Dipl.-Biol. Oliver Stehling

Institute of Anatomy and Cell Biology - Department of Immunobiology

Philipps-University of Marburg

Robert-Koch-Str. 6

35033 Marburg

The Department of Immunobiology at the Philipps-University of Marburg is investigating the role of  the monocyte - macrophage system during immune reactions in vivo. At the Annual Meeting of Immunology we are presenting some of our results about the functional role of monocytes during acute renal allograft rejection.
 

Workshop L
Allo- and Xenotransplantation
Abstract L.31 (Word97 or HTML)

Department of Immunobiology, Institute of Anatomy and Cell Biology, Philipps-University, Marburg, Germany

Cytotoxic activity of rat monocytes during acute renal allograft rejection

During acute rejection of fully allogeneic DA to  LEW renal grafts, monocytes and macrophages are thought to play an important role in orchestrating the alloreactive T cell response. However, only little is known about the direct contribution of activated monocytes to allograft destruction. Recently, we have shown that large numbers of monocytes accumulate inside allograft vessels, where they express the mRNA of genes the products of which might lead to intravascular coagulation and graft damage. In the present study, we perfused the vasculature of acutely rejecting rat renal allografts and analyzed the immunophenotype of monocytes by flow cytometry. Most of them exhibited an immunophenotype associated with activation. Particularly, they expressed CD8 and NKR-P1A, typical surface molecules of cytotoxic T lymphocytes and Natural Killer cells. Considering these features, we studied the ability of immunomagnetically purified monocytes to kill the standard tumor target Yac-1 in a non-radioactive, flow cytometry-based cytotoxicity assay in vitro. Using monocytes from allografts or from allograft recipients, specific lysis of Yac-1 after 6 hours coincubation at an effector-target-ratio of 1:100 was about 30 %. The cytotoxic effect of monocytes isolated from isograft recipients and untreated control animals was 1,5 and 3 fold lower, respectively. For comparison, the cytolytic activity of monocytes isolated from allografts or from allograft recipients was about half as high as that of Natural killer cells as determined by comparable assays. In conclusion, monocytes activated during acute renal allograft rejection express molecules associated with cell-mediated cytotoxicity and acquire a certain degree of natural cytolytic activity. Taking into account the high numbers of monocytes accumulating in the blood vessels of acutely rejecting renal allografts, monocyte cytotoxicity might play a considerable role in allogeneic kidney graft destruction.
 
 

Workshop L
Allo- and Xenotransplantation
Abstract L.8 (Word97 or HTML)

Institutes of Anatomy and Cell Biology and *Immunology, Philipps-University, Marburg, Germany

Do intravascular monocytes mediate acute renal allograft rejection in the rat?

In fully allogeneic (DA to LEW) rat kidneys necrotic patches and hemorrhagic lesions develop between day 4 and day 5 after transplantation. These lesions are probably due to destruction and obstruction of blood vessels. Intravascular graft leukocytes most likely mediate endothelial cell damage and intravascular coagulation. However, this cell population has not been thoroughly characterized so far. Non-adhering leukocytes and leukocytes adhering to endothelial cells were harvested from untreated control kidneys, renal isografts, and allografts on day 4 after transplantation by perfusion of the renal vasculature with large volumes of PBS/EDTA and the cellular composition of the perfusates was determined by flow cytometry. For comparison leukocytes isolated from the extrapulmonary vasculature were analyzed. The expression of typical products of activated monocytes was analyzed by RT-PCR and immunohistochemistry. About 100 million leukocytes were isolated from a single allogeneic kidney whereas the graft vasculature of isografts contained only about 10 million white blood cells. 73 percent of the allograft perfusate cells were monocytes. Most of them exhibited an activated immunophenotype. Intravascular allograft leukocytes expressed more TNF-a, iNOS, and tissue factor mRNA compared to cells isolated from isografts. Tissue factor expression was only detected in graft perfusates, whereas TNF-a and iNOS mRNA was also expressed by leukocytes of the extrapulmonary system. In conclusion, large numbers of monocytes accumulate inside allograft vessels. These cells might play a pivotal role in allograft destruction as they express genes the products of which are known to induce cell death and thrombosis.