Pulmonary Endothelial Protein Kinase C-Delta (PKCδ) Regulates Neutrophil Migration in Acute Lung Inflammation

Abstract

Excessive neutrophil migration across the pulmonary endothelium into the lung and release of oxidants and proteases are key elements in pathogenesis of acute lung injury. Previously, we identified protein kinase C-delta (PKCδ) as an important regulator of proinflammatory signaling in human neutrophils and demonstrated that intratracheal instillation of a TAT-conjugated PKCδ inhibitory peptide (PKCδ-TAT) is lung protective in a rat model of sepsis-induced indirect pulmonary injury (cecal ligation and puncture). In the present study, intratracheal instillation of this PKCδ inhibitor resulted in peptide distribution throughout the lung parenchyma and pulmonary endothelium and decreased neutrophil influx, with concomitant attenuation of sepsis-induced endothelial ICAM-1 and VCAM-1 expression in this model. To further delineate the role of PKCδ in regulating neutrophil migration, we used an in vitro transmigration model with human pulmonary microvascular endothelial cells (PMVECs). Consistent with in vivo findings, inhibition of PMVEC PKCδ decreased IL-1β-mediated neutrophil transmigration. PKCδ regulation was stimulus-dependent; PKCδ was required for transmigration mediated by IL-1β and fMLP (integrin-dependent), but not IL-8 (integrin-independent). PKCδ was essential for IL-1β-mediated neutrophil adherence and NF-κB-dependent expression of ICAM-1 and VCAM-1. In PMVECs, IL-1β-mediated production of ROS and activation of redox-sensitive NF-κB were PKCδ dependent, suggesting an upstream signaling role. Thus, PKCδ has an important role in regulating neutrophil-endothelial cell interactions and recruitment to the inflamed lung.

Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury

“…Under continued anesthesia and mechanical ventilation, the thorax was opened and the lungs were fixed by vascular perfusion according to standard protocols (Fehrenbach et al, 1999) or by gravity fixation intratracheally as previously described (Mondrinos et al, 2014). Lung tissue samples were obtained from different locations in the left and right lung to assess the uniformity of delivered peptide distribution.…”

“…Sepsis was induced by the cecal ligation and puncture (CLP) method as described previously (Rittirsch et al, 2009;Kilpatrick et al, 2011;Mondrinos et al, 2014). Sham controls underwent a laparotomy without cecal ligation or puncture.…”

“…Antigen retrieval was achieved by boiling slides for approximately 10 minutes in citrate buffer, pH 6.0 (Mondrinos et al, 2014). Following antigen retrieval steps, slides were blocked with 3% bovine serum albumin for 1 hour.…”

“…Lung tissue samples were obtained from different locations in the left and right lung to assess the uniformity of delivered peptide distribution. Lung tissue was paraffin-embedded, cut into 5-to 10-mm sections, H&E stained, and stained with specific antibodies for immunohistochemistry as described previously (Mondrinos et al, 2014).…”

“…Inflammatory pathologies such as ischemia-reperfusion (Chou et al, 2004) and endotoxin-induced lung injury (Chichger et al, 2012) are attenuated in PKCd 2/2 mice. We have shown that selective PKCd inhibition attenuates acute lung inflammation, reduces alveolar-capillary permeability and pulmonary edema, and preserves lung architecture in a rat model of sepsis-induced acute lung injury (Kilpatrick et al, 2011;Mondrinos et al, 2014). Therefore, targeting PKCd is a potential strategy for the treatment of indirect acute inflammatory lung injury.…”