Direct visualisation of cytomegalovirus-specific CD8+ T cells in renal transplant recipients.
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CD8-Positive T-Lymphocytes↗
Biomedical subjects
S H Sacks
Publications and source records attributed to S H Sacks.
Characteristics and outcome of membranous nephropathy in older patients.
Many patients with idiopathic membranous nephropathy are elderly, but little is known about the natural or treated history of these patients. We have studied a cohort of 155 patients with membranous nephropathy who were recruited and followed-up over a 20 year period. We have compared the clinical features and outcome of the older (>60 years) and younger age groups. There was a higher incidence of an identifiable cause for the nephropathy in older patients. At presentation with idiopathic disease, older patients were more often hypertensive and had worse renal impairment than the younger cohort, but had a similar levels of proteinuria, hypoalbuminemia and hematuria. Thrombotic complications and minor rheumatological complaints were more common in the older patients. Prognosis for life and renal survival was worse in the older onset patients. Treatment was well tolerated in selected older patients and was associated with a better outcome in those selected for treatment.
Adrenal Cortex Hormones↗
The allogeneic T and B cell response is strongly dependent on complement components C3 and C4.
BACKGROUND: The mechanisms controlling the production of antibodies against histocompatibility antigens are of prime importance in organ transplantation. METHODS: We investigated the role of complement in the response to allogeneic stimulation, using mice deficient in C3, C4, or C5 to dissect the role of the alternative, classical, and terminal complement pathways. RESULTS: After fully major histocompatibility complex disparate skin grafts, the allospecific immunoglobulin (Ig)G response was markedly impaired in C3- and C4-, but not in C5-deficient mice. This defect was most pronounced for second set responses. C3-deficient mice also demonstrated a decreased range of IgG isotypes. In contrast, there was no impairment of the allospecific IgM response. In functional T cell assays, the proliferative response and interferon-gamma secretion of recipient lymphocytes restimulated in vitro with donor antigen was decreased two- to threefold in C3-deficient mice. CONCLUSIONS: These data show impairment of allogeneic T cell and B cell function in mice with defective complement activation and suggest a predominant role for the classical pathway in stimulating alloimmunity. The terminal pathway seems unimportant in this regard. This extends the results reported for soluble protein antigens and demonstrates a surprisingly marked effect on the alloresponse despite the presence of a stringent antigenic stimulus. These results have implications for the prevention of sensitization in naïve transplant recipients.
Animals↗
Rapid rejection of HLA-A2 transgenic skin graft due to indirect allorecognition.
BACKGROUND: At present, it is not clear whether xenogeneic MHC molecules are recognized by T cells directly or indirectly through self-MHC-restricted presentation in a transplantation setting. METHODS: We have transplanted skin from HLA-A2 transgenic (B6.A2) to nontransgenic C57BL/6 (B6) mice and investigated the subsequent mouse T-cell responses to HLA molecules, in vivo and in vitro. RESULTS: Skin transplanted from transgenic B6.A2 to B6 mice was rejected rapidly, in 12-16 days. Although naive B6 mice did not respond to B6.A2 splenocytes in vitro, spleen cells from mice that underwent transplantation showed strong proliferative responses. An anti-B6.A2 T-cell line from mice that underwent transplantation made proliferative responses to B6.A2 splenocytes but did not recognize HLA-A2 on human cells or transfected allogeneic mouse cells. The indirect, self-H-2-restricted recognition of HLA-A2 implied by this was confirmed by the finding that lysates of HLA-A2-positive, but not HLA-A2-negative, human B cells were stimulatory when pulsed onto syngeneic antigen-presenting cells and by inhibition of anti-B6.A2 proliferation with both anti-mouse MHC class I and class II antibodies. CONCLUSION: Our results suggest that indirect recognition of xenogeneic MHC antigen plays a predominant role in graft rejection.
Animals↗
Protection and injury: the differing roles of complement in the development of glomerular injury.
The role of complement in autoimmune glomerulonephritis (as in other autoimmune diseases) is paradoxical, in that complement activation mediates acute inflammatory injury, yet inherited deficiency of complement may predispose to immune complex disease in particular immune complex glomerulonephritis. We have investigated the role of complement in experimentally induced glomerulonephritis in C3-deficient mice, using antibodies against the mouse glomerular basement membrane (GBM). In the acute phase of the disease, which is initiated by binding of heterologous antibody to the GBM, we confirmed that the inflammatory injury was positively complement dependent, with C3-deficient mice developing less severe injury. In contrast, in the autologous phase of the disease, mediated by the immune response against the heterologous antibody fixed in the GBM, the disease was negatively complement dependent. That is, by 14 days after disease induction the C3-deficient mice had heavier proteinuria and more severe uremia (p < 0.001) compared to the complement sufficient mice. The C3-deficient mice also showed a greater accumulation of electron-dense deposits in the GBM. These findings were reproduced in an accelerated model of this disease in which C3-deficient mice also develop more severe functional disturbance and demonstrate a higher rate of immune complex deposition. These data illustrate the potential for the net effect of complement to switch from a detrimental to a protective mode at different stages of autoimmune injury.
Animals↗
Epithelial secretion of C3 promotes colonization of the upper urinary tract by Escherichia coli.
To assess the role of complement in renal infection, we studied a model of Escherichia coli-induced pyelonephritis in mice deficient in complement components C3 and C4. Renal infection occurred less frequently in C3- and C4-deficient mice compared with wild-type mice. In vitro, renal epithelial cells internalized fewer bacteria in the absence of C3 or in the presence of blockade of C3 bound to the bacteria. Moreover, upregulation of epithelial C3 production by stimulation with lipopolysaccharide enhanced bacterial internalization. Here we provide evidence that uropathogenic E. coli might use host C3 to invade the renal epithelium and that local complement production is sufficient for the bacteria to achieve this effect.
Animals↗
Intrarenal synthesis of complement.
During the past decade, research has shown that the kidney has the capacity to synthesize most of the activation pathway components of the complement cascade. As well as implying physiological roles in local clearance of immune complexes and defense against invasive organisms, an increasing amount of evidence indicates that the intrarenal synthesis of complement makes an important contribution in the pathogenesis of renal injury. Here we review this evidence and present a case for more definitive investigation of these functions.
Animals↗
Transferrin but not albumin mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.
Complement is increasingly implicated in the pathogenesis of progressive renal disease resulting from persistent proteinuria. We have previously shown that apical serum proteins stimulate C3 in cultured human proximal tubular epithelial cells (PTECs), and that the stimulant is a nonalbumin compound of 30 to 100 kd. We postulated in this study that transferrin and apotransferrin, also important components of proteinuric urine in this molecular-weight range, might be the culprit. Human PTECs were obtained by differential sieving of renal cortical tissue from the normal pole of tumor nephrectomy specimens and characterized to be predominantly of proximal tubular origin. Complement C3 messenger RNA (mRNA) expression was analyzed in confluent growth-arrested PTEC monolayers in media containing different concentrations (2.5 to 20 mg/mL) of transferrin by reverse transcription and polymerase chain reaction. Pure human albumin was used as a control protein. C3 protein secretion was detected and quantified by a sandwich enzyme-linked immunosorbent assay on cell culture supernatants after distinct time points. Transferrin enhanced the rate of C3 secretion in a dose-dependent manner, reaching maximal stimulation at doses of 10 mg/mL. Selected experiments using the Transwell technique showed that C3 release was predominantly apical in the resting state. The addition of 10 mg/mL of transferrin apically but not basolaterally stimulated both apical and basolateral C3 secretion and increased the basolateral-apical ratio of C3 secretion from 0.45 +/- 0.16 to 0.93 +/- 0.24 (P: < 0.02). Constitutive C3 mRNA expression was upregulated by transferrin in a time- and dose-dependent fashion, reaching a peak after 24 hours. A similar degree of C3 upregulation was reproduced when iron-poor transferrin, apotransferrin, was used instead. These results indicate that C3 synthesis in PTECs is upregulated by transferrin, for which protein rather than iron moiety may account for the observed effects. These findings provide evidence linking proteinuria with overexpression of tubular complement.
Apoproteins↗
Local tissue complement synthesis--fine tuning a blunt instrument.
Complement is important to host defense and the regulation of inflammation. The liver is overwhelmingly the major source of circulating complement. However, many other organs are capable of synthesizing some or all of the complement components in a regulated tissue-specific manner. There is increasing evidence that this locally generated complement is biologically active and exerts powerful effects within the local environment. We review the role of local complement synthesis within different organs and speculate on its implication for immune and metabolic functions.
Adipocytes↗
Mechanisms of nephrotoxicity.
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Cyclosporine↗
In situ localization of C3 synthesis in experimental acute renal allograft rejection.
Recent evidence has implicated complement in renal transplant injury and identified the kidney as a source of complement components. We therefore investigated the local gene expression of complement component C3, pivotal to complement activation pathways and a mediator of inflammatory injury, in a rat renal transplant model. By reverse transcriptase-polymerase chain reaction, the expression of C3 mRNA increased in two phases. The first phase coincided with post-ischemic injury over 2 days post-transplantation and was localized by in situ hybridization to vessels and glomerular mesangial cells in allogeneic and syngeneic (control) kidney transplants. In allografts only, a second phase was found in tubular epithelial cells, glomerular parietal cells, vessel walls and some infiltrating cells, which peaked on day 4 together with rapid influx of leukocytes, tubule cell damage, the induction of interleukin-2 and interferon-gamma mRNA, and the up-regulation of tumor necrosis factor-alpha and interleukin-1beta mRNA in the graft. In vitro studies showed that interleukin-2 and interferon-gamma up-regulate C3 production in renal tubule cells. We conclude that post-ischemic injury led to transient up-regulation of glomerular expression of C3 mRNA. Subsequent cellular rejection was associated with tubulointerstitial/glomerular parietal cell expression of C3 mRNA. This differential expression of local C3, immediately post-transplant or associated with acute rejection, may have implications for putative therapeutic complement inhibition in clinical transplantation.
Acute Disease↗
Predominant role for C5b-9 in renal ischemia/reperfusion injury.
Previous work has indicated that complement is a mediator of ischemia/reperfusion (I/R) injury. To investigate the components of complement responsible for this effect, we examined a model of renal I/R injury in C3-, C4-, C5-, and C6-deficient mice. We occluded the renal arteries and veins (40-58 minutes) and, after reperfusion (0-72 hours), assessed renal structural and functional injury. C3-, C5-, and C6-deficient mice were protected from renal I/R injury, whereas C4-deficient mice were not protected. C6-deficient mice treated with antibody to block C5a generation showed no additional protection from I/R injury. Reconstitution with C6 alone restored the I/R injury in C6-deficient mice. Tubular epithelial cells were the main structures damaged by complement-mediated attack, and, in contrast, the renal vessels were spared. Neutrophil infiltration and myeloperoxidase activity were reduced in C-deficient mouse kidney, but by a similar extent in C3-deficient and C6-deficient mice. We conclude that the membrane attack complex of complement (in which C5 and C6 participate) may account for the effect of complement on mouse renal I/R injury. Neither C5a-mediated neutrophil infiltration nor the classic pathway, in which C4 participates, appears to contribute to I/R injury in this model. By contrast with other organs, such as the heart, the primary effect of complement in the ischemic area is on the parenchymal cell rather than the vascular endothelial cell. The membrane attack complex of complement is a potential target for prevention of I/R injury in this model.
Animals↗
Contribution of renal secreted complement C3 to the circulating pool in humans.
Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.
Adolescent↗
Accurate intracellular localization of HLA-DM requires correct spacing of a cytoplasmic YTPL targeting motif relative to the transmembrane domain.
HLA-DM is an MHC class II-related heterodimer that is targeted to lysosomal compartments by a tyrosine-based signal YTPL, present in the cytoplasmic tail of the beta chain. Similar signals in other proteins control transport to different intracellular locations and can be recognized at several sorting sites within the cell including the trans-Golgi network, the plasma membrane and the early or sorting endosome. Therefore, in addition to recognizing the basic tyrosine motif, the sorting machinery must be sensitive to additional features associated with these elements. Here we show that efficient trafficking of HLA-DM to lysosomal compartments is dependent upon the proximity of its tyrosine motif to the transmembrane domain. Constructs in which the spacing is altered are rapidly internalized but are expressed at the cell surface. We conclude that the spacing of the HLA-DMB-encoded tyrosine motif relative to the transmembrane domain is an important feature controlling DM sorting in endosomes.
Amino Acid Sequence↗
Altered distribution of intraglomerular immune complexes in C3-deficient mice.
We have studied the role of complement in a model of glomerular inflammation induced by the in situ formation of immune complexes along the glomerular basement membrane. In C3-deficient mice, produced by homologous recombination, immune complex formation occurs initially in the subendothelial site and progresses slowly to the subepithelial position, whereas wild-type mice do not develop subendothelial deposits. In addition, the accumulation of electron-dense deposits is greater in the complement-deficient mice. Complement therefore influences glomerular handling of immune complexes, possibly because of changes in the physiochemical characteristics of the immune complexes. However, despite evidence of complement activation in the wild-type mice, as demonstrated by immunohistochemical detection of C3, C4 and C9, the degree of proteinuria was similar in C3-deficient mice. We conclude that, although complement is required for the normal glomerular metabolism of immune complexes, other, complement-independent, factors are involved in the generation of glomerular injury in this model.
Animals↗
In vitro erythrophagocytosis by renal tubular cells and tubular toxicity by haemoglobin and iron.
BACKGROUND: Patients with gross haematuria of glomerular origin may develop acute tubular necrosis and reversible renal failure. Erythrocytes within the cytoplasm of proximal tubular epithelial cells (PTECs) can be seen on examination of renal biopsies from these patients. It is possible, therefore, that the tubular damage is a result of cytotoxic breakdown products released during erythrocyte degradation. METHODS: To test this hypothesis, we evaluated (i) by transmission electron microscopy, the capability of a PTEC line to phagocytose and degrade erythrocytes in vitro; and (ii) the effect on the viability of PTCEs in vitro both after erythrophagocytosis and after incubation with haemoglobin, free iron or both. RESULTS: Electron microscopic examination of PTECs exposed to erythrocytes for 96 h showed that 22% of PTECs contained one or more erythrocyte. These were within phagolysosomes and showed varying stages of degradation, with collapse and breakdown of the cell membrane and invasion by cytoplasmic organelles (the so-called haemolytic pathway of erythrocyte degradation). Despite the phagocytosis and degradation of the erythrocytes, no cytotoxicity could be demonstrated under the experimental conditions used. However, the presence of haemoglobin, free iron or both in the culture medium was toxic to the PTECs, resulting in a significant reduction in the number of viable cells present. CONCLUSIONS: PTECs are able to phagocytose and degrade erythrocytes, and haemoglobin and iron are toxic to proximal tubular cells in vitro.
Cells, Cultured↗
Rapamycin on trial.
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Clinical Trials as Topic↗
Targeting the complement system.
Interest has blossomed in the development of complement inhibitors, in parallel with a growth in our understanding of the biology of the complement cascade. The first generation of designed inhibitors was based on naturally occurring complement receptors and regulatory molecules. These agents provided useful tools for exploring the role of complement in experimental models of disease, but may have limited therapeutic application in humans because of their short half-lives, limited bioavailability and possible antigenicity. More recently, humanized antibodies and synthetic molecules that block the activation of complement have been developed, which look as though they may overcome some of these difficulties. The possibility for precision inhibition of a limited part of the complement cascade, or for inhibition confined to a single organ, may offer effective therapeutic results, while avoiding the disadvantages of nonselective complement blockade. This review examines the recent evidence that complement inhibition will reduce tissue damage resulting from organ transplantation, ischaemia-reperfusion injury, cancer, glomerulonephritis and the use of extracorporeal circuits.
Animals↗