Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation

Abstract

Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as “early activation” marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.

Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes

“…Previous data (Kinhult et al ., 2002) have shown that the expression of both CD63 and CD66b at the cell surface is low on resting cells. Consistent with previous work (Kuijpers et al ., 1991), we could show that stimulation with ionomycin induced secretion of both azurophilic and specific granules, fMLP induced the fusion of specific granules (CD66b), whereas in combination with cytochalasin B, fMLP induced the fusion of both specific (CD66b) and azurophilic (CD63) granules with the plasma membrane (data not shown). Next, we performed experiments using bacteria as the stimulus.…”

Expression of CD35 (CR1) and CD11b (CR3) on circulating neutrophils and eosinophils from allergic asthmatic children

“…The advantage of this technique is that neutrophils and eosinophils can be analysed separately without prior purification of cells. In this way the upregulation of receptors by purification procedures is avoided [22,25]. Double-staining with anti-CD16 to discriminate eosinophils from neutrophils is also avoided with this technique [24].…”

Neutrophil expression of tumour necrosis factor receptors (TNF-R) and of activation markers (CD11b, CD43, CD63) in rheumatoid arthritis

“…; 1995 Blackwell Science Ltd, Clinical and Experimental Immunology. 101: [25][26][27][28][29][30][31][32] resultant pellet was treated with 0 2% NaCI for 1 min to lyse residual erythrocytes. PMN were resuspended in Hanks' solution, Ca'"\ Mg^'^-free (Gificro, Paisley, UK).…”

“…However, this did not correlate with the disease activity state of these two patients, which was not particularly high (DAS < 3); neither of them were treated with steroids. CD63 is normally not present on blood PMN [31], while it is highly expressed on activated platelets [32]. Platelets often become activated and bind to PMN during blood collection, This may result in false detection of platelet markers on the neulrophil surface by FACS analysis [33][34][35].…”