Identification of an autoantibody to vascular endothelial cell-specific antigens in patients with systemic vasculitis.

PURPOSE: Although immunologic mechanisms have been postulated in the pathogenesis of vasculitis, an autoimmune process directed against specific autologous vascular wall antigens has not been previously documented. We examined the role of an immunologic response to vascular endothelial cell (VEC) antigens in patients with vasculitis, because of the observed immunogenicity of VECs in renal and cardiac allograft recipients. PATIENTS AND METHODS: The study patients included 21 with systemic vasculitis and four with hypersensitivity vasculitis. A healthy, normal control group consisted of 51 young subjects and 61 older subjects. Thirty-two patients with connective tissue diseases were also evaluated. The presence of autoantibody to autologous monocytes and other cell types was determined with previously described standard crossmatch techniques. Patient sera exhibiting autoantibody to autologous monocytes were screened against a panel of allogeneic cells. The cell panel consisted of concordant T and B lymphocytes, monocytes, and VECs from 16 umbilical cord donors. Sera from four patients were analyzed for specificity to the vascular endothelium of frozen sections of vessels. RESULTS: An autoantibody to VEC antigens was detected in 18 of the 21 patients (86%) with confirmed systemic vasculitis, not of the hypersensitivity type. This autoantibody was highly cytotoxic, complement-fixing, and specific for antigens on the surface of the VEC. Findings on allogeneic VEC panel analysis support the existence of multiple allotypes in the VEC antigen system. In three patients, fluctuations in the titer of the autoantibody generally correlated with clinical symptoms. In the four patients screened for the specificity of the autoantibody to anatomically different cadaveric blood vessels, specific anatomic patterns of reactivity were observed. Autoantibody to VEC antigens was not found in young controls and was documented in low frequency in older controls and in patients with connective tissue diseases. The autoantibody was seen in one of four patients (25%) with hypersensitivity vasculitis. CONCLUSION: Our results indicate that an autoantibody to VEC antigens may be involved in the pathogenesis of systemic vasculitis or may be a diagnostic marker for the disease process.

An immunological study of renal allograft rejection using the direct macrophage inhibition test.

The direct macrophage inhibition test was used to evaluate the relationship between cellular immune response and graft rejection in related renal allograft recipients. Forty recipients were evaluated before and at regular intervals after transplantation while on maintenance immunosuppressive therapy. Allograft recipients were classified into four categories: 1) Non-HL-A-identical with a rejection episode (14 patients); 2) non-HL-A-identical without a rejection episode (13 patients); 3) 3) HL-A-identical with a rejection episode (3 patients); 4) HL-A-identical without a rejection episode (10 patients). Ten HL-A-identical sibling pairs in good health were utilized as controls. Cellular immunity against donor antigen (macrophage inhibition greater than 20%) uniformly occurred in 16 of the 17 patients who experienced episodes of rejection in both the HL-A-identical and nonidentical graft recipient groups. Transplant recipients who did no experience any rejection episodes up to 2 years post-transplant, and members of the HL-A-identical control group had negative inhibition tests. In 5 cases changes in cellular immune response preceded rejection by several days. The two recipients with positive macrophage inhibition to their prospective donors before transplantation experienced irreversible accelerated rejection. Thus, the direct macrophage inhibition test can be used to screen prospective related donors and to monitor cellular immunity in recipients after transplantation. Preexistent cellular immunity to the donor detected before transplantation correlates with a very high incidence of rejection episodes. Graft recipients who experienced no rejection episodes failed to develop cellular immunity to their donors.

Significance of migration stimulatory factor in human renal allotransplantation.

Seventy-four recipients of related donor renal allografts were tested for the presence of cellular immunity to specific donor lymphocyte antigens using the direct migration inhibition factor (MIF) assay. Responses on the assay fell into one of the following three statistically distinct groups: (1) greater than 20% inhibition of macrophage migration, (2) nonresponsiveness, +/- 10% of control migration, and (3) greater than 12% stimulation of macrophage migration. Migration stimulation was shown to be reproducible and to correlate well with a very benign post-transplant clinical course. The production of migration stimulatory factor appears to be an immunological response analagous to the production of migration inhibition factor.

The vascular endothelial cell antigen system.

Vascular endothelial cells (VEC) are clearly immunogenic and express antigens unique to vascular endothelial cells. The observation that peripheral blood monocytes also express this VEC antigen system made prospective testing feasible. Preformed antibody to the VEC/monocyte antigen system of the donor usually leads to graft rejection in HLA-identical combinations, but antibody directed against donor monocytes exclusively (monocyte-specific antigens) appears to be benign. Clearly the VEC antigen system is an important immunogen in HLA-identical living-related donor combinations--and, in addition, this antigen system may be equally important in the non-HLA-identical combinations. The identification of antibody directed against the VEC/monocyte antigens of the donor is frequently masked by the concurrent development of anti-HLA antibody. Preliminary family segregation studies support the concept of genetic linkage between the VEC/monocyte antigen system and the major histocompatibility complex. A group of 153 consecutive, prospective monocyte crossmatches performed have yielded approximately a 7% incidence of positive monocyte crossmatches, with traditional crossmatches being negative. This frequency of patients sensitized to the VEC/monocyte antigens of the donor could conceivably account for up to 70% of the observed early graft loss in living-related donors. We think a positive monocyte crossmatch to the donor in the presence of positive reactivity to concordant VEC and monocytes remains a contraindication to transplantation.

Immunosuppressive metabolites of cyclosporine in the blood of renal allograft recipients.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.

The significance of a donor-specific vessel crossmatch in renal transplantation.

Very early graft rejection is usually attributed to pretransplant sensitization to HLA antigens represented on the lymphocyte. However, it is possible that such rejection episodes are secondary to antibody to a transplant-relevant system that is not represented on the lymphocyte but is represented within the allograft. This study suggests that sensitization to antigens on the VEC is detrimental to allograft success and can occur in the absence of sensitization to HLA antigens on the T or B cell or monocyte. When antibody to donor VEC is not present pretransplant, almost all patients (95%) will have a very benign posttransplant clinical course. When anti VEC antibody is present, early graft rejection or severe rejection episodes occur with a very high frequency (80%) in the nondiabetic patient. These observations, therefore, suggest that the pretransplant presence of antibody to VEC is detrimental to graft survival, and that such antibody can develop in the absence of anti HLA antibody. While the presence of such antibody is not an absolute contraindication to transplantation it does appear to represent a significant risk factor for immunological graft loss.

Clinical significance of the 1-hour biopsy in renal transplantation.

Biopsy specimens were obtained from 43 transplanted kidneys at the time of excision from the donor but prior to revascularization, and 1 hr after revascularization. Two independent laboratories evaluated specimens by immunofluorescent techniques for the presence of IgM, IgA, IgG, C'3, and fibrin. Results from the two laboratories examined for reproducibility and immunological specificity, and correlated with clinical course. Results show that immunoglobulin deposition seen in the 1-hr renal biopsy specimens is of little significance, because: (1) immunoglobulin deposition was difficult to quantitate reliably, (2) the presence of immunoglobulins did not correlate with clinical course, and (3) the majority of immunoglobulin deposition detected was not immunologically specific, since it was most often either present prior to vascularization or disappeared with vascularization.

Correlation of tissue typing, mixed lymphocyte culture, and related donor renal allograft survival.

Results of mixed lymphocyte culture reactions and tissue typing were correlated with the clinical courses of recipients of living related donor renal allografts. Forty-nine patients tested by the mixed lymphocyte culture technique were divided into two response groups: stimulation index greater than 5 and stimulation index less than 5. Seventy patients were tested by standard tissue typing methods and were categorized by the number of misstimulation in mixed lymphocyte culture than with mismatched antigens, suggesting that lymphocyte-defined histocompatibility is more important than serologically defined histocompatibility in selecting the best possible allograft donor.

Identification of the antibody to vascular endothelial cells in patients undergoing cardiac transplantation.

Acute cardiac dysfunction occurred in four cardiac allograft recipients with negative donor-specific lymphocyte crossmatches. In two recipients the transplanted heart was removed and the patients were maintained on bypass for several hours until a second cardiac allograft was available. In these patients the second transplanted heart also underwent acute dysfunction. The lymphocyte crossmatch was again negative in both second transplants. Two of the four recipients had no detectable antibody to a panel of lymphocytes. Examination of the hearts demonstrated histologic findings consistent with hyperacute rejection. Direct immunofluorescence performed on the transplanted hearts revealed the presence of immunoglobulin and complement deposited on the vascular endothelium. Pathology data was available on 3 of the 4 patients who experienced acute cardiac dysfunction. Pretransplant sera from these four recipients were screened for the presence of antivascular endothelial cell (VEC) antibody. The sera from all four recipients were found to contain antibody against an endothelial cell panel. In addition, donor-specific aorta and vena cava were available from one of the heart donors. The recipient was found to have donor-specific antibody to VEC. Thus, antibody directed against VEC specific antigens appears to be related to hyperacute rejection of heart allografts.

The significance of the monocyte crossmatch in recipients of living-related HLA identical kidney grafts.

Monocytes and vascular endothelial cells (VEC) carry a "cell specific" antigen system that has been found to play an important role in the pathogenesis of rejection. In a preliminary study utilizing a standard crossmatch technique with the peripheral blood monocyte as target, recipients of HLA identical living-related grafts who had either pretransplant or who developed posttransplant anti-monocyte antibody, had a high incidence of early graft rejection. This larger study of recipients of HLA identical living-related grafts represents the results of 21 patients from 13 separate transplant centers in the United States. Results from this study seem to indicate a positive monocyte crossmatch represented a degree of sensitization toward donor monocyte antigens which lead to a poor clinical course.

A reappraisal of the role of splenectomy in children receiving renal allografts.

Four renal allograft recipients between 8 and 14 years of age died of acute fulminating infections less than 72 hours after the onset of symptoms. These patients all had undergone splenectomy at the time of related donor renal transplantation. Because the therapeutic value of splenectomy in renal transplantation has not been established, the role of adjunctive splenectomy is evaluated, and the relationship between splenectomy and overwhelming infection is discussed. As a result of these acute deaths in 4 of 62 children undergoing renal transplantation at this institution (6.4%), splenectomy in conjunction with renal transplantation in children has been discontinued.

The detection and evaluation of migration stimulatory factor in renal allograft recipients.

Seventy-one recipients of related donor renal allografts were tested for cellular immunity with the direct migration inhibition factor assay. Patients were divided into three groups on the basis of their responses in the assay. It was shown that all patients whose lymphocytes stimulated macrophage migration in vitro experienced more benign clinical courses than did those patients whose lymphocytes inhibited macrophage migration in vitro. The significance of a correlation between in vitro macrophage stimulation and clinical course is discussed.

Antivascular endothelial cell antibody--its role in transplantation.

Sera samples from eight different groups of patients were tested for the presence of circulating antibody (IgG) directed against vascular endothelial cell antigens. The indirect immunofluorescent method which used either whole blood vessels or single endothelial cells as targets was compared to lymphocytotoxicity panels. The indirect immunofluorescent antibody test (IFA) consistently detected antibody in sera samples which were negative for lymphocytotoxic activity, and the presence of IFA antibody to vascular endothelial cells had a much better correlation with both clinical course and renal allograft rejection than did the lymphocytotoxicity panels. Single vascular endothelial cells appeared to be a more sensitive target for the detection of IFA antibody than did whole vessels. Preliminary absorption studies suggest that cell-specific as well as HL-A antigens play a role in the immunological response to vascular endothelial cells.

The significance of mixed lymphocyte culture in related renal transplantation.

Eighty-one donor-recipient pairs were evaluated prior to renal transplantation to obtain histocompatibility profiles. Standard tissue typing was used to detect serologically defined A and B locus antigens, and mixed lymphocyte cultures were employed to detect lymphocyte defined antigens. Results of both tests were correlated with each other and with allograft rejection. It was shown that as serologically defined histocompatibility at the A and B loci decreases, both the rate of graft rejection and the percentage of high mixed lymphocyte culture stimulation increase. Within each serologically defined category were found patients with a high and a low stimulation index in mixed lymphocyte culture. Regardless of the degree of serologically defined histocompatibility, patients with a high stimulation index had a statistically significant higher graft rejection rate than did patients with a low stimulation index. It appears that the mixed lymphocyte culture assay is a method superior to standard tissue typing in predicting renal allograft rejection with related donors, and therefore all potential donors for renal transplantation should be screened, utilizing the mixed lymphocyte culture technique.

The association of antivascular endothelial cell antibody with hyperacute rejection: a case report.

Early graft loss almost always occurs when recipients of a renal allograft develop antibody directed against antigens specific for donor vascular endothelial cells (VECs) and peripheral blood monocytes. In studies involving recipients of human leukocyte antigen identical, living-related grafts exhibiting preformed antibody to the VEC antigens of their donors, the median onset of rejection was 3 days after transplantation. Although preformed antibody to VEC antigens has been related in numerous articles to early graft loss, there has never been a published report of anti-VEC antibody leading to hyperacute rejection. We report a patient who hyperacutely rejected a renal allograft after undergoing a donor-specific transfusion protocol with her mother in which the kidney was removed in less than 24 hours. Nine months later the patient had a retransplantation with an allograft from a cadaveric donor. The cadaveric graft was again hyperacutely rejected, and this kidney was removed immediately. Anti-VEC/monocyte antibody directed against both donors was detected in the patient's pretransplant sera. With the exception of a positive B-lymphocyte crossmatch with her mother, all the standard crossmatches were negative.

Renal transplantation in patients with urinary tract abnormalities.

Patients with chronic renal failure and total diversion of the lower urinary tract have been considered poor transplant candidates, and post-transplant urinary diversion, i.e., Bricker loop, has been thought to be necessary. Our experience with nine patients clearly indicates that these patients are actually excellent transplant candidates and that post-transplant urinary diversion rarely is necessary. Ureteroneocystostomy of the allografted ureter was performed in seven patients with pretransplant total urinary diversion and all have completely normal bladder and renal function 10 to 66 months after transplantation; the two patients with Bricker loop procedures performed at transplantation died 7 months after transplantation of rejection and pancreatitis. The excellent results achieved with ureteroneocystostomy are attributed to (1) errors in diagnosis resulting in inappropriate bladder or ureteric surgery early in the course of the patient's disease; (2) confusion of immunologic of functional disorders with anatomic problems; (3) growth and development of the bladder, and (4) complete control of chronic bladder infection by pretransplant nephrectomy, ureterectomy, and antibiotics.

Induction of specific tolerance to small-bowel allografts.

BACKGROUND: The induction of specific tolerance would greatly improve survival and functional state of organ transplant recipients. One approach that has recently received attention is the creation of mixed hematopoietic chimerism through the transplantation of allogeneic and syngeneic T-cell-depleted (TCD) bone marrows. In these studies we examined whether tolerance to highly immunogenic small-bowel transplants could be induced by mixed allogeneic chimerism. Tolerance induction depends on the sharing of antigens between bone marrow cells and small-bowel tissue. METHODS: Adult Lewis rats were lethally irradiated and reconstituted with a mixture of 50 x 10(6) TCD bone marrow cells. Thirty days after reconstitution, animals were tested for chimerism by fluorescence-activated cell sorter analysis. Chimeric animals then received ACI heterotopic small-bowel allografts and were assessed daily for rejection. Small-bowel allograft survival was compared to three control groups: (1) untreated Lewis recipients, (2) irradiated TCD syngeneically reconstituted Lewis recipients, and (3) Lewis bone marrow recipients that did not develop chimerism. RESULTS: Median graft survival in control groups was 8 days. Graft survival in eight mixed chimeras ranged from more than 135 to more than 304 days (p < 0.0001), and no episode of rejection or graft-versus-host disease was observed. Mixed lymphocyte reactivity of chimeric lymphocytes confirmed in vivo observation of tolerance. CONCLUSIONS: Bone marrow cells share tissue-specific antigens with small-bowel cells to permit induction of tolerance.

Successful simultaneous renal transplantation and abdominal aortic aneurysmectomy.

At the time of related donor renal transplantation, a 49-year-old man with chronic glomerulinephritis was found to have a large fusiform aneurysm involving the internal and external iliac arteries, the abdominal aorta, and both common iliac arteries. Transplantation and abdominal aneurysmectomy using a standard Dacron bifurcation graft were successfully carried out. This patient has had no associated complications and is currently five years after transplantation and aneurysmectomy, with excellent renal function. It is believed that transplantation may now be offered to an older age group of patients with end-stage renal disease in whom atherosclerosis wll have developed as a natural process of aging.

Current trends in histocompatibility: clinical relevance versus laboratory phenomena.

The role of conventionally analyzed data in tissue typing is decreasing in clinical transplantation. Improved immunosuppression seems increasingly capable of overcoming the immune response to known histocompatibility differences. The introduction of the concept of public specificities may improve the correlation between matching and graft outcome. The further understanding of the roles of cell-specific antigen systems, particularly those on the vascular endothelial cells, may also improve the clinical relevance of matching. The VEC antigens are becoming more important both in our understanding of histocompatibility and of sensitization. The sensitization of potential transplant candidates needs to be prevented by new protocols for transfusion. The amelioration of sensitization to histocompatibility antigens similar to that achieved in transplantation across the ABO barrier seems theoretically possible and should be an important research effort. Most importantly, sensitization needs to be more accurately defined. Sensitization to specific antigens (cell-specific as well as HLA) must be more carefully correlated with graft survival, and the quantitative aspects of sensitization must be better understood. Thus, there are new trends in the field of histocompatibility that hold promise for further improving the results of clinical transplantation and our understanding of the rejection process.

Hodgkin's disease in human renal transplantation.

After transplantation Hodgkin's disease developed in two recipients of related donor renal allografts. Only one case of Hodgkin's disease had previously been reported in this specific patient population and these two cases demonstrate the very atypical biologic behavior of Hodgkin's disease in the immunosuppressed renal transplant patient.