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Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).

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Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression

Immunology > Immune cell function > Macrophage
Authors: Jennifer L. Larson-Casey
Jennifer L. Larson-CaseyAffiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Bio-protocol author page: a3716
 and A. Brent Carter
A. Brent CarterAffiliation 1: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Affiliation 2: Birmingham Veterans Administration Medical Center, Birmingham, AL, USA
For correspondence: bcarter1@uab.edu
Bio-protocol author page: a3717
Vol 6, Iss 22, 11/20/2016, 487 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2009

[Abstract] Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).

[Background] Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al., 2011). Our prior data demonstrate that alveolar macrophages are a critical source of TGF-β1 as mice harboring a conditional deletion of TGF-β1 in macrophages were protected from pulmonary fibrosis (Larson-Casey et al., 2016). The expression of α-SMA is a defining feature of myofibroblasts, and TGF-β1 is a well-characterized pro-fibrotic mediator that induces transformation of fibroblasts to myofibroblasts both in vitro (Desmoulière et al., 1993) and in vivo (Sime et al., 1997). Prior studies exposed fibroblasts to recombinant TGF-β1 to show its effect on differentiation and function (Horowitz et al., 2007). Here we have developed a protocol for determining the ability of mouse BAL fluid to alter the differentiation of human lung fibroblasts to myofibroblasts, the cells that produce extracellular matrix proteins.

Materials and Reagents

  1. 6-well cell culture plates (Corning, Costar®, catalog number: 3516 )
  2. Normal human fibroblasts (IMR-90) (ATCC, catalog number: CCL-186 )
  3. DMEM
  4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140095 )
  5. Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  6. Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  7. RPMI 1640 medium, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 11835030 )
  8. DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  9. NP-40
  10. Sodium chloride (NaCl)
  11. Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
  12. Phosphatase inhibitor (EMD Millipore, catalog number: 524625 )
  13. α-SMA antibody (American Research Products, catalog number: 03-61001 )
  14. β-actin antibody (Sigma-Aldrich, catalog number: A5441 )
  15. Tween 20
  16. Fibroblast culture media (see Recipes)
  17. Lysis buffer (see Recipes)

Equipment

  1. Cell culture incubator, 37 °C, 5% CO2: 95% air atmosphere (Thermo Fisher Scientific, FormaTM, model: Direct Heat CO2 Incubator )

Procedure

  1. Seed fibroblasts at a density of 1 x 106 per well of a 6-well cell culture plate in a total volume of 1 ml. Allow cells to adhere to cell culture plate (2-6 h).
  2. Harvest bronchoalveolar lavage (BAL) fluid from mice (Han and Ziegler, 2013). Mice can be treated with bleomycin (1.75 U/kg) or saline as a negative control. Determine the protein concentration of bronchoalveolar lavage (BAL) fluid. Using RPMI 1640 media, normalize all BAL samples to the same protein concentration in a total volume of 1 ml.
    Notes:
    1. Typically, 0.5-1 ml of BAL fluid is used and RPMI is used to normalize the protein concentration to a final total volume of 1 ml.
    2. Alternatively, this assay can be performed in vitro using conditioned media from macrophage cell lines or bone marrow derived macrophages. Harvest the conditioned media from macrophages post-transfection or post-treatment, using 1 million cells per 1 ml of media.
  3. Remove media from fibroblasts and rinse with 1.5 ml room temperature 1x PBS, add normalized BAL fluid to fibroblasts.
    1. Incubate fibroblasts with BAL fluid for 24 h in a cell culture incubator, 37 °C, 5% CO2: 95% air atmosphere.
    2. Harvest fibroblasts by lysing cells in lysis buffer (see Recipes).
  4. Determine α-SMA expression by immunoblot analysis. The expression of α-SMA protein was determined by Western blotting using 20 μg of total cellular protein. After blocking in 5% milk, the membranes were probed with mouse α-SMA primary antibody using a 1:3,000 dilution in Tris buffered saline containing 0.1% Tween 20, the membranes were incubated with the anti-mouse secondary antibody (1:2,000) in antibody dilution buffer for 1 h.

Data analysis

Results can be shown as a representative immunoblot (Figure 1); also see Larson-Casey et al. (2016).


Figure 1. Immunoblot analysis of α-SMA in IMR-90 fibroblasts cultured in BAL fluid (BALF) from saline or bleomycin (Bleo) exposed WT mice

Recipes

  1. Fibroblast culture media
    DMEM
    10% heat-inactivated FBS
    100 U/ml, 100 μg/ml penicillin-streptomycin
    1.25 μg/ml amphotericin B (fungizone)
  2. Lysis buffer
    1% NP-40
    0.15 M NaCl
    0.05 M Tris pH 7.4
    1 protease tablet
    Phosphatase inhibitor diluted 1:100

Acknowledgments

This work was supported by 2R01ES015981 & VA merit review BX001135.

References

  1. Desmoulière, A., Geinoz, A., Gabbiani, F. and Gabbiani, G. (1993). Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122(1): 103-111.
  2. Han, H. and Ziegler, S. F. (2013). Bronchoalveolar lavage and lung tissue digestion. Bio-protocol 3(16): e859.
  3. He, C., Murthy, S., McCormick, M. L., Spitz, D. R., Ryan, A. J. and Carter, A. B. (2011). Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H2O2 generation. J Biol Chem 286(17): 15597-15607.
  4. Horowitz, J. C., Rogers, D. S., Sharma, V., Vittal, R., White, E. S., Cui, Z. and Thannickal, V. J. (2007). Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts. Cell Signal 19(4): 761-771.
  5. Larson-Casey, J. L., Deshane, J. S., Ryan, A. J., Thannickal, V. J. and Carter, A. B. (2016). Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity 44(3): 582-596.
  6. Sime, P. J., Xing, Z., Graham, F. L., Csaky, K. G. and Gauldie, J. (1997). Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 100(4): 768-776.


How to cite: Larson-Casey, J. L. and Carter, A. (2016). Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression. Bio-protocol 6(22): e2009. DOI: 10.21769/BioProtoc.2009; Full Text



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