In vivo and in vitro assessment of porcine neutrophil activation responses to chemoattractants: flow cytometric evidence for the selective absence of formyl peptide receptors.
Interest in the role that activated granulocytes play in C5a-induced myocardial ischemia prompted us to investigate and compare activation responses of pig and human neutrophils. The responses of Hypaque-Ficoll purified porcine (P-PMN) and human neutrophils (H-PMN) to stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, phorbol myristate acetate (PMA), and calcium ionophore A23187 (A23187) were compared by flow cytometrically measured changes in the cells' forward (FWD-SC) (a measure of shape/volume change) and right angle (90 degrees-SC) light scatter (a measure of secretion), and in the distribution of the membrane potential sensitive fluorescent probe di-O-C (3). FMLP, C5a, and Zymosan-activated serum (ZAS stimulated chemotaxis and FMLP vs. PMA-stimulated adherence to plastic were also compared. Unstimulated P-PMN had lower FWD-SC and 90 degrees-SC than H-PMN (39.4 +/- 1.4 vs. 48.4 +/- 2.0 P less than 0.05, and 32.7 +/- 2.7 vs. 52.4 +/- 1.5 units, P less than 0.005, for FWD-SC and 90 degrees-SC of P-PMN vs. H-PMN, respectively). P-PMN selectively failed to increase their FWD-SC upon stimulation with FMLP (0.0 +/- 0.5% vs. 26.1 +/- 6.8%, P-PMN vs. H-PMN), or decrease their 90 degrees-SC when treated with cytochalasin B + FMLP (secretion) (2.4 +/- 0.1% vs. -35.8 +/- 4.6% change in 90 degrees-SC, P-PMN vs. H-PMN), while responding comparably to C5a, PMA, and A23187. P-PMN failed to depolarize in response to FMLP but responded similarly to H-PMN when activated by C5a, A23187, and PMA. P-PMN's chemotactic response to FMLP was selectively absent since the cells responded well to purified pig C5a. FMLP stimulated significant increases in H-PMN adherence to bovine serum albumin-coated plastic (44.1 +/- 6.7% vs. 12.6 +/- 3.7%, FMLP vs. buffer, P less than 0.025), but failed to increase adherence of P-PMN above baseline 0.68 +/- 0.20% vs. 2.12 +/- 1.90%, FMLP vs. buffer, P greater than 0.05. PMA (100 ng/ml) stimulated comparable increases in adherence in both PMN types (48.6 +/- 5.2% vs. 58.7 +/- 4.9%, P-PMN vs. H-PMN, P less than 0.025). Binding studies using the fluoresceinated N-formyl peptide f-met-leu-phe-lysine-fluorescein-isothiocyanate (FMLPL-FITC) in the absence and presence of excess non-fluoresceinated FMLPL indicated that P-PMN lack specific binding sites for the N-formyl peptides.(ABSTRACT TRUNCATED AT 400 WORDS)
Fletcher
M P
MP
Division of Rheumatology/Allergy and Clinical Immunology, School of Medicine, University of California, Davis 95616.
Stahl
G L
GL
Longhurst
J C
JC
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology; D002630:Chemotactic Factors / Q000276:immunology / Q000494:pharmacology*; D015936:Complement C5a / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013861:Thiocyanates / Q000378:metabolism; D015054:Zymosan / Q000494:pharmacology
D002630:Chemotactic Factors; D005452:Fluoresceins; D008024:Ligands; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013861:Thiocyanates; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D015054:Zymosan; D016650:Fluorescein-5-isothiocyanate; D013755:Tetradecanoylphorbol Acetate