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. 2006 Jan 23;203(1):131-40.
doi: 10.1084/jem.20051794. Epub 2005 Dec 27.

MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock (V体育ios版)

Qun Zhao  1 , Xianxi Wang, Leif D Nelin, Yongxue Yao, Ranyia Matta, Mary E Manson, Reshma S Baliga, Xiaomei Meng, Charles V Smith, John A Bauer, Cheong-Hee Chang, Yusen Liu
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MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock (V体育平台登录)

"VSports最新版本" Qun Zhao et al. J Exp Med. .

VSports app下载 - Abstract

Septic shock is a leading cause of morbidity and mortality. However, genetic factors predisposing to septic shock are not fully understood VSports手机版. Excessive production of proinflammatory cytokines, particularly tumor necrosis factor (TNF)-alpha, and the resultant severe hypotension play a central role in the pathophysiological process. Mitogen-activated protein (MAP) kinase cascades are crucial in the biosynthesis of proinflammatory cytokines. MAP kinase phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAP kinase. Thus, we hypothesize that knockout of the Mkp-1 gene results in prolonged MAP kinase activation, augmented cytokine production, and increased susceptibility to endotoxic shock. Here, we show that knockout of Mkp-1 substantially sensitizes mice to endotoxic shock induced by lipopolysaccharide (LPS) challenge. We demonstrate that upon LPS challenge, Mkp-1-/- cells exhibit prolonged p38 and c-Jun NH2-terminal kinase activation as well as enhanced TNF-alpha and interleukin (IL)-6 production compared with wild-type cells. After LPS challenge, Mkp-1 knockout mice produce dramatically more TNF-alpha, IL-6, and IL-10 than do wild-type mice. Consequently, Mkp-1 knockout mice develop severe hypotension and multiple organ failure, and exhibit a remarkable increase in mortality. Our studies demonstrate that MKP-1 is a pivotal feedback control regulator of the innate immune responses and plays a critical role in suppressing endotoxin shock. .

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Figures

Figure 1.
Figure 1.
Mkp-1 deficiency results in prolonged p38 and JNK activation. (A) The kinetics of MAP kinase activation in Mkp-1 +/+ and Mkp-1 −/− macrophages after LPS stimulation. Elicited peritoneal macrophages from both Mkp-1 +/+ and Mkp-1 −/− mice were stimulated with 100 ng/ml LPS for the indicated times, and cell lysates were analyzed for phospho-p38, phospho-JNK, phospho-ERK, phospho-MK2, and MKP-1 using Western blotting. (B) Detailed kinetics of p38, JNK, and ERK inactivation after LPS challenge. Mkp-1 +/+ and Mkp-1 −/− samples were processed together, with the exception of the initial electrophoresis in which Mkp-1 +/+ and Mkp-1 −/− samples were run on different gels. (C) Comparison of MK2 activities between Mkp-1 +/+ and Mkp-1 −/− macrophages using immune complex kinase assays. Peritoneal macrophages were treated with 100 ng/ml LPS for the indicated times, and MK-2 kinase activity was examined by immune complex kinase assays using [γ-32P]ATP and a mouse Hsp25 as substrate. Kinase activities were quantitated using a phosphorImager and expressed as fold-activation relative to controls.
Figure 2.
Figure 2.
IFN-γ enhances activation of p38 and JNK by inhibiting MKP-1 expression. Resident peritoneal macrophages were treated with or without 50 U/ml IFN-γ overnight before LPS stimulation (100 ng/ml) at the indicated times. The levels of MKP-1 protein and phosphorylated MAP kinases were examined using Western blotting. Please note that the order for the + IFN-γ samples is from right to left.
Figure 3.
Figure 3.
Knockout of Mkp-1 alters cytokine expression in macrophages. (A) Cytokine production by IFN-γ–primed resident peritoneal macrophages. Resident peritoneal macrophages primed with IFN-γ overnight were stimulated with LPS for 4 and 6 h. Cytokine concentrations in the medium were analyzed by ELISA. Data are presented as the mean ± SEM (n = 3 in each group). *, Mkp-1 −/− different from Mkp-1 +/+, P < 0.001. (B) Cytokine production by thioglycollate-elicited peritoneal macrophages after stimulation with 100 ng/ml LPS for 4 and 6 h. Data are presented as mean ± SEM (n = 4 in each group). *, Mkp-1 −/− different from Mkp-1 +/+, P < 0.05; #, Mkp-1 −/− different from Mkp-1 +/+, P < 0.005. (C) Cytokine production by unprimed resident peritoneal macrophages from both Mkp-1 +/+ and Mkp-1 −/− mice. Resident peritoneal macrophages were stimulated with 100 ng/ml LPS for the indicated times, and cytokine concentrations in the medium were assayed by ELISA. Data are presented as the mean ± SEM (n = 6 in each group). *, Mkp-1 −/− different from Mkp-1 +/+, P < 0.001. (D) Expression of TNF-α, IL-6, IL-12p35, and IL-12p40 mRNA in LPS-stimulated wild-type and Mkp-1 −/− resident peritoneal macrophages. Resident peritoneal macrophages were primed overnight with 50 U/ml IFN-γ and then stimulated with 100 ng/ml LPS for 4 h. qRT-PCR was performed to assess the levels of mRNA for TNF-α, IL-6, IL-12p35, and IL-12p40. The relative mRNA levels were normalized to the GAPDH mRNA. Values represent expression levels relative to those in wild-type cells. Data are means ± SEM of three independent experiments. *, different from Mkp-1 −/− cells, P < 0.05.
Figure 4.
Figure 4.
Knockout of Mkp-1 shifts the pattern of cytokine expression in splenocytes and dendritic cells. (A) Cytokine expression in LPS-stimulated splenocytes. Splenocytes isolated from Mkp-1 +/+ and Mkp-1 −/− mice were stimulated with 100 ng/ml LPS for the indicated times, and cytokine concentrations in the medium were assayed by ELISA. IFN-γ levels were measured 24 h after LPS treatment. Data are presented as the mean ± SEM (n = 3–9 for each group). *, Mkp-1 −/− different from Mkp-1 +/+, P < 0.05. (B) Cytokine production by bone marrow–derived dendritic cells from both Mkp-1 +/+ and Mkp-1 −/− mice. Cells were stimulated with 1 μg/ml LPS for 24 h. Data are presented as mean ± SEM (n = 3 in each group). *, Mkp-1 −/− different from Mkp-1 +/+ , P < 0.05; #, Mkp-1 −/− different from Mkp-1 +/+, P < 0.005.
Figure 5.
Figure 5.
Deficiency in Mkp-1 enhances LPS-triggered production of TNF-α, IL-6, and IL-10 in vivo. (A) Both Mkp-1+/+ and Mkp-1 −/− mice were injected i.p. with either vehicle (PBS) or LPS at indicated doses. Mice were killed 90 min later and plasma cytokine concentrations were measured by ELISA. Data are presented as mean ± SEM (n = 6 in each group). Two-way ANOVA demonstrates a significant effect of both genotype (P < 0.001) and LPS dose (P = 0.005) on TNF-α and IL-6 concentrations. (B) Mice were injected i.p. with LPS (1.5 mg/kg body weight) and killed at the indicated times. Serum cytokines were measured using ELISA. Data are presented as mean ± SEM (n = 10). Two-way ANOVA demonstrates a significant effect of both genotype and time after LPS challenge on TNF-α, IL-6, and IL-10 concentrations (P < 0.001).
Figure 6.
Figure 6.
Increased mortality in Mkp-1 knockout mice in response to LPS challenge. (A) Survival curves of Mkp-1 +/+ and Mkp-1 −/− mice after challenge i.p. with 5 mg LPS per kg body weight. Kaplan-Meier analysis demonstrates a significant difference in survival between Mkp-1 +/+ and Mkp-1 −/− mice (P < 0.001; n = 12 in each group). (B) Survival curves of Mkp-1 −/− mice and their wild-type littermates after challenge i.p. with 1.5 mg LPS per kg body weight (P < 0.0005; n = 12 in each group).
Figure 7.
Figure 7.
LPS causes renal, heptic, and pulmonary damages in Mkp-1−/− mice. Mice were challenged with LPS (1.5 mg/kg body weight) and killed 24 h later. Plasma BUN and ALT activity were measured. Lungs were perfused and fixed with formalin, and 4-μm sections were stained with hematoxylin and eosin. (A) BUN and ALT activities in vehicle- and LPS-challenged mice. Data are presented as mean ± SEM of 8–13 independent experiments. *, P < 0.001, one-way ANOVA comparing Mkp-1 +/+ with Mkp-1 −/− groups. (B) Representative images of lung sections from LPS-challenged wild-type and Mkp-1 −/− mice.
Figure 8.
Figure 8.
LPS challenge results in hypotension and increased nitric oxide production in Mkp-1−/− mice. Mkp-1 +/+ and Mkp-1 −/− mice were injected i.p. with LPS (1.5 mg/kg body weight). (A) Systolic blood pressure in mice injected with LPS. Data are presented as mean ± SEM of six independent experiments. *, 4 and 24 h different from control (0 h), P < 0.001; †, Mkp-1 −/− different from Mkp-1 +/+ at same time point, P < 0.05. (B) Plasma nitrate levels. Mkp-1 +/+ and Mkp-1 −/− mice were injected i.p. with LPS and killed 24 h later. Plasma nitrate levels were measured by chemiluminescence. Data are presented as the mean ± SEM of four independent experiments. *, Mkp-1 −/− different from Mkp-1 +/+, P < 0.05.
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References

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