Activation of extracellular signal-related protein kinases 1 and 2 of the mitogen-activated protein kinase family by lipopolysaccharide requires plasma in neutrophils from adults and newborns

Abstract

Neutrophils exposed to low concentrations of gram-negative lipopolysaccharide (LPS) become primed and have an increased oxidative response to a second stimulus (e.g., formyl-methionyl-leucyl-phenylalanine [fMLP]). In studies aimed at understanding newborn sepsis, we have shown that neutrophils of newborns are not primed in response to LPS. To further understand the processes involved in LPS-mediated priming of neutrophils, we explored the role of extracellular signal-related protein kinases (ERK 1 and 2) of the mitogen-activated protein kinase family. We found that LPS activated ERK 1 and 2 in cells of both adults and newborns and that activation was plasma dependent (maximal at > or =5%) through LPS-binding protein. Although fibronectin in plasma is required for LPS-mediated priming of neutrophils of adults assessed by fMLP-triggered oxidative burst, it was not required for LPS-mediated activation of ERK 1 and 2. LPS-mediated activation was dose and time dependent; maximal activation occurred with approximately 5 ng of LPS per ml and at 10 to 40 min. We used the inhibitor PD 98059 to study the role of ERK 1 and 2 in the LPS-primed fMLP-triggered oxidative burst. While Western blotting showed that 100 microM PD 98059 completely inhibited LPS-mediated ERK activation, oxidative response to fMLP by a chemiluminescence assay revealed that the same concentration inhibited the LPS-primed oxidative burst by only 40%. We conclude that in neutrophils, LPS-mediated activation of ERK 1 and 2 requires plasma and that this activation is not dependent on fibronectin. In addition, we found that the ERK pathway is not responsible for the lack of LPS priming in neutrophils of newborns but may be required for 40% of the LPS-primed fMLP-triggered oxidative burst in cells of adults.

FIG. 1

(A) Sample Western blots for activated ERK 1 and 2 (upper blot) and total ERK 2 (lower blot) from neutrophils of adults and newborns incubated with or without 5 ng of LPS per ml and the indicated plasma concentrations for 20 min. PMN, polymorphonuclear leukocytes. (B) Neutrophils from five adults and five newborns were incubated with or without plasma (5%) and LPS (5 ng/ml) for 20 min. The mean (± standard error of the mean) level of activated ERK 1 and 2 divided by total ERK 2 determined by densitometric scanning is indicated. (C) Autoradiograph showing phosphorylation of myelin basic protein (MBP) by ERK in the same adult samples shown in panel A.

FIG. 2

ERK 1 and 2 activation by LPS in neutrophils is plasma dependent. Neutrophils from adults and newborns were incubated with LPS (5 ng/ml) and the indicated plasma concentrations for 20 min. ERK 1 and 2 activation was quantified by densitometric scanning of the bands. Values for ERK 1 and 2 were combined, and percent activation was calculated relative to values obtained for 5% plasma.

FIG. 3

(A) Sample Western blot for activated ERK 1 and 2 from neutrophils of newborns incubated with LPS for the indicated times. (B) Neutrophils from adults and newborns were incubated with LPS (5 ng/ml) and 5% plasma for the indicated times. Total ERK 1 and 2 activation was quantified by densitometric scanning of the bands (A). Results are expressed as percent activation relative to values obtained for 20 min of incubation.

FIG. 4

LPS-mediated activation of ERK 1 and 2 is dose dependent in neutrophils. Four individual neutrophil preparations of adults and two of newborns were incubated with the indicated LPS concentrations for 20 min. ERK 1 and 2 activation was quantified by densitometric scanning of the bands. Values for ERK 1 and 2 were combined, and percent activation was calculated relative to values obtained for 5 ng of LPS per ml.

FIG. 5

Effects of PD 98059 on ERK activation and oxidative burst. (A) Sample Western blot for activated ERK 1 and 2 from neutrophils of adults incubated with PD 98059. (B) Neutrophils from adults were incubated with LPS (5 ng/ml) with 5% plasma and the indicated PD 98059 concentrations for 20 min. Activation of ERK 1 and 2 (⧫) was measured by Western blotting as described previously and expressed relative to values obtained for no inhibitor (plus LPS). For measurement of oxidative burst, neutrophils were incubated with (×) or without (□) LPS (5 ng/ml) with 5% plasma and a range of PD 98059 concentrations. After stimulation with fMLP, oxidative burst was measured as integrated chemiluminescence intensity for 4 min and expressed relative to values obtained for no inhibitor (plus LPS). Each data point represents the mean ± range or standard error of the mean of at least two independent measurements; oxidative burst (plus LPS) was significantly greater than ERK activity at PD 98059 concentrations of ≥10 μM (P < 0.05).

FIG. 6

Depleting plasma of fibronectin (FN) does not affect ERK activation in neutrophils from adults. (A) Equal protein amounts of control plasma (lane 1) and fibronectin-depleted plasma (FN-dep) (lane 2) were separated by SDS-PAGE along with fibronectin purified from the control plasma (lane 3) and purchased control fibronectin (lane 4). (B) Sample Western blot for activated ERK 1 and 2 from neutrophils of adults incubated with or without 5 ng of LPS per ml and with either 5% autologous plasma, control plasma, or fibronectin-depleted plasma. The blot is representative of two experiments.

FIG. 7

Requirement of LBP for ERK 1 and 2 (ERK1/2) activation. Sample Western blots for activated ERK 1 and 2 from neutrophils of adults incubated with LPS (5 ng/ml) are shown. (A) Neutrophils were resuspended in media supplemented with 5% plasma, human LBP (5 ng/ml), or control media followed by stimulation with or without LPS for 20 min at 37°C before lysis. (B) Plasma was preincubated with 20 μg of mouse anti-LBP monoclonal antibody (mAb) (IgG1), control mouse IgG1, or media alone for 1 h at room temperature before being added to neutrophils to a final plasma concentration of 5%. Cells were then incubated in the presence or absence of LPS for 20 min at 37°C before lysis. phospho-ERK1/2, phosphorylated ERK1 and ERK2 proteins identified using E-4 monoclonal antibody.