Contribution of Connexin Hemichannels to the Pathogenesis of Acute Lung Injury

Abstract

Connexin (Cx) family members form hemichannels (HCs) and gap junctions (GJs). Biological functions of Cx HCs have not been adequately characterized due to the inability to selectively target HCs or GJs. Recently, we developed a 6-mer peptide mimetic (P5) of the first extracellular loop of Cx43 and showed that it can block the permeability of HCs but not GJs formed by Cx43. In this study, we further characterized the HC blocking property of P5 and investigated the role of Cx HCs in acute lung injury (ALI). We found that P5 administration decreased HC permeability, in pulmonary microvascular endothelial cells, HepG2 cells, and even Cx43-deficient astrocytes, which express different sets of Cxs, suggesting that P5 is a broad spectrum Cx HC blocker. In addition, P5 reduced HC permeability of alveolar cells in vivo. Moreover, P5 decreased endotoxin-induced release, by vascular endothelial cells in vitro, of high mobility group box protein 1 (HMGB1), a critical mediator of acute lung injury (ALI), and reduced HMGB1 accumulation in bronchoalveolar lavage fluid (BALF) of mice subjected to intratracheal endotoxin instillation. Furthermore, P5 administration resulted in a significant decrease in the concentrations of ALT, AST, and LDH in the BALF, the accumulation of leukocytes in alveoli, and the mortality rate of mice subjected to ALI. Wright-Giemsa staining showed that P5 caused similar reductions of both neutrophils and monocytes in BALF of ALI mice. Together, these results suggest that Cx HCs mediate HMGB1 release, augment leukocyte recruitment, and contribute to ALI pathology.

Figure 1

Cx43 mimetic peptide P5 inhibited LY uptake in vitro. HPMVEC (a, b), HepG2 (c), and Cx43-deficient cell (e) cultures were divided into Cont (saline), P5 (20 μg/ml), LPS (0.5 μg/ml), and LPS+P5 (0.5 μg/ml LPS+20 μg/ml P5) groups. LY was applied to the culture medium for 10 min incubation. Then, cells were fixed with paraformaldehyde and stained with DAPI. The ratio of LY/DAPI fluorescence signals was used to determine HC permeability. The unit of the LY/DAPI ratio is arbitrary, and the values are not comparable between experiments using different cells. (d) Expression of Cx43 in HepG2 and NIH3T3 cells with or without LPS challenge was examined using Western blotting. a.u.: arbitrary unit.

Figure 2

The effects of P5 on HC permeability (a, b) and Cx expression (c, d) of alveolar cells in vivo. (a, b) At 20 hr after LPS challenge, saline or P5 was administered (ip) twice at 2 and 16 hr post-ALI induction. After 10 min, mouse lungs were perfused with PBS, fixed with PF, and then cut into sections that were subsequently stained with DAPI. LY-containing cells appeared in green, showing LY uptake through HCs. Nuclei of all alveolar cells were stained with DAPI and appeared in blue. The ratio of LY/DAPI fluorescence signals was used to determine HC activity. a.u.: arbitrary unit. (c, d) Saline or P5 was administered (ip) twice at 2 and 16 hr post-ALI induction. After BALF extraction, lungs were harvested and homogenated to determine the expression of Cx37, Cx40, and Cx43 by Western blotting.

Figure 3

P5 inhibited the extracellular release of HMGB1 in vitro and in vivo. (a) Cultured HPMVEC were divided into Cont, P5 (20 μg/ml), LPS (0.5 μg/ml), and LPS+P5 (0.5 μg/ml LPS and 20 μg/ml P5) groups. At 16 hr post-LPS challenge, culture medium was collected, concentrated, and subjected to Western blotting analysis of HMGB1. n = 4. (b) Balb/c mice were divided into 4 groups: Sham (surgery+saline), P5 (8 mg/kg P5), ALI (5 mg/kg LPS+saline), and ALI+P5 (5 mg/kg LPS + 8mg/kg P5). ALI was induced by intratracheal instillation of LPS. Saline or P5 was administered twice through ip at 2 and 16 hr post-ALI induction. BALF was collected at 4 hr after second saline or P5 administration, concentrated, and subjected to Western blotting analysis of HMGB1. a.u.: arbitrary unit; n = 10.

Figure 4

Effects of P5 on the levels of ALT (a), AST (b), and LDH (c) in BALF and survival rate (d) of ALI mice. (a–c) Mouse ALI induction and treatments were performed in the same fashion as in Figure 3(b). The relative levels of AST (a), ALT (b), and LDH (c) in BALF were assessed by Western blotting. n = 10. ^∗P < 0.05. Values are in arbitrary units (a.u.). (d) Same volume (200 μl) saline or P5 (8 mg/kg) was administered twice through ip at 2 and 16 hr post-ALI induction (5 mg/kg LPS, intratracheal instillation) in Balb/c mice. Survivors were counted daily for 10 days. Arrows indicate the time of saline or P5 injections, n = 30.

Figure 5

P5 reduced alveolar leukocyte recruitment. (a, b) Saline (Sham) or P5 (8 mg/kg, P5) was injected twice though ip at 2 and 16 hr post-ALI induction in mice. At 4 hr after second administration, mouse lungs were fixed in situ with PF, cut into 10 μm sections, and stained with H&E. The counts of alveoli with and without leukocytes were obtained in a double-blind manner. n = 10, ^∗P < 0.05. (c) Mouse BALF samples were obtained as described in Figure 3(b). Flow cytometry analysis of BALF was performed at Body Fluid mode to obtain total counts of alveolar leukocytes. n = 10, ^∗P < 0.05. (d) After centrifugation at 1,000 g, cell pellets from BALF were resuspended in serum and subjected to Wright-Giemsa staining on a glass slide. The counts of monocyte/macrophage (MN), neutrophil (PMN), and lymphocyte (Lym) and the ratio (%) of each cell type were obtained. N = 10.