Inhibition of myristoylated alanine-rich C kinase substrate (MARCKS) protein inhibits ozone-induced airway neutrophilia and inflammation

Abstract

Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 +/- 0.9-fold), interleukin-6 (IL-6; 12.7 +/- 1.9-fold), and tumor necrosis factor (TNF; 2.1 +/- 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% +/- 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% +/- 14%, 47% +/- 15%, and 71.1% +/- 14%, and IL-6 secretion by 69% +/- 12%, 40% +/- 7%, and 86.1% +/- 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% +/- 7%) and BIO-11006 (84% +/- 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.

FIGURE 1

Ozone differentially induces cytokine secretions in mice. Mice were exposed to ozone (100 ppb) or forced air for 4 hours. After 1 hour of recovery time, the mice were sacrificed, and cytokine concentration was then determined in BAL fluid by ELISA for KC (A), IL-6 (B), TNF (C) and IFN-γ (D). Each cohort consisted of 5 mice, and BAL fluid obtained from each mouse was performed in triplicate as described in Materials and Methods. Data represent mean ± SEM from 3 separate experiments. *Significantly different from FA when P < .05 by ANOVA.

FIGURE 2

Ozone exposure specifically increases neutrophil cell counts in BAL. Cohorts of 5 mice were exposed to ozone (100 ppb) or forced air for 4 hours. After 1 hour of recovery time, the mice were sacrificed and lavaged with sterile PBS as described in Materials and Methods. (A) Total cell numbers in BAL fluid in cohorts exposed to forced air or ozone after administration of diluent/PBS or scrambled peptide, RNS. (B) Differential cell counts expressed as a percent of total cell numbers within each cohort. Data represent group mean ± SEM from three separate experiments. *’ **Significantly different from FA when P < .05 by ANOVA.

FIGURE 3

MARCKS protein inhibitors attenuate ozone-induced cytokine enhancement in mice. Mice were i.t. injected with PBS, RNS, MANS, BIO-11000, or BIO-11006 and exposed to ozone (100 ppb) for 4 hours. Each cohort consisted of 5 mice, and BAL fluid obtained from each mouse was assessed in triplicate for KC (A), IL-6 (B), and TNF (C) by ELISA. MANS, BIO-11000, and BIO-11006 had no effect on KC, TNF, IL-6, or IFN-γ levels in animals treated with forced air (data not shown). Data represents group mean ± SEM from 3 separate experiments. *,**,***Significantly different from RNS when P < .05 by ANOVA.

FIGURE 4

MANS and BIO-11006 substantially abrogate ozone-induced neutrophilia. After i.t. instillation of PBS, RNS, MANS, BIO-11000, or BIO-11006, mice were exposed to ozone for 4 hours. Cell counts were performed for neutrophils, macrophages, eosinophils, and lymphocytes in BAL fluid. Data in the figure are representative of mean cell counts ± SEM from 3 separate experiments with each experimental group containing 5 animals. *,**Significantly different from RNS when P < .05 by ANOVA.

FIGURE 5

Representative lung histology of ozone-induced immune cell influx in murine lungs. H&E staining of left lung sections from mice following (A) i.t. PBS instillation and exposure to forced air and (B) i.t. PBS administration followed by ozone exposure at 100 ppb. (C) After ozone exposure, maximal infiltration of leukocytes was observed in the submucosa of mice intratracheally administered with RNS peptide and was comparable to that observed in PBS-instilled mice (B). Differential effects of MARCKS inhibitory peptides, MANS (D), BIO-11000 (E), and BIO-11006 (F), abrogated ozone-induced leukocyte migration in the submucosa. All sections were magnified 200×, and the scale bar (lower left corner of all figures) denotes 50 μm. The lungs of 5 mice from each treatment group were processed for histology, and results shown are representative of each group.

FIGURE 6

MARCKS peptide inhibitors attenuate ozone-induced inflammation. H&E-stained lung tissue sections from ozone-exposed animals treated with or without PBS, RNS, MANS, BIO-11000, and BIO-11006 were graded for immune cell infiltration following histopathological parameters described in Materials and Methods. Each point is representative of the mean score per animal calculated from a total of 15 airways in 5 to 7 tissue sections. Data are shown as individual values, and each horizontal line indicates the mean value per group (n = 4 animals). *Significantly different from the FA-exposed animals at P < .05.