Protein synthesis dependent and independent mechanisms of neutrophil emigration. Different mechanisms of inflammation in rabbits induced by interleukin-1, tumor necrosis factor alpha or endotoxin versus leukocyte chemoattractants.

Inflammation constitutes the body's principal mode of defense against infection and other harmful agents. Neutrophil leukocytes are the primary effector cells in this process. The role of protein synthesis in neutrophil emigration into acute inflammatory lesions was examined. Local intradermal injections of actinomycin D, cycloheximide or puromycin could inhibit in a dose- and time-dependent manner neutrophil emigration induced by interleukin-1, tumor necrosis factor alpha or endotoxin, but not by the leukocyte chemoattractants C5a des arg (zymosan-activated plasma), n-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. Maximal inhibition, measured at the time of peak emigration, was greater than 90%. The onset of neutrophil emigration induced by the cytokines or by endotoxin was delayed by 30 to 60 minutes in comparison to the leukocyte chemoattractants. These results demonstrate at least two mechanisms of neutrophil emigration: one with a slower onset and dependence on local RNA transcription and translation and the other rapid in onset and independent of protein synthesis.

Suppression of fluid pumping in isolated bovine mesenteric lymphatics by interleukin-1: interaction with prostaglandin E2.

In addition to local physiological forces, the modulation of lymphatic pumping by chemical mediators may play an important role in the regulation of extravascular water in inflammation and shock. Since Interleukin-1 (IL-1) appears to be of major importance in the host's response to infection by mediating many inflammatory events, we thought it important to determine if this cytokine could affect the lymphatic circulation and in particular to ask whether IL-1 was capable of altering lymphatic pumping in response to changes in transmural pressure. Bovine lymphatic segments (6 to 8 cm in length) were cannulated at both ends and suspended in an organ bath preparation. The vessels were provided with Krebs solution from a reservoir. With no net driving pressure, a transmural pressure applied to the ducts elicited contractile activity and fluid pumping with increases in pumping up to 8 cm H2O and reductions in flow above this level of distension. Human recombinant IL-1 alpha (10(-7) to 10(-9) M) administered into the lumina of the vessels depressed pumping activity approximately 5-30% at transmural pressures between 2 and 16 cm H2O. With limited supplies, we could only assess the effects of human recombinant IL-1 beta at 10(-8) M. However, it was more potent than IL-1 alpha, inhibiting pumping at all transmural pressures with maximum suppression in the range of 70% at peak flows. The ability of IL-1 to induce prostaglandin synthesis may be one of its most important biologic functions. It is likely therefore that IL-1 and PGE2 are closely linked and are probably present together in inflammatory lesions. With this in mind, we investigated the effects of PGE2 alone and in combination with IL-1 alpha. PGE2 by itself reduced pumping at concentrations between 10(-6) and 10(-9) M. When mixed with IL-1 alpha (both agents at a final concentration of 10(-9) M), the mixture had a marked inhibitory effect on flow, reducing pumping 50 to 70% at peak flows. The effect of the mixture of IL-1 alpha and PGE2 compared with the two agents administered separately was greater than the predicted additive effect at transmural pressures above 6 cm H2O. At lower transmural pressures, however, the level of real inhibition was less than the predicted additive effect.(ABSTRACT TRUNCATED AT 400 WORDS)

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 and a Shwartzman-like reaction induced by interleukin-1 and tumor necrosis factor. Synergistic action of the cytokines in the induction of inflammation and microvascular injury.

A Shwartzman-like reaction was elicited in rabbits by preparing the skin with intradermal injections of recombinant human tumor necrosis factor alpha (TNF alpha) and recombinant human interleukin-1 (IL-1 alpha or beta). The animals were challenged intravenously with endotoxin or by intravascular activation of complement with immune complexes or zymosan 18 hours later and were sacrificed after another 2 hours. Animals challenged with saline did not develop Shwartzman-like reactions. The sites prepared with endotoxin or with either form of IL-1 plus TNF alpha developed visible hemorrhage, whereas sites injected with either IL-1 or TNF alpha alone did not. Hemorrhage and microthrombosis were quantitated with 59Fe-labeled erythrocytes and 111In-platelets for 2 hours after the intravenous challenge, and the findings confirmed the observations made on gross inspection. Dermal sites prepared with the cytokines and challenged intravenously with endotoxin, immune complexes, or zymosan exhibited some diffuse hemorrhage and an intense erythrocyte extravasation around distended vessels, along skin appendages, and the panniculus carnosus muscle. The lumens of many large and postcapillary venules contained aggregates of platelets and leukocytes. These changes were superimposed on those seen at prepared sites (leukocyte infiltration). By electron microscopy fibrin was demonstrable in association with the formed elements of the blood. Histologic examination of the 18-hour-old preparative lesions or 20-hour-old lesions of saline-"challenged" animals revealed accumulation of leukocytes in the dermis, predominantly neutrophils. This accumulation was sparse at sites treated with only IL-1 or TNF alpha, but very intense at sites treated with both IL-1 and TNF alpha or with endotoxin. These observations were confirmed quantitatively by measuring the accumulation of 51Cr-labeled neutrophils for 2 hours after injection. The potency of IL-1 alpha was comparable to that in our earlier report, and TNF alpha was about three log times less potent. Sites treated with both IL-1 alpha and TNF alpha resulted in 69% greater neutrophil emigration than the additive response elicited by each cytokine. The reported findings implicate a synergism between IL-1 and TNF alpha in the induction of both the inflammatory reaction (preceding the Shwartzman reaction) and the thrombohemorrhagic component of the Shwartzman reaction proper.

The role of histamine and other mediators in microvascular changes in acute inflammation.

In addition to bronchial smooth muscle, histamine and other mediators act in bronchial asthma on the microcirculation of pulmonary connective tissue. The mediators induce enhanced blood flow, and by acting on the blood-tissue barrier, they induce increase in vasopermeability with edema and in more severe injury microhemorrhage and microthrombosis and infiltration of the connective tissue by leukocytes, predominantly neutrophils. The scheme proposed 10 years ago by K. F. Austen and co-workers in 1976 still holds true: a short-acting humoral-cellular phase is followed by a longer-acting pathopharmacologic or inflammatory phase. Some mediators, including histamine, serotonin, bradykinin, the sulfido leukotrienes and platelet activating factor, have a direct effect on endothelium and smooth muscle cells and more severe injury is due to mediators that exert their effect via neutrophils, which release lysosomal constituents. The intact complement-derived fragments C3a and C5a act directly and for a short period, as do histamine and the other direct-action mediators. The accumulation of neutrophils is brought about by C5a and its stable derivative C5ades Arg, leukotriene B4, PAF, and interleukin-1. Unlike the direct-action mediators, whose effect on the microcirculation does not extend beyond 20-25 min, those mediating via neutrophils have a delayed effect (peak 1-2 h), which parallels the neutrophil influx. The direct action mediators exert their effects on the microcirculation also in neutropenic animals. The most potent agents causing enhanced blood flow are the prostaglandins of the E class. Through the enhanced blood flow increase in vasopermeability, neutrophil emigration and hemorrhage are enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Relationship between increased vascular permeability and extravascular albumin clearance in rabbit inflammatory responses induced with Escherichia coli.

Intradermal injections of killed Escherichia coli are known to cause a variety of pathophysiological changes in the microcirculation that facilitate the extravasation of plasma constituents into the interstitium. In an attempt to learn more of the factors that regulate the magnitude and duration of inflammatory edema, we have focused on the relationship between the extravasation of protein into the interstitium and the removal of extravascular protein from the lesion sites. Vascular permeability changes have been assessed by the local accumulation of systemically administered [131I] or [125I]-albumin and extravascular protein clearance measured by monitoring the disappearance of [125I]-albumin from the same sites. Radioactivity was quantitated with an external gamma-scintillation probe or by punching out the lesion sites in sacrificed animals and counting in a gamma-spectrometer. Scintillation probe measurements of the net accumulation of intravenously administered [125I]-albumin in E. coli-induced skin lesions revealed that the extravasation of albumin was greater than the clearance of protein from the same sites. Comparisons of the removal rates of albumin injected directly into the E. coli sites revealed that, despite increases in vascular permeability amounting to 170 to 700% of control values, the mobilization of deposited albumin was no greater than that from control tissues that received saline; in fact with high concentrations of E. coli (10(8) injected/site) the mobilization of protein from the lesions was significantly reduced. The systemic administration of 055:B5 endotoxin (0.3, 1.6, or 3.3 micrograms/kg) also suppressed the clearance of albumin from skin. In contrast to these results, 300 to 1500% increases in vascular permeability induced with other inflammatory stimuli including thermal injury, high concentrations of bovine serum albumin, or bradykinin, resulted in enhanced clearance of extravascular protein from lesion or injection sites. These experiments suggest that an inability to effectively mobilize extravascular protein from the inflammatory focus could be a major contributing factor in regulating edema in inflammatory reactions induced with E. coli and may possibly contribute to the edema associated with septicemia.

The role of interleukin-1 in neutrophil leukocyte emigration induced by endotoxin.

Chemotactic factors induce neutrophil emigration into tissues. Interleukin-1 (IL-1) was found to be several log times more potent in this respect than C5a des Arg, leukotriene B4, and f-Met-Leu-Phe and of comparable potency to endotoxin. Kinetic studies revealed a rapid and transient neutrophil influx, with the peak rate at 30-90 minutes. Cross tachyphylaxis was observed between IL-1 and endotoxin; and this, together with its high potency and rapid onset of action, suggest that IL-1 mediates endotoxin-induced neutrophil emigration.

Neutropenic responses to intradermal injections of Escherichia coli. Effects on the kinetics of polymorphonuclear leukocyte emigration.

Killed Escherichia coli organisms injected intradermally into rabbits induced significant neutropenia and provoked a rapid rise in body temperature. Both the magnitude and the duration of the neutropenia were dose-dependent. After recovery from neutropenia, the rabbits became refractory to its redevelopment when subsequently given an equivalent dose of E coli. The influence of neutropenia and the subsequent refractory period on the rate of polymorphonuclear leukocyte (PMN) emigration into inflammatory sites was examined. Killed E coli organisms (6 X 10(8) per site) were injected into two groups of 20 intradermal sites in each rabbit. The first group (Group F) preceded the second (Group S) by 6 hours. The kinetics of PMN emigration, quantitated with 51Cr-labeled cells, differed in the two groups. In Group S sites an intense PMN influx was measured at 0-4 hours, and subsequently the extent of PMN emigration rapidly declined. In Group F sites a minute PMN influx was detected during the first 4 hours, coinciding with a marked neutropenia. The maximal PMN influx into Group F sites was measured between 6 and 10 hours. Microscopic sections at 4 hours showed a scanty PMN infiltrate and numerous bacteria in the dermis of Group F sites, while extensive phagocytosis of bacteria by PMNs was apparent in Group S sites. By comparing the extent of bacterial phagocytosis in 4-hour-old sites with the magnitudes of PMN emigration between 6 and 10 hours in both groups, we concluded that the phagocytic elimination of killed E coli was not a major mechanism regulating the cessation of local PMN emigration. Instead, we propose that tachyphylaxis or desensitization of sites to inflammatory factors released from E coli is the responsible mechanism.

Emigration and accumulation of PMN-leukocytes induced by endotoxin, interleukin 1 and other chemotactic substances.

This publication describes polymorphonuclear leukocyte (PMN) emigration and accumulation, which is prerequisite for their defensive function in infected tissues. The extravasated PMNs can kill microorganisms, but in this process they also release proteolytic enzymes and other cell constituents which can alter and even injure the tissues, primarily the microcirculation. In the first part of the paper in vivo quantitation of the acute inflammatory reaction is described with emphasis on PMN emigration and accumulation. With 51Cr-labeled PMNs the kinetics of their emigration induced by a number of chemotaxins and chemotaxinigens was found to be similar, peaking in 1-4 hour old lesions and returning to baseline values thereafter. The most potent substance tested was endotoxin, which induced a PMN influx at a molar concentration a least 3 orders of magnitude lower than the other substances tested, implying the these substances are not the primary endogenous mediators of endotoxin induced inflammation. Next we describe an observation which shed considerable light on the mechanisms underlying PMN emigration. When a chemotaxin or endotoxin was injected intradermally and after varying periods of time reinjected into the same site, the PMN influx into those sites was diminished, compared to sites not previously injected, i. e. injected for the first time. This tachyphylaxis or diminished responsiveness was attributed to a downregulation of receptors, presumably on endothelial cells, coupled to a facilitatory mechanism. Other mechanism proposed to terminate emigration of PMNs during inflammatory reaction were unlikely, based on our experimental findings. Endotoxin is not chemotactic in vitro but it induces PMN emigration when injected intradermally. Hence the third part of the publication deals with PMN emigration induced by interleukin 1 and its significance for endotoxin-induced inflammation. IL 1 is the only chemotaxin which induces PMN accumulation at a concentration comparable to that of endotoxin and considerably lower than the other chemotaxins. There was cross tachyphylaxis between endotoxin and IL 1 and vice versa. The PMN influx into IL 1 sites injected 6 hours earlier with IL 1 or with endotoxin was diminished compared to IL 1 sites injected into normal skin. Sites injected first with IL 1 and then with a low dose of endotoxin also exhibited cross tachyphylaxis. FMLP or LTB4 injected into sites pretreated with endotoxin did not exhibit cross tachyphylaxis, i. e. the PMN influx was similar to sites injected for the first time with these chemotaxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased permeability of microcarrier-cultured endothelial monolayers in response to histamine and thrombin. A model for the in vitro study of increased vasopermeability.

The permeability response of endothelial monolayers to some "direct-action" type mediators of vasopermeability were studied in vitro. Endothelial cells, cultured to confluence on denatured collagen-coated dextran microcarriers or gelatin microcarriers, prevented staining of the microcarriers with Evans blue dye. Increases in staining, as determined by the spectrophotometric quantitation of the dye after extraction from the microcarriers with formamide, occurred after treatment of human umbilical vein endothelium with histamine (10(-5) M) or thrombin (0.1 U/ml). These increases in monolayer permeability were reversible. Neither bradykinin nor serotonin had any effect in this system. Endothelial monolayers cultured this way consistently stained with silver nitrate at the cell junction areas. Monolayer response to histamine was characterized morphologically by small openings which occurred randomly along the cell junctions; while with thrombin, the spaces, which had developed at junctions, occurred to a greater extent. Prostaglandin E1 (30 microM) and isoproterenol (10 microM), in the presence of 3-isobutyl-1-methylxanthine (1 mM), partially inhibited histamine- and thrombin-mediated changes in permeability. This model responds to certain vasopermeability-altering agents in a manner similar to that of the microcirculation. These studies support the concept that the vasopermeability enhancing effect of histamine in vivo results, in part, from a direct effect on the endothelium.

Severe microvascular injury induced by lysosomal releasates of human polymorphonuclear leukocytes. Increase in vasopermeability, hemorrhage, and microthrombosis due to degradation of subendothelial and perivascular matrices.

The purpose of this study was to assess the nature of the lesions in the microcirculation of the dermis of rabbits induced with lysosomal releasates of human polymorphonuclear leukocytes (PMNs). No attempt was made in the studies presented in this publication to deal with the offending agent in the releasate. Four parameters of microvascular injury were quantitated: increase in vascular permeability with 125I-labeled serum albumin, hemorrhage with 59Fe-labeled erythrocytes, accumulation (aggregation) of platelets with 111In-labeled platelets. In one experiment accumulation of 51Cr-PMNs was investigated. The lysosomal releasate induced a rapid increase in vasopermeability, but both hemorrhage and exudate formation peaked 1 hour after intradermal injection. Platelet accumulation was also demonstrable in these lesions, and microthrombosis was a very prominent feature. The microvascular injury, including microthrombosis, could be elicited also in animals rendered leukopenic with nitrogen mustard. Simultaneous injection of prostaglandin E2 with the releasate enhanced the microvascular injury. The morphologic changes in the microcirculation of the rabbit's dermis were assessed in lesions 5 minutes to 5 hours old. Several changes were encountered, primarily in the wall of venules and small veins and to a lesser degree in small arteries and capillaries. Ultrastructurally very early lesions (up to 15 minutes) had gaps or spaces in the endothelium, resembling those induced by mediators such as histamine or bradykinin. Older lesions were different, quite characteristic, and represent the hallmark of these lesions. Lysis and disappearance of vascular basement membrane, of perivascular collagen, and of the internal elastic lamina were a frequent finding, best demonstrable when microthrombi did not abut on vessel walls. Cellular components of vessels (endothelium, pericytes, smooth muscle) showed fragmentation, leading to complete disappearance of cellular elements. These lesions were usually walled off by platelet aggregates and fibrin. At times microthrombi occluded an entire vessel. These changes were interpreted as hemostasis. The mild accumulation of PMNs at the site of injury did not contribute significantly to the microvascular injury. The findings indicate that the unique changes in the microcirculation, not described before, may occur quite frequently, when the microvascular injury is elicited primarily by release of lysosomal constituents by phagocytic or nonphagocytic stimuli. One can conclude that the hallmark of this type of injury is disappearance of basement membrane followed secondarily by disintegration of the vascular wall, followed in turn by hemo

Acute inflammation induced by immune complexes in the microcirculation.

The aims of the studies presented in this publication were to elucidate the morphology and quantitate the kinetics of an inflammatory reaction elicited by immune complexes and to ascertain the role of complement in the reaction. The hallmark of both the direct active (DAA) and reversed passive (RPA) Arthus reactions was the accumulation of immune precipitates and polymorphonuclear leukocytes (PMNs) in and around vessels. Using fluoresceinated antigen as a tracer, immune complexes localized in the lumina and walls of venules and small veins in the DAA and in the wall of vessels and perivascularly in RPA. PMNs accumulated at these same sites, phagocytosed the fluoresceinated complexes and became degranulated. The precise localization of immune complexes was achieved by examining the same tissue sections first by fluorescence microscopy, followed by conventional staining and examination by light microscopy. Marked stasis of the microcirculation was observed, particularly in DAA, in which a few immune complex-containing PMNs were entrapped in a mass of densely packed red blood cells. Some edema was observed in early lesions and definitive separation of collagen fibers was noted in lesions older than 2 hr. Hemorrhage became the dominant characteristic of both types of reactions from 2 hr onward. By administering radiolabeled cells, proteins, and microspheres as a "pulse," given at various times before sacrifice, the quantitation and kinetics of the inflammatory lesions elicited by immune complexes could be elucidated. In RPA all parameters quantitated reached a peak soon after elicitation of the reaction (2-4 hr), which is in keeping with other forms of acute inflammation. In DAA there was some difficulty in assessing the quantitation because of interanimal variations and because of progression of the inflammatory lesions, as the antigen diffused peripherally from the site of its injection. Peak activities occurred in 4- to 8-hr-old lesions. These observations and a comparison of the center and periphery of the lesions, strengthen the contention that the RPA and DAA have common features and features which differ. In common are immunological mechanisms (antigen-antibody interaction and complement activation) and cellular events (polymorphonuclear leukocyte chemotaxis, phagocytosis, and release of lysosomal contents). Different features are the site of immune complex formation and its sequelae. In RPA they form primarily in the wall of venules and small veins and hence have a marked effect on increase in vessel permeability. In the DAA most of the complexes form and the leukocytes accumulate in the lumen of the same vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of a fibrin(ogen)-derived vasoactive peptide on polymorphonuclear leukocyte emigration.

A vasoactive peptide known to increase vascular permeability and corresponding to residues 30-43 of the human fibrinogen B beta-chain induced polymorphonuclear leukocyte emigration in rabbit skin in vivo. The leukocyte emigration was much stronger after 2 h than after 0.5 h. Addition of prostaglandin E2 (PGE2) did not influence the chemotactic activity, which might possibly be explained by a known PGI2 releasing capacity of this peptide. PGE2 enhanced the leukocyte emigration due to leukotriene B4.

Desensitization of acute inflammatory lesions to chemotaxins and endotoxin.

Desensitization of the neutrophil inflammatory response to intracutaneous injection of chemotaxins and endotoxin was studied in rabbits. When restimulated 6 hr later with the same agent, inflammatory lesions initiated with platelet-activating factor (PAF), leukotriene B4 (LTB4), or endotoxin supported a diminished influx of neutrophils compared with responses in normal skin. In contrast, repeated stimulation of lesions with alpha-casein failed to lead to desensitization. The specificity of desensitization was investigated, and it was found that lesions initiated with formylmethionyl-leucyl-phenylalanine (FMLP) supported a normal response when restimulated with PAF, alpha-casein, or endotoxin. Initiation of lesions with LTB4, however, diminished the subsequent response to zymosan-activated plasma and PAF, but not endotoxin, alpha-casein, or FMLP. This result indicates that LTB4 is not a final common mediator of neutrophil infiltration of acute inflammatory lesions. Desensitization was detected irrespective of the concentration of chemotaxin used to investigate the response. Repeated stimulation of lesions with FMLP abolished the accumulation of neutrophils after the final stimulus, indicating that complete desensitization can occur and the presence of a chemotaxin within an inflammatory lesion is not sufficient stimulus for neutrophil infiltration of the site to proceed. Lesions initiated with endotoxin supported comparable responses when restimulated with a mixture of FMLP and endotoxin or FMLP alone, despite 93% inhibition of the response to restimulation with endotoxin alone. This indicates that a cell-directed inhibitor of neutrophil migration, such as lipomodulin or neutrophil-immobilizing factor, does not produce the diminished responses in desensitized lesions. It is proposed that desensitization occurs due to down-regulation of a receptor-coupled pathway that permits or facilitates neutrophil extravasation in acute inflammatory lesions. The chemotaxin receptors regulating neutrophil extravasation are probably located on endothelial cells of post-capillary venules.