The role of the extracellular matrix protein mindin in airway response to environmental airways injury

Abstract

Background: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored.

Objectives: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone.

Methods: We exposed C57BL/6J and mindin-deficient (-/-) mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies.

Results: After inhalation of LPS, mindin-/- mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin-/- mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin-/- mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin-/- mice demonstrated reduced constriction in response to carbachol.

Conclusions: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.

Figure 1

Airway responsiveness in C57BL/6J and mindin^–/– mice before (A) and after (B) challenge with inhaled LPS (n = 5/group). Baseline values with methacholine challenge were similar in unexposed animals (A); however, after LPS exposure (B), C57BL/6J mice showed significantly increased airway responsiveness compared with LPS-exposed mindin^–/– mice. *p < 0.05.

Figure 2

Cellular recruitment into the airspace (A,B) and total protein level (C) measured in whole-lung lavage fluid from C57BL/6J and mindin^–/– mice after exposure to saline or LPS (n = 5/group). Cellular recruitment into the airspace was evaluated by the number of total cells (A) and the number of neutrophils (B). Mindin^–/– mice demonstrate reduced cellular inflammation after exposure to LPS compared with C57BL/6J mice. Mindin^–/– mice do not have a detectable increase in total protein after exposure to LPS (C). *p < 0.05 for LPS-exposed C57BL/6J mice compared with either group of saline controls. **p < 0.05 for LPS-exposed C57BL/6J mice compared with LPS-exposed mindin^–/– mice.

Figure 3

Proinflammatory cytokines KC (A), IL-1β (B), MCP-1 (C), and TNF-α (D) measured in BALF from C57BL/6J and mindin^–/– mice after exposure to saline or LPS (n = 5/group). *p < 0.05 for LPS-exposed C57BL/6J mice compared with either group of saline controls. **p < 0.05 for LPS-exposed C57BL/6J mice compared with LPS-exposed mindin^–/– mice.

Figure 4

Airway responsiveness in C57BL/6J and mindin^–/– mice before (n = 4/group; A) and after (n = 5/group; B) challenge with ozone. *p < 0.05 compared with ozone-exposed mindin^–/– mice.

Figure 5

Cellular recruitment into the airspace, evaluated by the number of total cells (A) and the number of neutrophils (B), and total protein level (C) measured in whole-lung lavage fluid from C57BL/6J and mindin^–/– mice after exposure to FA or ozone (n = 5 per group). *p < 0.05 for ozone-exposed C57BL/6J mice compared with either group of FA-exposed controls. ^#p < 0.05 for ozone-exposed mindin^–/– mice compared with either group of FA-exposed controls.

Figure 6

Proinflammatory cytokines KC (A), IL-1β (B), MCP-1 (C), and TNF-α (D) measured in the BALF from C57BL/6 and mindin^–/– mice after exposure to FA or ozone (n = 5/group). *p < 0.05 for ozone-exposed C57BL/6J mice compared with either group of FA-exposed controls. **p < 0.05 for ozone-exposed C57BL/6J mice compared with ozone-exposed mindin^–/– mice.

Figure 7

Bronchial ring contractile response to carbachol in naive (A), LPS-exposed (B), and ozone-exposed (C) C57BL/6J and mindin^–/– mice.In A, n = 6 for C57BL/6J and n = 5 for mindin^–/–; in B, n = 6 for C57BL/6J and n = 6 for mindin^–/–; in C, n = 9 for C57BL/6J and n = 8 for mindin^–/–. *p < 0.05 for ozone-exposed C57BL/6J mice compared with ozone-exposed mindin^–/– mice.