Changes in neutrophil surface-receptor expression after stimulation with FMLP, endotoxin, interleukin-8 and activated complement compared to degranulation.
Neutrophil activation induces changes in the expression of surface receptors and may lead to degranulation. Surface expression of beta2-integrins, l-selectin, complement receptor 1 (CR-1), decay-accelerating factor (DAF), C5a receptor, intercellular adhesion molecule-1 (ICAM-1) and ICAM-3 was compared by flow cytometry on isolated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), endotoxin or interleukin-8 and on neutrophils in whole blood anti-coagulated with the thrombin inhibitor lepirudin and stimulated with cobra venom factor to induce complement activation. Myeloperoxidase and lactoferrin in the supernatants were quantified in enzyme immunoassays. With high enough doses, all stimulants induced significant upregulation of beta2-integrins, CR-1 and DAF and downregulation of l-selectin. ICAM-3 was either unchanged or somewhat downregulated. Only FMLP and PMA induced significant upregulation of ICAM-1. Combined measurement of beta2-integrins and l-selectin permitted graded evaluation of early neutrophil activation. Measurement of degranulation showed no differences compared to unstimulated controls due to substantial spontaneous degranulation of isolated neutrophils by rewarming from 4 degrees C and incubation at 37 degrees C. Spontaneous activation was less in ethylenediaminetetraacetic acid-anti-coagulated blood, but calcium chelation may also inhibit the stimulated responses. There was large activation of unstimulated neutrophils in lepirudin-anti-coagulated blood at 37 degrees C, obscuring changes induced by stimulation, which may render this anti-coagulant unsuitable for studies of neutrophils.
Videm
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Department of Laboratory medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway. vibeke.videm@medisin.ntnu.no
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D000925:Anticoagulants / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D015550:Cell Degranulation / Q000502:physiology*; D003169:Complement Inactivator Proteins / Q000494:pharmacology; D003165:Complement System Proteins / Q000494:pharmacology; D004546:Elapid Venoms / Q000494:pharmacology; D004731:Endotoxins / Q000494:pharmacology; D005434:Flow Cytometry; D006629:Hirudins / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D007781:Lactoferrin / Q000187:drug effects / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects / Q000276:immunology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D009195:Peroxidase / Q000187:drug effects / Q000378:metabolism; D011971:Receptors, Immunologic / Q000187:drug effects* / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation
D000925:Anticoagulants; D002273:Carcinogens; D003169:Complement Inactivator Proteins; D004546:Elapid Venoms; D004731:Endotoxins; D006629:Hirudins; D016209:Interleukin-8; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C016310:cobra venom factor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003165:Complement System Proteins; D009195:Peroxidase; D007781:Lactoferrin; D013755:Tetradecanoylphorbol Acetate; C083544:lepirudin