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Comparative Study
. 2010 Sep;299(3):L353-62.
doi: 10.1152/ajplung.00315.2009. Epub 2010 Jun 18.

TRPV4 channels augment macrophage activation and ventilator-induced lung injury

Kazutoshi Hamanaka  1 , Ming-Yuan Jian, Mary I Townsley, Judy A King, Wolfgang Liedtke, David S Weber, Fabien G Eyal, Mary M Clapp, James C Parker
Affiliations
Comparative Study

TRPV4 channels augment macrophage activation and ventilator-induced lung injury

Kazutoshi Hamanaka et al. Am J Physiol Lung Cell Mol Physiol. 2010 Sep.

Abstract

We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4(-/-) and TRPV4(+/+) mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (K(f)) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH(2)O PIP did not significantly increase in lungs from TRPV4(-/-) mice but increased >2.2-fold in TRPV4(+/+) lungs, TRPV4(+/+) lungs instilled with TRPV4(-/-) macrophages, and TRPV4(-/-) lungs instilled with TRPV4(+/+) macrophages after ventilation with 35 cmH(2)O PIP. Activation of TRPV4 with 4-alpha-phorbol didecanoate (4alphaPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4(+/+) macrophages compared with TRPV4(-/-) macrophages after 4alphaPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4(+/+) lungs compared with low PIP ventilated TRPV4(+/+) or high PIP ventilated TRPV4(-/-) lungs. Thus TRPV4(+/+) macrophages restored susceptibility of TRPV4(-/-) lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. K(f) increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.

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Figures

Fig. 1.
Fig. 1.
Time course for the increases in peak inflation pressure (PIP) and venous pressure for the high PIP (solid line) and low PIP (dashed line) protocols.
Fig. 2.
Fig. 2.
A: filtration coefficient (Kf) after the low PIP ventilation protocol in wild-type lungs (LP WT), WT lungs with knockout (KO) mouse alveolar macrophages added (LP WT+KO AM), KO lungs (LP KO), and KO lungs with WT macrophages added (LP KO+WT AM). *P < 0.05 vs. the same group after 30 min. #P < 0.05 vs. KO group within the same time period. B: filtration coefficient (Kf) after the high PIP ventilation protocol in groups of WT (HP WT) mouse lungs, WT lungs with KO macrophages added (HP WT+KO AM), KO lungs (HP KO), and KO lungs with WT macrophages added (HP KO+WT AM). *P < 0.05 vs. the same group after 30 min. #P < 0.05 vs. KO group within the same time period. C: wet-to-dry lung weight ratios after low and high PIP protocols in all groups. *P < 0.05 vs. same treatment group ventilated with the low PIP protocol. #P < 0.05 vs. KO group within the same time period.
Fig. 3.
Fig. 3.
Calcium response of transient receptor potential vanilloid 4 TRPV4−/− and TRPV4+/+ macrophages stained with fluo-4 dye treated with 4-α-phorbol didecanoate (4αPDD). A: fluorescence images of TRPV4−/− and TRPV4+/+ macrophages after treatment with 4αPDD. B: time courses of fluorescence intensities of individual TRPV4−/− and TRPV4+/+ macrophages after treatment with 4αPDD. C: mean fluorescence intensities of fluo-4 in TRPV4+/+ and TRPV4−/− macrophages with and without treatment with 4αPDD. *P < 0.05 vs. baseline value in the same genotype. #P < 0.05 vs. TRPV4−/− group after same treatment.
Fig. 4.
Fig. 4.
Superoxide production in TRPV4−/− and TRPV4+/+ macrophages with and without 4αPDD. A: TRPV4+/+ and TRPV4−/− macrophages stained with MitoTracker Red after treatment with 4αPDD. B: mean fluorescence intensities of MitoTracker Red in TRPV4+/+ and TRPV4−/− macrophages with and without treatment with 4αPDD. *P < 0.05 vs. baseline value in the same genotype. #P < 0.05 vs. TRPV4−/− group after same treatment.
Fig. 5.
Fig. 5.
Nitric oxide production in TRPV4−/− and TRPV4+/+ macrophages with and without 4αPDD. A: micrographs of TRPV4−/− and TRPV4+/+ macrophages stained with 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) dye after treatment with 4αPDD. B: mean fluorescence intensities of DAF-FM in TRPV4+/+ and TRPV4−/− macrophages with and without treatment with 4αPDD. *P < 0.05 vs. baseline value in the same genotype. #P < 0.05 vs. TRPV4−/− group after same treatment.
Fig. 6.
Fig. 6.
Scanning electron micrographs (SEM) of bronchoalveolar lavage macrophages showing freshly harvested TRPV4−/− (A) and TRPV4+/+ (C) macrophages and TRPV4−/− (B) and TRPV4+/+ (D) macrophages after 24-h incubation and treatment with 4αPDD. Note protrusions and spreading (arrow) of TRPV4+/+ macrophage treated with 4αPDD.
Fig. 7.
Fig. 7.
Bar graphs summarizing mean diameters (A) and mean cross-sectional areas (B) of TRPV4−/− (black bars) and TRPV4+/+ (gray bars) macrophages, freshly harvested (control), after 1 day in culture (baseline) and after challenge with 4αPDD. *P < 0.05 vs. baseline value in the same genotype. **P < 0.05 vs. baseline and 24-h culture groups of the same genotype. #P < 0.05 vs. TRPV4−/− group after same treatment.
Fig. 8.
Fig. 8.
Immunohistochemistry staining of mouse lungs for nitrotyrosine (brown color) in lung tissue counterstained with hematoxylin. A, TRPV4−/− lungs ventilated at low PIP; B, TRPV4+/+ lungs ventilated at low PIP; C, TRPV4−/− lungs ventilated at high PIP; D, TRPV4+/+ lungs ventilated with high PIP; E, high PIP ventilated TRPV4−/− lungs with TRPV4+/+ macrophages added; F, high PIP ventilated TRPV4+/+ lungs with TRPV4−/− macrophages added. Brown color indicates nitrotyrosine staining.
Fig. 9.
Fig. 9.
Regression analysis of group Kf values on values of nitrotyrosine staining (area × intensity) relative to the LP KO value in LP KO, HP KO, LP WT, HP WT, HP WT+KO AM, and HP KO+WT AM groups (r2 = 0.87).
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