Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.

This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran-sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules.

Red blood cells from patients with sickle cell disease exhibit an increased adherence to cultured endothelium pretreated with tumour necrosis factor (TNF).

Red blood cells (RBCs) from 24 patients with sickle cell disease were more adherent to cultured endothelium pretreated with the inflammatory cytokine, tumour necrosis factor (TNF) than RBCs from 22 healthy subjects. The enhanced sticking was apparent in RBC preparations from patients who were in crisis (mean 190% increase from controls) and out of crisis (mean 220% increase) and was not related to the number of circulating RBCs, reticulocytes, platelets, leucocytes or haemoglobin levels. When irreversibly sickled RBCs, enriched by centrifugation on density gradients, were added to TNF-treated endothelium they were found to be significantly more adherent (mean 411% increase; P < 0.001) than the unfractionated RBCs from the same patients. There was no difference between the adherent properties of sickle RBCs and normal RBCs for untreated endothelium. Contributing factors to the enhanced adhesion to TNF-treated endothelium may be the low surface change of sickle RBCs, and increased levels of fibrinogen and von Willebrand's factor (vWF) in the patients' plasma. By acting on vascular endothelium to increase its adhesiveness for sickled RBCs, it is concluded that inflammatory cytokines such as TNF may have a prominent role in mediating the events that lead to microvascular occlusions in sickle cell disease.

Blood polymorphonuclear leukocytes from the majority of sickle cell patients in the crisis phase of the disease show enhanced adhesion to vascular endothelium and increased expression of CD64.

There is increasing interest in the role of blood polymorphonuclear leukocytes (PMNs) in the pathogenesis of sickle cell crisis. We studied the adherence of PMNs from 18 sickle cell patients in crisis, 25 out of crisis, and 43 healthy subjects (controls) to monolayers of human umbilical cord endothelium that were either untreated or pretreated with tumor necrosis factor alpha (TNFalpha). Overall, the PMNs from patients in crisis were more adherent than control PMNs to untreated endothelial monolayers (mean 53% increase; P < .001) and TNFalpha-treated monolayers (mean 41% increase; P < .002). Increased adhesiveness was not associated with an abnormal expression of CD11a, CD11b, CD11c, CD18, CD62L, or CD15. There was an increase in the number of PMNs expressing CD64 in patients in crisis (median value, 44%) compared with patients out of crisis (median, 21%; P = .025) and controls (median, 6.5%; P < .001). Sera from patients in crisis had normal levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-gamma, TNFalpha, interleukin-1 (IL-1), IL-6, or IL-8 and did not modify the adherence of PMNs or their expression of CD64. Only IFN-gamma induced CD64 expression on PMNs, but this effect was not associated with enhanced binding to endothelium. Because PMNs bound to endothelial monolayers were CD64(+) and CD64-enriched PMNs were 7 times more adherent to endothelial monolayers than CD64-depleted PMNs, it is likely that CD64 is a marker of adherent PMNs. Two of the three anti-CD64 antibodies used in our antibody blocking studies (clones 32.2 and 197) partially inhibited the binding of sickle cell PMNs to untreated endothelium (mean inhibitions of 33% [P = .01] and 21% [P = .03], respectively), whereas only one (clone 197) inhibited binding to TNFalpha-treated endothelium (mean inhibition, 29%; P = . 004). In some patients with sickle cell disease, an enhanced PMN adhesion to vascular endothelium could contribute to the vascular occlusion that characterizes the acute crisis of the disease.