A new method for measuring eosinophil activating factors, based on the increased expression of CR3 alpha chain (CD11b) on the surface of activated eosinophils.
The observation that activation of eosinophils in vitro with PAF increases the surface expression of the alpha chain of the complement receptor CR3 (CD11b) has been extended to other eosinophil activating factors. CD11b may be detected on activated eosinophils by reaction with mouse monoclonal anti-human CD11b IgG, following the addition of urease-conjugated sheep anti-mouse IgG. CD11b levels were increased on eosinophils after incubation with (a) recombinant colony stimulating factors, IL-3, GM-CSF and IL-5, at concentrations of 100 U/ml, or (b) with eosinophil activating factors, recombinant TNF alpha (1000 U/ml), EAF purified from mononuclear cell supernatants and PAF (10(-6) M). CD11b levels were not affected by IL-1 alpha, IL-2 or IFN-gamma. Unstimulated neutrophils had higher levels of CD11b than unstimulated eosinophils, but neutrophil CD11b was unaffected by IL-3, GM-CSF and IL-5 and was only slightly affected by TNF, EAF and PAF. Polyclonal rabbit antibodies to IL-3 and TNF neutralised their CD11b enhancing activities. The PAF antagonists WEB 2086 and WEB 2170 neutralised the CD11b enhancing activity of PAF. We conclude that measurement of CD11b expression on eosinophils is a convenient method for the assay of eosinophil activating activity.
Thorne
K J
KJ
Department of Pathology, University of Cambridge, U.K.
Richardson
B A
BA
Mazza
G
G
Butterworth
A E
AE
D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D016207:Cytokines / Q000494:pharmacology; D004804:Eosinophils / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008222:Lymphokines / Q000494:pharmacology; D016177:Macrophage-1 Antigen / Q000096:biosynthesis*; D008722:Methods; D051379:Mice; D009504:Neutrophils / Q000187:drug effects; D010972:Platelet Activating Factor / Q000494:pharmacology; D011994:Recombinant Proteins
D000954:Antigens, Surface; D003115:Colony-Stimulating Factors; D016207:Cytokines; D008222:Lymphokines; D016177:Macrophage-1 Antigen; D010972:Platelet Activating Factor; D011994:Recombinant Proteins; C022227:eosinophil stimulating promoter