Khalil A. Aziz

Activation-dependent proteolytic degradation of polymorphonuclear CD11b.

CD11b/CD18 is the principal integrin of polymorphonuclear (PMN) leucocytes and is involved in their adhesion, migration and phagocytosis. In quiescent cells, the receptor is stored in intracellular granules from where it is translocated to the cell surface in response to a variety of stimuli. In this study, we demonstrated that strong stimulation of PMNs not only leads to the upregulation of CD11b surface expression, but also to the subsequent time‐dependent apparent loss of this receptor, as detected by fluorescence‐activated cell sorting (FACS) using a monoclonal antibody (mAb) against an N‐terminal CD11b epitope. This epitope loss was observed following either direct stimulation of protein kinase C (PKC) with phorbol 12‐myristate 13‐acetate (PMA) or after multiple receptor stimulation using a combination of the agonist N‐formylmethionyl‐leucyl‐phenylalanine (FMLP) and the priming agents granulocyte macrophage‐colony stimulating factor (GM‐CSF) and platelet factor (PF) 4. However, upregulation following weak stimulation with FMLP alone was not followed by subsequent epitope loss of the receptor. The increases and subsequent decreases in CD11b expression induced by PMA were paralleled by an increase and a decrease in PMN adhesion to CD11b‐specific ligands, fibrinogen and intercellular adhesion molecule (ICAM)‐1. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot analysis showed that this epitope loss of PMN CD11b was the result of proteolytic degradation of the N‐terminal region of the molecule. The use of a range of proteinase inhibitors indicated that this CD11b degradation involves a cell‐associated serine proteinase. This is the first demonstration of the proteolytic alteration of CD11b in response to strong PMN stimulation. Given the central role of CD11b/CD18 in all aspects of PMN function, this alteration of the CD11b molecule and its effect on PMN adhesion are probably of considerable pathophysiological importance.

The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming.

Because of uncertainties regarding the comparability of granulocyte‐macrophage and granulocyte colony‐stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl‐leucylphenylalanine (fMLP) was not influenced by G‐CSF (0.1–100 ng/ml), whereas GM‐CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM‐CSF and G‐CSF in combination was not different from that of PMNs treated with GM‐CSF alone. GM‐CSF (10 ng/ml) increased the rate of O2 ‐ production by 79%, caused a fivefold increase in fMLF‐induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G‐CSF (50 ng/ml) only slightly increased O2 ‐ production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM‐CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G‐CSF on mature PMNs is negligible compared with that of GM‐CSF. Our results are in conflict with previous reports of much more pronounced G‐CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM‐CSF. We therefore suggest that the primary role of G‐CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in viva J. Leukoc. Biol. 55: 612–616; 1994.