Attenuation of obliterative bronchiolitis by a CXCR4 antagonist in the murine heterotopic tracheal transplant model

Abstract

Background: Long-term success in lung transplantation is limited by obliterative bronchiolitis (OB), yet the mechanism for this disease is not well understood. Chemokine SDF-1 and its receptor, CXCR4, have been reported to be involved in several fibrogenic processes by recruiting inflammatory and fibroblast progenitor cells into injured tissues. We hypothesized that the SDF-1/CXCR4 axis also plays a role in the pathogenesis of OB.

Methods: Using the mouse heterotopic allogeneic airway transplant model, we transplanted mouse tracheas from BALB/c donors into C57BL/6 recipients. At Day 10 after transplant, we found high expression of SDF-1 in cells in the sub-epithelial layers of the allograft. Approximately 26% of cells infiltrating the allograft were CD45(+)CXCR4(+), as determined by flow cytometry analysis.

Results: Treatment of the recipients with a CXCR4 antagonist, TN14003, decreased cell infiltration into the grafts at Day 10 post-implantation. At Day 42, a significant reduction in luminal occlusion was found in the TN14003-treated animals compared with controls (57.40% vs 98.21%, p < 0.01). To demonstrate the relevance of the SDF-1/CXCR4 axis in OB, sections of lung tissue obtained from lung transplant patients with OB were examined for SDF-1 and CXCR4 expression. We found a higher number of CXCR4- and SDF-1-positive cells in samples from patients with OB as compared with normal lungs.

Conclusions: These findings provide new insights into the mechanisms of lung chronic rejection and may lead to new intervention tools for the treatment of OB.

Figures

Figure 1

SDF-1/CXCR4 expression at day 10 post-transplant. Mice were sacrificed day 10 post-transplant. (A) Frozen trachea sections from isograft and allograft groups were stained with a rabbit anti-SDF-1 antibody (red fluorescence). Photographs were taken using a fluorescence microscope at 20x magnification. (B) Frozen sections from isografts and allografts were stained with a rabbit anti-CXCR4 antibody. (C) CXCR4 protein expression as shown in western blot.

Figure 2

CXCR4 antagonist reduces epithelial loss and trachea occlusion. C57BL/6 mice were transplanted heterotopically with C57BL/6 (isograft) or BALB/c (allograft) trachea. In allograft group, half of the mice were injected daily with 160ng/g of CXCR4 antagonist TN14003, a 14 amino acid peptide with loop structure. Tracheal grafts were harvested on days 10 and 42 post-transplant, stained with H&E (figure 2A, 2B) and percentage of trachea occlusion determined on day 42 (figure 2C). Values represent mean ± standard error. n=4, *, p<0.05.

Figure 3. TN14003 reduces CD45+CXCR4+ cell infiltration

(A) Trachea cells from day 10 post-transplant in isograft (Iso), allograft (Allo) and allograft plus TN14003 groups were labeled with anti-mouse CXCR4 and CD45 and subjected to FACS analysis. (B) Paraffin sections from Isografts, allografts and allografts plus TN14003 groups were stained with an anti-CD4 or an anti-CD8 antibody. Cells with dark brown staining represent positive cells for CD4 or CD8.

Figure 4. Effect of TN14003 on cytokine levels

(A) TGF-β1 expression in trachea 10 days post-transplant was determined by immunohistochemistry. (B) Cytokine levels in the serum of recipient mice at day 10 post-transplant were determined using Luminex. Values represent mean ± standard error. *, **, p<0.05, n=4.

Figure 5

Increased SDF-1 and CXCR-4 in OB lung samples compared to normal human lungs. Lung tissues from OB patients (right) and normal control (left) were paraffin embedded and sectioned. Immunohistochemistry was performed for SDF-1 (A) and CXCR-4 (B). Cells stained dark brown were SDF-1 or CXCX4 expressing cells (see arrows).