Cells of the monocyte/macrophage system originate from the bone marrow, reach the organs via the blood, immigrate through postcapillary venules and further differentiate into organ-specific tissue macrophages. In rats and other species, activated monocytes/macrophages aggravate autoimmune reactions, rejection of non-vascularized allografts and chronic allograft rejection. It is very likely that they also contribute to acute allograft destruction. So far it has been impossible to distinguish the function of monocytes from that of macrophages, because cell phenotypes and their alterations upon activation are ill-defined. We have thus begun to characterize the ex vivo phenotype and function of rat monocytes in the normal state and during renal allograft rejection. Monocytes are recovered from both the central and the marginal blood pool by perfusing either the recipient's circulation or the allograft vasculature. Rat monocytes have a unique surface phenotype. During allograft rejection or after infusion of interferon-gamma they up-regulate class II MHC molecules, CD161 (NKR-P1A), CD62L and CD8, while CD4 and CD43 are down-modulated. Activated perfusate monocytes exert increased in vitro cytotoxicity against tumour targets, which differs from that of NK cells. We speculate that activated monocytes contribute to kidney allograft destruction by directly damaging endothelial cells or by promoting intravascular coagulation.

LEW rats were treated intravenously with recombinant rat interferon-γ (IFN-γ) for 3 days to achieve intravascular accumulation, proliferation, and activation of monocytes. Monocytes, defined by their expression of the ED1, ED9, and Ox41 antigens, were recovered from the vasculature by perfusion with PBS/EDTA, subsequently depleted of erythrocytes and granulocytes by Percoll density gradient centrifugation, and analyzed by flow cytometry and immunocytology. In untreated and control-infused specified pathogen-free (SPF) rats, lymphocytes and monocytes formed overlapping cell populations with respect to size and internal granularity. At least two intravascular monocyte subsets, probably central and marginating cells, were distinguished by their size and differential expression of CD43, CD4, CD11a, CD18, and L-selectin. It is interesting to note that a fraction of the monocytes in normal and control-infused animals carried the NKR-P1A molecule. IFN-γ treatment provoked a duplication of monocyte size and granularity. Both the number of positive monocytes and the level of expression of NKR-P1A strongly increased after IFN-γ infusion, whereas CD43 (leukosialin) and CD4 were impressively down-regulated. NKR-P1A+ L-selectin+CD43low CD4- monocytes also occur in the vasculature of rats during immune reactions in vivo. We speculate that these cells are involved in organ damage and that their number is controlled by activation-induced cell death within the vessels.

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Scriba A, Grau V, Steiniger B. Phenotype of Rat Monocytes During Acute Kidney Allograft Rejection: Increased Expression of NKR-P1 and Reduction of CD43. Scand J Immunol 1998;47;332-342 Monocytes and macrophages mediate cytotoxicity after appropriate activation and thus represent effectors secondary to T lymphocytes and natural killer (NK) cells. However, very little is known about the role of activated monocytes in organ allograft rejection. In this study, isogeneic (LEW to LEW) and fully allogeneic (DA to LEW) rat kidneys were grafted to bilaterally nephrectomized recipients. Four days after transplantation a comprehensive sample of leucocytes was recovered by perfusion of the recipients' vasculature with phosphate-buffered saline containing EDTA (PBS/EDTA). Monocytes were enriched by density gradient centrifugation and their physical parameters and immunophenotype were investigated by flow cytometry in comparison with untreated, specified pathogen-free (SPF) LEW rats. Isotransplantation and allotransplantation of kidneys dramatically increased the absolute number of intravascular monocytes in the recipient. Analysis of NKR-P1 (CD161), CD4, CD62L, CD43, CD11a, CD18 and MHC class II expression identified at least two monocyte populations in all experimental groups. In graft recipients it was evident that activated monocytes were able to express and upregulate NKR-P1 and CD8, which have not been detected in these cells to date. If activated monocytes utilize NKR-P1 and CD8, by analogy to NK cells and lymphocytes these cells may be endowed with additional pathways to upregulate cytolytic functions and effect allograft damage. Professor B. Steiniger,