Arto Nemlander

The inflammatory mechanisms of allograft rejection

If one wishes to describe the rejection process in biologicai terms, it may be divided into the following, largely overlapping components: (a) induction of rejection, i.e., how does the alien graft induce the antiallograft immune response, (b) the immune response towards the graft, and (c) the inflammatory response of rejection and regulation of the inflammation by the immune response. Finally one should consider (d) the effector mechanisms in situ, i.e., how do the inflammatory cells, together with the antibody, destroy the graft.

Effect of Cyclosporin A on the in Situ Inflammatory Response of Rat Renal Allograft Rejection

The impact of cyclosporin A (CyA) on a normal kidney parenchyma and on the in situ inflammatory response of rejection was investigated in normal DA rats and after transplantation of DA renal allografts to Lewis recipients. In a normal, non‐transplanted DA kidney more than 80 mg/kg/day of CyA induced light‐microscopic changes in the distal tubular cells of the renal cortex and outer medulla. These changes were not accompanied by any visible inflammation and were directly proportional to the dose of the drug and to the duration of drug administration. Treatment of a transplant recipient with 40 mg/kg/day of CyA abolished or at least efficiently reduced the in situ inflammatory response of rejection both as analysed from tissue sections and as quantified from the recovery of inflammatory cells after enzymatic digestion. It also reduced efficiently not only the number of T and B blast cells of the inflammatory infiltrate but also the number of other inflammatory cells, such as in situ lymphocytes, monocytes, and macrophages, and abolished or at least reduced the generation of (T) killer cells in situ and in the recipient spleen. These effects were inversely proportional to the time elapsed between grafting and initiation of treatment: although a complete suppression of all three features was obtained if the drug treatment was initiated already on the day of transplantation, a significant reduction of these functions was still found if the treatment was initiated later when the blastogenic response was already underway.

In situ effector pathways of allograft destruction: 1. Generation of the “cellular” effector response in the graft and the graft recipient

Inflammatory leukocytes of DA-to-WF rat renal allografts displayed significant cytolytic activity to natural killer (NK) target cells on Day 2 after transplantation. The NK activity, which was associated with large granular lymphocytes in discontinuous Percoll gradients, peaked on Day 4 and disappeared rapidly thereafter. Coincident with the presence of NK activity in the graft, a decrease in NK activity in the recipient spleen was observed. Low NK activity was also recorded in WF-to-WF autografts. The cells displaying direct cytotoxic activity to donor (but not to recipient) strain peritoneal exudate target cells (PEC) were associated with the T suppressor/killer lymphocytes in affinity chromatography. They appeared in the graft between Days 2 and 4, peaked between Days 6 and 8 and disappeared slowly thereafter. In the spleen the cytotoxic T lymphocyte (CTL) activity appeared later and it reached a maximum between Days 16 and 20 before decreasing. In the blood distinct CTL activity was seen only from Days 16-20 onwards, after the graft had been rejected. No CTL activity was recorded in the graft, blood, or spleen of an autograft recipient. Addition of donor-directed post-transplantation antibody (antibody-dependent cellular cytotoxicity, ADCC) had a slight enhancing effect on the cytotoxic activity of inflammatory leukocytes up to Day 5. After this time, added antibody had a blocking effect on direct CTL activity. No ADCC activity was recorded in the inflammatory population of an autograft. On the contrary, high levels of ADCC activity to donor strain PEC were recorded in the spleens of both autograft and allograft recipients throughout the period of follow-up. The results demonstrate that at least three cellular effector pathways exist in an allograft: a strong natural killer cell component, a strong cytotoxic T lymphocyte component, and (possibly) a weak cell component participating in an ADCC type of cytotoxicity.

Immunogenicity of allograft components: II. Relative immunogenicity of rat kidney parenchymal versus “passenger” cells

Abstract Well-perfused adult DA kidneys were enzymatically dispersed under conditions which do not affect the expression of cell surface major histocompatibility antigens. The kidney cell suspensions were separated via sedimentation at unit gravity into three fractions: I, rapidly sedimenting (>6.5 mm/hr) enriched for kidney tubular and glomerular cells and depleted of passenger leukocytes (76 and 8%, respectively); II, intermediate (5.1–6.0 mm/hr) mixed population equivalent to the unseparated kidney cell suspension (52% tubular and glomerular cells, 20% endothelial cells, and 28% passenger leukocytes); and III, slow sedimenting (

In situ effector pathways of allograft destruction: 2. Generation of the “humoral” response in the graft and the graft recipient

The frequency of both immunoglobulin (Ig)-synthesizing and Ig-secreting B cells have been analyzed in DA-to-WF rat renal allografts (and in control WF-to-WF autografts). We have correlated the in situ B-cell responses with corresponding events in the central lymphatic system of the recipient. Intracellular IgM- and IgG-containing plasma cells appeared in an allograft (but not in an autograft) very shortly after the transplantation. The numbers of both cell types in situ was approximately equal, the highest numbers of each being found on Day 4 after transplantation. A similar early response was observed in the recipients spleen, however, very few Ig-synthesizing cells were seen in the blood. Only a fraction of the Ig-synthesizing cells in the allograft were involved in immunoglobulin secretion. Thus, the recovery of IgG- and IgM-secreting cells from an allograft was 10 and 2% of intracellular IgG- and IgM-containing cells, respectively. It appears, therefore, that allograft-infiltrating Ig-synthesizing B cells either die or migrate elsewhere before secreting immunoglobulin. The B-cell response in the graft occurs very early and is disproportionally high when the very low frequency of B lymphocytes in the allograft is considered. The data provide no evidence for inflammatory B cells being an integral part of graft rejection. Indeed, the possibility remains that the inflammatory B-cell response observed during the rejection process represents a meaningless byproduct of the inflammatory response.

Redistribution of rat renal allograft-responding leukocytes during rejection: 1. Model

Abstract We describe a model that enables us to trace the traffic of allograft-responding inflammatory leukocytes to and from the graft without handling of these cells in vitro . At different times after transplantation, the kidney transplant pedicle—including the artery, vein, and draining lymphatics—is clamped. The allograft-responding leukocytes are labeled by a [ 3 H]thymidine pulse either in situ or in the systemic lymphoid organs of the recipient. Fifteen minutes later the pulse is chased with a 1000-fold excess of cold thymidine, and the clamp is opened. The animals are sacrificed 18 hr later, when a balance between the synthesis of new labeled leukocytes from the originally labeled ones and dilution of intracellular label has been achieved. This model was used to analyze the allograft-responding inflammatory cell traffic to and from a renal transplant performed across the major histocompatibility complex in the rat. A sizable traffic was observed to both directions: After systemic injection of label only 0.008 × 10 6 labeled cells × hr −1 were found to emigrate into a kidney allograft (control). Already on the third day after transplantation—when the in situ inflammatory response is still at its beginning—more than 0.3 × 10 6 labeled cells × hr −1 migrated from the host to the allograft and 1.6 × 10 6 labeled cells × hr −1 left the allograft to the recipient spleen. The first figure is several-fold higher than any previous estimate. The findings emphasize the systemic nature of the antiallograft inflammatory response.

Immunogenicity of allograft components: I. Assay for immunogenicity

Abstract We describe an assay for in vivo quantitation of the “immunogenicity” of isolated cell populations. The assay is based on the observation that if an AgB-incompatible recipient rat is primed with donor strain spleen cells 72 hr prior to transplantation, heart allograft survival is reduced from 6.2 to 3.0 days. The effect is independent of the priming cell dose at levels above 3 × 10 5 cells, whereas doses lower than 10 5 spleen cells are unable to reduce the survival. The effect is suboptimal if the priming-transplantation interval is less than 3 days, or is prolonged to 4–10 days. The effect is immunologically specific: priming with irrelevant AgB-incompatible spleen cells fails to reduce the survival. Priming with cell populations previously reported “less immunogenic,” such as ultrasonicated spleen cells, erythrocytes, spleen T cells, or spleen cells deriving from methotrexate or cyclophosphamide-treated rats, fails to reduce the survival, or reduces it only when given in 100-fold higher numbers than the minimal dose of intact spleen cells giving maximal reduction.

Evidence that thymectomized, bone marrow-reconstituted rats do not reject their allografts.

We have investigated the reasons why thymectomized, bone marrow—reconstituted (B) rats do not reject their allografts, by comparing the structure of inflammation and functions of inflammatory cells in nonrejecting allografts to rejecting allografts in normal control recipients. The results demonstrate that B recipients mount a specific cellular response towards the graft. The response in B recipients differs from that in normal controls by a smaller intensity of inflammation, fewer blast cells, and activated mononuclear phagocytes in the inflammatory infiltrate, as well as a delay in the appearance of specific donor-directed lytic activity in the graft. B rats also have fewer blast cells and an inverted CD4/8 ratio in the spleen. There is no obvious absence of any given cell type or cellular function in the graft inflammatory infiltrate. In light of these results no cell type responsible for allograft nonrejection can be pinpointed.

Effect of Irradiation on Rat Renal Transplant Rejection

Leucocytes were selectively eliminated either from a DA renal allograftor from a WF host by irradiation of either the host or the graft on different days after the transplantation. The recovery of inflammatory leucocytes and the generation of lymphoid killer cells—that is, the natural killer (NK) cells and the cytotoxic T lymphocytes (CTL)—were analysed separately in the two compartments. Early irradiation of the graft did not affect the recovery of leucocytes in either compartment. The NK activity was only slightly reduced in the graft but was distinctly reduced in the spleen. A delay in the generation of the CTL activity was observed in the spleen. Late irradiation of the graft reduced the recovery of leucocytes in both compartments. The disappearance of the NK activity increased in the graft but not in the spleen. The CTL activity in the spleen developed normally up to day 6, whereafter it declined. After selective irradiation of the host a fair number of leucocytes remained in the graft, compared with a nearly complete disappearance of leucocytes from the graft and blood. The NK and CTL activity declined rapidly in both compartments. The data demonstrate a bidirectional interdependence between the graft and the host during the rejection.

Natural Cytotoxicity and Allograft Rejection

This book describes the role of natural cytotoxicity in health and disease. As virtually every aspect of natural cytotoxicity is covered by other contributors, we will limit ourselves strictly to the role of natural cytotoxicity and natural killer cells in the rejection of parenchymal organ allografts.