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Deficient total cell content of CR3 (CD11b) in neonatal neutrophils
N Abughali, M Berger and MF Tosi
Department of Pediatrics, Case Western Reserve University School of
Medicine, Cleveland, OH 44106.
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.
Volume 83,
Issue 4,
pp. 1086-1092,
02/15/1994
Copyright © 1994 by The American Society of Hematology
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