Experimental transplantation
Anti-proliferative properties of the phosphodiesterase-4 inhibitor rolipram can supplement immunosuppressive effects of cyclosporine for treatment of obliterative bronchiolitis in heterotopic rat allografts

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Abstract

Background

Potent prevention and therapy of obliterative bronchiolitis may enhance long-term survival after lung transplantation. Phosphodiesterase-4 inhibitors have been established for anti-inflammatory treatment, particularly of pulmonary diseases. Using a heterotopic rat model, the effect of rolipram was investigated and compared with cyclosporine for epithelium disturbance and leukocyte infiltration and proliferation, which are key events in the development of obliterative bronchiolitis.

Methods

Tracheae were transplanted into the omentum of allo- and syngeneic animals. Four allogeneic groups were investigated: treatment with rolipram; treatment with cyclosporine; treatment with a combination of rolipram and cyclosporine; and untreated (60-day time course). Using histo- and immunohistochemical stainings, epithelium disturbance, leukocyte subsets, proliferating cells and luminal occlusion were quantified by digital morphometry.

Results

In rolipram-treated animals, the epithelium was completely disturbed until Day 14. It was temporarily preserved in rats that received cyclosporine until Day 60. In the acute phase (Day 5), infiltration of monocytes/macrophages was significantly inhibited by rolipram, but less effective than in cyclosporine-treated rats. At later timepoints (Days 28 and 60), rolipram significantly inhibited proliferation, in contrast to enhanced proliferation of fibroblast-like cells after cyclosporine treatment. The combination of rolipram and cyclosporine led to temporary epithelial preservation and effective inhibition of leukocyte infiltration (Day 5) and proliferation (Days 28 and 60). Luminal occlusion was significantly reduced in the combination group compared with the cyclosporine-only group.

Conclusions

Although cyclosporine temporary protects epithelial integrity by the inhibition of acute rejection, rolipram showed greater potency for long-term inhibition of mesenchymal-cell proliferation. The combination of both drugs may be useful for limiting chronic obliterative changes after lung transplantation.

Section snippets

Rats

Allogeneic and syngeneic transplantations were performed from Brown-Norway (BN) to Lewis (Lew) and from Lewis to Lewis strains of rats, respectively. Inbred BN (BN/Crl BR) and Lew (LEW/Crl BR) animals were purchased from Charles River (Sulzfeld, Germany). All animals were male, 7 to 10 weeks of age, and weighed 200 to 230 g. The rats were allowed free access to standard-pellet food and water. The rats received humane care in compliance with the Principles of Laboratory Animal Care and the Guide

Epithelium damage

One day after transplantation the respiratory epithelium was diminished in all groups, most probably caused by ischemia during and after transplantation (Figure 1). Thereafter, the coverage of the inner luminal surface layer was re-epithelized from Days 1 to 5. In non-immunosuppressed and rolipram-treated animals the integrity of the epithelial cell layer disappeared, starting on Day 5 FIGURE 1, FIGURE 2. First, the cell surface marker, CD44v6, normally expressed as membranous and luminal

Discussion

The purpose of this study was to compare the effects of CsA and the PDE-4 inhibitor, rolipram, on epithelium disturbance, leukocyte infiltration and proliferation—events suggested to be key steps in the sequential development of OB.

In untreated allografts typical rejection events occurred. The stepwise disturbance of the epithelium started with a lymphocyte-initiated loss of the epithelial cell-cell contacts, followed by a complete predominantly monocyte/macrophage-mediated disturbance until

Acknowledgements

The authors thank B. Jahnke, R. Hentschel and B. Nietsche for excellent technical assistance. We also thank Prof Szelenyi, Dr Marx and Dr Poppe (Arzneimittelwerk Dresden, Asta Medica) for their support in performing the animal studies and for providing rolipram. In addition, we are grateful to Dr H. Fehrenbach for helpful suggestions regarding the digital quantification of immunohistochemical data. This study was supported by Sächsische Aufbaubank.

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