Cross-linking of CD18 primes human neutrophils for activation of the respiratory burst in response to specific stimuli: implications for adhesion-dependent physiological responses in neutrophils.

Adhesion is known to prime neutrophils for physiological activation in response to cytokines and other stimuli. We have employed the technique of receptor cross-linking to study the potential role of CD18, the common beta-subunit of the beta 2-integrin family of adhesion molecules, in the regulation of the respiratory burst, as measured by luminol-enhanced chemiluminescence and iodination, in human neutrophils. CD18 cross-linking primed neutrophils to activate the respiratory burst after stimulation with tumor necrosis factor alpha (TNF-alpha) (100 units/mL), formylmethionyl-leucyl-phenylalanine (fMLP) (1 microM), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (1 micrograms/mL), but not granulocyte colony-stimulating factor (G-CSF) (1 micrograms/mL), interferon-gamma (IFN-gamma) (100 U/mL), or phorbol myristate acetate (100 nM). The maximal rate of chemiluminescence induced by fMLP, TNF-alpha, and GM-CSF was enhanced 8-, 6-, and 1.5-fold, respectively, following CD18 cross-linking. Priming of the respiratory burst by direct engagement of CD18 was confirmed in neutrophil-mediated iodination experiments, where iodination induced by TNF-alpha, fMLP, and GM-CSF was increased 15-, 20-, and 7-fold, respectively, by CD18 cross-linking. Immunoblot experiments demonstrated that TNF-alpha-induced tyrosine phosphorylation was both accelerated and more intense in neutrophils after cross-linking of CD18. Major tyrosine phosphoprotein products include proteins with approximate molecular masses of 40, 70, and 110 kDa. Genistein (50 microM), a selective tyrosine kinase inhibitor, reduced the TNF-alpha-stimulated respiratory burst by > 80% whether or not CD18 was cross-linked. These results affirm the importance of CD18 in adhesion-dependent priming of neutrophil functions and demonstrate that CD18 engagement per se is sufficient to prime neutrophils for cytokine-induced signal transduction mediated by tyrosine phosphorylation.

Cross-linking of CD45 enhances activation of the respiratory burst in response to specific stimuli in human phagocytes.

The phosphotyrosine phosphatase CD45 is expressed on the surface of all leukocytes and is known to play a critical role in the regulation of both T and B cell function. In contrast, relatively little information exists regarding the role of CD45 in the phagocyte lineage. We present evidence that CD45 modulates activation of the inducible respiratory burst in normal human neutrophils, monocytes, and eosinophils, as measured by luminol-enhanced chemiluminescence. In neutrophils, the respiratory burst induced by FMLP (1 microM), granulocyte-macrophage CSF (GM-CSF; 1 microgram/ml), or TNF-alpha (100 U/ml) was enhanced synergistically by CD45 cross-linking. This effect was most striking upon stimulation with TNF-alpha, in which cross-linking of CD45 resulted in a 30-fold increase in chemiluminescence. Chemiluminescence induced by PMA (100 nM), opsonized zymosan (1 mg/ml), LPS (1 microgram/ml), IFN-gamma (100 U/ml), or granulocyte CSF (1 microgram/ml) was not affected significantly by CD45 cross-linking. Similar results were obtained by using iodination for measurement of the respiratory burst. In monocytes, CD45 cross-linking significantly increased chemiluminescence stimulated by FMLP, GM-CSF, TNF-alpha, and LPS, and GM-CSF- and TNF-alpha-induced chemiluminescence was enhanced significantly by cross-linking of CD45 on eosinophils. Immunoblot analysis demonstrated that both the rate and intensity of TNF-alpha-induced tyrosine phosphorylation were increased by CD45 cross-linking in neutrophils. Major tyrosine-phosphorylated products include proteins with approximate molecular masses of 40 kDa, 70 kDa, 78 kDa, and 110 kDa. These results provide direct evidence that CD45 is capable of regulating the inducible respiratory burst in human phagocytes. On the basis of our findings, we postulate that CD45 may mediate coupling of specific cell surface receptors to downstream tyrosine kinase-dependent signal-transduction pathway(s) in activated phagocytes.