Inhibition of kinases impairs neutrophil activation and killing of Staphylococcus aureus

Abstract

Intracellular phosphorylations polymorphonuclear neutrophils are mediated by kinases, including mitogen activated-protein (MAP) kinases and phosphatidylinositol 3-kinase. In the present study we demonstrate their effector functions upon both ligation of cell-surface seven-transmembrane-spanning receptors by bacterial peptide formylmethionyl-leucylphenylalanine as well as in the process of destruction of Staphylococcus aureus. To regulate neutrophil MAP kinases p38 and p44/42, specifically, we made use of their specific inhibitors 10 microM SK&F 86002 (for p38) and PD 098059 (for activating kinase of p44/42). SK&F 86002 was a potent inhibitor (by 70%) of induced antimicrobial oxygen-radical generation compared with PD 098059 (by 20%). SK&F 86002 and PD 098059 inhibited mobilization of a dominant neutrophil adhesion molecule, beta2 integrin, from cytoplasmic granules to the plasma membrane by 40 and 10% respectively, and the combination of the two drugs resulted in a 90% effect. The combined effect of both drugs was moderate inhibition of bacterial destruction, despite the fact that neither compound had detectable effect on bactericidal activity if applied individually. Bacterial destruction was also inhibited by wortmannin (0.1 microM), the specific inhibitor of phosphatidylinositol 3-kinase, which had previously been described to target various other activations of the neutrophil, including oxygen-radical generation. Although the relative contribution of p38 and p44/42 MAP kinases varied, the marked effects of the combined inhibition of the kinases revealed their concerted actions to be critical for normal neutrophil function.

MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis

“…In our experiments we have observed that LPS-induced Rac activation occurs within less than 1 h of LPS stimulation, whereas enhanced phagocytosis of bacteria occurs after 12 tosis and chemotaxis [25][26][27]. A previous study even shows that CpG regulates the phagocytosis of bacteria through p38 [13].…”

Hypoxic regulation of neutrophil function and consequences for Staphylococcus aureus infection

“…Furthermore, in this study, a brief (15 min) period of reoxygenation restored both ROS formation and bactericidal capacity. Inhibition of neutrophil ROS production by selective inhibitors of p38 and p44/42 MAPK has also been shown to decrease S. aureus killing, although these inhibitors additionally reduced mobilisation of b 2 integrin to the plasma membrane, which may have contributed to the effect [89]. Neutrophils isolated from patients suffering recurrent pyogenic infections showed a strong correlation between impaired S. aureus killing and reduced superoxide anion production [90], and neutrophils from patients with chronic granulomatous disease (where ROS generation is absent) had significantly impaired ability to kill S. aureus [32].…”

Nonopsonic Binding of Type III Group B Streptococci to Human Neutrophils Induces Interleukin-8 Release Mediated by the p38 Mitogen-Activated Protein Kinase Pathway

“…These MAP kinases have an essential role in PMN intracellular signaling pathways in response to various external stimuli and environmental stresses (15,31,55). The p38 MAP kinase pathway is responsible for PMN superoxide generation, chemotaxis, and IL-8 production in response to TNF-␣ and FMLP, and the activation of MAP kinase pathways generally results in activation of transcription factors which regulate protein synthesis (36,55). The p38 MAP kinase pathway is involved in PMN apoptosis and gene expression (11,55).…”

“…The activities of p38 and extracellular signal-related kinase (ERK 1/2, p44/42) increase rapidly in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) or lipopolysaccharide, suggesting that these kinase cascades have a pivotal role in regulating PMN function (7,24). The intracellular signaling pathways which regulate key PMN functions have been partially characterized, but the extent to which they contribute to PMN activation by GBS is undefined (5,36).…”