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Viable infiltrating host leukocytes have been isolated from 10 rejected human renal allografts, removed 1 to 67 months after transplantation. The cell populations have been identified by surface characteristics and their cytotoxic capacities were assessed. A heterogenous population of cells of host origin accumulated in the grafts, including T and B lymphocytes, Fc+ cells, and macrophages. Using a 51Cr release assay, specific cytotoxicity against donor alloantigens was determined. Cytotoxicity of the infiltrating cells was almost invariably greater than cytotoxicity mounted by recipient peripheral blood lymphocytes. Deletion studies confirmed previous work and suggested that T cells were primarily responsible for cytolysis in early acute rejection; non-T cells more often in late chronic rejection. Antibodies eluted from the grafts demonstrated both specific antidonor and nonspecific activity as well as cross-reacting anti-HLA activity. Allograft morphology was examined and cellular and humoral host responses were assessed. These studies emphasize the complexities of immune responses produced by the host against transplanted tissues.

Nineteen patients with acute rejection of a renal allograft were treated with the monoclonal antibody anti-T12, directed against a determinant present on all post-thymic T cells. Seven patients had a good response, four had an equivocal response, and eight failed to respond. Histologic studies demonstrated that the good responders had primarily cellular rejection. The nonresponders included 4 patients with moderate-to-severe humoral rejection, one patient with an inadequate dose of antibody, one patient who withdrew before completing the study, and one patient with late end-stage rejection. All eleven patients with good or equivocal responses have functioning kidneys in a follow-up of 1-15 months (mean 7 months). Only one patient has had a subsequent acute rejection episode, which responded to a steroid pulse. No significant complications of anti-T12 therapy occurred.

To determine the identity and cytotoxic capacity of lymphoid cells involved in allograft rejection, we studied viable, monodispersed cells recovered from 10 rejected human renal allografts. A heterogeneous population of cells including macrophages and both bone-marrow (B) and thymus-derived (T) lymphocytes accumulate in rejected grafts. Infiltrating lymphocytes exerted a specific cytolytic effect on 51Cr-labeled peripheral blood lymphocytes bearing donor antigens, ranging from 7 to 44 per cent specific lysis in nine of 10 cases. Cytolysis was closely correlated (r equal 0.91, p less than 0.05) with the histologica grade of cellular rejection but not with humoral rejection, suggesting that cytotoxic lymphocytes are an important element in cellular rejection. Limited fractionation studies showed that both T cells (in early rejection) and non-T cells (in late rejection) may produce cytotoxicity. Since as many as 50 per cent of cells recovered bore Fc receptors, the rejection process may also involve antibody-dependent target-cell lysis.

Two antiplatelet drugs, pyridinolcarbamate and Sudoxicam, were tested separately and in combination with heparin for their ability to modify experimental hyperacute renal allograft rejection in primates. Pyridinolcarbamate delayed and amerliorated tissue injury and obstructive thrombosis but only minimally prolonged renal blood flow over that seen in untreated allografts, suggesting that graft failure was primarily due to vasoconstriction. Sudoxicam, an antiinflammatory agent, resulted in higher initial blood flow, but the duration of flow and graft survival were again only minimally prolonged. However, functional changes including C3, Factor II and X consumption were prevented, a net increase in Factor VIII activity was minimized, and fibrinolysis was inhibited. The combined effects of pyridinolcarbamate and heparin resembled those of heparin alone. Combination of Sudoxicam with heparin was more effective than heparin along in preventing intrarenal complement consumption, sequestration of formed elements, activation of coagulation, and inhibition of fibrinolysis. However, the latter combination also failed to prolong venous flow and graft survival over that seen with heparin alone.

The assays of lymphocyte-mediated cytotoxicity (LMC), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were correlated with histopathologic criteria of refection in 35 transplant biopsies. A positive LMC was seen with 6/8 biopsies showing moderate to severe cellular rejection and in 8/17 with mild cellular rejection. Positive ADCC and/or CDC assays were associated with 14/14 biopsies containing rejection vasculitis. These results suggest that selected in vitro assays may be useful in monitoring the immunologic events occurring in the rejecting allograft.

The purpose of the present experiments was to define a non-major-histocompatibility-complex (MHC) endothelial antigen system in the rat and to study the genetics of these antigens as well as their significance in renal transplantation. Several MHC-identical rat strain combinations underwent reciprocal immunization using spleen and lymph node cells and complete Freund's adjuvant. In one combination (MAXX anti-BN) alloantibodies were found against antigens on peritubular and venous endothelium of the kidney from the immunizing strain as well as from two other strains. Preliminary results suggested that the endothelial antigen is present on monocytes but not on nonstimulated T and B lymphocytes. With kidneys from 7 MHC-congenic lines it was shown that the endothelial antigens are encoded outside the MHC-region. The antigen seems to be expressed as a dominant trait. In an F2 population of 32 animals, segregation of the endothelial antigen or antigens appeared to be independent of the MHC, AgF, and tubular basement membrane antigens--as well as the locus for albinism. Transplantation of MHC-identical but endothelial-antigen-incompatible kidneys into nonimmunized recipients did not provoke acute rejection. Pretransplant immunity against donor endothelial antigens was, however, associated with accelerated acute rejection. The rejection was donor-specific because third-party MHC-incompatible but endothelial-antigen-compatible kidneys were rejected like first-set grafts. This model shows that graft rejection in presensitized recipients of an MHC-identical kidney can be mediated through immunity against non-MHC antigens.

Hyperacute renal allograft rejection is initiated by primary immune injury to vascular endothelium and is propagated by secondary vasoconstriction, platelet aggregation and intravascular coagulation. Previous dissociation of these primary and secondary events, with graft survival in one human, suggested that the more usual graft failure was due to secondary injury. As a basis for further modification studies, this primate model most closely resembled its counterpart in man, as the onset and intensity of functional, morphologic and biochemical alterations were similar. Unmodified allografts failed within 5 minutes. The earliest and most abnormal finding was marked reduction in renal blood flow affecting all compartments. By 5 minutes, histologic changes of hyperacute rejection as well as antibody and faint C3 deposits were noted, but biopsies suggested that the initial flow reduction was more likely due to vasoconstriction, which was then followed by vascular obstruction. Glomeruli appeared most damaged, but at the highest antibody titer arterial injury was more prominent. Early red cell sequestration and stasis was marked, followed by progressive platelet clumping and neutrophil infiltration. While the decline in renal venous C3 levels was prompt, as in man, early intrarenal activation of the coagulation, fibrinolytic and kinin-forming systems could not be demonstrated, and fibrin formation was sparse by light and fluorescence microscopy. Qualitatively similar histologic and functional alterations were noted in autograft controls. While the initiating event was unclear and may have been accentuated by the arteriovenous shunts utilized, the final mechanism was probably marked vasoconstriction with renal ischemia. Intrarenal C3 consumption was an important finding and was not associated with tissue deposits of antibody or complement; it may provide a parallel with the progressive complement-mediated injury associated with acute myocardial ischemia noted by others. Endothelial injury was not seen in arteries, and all alterations were delayed in onset and progressed more slowly than in allografts. These findings may elucidate the mechanism of early malfunction of most autografts. Treatment of additional autografts with increasing doses of heparin progressively reversed these changes and even prevented the initial reduction in blood flow. Therefore, many alterations consistent with hyperacute rejection which are probably immediately responsible for graft failure can also be initiated by nonspecific, nonimmunologic events and, where injury is less intense, can be prevented pharmacologically. This model provides a means of dissecting the injurious events and subsequent evaluation of the effectiveness and interaction of various agents on the damaging secondary alterations which occur during hyperacute rejection.

Pooled serum aliquots obtained from sensitized potential renal allograft recipients on chronic hemodialysis were evaluated for their lymphocytotoxicity titers against the lymphocytes and then for alloantibodies against the platelets of 7 random donors by 5 methods. Platelet donor specific lymphocytotoxicity was present in 93% of 42 combinations. Of the positive combinations, 57% had a positive test for antiplatelet activity by the 14C serotonin release assay, 16% by the platelet aggregation method, and 19% as judged by acid phosphatase availability on the platelet membrane. No serum tested released beta-glucoronidase or lactic dehydrogenase. No correlation of the height of the titer of antiplatelet activity with that for lymphocytoxicity was detected. Thus, even in sera demonstrating significant activity against donor lymphocyte antigens, detection of associated platelet antibody activity is not uniform. Thus, a positive lymphocytoxic titer does not necessarily predict detectable antiplatelet activity. Therefore, additional tests for detection of antiplatelet activity should also be considered. This study shows that of the tests evaluated, the 14C serotonin release assay is the most sensitive for detection of antiplatelet antibodies.

A prospective study of 31 cadaveric renal allograft recipients was performed to determine the significance of pretransplant presensitization undetected by the conventional microlymphocytotoxicity crossmatch. Donor-specific humoral presensitization revealed by the antibody-dependent cell-mediated cytotoxicity assay (ADCC) was associated with a high incidence of early graft rejection. Six-month graft survival was 20% in recipients with positive pretransplant ADCC and 75% in ADCC-negative recipients (P < 0.01). Among recipients highly presensitized to a random panel of HLA antigens, donor-specific humoral presensitization detected by chromium-51-release complement-dependent cytotoxicity (51Cr-CDC) was also highly correlated with accelerated rejection (P < 0.05). Pathologic study of the rejected allografts revealed antibody-mediated rejection vasculitis in all recipients. We conclude that humoral presensitization undetected by current conventional methods plays a cardinal role in early renal graft rejection and is a major factor responsible for low cadaveric renal transplant survival. This study suggests that use of the ADCC and 51Cr-CDC as routine adjunctive crossmatch procedures may contribute to improvement in renal transplant survival rates.

Inhibition of renal vasoconstriction during hyperacute rejection by phenoxybenzamine or methylprednisolone combined with either the antiplatelet agent pyridinolcarbamate or heparin was evaluted in primates. Phenoxybenzamine plus pyridinolcarbamate did not prolong kidney survival. Phenoxybenzamine plus heparin uniformly prolonged low rates of venous flow to 180 minutes and delayed secondary C3 consumption, sequestration of erythrocytes and platelets, coagulation, and fibrinolysis; neutrophil sequestration and vascular injury and obstruction were more marked than with heparin alone. Host pretreatment with methylprednisolone plus heparin also prolonged the low rates of venous flow to 180 minutes, further reduced secondary alterations, and resulted in the least vascular injury. When intact donor kidneys were also pretreated with methylprednisolone, persistently normal rates of venous flow were achieved. Despite marked consumption of Factor XII, the consumption of C3, other coagulation factors, prekallikrein, and sequestration of formed elements was minimal, and the histology appeared compatible with even more prolonged survival.

To define the composition and proliferative and functional activity of the rejection formed sheep erythrocyte rosettes (T cells), expressed surface Ig (B cells) or Fc receptors and specific lymphocyte-mediated cytotoxic activity. Small clumped lymphocytes (48.8 +/- 18.9 per cent) and plasma cells (18.8 +/- 10.9 per cent) were most common and correlated negatively (p less than 0.01). Percentages of surface Ig-bearing and small clumped lymphocytes correlated directly (p less than 0.05) as did the percentages of E rosette-forming and plasma cells (p less than 0.05). The percentages of surface Ig-bearing B cells correlated negatively with E rosette-forming T cells (p less than 0.05). Within this spectrum of rejection, three patterns could be identified. In the first pattern, humoral rejection (by immunofluorescence) was intense and cellular infiltration was primarily by surface Ig and Fc receptor-positive small clumped B lymphocytes. The second and most common pattern consisted of a mixed T and B cell infiltrate with intermediate to high lymphocyte-mediated cytotoxic activity and variable humoral rejection. In the third pattern, invitro studies showed primarily E rosette-forming T cells with paradoxically low lymphocyte-mediated cytotoxic activity. However, morphologic studies also revealed the highest percentages of plasma cells but only mild humoral rejection. Thus, combined morphologic and in vitro studies identified three patterns of protracted renal allograft rejection. Thus, combined morphologic and in vitro studies identified three patterns of protracted renal allograft rejection for which the percentages of plasma cells may provide a distinguishing morphologic marker. Furthermore, the degree of lymphocytic invasion of renal tubules and blood vessels may provide an estimate of lymphocyte-mediated cytotoxic activity.

Immunologic renal allograft rejection is the most common cause of diminished renal function in the transplant patient, yet it is difficult to distinguish from other etiologies, frequently requiring biopsy before therapy can be instituted. To determine the utility of ultrasound in the diagnosis of this type of rejection, the authors correlated histologic and echographic findings of 30 coded biopsies and sonograms in 25 patients. The results were encouraging, with a 71% rate of accuracy in indicating steroid therapy for acute rejection and an accuracy rate of 81% in contraindicating it. There was exact agreement of echographic and histologic evaluations as to extent of chronic rejection in 70% of cases.

Two groups of specifically presensitized Macaca speciosa monkeys received renal allografts via anastomosis to an indwelling arteriovenous (A-V) shunt. One group was pretreated with heparin (2 mg/kg) intravenously and the other was also treated with constant heparin infusion (1 mg/kg/hr) directly into the renal artery. These studies were performed to evaluate the effects of heparin within the kidney on total and compartmental blood flow, complement (C3) levels, sequestration of formed elements, and activation of the coagulation, fibrinolytic, and kinin-forming systems during the initial 3 hours of hyperacute rejection. The results are compared with those previously reported in unmodified control allografts. Heparin prolonged blood clotting time to infinity, markedly prolonged total renal venous blood flow, and normalized compartmental distribution in both groups despite antibody deposition similar to that in controls. With heparin pretreatment only, initial morphologic injury was much reduced but then progressed rapidly. Marked initial cortical cyanosis with mottling appeared to change constantly and was associated with fluctuations in renal turgor, total blood flow, and sequestration of formed elements, all of which suggested repetitive local cortical arterial spasm and incremental destruction of the grafts. Activation of coagulation Factors II and XII was also revealed and marked net Factor VIII activity was observed in the venous effluent. The latter reflects either formation and release of this factor by the injured kidney, or provides in vivo documentation of the "hyperactivation" of Factor VIII by thrombin known to occur in vitro. The addition of intrarenal artery heparin infusion resulted in greater improvement in early total blood flow rates and more uniformly progressive cyanosis and loss of turgor, but the diffuse initial morphologic injury suggested more uniform perfusion of injured areas. Intrarenal consumption of C3 and sequestration of formed elements was similar to that in controls. Paradoxically, prompt activation and consumption of all coagulation factors, plasminogen, and prekallikrein were observed, but formed fibrin was sparse. The exess amount of Factor XIIa present during heparin blockade may have been diverted to production of plasminogen activator and kallikrein formation. The enormous numbers of neutrophils observed within vessels of grafts which showed the greatest kallikrein activation provide the probable in vivo demonstration of the chemotactic properties of kallikrein noted by others in vitro. Heparin-induced platelet aggregation may have played an important role in the failure of these grafts. These studies elucidate the intrarenal effects of heparin during hyperacute rejection and again suggest that vasoconstriction is the most important early determinant of graft failure, as blood flow appeared unrelated to the degree of vascular injury and apparent obstruction. Also, heparin may exer a beneficial effect on blood flow by other than its known action on coagulation.

Nineteen renal allograft recipients experiencing rejection were infused with monoclonal mouse anti-human T12 directed to a T lymphocyte differentiation antigen expressed on mature post-thymic cells. Patients were offered this therapy as an alternative to high-dose methylprednisolone after diagnosis of an acute rejection episode within 3 months of transplantation. Seven patients had clear-cut reversals of rejection activity within the 10-day period of treatment. Four additional patients had delayed responses in association with acute tubular necrosis and/or cyclosporine toxicity, and they received other therapies according to protocol when anti-T12 alone had not caused a response after 5 days. All but one of these responding patients have been free of subsequent acute rejection over a 1-15 month follow-up period. Treatment failures appear to be related to the presence of allo-antibody-mediated vasculitis. Therapy was well tolerated. These preliminary studies have revealed the existence of a T12 negative subpopulation of extrathymic T cells, usually undetectable in peripheral blood.

36 renal biopsies from patients with nephritis were studied for glomerular localization of the heavy chain subgroups of immunoglobulin G (IgG or gammaG). The deposition pattern of these subgroups was selective and did not reflect the normal serum concentration of these proteins. gammaG2, which comprises 18% of normal serum gammaG, was the predominant or unique subgroup deposited in five cases of lupus nephritis and four biopsies with other forms of nephritis associated with granular gammaG deposits. gammaG3, which normally makes up only 8% of the serum gammaG, was the dominant subgroup seen in one biopsy of lobular glomerulonephritis. Patients with linear gammaG deposits generally had a selective absence of gammaG3 and often had large amounts of gammaG4 (normally 3% of the serum gammaG) deposited. The deposition of complement components C1q, C4, and C3 was variable. One biopsy had only gammaG2 and no complement components in the deposits and had no neutrophile leukocyte infiltration. This latter observation correlates well with the poor ability of gammaG2 to fix complement in vitro. Similarly, deposits containing large amounts of gammaG4, which does not fix complement, also tended to have less inflammatory infiltrate than deposits devoid of this subgroup. The selective deposition of monotypic or restricted gammaG subgroups on the glomerulus supports the likelihood that the gammaG represents antibody. The nature of the subgroup involved in the deposit may represent one variable in the determination of the inflammatory and morphological picture that evolves in human glomerulonephritis.