Neutrophil leukocyte emigration induced by endotoxin. Mediator roles of interleukin 1 and tumor necrosis factor alpha 1

The hypothesis that cytokines mediate neutrophil emigration induced by endotoxin (LPS) was studied by examining the potency, the kinetics of neutrophil emigration, and the tachyphylaxis of intradermal sites with IL-1, TNF-alpha and LPS. Human rIL-1 alpha and IL-1 beta, synthetic lipid A, and LPS were several orders of magnitude more potent than human rTNF. The kinetic profiles of neutrophil emigration induced by IL-1 alpha, TNF, and LPS were characterized by minimal emigration in the first 30 min, followed by rapid and transient emigration. After the injection of LPS, the onset and the time at which the rate of emigration was maximal consistently appeared 30 min later than IL-alpha or TNF, suggesting that neutrophil emigration in response to LPS was mediated by a locally generated cytokine. IL-1 and TNF were then examined as potential secondary mediators of LPS-induced emigration by comparing the patterns of tachyphylaxis between LPS and IL-1 alpha or TNF; i.e., the magnitude of neutrophil emigration into inflammatory sites was compared with sites injected 6 h previously (desensitizing injections) with a cytokine or with LPS. Tachyphylaxis was dose dependent with each and also between the IL-1 species; therefore, when tachyphylaxis between the cytokines and LPS was examined, relatively higher doses were selected for the desensitizing injections than for the test injections. With this approach, desensitizing injections of IL-1 alpha diminished the neutrophil accumulation after LPS, and LPS also desensitized sites to IL-1 alpha. However, tachyphylaxis was not observed between TNF and LPS, or between TNF and IL-1 alpha. These data suggest that IL-1, but not TNF, is a potential mediator of LPS-induced neutrophil emigration.     

Acute inflammation in gram-negative infection: endotoxin, interleukin 1, tumor necrosis factor, and neutrophils

Experimental bacterial infection of the dermis induced with gram-negative microorganisms is associated with an acute inflammatory reaction, which represents the principal local defense against spread of the infection. When the inflammatory reaction is quantitated with radiolabeled cells and proteins, the kinetics resemble acute inflammation induced with other agents, such as immune complexes or chemotaxins. There is an interrelationship between the components or events of the inflammatory reaction; inasmuch as vascular injury is neutrophil-dependent, neutrophils must migrate to the site where the bacteria multiply. In neutropenic animals there is no such emigration and bacterial multiplication is not inhibited. The microorganisms shed endotoxin, which in turn induces secretion of interleukin 1 (IL 1) and probably tumor necrosis factor. Endotoxin is the most potent agent (10(-15) mol vs. 10(-12) mol of C5ades Arg) capable of inducing a neutrophil influx. Desensitization or tachyphylaxis of the tissues (probably of postcapillary venular endothelium) to IL 1 seems to control cessation of the neutrophil influx (also in vitro evidence). Phagocytosis of the bacteria by neutrophils is associated with release of oxygen radicals and lysosomal proteases from the neutrophils. These are instrumental in eliciting microvascular injury, which is characterized by enhanced vasopermeability, hemorrhage, and thrombosis.     

The effects of heparin cofactor II-derived chemotaxins on neutrophil actin conformation and cyclic AMP levels

The serine proteinase inhibitor heparin cofactor II (HC) can be cleaved by polymorphonuclear leukocyte (PMN) elastase (LE) to yield potent chemotactic activity for PMN and monocytes. In contrast to the bacterially-derived chemotaxin formyl-Met-Leu-Phe (fMLP), the HC-derived chemotaxin does not stimulate PMN degranulation or oxidative burst activity. We compared the effects of HC-derived chemotaxins to the effects of fMLP on PMN actin conformation and on the cAMP levels. Both the HC chemotaxins and fMLP rapidly induced an increase in F-actin which was similar in magnitude and time-course. However, in contrast to fMLP, HC-derived chemotaxins did not elevate cAMP levels. HC-derived chemotaxins may be useful probes of chemotactic responses, since they do not have the mixed biological activities of fMLP.     

Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse. Prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor

The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.     

Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.     

Dissociation between alveolar transmigration of neutrophils and lung injury in hyperoxia

The objective of this study was to quantitatively assess changes in cell adhesion molecule (CAM) expression on the pulmonary endothelial surface during hyperoxia and to assess the functional significance of those changes on cellular trafficking and development of oxygen-induced lung injury. Mice were placed in >95% O(2) for 0-72 h, and pulmonary injury and neutrophil (PMN) sequestration were assessed. Specific pulmonary CAM expression was quantified with a dual-radiolabeled MAb technique. To test the role of CAMs in PMN trafficking during hyperoxia, blocking MAbs to murine P-selectin, ICAM-1, or platelet-endothelial cell adhesion molecule-1 (PECAM-1) were injected in wild-type mice. Mice genetically deficient in these CAMs and PMN-depleted mice were also evaluated. PMN sequestration occurred within 8 h of hyperoxia, although alveolar emigration occurred later (between 48 and 72 h), coincident with rapid escalation of the lung injury. Hyperoxia significantly increased pulmonary uptake of radiolabeled antibodies to P-selectin, ICAM-1, and PECAM-1, reflecting an increase in their level on pulmonary endothelium and possibly sequestered blood cells. Although both anti-PECAM-1 and anti-ICAM-1 antibodies suppressed PMN alveolar influx in wild-type mice, only mice genetically deficient in PECAM-1 showed PMN influx suppression. Neither CAM blockade, nor genetic deficiency, nor PMN depletion attenuated lung injury. We conclude that early pulmonary PMN retention during hyperoxia is not temporally associated with an increase in endothelial CAMs; however, subsequent PMN emigration into the alveolar space may be supported by PECAM-1 and ICAM-1. Blocking PMN recruitment did not prevent lung injury, supporting dissociation between PMN infiltration and lung injury during hyperoxia in mice.     

New pharmacological studies with pentoxifylline

Polymorphonuclear (PMN) overactivation plays a critical role in microcirculation as well as in conditions such as multiorgan failure (MOF). Pentoxifylline has been shown to prevent PMN activation by endotoxin and cytokines such as TNF alpha and IL-1. In addition, MOF induced by IL-2 in animals can be prevented by pentoxifylline. The present studies evaluated two aspects of PMN activation and pentoxifylline interaction. The first was the time sequence for pentoxifylline prevention of TNF alpha activation and the second was the activity of pentoxifylline on amphotericin B activation of PMNs. TNF alpha activation of PMNs is blocked by pentoxifylline when cells are exposed to pentoxifylline prior to TNF alpha or after TNF alpha. Amphotericin B activation of PMNs was demonstrated by a decreased chemotaxis, increased chemiluminescence, and increased PMN spreading. In all conditions, pentoxifylline decreased amphotericin B activation of PMNs. These results suggest that pentoxifylline can reverse cytokine activation of PMNs and that pentoxifylline may alter some of the toxic effects of amphotericin.     

CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits

Neutrophil (PMN) migration in the systemic and pulmonary circulation of rabbits was compared by using different inflammatory stimuli to determine the role of the leukocyte adhesion complex, CD11/CD18, in each of these vascular beds. The adhesion complex was blocked by administering the anti-CD18 mAb 60.3. The data show that mAb 60.3 blocks PMN emigration into inflammatory foci in the abdominal wall produced by implanting sponges containing either hydrochloric acid, Streptococcus pneumoniae, Escherichia coli endotoxin, or PMA. mAb 60.3 also inhibited PMN emigration in response to peritoneal instillation of S. pneumoniae. The effect of mAb 60.3 on PMN emigration in the lungs varied depending upon the stimulus. PMN failed to migrate into the PMA-induced pneumonia; however, mAb 60.3 pretreatment only partially inhibited endotoxin-induced pneumonia and did not inhibit S. pneumoniae or hydrochloric acid-induced pneumonias. PMN lavaged from the alveolar spaces in the Streptococcal pneumonia had similar quantities of mAb 60.3 bound to their surfaces as the circulating PMN. We conclude that the CD11/CD18 complex mediates PMN adherence in the systemic circulation. However, PMN adherence in the pulmonary circulation may occur by either CD18-dependent or -independent mechanisms that are specific to the inciting stimulus.     

Neutrophil kinetics in O2-exposed rabbits

Hyperoxic injury results in an influx of polymorphonuclear leukocytes (PMN) into the lung. To better understand the role of the PMN in this injury, kinetic studies were used to assess the survival of PMNs in the circulation. The rate of deposition of PMNs in the lungs of rabbits exposed to hyperoxia was also examined. The half-lives (T1/2) of [3H]thymidine-labeled PMNs in the circulation in rabbits exposed to air or to 95% O2 for less than or equal to 48 h varied between 3.9 and 4.5 h. After 72 h of hyperoxic exposure, T1/2 fell to 2.2 h, the marginal and circulating PMN pool increased and 3H deposition in the lung increased 10-fold. Autoradiographs confirmed that [3H]thymidine was initially nuclear- and cellular-associated but, with time, [3H]thymidine dispersed throughout the lung, suggesting PMN disintegration. These PMN events seem to occur in the later phases of O2 toxicity, and because PMNs are an additional source of oxyradicals, they may further amplify oxidant injury.     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

Neutrophil recruitment and increased permeability during acute lung injury induced by lipopolysaccharide

The intranasal administration of lipopolysaccharide (LPS) to mice triggers a huge influx of polymorphonuclear neutrophils (PMNs) into the airway spaces, with a peak at 48 h. The increase in protein concentration, an index of microvascular permeability, displayed a different pattern, i.e., a first increase with a plateau between 3 and 24 h followed by a second increase peaking at 72 h. When mice were depleted of circulating PMNs, the increase in protein concentration was inhibited at 3 h but not at 24 h. The lack of PMN involvement at 24 h was confirmed by 1) in situ activation of exudated PMNs present in the air spaces on intranasal administration of LPS and 2) induction of the migration of PMNs sequestered in lung vessels on intraperitoneal administration of LPS. These findings show that the increase in microvascular permeability during lung inflammation is due to at least two distinct mechanisms, an early one related to the neutrophil influx and a delayed one occurring even under neutropenic conditions.     

Interleukin 1: a common endogenous mediator of inflammation and the local Shwartzman reaction

In this study we investigated the role of interleukin 1 (IL 1) in the induction of inflammatory lesions and in the preparation and provocation of the local Shwartzman reaction. Both of these phenomena can be induced with a variety of agents. This suggested to us that a common endogenous mediator may be crucial to the development of these two lesions. When IL 1 was injected intradermally into shaved rabbit backs, 51Cr-labeled neutrophils accumulated at the injection site. Neutrophils began to accumulate less than 1 hr after injection, and the maximum rate of accumulation was observed by 4 hr. This activity was dose dependent. It was calculated that in all animals, 10(-14) mol of IL 1 induced significant neutrophil accumulation, whereas in many animals, as little as 10(-15) mol of IL 1 sufficed. When 4.2 X 10(-9) mol of E. coli 0111:B4 lipopolysaccharide W was injected i.v. 24 hr after an intradermal injection of IL 1 (2.9 X 10(-13) mol), a local Shwartzman reaction was seen 4 hr later at the intradermal injection site. IL 1 injected i.v. 24 hr after an intradermal injection of either IL 1 or lipopolysaccharide also produced a local Shwartzman reaction. These data indicate that IL 1 may be the common endogenous mediator of the inflammatory response, and IL 1 may serve in the same role for the preparation and provocation of the local Shwartzman reaction.     

A novel effect of polymorphonuclear leukocytes in the facilitation of angiogenesis

The purpose of this study was to examine whether the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells and/or reactive oxygen species (ROS) released from PMNs are responsible for inducing angiogenesis. Angiogenesis was assessed by tube formation using endothelial cells obtained from bovine thoracic aorta (BAECs) grown on a layer of collagen type I. Addition of PMNs to BAECs weakly induced angiogenesis. The angiogenesis induced by PMNs alone was further enhanced by treatment of the PMNs with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a selective activator of PMN. The involvement of PMN adhesion to BAECs via adhesion molecules in angiogenesis was investigated by using monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1). These antibodies blocked both the PMN adhesion to BAECs and the enhancement of angiogenesis induced by FMLP-treated PMNs. Furthermore, the enhancement of angiogenesis by FMLP-treated PMNs was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). These results suggest that PMNs induce angiogenesis in vitro, and that the mechanism of stimulation of angiogenesis by PMNs may involve the adherence of PMNs to endothelial cells via E-selectin and ICAM-1, and H2O2, but not superoxide. Thus, activated PMNs in pathological states may not only induce tissue injury, but may also function as regulators of angiogenesis.     

Cessation of neutrophil influx in C5a-induced acute experimental arthritis is associated with loss of chemoattractant activity from the joint space

Neutrophil emigration is a critical component of the inflammatory process and is generally thought to play a role in host defense as well as in the tissue injury that often accompanies inflammation. Most inflammatory reactions exhibit a sequence of emigrating cell types, thus clearly demonstrating that the neutrophil influx eventually ceases and that the neutrophils are then removed from the lesion. It has been our premise that in order to understand the processes that lead to the progressive inflammatory reactions that underly so many disease processes, it is important to determine the mechanism by which the "normal" inflammatory response resolves. The purpose of this study was to identify the time of cessation of neutrophil influx in experimental arthritis induced by the injection of C5 fragments (C5f) and to investigate mechanisms underlying the cessation process. The migration of i.v. delivery pulses to inflamed joints was assessed by lavage of the joint space and by external scintigraphy. We found no evidence for the development of inhibitory systems against chemotactic factors or "desensitization" of the inflamed site, because a second injection of C5f into joints which had been injected previously with C5f resulted in enhancement rather than inhibition of migration. Neither was evidence found for altered tissue barriers to migration or for desensitization of neutrophils as possible explanations for cessation of influx. The major mechanism appeared to be a loss of chemoattractant activity in the joint space between 2 h and 6 h after C5f injection which was detected by transfer into a fresh joint. Radiolabeled C5a des-Arg had a t1/2 of disappearance from the joint of less than 1 h, which suggested that the transferred chemoattractant must, in part, have been due to the generation of new chemotaxins by C5f injection. These observations suggest that continued generation of chemoattractants or failure of their subsequent removal may be mechanisms leading to persistent neutrophil influx in chronic inflammation.     

CR3 (CD11b/CD18) activation of nasal neutrophils: a measure of upper airway endotoxin exposure

Inhaled endotoxin (lipopolysaccharide, LPS) initiates an inflammatory response and leads to the expression of CR3 (CD11b/CD18) receptors on polymorphonuclear leukocytes (PMNs). We determined if PMN activation in nasal lavage fluid (NLF) is a possible biomarker of occupational endotoxin exposure. Seven subjects exposed to endotoxin provided NLF samples that were split into three aliquots (negative control – 1?M nicotinamide; sham; positive control – 11 ηg of exogenous LPS) and PMN activation was measured using a chemiluminometer. Differences in mean PMN activation were apparent, negative control: 548?±?15.65 RLU 100 μl^?1; sham: 11469?±?2582 RLU 100 μl^?1; positive control: 42026?±?16659 RLU 100 μl (n?=?7; p <0.05). This technique shows promise as a diagnostic method for measuring upper airway LPS exposure.     

Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40–70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro , had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.     

Effects of oxygen radicals on the conformation of sulfhydryl groups on human polymorphonuclear leukocyte membranes.

The healthy intact polymorphonuclear leukocytes (PMNs) were labeled with 4-maleimide-TEMPO spin labeling compound (MAL) to study the effects of oxygen radicals produced by phorbol myristate acetate (PMA)-stimulated PMNs on the conformation of sulfhydryl (SH) groups of PMN membrane proteins. The lipid peroxidation induced by PMA-stimulated PMNs was detected by evaluating the formation of malonaldehyde (MDA) with the thiobarbituric acid (TBA) test. From the experiments of luminol-dependent chemiluminescence (CL) and fluorometry, it was found that Chinese herbs schizandrin B (Sin B) and quercetin (Q) possessed scavenging properties for oxygen radicals produced during the PMN respiratory burst. These two herbs can also inhibit the conformation changes in SH binding sites on the PMN membrane proteins caused by oxygen radicals produced by the PMNs themselves. They also decreased the amount of MDA, which was a final product formed during lipid peroxidation.     

Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these interactions. Whether platelets also promote PMN transmigration across the endothelium is less clear. We tested the hypothesis that platelets enhance PMN transmigration across the inflamed endothelium and that PSGL-1 is involved. We studied the effects of platelets on PMN transmigration in vivo and in vitro using a well-characterized corneal injury model in C57BL/6 mice and IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) under static and dynamic conditions. In vivo, platelet depletion altered PMN emigration from limbal microvessels after injury, with decreased emigration 6 and 12 h after injury. Both PSGL-1-/- and P-selectin-/- mice, but not Mac-1-/- mice, also had reduced PMN emigration at 12 h after injury relative to wild-type control mice. In the in vitro HUVEC model, platelets enhanced PMN transendothelial migration under static and dynamic conditions independent of firm adhesion. Anti-PSGL-1 antibodies markedly inhibited platelet-PMN aggregates, as assessed by flow cytometry, and attenuated the effect of platelets on PMN transmigration under static conditions without affecting firm adhesion. These data support the notion that platelets enhance neutrophil transmigration across the inflamed endothelium both in vivo and in vitro, via a PSGL-1-dependent mechanism.