Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Abstract

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins “activation reporter.” When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP-or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.

Effect of trans‐resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function

“…PMN were incubated for 30 min at room temperature with a fluorescein isothiocyanate (FITC)‐ conjugated anti‐MAC‐1 mouse MoAb or with the CBRM1/5 mouse MoAb, recognizing an activation‐dependent epitope on MAC‐1 α‐chain, followed by a FITC‐conjugated anti‐mouse IgG. PMN fluorescence was analysed by a FACStar flow cytometer (Becton Dickinson), as previously described (Evangelista et al ., 1996). Labelling with anti‐MAC‐1 and CBRM1/5 MoAbs was evaluated as mean fluorescence (MF) intensity in arbitrary units and presented as percentage of the value expressed by resting PMN.…”

“…A previously described methodology has been used (Evangelista et al ., 1996). Briefly, PMN were stained with the vital red fluorescent dye hydroethidine (HE, 20μg/5 × 10 7 cells ml −1 ) for 30 min at 4°C.…”

“…The percentage of PMN showing green fluorescence above the threshold represents the percentage of PMN binding platelets (%[+] PMN). Platelet adhesion was also quantified by evaluating the relative number of platelets bound to one hundred PMN (platelets/100 PMN), as described by Evangelista et al (1996).…”

Platelet Activation and Platelet-Leucocyte Interaction in Patients with Migraine. Subtype Differences and Influence of Triptans

“…Various different platelet–leucocyte interactions are known: recognition and adhesion is mediated between P‐selectin and PSGL‐1 on leucocytes, as there is a β 2 ‐integrin (MAC‐1)‐dependent adhesion step between both cell types (26, 27). Different, in part antipodal reactions follow: ploymorphonuclear lymphocytes (PMN) can, depending on their activation to a variable extent, inhibit platelet aggregation (28, 29).…”

Influence of colloid fluids on polymorphonuclear granulocyte function in vivo

“…The surface receptors evaluated in our study are responsible for different PMN functions. CD62L, a selectin, plays an important role in interaction of granulocytes with endothelial cells and thrombocytes in inflammatory processes (13), mediating the primary contact (14–16). CD11b is a component of the complement receptor CR3 (CD11b/CD18) activated by very small amounts of complement factor iC3b.…”