Immunological consequence of renal transplantation and immunosuppression. I. Alterations in human natural killer-cell activity.

The role and activity of natural killer (NK) cells following renal transplantation remain unknown. To monitor NK activity, a 51Cr release of K-562 targets in prednisone- and azathioprine-treated patients receiving renal allografts was utilized. In 18 patients in whom NK activity was measured prior to and after transplantation, a significant diminution in NK activity within 3 weeks following transplantation was demonstrated compared to pretransplant values (34.71 vs 12.20%, respectively; P less than 0.001). In 11 subjects who had NK activity assayed at various intervals after transplantation but not prior to allografting, mean NK values were markedly lower (mean, 14.2%) than those of normal volunteers or patients maintained on hemodialysis (P less than 0.001). The latter two control groups demonstrated no difference (P = NS) in mean NK activity (39.46 vs 35.82%, respectively). In 5 of the 29 patients evaluated with good long-term graft function (mean, 2.7 years), restitution of normal NK activity was demonstrated. In two patients with bacterial infections, NK activity increased from 39.29 to 51.7% and from 13.54 to 20.00%. After infection, these values were 35.3% in the former and 3.39% in the latter. Viral infection did not appear to affect NK activity significantly. NK activity was increased in only one of seven patients with documented rejection episodes. In three of such patients, NK activity declined significantly following pulse methylprednisolone therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced resistance to the effects of hypothermic ischemia in the preserved canine kidney.

Addition of trifluoperazine (TFP), a powerful calmodulin inhibitor to Collins' flush solution has exerted a significant protective effect on the cold-preserved kidney, with successful autotransplantation of 80% of preserved kidneys (4 of 5) after 72 hr of storage. In contrast, use of Collins' solution alone resulted in successful autotransplantation of only 33% (2 of 6) of kidneys after a similar period of preservation. In an attempt to analyze the significance of this result, the microcirculation of preserved kidneys was studied with injections of technetium-labeled microspheres into the kidneys, followed by study with a noninvasive radionuclide scintiphotography (RNS) technique that does not interfere with subsequent transplantation of the kidney. Such studies demonstrate that prolonged cold preservation after flush-cooling with Collins' solution is associated with a progressive deterioration of the integrity of the microcirculation, resulting in poor flow to the renal cortex. In contrast, when TFP is added to the Collins' solution, there are uniform increases in renal cortical flow in kidneys stored for 48 and 72 hr, with preservation of the integrity of the renal microcirculation. Biological testing shows a clear-cut correlation between these observations and the capacity of the tested kidneys to sustain life after retransplantation. Further experiments suggest that the decreases observed in cortical flow in kidneys preserved in the cold for 72 hr are a consequence of cellular swelling, and not of a vasospastic response. The data support the conclusion that TFP exerts its protective effect on the basis of its membrane stabilizing capacity as a calmodulin inhibitor, and not through direct vasodilatation.

Immunological monitoring in patients with end-stage renal disease.

Parameters of cell-mediated immune function were determined in 76 patients with end-stage renal disease. Lymphocyte subpopulations (OKT3, OKT4, OKT8, OKIa1, OKM1, OKT9, OKT10), natural killer (NK)-cell activity (percentage 51Cr release from K562 targets), and delayed cutaneous hypersensitivity were measured and correlated with other variables. The results indicate that (1) uremic patients have a significant diminution in the OKT4-lymphocyte subpopulation and OKT4/OKT8 (helper/suppressor) ratio compared to normal controls; (2 blood transfusions do not induce significant alterations in the helper/suppressor-cell ratio; (3) uremic patients have a significant increase in OKM1 cells compared to normal controls; (4) the majority of uremic patients in this series developed delayed cutaneous hypersensitivity responses to recall antigens and could be de novo sensitized to 2,4-dinitrochlorobenzene (DNCB); (5) skin-test reactivity could not be correlated with total circulating T cells or levels of any lymphocyte subpopulations; and (6) NK-cell activity in uremic patients is not significantly different from that in normal controls. These results highlight the varying levels and function of different lymphocyte subsets in patients with end-stage renal disease when they are treated with chronic maintenance hemodialysis.

The role of calmodulin in the regulation of human lymphocyte activation.

Calmodulin (Cam) was isolated from normal and from transformed human lymphocytes by affinity chromatography on CAPP-Sepharose 4B, followed by chromatofocusing. In the presence of Ca2+, lymphocyte Cam migrated as a single protein on 2-DE, and was located on the same position as Cam extracted from dog brain and rat testis; its MW was 17,500, with a pI of 3.9. In the presence of Ca2+, lymphocyte Cam stimulated activator-depleted dog brain phosphodiesterase; this effect was inhibited by trifluoperazine (TFP) or by EGTA. By the RIA technique, the EGTA-soluble Cam content of resting lymphocytes constituted 0.58% of the total protein; the total Cam was comparable to the content of other major proteins in lymphocytes, such as actin, tubulin, and intermediate filament protein. The amount of Cam per cell and the rate of incorporation of L-[35S]methionine into Cam increased after mitogen-induced transformation. Immunofluorescence labeling of normal, mitogen-transformed, and EBV-genome-positive lymphocytes with affinity-purified anti-Cam antibodies showed bright fluorescence in the region of the Golgi apparatus, as well as diffuse cytoplasmic but scant nuclear staining. Similar patterns were observed in T suppressor, T helper, and B cells. Normal lymphocytes cultured in the presence of 2 X 10(-5) M TFP remained viable, but failed to undergo blastogenic transformation after stimulation with allogeneic cells, concanavalin A (Con A), or pokeweed mitogen (PWM). The same concentration of TFP inhibited the replication of EBV-genome-positive and of leukemia cells. Exposure of natural killer cells or allospecific killer cells to this concentration of TFP inhibited the effector phase of killing in a dose-dependent manner. In the presence of lower concentrations of TFP (0.25-1.0 X 10(-5) M) strong MLC responses were inhibited, while weak reactions were markedly amplified. A similar effect was not observed in lymphocytes stimulated into blastogenesis by lectins, suggesting that different Cam-dependent secondary messenger(s) may be involved in the blastogenic responses evoked by alloantigenic determinants. The amplification of weak MLC responses by 0.25-1.0 X 10(-5) M TFP constitutes the first biological illustration of the capacity of a Cam-binding agent to enhance as well as to inhibit cellular activation. The paradoxical effect may have been a consequence of a shift in the relative concentrations of the four known molecular Cam X Ca2+ conformers. The results are also consistent with the suggested capacity of Cam X Ca2+ conformers to activate different enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetics of natural resistance to thermal injury.

The possible influence of genetic factors in conditioning the host's natural resistance to the lethal effects of severe thermal injury has been studied in 908 rats of comparable age and weight, originating from two outbred, eight inbred, and two congenic strains of animals of defined genetic background. Each animal was exposed to a standard, full-thickness, 40% body surface area skin burn by controlled contact with a heated metal plate. The 21-day postburn mortality was 100% in 217 Fisher (F-344) and 97 ACI male and female rats. The mortality was reduced to 49-63% in an intermediate group of 84 Lewis, 98 Wistar, 48 Sprague-Dawley, 96 Wistar-Furth (WF), and 48 Osborne-Mendel (OM) male rats; 48 female OM rats had a mortality of 86% at 21 days after injury. The same injury produced a mortality in 4% in 90 Buffalo (BUF) and 22% in 41 Brown-Norwegian (BN) males, while females of the same strains exhibited a 21-day mortality of 23% and 54%, respectively. Further studies of the effects of similar injury in two congenic strains of rats derived from some of the inbred lines of animals listed above yielded a 21-day mortality of 50% in 18 BN.1B(BUF) and 20% in 15 BN.1U(WF) male rats, and 65% and 36%, respectively, in females of the same lines. These data point to the importance of genetic factors as a key determinant of host resistance or susceptibility to the effects of severe thermal injury. The segregation of responses to thermal injury in inbred rats into susceptible, intermediate, and resistant groups on the basis of strain origin indicates that such natural resistance may be a quantitative trait. One of the genetic components affecting host resistance is sex-linked. The existence of genetically controlled variations in natural resistance to trauma may be an important determinant of survival and may be a source of guidelines for the triage and clinical care of injured patients. It may also be an important selective factor in evolution.

Appearance of cytoskeletal components on the surface of leukemia cells and of lymphocytes transformed by mitogens and Epstein-Barr virus.

Lactoperoxidase iodination and two-dimensional electrophoresis of the labeled proteins have demonstrated well-characterized cytoskeletal proteins (actin and tubulins) on the surface of human lymphocytes undergoing blastogenic transformation and of certain malignant human cells. Such proteins could not be detected on the surface of normal resting human lymphocytes. The most prominent cytoskeletal protein identified on the surface membrane of mitogen-transformed T and B lymphocytes was actin. In Epstein-Barr virus genome-positive Burkitt's lymphoma and lymphoblastoid cell lines and in two leukemia cells, the major iodinated membrane protein components were actin and alpha 1-, alpha 2-, and beta-tubulins. These proteins were firmly connected to the cytoplasmic skeleton and could not be removed by Triton X-100. Concurrent immunofluorescence studies with specific antibodies and F(ab')2 fragments confirmed the appearance of cytoskeletal components on the biochemical data, and indicated that such cytoskeletal proteins formed distinctive patterns on the cell surface, ranging from small patches to large projections. Five-hour labeling with [35S]methionine indicates that all such cells released large quantities of labeled actin and tubulins into the culture medium. These materials were not readsorbed to the membrane surfaces of the cells.

The major histocompatibility complex (HLA) as a genetic marker in human craniofacial anomalies.

Study of the incidence and segregation of the serologically detectable A and B products of the HLA complex in 140 family units in which one or more offspring was afflicted with a developmental craniofacial anomaly has uncovered no evidence of an association between HLA-A or B antigens or haplotypes and the malformations under study. Further analysis of HLA-D products in the same family units by the mixed leukocyte culture (MLC) technique has, however, uncovered a relatively high incidence of non-reactivity between the cells of one (or both) parent(s) and cells of some offspring in 41 of the 140 families included in this study. The parent couples involved in this finding were unrelated and generally did not share any HLA-SD haplotypes. When this finding was studied further by Primed LD Typing techniques, the results in six families suggested that such MLC non-reactivity is a consequence of the sharing of LD alleles by each pair of parents in these families. The known polymorphism of the HLA-D locus (or loci) and the low incidence of comparable findings in the normal population suggest that LD allele sharing in this particular population may be related to the selection of certain particular HLA-D products in families afflicted with developmental craniofacial anomalies. This result may be relevant to the possible existence in man of an analogue of the murine T/t complex which may occur in linkage with the HLA complex, in the same manner as the linkage disequilibrium which is been documented between the t complex and H-2 in chromosome 17 of the mouse.

Induction of unresponsiveness to major transplantable organs in adult mammals: a recapitulation of ontogeny by irradiation and bone marrow replacement.

Transplantation of renal allografts obtained from prospectively selected genotypically DLA-identical donors into supralethally irradiated dogs reconstituted with their own stored bone marrow has produced a state of unresponsiveness to these kidneys in the recipients. Eleven of 18 kidneys transplanted at 12 hours after marrow replacement currently survive with normal function and maintain life in the recipients for 757, 800, 825, 978, 1062, 1092, 1136, 1282, 1373, 1380, and 1381 days, respectively. Similar results occurred in eight of 13 allografts transplanted at 28 hours after marrow replacement, which currently survive for 349, 363, 377, 407,436,470, 485, and 513 days, respectively, and in eight of 13 kidneys grafted at 36 hours after marrow replacement, which are surviving for 197, 247, 298, 324, 337, 396, 443, and 472 days, respectively. Achievement of optimal results is dependent on the specific timing and sequence of each procedure. Only four of 16 recipients of kidneys transplanted at the time of marrow replacement were unresponsive to their allografts. Similarly, only five of 19 recipients of kidneys placed in irradiated dogs at 40 hours before marrow replacement accepted such allografts. When kidney transplants were placed into the recipients 20 hours before removal of marrow, irradiation, and reconstitution with stored marrow, only three of 21 dogs became unresponsive to such ailografts. In five of 12 instances, the recipients were also unresponsive to skin allografts obtained from their respective kidney donors. Such skin grafts currently survive for 606, 673, 687, 701, and 708 days, respectively. The remaining seven skin grafts were rejected at 28, 39,42, 84, 90, 92, and 115 days, respectively. Second- and third-set skin grafts from the same kidney donor were rejected by six of these dogs at 19, 20, 21, 29, 29, and 30 days, and at 21, 22, 23, 24, 27, and 27 days, respectively. Rejection of these skin grafts had no detectable effect on the function and survival of kidney allografts from the same source. Seven of eight skin grafts obtained from other DLA-identical donors were rejected at 13,14,16,25,28,38, and 84 days, respectively; one allograft continues to survive for 708 days. Eleven DLA-incompatible skin allografts placed on the recipients at the same time were rejected within 11-20 days. Supralethal total body irradiation and bone marrow replacement can establish in the adult canine host a privileged phase of immunological reactivity during which exposure to alloantigens produces specific long-term unresponsiveness rather than sensitization. The use of stored autologous rather than allogeneic bone marrow for reconstitution of the irradiated recipient eliminates the hazards of GVH complication usually associated with this procedure. This consideration and the apparent capacity of the tolerant host to maintain a long-term state of unresponsiveness without any further immunosuppressive therapy point to the potential relevance of the results to human transplantation.