Resistin-like molecule α1 (Fizz1) recruits lung dendritic cells without causing pulmonary fibrosis

Abstract

Background: Resistin-like molecule alpha or found in inflammatory zone protein (Fizz1) is increased in pulmonary epithelial cells and also in limited amounts by other lung cells during various lung injuries and fibrosis. However, the direct role of Fizz1 produced in the pulmonary epithelium has not been determined.

Methods: Fizz1 Transgenic mice (CCSP/Fizz1) were generated that overexpress Fizz1 in the lung epithelium under the control of a doxycycline (Dox) inducible lung epithelial cell specific promoter Scgb1a1 (Clara cell secretory protein, CCSP). Histology and FACS analysis of lung cells were used to identify the direct effects of Fizz1 in the transgenic mice (Dox treated) when compared with control (CCSP/-) mice. Intratracheal bleomycin sulfate or silica in saline and saline alone were used to study the role of Fizz1 during bleomycin- and silica-induced pulmonary fibrosis in CCSP/Fizz1 and CCSP/- mice. Weight change, pulmonary inflammation, and fibrosis were assessed 10 days post bleomycin or 28 days post silica challenge.

Results: When CCSP/Fizz1 mice were fed Dox food, elevated Fizz1 protein was detected in lung homogenates by western blot. Lungs of mice in which Fizz1 was induced in the epithelium contained increased lung cells staining for CD11c and F4/80 by FACS analysis consistent with increased dendritic cells however, no changes were observed in the percentage of interstitial macrophages compared to CCSP/- controls. No significant changes were found in the lung histology of CCSP/Fizz1 mice after up to 8 weeks of overexpression compared to CCSP/- controls. Overexpression of Fizz1 prior to challenge or following challenge with bleomycin or silica did not significantly alter airway inflammation or fibrosis compared to control mice.

Conclusions: The current study demonstrates that epithelial cell derived Fizz1 is sufficient to increase the bone-marrow derived dendritic cells in the lungs, but it is not sufficient to cause lung fibrosis or alter chemical or particle-induced fibrosis.

Figure 1

Generation of CCSP/Fizz1 transgenic mice. (A) Gene targeting strategy for generating conditional CCSP/Fizz1 transgenic mice. TetO-CMV Fizz1^+/+ mice were generated by cloning full length Fizz1 cDNA from mouse lung RNA and inserting into the (TetO)₇-CMV promoter. CCSP/Fizz1 transgenic mice were produced by mating single transgenic CCSP/- mice with (TetO)₇-CMV Fizz1^+/+ mice. To induce Fizz1 expression in bitransgenic mice were administered doxycycline (Dox) as described in methods. (B) Western blot shows Fizz1 overexpression in the lungs of CCSP/Fizz1 mice on Dox for two weeks. rFizz1 is a lane containing purified Fizz1 (Peprotech) loaded as a positive control. Lanes 1, 2 are duplicate samples. The expressed Fizz1 in Dox treated transgenic mice migrated identically to the Fizz1 marker compared to CCSP/Fizz1 mice without Dox treatment. The relative intensity of the bands in the On Dox lanes was increased about 14.1 fold.

Figure 2

The lung epithelial specific Fizz1 overexpression increases the percentage of dendritic cells in the lung. (A) CCSP/Fizz1 mice were fed doxycycline food for 14 days. Total BAL cells increased with Fizz1 overexpression in CCSP/Fizz1 mice. Statistical significance is measured using unpaired student’s t test (n = 11/group). (B) CCSP/Fizz1 mice were fed doxycycline food for 14 days and total lung cells were stained with anti-CD11b and anti-CD45 antibodies. Gating strategy and representation of CD45+ and CD11b + cells in the lungs of Fizz1 overexpressing mice compared to CCSP/Fizz1 mice on no Dox. (C) Fizz1 overexpression has caused increase in the percentage of CD45 and CD11b double positive cells. Statistical significance is measured using unpaired student’s t test (n = 3/group). (D) CCSP/Fizz1 mice were fed doxycycline food for 14 days and total lung cells were stained with anti-CD11c and anti-F4/80 antibodies. Gating strategy and representation of DC, AM and IM in the lungs of Fizz1 overexpressing mice compared to CCSP/Fizz1 mice on no Dox. (E) Mice overexpressing Fizz1 demonstrate increases in the percentage of cells staining for CD 11c that do not stain for F4/80 consistent with changes in dendritic cells. Statistical significance is measured using unpaired student’s t test (n = 3/group). Data represent one of two independent experiments showing similar results.

Figure 3

The effect of Fizz1 overexpression on the lung histology. H&E stained lung sections from CCSP/- and CCSP/Fizz1 mice on Dox for 8 weeks at 10X (top) & 20X (bottom) magnification. Histological examination reveals no detectable changes in the morphology of airways, blood vessels and also inflammatory cells in the lung tissues of CCSP/- and CCSP/Fizz1 mice on Dox for 8 weeks.

Figure 4

Fizz1 overexpression in the lung has no effect on bleomycin-induced inflammation. (A) CCSP/- mice lungs were exposed to saline or bleomycin via the I.T. route. Fizz1 transcripts were measured using RT-PCR at day10 post-bleomycin. Statistical significance is measured using unpaired student’s t test (n = 5/group). (B) Schematic representation of Fizz1 induction by feeding doxycycline food and administration of 0.15U of bleomycin via the I.T. route. (C) CCSP/Fizz1 mice exposed to bleomcyin demonstrate accelerated weight loss compared with CCSP/- controls receiving bleomycin. Data shown are mean ± SEM (n = 7-8/group). (D) Total BALF cells quantified at day10 post bleomycin or saline. Data shown are mean ± SEM (n = 7-8/group). Statistical significance is measured using unpaired student’s t test. (E) Absolute counts of macropahges in BALF cells were obtained from cell differentials. Data shown are mean ± SEM (n = 7-8/group). Data represent one of two independent experiments showing similar results.

Figure 5

Fizz1 overexpression has no effect on bleomycin-induced fibrosis in the lung. CCSP/- and CCSP/Fizz1 mice were exposed to saline or bleomycin (0.15U) via the I.T. route, with pulmonary fibrosis assessed at day 10. (A) Masson Trichrome stained lung sections at 10X magnification. Scale bar 200 μm. (B) Total lung weights were measured in all groups at day10 post-bleomycin. (C) The lung collagen deposition, expressed as micromoles of hydroxyproline per lung. The above data is an average of two independent experiments with the total 7–14 mice per group. Statistical significance is measured using one way ANOVA (n = 7-14/group).

Figure 6

Fizz1 overexpression has no effect on silica-induced granulomatous inflammation in the lung. CCSP/- and CCSP/Fizz1 mice were exposed to saline or silica (3 mg/mice) via the I.T. route, pulmonary inflammation assessed on day 28 after silica instillation. Data represent one of two independent experiments showing similar results with five animals per group. Data shown are mean ± SEM. Statistical significance is measured using one way ANNOVA (n = 5/group). (A) H&E stained lung sections at 10X magnification. Scale bar 200 μm. CCSP/- and CCSP/Fizz1 mice exposed to silica develop granulomatous nodules of similar size (B) Total BAL cells counted at day 28 post silica or saline. (C) Absolute counts of macrophages in BALF cells were obtained from cell differentials.

Figure 7

Fizz1 overexpression has no effect on silica-induced fibrosis in the lung. CCSP/- and CCSP/Fizz1 mice were exposed to saline or silica (3 mg/mice) via the I.T. route, with pulmonary fibrosis assessed on day 28 after silica instillation. One of two independent experiments is shown with five animals per group. Data shown are mean ± SEM. Statistical significance is measured using one way ANOVA . (A) Masson Trichrome stained lung sections at 10X magnification. CCSP/- and CCSP/Fizz1 mice exposed to silica develop granulomatous nodules of similar size. Scale bar 200 μm. (B) Total lung weights were measured in all groups at day 28 post-silica. CCSP/- and CCSP/Fizz1 mice exposed to silica showed increases in the lung weights and no difference with Fizz1 overexpression. (C) The lung collagen deposition, expressed as micromoles of hydroxyproline per lung. CCSP/- and CCSP/Fizz1 mice exposed to silica showed increases in the lung weights and no difference with Fizz1 overexpression.