Anti-TNF-alpha treatment down-regulates the expression of fibronectin and decreases cellular infiltration of cardiac allografts in rats.

Extracellular matrix (ECM) components provide costimulatory signals for T cell activation in vitro, and may be critical for lymphocyte migration and tissue positioning in vivo. We conducted a series of studies in rat recipients of cardiac allografts to evaluate intragraft expression of a prominent ECM protein, fibronectin (FN), and to analyze the effects of infusing a neutralizing anti-TNF-alpha serum on FN expression and lymphocyte migration into the transplants. LBNF1 cardiac allografts were rejected within 8 days in control LEW rats. A prominent immunohistochemical feature of this immune response was the dense deposition of FN at the graft site as early as 3 h, which then peaked at 4 to 6 days. The early 3-h FN deposition (likely plasma FN) was noted before cellular infiltration. Northern blot analysis established that a marked induction of FN mRNA expression occurred in rejecting cardiac allografts at day 4 after transplantation. To determine the source of FN mRNA, we conducted a series of in situ hybridization studies with probes for FN and lysozyme, a macrophage-specific marker. Indeed, the majority of graft-infiltrating cells expressed lysozyme mRNA and FN mRNA. Administration of anti-TNF-alpha serum into LEW hosts (0.5 ml i.v. at days 1 and 3 only) abrogated acute rejection and prolonged cardiac allograft survival to approximately 13 days. This was accompanied by depressed circulating and intragraft TNF-alpha levels, and markedly down-regulated FN mRNA/protein expression patterns, as compared with those in recipients given nonimmune rabbit serum. Anti-TNF-alpha treatment also markedly decreased graft infiltration by ED1+ monocytes/macrophages, OX-8+, and VLA-4+ cells, normally peaking at 4 days. Moreover, we found that the migration of 111In-labeled specifically sensitized lymph node lymphocytes to cardiac allografts in secondary rat recipients conditioned with anti-TNF-alpha serum was significantly decreased, as compared with that in controls. Thus, FN expression by intragraft macrophages occurs within the same interval as cellular infiltration, and may act as an ECM component "signal" for selective homing of recirculating lymphocytes in graft recipients. The results of this study support the notion that in vivo interactions between mononuclear cells and ECM may be vital for the ingress of alloreactive lymphocytes at the graft site, and offer potential novel sites for therapeutic intervention in the control of transplant rejection.