Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury

Abstract

The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ(-/-)) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients (k(f)) or wet-to-dry weight ratios between PKCδ(+/+) and PKCδ(-/-) mice. However, at 24 h after exposure to LPS, the k(f) values were significantly higher in lungs isolated from PKCδ(+/+) than PKCδ(-/-) mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ(+/+) mice displayed increased protein and cell content compared with LPS-exposed PKCδ(-/-) mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ(+/+) mouse lung parenchyma relative to PKCδ(-/-) mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased k(f) lung values in PKCδ(+/+) mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ(-/-) mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.

Fig. 1.

PKCδ-deficient (PKCδ^−/−) mice demonstrate attenuated hydrostatic lung edema formation following LPS exposure. A: lung vascular permeability was assessed by measuring capillary filtration coefficient (k_f) in ex vivo lungs isolated from PKCδ^+/+ and PKCδ^−/− mice. B: after jugular vein catheterization, sedated PKCδ^+/+ and PKCδ^−/− mice were subjected to a hydrostatic challenge, i.e., infusion of NaCl at 40 μl/g mouse over a 2-min period. Lungs were harvested after 30 min, and lung edema was determined as change in wet-to-dry lung weight ratio. C: lungs of PKCδ^+/+ and PKCδ^−/− mice that had been injected intraperitoneally with saline or LPS (5 mg/kg) were collected at 24 h postinjection, and k_f was determined. Values are means ± SE; n = 8–12 (A), 6–14 (B), and 3–8 (C). *P < 0.001 vs. vehicle. #P < 0.001 vs. LPS-treated PKCδ^+/+ mice.

Fig. 2.

Diminished cell infiltration, protein accumulation, and cellularity in PKCδ^−/− mouse lungs upon LPS instillation. A and B: bronchoalveolar lavage (BAL) fluid was collected from PKCδ^+/+ and PKCδ^−/− mice treated with LPS (2.5 mg/kg) intratracheally for 4 h (i) or 24 h (ii) and analyzed for protein concentration and cell count. Values are means ± SE; n = 4–17. *P < 0.01 vs. vehicle. C: representative images of lungs from PKCδ^+/+ and PKCδ^−/− mice treated with LPS (5 mg/kg ip) for 24 h and inflation-fixed and immunohistologically stained with Leder stain and hematoxylin. Scale bars, 50 μm.

Fig. 3.

Disruption of PKCδ attenuates LPS-induced increases in pulmonary vascular damage. A: equal numbers of lung microvascular endothelial cells (LMVEC) were transfected with eukaryotic vectors encoding catalytically inactive PKCδ (PKCδ^K376R) or green fluorescent protein (GFP) cDNA. Resistance across monolayers was measured at 48 h posttransfection. PKCδ protein overexpression was confirmed by immunoblot (IB) analysis of lysates of transiently transfected LMVEC (inset), with relative levels of PKCδ determined via densitometry and expressed as means ± SE. ru, Resistance units. B: normalized resistance of LMVEC overexpressing PKCδ^K376R or GFP cDNA in the presence and absence of LPS (1 μg/ml). Arrow indicates point of addition. A representative trace is shown. C: percent drop in transendothelial resistance (TER) of LMVEC overexpressing PKCδ^K376R or GFP cDNA relative to addition of LPS. Values are means ± SE; n = 8–11. *P < 0.05 vs. GFP vehicle.

Fig. 4.

Acute expression of PKCδ^K376R confers protection against LPS-induced acute lung injury (ALI) in PKCδ^+/+ mice, but acute expression of PKCδ^wt does not influence onset of ALI in LPS-treated PKCδ^−/− mice. PKCδ^+/+ (B) and PKCδ^−/− (A and C) mice were injected with liposomes encapsulating plasmid cDNA encoding PKCδ^K376R, PKCδ^wt, or GFP. At 24 h after liposome injection, LPS was administered (5 mg/kg ip). After an additional 24 h, k_f was assessed. A: to prove overexpression of cDNA in lung endothelium, lungs were harvested from PKCδ^−/− mice that had been injected with liposomes encapsulating GFP or PKCδ^wt cDNA for 24 h, and endothelial cells were isolated from lung homogenate using platelet-endothelial cell adhesion molecule (PECAM-1) antibodies conjugated to magnetic beads and plated for an additional 24 h. Lysates of harvested lung endothelial cells were immunoblotted for PKCδ. Membranes were stripped and reprobed for vascular endothelial (VE)-cadherin expression to confirm that harvested cells were endothelium-derived. Values are means ± SE; n = 3–8. *P < 0.05 vs. GFP vehicle.