Increased tissue survival in experimental skin flaps in mast cell-deficient rats.

AIM: The role of mast cells and their principal mediator, histamine, in surgical skin flap survival was investigated using mast cell-deficient (Ws/Ws); their congenic littermates wild-type (+/+), and Wistar rats. METHODS: A standardized dorsal skin flap was raised and sutured back into position, and 6 days later the percentage of flap survival was assessed. Moreover, endogenous histamine concentration in the dorsal skin during the surgical preparation was determined using in vivo microdialysis technique together with high performance liquid chromatography-fluorometry. Accumulation of skin flap myeloperoxidase (MPO) (reflecting leucocyte recruitment) was determined spectrophotometrically. RESULTS: The experimental skin flaps in genetically mast cell-deficient rats exhibited increased tissue survival and showed little accumulation of MPO and rather low and stable level of histamine output in comparison with skin flaps in the wild-type (+/+) littermates or normal Wistar rats. Antihistamine treatment inhibited but did not prevent leucocyte recruitment in the skin flaps post-surgery in +/+ and Wistar rats. CONCLUSION: It is suggested that mast cell derived histamine plays an important role in leucocyte recruitment in skin flaps. However, mast cell-independent factors should be taken into consideration and needs further investigation as even in mast cell-deficient animals there was some accumulation of leucocytes and tissue necrosis in the skin flaps post-surgery.

Absence of mast cell involvement in active systemic anaphylaxis in rats.

This study investigates the role of mast cells in the hypotension induced by antigen-mediated anaphylaxis, compound 48/80 and dextran in mast cell-deficient white spotting (Ws/Ws) and normal wild type (+/+) rats. Rats were sensitized with 10 microg of intraperitoneal ovalbumin in saline or saline alone (sham-sensitized). Sensitized rats, both Ws/Ws and +/+ but not sham-sensitized rats, challenged intravenously with ovalbumin exhibited hypotensive responses. There was no evidence of mast cell activation in rat mesentery 20 min after intravenous antigen challenge in sensitized +/+ rats. Hypotension induced by intravenous injection of dextran (Dextran-162, 6%, 2 ml kg(-1)) or compound 48/80 (1 mg kg(-1)) occurred in +/+ rats, but not in Ws/Ws rats, and was inhibited by pretreatment with a combination of chlorpheniramine and cimetidine. Taken together, these data indicate that the hypotensive response induced by antigen-mediated anaphylaxis is independent of mast cell activation, whereas mast cell amines play the main role in the hypotensive response induced by dextran or compound 48/80.

Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability.

Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic beta2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.

Engagement of beta2 integrins induces surface expression of beta1 integrin receptors in human neutrophils.

Induction of beta1 integrin (CD49/CD29) expression in polymorphonuclear leukocytes (PMN) has been shown to be associated with transendothelial migration recently. Yet, beta1 integrin expression is relatively insensitive to cell activation with soluble agonists, such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). We hypothesized that beta2 integrins (CD11/CD18), critically involved in PMN adhesion and extravasation, may play a role in regulating 1 integrin expression in PMN. Antibody cross-linking of CD18, mimicking adhesion-dependent engagement of beta2 integrins, resulted in rapid, tyrosine kinase-dependent upregulation of beta1 integrins. This response was potentiated by simultaneous chemoattractant (fMLP) stimulation of PMN. Moreover, upregulation of beta1 integrins evoked by CD18 cross-linking was found to support adhesion of fMLP-stimulated PMN to matrix proteins and also was critical for the ability of PMN to migrate in collagen gels in response to a gradient of fMLP. Taken together, these data demonstrate that engagement of beta2 integrins in human PMN induces beta1 integrin expression in these cells of significance for their migration in the extravascular tissue. Thus, beta2 integrins may serve the function to regulate PMN locomotion in extravascular tissue via receptor crosstalk with beta1 integrins.

Spontaneous leukocyte rolling in rat and mouse microvessels is independent of mast cell activity.

OBJECTIVE AND DESIGN: The role of mast cells in spontaneous leukocyte rolling in venules of the mouse cremaster muscle and rat mesentery was investigated. MATERIALS: The experiments were carried out using mast cell-deficient rats (Ws/Ws), WBB6F1 mice (W/Wv), and their congenic littermates (wild type). TREATMENT: Administration of compound 48/80 intraperitoneally (50 microg) in rats and intrascrotally (5 microg) in mice, 4 h prior to the experiments. METHODS: Intravital microscopy of the terminal vascular beds in mouse cremaster muscle and rat mesentery. RESULTS: The level of spontaneous leukocyte rolling and the rolling velocity in venules of mast cell-deficient animals exactly matched that seen in wild-type animals. Challenge with compound 48/80 markedly increased leukocyte adhesion and emigration in venules of wild-type animals. In contrast, the number of adherent and extravascular leukocytes was very low in compound 48/80-challenged animals lacking mast cells and did not differ from that seen in control animals treated with phosphate-buffered saline. CONCLUSIONS: The presence or activation of mast cells has no bearing on spontaneous leukocyte rolling, at least not in rat and mouse microvessels.

Signaling via beta(2) integrins triggers neutrophil-dependent alteration in endothelial barrier function.

Activation of polymorphonuclear leukocytes (PMNs) and adhesion to the endothelial lining is a major cause of edema formation. Although known to be dependent on the function of beta(2) integrins (CD11/CD18), the precise mechanisms by which adherent PMNs may impair endothelial barrier capacity remain unclear. Here, the role of transmembrane signaling by beta(2) integrins in PMN-induced alterations in tight junctional permeability of cultured endothelial cell (EC) monolayers was investigated. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD11b/CD18, mimicking adhesion-dependent receptor engagement. CD18 cross-linking in PMNs added to the EC monolayer provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca(2+) and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cell-free supernatant obtained after CD18 cross-linking in suspended PMNs triggered an EC response indistinguishable from that induced by direct PMN activation, and caused clear-cut venular plasma leakage when added to the hamster cheek pouch in vivo preparation. The PMN-evoked EC response was specific to beta(2) integrin engagement inasmuch as antibody cross-linking of l-selectin or CD44 was without effect on EC function. Our data demonstrate a causal link between outside-in signaling by beta(2) integrins and the capacity of PMNs to induce alterations in vascular permeability, and suggest a paracrine mechanism that involves PMN-derived cationic protein(s) in the cellular crosstalk between PMNs and ECs.

Integrin alpha(2)beta(1) (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue.

Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the alpha(2)beta(1) (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the alpha(2)beta(1) integrin reduced PMN migration velocity in the extravascular tissue by 73% +/- 3% and 70% +/- 10%, respectively (means +/- SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-alpha(2) mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the alpha(4)beta(1) or alpha(5)beta(1) integrins. Further evidence for a critical role of expression of alpha(2)beta(1) integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-alpha(2) mAb treatment. Thus, expression of alpha(2)beta(1) integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated. (Blood. 2000;95:1804-1809)

Influence of local haemodynamics on leucocyte rolling and chemoattractant-induced firm adhesion in microvessels of the rat mesentery.

Tissue hyperaemia, oedema formation and leucocyte accumulation are characteristic features of the inflammatory process referable to changes at the microcirculatory level. Here, we used intravital fluorescence video microscopy to assess relationships among haemodynamical parameters, leucocyte rolling, and chemoattractant-induced firm adhesion in small venules (13-24 microM) of the rat mesentery. The rolling leucocyte flux in these vessels was directly proportional to the total leucocyte flux (r = 0.76, P < 0.001), which in turn closely correlated to the venular blood flow (r = 0.77, P < 0.001). Consequently, the rolling to total leucocyte flux fraction, averaging 39 +/- 15%, did not vary with the blood flow and showed no correlation to either blood flow velocity (r = -0.15, P = 0.42) or wall shear rate (r = -0.06, P = 0.77), indicating that the extent of leucocyte rolling is not primarily dependent on the fluid viscous drag at physiological blood flow rates in vivo. Stimulation of the mesentery with the chemoattractant fMLP (10(-6) M) induced firm adhesion of rolling leucocytes. It was found that the number of adherent leucocytes in individual vessels was directly related to the rolling leucocyte flux (r = 0.78, P < 0.001) and hence to the venular blood flow (r = 0.47, P < 0.05), while there was no correlation to the wall shear rate (r = 0.27, P = 0.24). The dependence of the firm adhesive response on the blood flow level and the delivery rate of leucocytes was confirmed at the whole organ level. Thus, leucocyte accumulation in rat skin lesions was markedly enhanced when a vasodilator was co-administered with the chemotactic stimulus compared with chemotactic stimulation alone. The data indicate that, within a physiological blood flow range, the leucocyte response to chemotactic stimulation is largely independent of the prevailing hydrodynamic shear forces. Instead, manifestation of the firm adhesive response, because of its dependence on the preceding rolling interaction, is clearly related to the blood flow level in the microvessels, which emphasizes the significance of tissue hyperaemia in inflammation.

Kinetics of leukocyte-induced changes in endothelial barrier function.

1. Extravasation of polymorphonuclear leukocytes (PMN) and associated plasma leakage are key events in the inflammatory process. The kinetics of PMN-induced changes in endothelial barrier function were studied by means of confluent monolayers of bovine aorta or human umbilical vein endothelial cells (EC), cultured on permeable membranes and mounted in a two-compartment diffusion chamber. The model permitted continuous measurement of transendothelial electrical resistance (TEER), and analysis of protein efflux and PMN migration across the EC monolayer. 2. Transendothelial chemotactic stimulation (fMLP or LTB4) of PMN resting on EC in the upper compartment induced a prompt decline in TEER, followed by an increase in protein flux and transmigration of PMN. Adding the chemoattractant together with PMN in the upper compartment provoked adhesion of PMN, fall in TEER and increase in protein permeability, but no transmigration of PMN, whereas inhibition of PMN adhesion to EC by pretreatment with anti-CD18 mAb prevented all responses to chemotactic stimulation. 3. Chemoattractant-induced adhesion of PMN to the EC monolayer induced a rapid rise in EC cytosolic free Ca2+, similar to that obtained by direct stimulation of EC with histamine, indicating an active response of EC to PMN activation and adhesion. 4. In summary, continuous recording of transendothelial electrical resistance in the in vitro model described permits rapid and sensitive analysis of leukocyte activation-induced effects on EC barrier function. The kinetics and specificity of the EC and PMN responses to chemoattractant stimulation suggest that activated PMN, via adhesion-dependent events, have a direct effect on EC junctional integrity independent of whether transmigration occurs or not.

beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo.

Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.

An approach for studies of mediator-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.

1. Although intravital microscopy is the method of choice for observation of inflammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2. By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3-6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte-endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. 3. Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not firm adhesion per se), or by use of intravital microscopy immediately before tissue fixation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. 4. Intraperitoneal injection of 10(-3) M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs firmly adherent cells to the PMNL, accumulation was again approximately 90%. Intraperitoneal injection of leukotriene C4, but not prostaglandin E2, caused a significant increase in venular PMNL concentration. 5. Systemic treatment with the anti-P-selectin monoclonal antibody PB1.3 had no effect on the histamine-induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rats. On the other hand, identical treatment with PB1.3 very effectively inhibited the histamine-induced PMNL response in the mesentery of rabbits. 6. In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and firm leukocyte adhesion is first determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the 'spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.

Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.

1. The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte-endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediator-induced venular leukocyte accumulation, reflecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. 2. We first examined the relative importance of histamine and 5-hydroxytryptamine (5-HT) in acute mast cell-dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine1 (H1)- and histamine2 (H2)-receptor blockade. In contrast, the 5-HT-receptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5-HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose-dependent; 5-HT response bell shaped). Prostaglandin D2 did not cause PMNL accumulation. 3. The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still significantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine-induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not firm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than firm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). 4. To examine the role of histamine-receptors in the histamine-induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H1-receptor antagonist), cimetidine, or ranitidine (H2-receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to histamine by 52%, while both H2-receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine-induced PMNL rolling by 82%. Furthermore, in contrast to an H3-receptor agonist, challenge with either the H1-receptor agonist 2-thiazolylethylamine or two different H2-receptor agonists (impromidine, dimaprit) was sufficient to provoke significant venular PMNL accumulation. 5. Treatment with the nitric oxide-synthase inhibitor L-NAME did not affect the histamine-induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and flow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P-selectin to rat isolated PMNLs. 6. We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was specifically due to PMNL rolling, involved activation of both H1- and H2-receptors, and lasted for 2 3 h. Moreover, the histamine-induced PMNL rolling was not dependent on nitric oxide synthesis, but was sensitive to glucocorticoid treatment, possibly via inhibition of expression or function of leukocytic P-selectin ligand(s).

In vivo rolling and endothelial selectin binding of mononuclear leukocytes is distinct from that of polymorphonuclear cells.

In inflammation, rolling of leukocytes along the microvascular endothelium is a precondition for subsequent integrin-mediated firm adhesion and extravasation. Rolling characteristics of polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL) in small venules (15-25 microns) of the rat mesentery were studied by intravital fluorescence microscopy under basal conditions and after intravenous treatment with an anti-rat neutrophil serum (ANS). The baseline rolling fraction of the venular total leukocyte flux was 36 +/- 15% (mean +/- SD). The PMNL fraction of the systemic leukocyte count was 27 +/- 9%. Treatment with ANS resulted in total depletion of circulating PMNL and reduced the leukocyte rolling fraction to 12 +/- 5%, in this situation represented only by MNL. In rats treated intraperitoneally with interleukin (IL)-1 beta for 4 h, the leukocyte rolling fraction was 53 +/- 13% and was reduced to 33 +/- 11% after ANS treatment. These data indicated that most, if not all, circulating PMNL rolled along the venular endothelial lining in the rat mesentery prepared for intravital microscopy, whereas MNL rolling was minor (approximately 10%) under the same basal condition. In cytokine-activated tissue, on the other hand, the number of rolling MNL was greatly increased. While PMNL rolling is known to be entirely selectin dependent, the increased MNL rolling after IL-1 stimulation was likely mediated by alpha 4 integrins, inasmuch as the rolling fraction of isolated peripheral blood lymphocytes injected into the microcirculation of the cytokine-stimulated mesentery was reduced from 31 +/- 14% to 6 +/- 2% by pretreatment of the cells with a monoclonal antibody against the rat integrin alpha 4 chain. In accordance with the in vivo rolling characteristics of the two cell populations, binding of soluble P- or E-selectin (selectin/IgG chimeras) was less intense for blood lymphocytes than for granulocytes, as determined by flow cytometric analyses of rat and human leukocytes. Taken together, our findings in vivo indicate that the adhesive interactions responsible for rolling of PMNL and MNL, respectively, are distinct in terms of receptor occupancy, and may help explain the temporal selectivity in recruitment of different leukocyte subpopulations in inflammatory or immune reactions.

Microvascular actions of histamine: synergism with leukotriene B4 and role in allergic leucocyte recruitment.

BACKGROUND: Previous studies have shown that antihistamines provide little or no protection against the recruitment of leucocytes in allergic inflammation. OBJECTIVE: We wanted to examine if threshold doses of histamine can potentiate chemoattractant-induced leukocyte adhesion and if complete inhibition of histamine-induced microvascular effects is necessary to reduce allergic leucocyte recruitment. METHODS: The role of histamine in allergic leucocyte recruitment was examined by use of intravital microscopy of the hamster cheek pouch microcirculation. RESULTS: We found that topical administration of histamine caused a concentration-dependent increase in microvascular permeability in the cheek pouch; i.e. 0.3 microM histamine caused no detectable plasma leakage, while 1 microM and 10 microM histamine resulted in 29 +/- 9.3 and 356 +/- 47 leakage sites/cm2 cheek pouch area, respectively. The percentage of postcapillary venules with more than five adherent leucocytes (an index of early leucocyte recruitment) was 1.1 +/- 0.51% in the control situation, and did not increase significantly after stimulation with histamine alone (0.3-10 microM) or with 1 nM leukotriene B4 (LTB4). On the other hand, coapplication of 10 microM histamine and 1 nM LTB4 increased leucocyte adhesion 24-fold. In fact, the 10 times lower dose of histamine (1 microM) together with 1 nM LTB4 increased leucocyte adhesion to a similar extent (20 fold). The increase in vascular permeability evoked by exogenous 10 microM histamine (with or without LTB4), or by histamine released from activated mast cells (antigen challenge), was completely reversed by local pretreatment with the H1-receptor antagonist mepyramine. This mepyramine treatment also abolished the enhanced leucocyte adhesion in response to coapplication of histamine and LTB4. Moreover, mepyramine, which had no effect on leucocyte recruitment evoked by 3 nM LTB4 per se, reduced antigen-induced recruitment of leucocytes to the extravascular tissue by 79.5 +/- 14.8%. CONCLUSION: We conclude that threshold concentrations of histamine can strikingly potentiate chemoattractant-induced leucocyte responses, and that in order to reduce allergic leucocyte recruitment it may be necessary to use antihistamines in doses high enough to abolish the microvascular actions of histamine.

Tumor cell arrest in the microcirculation: lack of evidence for a leukocyte-like rolling adhesive interaction with vascular endothelium in vivo.

Hematogenous spread of tumor cells and metastasis formation in secondary organs are insidious aspects of cancer. In the present intravital microscopic study in the rabbit mesentery, we examined the in vivo flow behavior of six human tumor cell lines of different histological origin. The tumor cells and human neutrophils were injected locally into a side branch of the superior mesenteric artery upstream of the observed microvascular area in the mesentery. None of the tumor cells behaved similar to the leukocytes of which a substantial fraction rolled along the endothelium of small venules. Thus, the tumor cells passed the same venular segments without interacting with the endothelial lining. Yet, three of the tumor cell lines (HT-29, DLD-1, and HCT-8) were strongly positive for the oligosaccharides Lewis(x), sialyl-Lewis(x), and sialyl-Lewis(a) which are recognized by the endothelial selectins that mediate leukocyte rolling. On the other hand, some tumor cells were trapped in the smallest vessels and remained so throughout the experimental period, apparently due to a discrepancy in size between tumor cells and microvessel lumen. Taken together, our in vivo findings suggest that initial microvascular arrest of metastasizing tumor cells is dependent primarily on mechanical factors rather than on receptor-mediated leukocyte-like adhesive interactions.

Inhibitory effect of locally administered heparin on leukocyte rolling and chemoattractant-induced firm adhesion in rat mesenteric venules in vivo.

1. Anti-inflammatory actions of heparin and related glycosaminoglycans have been described in the literature. Here, we used intravital microscopy of the rat mesentery microcirculation to examine effects of locally administered heparin on leukocyte rolling and chemoattractant-induced firm adhesion. 2. It was found that topical application of heparin reduced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion. Notably, the inhibitory action of heparin was not dose-dependent, but rather a biphasic dose-response was found, i.e. low (2 and 20 iu ml(-1)) and high (1000 iu ml(-1)) concentrations of heparin significantly reduced adhesion, whereas an intermediate dose (200 iu ml(-1)) was less effective. 3. Heparin, 2 and 20 iu ml(-1), decreased rolling leukocyte flux, while having no effect on blood flow or total leukocyte flux. By contrast, heparin, 200 and 1000 iu ml(-1), increased total leukocyte flux in parallel with a rise in volume blood flow resulting in recovery of the rolling leukocyte flux at these doses. Thus, the biphasic inhibitory action of heparin on fMLP-induced firm adhesion could in part be attributed to changes in leukocyte delivery (i.e. blood flow) and rolling leukocyte flux induced by heparin. 4. When compensating for the influence of different rolling levels on fMLP-evoked adhesion, a dose-related inhibitory effect of heparin on the firm adhesive response per se was revealed. Similar results were obtained in a static adhesion assay in vitro where heparin 200 and 1000 iu ml(-1) (but not 2 and 20 iu ml(-1)) significantly inhibited fMLP-induced leukocyte adhesion in the absence of any modulatory influence on changes in rolling. 5. Our data show that locally administered heparin inhibits leukocyte rolling as well as chemoattractant-induced firm adhesion in vivo which thus may contribute to the postulated anti-inflammatory activity of this compound. However, because of interference with several microvascular functions, strict dose-dependent responses to heparin treatment were not found, which illustrates the complex interplay between local blood flow, leukocyte rolling and chemoattractant-induced adhesion as determinants of leukocyte recruitment to tissues in inflammation.

Propentofylline inhibits polymorphonuclear leukocyte recruitment in vivo by a mechanism involving adenosine A2A receptors.

Propentofylline is an atypical xanthine derivative that blocks adenosine uptake and has been shown to protect against ischemia-induced cerebral damage. We have studied the effect of propentofylline on recruitment of polymorphonuclear leukocytes during acute peritonitis induced by zymosan in mice. Following i.p. injection of zymosan, recruitment of polymorphonuclear leukocytes, reflected by myeloperoxidase activity in the peritoneal cavity, increased from 2 h onwards, peaked at 4 h and then decreased gradually. Propentofylline antagonized the zymosan-induced peritoneal myeloperoxidase accumulation in a concentration-dependent manner. This effect of propentofylline was counteracted by the non-selective adenosine receptor antagonist theophylline (50 mg/kg), and by the selective adenosine A2A receptor antagonists, 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo[4,3-a]quinoxaline (CP 66713) and 1,3-dipropyl-8-[3,4-dimethoxystyryl]-7-methylxanthine (KF 17387) (both at 2 mg/kg). The results indicate that propentofylline can reduce polymorphonuclear leukocyte recruitment in vivo and that this effect is related to an action on adenosine A2A receptors.

Anti-IgE-induced accumulation of leukocytes, mediators, and albumin in skin chamber fluid from healthy and atopic subjects.

The aim of this study was to examine potential differences between healthy and atopic subjects with regard to IgE-mediated cutaneous inflammation. For this purpose, we analyzed histamine, tryptase, leukotriene B4, albumin, eosinophils, and total leukocytes in skin chamber fluid after challenge with anti-human IgE. We also measured gross skin reactivity (wheal, flare, and late-phase reactions), circulating IgE, and eosinophils, as well as the state of eosinophil activation. It was found that despite having more circulating IgE, the skin responsiveness of the atopic subjects did not differ significantly from that of the nonatopic subjects with respect to mediator release, albumin extravasation, or total recruitment of leukocytes. Moreover, the sizes of anti-IgE-induced wheal, flare, and late-phase reactions were very similar in the two groups. On the other hand, significant recruitment of eosinophils during the IgE-mediated reaction was more or less restricted to the atopic group. Yet the recruited eosinophils, of which the majority was in an early state of activation before degranulation, did not seem to contribute significantly to the IgE-mediated delayed skin edema. Furthermore, the eosinophil count in anti-IgE chambers of the atopic subjects did not correlate with any of the other parameters monitored. Thus because the anti-IgE-induced recruitment of eosinophils appeared to be unrelated to factors such as the number of peripheral blood eosinophils, the degree of mast cell activation, the intensity of inflammatory skin changes, and the level of circulating IgE, it is apparent that the mechanisms for and pathophysiologic role of IgE-mediated dermal eosinophil accumulation in atopic subjects require further investigation.