Hyperacute Renal Allograft Rejection Research Articles

Platelets and capillary injury in acute humoral rejection of renal allografts

Platelet accumulation in glomerular capillaries (GC) and peritubular capillaries (PC) has pathogenetic importance in antibody-mediated hyperacute renal allograft rejection. CD61 is expressed constitutively by platelets, by platelet microparticles arising from platelet activation, and is readily detectable by immunohistochemistry. This study examined the immunohistochemical localization of CD61 in acute humoral rejection (AHR) of renal allografts to explore the relationship of platelet accumulation to antibody-mediated rejection. Two observers graded the extent of CD61 staining in PC and GC from 0 (none) to 2+ (>50%) in 15 renal allograft biopsy specimens with AHR and compared these with tissues from allografts with acute cellular rejection (ACR) (n = 23); acute calcineurin inhibitor toxicity (ACIT) (n = 21) with thrombotic microangiopathy (TMA) (n = 11) and tubular toxicity only (n = 10); acute tubular necrosis (ATN) (n = 16); acute renal vein thrombosis (RVT) (n = 4); and histologically unremarkable native kidneys (n = 26). Selected tissues were examined by electron microscopy and stained for CD34 by immunohistochemistry. Histologically unremarkable native kidney tissues had CD61 only in scattered small lumenal granules in GC and PC. Mural and occlusive lumenal CD61 deposits (>0.5+) were observed in 13 of 13 (100%) allograft tissues with GC thrombi due to AHR (1) and ACIT TMA (9) and RVT (3). Twenty-seven of 66 allografts (40.9%) without glomerular thrombi had >0.5+ GC CD61 in AHR (60%), ACR (26%), tubular ACIT (60%), and ATN (44%). More than trace (>0.5+) PC mural and lumenal CD61 deposits were seen only in AHR (53.3%) and ACR (30%). PC CD61 correlated with interstitial hemorrhage (r = 0.64), neutrophilic capillaritis (r = 0.47), and interstitial inflammation (r = 0.47) ( P <0.001 for each). PC CD61 was observed in 11 of 11 foci of necrosis due to AHR, RVT, and ischemia. In AHR, capillaries with CD61 deposits had few platelets, numerous microvesicles and membrane fragments, severe endothelial injury seen on electron microscopy, and reduced capillary CD34 expression. CD61 detection by immunohistochemistry revealed products of capillary platelet activation in allograft biopsy specimens without light microscopic thrombi. Observations in this study suggest that intracapillary platelet activation occurs in response to graft capillary injury from many causes and may not be specific for antibody-mediated rejection.

Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins.

Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes. Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished. These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.

Reversal of Delayed Hyperacute Renal Allograft Rejection With a Tacrolimus-Based Therapeutic Regimen

Reversal of Delayed Hyperacute Renal Allograft Rejection With a Tacrolimus-Based Therapeutic Regimen

Hyperacute renal allograft rejection from anti-HLA class 1 antibody to B cells -antibody detection by two color FCXM was possible only after using pronase-digested donor lymphocytes

Abstract We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti-HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement-dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two-color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipient's serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (FcyR) on non-T-cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti-HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two-color FCXM lacks the specificity to detect such antibodies.

The platelet-activating factor antagonist WEB 2170. Its beneficial effect on dog renal allograft survival.

Platelet-activating factor is a phospholipid mediator of inflammation produced both by inflammatory cells (e.g., basophils, neutrophils, monocytes, platelets) and endothelial cells (1); its production is stimulated by several cytokine mediators, including TNF. The role of PAF in allograft rejection includes increasing endothelial permeability leading to reduced blood flow and margination of leukocytes; recruitment of cells into the allograft at the initiation of rejection; and intercellular signaling within the graft (2–4). In vivo evidence of PAF's role in rejection comes from a rabbit model, where a sustained local release of PAF was observed during hyperacute renal allograft rejection, with associated platelet and neutrophil accumulation within 5 min of revascularization (5).

Lymphocytotoxins in sera from highly sensitized multiparous dialysis patients: antibody class, relationship with the HLA and with paternal antigens

1. Sera from 11 highly sensitized multiparous dialysis patients were studied in order to define the target antigens, antibody class and relationship with paternal HLA class I antigens of the underlying lymphocytotoxic antibodies. All sera contained lymphocytotoxic antibodies to over 70% of a panel of lymphocytes from 24 donors (panel reactivity greater than 70%). 2. Inhibition of cytotoxic activity against paternal lymphocytes by monoclonal antibodies to HLA framework determinants indicated that all 11 sera contained lymphocytotoxic antibodies to paternal class I antigens. In addition, five sera contained lymphocytotoxic antibodies to paternal class II antigens. 3. In order to determine the extent to which lymphocytotoxic antibodies were directed to paternal antigens, the panel reactivity of sera was compared before and after absorption with paternal peripheral blood lymphocytes. Over 50% of panel reactivity was absorbed from eight out of 11 sera, and in three of these 11 over 80% was absorbed. In the majority of patients this change in panel reactivity could be ascribed to binding of lymphocytotoxic antibodies to specific paternal class I antigens. 4. Digestion of sera with dithiothreitol had no significant effect on panel reactivity, indicating that the lymphocytotoxic antibodies were of immunoglobulin G class. 5. No sera reacted with either autologous lymphocytes or K562 cells, indicating an absence of autoantibodies. 6. These studies imply that panel-reactive lymphocytotoxic antibodies in the sera of highly sensitized multiparous patients are those which mediate hyperacute renal allograft rejection. Their development may be related to secondary humoral responses to antigens in blood transfusions from donors who share paternal class I specificities.

FK506—ITS INFLUENCE ON ANTI-CLASS 1 MHC ALLOANTIBODY RESPONSES TO BLOOD TRANSFUSIONS

The influence of FK506 on in vivo alloantibody responses to major histocompatability class 1 antigens was investigated in inbred rat strains, and compared with the effect of cyclosporine. AO rats received transfusions of DA blood on days 0 and 7. From days 0 to 14 the rats also received, daily, either FK506 0.3 mg/kg suspended in saline or dissolved in olive oil, or CsA 10 mg/kg. The administration of FK506 suspended in saline at the time of blood transfusion completely abrogated the development of anti-MHC class 1 alloantibodies as detected by indirect hemagglutination (IHA)* and 51Cr release complement dependent cytotoxicity assays (CDC). Isotyping studies showed that FK506 suspended in saline suppressed IgM production and inhibited the switch to IgG production. Similar responses were seen in CsA-treated animals. In contrast, rats treated with FK506 dissolved in olive oil developed high titers of anti-class 1 alloantibodies. On days 49 and 56 the rats were challenged with further DA blood transfusions given without immunosuppression. In the groups given FK506 suspended in saline or CsA, cytotoxic antibodies did not develop; low titer antibodies were, however, detected by IHA in the animals that had previously received FK506 suspended in saline. The results indicate that FK506, in common with CsA, inhibits anti-class 1 MHC alloantibody production, and at the same time enables the development of tolerance. The vehicle in which FK506 is administered is, however, critical to its efficacy at the low doses used. These results may be of relevance to clinical transplantation as similar antibodies mediate hyperacute renal allograft rejection in man.

THE SIGNIFICANCE OF THE ANTI-CLASS I ANTIBODY RESPONSE

In renal transplantation, preformed cytotoxic antibody against donor HLA class I antigens causes hyperacute rejection of renal allografts, but its pathogenic significance when it develops in the posttransplant period is unknown. In the present studies we describe the clinical and pathologic features of patients with rejection associated with anti-class I. In the course of 400 consecutive cadaveric renal transplants, 7 patients were identified who had antibody against donor class I HLA antigens in association with atypical but distinctive patterns of rejection. All 7 were presensitized. In 3 patients, the transplant had been inadvertently performed with a positive donor-specific T cell crossmatch. In the remaining 4, the T cell crossmatch on current sera was negative but became positive posttransplant. The clinical picture was deterioration of graft function with rapid onset of oliguria, apparently due to acute tubular necrosis, but with persistence of blood flow demonstrable by radioisotope scan studies. Renal histology showed that the typical lesions observed in cell-mediated rejection, such as tubulitis and interstitial infiltration, were absent. Granular complement deposition (6), polymorphonuclear infiltration (6), and endothelial injury in the microvasculature (6) were common, and mononuclear infiltrates were absent (2) or not prominent (4). In 3 patients the glomerular changes resembled a picture of hemolytic uremic syndrome, with capillary fibrin thrombi and widening of the subendothelial space. IgG staining was negative. The pathologic features suggest that anti-class I antibody appearing or persisting in the early posttransplant period injures the endothelium of the microvasculature, with the clinical presentation different from that of hyperacute rejection. Particularly in sensitized patients, rapid deterioration in function, leading to a picture of acute tubular necrosis, with pathologic features of endothelial injury in the microcirculation, should suggest the diagnosis of anti-class I-mediated rejection.

Hyperacute renal allograft rejection in HLA-AB identical situations: [formula omitted] Dept. of Surgery and Pathobiology, Harvard Medical School at the Massachusetts General Hospital, Boston

Hyperacute renal allograft rejection in HLA-AB identical situations: [formula omitted] Dept. of Surgery and Pathobiology, Harvard Medical School at the Massachusetts General Hospital, Boston

Passive enhancement and hyperacute rejection of renal allografts in the rat.

Attempts to induce passive enhancement of renal allografts with donor-specific cytotoxic antibody can result in antibody-mediated damage to the grafted organ. Hyperacute renal allograft rejection occurred in the homozygous DA to Lewis strain combination when Lewis anti-DA serum was administered with guinea pig complement. Hyperacute rejection was prevented in four of five rats when Lewis anti-DA F(ab')2 was injected before the administration of Lewis anti-DA serum and complement and these grafts had enhanced survival. Red blood cell absorption of the Lewis anti-DA serum to produce an anti-Ia-like serum, also removed its ability to produce hyperacute rejection without removing its ability to enhance. With appropriate modifications both of these techniques would be applicable to human transplantation.

Short term suppression of hyperacute renal allograft rejection in presensitised dogs with prostacyclin

One of the main features of hyperacute renal allograft rejection in presensitised dogs is platelet aggregation within the kidney as detected by light microscopy and renal arterio-venous platelet counts. Graft failure, as determined by reduction and ultimate cessation of renal blood flow and urine production, can be abrogated in the short term by prostacyclin which is the most potent inhibitor of platelet aggregation yet discovered. After 4 h of extracorporeal perfusion, by which time all control kidneys had been rejected, all prostacyclin treated kidneys had normal or above normal blood flow rates, were producing urine and were similar histologically (light microscopy) to 4-hour autografts.

PLATELET SURVIVAL TEST

SUMMARY The platelet survival test (PST) was evaluated as a means of predicting hyperacute renal allograft rejection. Renal allografts were performed in 12 control and 22 actively preimmunized animals within 1 week of completing immunization. Immunological reactivity assessed immediately prior to renal transplantation was accurately measured by the PST. No hyperacute rejection developed in the control animals or in 10 animals with a slight reduction of platelet survival. Of 12 animals with a moderate or severe reduction of platelet survival, gross and clinical signs of hyperacute rejection were present in 10, and histological evidence of hyperacute rejection was present in 7. Presence or absence of cytotoxic antibody was poorly correlated with eventual outcome. Hyperacute rejection developed in only 1 of 5 animals with a moderate or strong cytotoxic antibody titer; 2 animals with hyperacute rejection had no detectable cytotoxic antibody.

Hyperacute renal allograft rejection in the rabbit. Passive transfer, specificity, and prevention.

SUMMARY The pathogenesis of hyperacute rejection of renal allografts was investigated in randomly bred rabbits sensitized by ear skin allografts. Four of six kidneys from the skin donor and 3 of 4 kidneys from unrelated nonspecific rabbits transplanted in sensitized recipients developed diffuse cortical necrosis. Serum from 10 other skin-sensitized rabbits was injected into the left renal artery of the skin donor (specific) and into the left renal artery of previously unused rabbits (nonspecific). Diffuse cortical necrosis developed in 3 of 10 specific skin donors whose kidneys were injected with serum from skin-sensitized rabbits. Of 10 nonspecific rabbits, kidneys injected with serum from sensitized rabbits, 2 developed cortical necrosis. Heparin treatment at a dose of 2 mg/kg of skin-sensitized renal allograft recipients did not entirely prevent the development of diffuse cortical necrosis. None of the sensitized recipients treated with heparin in a dose of 10 or 20 mg/kg manifested cortical necrosis (P<0.01). Four of six recipients treated with papaverine developed cortical necrosis, and one had diffuse tubular necrosis, indicating the inefficacy of this treatment approach.

Intraoperative blood transfusions and hyperacute renal allograft rejection in a presensitized recipient.

A maternal renal allograft was rejected in a hyperacute manner by the presensitized recipient whose lymphocytotoxic antibodies did not react with lymphocytes of the kidney donor but did react with those of all 3 blood donors whose blood was transfused into the kidney recipient during the transplant operation. The resulting hyperacute rejection is believed to have been precipitated by the interaction of the recipient's cytotoxic antibody with blood donors' lymphocytes and platelets and/or an additional kidney donor antigen detected only after treatment with trypsin. The use of blood with a negative lymphocyte cross‐match for renal allograft recipients having preformed lymphocytotoxic antibody should be considered.

Hyperacute rejection of renal allografts in the primate: protective effect of F(ab') 2 fragment of hyperimmune serum.

Hyperacute (HA) renal allograft rejection and its prevention were studied in a presensitized primate model. The results indicated that: (1) adequate perfusion of the renal allograft with recipient-specific F(ab')2 ameliorated rejection; (2) recipient-nonspecific F(ab')2 derived from high cytotoxic titer serum could have a similar protective effect; (3) prior perfusion of other donor organs had only a temporary effect of lengthening the graft survival; while (4) i.v. heparin and/or cobra venom factor (Co F) administration had no clinical effect.

Complement-Mediated Vasoconstriction and Graft Rejection

The hyperacute rejection of renal allografts has much in common with pig-to-dog renal xenograft rejections. An analysis of the xenograft model revealed new functional aspects of graft rejection that appear to be an integral part of the humoral immune injury. Anaphylatoxin or C5a, a cleavage product of the fifth complement component was shown to play a major role in the rejection of isolated kidneys; it was generated in pig and dog serum by activation of the complement system and caused severe Vasoconstriction in the renal vasculature. It is suggested that histocompatibility antibodies and complement may affect allograft survival via this potent mediator.

Coagulation findings in the hemolytic-uremic syndrome of infancy: Similarity to hyperacute renal allograft rejection

Sixteen infants with the hemolytic-uremic syndrome were investigated with coagulationand fibrinolytic studies to elucidate the possible role of disseminated intravascular coagulation in the pathogenesis of the disease. Coagulation factors were not reduced in all 10 patients in whom they were assayed. In 3 of the 10 infants elevated levels of factors V, VIII, and fibrinogen were noted, but similar changes have been recorded in acute renal failure from other causes. Fibrinolysis as measured by serial thrombin times, euglobulin lysis times, and fibrin plate lysis studies were normal. Fibrinogen degradation products were demonstrated in all 5 infants studied by the hemagglutination-inhibition immunoassay. Platelet survival time was reduced only mildly to moderately. Comparable studies were carried out in 4 patients with hyperacute renal allograft rejection; the findings were similar to those in the hemolytic-uremic syndrome. It is suggested that a similar pathogenic mechanism may be involved and that in the hemolytic-uremic syndrome an antigen antibody-mediated reaction occurs with the kidney as the “target organ” and that the hematologic abnormalities are secondary. Thus the coagulation studies have failed to substantiate a disseminated intravascular coagulation state.

Heparin and aspirin in the treatment of hyperacute rejection of renal allografts in presensitized dogs.

SUMMARY Eleven pairs of dogs exchanged at least three skin grafts within the pairs until a “white graft” occurred and serum lymphocytotoxic antibody was demonstrated. Kidneys were then exchanged within the pairs. Six of eight kidneys in untreated dogs rejected by 12 hr, two within 36 hr. Four azathioprine-treated dogs rejected at 2, 4, 12, and 44 hr. Four dogs treated with heparin before and after transplantation did not reject hyperacutely. One animal received heparin at 15 min posttransplantation, but irreversible hyperacute rejection had already begun. Five aspirin-treated recipients did not reject acutely but were oliguric. Control grafts showed red cell aggregates in dilated peritubular and glomerular capillaries as early as 5 min. Fibrin-platelet microthrombi, swollen and vacuolated endothelial cells, and damage to arterial media were also early findings. By 24 hr, vascular thrombosis and cortical necrosis were evident. Aspirin-treated dogs showed similar but less severe changes. Heparin-treated dogs had only occasional intravascular red cell microaggregates but, by 4 days, cellular rejection was evident.