Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma

Abstract

Gastrin-releasing peptide (GRP) is synthesized by pulmonary neuroendocrine cells in inflammatory lung diseases, such as bronchopulmonary dysplasia (BPD). Many BPD infants develop asthma, a serious disorder of intermittent airway obstruction. Despite extensive research, early mechanisms of asthma remain controversial. The incidence of asthma is growing, now affecting >300 million people worldwide. To test the hypothesis that GRP mediates asthma, we used two murine models: ozone exposure for air pollution-induced airway hyperreactivity (AHR), and ovalbumin (OVA)-induced allergic airway disease. BALB/c mice were given small molecule GRP blocking agent 77427, or GRP blocking antibody 2A11, before exposure to ozone or OVA challenge. In both models, GRP blockade abrogated AHR and bronchoalveolar lavage (BAL) macrophages and granulocytes, and decreased BAL cytokines implicated in asthma, including those typically derived from Th1 (e.g., IL-2, TNFα), Th2 (e.g., IL-5, IL-13), Th17 (IL-17), macrophages (e.g., MCP-1, IL-1), and neutrophils (KC = IL-8). Dexamethasone generally had smaller effects on all parameters. Macrophages, T cells, and neutrophils express GRP receptor (GRPR). GRP blockade diminished serine phosphorylation of GRPR with ozone or OVA. Thus, GRP mediates AHR and airway inflammation in mice, suggesting that GRP blockade is promising as a broad-spectrum therapeutic approach to treat and/or prevent asthma in humans.

Fig. 1.

GRP blockade abrogates O₃-induced AHR and inflammation. (A and B) PFTs on BALB/C mice exposed to O₃ or FA, using the flexiVent system. Mice given 77427 (A) or 2A11 (B) before O₃ had decreased airway resistance at higher MCh doses (25 or 100 mg/mL). *P < 0.01, **P < 0.05, n = 8. (C and D) Pretreatment with 77427 (C) or 2A11(D) reduced numbers of BAL macrophages and PMN induced by O₃.*P < 0.01, n = 8.

Fig. 2.

GRP blockade abrogates OVA-induced AHR and inflammation. (A and B) Pretreatment with 77427 (A) reduced airway resistance to MCh induced by OVA down to baseline. *P < 0.01, n = 8. (B) Treatment with 2A11 showed a similar pattern of responsiveness that was a statistical trend (0.10> P > 0.05). (C and D) OVA/OVA mice pretreated with 77427 (C) or 2A11 (D) had fewer BAL macrophages, PMN, and eosinophils. *P < 0.01, **P < 0.05, n = 8.

Fig. 3.

Lung histopathology and IHC in mouse lungs exposed OVA with or without GRP blockade. (A) Mice treated with OVA/OVA had inflammatory infiltrates with PMN, mononuclear cells, and eosinophils (short green arrows) throughout the airway epithelium (asterisks) and smooth muscle (long arrows) (H&E). (B) In contrast, OVA-immunized mice given 77427 before the first OVA aerosol challenge had rare inflammatory cells (green arrows) in airway epithelium (asterisks) or smooth muscle (long black arrows), except for perivascular infiltrates and a few cells outside airway smooth muscle (H&E). (C) GRP IHC in control mouse lungs treated with Alum/PBS/Saline. (D) GPR IHC of mouse lungs exposed to OVA/PBS/OVA, showing strong GRP signal in cells lining the alveolar ducts (black arrows). The same distribution of staining was observed for the general NE marker, PGP9.5. (E) In parallel to Fig. 3D, GRP IHC was performed by using anti-GRP antibody preabsorbed with excess GRP peptide. (Scale bars: 25 μm.) L, airway lumen; V, vascular lumen.

Fig. 4.

GRP blockade decreases O₃- or OVA-induced BAL cytokine levels. Quantitative multiplex immunoassays show that GRP blockade by 77427 or 2A11 decreased O₃- or OVA-induced BAL fluid cytokine levels, represented here by a Th1 cytokine, TNFα (A); a Th2 cytokine, IL-5 (B); a TH17 cytokine, IL-17 (C); and PMN-derived cytokine, KC (IL-8) (D). **P < 0.001, n = 5.

Fig. 5.

GRPR gene expression in macrophages, PMN, and T cells. Positive controls for QRT-PCR included normal 5-wk-old BALB/c lung (low positive levels) and small cell carcinoma cell line, H345 (high positive levels) (42).

Fig. 6.

GRP blockade decreases O₃- or OVA-induced GRPR serine phosphorylation. (A) Immunoprecipitation and Western blot for pGRPR in mouse lungs exposed to O₃ with or without 77427. One representative blot out of four total. (B) Similarly, GRPR serine phosphorylation in the OVA model. One representative blot out of four total. Statistical analysis of densitometry for Fig. 6 A and B is in Tables S3 and S4.