Tumor Necrosis Factor-α Activation of the c-Jun N-terminal Kinase Pathway in Human Neutrophils

Abstract

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-␣ (TNF␣). Although TNF␣ has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNF␣ is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNF␣ on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNF␣ was found to result in ␤ 2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to ␤ 2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNF␣ led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNF␣؊induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to ␤ 2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNF␣ leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.The rapid influx of polymorphonuclear leukocytes (neutrophils) into sites of injury is an important component of the acute inflammatory response in humans (1). The resulting adherence and chemotaxis of leukocytes in the appropriate cytokine milieu are not only necessary for efficient clearance of microorganisms (2) but may also participate in appropriate resolution of the inflammatory response through the subsequent induction of cell death by apoptosis.Tumor necrosis factor-␣ (TNF␣), 1 a pluripotent cytokine, is produced by a variety of leukocytes in response to stimulation by lipopolysaccharide (3). In turn, exposure to TNF␣ induces wide ranging biological effects including cell differentiation, proliferation, apoptosis, and multiple pro-inflammatory effects. TNF␣ exerts potent effects on neutrophils that have been suggested to contribute to the inflammatory response in sepsis (4, 5) and the adult respiratory distress syndrome (6). However, TNF␣ also exerts anti-inflammatory effects as demonstrated by an enhanced inflammatory response in TNF␣ Ϫ/Ϫ mice after infection (7), an effect that has been ascribed to the ability of TNF␣ to induce apoptosis in neutrophils (8).Whereas TNF␣ can prime neutrophils in suspension (9, 10), many TNF␣-induced cellular responses, including oxidant release, have been repo...

Lipopolysaccharide-induced c-Jun NH2-terminal Kinase Activation in Human Neutrophils

“…In this study we have identified Syk and PI3K as two new components of LPS signaling and demonstrated that both Syk and PI3K participate in JNK activation. The activation of Syk has been shown to occur in neutrophils upon initiation of phagocytosis (65) and after TNF-␣ stimulation (37), and PI3K activation has been shown in neutrophils upon exposure to IL-8, leukotriene B4, and formylmethionylleucylphenylalanine (58 -60); however, the activation of Syk or PI3K after LPS stimulation has not been previously described. In addition, although Syk has been shown to play a role in JNK activation in T cells (66) and in neutrophils B, in separate experiments, neutrophils were incubated with DFP (1 mM) for 10 min prior to LPS stimulation (100 ng/ml) for the indicated times.…”

“…LPS-stimulated JNK activation is not observed in human neutrophils in suspension. The activation of JNK after LPS stimulation is only seen in neutrophils under non-suspended conditions, as was the case in analyzing the activation of JNK after TNF-␣ stimulation (37).…”

“…Activation of JNK has been linked to induction of apoptosis, as seen after TNF-␣ (37) and IL-1␤ stimulation (38), and has been suggested to be important in the transcriptional regulation of several inflammatory mediators, including IL-2 (39), Cox-2 (40), metalloproteinase 1 (39), vascular endothelial growth factor (41), and monocyte chemoattractant protein 1 (MCP-1) (39). JNK is activated upon exposure to osmotic stress (42), irradiation (43), growth factors (44), heat shock (42), IL-1␤ (39), and TNF-␣ (37,42,45). In addition, JNK activation has been shown to occur in macrophages upon LPS stimulation (46 -48), however, the activation of JNK in LPS-stimulated neutrophils has not been previously described.…”

“…We (49,50) and others (51) have reported that LPS stimulation of suspended neutrophils results in the activation of p38 MAPK, but not ERK or JNK. The absence of JNK activation in our previous studies may relate to the use of suspended neutrophils, which might not replicate events that occur in a complex microenvironment, because we have recently shown the activation of JNK in adherent neutrophils upon TNF-␣ stimulation (37).…”

“…Pathways have been described that depend on the small G-proteins Cdc42 and Rac1 (30,31), PI3K (34,43,44), and recently we have identified Syk as an upstream activator of JNK in adherent neutrophils after TNF-␣ stimulation (37). Activation of JNK after LPS stimulation in macrophages requires PI3K (46), although upstream components in the JNK pathway in neutrophils are unknown.…”