Lipopolysaccharide-binding protein- and CD14-dependent activation of mitogen-activated protein kinase p38 by lipopolysaccharide in human neutrophils is associated with priming of respiratory burst

Abstract

Neutrophil (PMN) functions can be primed for greatly increased oxidative radical release by exposure to certain agents such as lipopolysaccharide (LPS). Although a variety of signaling pathways involving both tyrosine kinases and mitogen-activated protein (MAP) kinases may be operative, the mechanisms of PMN priming are still not understood. We found that PMN priming was not achieved by treatment of cells with a very low concentration (5 ng/ml) of LPS unless additional "helper" factors were present in plasma (5%). Under these conditions, LPS induced tyrosine phosphorylation of a 38-kDa protein, which was coincident with the MAP kinase p38 action in this situation. LPS-mediated activation of p38 in human PMNs was dependent on the presence of LPS binding protein from plasma and CD14 on the surfaces of the cells. Phosphorylation of p38 was highly correlated with LPS priming of a formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN respiratory burst. Treatment of PMN with the p38-specific inhibitor SB203580 significantly attenuated the respiratory burst in cells primed by LPS and stimulated by fMLP. These results suggest that the LPS signaling pathway leading to p38 activation may be an important mechanism in regulation of PMN priming. The mediator(s) linking CD14 to p38 involves proteins that are functionally sensitive to genistein but insensitive to tyrphostin AG126 and to Src- and Syk-family kinase, protein kinase C, and phosphatidylinositol 3-kinase inhibitors. Elucidating this pathway will provide insight into possible regulation of PMN priming by LPS.

FIG. 1.

LPS increases tyrosine phosphorylation of a cellular 38-kDa protein (indicated by the arrow in panel A) that is coincident with the activation of p38 in human PMN. Purified PMN were resuspended in H-medium alone or supplemented with 5% plasma at a density of 10 × 10⁶/ml and were incubated at 37°C for 20 min in the absence or presence of 5 ng of LPS/ml. Cells were washed once with ice-cold phosphate-buffered saline-2.5 mM DFP, followed by lysis with RIPA buffer. Cellular proteins were analyzed by SDS-PAGE and Western blotting (WB) for tyrosine phosphorylation (A) and p38 activation (B). Equal loading was confirmed by stripping the membrane and reprobing for p38 protein (bottom panel).

FIG. 2.

Size fractionation of the plasma factor(s) that supports LPS activation of p38. PMN were incubated in H-medium supplemented with 5% whole or fractionated (on a 30-ml Sephacryl S-200 column) plasma in the absence or presence of 5 ng of LPS/ml at 37°C for 20 min. Cellular proteins were prepared and analyzed for p38 activation as described in the legend to Fig. 1.

FIG. 3.

LBP is the plasma factor that supports LPS activation of p38 and priming of the PMN respiratory burst. (A) PMN were resuspended in H-medium supplemented with 5% plasma pretreated with a control mouse IgG (mIg) or a mouse anti-LBP Ab and were incubated in the absence or presence of 5 ng of LPS/ml at 37°C for 20 min. Some cells were incubated in H-medium supplemented with 5 ng of purified LBP/ml. Cellular proteins were prepared and analyzed for p38 activation as described in the legend to Fig. 1. (B) PMN were resuspended at a density of 10⁶/ml in H-medium supplemented with 80 μM cytochrome c and 5 ng of purified LBP/ml or 5% plasma pretreated with a control mIg or a mouse anti-LBPAb. Cells were incubated in the absence or presence of 5 ng of LPS/ml at 37°C for 20 min and then with fMLP (10⁻⁶ M) for another 5 min. The amount of O₂⁻ produced by the cells was calculated from the absorbance of the incubation medium at 550 nm.

FIG. 4.

CD14, but not β integrins, mediates the LPS activation of p38 and priming of the respiratory burst. PMN either were not pretreated or were pretreated with either control mouse IgG (mIg) or a mouse anti-CD29, anti-CD18, or anti-CD14 Ab for 1 h at 4°C. Cells were then incubated with 5% plasma with or without LPS at 5 ng/ml and were analyzed for activation of p38 (A) or production of O₂⁻ (B) as described in the legends to Fig. 1 and 3.

FIG. 5.

p38 activation is partially required for LPS priming of the PMN respiratory burst. PMN in H-medium supplemented with 5% plasma were pretreated or not with 5 μM SB203580 for 10 min at room temperature and then assayed for O₂⁻ production as described in the legend to Fig. 3.

FIG. 6.

p38 activation in LPS-treated human PMN is mediated by a genistein-sensitive, tyrphostin AG126-insensitive signaling molecule(s) that does not belong to families of Src, Syk, PKC, and PI 3-kinase. (A) PMN were incubated in H-medium supplemented with 5% plasma with or without LPS at 5 ng/ml and were lysed with RIPA buffer as described in the legend to Fig. 1. The activity of Src-family kinases (Lyn, Fgr, and Hck) was analyzed by an in vitro immunocomplex kinase assay as described in Materials and Methods. (B) PMN were pretreated with either solvent (Nil), 5 μM PP2, 5 μM piceatannol (Pic), 1 μM calphostin C (Cal), 100 nM wortmannin (Wort), 50 μM tyrphostin AG126 (AG), or 50 μM genistein (Gen) for 10 min at room temperature. Cells were then incubated in the presence of 5% plasma with or without LPS at 5 ng/ml, lysed, and analyzed for p38 activation as described in the legend to Fig. 1. (C) PMN were treated with the indicated inhibitors as described above and assayed for O₂⁻ production as described in the legend to Fig. 3 by using TNF-α (20 ng/ml) and PMA (10 ng/ml) as stimuli (for 30 min at 37°C). Results are expressed as means ± standard errors of the means (n = 3) of percentages of changes in O₂⁻ production from that in the non-inhibitor-treated control (nil).