-selectin may contribute substantially to adhesion events within the microcirculation. This assertion is based on our observation of a similar magnitude of E- and P-selectin expression in most LPS-activated tissues. The data clearly illustrate the overlapping time course of E- and P-selectin upregulation in response to LPS. This is consistent with the observation in some models that simultaneous blockade of E- and P-selectin is required to detect changes in WBC kinetics in postcapillary venules. The overlapping expression of E- and P-selectin also agrees favorably with in vivo observations that E-selectin–deficient mice injected with thioglycolate exhibit normal WBC emigration compared with treated wild-type mice and that WBC emigration is completely abolished by an anti–P-selectin mAb, which has no effect in wild-type mice.16 The present study suggests that the dual radiolabeled antibody technique should provide a powerful tool for establishing the potential contribution of selectins and other CAMs in mediating the recruitment of leukocytes in different experimental inflammatory states.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the National Institutes of Health (PO1 DK-43785).
Received February 20, 1996; accepted May 29, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 P. Bauer, T. Welbourne, T. Shigematsu, J. Russell, and D. N. Granger Endothelial expression of selectins during endotoxin preconditioning Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2000; 279(6): R2015 - R2021. [Abstract] [Full Text] [PDF]
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M. J. Eppihimer and R. G. Schaub P-Selectin-Dependent Inhibition of Thrombosis During Venous Stasis Arterioscler Thromb Vasc Biol, November 1, 2000; 20(11): 2483 - 2488. [Abstract] [Full Text] [PDF]
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 K. Fukatsu, A. H. Lundberg, M. K. Hanna, Y. Wu, H. G. Wilcox, D. N. Granger, A. O. Gaber, and K. A. Kudsk Increased Expression of Intestinal P-Selectin and Pulmonary E-Selectin During Intravenous Total Parenteral Nutrition Arch Surg, October 1, 2000; 135(10): 1177 - 1182. [Abstract] [Full Text] [PDF]
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D. E. Swartz, A. J. E. Seely, L. Ferri, B. Giannias, and N. V. Christou Decreased Systemic Polymorphonuclear Neutrophil (PMN) Rolling Without Increased PMN Adhesion in Peritonitis at Remote Sites Arch Surg, August 1, 2000; 135(8): 959 - 966. [Abstract] [Full Text] [PDF]
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F. M. Akgur, M. F. Brown, G. B. Zibari, J. C. McDonald, C. J. Epstein, C. R. Ross, and D. N. Granger Role of superoxide in hemorrhagic shock-induced P-selectin expression Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H791 - H797. [Abstract] [Full Text] [PDF]
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S. Komatsu, R. D. Berg, J. M. Russell, Y. Nimura, and D. N. Granger Enteric microflora contribute to constitutive ICAM-1 expression on vascular endothelial cells Am J Physiol Gastrointest Liver Physiol, July 1, 2000; 279(1): G186 - G191. [Abstract] [Full Text] [PDF]
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J. Russell, C. J. Epstein, M. B. Grisham, J. S. Alexander, K.-Y. Yeh, and D. N. Granger Regulation of E-selectin expression in postischemic intestinal microvasculature Am J Physiol Gastrointest Liver Physiol, June 1, 2000; 278(6): G878 - G885. [Abstract] [Full Text] [PDF]
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P. Bauer, C. W. Lush, P. R. Kvietys, J. M. Russell, and D. N. Granger Role of endotoxin in the expression of endothelial selectins after cecal ligation and perforation Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2000; 278(5): R1140 - R1147. [Abstract] [Full Text] [PDF]
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T. J. Vachharajani, J. Work, A. C. Issekutz, and D. N. Granger Heme oxygenase modulates selectin expression in different regional vascular beds Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1613 - H1617. [Abstract] [Full Text] [PDF]
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O. A. Harari, J. F. McHale, D. Marshall, S. Ahmed, D. Brown, P. W. Askenase, and D. O. Haskard Endothelial Cell E- and P-Selectin Up-Regulation in Murine Contact Sensitivity Is Prolonged by Distinct Mechanisms Occurring in Sequence J. Immunol., December 15, 1999; 163(12): 6860 - 6866. [Abstract] [Full Text] [PDF]
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J. F. McHale, O. A. Harari, D. Marshall, and D. O. Haskard TNF-{alpha} and IL-1 Sequentially Induce Endothelial ICAM-1 and VCAM-1 Expression in MRL/lpr Lupus-Prone Mice J. Immunol., October 1, 1999; 163(7): 3993 - 4000. [Abstract] [Full Text] [PDF]
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M. J. Hickey, D. N. Granger, and P. Kubes Molecular Mechanisms Underlying IL-4-Induced Leukocyte Recruitment In Vivo: A Critical Role for the {alpha}4 Integrin J. Immunol., September 15, 1999; 163(6): 3441 - 3448. [Abstract] [Full Text] [PDF]
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R. R. Langley, J. Russell, M. J. Eppihimer, S. J. Alexander, M. Gerritsen, R. D. Specian, and D. N. Granger Quantification of murine endothelial cell adhesion molecules in solid tumors Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H1156 - H1166. [Abstract] [Full Text] [PDF]
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S. P. Jones, W. G. Girod, D. N. Granger, A. J. Palazzo, and D. J. Lefer Reperfusion injury is not affected by blockade of P-selectin in the diabetic mouse heart Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H763 - H769. [Abstract] [Full Text] [PDF]
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 V. H. Thomas, Y. Yang, and K. G. Rice In Vivo Ligand Specificity of E-selectin Binding to Multivalent Sialyl Lewisx N-linked Oligosaccharides J. Biol. Chem., July 2, 1999; 274(27): 19035 - 19040. [Abstract] [Full Text] [PDF]
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T. J. Murphy, G. Thurston, T. Ezaki, and D. M. McDonald Endothelial Cell Heterogeneity in Venules of Mouse Airways Induced by Polarized Inflammatory Stimulus Am. J. Pathol., July 1, 1999; 155(1): 93 - 103. [Abstract] [Full Text] [PDF]
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 N Mori, Y Horie, M E Gerritsen, D C Anderson, and D N Granger Anti-inflammatory drugs and endothelial cell adhesion molecule expression in murine vascular beds Gut, February 1, 1999; 44(2): 186 - 195. [Abstract] [Full Text] [PDF]
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P. Bauer, J. M. Russell, and D. N. Granger Role of endotoxin in intestinal reperfusion-induced expression of E-selectin Am J Physiol Gastrointest Liver Physiol, February 1, 1999; 276(2): G479 - G484. [Abstract] [Full Text] [PDF]
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J. F. McHale, O. A. Harari, D. Marshall, and D. O. Haskard Vascular Endothelial Cell Expression of ICAM-1 and VCAM-1 at the Onset of Eliciting Contact Hypersensitivity in Mice: Evidence for a Dominant Role of TNF-{alpha} J. Immunol., February 1, 1999; 162(3): 1648 - 1655. [Abstract] [Full Text] [PDF]
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M. J. Hickey, S. Kanwar, D.-M. McCafferty, D. N. Granger, M. J. Eppihimer, and P. Kubes Varying Roles of E-Selectin and P-Selectin in Different Microvascular Beds in Response to Antigen J. Immunol., January 15, 1999; 162(2): 1137 - 1143. [Abstract] [Full Text] [PDF]
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 J. Alferink, A. Tafuri, D. Vestweber, R. Hallmann, G. J. Hämmerling, and B. Arnold Control of Neonatal Tolerance to Tissue Antigens by Peripheral T Cell Trafficking Science, November 13, 1998; 282(5392): 1338 - 1341. [Abstract] [Full Text]
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A. J. Palazzo, S. P. Jones, D. C. Anderson, D. N. Granger, and D. J. Lefer Coronary endothelial P-selectin in pathogenesis of myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, November 1, 1998; 275(5): H1865 - H1872. [Abstract] [Full Text] [PDF]
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N. M. Bless, S. J. Tojo, H. Kawarai, Y. Natsume, A. B. Lentsch, V. A. Padgaonkar, B. J. Czermak, H. Schmal, H. P. Friedl, and P. A. Ward Differing Patterns of P-Selectin Expression in Lung Injury Am. J. Pathol., October 1, 1998; 153(4): 1113 - 1122. [Abstract] [Full Text] [PDF]
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M. M. Teixeira and P. G. Hellewell Contribution of Endothelial Selectins and {alpha}4 Integrins to Eosinophil Trafficking in Allergic and Nonallergic Inflammatory Reactions in Skin J. Immunol., September 1, 1998; 161(5): 2516 - 2523. [Abstract] [Full Text] [PDF]
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Y. Horie, R. Wolf, D. C. Anderson, and D. N. Granger Nitric oxide modulates gut ischemia-reperfusion-induced P-selectin expression in murine liver Am J Physiol Heart Circ Physiol, August 1, 1998; 275(2): H520 - H526. [Abstract] [Full Text] [PDF]
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Z. Morise, S. Komatsu, J. W. Fuseler, D. N. Granger, M. Perry, A. C. Issekutz, and M. B. Grisham ICAM-1 and P-selectin expression in a model of NSAID-induced gastropathy Am J Physiol Gastrointest Liver Physiol, February 1, 1998; 274(2): G246 - G252. [Abstract] [Full Text] [PDF]
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E. J. Kunkel, J. E. Chomas, and K. Ley Role of Primary and Secondary Capture for Leukocyte Accumulation In Vivo Circ. Res., January 23, 1998; 82(1): 30 - 38. [Abstract] [Full Text] [PDF]
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A. Kumar, J. L. Hoover, C. A. Simmons, V. Lindner, and R. J. Shebuski Remodeling and Neointimal Formation in the Carotid Artery of Normal and P-Selectin–Deficient Mice Circulation, December 16, 1997; 96(12): 4333 - 4342. [Abstract] [Full Text]
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M. J. Eppihimer, J. Russell, D. C. Anderson, C. J. Epstein, S. Laroux, and D. N. Granger Modulation of P-selectin expression in the postischemic intestinal microvasculature Am J Physiol Gastrointest Liver Physiol, December 1, 1997; 273(6): G1326 - G1332. [Abstract] [Full Text] [PDF]
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D. N. Granger II. Leukocyte-endothelial cell adhesion in the digestive system Am J Physiol Gastrointest Liver Physiol, November 1, 1997; 273(5): G982 - G986. [Abstract] [Full Text] [PDF]
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M. J. Eppihimer, J. Russell, D. C. Anderson, B. A. Wolitzky, and D. N. Granger Endothelial cell adhesion molecule expression in gene-targeted mice Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1903 - H1908. [Abstract] [Full Text] [PDF]
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D. D. Henninger, M. E. Gerritsen, and D. N. Granger Low-Density Lipoprotein Receptor Knockout Mice Exhibit Exaggerated Microvascular Responses to Inflammatory Stimuli Circ. Res., August 19, 1997; 81(2): 274 - 281. [Abstract] [Full Text]
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