Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

Abstract

Rationale: Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition.

Methods: We investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF).

Results: Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF.

Conclusion: These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

Figure 1

(A) RT-PCR analysis of human Cx43, Cx26, Cx32 mRNA expression in cultured lung fibroblasts. Representative results obtained by analysis of RNA extracted from 5 normal lung fibroblast cultures (N1-5) and 3 fibrotic fibroblast cultures (F1-3). (B) densitometry analysis of RT-PCR analysis of human Cx43 mRNA expression in cultured lung fibroblasts. RNA samples from human liver (Li) and heart (He) were also analyzed as positive controls. L: 100-bp ladder. (C) Human Cx43 mRNA level measured by quantitative real-time RT-PCR in cultured normal and fibrotic fibroblasts. Results obtained from commercially available human lung RNA sample has been reported as control. Relative quantification was performed by the 2^-ΔΔCt method using as calibrator the value obtained from human normal lung. Data represent mean ± SD of 5 primary normal fibroblast lines and 3 fibrotic fibroblast lines. NF normal lung fibroblast, FF fibrotic lung fibroblast. §p = NS, * p < 0.05 in comparison to normal fibroblasts.

Figure 2

Western blots for detection of Cx43 and β-actin in normal lung fibroblast cultures and fibrotic fibroblast cultures.

Figure 3

Time course of homologous gap junctional intercellular communication (GJIC) and effect of carbenoxolone on the calcein dye transfer of normal human lung fibroblasts. Data represent the number of calcein-positive recipient cells as percentage of total recipient cells and are the averages of four different normal lines ± SD. ◆ Normal fibroblasts, □ Normal fibroblasts treated with 100 μM carbenoxolone.

Figure 4

Dose-response curve of CBX on gap junctional intercellular communication in normal human lung fibroblasts. Data are expressed as the percentage of maximal inhibition of calcein dye transfer and are the averages of four dishes ± SD.

Figure 5

Representative flow cytometry dot plots showing calcein dye transfer trough gap junctions at different time points in a normal fibroblast line in the absence (A) and in the presence (B) of CBX (100 uM). At time 0 the recipient cells (DiI and calcein negative) are in the left-bottom box and donor cells (DiI and calcein positive) in the right-upper box. Note in A the time-dependent increase in calcein positive cells in the recipient cell populations (right-bottom box). This increase is almost completely blocked by CBX in B.

Figure 6

Homologous GJIC assayed by means of the calcein dye transfer technique on normal and fibrotic human lung fibroblast at different time points. Open bar represents normal human lung fibroblasts, N = 5; Filled bar represents fibrotic human lung fibroblasts N = 3; All the experiments were conducted in triplicate * p < 0.01 vs. normal fibroblasts.

Figure 7

Heterologous GJIC in normal and fibrotic human lung fibroblasts. A single normal fibroblast cell line was used as dye donor over all of the other recipient cell lines. Normal fibroblasts (N = 5) vs. fibrotic fibroblasts (N = 3) at 120 minutes. All the experiments were conducted in triplicate. * p < 0.05.