Endogenous annexin A1 counter-regulates bleomycin-induced lung fibrosis

Abstract

Background: The balancing functions of pro/anti-inflammatory mediators of the complex innate responses have been investigated in a variety of experimental inflammatory settings. Annexin-A1 (AnxA1) is one mediator of endogenous anti-inflammation, affording regulation of leukocyte trafficking and activation in many contexts, yet its role in lung pathologies has been scarcely investigated, despite being highly expressed in lung cells. Here we have applied the bleomycin lung fibrosis model to AnxA1 null mice over a 21-day time-course, to monitor potential impact of this mediator on the control of the inflammatory and fibrotic phases.

Results: Analyses in wild-type mice revealed strict spatial and temporal regulation of the Anxa1 gene, e.g. up-regulation in epithelial cells and infiltrated granulocytes at day 7, followed by augmented protein levels in alveolar macrophages by day 21. Absence of AnxA1 caused increases in: i) the degree of inflammation at day 7; and ii) indexes of fibrosis (assessed by deposition of hydroxyproline in the lung) at day 7 and 21. These alterations in AnxA1 null mice were paralleled by augmented TGF-β1, IFN-γ and TNF-α generation compared to wild-type mice. Finally, treatment of wild type animals with an AnxA1 peptido-mimetic, given prophylactically (from day 0 to 21) or therapeutically (from day 14 onward), ameliorated both signs of inflammation and fibrosis.

Conclusion: Collectively these data reveal a pathophysiological relevance for endogenous AnxA1 in lung inflammation and, more importantly, fibrosis, and may open new insights for the pharmacological treatment of lung fibrosis.

Figure 1

Analysis of endogenous AnxA1 protein and Anxa1 gene expression during lung fibrosis. (A-H;O-P) Wild type and (I-N) AnxA1 null mice received bleomycin i.t. at time 0. The AnxA1 protein content was analyzed by immunohistochemistry. At 0 time-point, wild type mice exhibit a basal immunostain for AnxA1 protein (A and B). After 7 days post-bleomycin administration, this protein expression was greatly increased (C and D). And, after 21 days, the AnxA1 expression was reduced (E and F) in epithelial cells (arrow) and polymorphonuclear (PMN) (arrowheads) and MPC (curve arrow)(G and H). Control of immunogold reaction (CR) showing no cellular immunostaining. Anxa1 gene promoter activity was visualized by X-Gal staining reaction. AnxA1 null mice lung showing Anxa1 gene expression (I and J) on the epithelial cells (arrow), intravascular PMN (arrowhead) and MPC (curve arrow); an intense positive reaction was attained (K-N) on day 7 and 21 post-bleomycin administration. Control for LacZ reaction (CR) showing wild type mice negative to the X-Gal staining (O and P). Haematoxylin counterstain. Bars, 10 μm.

Figure 2

Leukocyte influx into the lung tissue. Semiquantitative analysis of the histological sections showing intravascular polymorphonuclear (PMN) and monocytes (mono), and transmigrated PMN and MPC in lung tissue. Wild type and AnxA1 null mice were analyzed at 0, 7 and 21 days post-bleomycin i.t. administration as described in Methods section. Data are mean ± SEM from 5 mice per time point. *P < 0.05 versus 0 time point wild type group values; **P < 0.01 versus 0 time point wild type group values; ***P < 0.001 versus 0 time point wild type group values; #P < 0.05 versus corresponding wild type group values; ###P < 0.01 versus corresponding wild type group values.

Figure 3

Lung histopathology. (A-C) Wild type and (D-F) AnxA1 null mice lung were analyzed at 0, 7 and 21 days post-bleomycin i.t. administration as described in Material and Methods section. (A and D) Histological analysis of wild type and AnxA1 null mouse lungs showing presence of collagen in the connective tissue in the lung parenchyma (a), near the vessel (v) and bronchiole (b). (B) No major changes in the connective tissue of wild type lung parenchyma, as observed at day 7 post-bleomycin administration. (C) At day 21 post-bleomycin, the alveolar septa thicken because of a significant increase in connective tissue deposit (arrow). (E and F) In the AnxA1 null mice, the fibrosis was evident already at day 7; by day 21 there is evidence of increased connective tissue (arrows). Masson trichrome stain. Bars, 100 μm.

Figure 4

Bleomycin lethality in AnxA1 null mice. Wild type and AnxA1 null mice received 0.15 U/Kg bleomycin i.t. at time 0 and survival rate was monitored daily up to 21 days. Results are cumulated from three experiments with a total of 15 mice per group.

Figure 5

Serum and BAL supernatant cytokine levels. Wild type and AnxA1 null mice received bleomycin i.t. at time 0, with blood and BAL samples being collected at the reported time points. The serum and cell-free supernatant BAL concentration of TGF-β1, IFN-γ and TNF-α was determined by ELISA. Results are mean ± SEM from two separate experiments with 5 mice per group. *P < 0.05,**P < 0.01 and ***P < 0.001 versus 0 time point wild type group values; #P 0.05 versus corresponding wild type group values.

Figure 6

Effects of treatment with peptide Ac2-26 on lung fibrosis and hydroxyproline content. Lung were analyzed at 0 and 21 days post-bleomycin i.t. administration. Also a group of mice were daily treated i.p. with the peptide Ac2-26 (1 mg/kg) or between the days 14 to 21 post-bleomycin as described in Methods section. (A) Morphological analysis of sham group indicated the reduced content of collagen in the lung parenchyma (a), near the vessels (v) and bronchiole (b). (B) At day 21 after bleomycin administration, the alveolar septa thickening indicated the significantly increasement of collagen deposit (arrows). (C) The daily treatment with peptide Ac2-26 prevents bleomycin-induced lung fibrosis (curve arrows). Masson trichrome stain. Bar, 100 μm. (D) Pulmonary fibrosis was biochemically assessed by measurement of lung hydroxyproline content. Results are expressed as means ± SEM of μg of hydroxyproline per mg of lung tissue. **P < 0.01 versus 0 time point values; #P < 0.05 versus day 21 post-bleomycin time point values.

Figure 7

Analysis of endogenous AnxA1 expression in wild type mice after treatment with peptide Ac2-26. Mice received bleomycin i.t. at time 0. Immunohistochemistry was performed to visualize the protein expression in epithelial cells (arrows), PMN (arrowheads) and alveolar macrophages (curved arrow). (A and B) Sham mice exhibit a basal immunostain for AnxA1 protein. (C and D) Lung fibrosis induced by bleomycin reduced the AnxA1 expression in epithelial cells (arrow) and PMN (arrowheads). (E and F) Daily treatment with peptide Ac2-26 (1 mg/kg) increased the AnxA1 expression during lung fibrosis. Haematoxylin counterstain. Bar, 10 μm. (G) The AnxA1 protein content was also analyzed by Western blotting (seen as both the native 37 kDa and 33 kDa cleavage products). AnxA1 was down regulated during lung fibrosis. Daily i.p. treatment with peptide Ac2-26, or given only between day 14 and day 21 post-bleomycin, increased expression of this endogenous protein.