Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide

Abstract

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.

Figure 1

Effect of increasing doses of LPS on the dye leakage in the skin of iNOS deficient and wild-type mice. Topical dye leakage in the skin was assessed 2 h after s.c. injection of LPS or saline (10 ml kg⁻¹). Symbols and vertical bars represent means±s.e.mean of five experiments. ^*P<0.05 vs wild-type mice, †P<0.05 vs respective saline-treated mice (0 μg LPS site⁻¹).

Figure 2

Effect of iNOS inhibitors on LPS-induced dye leakage in mouse skin. Mice were treated with i.v. aminoguanidine (10 mg kg⁻¹) or L-NAME (10 mg kg⁻¹) 5 min prior to LPS (400 μg site⁻¹ s.c.). Control mice received saline instead of LPS. The amount of dye leakage in the skin was assessed 2 h later. Columns and bars represent means±s.e.mean of five experiments. ^*P<0.05.

Figure 3

Effect of COX inhibitors on LPS-induced dye leakage in mouse skin. Mice were treated with i.p. NS-398 (1 mg kg⁻¹) or indomethacin (5 mg kg⁻¹) 35 min prior to LPS (400 μg site⁻¹ s.c.). Control mice received saline instead of LPS. The dye leakage was assessed 2 h later. Columns and bars represent means±s.e.mean of five experiments. ^*P<0.05.

Figure 4

Effect of H₁ blocker on LPS-induced dye leakage in mouse skin. Mice were pretreated with diphenhydramine (10 mg kg⁻¹ s.c.) 20 min prior to LPS (400 μg site⁻¹ s.c.). Control mice received saline instead of LPS. The dye leakage was assessed 2 h later. Columns and bars represent means±s.e.mean of five experiments. ^*P<0.05.

Figure 5

Effect of anti-TNF-α antibody on LPS-induced dye leakage in mouse skin. Mice were treated with anti-TNF-α antibody (dilution 1 : 400, 10 ml kg⁻¹ s.c.) 24 h prior to LPS (400 μg site⁻¹ s.c.). The dye leakage was assessed 2 h later. Columns and bars represent means±s.e.mean of five experiments. ^*P<0.05.

Figure 6

Effect of systemic pretreatment with LPS on the LPS-induced dye leakage in mouse skin. LPS (0.15 mg kg⁻¹) or saline (10 ml kg⁻¹) was i.p. administered to mice 4 h previously. The mice were given LPS (400 μg site⁻¹ s.c.) or saline (10 ml kg⁻¹ s.c.) and the dye accumulation in the skin was determined 2 h later. Columns and bars represent means±s.e.mean of five experiments. ^*P<0.05.