Elevated levels of NO are localized to distal airways in asthma

Abstract

The contribution of nitric oxide (NO) to the pathophysiology of asthma remains incompletely defined despite its established pro- and anti-inflammatory effects. Induction of the inducible nitric oxide synthase (iNOS), arginase, and superoxide pathways is correlated with increased airway hyperresponsiveness in asthmatic subjects. To determine the contributions of these pathways in proximal and distal airways, we compared bronchial wash (BW) to traditional bronchoalveolar lavage (BAL) for measurements of reactive nitrogen/oxygen species, arginase activation, and cytokine/chemokine levels in asthmatic and normal subjects. Levels of NO were preferentially elevated in the BAL, demonstrating higher level NOS activation in the distal airway compartment of asthmatic subjects. In contrast, DHE(+) cells, which have the potential to generate reactive oxygen species, were increased in both proximal and distal airway compartments of asthmatics compared to controls. Different patterns of cytokines and chemokines were observed, with a predominance of epithelial cell-associated mediators in the BW compared to macrophage/monocyte-derived mediators in the BAL of asthmatic subjects. Our study demonstrates differential production of reactive species and soluble mediators within the distal airways compared to the proximal airways in asthma. These results indicate that cellular mechanisms are activated in the distal airways of asthmatics and must be considered in the development of therapeutic strategies for this chronic inflammatory disorder.

Figure 1. Bronchial wash (BW) and Bronchoalveolar lavage (BAL) fluids have significantly different levels of SP-D and RAGE

(A) Total number of picomoles of SP-D (measured by ELISA) comparing BW and BAL samples collected from asthmatic and control subjects, *p=0.01 BW vs. BAL from normals and BW vs. BAL from asthmatics; (B) Comparison of RAGE levels in picomoles (measured by ELISA) between BW and BAL samples from normal and asthmatic subjects. Data are means ± S.D. ^#p=0.02 BW vs. BAL fluid from normals and *p=0.01 BW vs. BAL from asthmatics.

Figure 2. Total NOx levels are increased in the BAL fluid from asthmatics and not in the BW fluid compared to controls

Total levels of nitrite and nitrate in BAL and BW fluid of normal and asthmatic subjects were measured by methods of chemiluminiscence and liquid chromatography. **p<0.001 comparing total nitrite and nitrate levels sampled by BAL from normal vs. asthmatics; *p=0.01 comparing total nitrite and nitrate levels in BAL vs BW fluid from asthmatics; ^#p=0.05 comparing total nitrite and nitrate levels in normal BW vs. BW fluid from asthmatics. Both nitrite and nitrate levels are reported as saline subtracted values.

Figure 3. Urea, a metabolite of the arginase pathway, is increased, whereas, ornithine is not enhanced significantly in the BAL fluid of asthmatics

(A) Total urea levels were measured in BAL and BW fluid of normal and asthmatic subjects as an indicator for arginase activity. *p=0.01 comparing BAL fluid from normal vs. asthmatic subjects. The arginase activity in BAL fluid compared to BW fluid of asthmatics was p=0.06. (B) Total levels of ornithine and ornithine-lactam detected in BW and BAL fluids from asthmatic and normal subjects by LC-MS/MS.

Figure 4. DHE⁺ cells with the potential to produce ROS are present in increased numbers in BAL and BW of asthmatic subjects compared to normal controls

(A) Representative overlaid histogram plots of BW and BAL cells from normal and asthmatic subjects stained with the indicator for ROS, Dihydroxyethidium (DHE). The percentages of DHE⁺ cells were determined by flow cytometry. *p<0.05 comparing BW cells from asthmatics with normal controls and BAL cells from asthmatics vs. normal controls. (B) Stacked histograms showing total cell numbers and numbers of DHE⁺ cells in pooled BAL and BW samples from 6 asthmatics compared to 5 normal control subjects.

Figure 5. BW and BAL sample different cell populations

(A) Representative images (1000 X) of DIFF Quik stained cytospin preparations of BW and traditional BAL cells from asthmatic and control subjects. White arrows designate eosinophils and black arrows highlight ciliated columnar epithelial cells (B) Blinded analysis of cell differentials (expressed as percents of total) from asthmatic and control subjects sampled using the BW and BAL techniques. ^#p=0.04 comparing the number of epithelial cells in BAL vs. BW fluid obtained from asthmatic subjects and ^##p=0.008 comparing epithelial cells recovered by BAL vs. BW from normal subjects. #p=0.04 comparing the number of macrophage/monocyte cells in BAL vs. BW fluid obtained from asthmatic subjects. ^$$p=0.005 comparing the number of macrophage/monocyte cells in BAL vs. BW fluid obtained from normal subjects. Eosinophils are represented as % Other WBC excluding monocytes and macrophages. p=0.15 comparing numbers of eosinophils in BW fluid of asthmatics vs. controls and p=0.66 comparing BAL fluid from asthmatics vs. controls.

Figure 6

Schematic representation of the inflammatory cytokine/chemokine signaling and inflammatory cellular contributions in larger, proximal versus smaller, distal airways in asthmatic subjects.