Abstract
Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-β1 levels, but not on IFN-γ and total TGF-β1, and enhanced granulocyte macrophage–colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-β1 and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8–induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.
Bleomycins are a family of glycopeptides antibiotics (4) with potent anti-tumor activity against a range of lymphomas, head and neck cancers, and germ-cell tumors (5). The therapeutic efficacy of bleomycin is limited by development of lung fibrosis (6, 7). Bleomycin instillation in the lung is the most frequently used experimental model to investigate cellular and biochemical mechanisms relevant for the pathogenesis of pulmonary injury and fibrosis. Bleomycin is usually administered in a high dose by single intratracheal instillation, and induces morphologic and biochemical changes in animal lungs that resemble those observed in human IPF (8). The pulmonary response to bleomycin injury is complex, requiring the coordinated expression of different cytokines and chemokines, as well as proliferation and migration of multiple cell types, including leukocytes, epithelial cells, endothelial cells, and fibroblasts (9).
Chemokines are small chemoattractant molecules that act on specific seven-transmembrane chemokine receptors and induce the activation and recruitment of leukocytes (10, 11). Chemokines may also modulate angiogenesis, wound healing, and tumorogenesis via interaction with leukocytes, endothelial cells, and fibroblasts (12), and are grouped into four categories—CXC, CC, C, and CX3C—based upon the presence of conserved cysteine residues within the mature peptides (10). It has been suggested that activation of the chemokine receptor CXCR2 by the chemokines CXCL1/KC (keratinocyte-derived chemokine) and CXCL2/MIP-2 (macrophage inflammatory protein-2) may play a relevant role in angiogenesis (13, 14), neutrophil recruitment into sites of inflammation (15–17), and hypernociception (18). Although neutrophils play an essential role in host defense against several pathogens (19), neutrophilic inflammation and angiogenesis process may also contribute to the pathogenesis of a range of chronic syndromes, including pulmonary fibrosis (20–22). The present study was performed to test the hypothesis that CXCR2-mediated neutrophil recruitment plays an important role in the development of pulmonary fibrosis. To this end, we evaluated the effect of a long-acting orally bioavailable antagonist of the receptor CXCR2, DF2162 (17, 18), in a model of pulmonary fibrosis induced by bleomycin in mice.
Eight- to 10-week-old male C57Bl/6J (WT) mice obtained from Centro de Bioterismo (CEBIO) of the Universidade Federal de Minas Gerais (UFMG, Brazil) and maintained in the animal facilities of Laboratório de Imunofarmacologia, Department of Biochemistry and Immunology (UFMG, Brazil), with filtered water, food ad libitum, and in a controlled environment (temperature and humidity) were used in the present study. All experiments were conducted under conditions approved by the local animal ethics committee, CETEA/UFMG (Protocol number 146/06). Bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY); ketamine (Dopalen; Vetbrands, São Paulo, Brazil); xylazine (Calmiun; Agener União Saúde Animal S.A., São Paulo, Brazil); carboxymethylcellulose (Sigma Chemical Co., St. Louis, MO); PBS (phosphate buffered saline, pH 7.4); recombinant human CXCL-8/IL-8 (R&D Systems, Minneapolis, MN); endothelial cell basal medium-2 (EBM-2; Cambrex BioScience, Walkersville, MD); Growth factor-reduced Matrigel matrix (BD Bioscience, Bedford, MA). DF2162 was synthesized at Dompé Research and Development (Dompé S.p.A., L'Aquila, Italy).
Animals were anesthetized intraperitoneally with 80 μl of a ketamine and xylazine solution (3.2 mg/kg and 0.16 mg/kg, respectively) before tracheotomy. A single 25-μl injection containing 0.125 U of bleomycin (Blenoxane; Bristol-Meyers) diluted in PBS, or PBS only (control group), was instilled intratracheally using Hamilton syringes. In each time-point after bleomycin, or PBS administration, animals were killed by a lethal dose of ketamine and xylazine, to perform bronchoalveolar lavage and collect lung samples for biochemical and histologic analysis.
Bleomycin-instilled mice received oral twice-daily treatment with vehicle or DF2162 (17, 18), a CXCR2-selective functional blocker derivative of reparexin (15, 16). DF2162 was given by oral gavage in 100 μl of 0.25% carboxymethylcellulose diluted in PBS. The vehicle group received 100 μl of 0.25% carboxymethylcellulose only. First of all, we performed a dose–response study with DF2162 (1.2, 6, and 30 mg/kg/dose) from Day 0 (the day of bleomycin instillation) to evaluate lung inflammation 2 days after. At a later moment, DF2162 was given at 6 mg/kg/dose from Days 0 to 16, and lung inflammation and fibrosis were evaluated at Days 2, 8, and 16 after bleomycin challenge. Finally, we evaluated the effects of late administration of 6 mg/kg/dose DF2162 from Days 8 to 16 on lung inflammation and fibrosis; evaluation was on Day 16 after bleomycin challenge.
Bronchoalveolar lavage (BAL) was performed to obtain leukocytes from alveolar spaces. The trachea was exposed and a 1.7-mm-outside-diameter polyethylene catheter was inserted. BAL was performed by washing the lungs three times with three different 1-ml aliquots of PBS. BAL samples (∼ 2.7–3.0 ml each) were centrifuged at 600 × g for 10 minutes at 4°C. The supernatant was used for enzyme-linked immunosorbent assay (ELISA) and the cell pellet used to evaluate the number of infiltrating leukocytes. The total number of leukocytes was determined by counting leukocytes in a modified Neubauer chamber. Differential counts were obtained from cytospin (Shandon III) preparations by evaluating the percentage of each leukocyte on a slide stained with May-Grünwald-Giemsa.
The extent of neutrophil accumulation in lung tissue was measured by assaying myeloperoxidase activity, as previously described (23). Using the conditions described below, this methodology is selective for the determination of neutrophils over macrophages (data not shown). Before lung removal, the pulmonary vasculature was perfused with 3 ml of PBS through the right ventricle, and the organ was removed and frozen at −70°C. Upon thawing, the tissue (0.1 g of tissue per 1.9 ml of buffer) was homogenized in a pH 4.7 buffer (0.1 M NaCl, 0.02 M Na2PO4, 0.015 M Na2EDTA), centrifuged at 3,000 × g for 10 minutes, and the pellet subjected to hypotonic lyses (1.5 ml of 0.2% NaCl solution followed by an addition 30 seconds later of an equal volume of a solution containing NaCl 1.6% and glucose 5%). After a further centrifugation, the pellet was resuspended in 0.05 M Na2PO4 buffer (pH 5.4) containing 0.5% hexadecyl-trimethylammonium bromide (HTAB) and re-homogenized. One-milliliter aliquots of the suspension were transferred into 1.5-ml microtubes followed by three freeze-thaw cycles using liquid nitrogen. The aliquots were then centrifuged for 15 minutes at 3,000 × g to perform the assay.
The assay employed 25 μl of 3,3′-5,5′-tetramethylbenzidine (TMB; Sigma), dissolved in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) at a final concentration of 1.6 mM, 100 μl of H2O2, dissolved in phosphate buffer (pH 5.4) containing HTAB in a final concentration of 0.002% vol/vol and 25 μl of supernatant from tissue sample processed. The reaction was started at 37°C for 5 minutes in a 96-well microplate by adding the supernatant and the TMB solution. After that, H2O2 was added and followed by a new incubation at 37°C for 5 minutes. The reaction was stopped by adding 100 μl of 1 M H2SO4 and quantified at 450 nm in a spectrophotometer (Emax; Molecular Devices, Sunnyvale, CA). Neutrophil number in each sample was calculated from a standard curve of neutrophils obtained from the peritoneal cavity of 5% casein-treated animals and processed in the same way. The results were expressed as relative number of neutrophils per mg of wet tissue.
Fragments (100 mg) of lungs were removed for hydroxyproline determination, as an indirect measurement of collagen content (24). Briefly, tissues were homogenized in 0.2% saline, frozen, and lyophilized. The assay was performed with 20 mg of the lyophilized material subjected to alkaline hydrolysis in 300 μl of H2O plus 75 μl of 10 M NaOH at 120°C for 20 minutes. An aliquot of 50 μl of the hydrolyzed tissue was added to 450 μl of Chloramine T oxidizing reagent (0.056 M Chloramine T and n-propanol 10% in acetate-citrate buffer [pH 6.5]) and allowed to react for 20 minutes. A hydroxyproline standard curve was prepared likewise. Color was developed by the addition of 500 μl of 1 M p-dimethylaminebenzaldehyde diluted in n-propanol–perchloric acid (2:1 [vol/vol]). The absorbance was quantified at 550 nm in a spectrophotometer (Emax; Molecular Devices).
The concentration of the cytokines IFN-γ, IL-10, IL-13, GM-CSF, and TGF-β1 (active and total forms), and chemokines CXCL1/KC and CCL5/RANTES was measured in lungs; and CXCL1/KC, CXCL2/MIP-2, CXCL9/MIG, and CXCL10/IP-10 was measured in BAL by ELISA. The assays were performed using Kits from R&D Systems (DuoSet kits) and according to the manufacturer's instructions.
One hundred milligrams of lung tissue from both controls and treated animals were homogenized in 1 ml of PBS (0.4 M NaCl and 10 mM de NaPO4) containing anti-proteases (0.1 mM phenylmethilsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KI aprotinin A) and 0.05% Tween 20. The samples were then centrifuged for 10 minutes at 3,000 × g and the supernatant immediately used for ELISA. All samples were assayed in duplicate.
The left lung was removed and fixed in 10% neutral phosphate-buffered formalin (pH 7.4). The tissues were dehydrated gradually in ethanol, embedded in paraffin, cut into 4-μm sections, stained with hematoxilin and eosin (H&E) or Gomori's Trichrome, and examined under light microscopy. Slides with lung sections stained with H&E were analyzed by a pathologist blinded to the experiment. For the quantitative analysis of Gomori's Trichrome area, images covering 326,000 μm2 of lung sections were captured with a digital camera (Optronics DEI-470; Goleta, CA) connected to a microscope (Olympus IX70; Hamburg, Germany) with a magnification of ×200, and collagen (green areas stained by Gomori's trichrome) was measured with the software Image Pro-Plus (Media Cybernetics, Silver Spring, MD). At least 20 images were obtained for each animal, with n = 6 per group. Results were expressed as the mean green area per μm2. This morphometrical index was used to describe lung collagen deposition.
Lungs of vehicle-treated or DF2162-treated mice were removed at Day 16 after intratracheal bleomycin instillation for analysis of von Willebrand Factor (vWF) mRNA expression. Total RNA was isolated from lungs by using an Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). The RNA obtained was suspended in diethyl pyrocarbonate–treated water and stocked at −70°C until use. Real-time RT-PCR was performed on an ABI PRISM 7900 sequence-detection system (Applied Biosystems, Warrington, UK) by using SYBR Green PCR Master Mix (Applied Biosystems) after a reverse transcription reaction of 1 μg of RNA by using M-MLV reverse transcriptase (Promega, Madison, WI). The relative level of gene expression was determined by the comparative threshold cycle method as described by the manufacturer, whereby data for each sample were normalized to hypoxanthine phosphoribosyltransferase (HPRT) and expressed as a fold change compared with PBS-instilled control mice. The following primer pairs were used: HPRT, 5′-GTTGGTTACAGGCCAGACTTTGTTG-3′ (forward) and 5′-GAGGGTAGGCTGGCCTATAGGCT-3′ (reverse); and vWF, 5′-GCGCTATCATGTATGAAGTCAGGTT-3′ (forward) and 5′-CACAGATTCCGCAAAGACCAT-3′ (reverse).
Primary endothelial cells were obtained from human umbilical veins (HUVEC) as described previously (25). HUVECs were seeded on 0.2% wt/vol gelatin-coated 25-cm2 tissue culture flasks and cultured in endothelial cell basal medium-2 (EBM-2) containing 20% fetal calf serum (FCS), 50 mg/ml endothelial cell growth supplement (ECGS) (Collaborative Research, Inc, Bedford, MA), and 100 mg/ml heparin (Sigma) until they reached confluence. Cells were passaged up to two times before use and then placed in suspension by trypsinization.
The proliferation assay was performed as described previously (26). Briefly, 96-well culture plates were with 0.2% wt/vol gelatin. Approximately 5 × 103 HUVECs were plated in EBM-2 supplemented with 20% FCS, 50 μg/ml ECGS, and 100 μg/ml heparin in gelatin-precoated 96-well, and allowed to adhere for 6 hours. The medium was then removed and the cells were washed twice with serum-free medium. Serum-free EBM-2 containing 1% Nutridoma (Roche diagnostics GmbH, Mannheim, Germany), ECGS, heparin, and 0.037 MBq/ml (1 μCi/ml) methyl 3(3H)-thymidine (GE Healthcare GmbH, Milan, Italy) in the presence or absence of human recombinant IL-8 (10 ng/ml) and/or DF2162 (as described bellow) was then added to the wells and left incubating for 24 h at 37°C in the presence of 5% CO2. After incubation time, nonadherent cells were removed by washing twice with ice-cold PBS. Then, 50 μl 10% ice-cold trichloroacetic acid (TCA) was added to each well and the precipitates were collected on a filter using a filtration manifold. Filters were extensively washed with distilled water and then suspended in scintillation fluid. Counts per minute (CPM) were quantified using a scintillator counter. For the blockade approach, DF2162 was diluted in EBM-2 medium containing 0.01% of dimethyl sulfoxide (DMSO; Sigma) to three different final concentrations: 0.01, 0.1, and 1 μM. The cells were pretreated for 1 hour with either vehicle (EBM-2+DMSO) or DF2162 before the addition of human IL-8.
For the scratch assay, 5 × 105 HUVECs were plated in gelatin-precoated 24-well culture plate and incubated in EBM-2 medium supplemented with 20% FCS, 50 μg/ml ECGS, and 100 μg/ml heparin for 24 hours at 37°C in presence of CO2 5%. The confluent cells were starved for 1 hour in EBM-2 medium with 5% FCS before starting the experiments. Confluent cell monolayer was then scraped with a yellow pipette tip to generate scratch wounds and rinsed twice with serum-free EBM-2 to remove debris. EBM-2 medium with 5% FCS was added to the cells, which had been pretreated for 1 hour with vehicle (0.01% DMSO in EBM-2) or DF2162 (0.1 μM) before the addition of human IL-8 (10 ng/ml). The cells were then incubated at 37°C in presence of 5% CO2. Scratches were photographed immediately and 12 hours after the scraping. Images were obtained using a Nikon digital camera coupled to a ×10 objective on a Zeiss Axiovert microscope (Zeiss, Thornwood, NY) and the wound area was measured using the software NIH ImageJ.
An in vitro capillary-like structure formation assay was performed as described earlier (27). Briefly, forty-eight-well culture plates (Nunc, Nalge Europe; Neerijse, Belgium) were pre-coated at 4°C with 150 μl of Growth factor–reduced Matrigel matrix, which was then allowed to solidify at 37°C for 30 minutes. HUVECs, 5 × 104 per well, were suspended in EBM-2 medium supplemented with 20% FCS, 50 μg/ml ECGS, and 100 μg/ml heparin and seeded on the Matrigel-coated plates. The cells had been pretreated for 1 hour with either vehicle (0.01% DMSO in EBM-2) or DF2162 (0.1 μM) before the addition of human IL-8 (10 ng/ml). The plates were incubated for 24 hours at 37°C in presence of 5% CO2, and the wells were then photographed using a Nikon digital camera coupled to a ×10 objective on a Zeiss Axiovert microscope to determine qualitative influence of DF2162 on the ability of the HUVECs to form capillary-like structures.
All results are presented as the mean ± SEM. Normalized data were analyzed by one-way ANOVA and differences between groups were assessed using the Student-Newman-Keuls post hoc test, using Software GraphPad Prism 3.0. Differences were considered significant at P < 0.05.
BAL and lung tissue were obtained at various time points after bleomycin or saline instillation. The chemokine CXCL1/KC peak was already detectable in lungs of bleomycin-instilled animals at 6 hours, remained elevated till 24 hours, and dropped slightly by 2 days after bleomycin challenge. There was a secondary peak of the chemokine at Day 4, which dropped again at Day 8 after challenge. Thereafter, the levels of CXCL1/KC returned to baseline (Figure 1A). At 12 hours, neutrophils started to accumulate progressively in the lungs, as quantified by MPO, with a peak at Day 8 (Figure 1B). An increase in lung macrophages number was observed at Day 24 (data not shown). In BAL, the total leukocytes count showed a biphasic fashion, with a first peak at Day 2 and a second one at Day 8 (Figure 1C). Neutrophils were detected in BAL 12 hours after bleomycin instillation. Neutrophil influx increased then peaked on Day 2, dropped slightly by Day 4, and reached a greater peak at Day 8, thus having biphasic recruitment similar to that observed with total cells (Figure 1D). The deposition of collagen in lung interstitium, as determined by hydroxyproline quantification, was first noticed at Day 8 after bleomycin instillation and increased steadily thereafter (Figure 1E). Therefore, the progressive collagen deposition in lungs is preceded by an increase in neutrophil influx associated with high levels of CXCL1/KC production. Our next question was whether blockade of CXCR2, the receptor for CXCL1/KC, could reduce lung neutrophil inflammation and subsequent fibrosis.
Figure 1. Time course of bleomycin-induced lung inflammation and fibrosis. The following parameters were evaluated: (A) CXCL1/KC production in lungs; (B) neutrophil influx into the lungs, as assessed by myeloperoxidase (MPO) assay; transmigration of (C) total leukocytes and (D) neutrophils into bronchoalveolar lavage (BAL) fluid; and (E) lung collagen deposition, as assessed by hydroxyproline measurement. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (control, open bars) and evaluated at the given times after challenge. Results are representative of two experiments and are shown as the mean ± SEM of seven to eight animals in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, when compared with PBS.
[More] [Minimize]To obtain an optimal dose of DF2162 for the chronic experiments, mice were treated orally with vehicle or DF2162 (1.2, 6, and 30 mg/kg/dose, twice daily). Neutrophil accumulation and lung CXCL1/KC production were evaluated 2 days after bleomycin challenge. Neutrophil recruitment into airway space was inhibited in a dose-dependent manner, with maximal inhibition observed with 6 mg/kg/dose (Figure 2A). The compound did not affect the pulmonary levels of CXCL1/KC (Figure 2B). Despite its ability to prevent neutrophil influx into BAL, DF2162 had no effect on neutrophil accumulation in lung tissue, as assayed by MPO (Figure 2C). The dose of 6 mg/kg/dose, twice daily, was then used in all further studies.
Figure 2. Dose-dependent effects of treatment with DF2162 in a model of bleomycin-induced lung injury. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (control, open bars) and evaluated 2 days after challenge. DF2162 was given orally (twice daily) at the doses of 1.2, 6, or 30 mg/kg (cross-hatched bars). The following parameters were evaluated: (A) Number of neutrophils in BAL fluid; (B) CXCL1/KC levels in BAL; and (C) neutrophil influx into the lung, as assessed by MPO assay. The dotted lines across the graphs represent values in naïve animals. Results are representative of two experiments and are shown as the mean ± SEM of seven to eight animals in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, when compared with vehicle-given bleomycin-challenged mice.
[More] [Minimize]Treatment with DF2162 (6 mg/kg/dose, twice daily, from Day 0) led to a significant inhibition of the accumulation of total leukocytes in alveolar fluid at Days 2 and 8 after bleomycin instillation (Figure 3A). This appeared to be resultant of a significant reduction in the recruitment of neutrophils at Days 2 and 8 (Figure 3B). There were few neutrophils in BAL fluid at Day 16 after bleomycin instillation, and the compound failed to affect the total number of cells at that time point (Figures 3A and 3B). DF2162 was not able to prevent neutrophil accumulation in lung tissues at Days 2 and 8 after bleomycin instillation. Actually, neutrophil accumulation in lung tissue was greater in DF2162 than in vehicle-treated animals at Day 16 (Figure 3C).
Figure 3. Effects of DF2162 treatment on lung inflammation induced by bleomycin. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (Control, open bars) and evaluated 2, 8, and 16 days after challenge. DF2162 was given orally (twice daily) at the dose 6 mg/kg (cross-hatched bars) from Day 0 to Day 16. The following parameters were evaluated: (A) total number of leukocytes and (B) neutrophils in BAL fluid; (C) neutrophil influx into the lung, as assessed by MPO assay. The dotted lines across the graphs represent values in naïve animals. Results are representative of two experiments and are shown as the mean ± SEM of seven to eight animals in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, when compared with vehicle-given bleomycin-challenged mice. Representative sections of hematoxylin and eosin–stained lungs of (D) control and (E–H) bleomycin-challenged animals are also shown. At Day 2 (bars = 50 μm) note the bleomycin-induced lung infiltrate (E) that was diminished in DF2162-treated animals (F). At Day 16 (bars = 25 μm), note the presence of mononuclear cell infiltrate (G, arrowhead) after bleomycin challenge. After treatment with DF2162 there was persistence of neutrophils in lung parenchyma (H, arrows).
[More] [Minimize]Histopathologic analysis by H&E, 2 days after bleomycin challenge, showed that there was an increase in thickness of the alveolar wall that was associated with interstitial edema, hyperemia, and characteristic leukocyte exudation (Figure 3E). In DF2162-treated mice, there was a reduction of alveolar wall edema, hyperemia, and leukocyte influx into the alveolar spaces at Day 2 after bleomycin (Figure 3F). At Day 16, lungs of vehicle-treated mice showed intense interstitial infiltration of lymphocytes and mononuclear cells (Figure 3G, arrowhead). In DF2162-treated mice, there were fewer lymphocytes, mononuclear cells, and overall number of cells, but an increase in the number of neutrophils in lung parenchyma (Figure 3H, arrows), concurring with MPO data (Figure 3C).
Bleomycin administration caused a progressive increase in collagen deposition at Days 8 and 16 after challenge in the vehicle- but not in the DF2162-treated mice, as assessed by measurement of hydroxyproline content (Figure 4A). Likewise, DF2162 treatment significantly diminished collagen deposition as assessed by morphometric analysis in Gomori's Trichrome–stained sections (Figure 4B). The pattern of collagen deposition between vehicle- and DF2162-treated mice was different; at Day 8, vehicle-treated mice exhibited diffuse interstitial pneumonitis, increased alveolar wall thickness, and mild fibrosis (Figure 4D), whereas DF2162-treated animals presented focal interstitial pneumonia and reduced septal thickness and fibrosis (Figure 4E). At Day 16, vehicle-treated mice presented diffuse and dense pulmonary fibrosis with intense remodeling and loss of normal pulmonary architecture (Figure 4F). These changes were much less intense in DF2162-treated mice, which developed focal lung fibrosis with preserved areas of parenchyma (Figure 4G).
Figure 4. Effects of DF2162 treatment on lung fibrosis induced by bleomycin. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (Control, open bars) and evaluated 8 and 16 days after challenge. DF2162 was given orally (twice daily) at the dose 6 mg/kg (cross-hatched bars) from Day 0 to Day 16. The following parameters were evaluated: lung collagen deposition, as assessed by (A) hydroxyproline measurement and (B) morphometric analysis of Gomori's trichrome–stained areas. The dotted lines across the graphs represent values in naïve animals. Results are representative of two experiments and are shown as the mean ± SEM of seven to eight animals in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, when compared with vehicle-given bleomycin-challenged mice. Representative sections of Gomori's trichrome–stained lungs of (C) control and (D–G) bleomycin-challenged animals are also shown. Note the large areas of collagen deposition (shown in green) at Days 8 (D) and 16 (F) after bleomycin challenge. DF2162 treatment decreased the amount and modified the pattern of collagen deposition at Days 8 (E) and 16 (G) after challenge (bars = 100 μm).
[More] [Minimize]Next, the effects of DF2162 on the levels of chemokines and cytokines induced by bleomycin were determined in lung tissue and BAL. The CXCR2 antagonist had no effect on levels of the neutrophil-active chemokine CXCL1/KC in lungs (Figure 5A), or CXCL1/KC and CXCL2/MIP-2 in BAL (Table 1). Levels of CCL5/RANTES in lungs of DF2162-treated mice were significantly lower than those of vehicle-treated animals (Figure 5B). In contrast, the concentration of CXCL9/MIG in BAL fluid was greater in DF2162 than vehicle-treated mice at Day 2 after instillation of bleomycin (Table 1).
Figure 5. Effects of DF2162 treatment on the cytokines and chemokines production in lung induced by bleomycin. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (Control, open bars) and evaluated at various times after challenge. DF2162 was given orally (twice daily) at the dose 6 mg/kg (cross-hatched bars) from Day 0 to Day 16. The following soluble mediators were evaluated by enzyme-linked immunosorbent assay in lung tissue: (A) CXCL1/KC, (B) CCL5/RANTES, (C) IFN-γ, (D) IL-10, (E) granulocyte macrophage–colony-stimulating factor (GM-CSF), (F) IL-13, (G) total transforming growth factor (TGF)-β1, and (H) active TGF-β1. The dotted lines across the graphs represent values in naïve animals. Results are representative of two experiments and are shown as the mean ± SEM of seven to eight animals in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, when compared with vehicle-given bleomycin-challenged mice.
[More] [Minimize]Bronchoalveolar Lavage Fluid | ||||||
|---|---|---|---|---|---|---|
| Groups | Chemokine | Day 2 | Day 8 | |||
| Control | CXCL1/KC | 22.3 ± 6.30 | ||||
| Bleo+vehicle | 89.8 ± 22.29* | 34.2 ± 7.20 | ||||
| Bleo+DF2162 | 75.8 ± 6.31* | 25.4 ± 3.36 | ||||
| Control | CXCL2/MIP-2 | 39.0 ± 4.33 | ||||
| Bleo+vehicle | 83.3 ± 4.18* | 38.9 ± 3.58 | ||||
| Bleo+DF2162 | 73.8 ± 6.33* | 38.6 ± 3.24 | ||||
| Control | CXCL9/MIG | 136.7 ± 25.63 | ||||
| Bleo+vehicle | 355.9 ± 57.19* | 399.8 ± 105.4 | ||||
| Bleo+DF2162 | 570.8 ± 33.26*† | 373.7 ± 18.09 | ||||
Levels of the proinflammatory cytokine IFN-γ were significantly different from background in vehicle- or DF2162-treated mice (Figure 5C). There was a significant increase in IL-10 production in lung tissue 16 days after bleomycin challenge, which was blocked by DF2162 treatment (Figure 5D). In contrast, DF2162 administration caused an increase in lung levels of GM-CSF at Day 16 after bleomycin challenge when compared with vehicle-treated mice (Figure 5E). No differences on VEGF levels were observed between DF2162-treated versus vehicle-treated mice (data not shown).
Lung levels of the profibrogenic cytokines IL-13 and total and active TGF-β1 were also evaluated. DF2162 administration caused a reduction in lung levels of IL-13, when compared with vehicle-treated mice, at Day 16 after bleomycin challenge (Figure 5F). Levels of total and active TGF-β1 were increased after bleomycin instillation. Although DF2162 treatment did not affect the total levels of TGF-β1, the production of active form of TGF-β1 was drastically reduced at Day 16 when compared with vehicle-treated mice (Figures 5G and 5H).
To determine whether DF2162 treatment have any therapeutic benefit, we investigated the effects of delayed DF2162 administration (6 mg/kg/dose, twice daily, from Days 8 to 16 after bleomycin instillation). Experiments were started on Day 8 because this is after the initial acute inflammatory phase. No significant changes were observed either in total leukocytes in BAL or neutrophil migration into alveolar space (Figures 6A and 6B, respectively) as well as interstitium at Day 16 after bleomycin instillation (Figure 6C). Lung levels of the proinflammatory GM-CSF (Figure 6D) and profibrogenic cytokines, active TGF-β1 (Figure 6F) and IL-13 (Figure 6G), were significantly reduced after late DF2162 administration when compared with vehicle-treated mice at Day 16 after bleomycin challenge. No differences on total TGF-β1 levels were observed between DF2162-treated versus vehicle-treated mice (Figure 6E).
Figure 6. Effects of the delayed administration of DF2162 on inflammation and fibrosis induced by bleomycin. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (Control, open bars) and evaluated at Day 16 after challenge. DF2162 was given orally (twice daily) at the dose 6 mg/kg (cross-hatched bars) from Day 8 to Day 16. The following parameters were evaluated: (A) number of total leukocytes and (B) neutrophils in BAL fluid; (C) neutrophil influx into the lung, as assessed by MPO assay; (D) GM-CSF, (E) total TGF-β1, (F) active TGF-β1, and (G) IL-13 levels in lungs; and lung collagen deposition, as assessed by (H) hydroxyproline measurement and (I) morphometric analysis of Gomori's trichrome–stained areas. The dotted lines across the graphs represent values in naïve animals. Results are representative of two experiments and are shown as the mean ± SEM of seven to eight animals in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, when compared with vehicle-given bleomycin-challenged mice. Representative sections of Gomori's trichrome–stained lungs of bleomycin-challenged (J and K) animals are also shown. Note the large areas of collagen deposition (shown in green) at Day 16 (J) after bleomycin challenge. DF2162 treatment decreased the amount and modified the pattern of collagen deposition at Day 16 (K) after challenge (bars = 100 μm).
[More] [Minimize]Lung fibrosis was assessed by hydroxyproline deposition and morphometry of Gomori's Trichrome–stained lung sections. The delayed treatment with DF2162 significantly diminished lung collagen deposition at Day 16 when compared with vehicle-treated mice (Figures 6H and 6I). In Gomori's Trichrome–stained section, diffuse and dense pulmonary fibrosis was observed in vehicle-treated mice (Figure 6J). In DF2162-treated animals, there was focal lung fibrosis with preserved areas of lung parenchyma and reduced septal thickness (Figure 6K).
BAL fluid obtained from DF2162- or vehicle-treated mice were used to measure the levels of pro-angiogenic and angiostatic chemokines in airway spaces. We found same levels of the pro-angiogenic chemokines CXCL1/KC and CXCL2/MIP-2 in vehicle-treated and DF2162-treated mice, whereas there was a significant increase in the levels of the angiostatic chemokines CXCL9/MIG and CXCL10/IP-10 in samples of DF2162-treated when compared with vehicle-treated mice (Table 2).
Bronchoalveolar Lavage Fluid | ||||
|---|---|---|---|---|
| Groups | Chemokine | Day 16 | ||
| Control | CXCL1/KC | 35.35 ± 2.23 | ||
| Bleo+vehicle | 80.51 ± 24.27* | |||
| Bleo+DF2162 (0–16 d) | 91.14 ± 15.89* | |||
| Bleo+DF2162 (8–16 d) | 95.57 ± 23.28* | |||
| Control | CXCL2/MIP-2 | 39.00 ± 2.72 | ||
| Bleo+vehicle | 71.49 ± 16.22* | |||
| Bleo+DF2162 (0–16 d) | 85.06 ± 10.10* | |||
| Bleo+DF2162 (8–16 d) | 84.29 ± 18.04* | |||
| Control | CXCL9/MIG | 121.60 ± 11.01 | ||
| Bleo+vehicle | 110. 60 ± 7.22 | |||
| Bleo+DF2162 (0–16 d) | 183.50 ± 31.81*† | |||
| Bleo+DF2162 (8–16 d) | 205.80 ± 34.89*† | |||
| Control | CXCL10/IP-10 | 50.38 ± 2.25 | ||
| Bleo+vehicle | 69.19 ± 5.30* | |||
| Bleo+DF2162 (0–16 d) | 107.90 ± 10.95* | |||
| Bleo+DF2162 (8–16 d) | 115.90 ± 21.38*† | |||
Next, to investigate a possible effect of DF2162 treatment in lung angiogenesis, we examine the expression of the endothelial-specific marker vWF (25). Bleomycin administration enhanced vWF gene expression at Day 16, as demonstrated by quantitative PCR. DF2162 administration significantly reduced the expression of vWF mRNA in lungs (Figure 7A).
Figure 7. Effects of DF2162 on a maker of angiogenesis in vivo and on endothelial cells in vitro. Animals were given a single intratracheal challenge of bleomycin (0.125 U) (solid bars) or PBS (Control) and evaluated at Day 16 after challenge. DF2162 was given orally (twice daily) at the dose 6 mg/kg (cross-hatched bar) from Day 0 to Day 16 (0–16), and lung expression of von Willebrand Factor was measured by quantitative PCR after bleomycin challenge (A); results are expressed as fold increase over control mice challenged with PBS. Results are representative of one experiment and are shown as the mean ± SEM of five animals in each group. (B) In vitro human umbilical vein endothelial cells (HUVEC) proliferation induced by IL-8 (10 ng/ml), in the absence or presence of DF2162 (0.01, 0.1, and 1 μM), measured by 3H-thymidine incorporation after 24 hours. (C) In vitro HUVEC scratch assay induced by IL-8 (10 ng/ml) in the absence or presence of DF2162 (0.1 μM) revealed by wound closure area after 12 hours. Results are expressed as % closure area in relation to t = 0, under an optical microscope with ×10 objective. Results are representative of two experiments and are shown as the mean ± SEM of four wells in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively. Effect of DF2162 on HUVEC capillary-like organization assay induced by IL-8 (10 ng/ml). HUVEC were plated on a Matrigel-coated 24-well plate with (D) medium alone, (E) medium containing DMSO, (F) IL-8 alone, or (G) IL-8 plus DF2162 (0.1 μM). After 24 hours of incubation, plates were examined for capillary-like organization under optical microscope (bars = 1 mm). Each assay was done in duplicate.
[More] [Minimize]To determine the effect of DF2162 on IL-8–induced HUVEC proliferation, cells were cultured in medium containing 10 ng/ml of human IL-8 in the absence or presence of DF2162 (0.01, 0.1, and 1 μM). Cell proliferation induced by IL-8 was significantly reduced in DF2162-treated cells, with the maximal inhibition of approximately 50% observed at 1 μM when compared with medium alone plus DMSO (Figure 7B).
Next, to investigate the effect of DF2162 on IL-8–induced HUVEC, we performed a scratch assay and compared the migration of HUVECs in presence of IL-8 (10 ng/ml) or IL-8 (10 ng/ml) plus DF2162 (0.1 μM). We found that HUVEC migration induced by IL-8 was significantly decreased in the DF2162-treated group (Figure 7C). We also examined the effect of DF2162 on endothelial cell organization into capillary-like structures. Endothelial cells were plated on Matrigel-coated 24-well plate with medium containing IL-8 (10 ng/ml) or IL-8 (10 ng/ml) plus DF2162 (0.1 μM). After 24 hours of incubation, we observed in endothelial cells incubated with medium containing IL-8 an increased of long and thin capillary-like formation without cell agglomerates into the capillary junctions (Figure 7F). HUVECs treated with both IL-8 and DF2162 (0.1 μM) displayed cell agglomerates into the capillary junctions and thickness (Figure 7G), and demonstrated similar morphology observed in DMSO-treated cells (Figure 7E).
In this study, we showed the protective and therapeutic effects of DF2162, a long-acting antagonist of the chemokine receptor CXCR2 (17, 18), on the progression of lung fibrosis in bleomycin-instilled mice. Our main findings were that blockade of CXCR2 resulted in: (1) reduction of neutrophil migration into the airways, but not lung parenchyma; (2) reduction of the production of the fibrogenic cytokines IL-13 and active TGF-β1; (3) amelioration of overall lung pathology and reduction of collagen accumulation; (4) decreases expression of vWF expression, a marker of angiogenesis, in the lung; and (5) decreased proliferation, migration and capillary-like organization induced by IL-8 on endothelial cells in vitro.
Initial studies designed to understand IPF pathogenesis have primarily focused on mechanisms related to fibroplasia and deposition of extracellular matrix (28, 29). However, multiple disorders associated with changes in extracellular matrix deposition are also associated to excessive inflammatory response and lung injury (30). Up to date, the real contribution of inflammation to trigger this pathology is controversial and still in constant discussion (31, 32). Pulmonary fibrosis can be either caused by chronic inflammation (33) or by a disrupted cross-talk between epithelial cells and fibroblast (28), which can occur in the absence of inflammation (34). Tissue damage after lung injury and inflammation seems to be a pivotal process, necessary to trigger the excessive and progressive scaring of lungs (33). Some studies have related an important role for neutrophils in the inflammatory process that precedes lung fibrosis induced by bleomycin (35, 36), but a recent study showed that neutrophil depletion with anti-PMN antibodies did not alter the susceptibility to bleomycin-induced pulmonary fibrosis (37). Therefore, the association between pulmonary fibrosis and inflammation is incompletely understood and remains unsolved.
In our study, we demonstrated a temporal relation between early lung CXCL1/KC production, neutrophil migration into airway space, and later lung fibrosis induced by bleomycin instillation. It was observed that the inflammatory response, especially the neutrophil influx, preceded the fibrotic process. Therefore, our objective was to block PMN recruitment with a CXCR2 receptor antagonist to evaluate whether CXCR2-mediated neutrophil influx could play a relevant role in the induction of the fibrotic response induced by bleomycin. CXCR2 is a very important receptor associated with neutrophil recruitment during infective or inflammatory processes (15–18). In the model of bleomycin-induced pulmonary inflammation and fibrosis, treatment with the CXCR2 antagonist diminished neutrophil transmigration into airway space, even in the presence of large concentrations of the CXCR2 ligands CXCL1/KC and CXCL2/MIP-2. These results show that blockade of CXCR2 is sufficient to prevent neutrophil influx into BAL fluid. Interestingly, the CXCR2 receptor antagonist administration did not modify the accumulation of neutrophils in lung parenchyma, as assessed by MPO measurements and confirmed by histology. The present study did not investigate in any detail mechanisms underlying this differential inhibition of accumulation in lung parenchyma or airway spaces. However, it has been demonstrated that intravascular, interstitial, and intra-alveolar accumulation of neutrophils may be differentially regulated; indeed, PMNs can enter the pulmonary interstitium without advancing to the alveolar airway space and crossing the epithelial barrier seems to be pivotal for inducing lung injury (36). Thus, blockade of CXCR2 prevented neutrophil influx into the airways but not their accumulation into lung parenchyma. Thus, although there was a good correlation between neutrophil influx into BAL and prevention of fibrosis, the presence of neutrophils in lung parenchyma and previous studies showing the lack of effect of anti-PMN antibodies in the model suggests that neutrophils may not be the major site of action for CXCR2 antagonists in the system.
Although CXCR2 has been identified as a receptor directing neutrophil migration and activation, other cell types, including lymphocytes, airway epithelial cells, and endothelial cells, express CXCR2 and may also be activated by ligands of this receptor (10, 13, 38, 39). It is, therefore, possible that blockade of CXCR2 on these other cell types may contribute to the protective effects of DF2162 on bleomycin-induced lung injury. Indeed, attenuation of alveolar wall thickness could be secondary to inhibition of CXCR2-dependent proliferation of airway epithelial cells (38). On endothelial cells, the activation of CXCR2 by CXCL1/KC or CXCL2/MIP-2 may induce angiogenesis, an effect counteracted by angiostatic CXCR3 ligands, such as CXCL9/MIG and CXCL10/IP-10 (13, 14). Interestingly, we found that blockade of CXCR2 was associated with an elevated production of CXCL9/MIG at Day 2 and CXCL9/MIG and CXCL10/IP-10 at Day 16 after bleomycin challenge. In addition, DF2162 administration led to a reduction in the expression of the endothelial cell marker vWF (25) in injured lungs. IL-8 has an important role on angiogenesis by promoting endothelial cell proliferation, survival, and migration and also the expression of matrix metalloproteinases through CXCR1 and CXCR2 signaling (27). Here, we report direct effects of DF2162 on HUVECs stimulated with IL-8 by reducing HUVEC proliferation, migration, and its ability in organizing capillary-like structures in vitro. Overall, these results suggest an anti-angiogenic effect of DF2162 on the setting of pulmonary fibrosis. The effects of DF2162 on angiogenesis could potentially be secondary to a direct effect of the compound on pulmonary endothelial cells or via the induction of high levels of CXCR3 agonists (CXCL9/MIG and CXCL10/IP-10), chemokines that generate a microenvironment with angiostatic characteristics.
It is noteworthy that we found that DF2162 administered mice had reduced levels of IL-10, IL-13, and active TGF-β1 and increased GM-CSF production, although they had normal levels of IFN-γ. IL-13 is a pleiotropic cytokine produced in large quantities by stimulated CD4 Th2 cells (40), CD8 T cells (41), and airway epithelial cells in lungs during asthma (42). Recent studies in macrophages demonstrated that IL-13 is an enhancer of the production of the pro-fibrotic cytokine TGF-β1 which play a major role in pulmonary fibrosis induced by bleomycin in mice (43). The reduction in lung collagen content was associated with lower levels of IL-13 and active TGF-β1 in lungs of DF2162-treated animals, suggesting that blockade of CXCR2 could be preventing fibrosis in this model via control of IL-13 production and TGF-β1 activation without change IFN-γ levels after bleomycin challenge. As CXCR2 receptors are present on activated T cells (10), inhibition of this receptor by DF2162 could prevent their in vivo recruitment and production of IL-13. Consistently, we found lower levels of infiltrating lymphocytes in lungs of DF2162-treated animals. DF2162-treated animals had lower levels of CCL5/RANTES, which signals through CCR5 receptor (10, 44), and is a lymphocyte chemoattractant factor that mediates T cell influx in fibrosing alveolitis (45). Thus, the blockade of CXCR2 on airway epithelial cells and lymphocytes could also contribute to the protective effects observed.
An intriguing finding was the elevated number of neutrophils in the lung interstitium of animals treated from Days 0 to 16 with DF2162 when compared with vehicle-treated mice at Day 16 after bleomycin instillation. This increase did not occurred when DF2162 was given from Day 8 to Day 16 after bleomycin. Those results are in accordance with those of Keane and coworkers, who found that the administration of anti–MIP-2/anti-CXCL2 antibodies did not reduce MPO levels (22). It is unclear, however, why neutrophils would persist for longer in the group of animals that received the CXCR2 receptor antagonist from Days 0 to 16. One possibility to explain such findings stems from the observation that there were reduced levels of IL-10 and increased levels of GM-CSF in lungs of DF2162-treated mice. As IL-10 possesses relevant anti-inflammatory and pro-apoptotic actions on neutrophils (46, 47), and GM-CSF has an opposite effect, prolonging neutrophil survival and activation (48), the balance between IL-10 and GM-CSF levels in drug-treated animals could account for the greater number of neutrophils in lung parenchyma at the later stages of DF2162-treated animals. Another putative mechanism contributing to leukocyte clearance and eventual resolution of inflammatory response is the transmigration of cells into airway spaces. The transmigrated granulocyte can then be safely eliminated by apoptosis and subsequent phagocytosis, or be trapped by secretions and removed by mucociliary escalator mechanisms (49, 50). As the blockade of CXCR2 inhibits neutrophil transmigration into airway space, the drug would, hence, promote their arrest and interstitial accumulation after bleomycin challenge. Whatever the mechanisms underlying the enhanced number of neutrophils found in lung interstitium after DF2162 treatment, these results would suggest that it is the neutrophils' transmigration into airway space that is relevant for the induction of lung injury by bleomycin instillation. During transmigration from interstitium to airway space, activated neutrophils are able to secrete proteases, such as elastase and matrix metalloproteinases. These proteases facilitate neutrophil transmigration by digesting extracellular matrix and promoting increase in vascular permeability, thus potentially enhancing lung damage and cleavage of inactive TGF-β1 into the active form (35, 36). We found identical levels of total TGF-β1 when comparing DF2162- and vehicle-treated-mice at Day 16 after bleomycin. However, DF2162-treated mice displayed reduced levels of active TGF-β1. The latter results suggest that DF2162 treatment may regulate not only the transmigration of neutrophils into airspaces but also their activation state. Further studies are necessary to confirm this possibility.
Therapeutic benefits of DF2162 administration were tested by giving the drug from day 8 after bleomycin challenge. Using this therapeutic schedule, the drug decreased the pulmonary levels of IL-13 and TGF-β1 and pulmonary fibrosis. There were also increased levels of angiostatic chemokines and reduced angiogenic activity. In summary, our work showed that CXCR2 has an important role in mediating pulmonary fibrosis after bleomycin instillation in mice. Blockade of CXCR2 was associated with inhibition of neutrophil influx into airspaces, inhibition of the fibrogenic cytokines IL-13 and TGF-β1 and decreased pulmonary angiogenesis and fibrosis. It is possible that CXCR2 may represent an interesting target for therapeutic intervention in idiopathic pulmonary fibrosis.
The authors thank Valdinéria Borges and Ilma Marçal for technical assistance. The authors are grateful to Dr. Massimo Locati and Dr. Alberto Mantovani, from Istituto Clinico Humanitas (ICH), for help with experiments in vitro and reagents.
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