CXCR1/CXCR2 antagonist CXCL8(3-74)K11R/G31P blocks lung inflammation in swine barn dust-instilled mice

Abstract

Inhalation of agricultural occupational dusts from swine confinement facilities can result in lung inflammation. The innate immune response to organic barn dusts results in production of a number of pro-inflammatory factors in the lungs of barn workers such as cytokines, chemokines, and an influx of neutrophils. Many of these inflammatory factors are influenced by the chemokine CXCL8/IL-8 (KC or MIP-2 in mice). Previously, we have demonstrated that an endotoxin-independent component of swine barn dust extract (SBE) elevates lung chemokines in a protein kinase C (PKC)-dependent manner resulting in the significant formation of lung inflammatory cell infiltrates in a mouse model of SBE injury. In this study we test the ability of a CXCR1/CXCR2 antagonist, CXCL8(3-74)K11R/G31P (G31P) to block many of the features of lung-inflammation in response to challenge with SBE in an established mouse exposure system. Injection of G31P concurrent with SBE nasal instillation over a course of 3 weeks significantly reduced neutrophil accumulation in the lungs of barn dust exposed animals compared to those given SBE alone. There was a similar reduction in pro-inflammatory cytokines and chemokines IL-6, KC, and MIP-2 in SBE plus G31P-treated mice. In addition to excreted products, the receptors ICAM-1, CXCR1, and CXCR2, which all were elevated with SBE exposure, were also decreased with G31P treatment. SBE activation of PKCα and PKCε was reduced as well with G31P treatment. Thus, G31P was found to be highly effective at reducing several features of lung inflammation in mice exposed to barn dust extracts.

Keywords: Chemokines; Cytokines; G31P; IL-8; Occupational dusts.

Figure 1

Mean total of lung lavage fluid cells (A) and percent composition of macrophage (mac) and neutrophils (PMN) in lavage cells (B) after repeated nasal instillation with saline, SBE, G31P, or SBE+G31P. Error bars are SE (n=8 mice/group). ***P < 0.001, ****P < 0.0001

Figure 2

Mean lung lavage fluid cytokine expression of KC (A), MIP-2 (B), and IL-6 (C). Error bars are SE (n=8 mice/group). **P < 0.01, ***P < 0.001

Figure 3

PKC activity. Lung trachea (A and B) and lung slices (C and D) were measured for PKCa and PKCε activity from saline, SBE, G31P and SBE+G31P treated mice. **P < 0.01

Figure 4

Hematoxylin and eosin staining of mouse lung from mice treated with saline (A), G31P (B), SBE (C), and SBE+G31P (D). No change was apparent in G31P treatment compared to Saline. SDE induced. SBE induced increased cellularity and peribronchial foci of mononuclear cells (arrow), both of which were reduced with G31P co-administration.

Figure 5

ICAM-1 staining of mouse lung from mice treated with saline (A), G31P (B), SBE (C), and SBE+G31P (D). ICAM-1 (brown stain) was expressed on bronchial epithelium of SDE treated mouse lung, but eliminated with co-administration of G31P.

Figure 6

Mouse lung staining for CXCR1 (A,C,E,G) and CXCR2 (B,D,F,H). Both receptors were clearly expressed in alveolar epithelium, bronchial epithelium, and alveolar macrophages. Levels of both receptors in Saline (A,B) appeared reduced in G31P treated animals (C,D), particularly in the bronchial epithelium (inset images). SDE treated animals (E,F) showed increased expression of both receptors, and G31P treatment (G,H) was able to reduce this, particularly in bronchial epithelium (inset images).