Activation of bovine neutrophils by Pasteurella haemolytica leukotoxin is calcium dependent

Abstract

In this study, we used the fluorescent probe Fluo-3 to show that an increase in cytosolic free calcium, [Ca2+]i, occurred when suspensions of bovine neutrophils were incubated with sublethal concentrations of P. haemolytica leukotoxin. This increase in [Ca2+]i was dependent on the concentration of leukotoxin present in the medium and, at a given concentration of leukotoxin, dependent on the external calcium concentration. The calcium channel blocker verapamil and the beta-adrenergic antagonist propranolol inhibited leukotoxin-stimulated Ca2+ gain, as did a neutralizing antileukotoxin monoclonal antibody. As reported previously, incubation of bovine neutrophils with partially purified leukotoxin stimulated a vigorous luminol-dependent chemiluminescence response (LDCL). The present study shows that LDCL stimulation was dependent on the presence of extracellular calcium and was inhibited by the addition of verapamil and propranolol. These data indicate that bovine neutrophils exhibit a considerable increase in cytoplasmic free calcium when they are incubated with P. haemolytica leukotoxin in the presence of external calcium. They also provide evidence that an increased [Ca2+]i is required for functional activation of the bovine neutrophil oxidative burst by P. haemolytica leukotoxin.

How Mannheimia haemolytica defeats host defence through a kiss of death mechanism

“…However, activation of bovine neutrophils by M. haemolytica leads in bulk to elevation of intracellular calcium [139], oxidative burst [114] and production of several lipid mediators [30,69] and proinflammatory cytokines [193] (Tab. I).…”

“…1 to 4), but not in porcine leukocytes (LKT binds to porcine LFA-1 without eliciting any effects). This binding is known to involve G proteins [79,90] and to cause, in a dose dependent way, sustained elevation in intracellular calcium in bovine leukocytes [36,89,139] that results mainly from an incoming flux from the extracellular medium via voltage-gated channels (Fig. 2) [61,79,80,139].…”

“…This binding is known to involve G proteins [79,90] and to cause, in a dose dependent way, sustained elevation in intracellular calcium in bovine leukocytes [36,89,139] that results mainly from an incoming flux from the extracellular medium via voltage-gated channels (Fig. 2) [61,79,80,139]. This calcium entrance is clearly involved in cytolysis [61] and is essential for triggering the NF-κB translocation into the nucleus (detectable after five minutes of exposure), as well as the production of proinflammatory cytokines such as TNF-α, IL-1β and IL-8, since calcium chelation blocks both phenomena (Fig.…”

Recombinant Bovine Interleukin-1β Amplifies the Effects of Partially PurifiedPasteurella haemolyticaLeukotoxin on Bovine Neutrophils in a β2-Integrin-Dependent Manner

“…Anti-␤ 2 -integrin MAbs have also been shown to block binding of LKT to blotted lysates of BL-3 cells and neutrophils (14,18). The mechanism by which the RTX toxins exert their lethal effect on target cells is only partially understood but is thought to involve changes in selective permeability of the cytoplasmic membrane that result in Ca 2ϩ influx and ATP efflux (23,34,36,37).The proinflammatory cytokine interleukin-1␤ (IL-1␤), which is produced by alveolar macrophages and other cells, can stimulate migration and functional activation of polymorphonuclear leukocytes (PMNs) in the lung (10). The ␤ 2 -integrins, which are found on most circulating leukocytes, play an important role in the extravasation of leukocytes during the inflammatory response (11).…”

“…Anti-␤ 2 -integrin MAbs have also been shown to block binding of LKT to blotted lysates of BL-3 cells and neutrophils (14,18). The mechanism by which the RTX toxins exert their lethal effect on target cells is only partially understood but is thought to involve changes in selective permeability of the cytoplasmic membrane that result in Ca 2ϩ influx and ATP efflux (23,34,36,37).…”