INVIVO AND INVITRO 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°-SC) light scatter (a measure of secretion), and in the distribution of the membrane potential sensitive fluorescent probe di-O-C((5)) (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°-SC than H-PMN (39.4 ± 1.4 vs. 48.4 ± 2.0 P < 0.05, and 32.7 ± 2.7 vs. 52.4 ± 1.5 units, P < 0.005, for FWD-SC and 90°-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°-SC when treated with cytochalasin B + FMLP (secretion) (2.4 ± 0.1% vs. -35.8 ± 4.6% change in 90°-SC, P-PMN vs. H-PMN), while responding comparably to C5a, PMA, and A23187. P-PMN failed 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 < 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 > 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 < 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. Intracoronary (LAD) infusion of FMLP in the instrumented intact pig produced no change in neutrophil extraction, LAD regional blood flow, or myocardial contractility while infusion of purified porcine C5a induced a rapid and marked increase in myocardial neutrophil extraction, a decrease in LAD coronary blood flow, and diminished contractility. It is concluded that P-PMN and H-PMN respond comparably to C5a, PMA, and A23187, but P-PMN are selectively unresponsive to activation by FMLP both in vitro and in vivo due to the absence of FMLP binding.