Characterization of Quin-C1 for its anti-inflammatory property in a mouse model of bleomycin-induced lung injury

Abstract

Aim: To study the in vivo effects of Quin-C1, a highly specific agonist for formyl peptide receptor 2 (FPR2/ALX), in a mouse model of bleomycin (BLM)-induced lung injury.

Methods: Male ICR mice were injected intratracheally with BLM (d 0), and intraperitoneally with Quin-C1 (0.2 mg/d) or vehicle between d 1 and d 28, during which pulmonary inflammation was monitored. A similar regimen was carried out between d 5 and d 28 to differentiate anti-inflammatory from anti-fibrotic effects. During the treatment, leukocyte numbers in bronchoalveolar lavage fluid (BALF) were counted, and FPR2/ALX transcripts, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), the mouse keratinocyte-derived chemokine (KC), transforming growth factor β1 (TGF-β1) and C-X-C motif chemokine 10 (CXCL10) expression levels in the lung tissue were also measured. Both hydroxyproline content and histological changes were examined on d 28 to assess the severity of lung fibrosis.

Results: BLM caused a significant increase in expression levels of all the selected cytokines and chemokines, as well as a thickening of the alveolar wall. Treatment with Quin-C1 significantly reduced the neutrophil and lymphocyte counts in BALF, diminished expression of TNF-α, IL-1β, KC, and TGF-β1, and decreased collagen deposition in lung tissue. The treatment also lowered the content of lung hydroxyproline. Quin-C1 did not ameliorate lung fibrosis when the treatment was started 5 d after the BLM challenge, suggesting that the protection may be attributed to its anti-inflammatory effects. Exposure to BLM or BLM plus Quin-C1 did not change the level of FPR2/ALX transcripts (mFpr1, mFpr2, and Lxa4r) in the lung tissue.

Conclusion: The results demonstrate an anti-inflammatory role for Quin-C1 in bleomycin-induced lung injury, which may be further explored for therapeutic applications.

Figure 1

Receptor expression and Quin-C1-induced calcium mobilization in mice. Bone marrow-derived neutrophils (5×10⁵/sample) were loaded with the FLIPR Calcium 5 dye and stimulated with the FPR2/ALX agonist WKYMVm (A) or with Quin-C1 (B) at indicated concentrations. Real-time changes in intracellular calcium concentrations were determined using a FlexStation II and are shown in relative fluorescence units (RFU). One set of calcium tracings, representative of 3 repeating experiments, is shown. C and D, representative figures showing Quin-C1 induced calcium mobilization in transfected RBL cells expressing mouse Fpr1 (C) or mouse Fpr2 (D). The experiments were conducted as described in (A) and (B). E, effect of 0.2 mg Quin-C1 on the expression of the transcripts for 3 Fpr family members in mouse lung tissue on d 0, 5, 7, 9, and 28 after bleomycin (BLM) administration. Reverse transcription products were PCR-amplified for 30 cycles for detection of mouse Fpr1, Lxa4r and Fpr2 transcripts, respectively. The PCR products were analyzed on an agarose gel, and β-actin was used as an internal control. Marker: 100 bp DNA ladder.

Figure 2

BLM induced changes in total and differential cell counts in the BALF and effects of Quin-C1. Mice were given intratracheal bleomycin (BLM) or saline injections (d 0), with or without daily Quin-C1 administration (0.2 mg or vehicle ip) starting on d 1. On d 0, 5, 7, and 9, mice from each group were sacrificed and the BALF was collected. Total cell count (A) was determined using a cell counter (Beckman Coulter, Fullerton CA, USA) and cell subset numbers were determined with Diff-quick staining (200 cells per animal were examined). Cell numbers for macrophages (B), lymphocytes (C) and neutrophils (D) in the BALF were calculated based on their respective percentage of the total cell count (A). Values are expressed as mean±SEM (n≥6 per group). ^bP<0.05, ^cP<0.01 compared to saline control; ^eP<0.05, ^fP<0.01 compared to BLM control.

Figure 3

BLM induced expression of cytokines and chemokines in lung tissue and effects of Quin-C1. Bleomycin (BLM)-challenged mice were treated with Quin-C1 (0.2 mg) or vehicle as described above. On d 0, 5, 7, and 9, the mice were sacrificed, and lung tissue homogenates were examined by ELISA for levels of TNF-α (A), IL-1β (B), KC (C), TGF-β1 (D), and CXCL10 (E). Values are expressed as mean±SEM (n≥5 per group). ^bP<0.05, ^cP<0.01 compared to saline control; ^eP<0.05, ^fP<0.01 compared to BLM control.

Figure 4

Effects of Quin-C1 on BLM induced body weight change and hydroxyproline content. The body weights of mice treated with saline, BLM and BLM plus Quin-C1 (0.2 mg) were monitored on d 0 and 28. The lung samples were homogenized and the contents of hydroxyproline measured on d 28 as described in the methods. Values are expressed as mean±SEM (n≥10 per group). ^bP<0.05, ^cP<0.01 compared to saline control; ^fP<0.01 compared to BLM control.

Figure 5

Histological analysis of BLM treated mouse lung tissue and effects of Quin-C1. Five-micrometer sections of lung tissue from mice receiving saline (A), BLM (B) and BLM plus Quin-C1 (0.2 mg) (C) were stained with Masson's trichrome for histological examination under a light microscope. (D) Alveolar wall thickness was determined using Image-Pro Plus 6.0 software and expressed as mean±SEM (n=3 mice from each experimental group). Images were taken at 400× except in insert (B), which was taken at 100× to show collagen deposition in a larger area of lung tissue. Bar=50 μm; ^cP<0.01 compared to saline control; ^fP<0.01 compared to BLM control.

Figure 6

Effects of different doses of Quin-C1 on inflammatory cell counts in the BALF, expression of cytokines and chemokines in lung tissue and body weight change in BLM treated mice. Mice were challenged with BLM or saline on d 0, with or without daily Quin-C1 administration (0.04, 0.2, or 1 mg ip) starting on d 1. On d 9, mice were sacrificed to examine the total and differential cell counts in the BALF (A), TNF-α, IL-1β, KC, TGF-β1, and CXCL10 levels in lung homogenates (B) and body weight changes (C). Values are expressed as mean±SEM (n≥8 per group). ^bP<0.05, ^cP<0.01 compared to saline control; ^eP<0.05, ^fP<0.01 compared to BLM control.