Rac2-deficient mice (rac2 ^-/- ) were established by gene disruption 10 and were backcrossed for >11 generations on the C57BL/6 strain background. Rac2-deficient mice were bred in our animal facility and housed under specific virus antigen-free conditions on a 12:12-h light–dark cycle and fed autoclaved food and water as needed. Wild type (WT) C57BL/6 mice were purchased from Charles River Laboratories and were housed and maintained in the same manner as rac2 ^-/- mice. For our experiments, we used female rac2 ^-/- or C57BL/6 WT mice at 10–14 wk of age. Animal experiments were approved by the University of Alberta Health Sciences Laboratory Animal Ethics Committee (Edmonton, AB, Canada).

After general anesthesia with 4% isoflurane inhalation, the trachea was exposed and a single intratracheal injection of BLM sulphate (0.125 U/100 g body weight dissolved in normal saline, Sigma-Aldrich Co., MO, USA) or saline as control was administrated to C57BL/6 WT and rac2 ^-/- mice. Following the injection, mice were sutured, allowed to recover and daily monitored for morbidity and mortality.

On day 21 after BLM intratracheal instillation, mice were weighed and lung function was measured. Each mouse was anesthetized with 0.1 ml/10 g body weight of a mixture containing xylazine (1.6 mg/ml; Bayer) and ketamine (8 mg/ml; Parnell). Following anesthesia, a tracheotomy was performed, and a polyethylene cannula was inserted. Pancuronium amide (2 mg/kg) was administered intravenously before animals were attached to a ventilator. Mice were mechanically ventilated on a computed controlled piston ventilator FlexiVent® Systems (SCIREQ, Montreal, QC, Canada) with a tidal volume of 5.5 ml/kg at a rate of 150 breaths/min.

On days 7 or 21 after BLM administration, left lungs were cannulated, inflated with 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO) for 5 min under a constant pressure of 20 cm of H₂O, removed from animals, and placed in fresh 10% neutral buffered formalin for 24 h. In the case of the 21 day group, lung resection was done after lung function testing. Tissues were then embedded in paraffin blocks, and sections were cut (4 μm) and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff-diastase stain (PAS-D). Staining of lung sections with Masson’s trichrome, or with antibodies for α-smooth muscle actin (αSMA) and TGFβ were performed to obtain histological evidence of fibrotic activity. Lung fibrosis was scored according to Ashcroft’s criteria as previously published 15 .

Mice were euthanized on days 3 and 7 after saline or BLM intratracheal instillation. Following blood collection by cardiac puncture, the trachea was exposed and intubated with a polyethylene catheter. Lungs were lavaged twice with 1 ml of isotonic phosphate-buffered saline (pH 7.4) and the bronchoalveolar lavage (BAL) fluid was collected. The BAL fluid was centrifuged at 300 g for 5 min. Total cells in the resulting pellet were counted, and cytospins of 5000 cells/sample were prepared and stained with Diff-Quick (Fisher Scientific Co, Kalamazoo, MI). Airway inflammation was assessed by counting the number of inflammatory cells in the BAL fluid as previously described 16 . BAL supernatants were kept frozen in -80°C for MMP and MPO evaluation. MMPs were evaluated by gelatin zymography as previously described 17 . MPO activity was assessed using tetramethylbenzidine (TMB) as the substrate, as previously described 9 . Cytokine and chemokine levels were also evaluated in BAL fluid at day 7 after BLM instillation.

After lung function measurements, right lungs from each animal were removed, weighed and immediately frozen on liquid nitrogen and stored at -80°C for biochemical analysis as described below.

Hydroxyproline determination was done as described 18 . In brief, lung tissue was thawed and homogenized in 1 ml of water. To precipitate protein, 125 μl of 50% trichloroacetic acid (Sigma-Aldrich) was added to the homogenate and samples were incubated on ice for 20 min. Samples were centrifuged at 300 g for 5 min at 4°C. Supernatants were discarded and 1 ml 12 N HCl was added to the pellet. The pellet was baked at 110°C for 24 h. The dried pellet was reconstituted with 2 ml of distilled water and 500 μl of chloramine T solution (1.4% chloramine T in 0.5 M sodium acetate and 10% isopropanol, Sigma-Aldrich) and incubated for 20 min at 24°C. Ehrlich’s/pDMAB (1 M p-diamethylaminobenzaldehyde in 70% isopropanol and 30% perchloric acid) was also added and incubated at 65°C for 15 min. A sample of 100 μl of the final reaction solution was transferred to a 96-well plate, and the absorbance for each sample was read at 550 nm in a Power Wave XS (BioTek Instruments Inc., Winooski, VT) ELISA reader. The concentration of lung hydroxyproline was calculated from a hydroxyproline standard curve and expressed as μg/gram of lung tissue.

The total collagen content of lungs was determined using homogenized right lungs and a Sircol™ Soluble Collagen Assay kit (Biocolor, Carrickfergus, UK) according to manufacter’s instructions. Data are expressed as the collagen content of the entire right lung.

Values are expressed as mean ± SEM. Statistical differences in the mean values among treatment groups were determined by using a one-way ANOVA test with post-hoc analysis using Tukey’s multiple comparison test. In all cases, a value for p < 0.05 was considered statistically significant.

Rac2 is expressed primarily in hematopoietic cells, and has an important role in cell motility, superoxide release and degranulation in neutrophils and macrophages 9 10 19 . Rac2 also mediates acute lung injury due to immune complexes in murine models 13 . The aim of this study was to understand the role of Rac2 in BLM-induced lung injury, a model with a more prolonged time course than immune complex-mediated injury. BLM induces reversible fibrosis over 2–3 weeks; early accumulation of neutrophils 20 and macrophages 21 has been shown to be important for the transition from inflammation to fibrosis in this model.

To understand the role of Rac2 in BLM-induced lung injury, we first examined the accumulation of inflammatory cells and inflammatory mediators in the airways of mice 3 and 7 days after induction of bleomycin-induced lung injury. To do that we performed BAL on days 3 and 7 after BLM administration, and evaluated the numbers of neutrophils and macrophages, and the presence of MPO, MMP-2 and MMP-9 activity, as well as cytokine and chemokine levels.

BAL performed on day 3 showed no significant differences in total cells, neutrophils, macrophages or lymphocytes between BLM- and saline-treated mice in WT and rac2 ^-/- groups (Figure  1A-D, n = 5). WT mice receiving BLM showed accumulation of inflammatory cells in the BAL on day 7 compared to saline-treated mice (Figure  1A). In contrast, BLM-treated rac2 ^-/- mice showed no significant accumulation of cells in BAL on day 7 (Figure  1A). While BLM induced the recruitment of neutrophils to the airways of WT mice o day 7, there were no increased numbers of neutrophils in BLM-treated rac2 ^-/- mice compared to saline treated mice on day 7 (Figure  1B). For both total cells and neutrophils there was a statistically significant difference between BLM-treated WT and rac2 ^-/- mice. The numbers of macrophages increased in BLM-treated WT and rac2 ^-/- mice on day 7 (Figure  1C) but the number of lymphocytes showed only a trend towards increase by BLM in both backgrounds (Figure  1D). There was no difference between BLM-treated WT and rac2 ^-/- mice in the number of macrophages and lymphocytes in the BAL, indicating that lack of Rac2 affects primarily neutrophil accumulation in the airways of BLM-treated mice.

We next measured MPO activity in BAL samples. WT mice treated with BLM showed higher levels of MPO in BAL on day 3 and day 7 compared to saline-treated mice (Figure  2A and B n = 5). Rac2 ^-/- mice exhibited increased MPO activity on day 3 but not on day 7 (Figure  2A and B). There was no statistically significant difference in MPO between BLM-treated WT and rac2 ^-/- mice on day 3 or day 7. Histological analysis of the lungs of saline and BLM-treated WT and rac2 ^-/- mice on day 7 showed diffuse inflammatory cell infiltration in both groups of BLM-treated mice, but there were no clear differences between the two groups in the pattern or degree of inflammation (Figure  3).

We also examined the levels of matrix metalloproteinases in the BAL samples from these mice. BLM treatment increased MMP-9 levels in WT mice, but not in rac2 ^-/- mice, on day 3 (Figure  2C). MMP-9 levels returned to baseline by day 7 (Figure  2D). In contrast MMP-2 levels were increased in both WT and rac2 ^-/- mice on day 7, but not day 3, and there was no difference in MMP-2 levels between BLM-treated WT and rac2 ^-/- mice on day 3 (Figure  2E and F).

In order to further understand the mechanisms for the defect in neutrophil migration to the airways in rac2 ^-/- mice treated with BLM, we evaluated the levels of various cytokines and chemokines in the BAL fluid. TNF was increased in WT mice following BLM administration, but not in rac2 ^-/- mice (Figure  4A, n = 5). IL-1β and CXCL3/KC showed a trend towards an increase in WT mice following BLM administration, but there were no statistically significant differences between any of the groups studies (Figure  4B and C, n = 5). CCL3/MIP-1α showed similar pattern with TNF (Figure  4D, n = 5). Finally CCL11/eotaxin and CXCL10/IP-10 were increased in both WT and rac2 ^-/- mice following BLM treatment and there was no difference between BLM-treated WT and rac2 ^-/- mice (Figure  4E and F, n = 5).

BLM-induced lung injury in mice is characterized by the development of extensive reversible fibrosis 15–20 days after a single treatment 22 . Fibrosis alters lung physiology and leads to significant morbidity and mortality in this model, before the process resolves. Since rac2 ^-/- mice showed altered early inflammatory response after lung injury, we next studied whether later events of BLM injury, including mortality and fibrosis, were also affected.

To understand the function of Rac2 in the long-term effects of BLM-induced lung injury, WT and rac2 ^-/- mice were given BLM intratracheally and followed for 21 days. WT mice showed 70% mortality by day 21, while rac2 ^-/- mice showed significantly lower mortality (p < 0.005) (Figure  5). In order to further understand the functional role of Rac2 in BLM-induced lung injury, we analyzed lung physiology and markers of fibrosis in the mice that survived until day 21.

WT mice that received BLM showed significantly increased airway resistance and elastance compared to saline-treated mice (p < 0.05) (Figure  6A and B, n = 8). In contrast, rac2 ^-/- mice that received BLM showed resistance and elastance levels that were similar to rac2 ^-/- mice receiving saline. There was also significant difference in resistance and elastance between BLM-treated WT and rac2 ^-/- mice.

The mortality and pulmonary physiology results indicate that BLM-induced injury is attenuated in rac2 ^-/- mice, and that in contrast to WT mice surviving to day 21, the injury still present in the rac2 ^-/- mice still surviving to day 21 does not affect lung physiology.

We also performed histological analysis of lungs from surviving BLM-treated and saline-treated mice on day 21. Lungs were excised, fixed and then stained with H&E for morphology and inflammatory cell infiltration and PAS-D to identify mucin.

Saline-treated WT and rac2 ^-/- mice (Figure  7A) showed normal architectural pattern of airways and alveoli, with an apparent increase in interstitial leukocytes evident in rac2 ^-/- mice. We hypothesize that the latter observation is the result of the modest peripheral neutrophilia that has been described in these animals 10 , but we have no data to support this conclusion. Lung parenchyma of WT BLM-treated mice exhibited prominent perivascular, peribronchiolar and intra-alveolar inflammation, with increased numbers of alveolar macrophages (Figure  7A). The lungs of rac2 ^-/- BLM-treated mice showed a similar picture except that interstitial inflammation often had a more focal distribution. A semi-quantitative analysis (perivascular, peribronchial and intra-alveolar infiltrates were graded on a score of 0–3 for all the mice and these 3 numbers of every mouse were averaged and are presented as the inflammation score) showed no significant difference in inflammatory infiltrates between BLM-treated WT and rac2 ^-/- mice (Figure  7B).

We also stained the lungs with PAS-D to identify mucin (Figure  8). WT BLM-treated mice displayed airway epithelium exhibiting increased numbers of mucin-containing epithelial cells consistent with goblet cell hyperplasia (arrow, fuchsia colored cells). In contrast, rac2 ^-/- BLM-treated mice showed airway epithelium with reduced numbers of mucin-containing epithelial cells compared to WT mice treated with BLM. In control saline-treated mice, there were essentially no mucin-containing epithelial cells in WT and rac2 ^-/- airway epithelia.

To further assess the degree of fibrosis histologically, we analyzed lung sections from WT and rac2 ^-/- mice after Masson’s Trichrome staining to evaluate collagen deposition and measured total collagen in the lungs of these mice. We also performed immunohistochemical analysis with staining for αSMA and TGFβ.

WT and rac2 ^-/- saline-instilled mice presented with normal lung parenchyma with an expected minimal level and distribution of collagen fibrils. In contrast, the lung parenchyma of WT and rac2 ^-/- BLM-treated mice showed increased collagen deposition compared saline-treated controls (arrows, Figure  9A lower panels). In general, BLM-treated rac2 ^-/- mice showed smaller patchy regions of collagen deposition compared to BLM-treated WT mice. However, evaluation of fibrosis by Ashcroft scoring as done before 15 showed no significant difference between BLM-treated WT and rac2 ^-/- mice (Figure  9B).

In order to determine fibrosis using biochemical approach, we estimated total lung collagen content using the Sircol Dye Reagent and hydroxyproline content using lung homogenates from saline and BLM-treated WT and rac2 ^-/- mice. We detected a significant increase in total lung collagen content and in hydroxyproline content in BLM-treated WT mice compared to saline-treated mice (p < 0.05, n =6), while rac2 ^-/- BLM-treated mice did not show significant increase in lung collagen or hydroxyproline content compared to saline-treated rac2 ^-/- animals (Figure  9C and D, n = 7-8 per group). However, BLM-treated rac2 ^-/- mice also showed no statistically significant difference in collagen or hydroxyproline content compared to BLM-treated WT mice.

To further study the role of Rac2 in pulmonary fibrosis was also performed immunohistochemistry for αSMA and TGFβ. As shown in Figure  10, there were no differences in αSMA and TGFβ staining between BLM-treated WT and rac2 ^-/- mice on day 21.