Sequential responses of mouse spleen T cells in mixed lymphocyte culture-induced cytolysis.

T cells triggered to blast transformation and proliferation by histoincompatible cells have the capacity of reverting "back" to lymphocytes. These "secondary" lymphocytes and their progeny cells are able to respond repeatedly to the same allogeneic stimulus in vitro.

Proliferation of B and T cells in mixed lymphocyte cultures.

Electrophoretically fractionated CBA/Ca spleen T cells alone respond to allogeneic cells in one-way MLC and to PHA. They do not respond to E. coli LPS. B cells alone do not respond to allogeneic cells nor to PHA, but do respond to LPS. When karyotypically distinguishable syngeneic mixtures of T and B lymphocytes are stimulated with allogeneic cells, at the most 5% of mitoses on 5-9th culture day are of B cell origin. This indicates that B cells are not substantially recruited to proliferate in the MLC.

Redistribution of renal allograft-responding leukocytes during rejection. II. Kinetics and specificity.

We investigated the traffic of allograft-responding leukocytes between the host and graft without handling of these cells in vitro. The blood flow between the host and graft was disconnected, the proliferating cells were labeled with [3H]thymidine selectively in the graft or in the host, the label was chased with cold thymidine, and the circulation was reestablished. The localization of labeled cells was quantitated by autoradiography. The first host-derived labeled cells appeared in the graft and graft-derived labeled cells in the host, already on the 1st d after transplantation. This was followed by an exponential increase in the labeled cell traffic in both directions. The peak of traffic was observed on day 4 after transplantation, whereafter the traffic rapidly declined and tapered off. This decline was not due to exhaustion of supply, as the labeled cells continued to proliferate in their original compartments, nor to a slowdown of blood circulation, which took place 2-3 d later. We consider the decline to indicate that the rejection has proceeded to a (irreversible) stage autonomous of the host lymphatic and hematopoietic system. During the exponential increase, nearly one-third of the graft-infiltrating inflammatory cells were replaced as a consequence of relocalization during each 18-h-period. All mononuclear white cell types, with the exception of granulocytes, participated in the traffic. Most lymphoid cells entrapped in the graft were descendents of recent cell divisions; most of the mononuclear phagocytes derived from a preexisting phagocyte pool. The entrapment of labeled leukocytes in a relevant graft was specific: when an allograft and an autograft were simultaneously transplanted, a more than 50-fold entrapment was observed in the allograft, compared with the autograft. Very few of the cells localized in irrelevant positions, such as the liver and lung, of the recipient.

Antagonistic effects of gamma interferon and steroids on tissue antigenicity.

A single injection of 10(5) U/kg of recombinant rat IFN-gamma increases the amount of tissue dendritic cells up to sixfold, and concomitantly induces the (capillary) endothelial cells to express class II MHC antigens. Both responses peak on the third day after IFN-gamma injection, and the antigen expression returns to basic levels on day 7. Simultaneous administration of 1 mg/kg/d of methylprednisolone entirely abolishes both responses. These observations demonstrate, for the first time, that IFN-gamma and steroids have antagonistic effects on class II MHC antigen presentation in tissue, and suggest that one immunosuppressive mechanism of glucocorticosteroids in organ transplantation is downregulation of graft antigenicity.

Characterization of surface glycoproteins of mouse lymphoid cells.

We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.

Mixed lymphocyte cultures produce effector cells: model in vitro for allograft rejection.

Mouse peripheral lymphocytes sensitized in vitro by culturing with allogeneic lymphocytes produced immunospecific destruction of target cells, as measured by release of chromium-51. Thus the sensitizing and effector phases of the cell-bound immune response can both be studied in an in vitro system.

Thymus-dependent and thymus-independent lymphocyte separation: relation to exposed sialic acid on cell surface.

On preparative cell electrophoresis mouse lymph node lymphocytes separate into fast-moving (T, thymus-dependent) and slow-moving (B, thymus-independent) fractions. After treatment with neuraminidase all lymphocytes move as one very slow fraction, indicating that the difference in the mobility of the two kinds of cells is due to differences in the density of exposed sialic acid on their surfaces.

Cytomegalovirus infection enhances smooth muscle cell proliferation and intimal thickening of rat aortic allografts.

Inbred DA (AG-B4, RT1a) and WF (AG-B2, RT1v) rats were used as donors and recipients of aortic allografts. The recipient rats were inoculated i.p. either on day 1 (early infection) or on day 60 (late infection) with 10(5) plaque-forming units of rat cytomegalovirus (RCMV). The control rats were left noninfected. The presence of viral infection was demonstrated by plaque assays from biopsies of the salivary glands, liver, and spleen at sacrifice. The rats received 300 microCi[3H]thymidine by i.v. injection 3 h before sacrifice, and the grafts were removed at various time points for histology, immunohistochemistry, and autoradiography. RCMV infection significantly enhanced the generation of allograft arteriosclerosis. Infection at the time of transplantation had two important effects. First, the infection was associated with an early, prominent inflammatory episode and proliferation of inflammatory cells in the allograft adventitia. Second, the viral infection doubled the proliferation rate of smooth muscle cells and the arteriosclerotic alterations in the intima. In late infection the impact of RCMV infection on the allograft histology was nearly nonexistent. RCMV infection showed no effect in syngeneic grafts. These results suggest that early infection is more important to the generation of accelerated allograft arteriosclerosis than late infection, and that an acute alloimmune response must be associated with virus infection, to induce accelerated allograft arteriosclerosis. RCMV-infected aortic allografts, as described here, provide the first experimental model to investigate the interaction between the virus and the vascular wall of the transplant.

Prevention of cardiac allograft arteriosclerosis by protein tyrosine kinase inhibitor selective for platelet-derived growth factor receptor.

BACKGROUND: Increased immunoreactivity of platelet-derived growth factor (PDGF)-AA, -Ralpha, and -Rbeta in intimal cells correlates with the development of cardiac allograft arteriosclerosis, a condition for which there is little or no current therapy. Therefore, we hypothesized that PDGF may have a rate-limiting role in the development of this disease. METHODS AND RESULTS: The hypothesis was tested in a rat model of heterotopic cardiac and aortic allografts using dark agouti (AG-B4, RT1(a)) donors and Wistar-Furth (AG-B2, RT1(u)) recipients. The recipients received CGP 53716, a selective PDGF-R protein tyrosine kinase inhibitor, 50 mg. kg-1. d-1, or vehicle for 60 days. Cardiac allograft recipients also received background cyclosporin A immunosuppression. Our results demonstrate that CGP 53716 significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac and aortic allograft recipients. When rat coronary smooth muscle cells were stimulated in vitro with PDGF-AA or -BB in the presence of interleukin-1beta or tumor necrosis factor-alpha, CGP 53716 significantly inhibited only AA-ligand-induced but not BB-ligand-induced replication. Concomitantly, in quantitative reverse transcriptase-polymerase chain reaction, interleukin-1beta or tumor necrosis factor-alpha stimulation specifically upregulated the expression of PDGF-Ralpha mRNA but not of other ligand or receptor genes in cultured smooth muscle cells. CONCLUSIONS: We conclude that a PDGF-AA/Ralpha-dependent cycle is induced in the generation of allograft arteriosclerosis that may be inhibited by blocking of signaling downstream of PDGF-R.

Organ transplantations: from basic research to clinical applications. A personal odyssey.

This article is a summary of the impact on contemporary medicine of organ and tissue transplantation. The article describes how, via trial and error, and beginning from basic research, the results of organ transplantation have steadily increased as has the number of organs that can be transplanted. Currently, the short-term results of most organ transplants, with the notable exception of the pancreas and the lung, are close to perfect; very few organs are lost any longer due to acute rejection. There is, however, little information on long-term results using the current modalities of immunosuppression, particularly on the effect of chronic rejection on late graft survival.

Eicosanoids are regulatory molecules in gamma-interferon-induced endothelial antigenicity and adherence for leucocytes.

When the endothelial cells (ECs) were stimulated with gamma-interferon (gIFN) in the presence of methylprednisolone (MP) or prostaglandin E2 (PGE2), MP enhanced gIFN-induced Ia antigen expression, whereas PGE2 inhibited it. On the other hand, while PGE2 had no effect on leucocyte binding to ECs, MP entirely inhibited it. By using selective inhibitors of the cyclo-oxygenase pathway (indomethacin, IM) and the 5-lipoxygenase pathway (L651.392), we found that addition of IM to gIFN-stimulated ECs enhanced Ia expression but had no effect on leucocyte adherence to ECs. Instead, addition of L651.392 to gIFN-stimulated ECs partially reduced leucocyte adherence to ECs but had no effect on Ia expression. Pretreatment of the ECs or leucocytes or both with monoclonal anti-class II antibody, had no effect on gIFN-induced leucocyte binding to ECs. These findings suggest that gIFN-induced endothelial cell antigenicity and leucocyte adherence are regulated independently of each other by different molecular pathways. Moreover, arachidonic acid metabolites appear to be the regulatory molecules in gIFN effects on the ECs.

Protein kinase C is crucial in signal transduction during IFN-gamma induction in endothelial cells.

We have demonstrated that IFN-gamma, a potent peptide mediator in inflammatory responses, operates via the protein kinase C dependent transduction pathway in the induction of class II MHC antigens on rat microvascular endothelial cells. Stimulators of protein kinase C, like PMA, replaced IFN-gamma in the induction of MHC class II on endothelial cells in a dose-dependent manner. Selective enzyme inhibitors of protein kinase C, H-7 as well as sphingosine down-regulated the IFN-gamma induced class II expression in a dose-dependent manner. Addition of cAMP or cGMP in the culture, had no effect on the class II expression on the endothelial cells. Transient rise of cytosolic Ca2+ by calcium ionophore A23187, or a calmodulin antagonist W-7, had no effect on the IFN-gamma induced class II expression.

Leukotriene B4 increases the lymphocyte binding to endothelial cells.

Leukotrienes are potent mediators of local microvascular environment. Leukotriene B4 treatment of cultured endothelium increases the binding of lymphocytes to endothelial cell monolayers within minutes. This effect is dose-dependent and reversible upon removal of the leukotriene. Pretreatment of lymphocytes slightly decreases the binding and pretreatment of both lymphocytes and endothelium with leukotriene B4 prior to the adherence assay did not alter the binding. These results suggest that leukotriene B4 regulates exclusively the vascular side, but not the white cell side of this interaction.

The impact of acute episodes of rejection on the generation of chronic rejection in rat renal allografts.

We have investigated the impact of the frequency of acute rejection episodes on the generation of chronic rejection in long-surviving rat renal allografts. A total of 33 renal transplantations was performed from DA to WF rats, receiving different cyclosporine-based immunosuppressive regimens. As a consequence, different numbers of acute rejection episodes were recorded in the recipients, all of which were successfully treated with cyclosporine. Upon sacrifice at 12 weeks posttransplantation, the frequency of acute rejection episodes was correlated with the major histological parameters of chronic rejection and with graft function. The intensity of the major histological parameters of chronic rejection, exemplified by vascular intimal proliferation and glomerular mesangial matrix increase, and the decline in graft function between the no-rejection vs. rejection groups, were directly proportional to the number of acute rejection episodes. Vascular intimal proliferation increased from 0.5 +/- 0.4 (arbitrary units) in the no-rejection group to 1.7 +/- 0.9 (P = 0.0093) after one rejection episode, to 2.2 +/- 0.3 (P = 0.0001) after two, and to 2.2 +/- 0.5 (P = 0.0014) after three or four rejection episodes. Glomerular mesangial matrix increased from 1.2 +/- 0.3 (arbitrary units) in the no-rejection group to 1.9 +/- 0.6 (P = 0.017) after one, to 2.2 +/- 0.5 (P = 0.0005) after two, and to 2.1 +/- 0.4 (P = 0.003) after three or four rejection episodes. Serum creatinine increased from 93 +/- 24 mumol/L in the no-rejection group to 196 +/- 92 mumol/L (P = 0.016) after one, to 238 +/- 38 mumol/L (P = 0.0001) after two, and to 308 +/- 85 mumol/L (P = 0.0016) after three or four rejection episodes. Prolongation of the preoperative ischemia time from 30-60 min correlated with an increase in the number of acute rejection episodes as well as an increase in chronic changes. To conclude, in the rat renal allograft model, acute allograft rejection carries a highly significant correlation with the development of chronic rejection.

Prolongation of rat cardiac allograft survival by donor pretreatment. Screening of antineoplastic drugs.

We have made preliminary investigations into the effect of 33 different drugs on the survival of rat cardiac allografts, the drugs being administered to the allograft donor. All drugs were administered at LD50 i.v. to the graft donor 6 hr prior to removal of the organ. Especially effective were alkylating agents and antimetabolites. Pretreatment with cyclophosphamide, busulfan, methotrexate, azauridine, or bromodeoxyuridine prolonged the survival from 7 to more than 20 days. Pretreatment with chlorambusil, mannomustine, mannosulfan, 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea, DTC, fluoruracil, or hudroxyurea prolonged the survival to more than 14 days. Several other alkylating agents and antimetabolites prolonged the survival moderately, i.e., to approximately 10 days or more. Purine antagonists, mercaptopurine and azathioprine, were totally ineffective as were also anticancer antibiotics and vinca alkaloids. Pretreatment with procarbazine or methylprednisolone alone increased the survival only moderately, whereas pretreatment with both of these drugs together increased the survival up to 27 days.

Reversibility of allograft arteriosclerosis after retransplantation to donor strain.

The rat aortic transplant model was modified to investigate whether the vascular wall changes on chronic rejection are reversible. DA (RT1a) aortas were first transplanted to WF (RT1a) recipients. The first transplantation was accompanied, as described earlier, by an increase in intimal cellularity and thickness and typical arteriosclerotic changes of chronic rejection in the allograft intima. A second transplantation was made to DA, WF or to (DAxWF)F1 recipients 10 days-2 months after the first transplantation. In all retransplantations performed at any one of the indicated timepoints, the thickness and number of nuclei of the intima continued to increase. These observations demonstrate that, after an initial trigger, allograft arteriosclerosis proceeds and is irreversible despite elimination of histoincompatibility.

Histological chronic allograft damage index accurately predicts chronic renal allograft rejection.

Chronic rejection has emerged as a major problem in renal transplantation, and clinical trials for prophylaxis and therapy are underway. Use of graft and patient survival as endpoints in prophylactic studies requires long follow-ups to read the endpoint. There is also an obvious need for a starting point for intervention studies. Previously, we formed a histological chronic allograft damage index (CADI), based on numerical scoring of histological alterations compatible with chronic rejection. Using protocol core needle biopsies of 89 functioning grafts 2 years after transplantation and a follow-up of 6 years, we demonstrate now that (a) the CADI at 2 years correlates significantly (r = 0.717, P = 0.0001) with transplant function at 6 years, and (b) that the CADI at 2 years reliably (P = 0.001) predicts the patients who will proceed to clinical chronic rejection later. As protocol core biopsy is an early predictive parameter for chronic rejection, our results suggest that a protocol core biopsy (at 2 years or possibly even earlier) should be included in all clinical investigative protocols dealing with chronic renal allograft rejection.

Bone marrow transplantation in the rat. B lymphocyte activation in acute graft-versus-host disease.

We have isolated the white cells from the bone marrow, spleen, and blood of a rat recipient of a bone marrow allograft and the inflammatory leukocytes from the recipient skin, lung, gut, and liver (the parenchymal target organs for acute graft-versus-host disease (aGVHD)) and compared the number of immunoglobulin-synthesizing and releasing cells in these cell populations to corresponding compartments of a syngeneic graft recipient. Bone marrow transplantation was associated in the early phase with marked immunoglobulin production in the cells of bone marrow, spleen, and blood of the allograft recipient; as, however, a similar response occurred in the syngeneic graft recipient we conclude that this is related to reconstitution rather than to aGVHD. Later, during aGVHD, the number of immunoglobulin releasing cells decreased significantly in the spleen and bone marrow of the allografted animal. In clear contrast, in the liver--but not in skin, lung, or gut--very few immunoglobulin-releasing cells were observed in the syngeneic graft recipient, whereas in the allograft recipient a very strong and significantly higher immunoglobulin synthesis and release was seen coinciding with the inflammatory episode of aGVHD in the liver.

The influence of the pattern of inflammation and administration of steroids on class II MHC antigen expression in renal transplants.

We have investigated the expression of class II major histocompatibility complex (MHC) antigens in human renal allografts before, during, and after the first episode(s) of rejection, and correlated the antigen expression with the cytological pattern of inflammation as well as with the extent of steroid administration. The results confirm that the class II MHC antigens are rapidly lost from a renal transplant after successful transplantation. Downregulation of graft class II antigenicity was observed in all three immunosuppressive regimens employed, with a steroid dose ranging from 0.5 +/- 0.2 mg/kg/day to 1.8 +/- 0.3 mg/kg/day of methylprednisolone. During rejection the class II MHC antigens reappear in the graft parenchymal (vascular endothelial and tubular) cells, whereas after the successfully treated episode they again disappear from the graft. The upregulation of graft antigenicity is associated only with inflammatory patterns with a distinct blastogenic component; nonblastogenic patterns of inflammation are not associated with upregulation of class II antigens. During blastogenic inflammation, the extent of class II antigen expression was inversely proportional to the amount of steroid administered. The results support the suggestion that upregulation of class II antigen contents in a graft is due to (gamma-interferon released by) the inflammatory (T) blast cells, and suggest that a major downregulating mechanism of class II antigen expression is administration of glucocorticosteroids.

Sponge matrix allografts. A model for analysis of killer cells infiltrating mouse allografts.

A method for isolation of allograft-infiltrating cells in a functionally viable state is described in this article. The method is based on the use of a spongious tissue into which cells of strain A (e.g., fibroblasts or tumor cells) are grown. The resulting graft is then transplanted to a strain B animal, and the infiltrating cells are released from it by gentle compression. The graft-infiltrating cells are completely recovered, and they may be processed for further experimentation and analysis by employing exclusively physical methods of cell preparation. As an example of future applications, some preliminary results on density- and charge fractionation of the graft-infiltrating cells are also reported.