Deficient total cell content of CR3 (CD11b) in neonatal neutrophils

Abstract

Neonatal neutrophils (PMN) show a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b), a critical adhesion molecule, on chemoattractant-activated PMN. After activation of PMN with additional stimuli including calcium ionophores, we also found deficient surface CR3 (but normal CR1) expression on neonatal PMN suggesting that abnormal signaling mechanisms are not likely to explain the deficient CR3 expression on activated neonatal PMN. Therefore, we hypothesized that deficient surface expression of CR3 on stimulated neonatal neutrophils is caused by a deficiency in total cell content of CR3. We tested this hypothesis using three different methods to compare the total quantity of CR3 in neonatal versus adult PMN. Western blotting of serial twofold dilutions of PMN lysates from five adult and neonatal pairs, using a monoclonal antibody (MoAb) against CR3 (21PM19C), consistently showed diminished CR3 content in neonatal PMN. A sandwich enzyme-linked immunosorbent assay, in which the CR3 heterodimers in PMN lysates were captured by MoAb to the beta-chain, CD18 (R15.7), then detected with a biotinylated MoAb to the alpha-chain, CD11b (anti-Mac-1), showed that neonatal PMN lysates contain about 66% of adult PMN levels of CR3 (P < 0.03; n = 6). PMN fixed with paraformaldehyde and permeabilized with saponin were studied by immunofluorescence flow cytometry to determine total (surface plus intracellular) CR3 content using phycoerythrin-conjugated MoAb to CR3 (anti-Leu15). Mean total cell CR3 content (in relative fluorescence units) was 58 +/- 14 for adult PMN and 27 +/- 6 for neonatal PMN (n=5; P=0.013). In each method, total cell content of CR1 was equivalent for neonatal versus adult PMN. We conclude that neonatal PMN are markedly deficient in total cell CR3 content compared with adult PMN. This result provides a primary explanation for deficient CR3 surface expression on activated neonatal PMN that, in turn, may be important in the chemotactic defect of these cells.