Leukocyte CD11b Research Articles

Changes in Immune Cell Distribution and IL‐10 Production are Regulated through Endometrial IP‐10 Expression in the Goat Uterus

Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon-gamma (IFN-gamma)-inducible protein 10 kDa (IP-10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP-10 during the period of pregnancy establishment. Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of IP-10, IFN-gamma, tumor necrosis factor-alpha, interleukin-10 (IL-10), CXCR3 mRNA and leukocyte cell surface markers, CD4, CD8, CD11b and CD45 mRNA during the caprine early pregnancy was investigated. The ability of IP-10 to stimulate peripheral blood mononuclear cells (PBMCs) migration was demonstrated using a chemotaxis assay. Changes in migration of PBMCs' immune cell population and cytokine expressions with IP-10 stimulation were investigated using flow cytometry and RT-PCR respectively. Levels of IP-10, IL-10, CD4 and CD11b mRNA, and the number of CD4 and CD11b positive cells in pregnant goat endometrium were higher than those of cyclic goat endometrium. Migration of PBMCs was stimulated by recombinant caprine IP-10, and the effect was significantly reduced by neutralization with the use of an anti-IP-10 antibody. In the flow cytometric and RT-PCR analyses, migrated cells stimulated by IP-10 increased the expression of IL-10 and CD11b mRNA. Furthermore, IP-10 could stimulate the expression of IL-10 mRNA from PBMCs. Endometrial chemokine IP-10 could regulate IL-10 production by resident and possibly migrated cells expressing CD11b, probably natural killer cells, and these changes may result in immune environments of the uterus suitable for conceptus implantation in ruminants.

Contrast medium attenuates platelet activation and platelet-leukocyte cross–talk

The influence of ionic and non-ionic contrast media (CM) on platelet and leukocyte activation and platelet-leukocyte crosstalk was investigated in hirudinized whole blood. The blood was incubated with and without the ionic CM ioxaglate and the nonionic CM iodixanol at 37 degrees C for 5 min, without or with stirring. Platelet and leukocyte activation and platelet-leukocyte aggregation were measured using whole blood flow cytometry. When blood samples were pre-incubated in the presence of 2%, 5%, and 10% of CM without stirring, both ioxaglate and iodixanol had little effect on unstimulated samples, but dose-independently decreased 1 microM ADP-induced platelet P-selectin expression and fibrinogen binding, and thus platelet-leukocyte aggregate formation. Ioxaglate had little effect on leukocyte CD11b expression, whilst iodixanol slightly enhanced resting and N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 microM)-stimulated leukocyte CD11b expression. Blood samples were also incubated with stirring to investigate the impact of CM (5% of ioxaglate or iodixanol) on platelet-leukocyte cross-talk. Collagen induced marked platelet activation and platelet-leukocyte aggregation, and subsequently elevated leukocyte CD11b expression. The latter was attenuated by ioxaglate and iodixanol, and was accompanied by reduced platelet-leukocyte aggregation. In conclusion, the CM ioxaglate and iodixanol attenuate platelet activation and platelet-leukocyte cross-talk. Inhibitory effects of the contrast agents on this cross-talk are apparently exerted by reducing heterotypic conjugation, and may be beneficial in connection with PCI.

Long-distance running modulates the expression of leucocyte and endothelial adhesion molecules.

There still exist many unanswered questions whether physical exercise is beneficial or harmful to the immune system. The 'open-window' post-exercise hypothesis states that athletes are more susceptible to infections after exercise, but there is a need for further elucidation. The aim of the present study was to investigate the effect of long-distance running on leucocyte expression of selected adhesion molecules as well as the plasma levels of soluble leucocyte- and endothelium-derived adhesion molecules. Twenty-seven men participating in Oslo marathon together with 16 entrants (eight men and eight women) in the Oslo half-marathon were recruited to this study. Venous blood was collected before and immediately after the races for analysing the leucocyte expression of CD62L, CD11b and CD14 with the help of flow cytometry, and plasma concentrations of soluble (s) sE-selectin, sL-selectin, sP-selectin, sVCAM-1, sICAM-1 and sCD14 were assessed by means of enzyme-linked immunosorbent assays. A significant increase of leucocyte CD11b expression was observed following both races, compared to the pre-race situation. Monocyte CD14 expression increased only after the marathon race. After both races, CD62L expression was significantly lowered on all leucocyte subsets, whereas the plasma levels of sE-selectin, sP-selectin, sL-selectin, sVCAM-1, sICAM-1 and sCD14 were all increased. Altogether, these changes negatively influence the ability of leucocytes to adhere to and actively transmigrate the endothelium to reach the tissues. Our study thus supports the 'open-window' hypothesis, indicating a reduced capacity to combat infectious agents during the immediate post-exercise period.

Activation of leukocytes by postprandial lipemia in healthy volunteers

Activation of leukocytes is obligatory for inflammation and atherogenesis by adhering to the endothelium via specific ligands. Although in vitro studies have shown that triglycerides (TG) can activate leukocytes, it is unknown whether this occurs in vivo . Using flowcytometry, we studied the expression of leukocyte activation markers CD11A, CD11B, CD62L (all involved in endothelium adhesion) and CD66B (a neutrophil degranulation marker) during a 6 h fat challenge (50 g/m 2) and a water test in 10 healthy males (52 ± 3 years). After fat, neutrophil counts were increased between t = 1 and t = 6 h, with a maximum at t = 3 h (+32% versus t = 0, P < 0.05), while they remained unchanged after water. Both tests showed gradual lymphocyte count increments. The expression of activation markers on lymphocytes was low and showed comparable responses after both tests. After fat, a significant increase up to a maximum at t = 6 h was seen for CD11B on monocytes and on neutrophils for CD11B, CD62L and CD66B. Postprandial activation of monocytes and neutrophils was higher after fat than after water. The maximal postprandial TG increment was significantly related to the increase of CD11B on monocytes (Pearson’s R = 0.64, P < 0.05). In conclusion, postprandially there is a TG-specific increase of neutrophil counts and increased activation of monocytes and neutrophils. These results are suggestive of a pro-inflammatory situation that may correspond with increased adhesive capacity of these cells contributing to the inflammatory component of atherosclerosis.

Exercise-induced platelet and leucocyte activation is not enhanced in well-controlled Type 1 diabetes, despite increased activity at rest.

Stress can evoke a prothrombotic state with activated platelets and leucocytes, and increased platelet activation at rest has been reported for diabetic subjects. Thus, prothrombotic responses to stress may be enhanced in diabetes mellitus. We therefore evaluated platelet and leucocyte responses to exercise in Type 1 diabetic patients. Type 1 diabetes mellitus patients with good metabolic control and healthy subjects matched for age and body mass index ( n=15 for both) performed a maximal exercise test. Platelet and leucocyte activation and their heterotypic aggregation were monitored by whole blood flow cytometry. Diabetic platelets did not show higher basal levels of P-selectin expression, but were more reactive to ADP and thrombin stimulation. Diabetic patients had increased lymphocyte and monocyte CD11b expression, and increased circulating platelet-monocyte aggregates. Exercise evoked thrombocytosis, increased circulating platelet P-selectin expression, enhanced platelet sensitivity to ADP and thrombin, and elevated plasma levels of soluble P-selectin to a similar degree in diabetic patients and healthy subjects. Exercise induced marked leucocytosis and elevated plasma elastase, but only slightly increased leucocyte CD11b expression and leucocyte reactivity to stimulation by N-formyl-methionyl-leucyl-phenylalanine. In all of these there was no difference between diabetic patients and healthy subjects. The numbers, but not percentages of circulating platelet-leucocyte aggregates were markedly increased by exercise, but the increase was less prominent among diabetic patients. Strenuous exercise evokes platelet and leucocyte activation in Type 1 diabetic patients and healthy subjects. Platelet and monocyte hyperactivity were found at rest, but responses to stress were not augmented in metabolically well-controlled Type 1 diabetes mellitus patients.

Assessment of inflammatory response and sequestration of blood iron transferrin complexes in a rat model of lung injury resulting from exposure to low-frequency shock waves.

Impact of air blast overpressure waves (OPW), or shock wave, with the body wall or body armor produces two types of energy waves: high-frequency low-amplitude stress waves and long-duration low-frequency share waves. These types of energy waves are characterized by different mechanisms of primary tissue injury that mostly affect lung. Systemic inflammation and resultant acute respiratory distress syndrome are known major secondary causative agents of delayed multiple organ failure and subsequent death after OPW exposure. However, association of each pattern of the blast OPW-produced energy waves with postexposure inflammatory events has not yet been delineated. The objectives of the present research were a) establishment of a rat model for assessment of the inflammatory response following lung injury produced by exposure to medium-amplitude (approximately 120 kPa) low-frequency (260+/-5 Hz) OPWs; and b) assessment of the dynamics of alteration in polymorphonuclear leukocyte counts and expression of CD11b adhesion molecules on the surface of polymorphonuclear leukocytes and status of iron-transferrin complexes in peripheral blood after OPW exposure. This study focused on the OPW effects at different time periods, using a sequential approach to postexposure events. Lung injury in rat was induced by OPW generated in a laboratory shock tube. Animals were exposed to OPW (at peak overpressure of 118+/-7 kPa) that produced "moderate" lung injury. Military research institute. Twenty-seven CVF Sprague-Dawley rats were subjected to OPW exposures, and 17 sham-treated animals were used as control. Lung tissue and blood samples were collected at 1, 3, 6, 12, and 24 hrs following OPW exposures and compared with samples collected from nonexposed animals. OPW-induced lung injury caused a 2.7-fold increase in the number of circulatory polymorphonuclear leukocytes as early as 1 hr postexposure, which is indicative of mobilization of the pool of marginated polymorphonuclear leukocytes into the free circulation. Polymorphonuclear leukocyte counts increased through the following 3- and 6-hr periods, when they were, respectively, 5-fold and 3.5-fold higher than in controls. These effects were accompanied by a pronounced expression of CD11b in polymorphonuclear leukocytes and tissue sequestration of blood iron-transferrin complexes during the entire 24-hr period of observations. The increase in circulatory polymorphonuclear leukocytes was accompanied by a decrease in iron-transferrin complex concentrations that apparently reflected implication of blood plasma iron in the inflammatory cell response to OPW-induced injury. The observed dynamics in polymorphonuclear leukocyte alterations in peripheral blood after OPW exposure were similar to those found recently in clinical observations of nonpenetrating injury and in animal models of infectious insults. Therefore, our data suggest that the main pattern of proinflammatory alterations in the rat model of lung injury induced by exposure to long-duration shock wave is similar to patterns that are characteristic of major trauma. The data further suggest that the expression of polymorphonuclear leukocyte CD11b and the response of iron-transferrin complex can be considered as potential surrogate markers in blood for systemic alterations following OPW-induced injury and, therefore, warrant further investigation in a human pilot study.

Interactions between bradykinin (BK) and cell adhesion molecule (CAM) expression in peptidoglycan-polysaccharide (PG-PS)-induced arthritis.

Bradykinin (BK), a vasoactive, proinflammatory nonapeptide, promotes cell adhesion molecule (CAM) expression, leukocyte sequestration, inter-endothelial gap formation, and protein extravasation in postcapillary venules. These effects are mediated by bradykinin-1 (B1R) and-2 (B2R) receptors. We delineated some of the mechanisms by which BK could influence chronic inflammation by altering CAM expression on leukocytes, endothelium, and synovium in joint sections of peptidoglycan-polysaccharide-injected Lewis rats. Blocking B1R results in significantly increased joint inflammation. Immunohistochemistry of the B1R antagonist group revealed increased leukocyte and synovial CD11b and CD54 expression and increased CD11b and CD44 endothelial expression. B2R antagonism decreased leukocyte and synovial CD44 and CD54 and endothelial CD11b expression. Although these findings implicate B2R involvement in the acute phase of inflammation by facilitating leukocyte activation (CD11b), homing (CD44), and transmigration (CD54). Treatment with a B2R antagonist did not affect the disease evolution in this model. In contrast, when both BK receptors are blocked, the aggravation of inflammation by B1R blockade is neutralized and there is no difference from the disease-untreated model. Our findings suggest that B1R and B2R signaling show physiologic antagonism. B1R signaling suggests involvement in down-regulation of leukocyte activation, transmigration, and homing. Further studies are needed to evaluate the B1 receptor agonist's role in this model.

Effect of complement inhibition and heparin coating on artificial surface–induced leukocyte and platelet activation

Background Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. Methods A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. Results Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs–C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs–C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. Conclusions Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.

Leukocyte CD11b and CD62L Expression in Response to Interstitial Inflammation in CAPD Patients

Very little is known about the kinetics of leukocyte recruitment and the modulation of adhesion molecules on leukocytes in the interstitium at the site of inflammation outside the peritoneal cavity in patients on peritoneal dialysis. These issues were addressed in the present study. Two skin blisters were raised in 10 patients on peritoneal dialysis and in 19 healthy subjects. After 12 hours, blister exudates were collected and the blisters were thereafter challenged with buffer or autologous serum in order to establish an intermediate and an intense cutaneous inflammation. Leukocyte count, leukocyte CD11b/CD62L expression, monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8) were determined in blood and at the three sites of interstitial inflammation by immunostaining, flow cytometry, and ELISA. In monocytes and granulocytes, expression of CD11b increased and CD62L decreased significantly during the transmigration process from the peripheral blood into the three sites of interstitial inflammation. In the nonstimulated blister, expression of CD11b on both monocytes and granulocytes was similar in patients and healthy subjects. At the site of intermediate inflammation, expression of CD11b on both monocytes and granulocytes was significantly higher in healthy subjects compared to patients (p < 0.05 and p < 0.001 respectively). At the site of intense inflammation, expression of CD11b on granulocytes was significantly lower (p < 0.001), and CD62L significantly higher (p < 0.001) in patients. Interstitial concentrations of MCP-1 and IL-8 at the sites of intermediate inflammation were significantly higher in healthy subjects compared to patients (p < 0.05 and p < 0.05 respectively). However, at the site of intense inflammation, similar concentrations of MCP-1 and IL-8 were observed. Furthermore, there were no significant correlations between concentrations of MCP-1 and IL-8 in blister exudates and expression of CD11b on monocytes and granulocytes at the sites of interstitial inflammation. The ability of monocytes and granulocytes to modulate the expression of adhesion molecule in response to interstitial inflammation was significantly impaired in patients on peritoneal dialysis. Furthermore, these data suggest that the expression of CD11b on leukocytes is not dependent on concentrations of IL-8 and MCP-1 in interstitium in this patient group.

Enhanced leukocyte–platelet cross‐talk in Type 1 diabetes mellitus: relationship to microangiopathy

Platelets and leukocytes may influence each others' function, i.e. platelet-leukocyte cross-talk. Diabetes mellitus (DM) is associated with platelet and leukocyte dysfunction. To evaluate platelet-leukocyte cross-talk, and if this might contribute to platelet and leukocyte dysfunction and microangiopathy in DM patients. We evaluated platelet and leukocyte function, and cross-talk between these cells in Type 1 DM patients without (n = 19) and with (n = 20) microangiopathy, and healthy subjects (n = 27), using whole blood flow cytometry. Platelet-leukocyte cross-talk was studied in hirudinized whole blood incubated at 37 degrees C with stirring. Basal single platelet P-selectin and leukocyte CD11b expression were similar in DM patients and healthy subjects, whilst circulating platelet-leukocyte aggregates and plasma elastase levels were elevated in DM patients. The thromboxane A2 analog U46619 (3 x 10(-7) m) induced more marked increases of platelet P-selectin expression and platelet-leukocyte aggregation in DM patients than in healthy subjects. The leukocyte-specific agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-7) m) induced more marked CD11b expression in DM patients with microangiopathy, compared with healthy subjects. Platelet-leukocyte cross-talk induced by U46619 (10(-6) m) showed no difference between DM patients and healthy subjects. fMLP (10(-6) m) evoked marked leukocyte activation, which subsequently caused mild platelet P-selectin expression. This leukocyte-platelet cross-talk was more pronounced in DM patients than in healthy subjects. Furthermore, enhanced leukocyte-platelet cross-talk was correlated to platelet hyperreactivity among DM patients with microangiopathy only. Type 1 DM is associated with platelet and leukocyte hyperactivity, and enhanced leukocyte-platelet cross-talk, which may contribute to platelet hyperactivity and the microvascular complications seen in Type 1 DM.

Glycoprotein IIb/IIIa inhibition attenuates platelet-activating factor-induced platelet activation by reducing protein kinase C activity

Glycoprotein (GP)IIb/IIIa inhibition may abolish activated leukocyte-induced platelet activation, in which leukocyte-released platelet-activating factor (PAF) is a major mediator. The present study thus investigated if and how GPIIb/IIIa inhibitors interfere with PAF-induced platelet activation. Platelet and leukocyte activation were monitored by flow cytometry and immunoblotting. GPIIb/IIIa inhibitors (c7E3, non-peptide SR121566, and MAb RFGP56) attenuated PAF-induced, but not adenosine diphosphate (ADP)- or thrombin receptor activating peptide (TRAP)-induced platelet P-selectin expression in whole blood. GPIIb/IIIa blockade enhanced ADP- or TRAP-induced leukocyte CD11b expression, but not the response to PAF. GPIIb/IIIa blockade attenuated PAF-induced, but enhanced ADP- or TRAP-induced platelet-leukocyte aggregation. Under the present experimental conditions, thromboxane A2 receptor antagonism did not significantly influence PAF-induced platelet activation, and GPIIb/IIIa inhibition did not interfere with calcium mobilization/influx in platelets. Protein kinase C (PKC) blockade inhibited PAF-induced platelet P-selectin expression, and PAF-induced PKC activity was reduced by GPIIb/IIIa inhibition. PAF (=1 micro m) did not induce MEK 1/2 or ERK 1/2 phosphorylation, whilst thrombin induced marked responses, which were enhanced by GPIIb/IIIa blockade. Thus, GPIIb/IIIa inhibition attenuates PAF-induced platelet activation via inhibiting PKC activity. GPIIb/IIIa blockade enhances thrombin-induced platelet MEK 1/2 and ERK 1/2 activation, and augments ADP- and TRAP-induced leukocyte activation by enhancing platelet-leukocyte aggregation.

The effect of methylprednisolone on cytokine concentration and leukocyte adhesion molecule expression in an isolated cardiopulmonary bypass system

This study examines the effect of methylprednisolone on cytokine balance and adhesion molecule expression within an isolated cardiopulmonary bypass (CPB) system. This isolated CPB system is an in vitro model which simulates the pro-inflammatory immune response. Whole blood from 10 volunteers was obtained in two equal amounts. Heparin and saline were added to the control group while heparin and methylprednisolone were added to the methylprednisolone group. The blood was added to two identical CPB circuits and bypass commenced by a trained perfusionist. Samples were taken at blood donation (Sample 0), 10 min after the addition of drugs (Sample 1) and after 30, 60 and 90 min of CPB (Samples 2, 3 and 4, respectively). Cytokines interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1ra) and tumour necrosis factor soluble receptor 2 (TNFsr2) and the leucocyte adhesion molecules L-selectin, HLA DR, CD18 and CD11b were determined. IL-8 increased in both groups. This increase was significantly less in the methylprednisolone group. Increases in granulocyte CD11b and CD18 expression were less in the methylprednisolone group than in the control group but did not reach statistical significance. These results indicate that methylprednisolone significantly reduces the production of IL-8 in an isolated CPB system. This effect occurs in the absence of IL-10.

Biphasic pro-thrombotic and inflammatory responses after coronary artery bypass surgery

Early graft failure after coronary artery bypass grafting (CABG) is related to thrombosis and inflammation in the grafted vessel(s). The time courses of, and relationships between, pro-thrombotic and inflammatory responses to CABG surgery have, however, not been well defined. Fifteen patients undergoing CABG were examined before, and 1 h, 1 day, 7 days, and 3 months after surgery. Cellular markers of platelet and leukocyte activation were monitored by whole blood flow cytometry, and plasma markers of pro-thrombotic and inflammatory responses were analyzed by immunoassays. CABG immediately increased circulating P-selectin-positive platelets, leukocyte CD11b expression, and platelet-leukocyte aggregates (PLAs). Thrombin generation (F1 + 2 levels) and cytokine release [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-8, and IL-10], soluble P-selectin, and soluble E-selectin also increased immediately. These alterations persisted during the first week after surgery, with re-bound increases of circulating activated platelets and PLAs, TNF-alpha, and F1 + 2 on day 7. Platelet and PLA responsiveness to in vitro stimulation was suppressed immediately after CABG, but markedly enhanced 1 week after surgery. After 3 months, plasma soluble P-selectin, F1 + 2, and IL-10, and monocyte CD11b expression were still slightly elevated compared with baseline. In conclusion, CABG induces marked pro-thrombotic and inflammatory responses, which persist for at least 1 week. Platelet activation, platelet reactivity, PLA formation, thrombin generation, and TNF-alpha release show a second peak 1 week after surgery. These findings suggest that intensified and prolonged antithrombotic/inflammatory treatment should be considered after CABG surgery.

Stimulation of inflammatory markers after blunt trauma.

Inflammatory mediators are released after trauma and may be related to the pathogenesis of sepsis. A prospective combined study of the pattern of release of an inflammatory mediator, interleukin (IL) 6, leucocyte activation (polymorphonuclear leucocyte (PMN) CD11b receptor expression and plasma elastase-alpha1 proteinase inhibitor complex (E-alpha1PI)) and soluble endothelial adhesion molecule expression (soluble E-selectin (sE-selectin) and soluble intracellular adhesion molecule 1 (sICAM-1)) was performed in patients suffering blunt trauma without adult respiratory distress syndrome (ARDS) or multiple organ failure syndrome (MOFS). Thirty-one patients with a mean Injury Severity Score (ISS) of 14 (range 9-57) were studied. Venous blood samples were collected within 6 h of injury and then at 1, 3, 5 and 7 days. Leucocyte CD11b expression was quantified by flow cytometry. Serum IL-6, plasma E-alpha1PI, sE-selectin and sICAM-1 were measured by enzyme-linked immunosorbent assay. Serum IL-6, CD11b expression and E-alpha1PI levels were significantly raised above control values (P < 0.0001) on admission, slowly returning towards control values over the study period (median IL-6, 140 pg/ml versus undetectable; CD11b, 14.8 versus 6.4 mean channel fluorescence units; E-alpha1 PI, 208 versus 52 microg/l). The sICAM-1 level rose to a median of 539 ng/ml at 5 days (control 243 ng/ml). The median sE-selectin level also progressively increased to a maximum level of 80 ng/ml at 5 days (control 49 ng/ml). Eleven patients developed postoperative sepsis. Significant differences in CD11b expression were seen at days 3, 5 and 7 and in E-alpha1 PI at 6 h, 24 h and 3 days in patients who subsequently developed sepsis (P < 0.05). Severe injury (ISS 16 or greater) was associated with significantly greater responses in these measurements. These data show that markers of inflammation are specifically stimulated by trauma even when ARDS and MOFS do not occur. The CD11b receptor on PMNs may be useful in screening patients destined to develop post-traumatic sepsis.

Platelet-leukocyte aggregation under shear stress: differential involvement of selectins and integrins.

Platelets and leukocytes can form heterotypic aggregates. We studied how shear stress influences platelet-leukocyte aggregation (PLA). Shear stress was applied to hirudinized blood, using a cone-and-plate(let) analyzer. Platelet and leukocyte activation and PLA formation were monitored by flow cytometry. Shear stress per se increased platelet P-selectin expression, leukocyte CD11b expression, and PLA formation. Shear-induced heterotypic aggregation was seen among monocytes and neutrophils, but not lymphocytes. ADP induced marked PLA formation, but this was reduced by shear stress. fMLP mildly increased PLA formation, and this effect was enhanced by shear stress. P-selectin blockade abolished, whilst GPIIb/IIIa blockade enhanced shear- and agonist-induced PLA formation. At 1800 s(-1), shear-and agonist-induced PLA formation were maintained or further enhanced with GPIIb/IIIa blockade alone, but reduced by simultaneous blockade of GPIIb/IIIa, CD11b, and CD18. In conclusion, shear stress per se enhances PLA formation. With agonist stimulation, shear stress enhances PLA formation primarily mediated by integrins, but attenuates PLA formation primarily mediated by P-selectin. The present results indicate that P-selectin-mediated bridging is essential for the initiation of PLA formation, while integrin-bridgings contribute importantly to the stability of heterotypic conjugates under high shear stress.

Aspirin treatment does not attenuate platelet or leukocyte activation as monitored by whole blood flow cytometry

Despite undoubtful clinical evidence of anti-platelet effects of aspirin, previous studies demonstrate little inhibition by aspirin treatment of single platelet activation as measured by flow cytometry. This discrepancy was further evaluated using flow cytometric measurements of circulating platelet–leukocyte aggregates (PLAs), which may be a more sensitive marker of platelet activation in vivo than measurements of activation markers on single platelets. Blood samples were obtained from 15 healthy subjects before and after aspirin treatment (75 and 500 mg daily for 1 week). Platelet (P-selectin expression) and leukocyte (CD11b expression) activation and platelet–leukocyte aggregation were monitored by whole blood flow cytometry. Approximately 1% platelets in unstimulated samples were P-selectin-positive, i.e., circulating activated platelets, and about 3% of leukocytes were circulating as PLAs; neither of these parameters were reduced by aspirin treatment. Circulating platelet micro-aggregates were not influenced by aspirin either. In vitro stimulation with ADP, thrombin, or PAF increased platelet P-selectin expression and thus PLA formation, but these responses were not affected by aspirin. Leukocyte CD11b expression, a marker of leukocyte secretion, was not significantly influenced by aspirin either in unstimulated samples or upon in vitro stimulation. Thus, the present data support the concept that thromboxane generation is of little importance for the activation of single platelets; the platelet inhibiting effect of aspirin is seen mainly in the presence of close cell–cell contact, which enhances the importance of thromboxane. Multiple mechanisms may contribute to the remarkable clinical anti-thrombotic effect of aspirin.

Effects of insulin on platelet and leukocyte activity in whole blood

Diabetes mellitus (DM) is associated with platelet and leukocyte dysfunction. Previous observations with regard to insulin effects on platelet and leukocyte function are less than consistent. We thus investigated the effects of insulin on platelets and leukocytes, as well as on platelet–leukocyte interactions in whole blood. Hirudinized whole blood from 20 healthy subjects was preincubated at 37 °C in the absence or presence of insulin (30 and 300 μU/ml), and further incubated without or with adenosine diphosphate (ADP) (10 −5 M) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 −7 M), respectively. Platelet P-selectin expression, platelet fibrinogen binding, single platelet and platelet–platelet aggregate (PPA) counts, and leukocyte CD11b expression and superoxide anion production were monitored by flow cytometry. Insulin decreased single platelet counts ( P<0.05) and increased PPAs ( P<0.01) at 300 μU/ml in unstimulated samples, but did not significantly influence the P-selectin expression or fibrinogen binding of single platelets. Insulin also enhanced ADP-induced platelet aggregation, seen as an augmented decrease of single platelet counts. Insulin (30 μU/ml) increased leukocyte CD11b mean fluorescence intensity (MFI) in unstimulated, as well as fMLP- and ADP-stimulated samples ( P<0.05 for all). fMLP-induced superoxide anion (O 2 −) production was, however, attenuated by insulin. Furthermore, fMLP-activation of leukocytes was associated with enhanced platelet fibrinogen binding and P-selectin expression. In conclusion, clinically relevant concentrations of insulin enhance platelet aggregability and leukocyte CD11b expression, but attenuate leukocyte respiratory burst activity. Our results suggest that insulin may modulate thrombotic and inflammatory processes in vivo in a complex manner.

Outer membrane vesicles from Neisseria meningitidis.

Flow cytometry was used to study the expression of leukocyte adhesion molecules CD11a, CD11b, CD11c, CD14, and CD62L (L-selectin) and production of reactive oxygen species (ROS) in an ex vivo human whole-blood system stimulated with lipopolysaccharide-containing outer membrane vesicles (LPS-OMV) from N. meningitidis. Results demonstrated a dose-dependent increase in surface expression of CD11a, CD11b, CD11c and CD14 in granulocytes and monocytes (maximal at 30-120 min) upon OMV-LPS challenge, whereas CD62L expression was heavily downregulated (maximal at 30-120 min). The OMV-associated LPS was almost as potent (on a weight basis) as purified LPS from E. coli in inducing adhesion molecule modulation but the response was delayed. Upon stimulation with OMV-LPS or E. coli-LPS, the production of intracellular ROS increased in both granulocytes and monocytes when dihydroethidium (DHE, mainly reflecting superoxide anion) was used as a probe, whereas peroxynitrite production monitored with dihydrorhodamine 123 (DHR) was not significantly changed. The OMV-mediated modulation of leukocyte adhesion molecule expression and increased ROS production may certainly lead to increased entrapment of leukocytes in the microcirculation and contribute to untoward inflammatory reactions as seen in systemic meningococcal disease.

Role of angiotensin II type 1 receptor in the regulation of cellular adhesion molecules in atherosclerosis

Background Inflammation is a central feature of coronary artery disease (CAD) that is characterized by increased expression of cellular adhesion molecules with the exception of L-selectin. L-selectin is a leukocyte adhesion molecule that is rapidly shed after leukocyte activation so that it appears to be decreased in CAD. The renin-angiotensin system (RAS) is implicated in atherogenesis and up-regulates these molecules. Objectives The aim of this study was to investigate the effect of angiotensin type 1 (AT1) receptor antagonism on serum and leukocyte adhesion molecule expression in patients with CAD. Blood samples were collected from 31 patients before and after 8 weeks of treatment with losartan (44 ± 2 mg/d, mean ± SE), an AT1 receptor antagonist. We measured serum intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial-leukocyte adhesion molecule, and C-reactive protein (CRP). By flow cytometry, we also measured the expression of leukocyte CD11a, CD11b, CD11c, CD18, CD31, CD49d, and CD62L (L-selectin) in 13 patients. Results Treatment with losartan decreased systolic blood pressure (141 ± 3 vs 135 ± 4 mm Hg, P =.04) and increased plasma renin activity (1.2 ± 0.4 vs 2.7 ± 0.5 ng/mL/h, P =.001). There was a significant increase in L-selectin expression on monocytes (86 ± 6 vs 118 ± 10 MESF units, P =.007), lymphocytes (52 ± 10 vs 79 ± 8, P =.01), and granulocytes (124 ± 7 vs 156 ± 18, P =.056). However, there were no changes in the other leukocyte and serum adhesion molecules or CRP. Conclusions These findings suggest that AT1 receptor antagonism selectively modulates L-selectin expression on leukocytes and that endogenous stimulation of AT1 receptors by the RAS contributes to the activation of leukocytes and decreased expression of L-selectin in CAD. (Am Heart J 2001;142:248-53.)

Blood Cell Activation, Coagulation, and Inflammation in Men and Women With Coronary Artery Disease

We investigated in some detail the immunologic and procoagulant activation patterns in men and women with unstable (UA, n=26) versus stable (SA, n=40) coronary artery disease (CAD). Leukocyte activation and platelet–leukocyte complex formation were assessed by flow cytometry. Plasma markers of coagulation and inflammation were analyzed. Unstable patients displayed higher levels of platelet–leukocyte complexes ( P<.001), of leukocyte CD11b ( P<.01), and of T cell HLA-DR ( P<.05) than healthy controls did. Female UA patients presented the highest degree of complexes. SA patients only differed significantly from controls with respect to HLA-DR ( P=.02). UA patients had higher levels of C-reactive protein ( P<.01), IL-6 ( P<.001), IL-10 ( P<.01), and soluble fibrin ( P<.001) than did stable ones. Regarding P-selectin levels, 25% of SA and 50% of UA patients were above normal range. Again, UA women presented the highest marker amounts. As for soluble tissue factor, women had higher levels than men regardless of the severity of disease ( P<.001 in SA). We conclude that in unstable coronary syndromes, there is an activation of both coagulation and inflammation that coincides with an increased activation of platelets and leukocytes. Cellular interactions may contribute to the systemic responses observed. Women have different patterns of cellular activation than men, indicating differences in pathogenetic mechanisms.