THE PARTICIPATION OF TUMOR-NECROSIS-FACTOR IN THE PATHOGENESIS OF LUNG ALLOGRAFT-REJECTION IN THE RAT

Tumor necrosis factor alpha and beta are polypeptide cytokines with a wide range of metabolic, immunologic, and inflammatory activities. TNF is known to participate in immune meditated injury of native lungs, b ut a role for TNF in mediating lung allograft rejection (AR) has not b een established. In experiments reported here, we assessed the role of TNF in mediating lung AR in a rat model of lung transplantation (BN-- >Lew) (RT1n-->RT1l). This model shows florid AR with all grafts comple tely destroyed by day 6 posttransplant. Graft pathology is characteriz ed by massive lymphocytic infiltrates and hemorrhagic necrosis. Initia lly, 5 lung allograft recipients in each group were sacrificed on days 1 to 6 posttransplant. Allografts were removed, mRNA isolated, and No rthern blotting or RT-PCR performed with blots probed with cDNAs or ol igos specific for rat TNF-alpha cyclophylin and gamma-actin. Data were compared with syngeneic transplants (Lew-->Lew) and with normal contr ols. In addition, frozen lung allograft tissue was examined by indirec t immunofluorescence, using antibodies specific for TNF. TNF-alpha mRN A levels were detectable on day 2 posttransplant, and peaked on days 6 -7 posttransplant. IF studies showed TNF protein expression in mononuc lear cells of rejecting allografts on day 3, peaking on day 6. Both TN F-alpha mRNA and protein levels correlated with maximal AR and hemorrh agic necrosis of grafts. Minimal TNF-alpha mRNA or protein was detecte d in syngeneic grafts or in contralateral native lungs. We than examin ed the ability of a rabbit polyclonal anti-TNF-alpha (7000 U/day) and anti-TNF-beta (5000 U/day) with 30% crossreactivity with rat TNF to mo dify the AR response. For each group, 4-5 left lung transplants were p erformed as described, and animals treated with anti-TNF-alpha, anti-T NF-beta, (anti-TNF-alpha + anti-TNF-beta) or with preimmune rabbit ser a. All animals were sacrificed on day 6 posttransplant. Several pathol ogical categories of inflammation were examined and scored (0-4), with a score of 0=0% involvement; 1=1-25% involvement; 2=26-50% involvemen t; 3=51-75% involvement; and 4=76-100% involvement. The mean and SD sc ores were obtained for all animals in the treatment categories mention ed above, and compared with preimmune-treated controls. Briefly, no di fferences in perivascular, peribronchial, or peribronchiolar cell infi ltrates or edema were seen in treatment groups compared with controls. However, profound differences were seen in scores for vasculitis (1.6 +/-0.5 vs. 3+/-0), (P< 0.0001), necrosis (0+/-0 vs. 3.0+/-0.71) (P<0.0 0001), and interalveolar hemorrhage (0.6+/-0.5 vs. 3.5+/-0.71) (P< 0.0 0001) in the (anti-TNF-alpha + anti-TNF-beta) treatment group compared with preimmune controls. Both anti-TNF-alpha and anti-TNF-beta treatm ent groups showed ameliorative effects on hemorrhagic necrosis and vas culitis, but not to the degree seen with the combination of both antib odies. These data suggest that TNF-alpha and beta are important mediat ors of lung AR in the rat. Although the intensity of cellular infiltra tion was not affected by anti-TNF therapy, there were profound reducti ons in vasculitis and hemorrhagic necrosis of the grafts.