Role of CXC chemokines in the enhancement of LPS-induced neutrophil accumulation in the lung of mice by dexamethasone

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Abstract

Lipopolysaccharide (LPS)-induced multiple organ injury was mediated in part by a transcription factor, nuclear factor-κB (NF-κB). Mice were pretreated with dexamethasone (DEX), an inhibitor of NF-κB activation, to elucidate its effects on LPS-induced early responses in vivo. Early responses measured 1 h after intraperitoneal LPS administration at a dose of 1 mg/kg were (1) neutrophil accumulation in the tissues, (2) neutrophil degranulation, and (3) protein and mRNA expressions of tumor necrosis factor-α (TNF-α) and ELR+ CXC chemokines [macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC)]. Treatment with DEX before LPS administration suppressed NF-κB activation and plasma TNF-α levels almost to undetectable levels, but enhanced neutrophil accumulation and augmented MIP-2 levels in the lung. The suppression of plasma TNF-α levels by pretreatment with an anti-TNF-α antibody did not enhance LPS-induced neutrophil accumulation in the lung. These results demonstrate that the enhancement of LPS-induced neutrophil accumulation by DEX might be mediated by MIP-2 and not by TNF-α.

Section snippets

Materials and methods

Reagents. Lipopolysaccharide (LPS, Escherichia coli, serotype 011:B4), gelatin type A (from porcine skin), o-dianisidine dihydrochloride, tetramethylbenzidine, leupeptin, aprotinin, and Coomassie brilliant blue R-250 were purchased from Sigma (MO, USA). Dexamethasone phosphate (DEX) was from Tokyo Chemical (Tokyo, Japan). Pepstatin A was from Peptide Institute (Osaka, Japan). Vinblastine sulfate and phenylmethylsulfonyl fluoride (PMSF) were from Wako (Tokyo, Japan). ISOGEN was from Nippon Gene

Effects of DEX on LPS-induced lung MPO, lung MMP-9, and plasma MMP-9 activities

The lung and the liver MPO activities (Fig. 1) and plasma gelatinase activities (Fig. 2) were measured 1, 2, 4, and 24 h after LPS administration. MPO activities of the lung and the liver reached maximum levels 2 and 4 h after LPS administration, respectively. A gelatin zymogram (Fig. 2A) shows two kinds of protein with gelatinase activities corresponding to pro-matrix metalloproteinases-9 (proMMP-9) and proMMP-2 by molecular weights (101 and 62 kDa) and activation profiles with APMA. Only

Discussion

The focus of the present study was to examine the effects of DEX, as an inhibitor of NF-κB activation, on early responses of mice administered LPS intraperitoneally. Our results show that DEX augmented LPS-induced neutrophil accumulation in the lung but had no effects on LPS-induced neutrophil degranulation. The enhancement of neutrophil accumulation in the lung by DEX was not related to TNF-α, but dependent on the MIP-2 in the lung.

MPO and gelatinase are contents of granules within neutrophils

Acknowledgements

Thanks are given to Ms. Yoshiko Sasaki, Ms. Yukie Iwata, Ms. Aiko Kimura, Ms. Michiyo Tamada, Ms. Youko Hirayama, and Ms. Kana Izutu for their assistance in the experimental work.

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