MARCKS-related peptide modulates in vivo the secretion of airway Muc5ac

Abstract

In a mouse model of neutrophil elastase-induced bronchitis that exhibits goblet cell metaplasia and inflammation, we investigated the effects of intratracheal instillation of the MANS peptide, a peptide identical to the NH(2) terminus of the myristoylated alanine-rich C kinase substrate (MARCKS) on mucin protein airway secretion, inflammation, and airway reactivity. To induce mucus cell metaplasia in the airways, male BALB/c mice were treated repetitively with the serine protease, neutrophil elastase, on days 1, 4, and 7. On day 11, when goblet cell metaplasia was fully developed and profiles of proinflammatory cytokines were maximal, the animals were exposed to aerosolized methacholine after intratracheal instillation of MANS or a missense control peptide (RNS). MANS, but not RNS, attenuated the methacholine-stimulated secretion of the major respiratory mucin protein, Muc5ac (50% reduction). Concurrently, elastase-induced proinflammatory cytokines typically recovered in bronchoalveolar lavage (BAL), including KC, IL-1beta, IL-6, MCP-1, and TNFalpha, were reduced by the MANS peptide (mean levels decreased 50-60%). Secondary to the effects of MANS on mucin secretion and inflammation, mechanical lung function by forced oscillation technique was characterized with respect to airway reactivity in response to cumulative aerosol stimulation with serotonin. The MANS peptide was also found to effectively attenuate airway hyperresponsiveness to serotonin in this airway hypersecretory model. Collectively, these findings support the concept that even in airway epithelia remodeled with goblet cell metaplasia and in a state of mucin hypersecretion, exogenous attenuation of function of MARCKS protein via the MANS peptide decreases airway mucin secretion, inflammation, and hyperreactivity.

Fig. 1.

A: bronchoalveolar lavage (BAL) fluid total cell count from mice treated with neutrophil elastase (NE) or Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol] and inhibited with myristoylated amino-terminal sequence peptide (MANS), scrambled missense control peptide (RNS), or saline. Count data are means ± SE (n = 4–10 mice per group). For all NE treatment groups, total BAL cells were increased compared with Vehicle treatment; *differences between NE- and Vehicle-treated were significant at P < 0.05. In NE-treated mice, pretreatment with MANS reduced BAL total cells compared with NE, NE + RNS, and NE + saline treatment groups; #differences between groups were significant at P < 0.05. B: BAL differential cell counts of mice treated with NE or Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol]. Cell differential data are mean cell counts ± SE (n = 4–10 mice per group). Macrophage, polymorphonuclear neutrophil (PMN), lymphocyte, and eosinophil cell counts for all NE treatment groups were increased compared with the respective cell counts of Vehicle-treated mice, and differences were significant at P < 0.05. NE-treated mice that were pretreated with MANS had reductions in BAL cell differential counts for PMNs, lymphocytes, and eosinophils, respectively, compared with NE-treated mice pretreated with RNS or saline; #differences in cell counts were significant at P < 0.05. Avg, average.

Fig. 2.

Effect of NE treatment on BAL cytokine profile and inhibition by MANS peptide. BAL levels of proinflammatory cytokines KC, IL-1β, IL-6, MCP-1, and TNFα for indicated treatment groups (each datum point expressed in picograms per milliliter represents the cytokine level for an individual mouse, and horizontal bar through the points indicates the mean of the values). Mean cytokine levels in the BAL collected from all NE-treated mice groups were elevated compared with respective groups treated with Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol], and cytokine differences were significant at P < 0.05; pretreatment with MANS peptide reduced BAL cytokine level for each cytokine compared with mice treated with NE alone, NE + RNS, or NE + saline, and reduced levels were significant at P < 0.05.

Fig. 3.

Effect of NE treatment on Alcian blue/periodic acid-Schiff (AB/PAS) glycoprotein staining of airway epithelium. Representative histological tissue images of mice airways from: A, Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol]-treated; B, Vehicle + saline-treated; C, NE + saline-treated; D, NE + MANS peptide-treated; and E, NE + RNS-treated. Airway image of NE + MANS-treated mouse (D) demonstrates enhanced epithelial staining with AB/PAS consistent with retention of mucin glycoproteins within epithelial goblet cells. Scale bar = 50 μm.

Fig. 4.

Influence of methacholine stimulation on Muc5ac mucin secretion. Muc5ac mucin protein level in BAL fluids collected after in vivo stimulation with methacholine aerosol for Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol] and NE-treated mice (mean level ± SE, expressed as nanograms per milliliter, n = 10–12 mice per group). Each group was exposed to methacholine (60 mM) aerosol for 3 min to stimulate airway mucin secretion (see materials and methods). BAL samples from all NE-treated mice groups had higher levels of Muc5ac than Vehicle-treated controls. #Levels exceeded those of Vehicle controls, and differences were significant at P < 0.05; *Muc5ac was decreased compared with NE + saline and NE + RNS groups, and differences were significant at P < 0.05.

Fig. 5.

MANS peptide inhibits mucin glycoprotein secretion from airway epithelium. Histogram summary of AB/PAS scoring of large airways (mean index score ± SE for 8–10 mice per group); representative staining of airways are illustrated in Fig. 3. No evidence of staining with AB/PAS was apparent in airway tissue images sampled from mice treated with Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol] or Vehicle + saline.

Fig. 6.

Airway responsiveness of NE-treated mice to serotonin bronchoprovocation. Means ± SE of total pulmonary resistance (R_T) at indicated serotonin concentration (R_T is expressed as cmH₂O·ml⁻¹·s⁻¹; 4–12 mice per group). *R_T response to cumulative aerosol concentration of serotonin was elevated in mice treated with NE + saline compared with mice treated with Vehicle [1:1 sodium acetate (NaOAc)-to-glycerol] + MANS and significant at P < 0.05; #changes in R_T in mice treated with NE + MANS were decreased compared with mice treated with NE + saline, and the differences were significant at P < 0.05; $changes in R_T in NE + RNS mice were elevated at indicated concentrations compared with mice treated with Vehicle + RNS, and difference was significant at P < 0.05.