Pasteurella haemolytica leukotoxin-induced increase in phospholipase A2 activity in bovine neutrophils

Abstract

Exposure of bovine neutrophils to Pasteurella haemolytica leukotoxin (LKT) stimulates the production of leukotriene B4 (LTB4), which is believed to be an important chemotactic agent in the development of acute fibrinopurulent pneumonic infection in cattle. The involvement of phospholipase A2 (PLA2) in LKT-induced synthesis of LTB4 was studied by using bovine neutrophils labeled with 3H-arachidonate ([3H]AA). Incubation of isolated neutrophils with [3H]AA resulted in incorporation of radioactivity in the PLA2 substrates phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Exposure of radiolabeled neutrophils to LKT caused concentration- and time-dependent release of radioactivity and redistribution of radioactivity in neutrophil membranes consistent with utilization of phosphoglyceride substrate and release of free fatty acid and eicosanoid products. These LKT-induced effects could be inhibited by pretreatment with arachidonyl trifluoromethyl ketone, an inhibitor of type IV cytoplasmic PLA2, and were dependent on extracellular calcium. These results support the conclusion that LKT-induced synthesis of LTB4 involves a calcium-mediated increase in PLA2 activity.

FIG. 1

Effects of LKT and LKT(−) control preparations on release of ³H and LDH. Isolated neutrophils, loaded with [³H]AA, were exposed to dilutions of LKT or LKT(−) for 60 min (n = 4). Mean values describing release of ³H caused by LKT dilutions of ≤1:1,000 were significantly different from corresponding LKT(−) values. Except for that obtained with the 1:10,000 dilution, all percent specific LDH release values were significantly different from corresponding negative control values.

FIG. 2

Time-dependent effects on release of ³H and LDH after exposure of isolated bovine neutrophils to LKT (1:10 dilution), LKT(−) (1:10 dilution), or A23187 (A23; 2.5 μM). All LKT and A23187 values derived from samples incubated for ≥5 min were significantly different from corresponding LKT(−) values.

FIG. 3

Mean (±SD) percent decrease in radioactivity released from isolated bovine neutrophils exposed to LKT or A23187 (A23) in the presence of different concentrations of AACOCF₃ (n = 4). ∗, radioactivity (disintegrations per minute) values are significantly different from corresponding inhibitor-free (0 μM AACOCF₃) values.

FIG. 4

Effect of AACOCF3 (Inh.) on synthesis of LTB₄ induced by exposure of isolated bovine neutrophils to LKT (1:10) or A23187 (5 μM) for 120 min (n = 4).

FIG. 5

Extracellular calcium dependence of LKT-induced effects on percent specific ³H release and percent specific LDH release. Isolated neutrophils were exposed to a 1:10 dilution of LKT in buffer suspensions containing 1 mM CaCl₂ (1 mM Ca), no Ca²⁺ (Ca-free), 1 mM EGTA and no Ca²⁺ (EGTA), or 1 mM EGTA and 3 mM CaCl₂ (EGTA + 3 mM Ca). All treatments within each of the response variables were significantly different from one another.