Differentiation of the roles of NO from airway epithelium and inflammatory cells in ozone-induced lung inflammation

Abstract

Mice lacking inducible nitric oxide synthase (NOS2-/-) are more susceptible to ozone-induced lung inflammation and injury than their isogenic wild-type (NOS2+/+) counterparts, demonstrating an apparent protective effect for NOS2 in murine lungs. We hypothesized that nitric oxide (NO) generated from either NOS2 in the airway epithelial cells or the bone-marrow-derived inflammatory cells was responsible for the protective effect of NOS2. To test this hypothesis, we prepared chimeric mice by killing their endogenous bone marrow cells by whole body irradiation followed by bone marrow transplantation from a heterologous donor mouse. We exposed C57BL/6 (NOS2+/+), NOS2-/-, and chimeric NOS2 mice (NOS2-/+, NOS2+/-) to 1 ppm of ozone for 3 consecutive nights. NOS2-/- mice were more severely injured after exposure to ozone than C57BL/6 mice, including a more robust inflammatory cell influx (4.14 x 10(5) +/- 2.19 x 10(5) vs. 2.78 x 10(5) +/- 1.36 x 10(5) cells respectively; P = 0.036) and greater oxidation of total protein sulfhydryls (R-SH) in their blood plasma. Chimeric NOS2-/+ mice, which had bone marrow from NOS2-/- mice transplanted into C57BL/6 recipients, had a significantly greater response to ozone (increased numbers of neutrophils in lung lavage and decreased concentrations of exhaled NO) as compared to the reciprocal chimeric strain (NOS2+/-). We conclude that NOS2 has a protective effect against acute lung injury caused by ozone inhalation, which may be mediated, in part, by NO generated by NOS2 from inflammatory cells, predominantly neutrophils, recruited into the lung.

Figure 1

Total R-SH content of 10 ul aliquots of blood plasma determined by a colorimetric reaction using Ellman’s reagent. The responses of C57BL (NOS2+/+) and NOS2−/− mice, as well as of the reciprocal chimeric strains prepared from these mice, to exposure to ozone are compared. * Symbol denotes p<0.02 compared to the corresponding group exposed only to filtered air.

Figure 2

Total cell counts from lung lavage fluid. The number in parentheses is number of experimental animals in each group. * Symbol denotes p<0.05 compared to C57BL/6 (NOS2+/+) mice exposed to ozone; + symbol denotes p<0.05 compared NOS2−/− group exposed to ozone. There are significantly more total cells in each group exposed to ozone than in their corresponding filtered air controls (not indicated on figure).

Figure 3

Percentage of neutrophils in lung lavage fluid. Air controls for each of the four mouse strains studied contained 0% neutrophils (the only cell type present in the air controls was pulmonary alveolar macrophages), so the air control results are not shown on the figure. * Symbol denotes p<0.05 compared to the NOS2−/− group.

Figure 4

Dynamic lung compliance measured in anesthetized, tracheotomized, and ventilated mice after exposure to air or ozone. Mice were deeply anesthetized and sedated with medetomidine, 0.5 mg/kg, and tiletamine/zolpidem, 50 mg/kg and ventilated at 7–8 cc/kg with a mouse ventilator for the duration of the procedure. Measurements were made with the mouse in the supine position using a plethysmograph. * Symbol denotes P=0.005 as compared to the corresponding value in the reciprocal chimeric strain.

Figure 5

Exhaled nitric oxide concentration in ventilated mice after exposure to air and ozone. Exhaled gas was collected during the first 5 minutes the mouse was on the ventilator with a collection bag connected to the exhalation port. Exhaled NO concentrations were measured from this sample with a Sievers NO Analyzer. + Symbol denotes a p value < 0.05 compared to NOS2+/+ mice exposed to ozone. # Symbol denotes p value < 0.05 compared to NOS2−/− mice after exposure to ozone.

Figure 6

Nitrate and nitrite (NOx) concentrations found from lung lavage cells stimulated with LPS + IFN-α from mice exposed to ozone. * Symbol denotes p value <0.05 compared to NOS2+/+ mice. + Symbol denotes p value <0.05 compared to NO2−/− mice. # Symbol denotes p value < 0.05 compared to NOS2+/+ mice.

Figure 7

Nitrate and nitrite concentration found from bone marrow cells stimulated with LPS + IFN-α from mice exposed to ozone. * Symbol denotes p value < 0.03 compared to both of the other groups.

Figure 8

NOS3 Western blot band density from the airways of each of the four groups of mice studied after exposure to ozone (n=5–7 each). *^, + Symbols denote p value < 0.02 compared to C57BL (NOS2+/+) and NOS2−/− mice, respectively.

Figure 9

NOS1 Western blot band density from the airways of each of the four groups of mice studied after exposure to ozone (n=5–7 each). Data were log-transformed to better fit the figure. * Symbol denotes p value <0.05 compared to C57BL/6 (NOS2+/+) mice.