Regulatory effects of iNOS on acute lung inflammatory responses in mice

Abstract

The role of endogenous NO in the regulation of acute lung injury is not well defined. We investigated the effects of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the acute inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) into wild-type (WT) mice and mice deficient in iNOS (iNOS(-/-)) or eNOS (eNOS(-/-)). Endpoints of inflammatory injury were myeloperoxidase (MPO) content and leak of albumin into lung. Inflammatory injury was similar in WT and eNOS(-/-) mice but was substantially increased in iNOS(-/-) mice. Bronchoalveolar lavage (BAL) fluids of iNOS(-/-) and WT mice showed similar levels of CXC chemokines (MIP-2, KC) but enhanced levels of CC chemokines (MCP-1, MCP-3). Increased lung content of MPO in iNOS(-/-) mice was reduced by anti-MCP-1 to values found in WT mice. In vitro stimulation of microvascular endothelial cells with LPS and IFN gamma revealed elevated production of CXC and CC chemokines in cells from iNOS(-/-) mice when compared to endothelial cells from iNOS(+/+) mice. Peritoneal macrophages from iNOS(-/-) donors also revealed increased production of CC chemokines after stimulation with LPS and interferon (IFN gamma). These data indicate that absence of iNOS causes enhanced lung inflammatory responses in mice which may be related to enhanced production of MCP-1 by endothelial cells and macrophages. It appears that iNOS affects the lung inflammatory response by regulating chemokine production.

Figure 1.

Lung vascular permeability and neutrophil accumulation (MPO content) in iNOS^−/− (EXP 1) and eNOS^−/− (EXP 2) mice compared to wild-type (WT) mice after LPS-induced lung injury. Mouse albumin levels were measured in BAL fluids (A) as an index for vascular leakage and MPO activity in whole lungs (B) as an index for neutrophil influx. Results are means ± SEM from at least five mice, where # is P < 0.05 compared to uninjured control and * is P < 0.05 compared to WT treated mice.

Figure 2.

Hematoxylin and eosin staining of lung tissues obtained from iNOS^+/+ mice treated with saline (A) or LPS (C), and iNOS^−/− mice treated with LPS (E). Interstitial neutrophil infiltration is present in lungs from iNOS^+/+ treated mice (C and D), and substantially increased in the iNOS^−/−-treated mice (E and F). Magnification: A, C, and E, ×20; B, D, and F, ×40.

Figure 3.

CXC (MIP-2, KC) and CC (MCP-1, MCP-3) chemokine levels in BAL fluid of iNOS^−/− and eNOS^−/− mice compared to WT mice after LPS-induced lung injury. Results are the mean ± SEM from at least five mice where # is P < 0.05 compared to uninjured and * is P < 0.05 compared to WT treated.

Figure 4.

Ability of anti-MCP-1 to reduce lung neutrophil influx in iNOS^−/− mice after LPS-induced lung injury determined by MPO activity in whole lungs. Results are the mean ± SEM from at least five mice where # is P < 0.05 compared to uninjured and * is P < 0.05 compared to WT treated.

Figure 5.

NO production (A) and NOS protein expression (B) in dermal microvascular endothelial cells after 24-hour exposure to LPS (10 μg/ml) and/or IFNγ (25 U/ml). Protein expression was determined by Western blot analysis. Values in (A) are means ± SEM based on three separate experiments performed using triplicate samples. The data in (B) are representative of results from two separate and independent experiments.

Figure 6.

Time course for NO production and iNOS protein expression in dermal microendothelial cells stimulated with LPS and IFNγ (10 μg/ml and 25 U/ml, respectively). A: NO production in endothelial cells derived from iNOS^+/+ and iNOS^−/− mice as a function of duration of stimulation. B: Western blot analysis of iNOS protein in iNOS^+/+ dermal microendothelial cells and peritoneal macrophages. C: Lack of iNOS protein expression in iNOS^−/− endothelial cells compared to iNOS^+/+ endothelial cells after 24-hour stimulation. iNOS protein is also shown in peritoneal macrophages from iNOS^+/+ mice. Values in A are means ± SEM based on three experiments performed in triplicate. Data in B and C are representative of two separate and independent experiments, where * is P < 0.05.

Figure 7.

Time-course analysis of LPS- and IFNγ-induced (10 μg/ml and 25 U/ml, respectively) CC and CXC protein expression in dermal microendothelial cells derived from iNOS^+/+ and iNOS^−/− mice. Results are the mean ± SEM based on three separate and independent experiments with triplicate samples for each data point, where * is P < 0.05, compared to stimulated iNOS^+/+ cells.

Figure 8.

Analysis of LPS- and IFNγ-induced (10 ng/ml and 25 U/ml, respectively) CXC and CC protein expression in peritoneal macrophages derived from iNOS^+/+ and iNOS^−/− mice. Results are the mean ± SEM based on three separate and independent experiments with triplicate samples for each data point where # is P < 0.05 compared to uninjured and * is P < 0.05 compared to iNOS^+/+ treated.