Complement-here, there and everywhere, but what about the transplanted organ?

The part of the innate immune system that communicates and effectively primes the adaptive immune system was termed "complement" by Ehrlich to reflect its complementarity to antibodies having previously been described as "alexine" (i.e protective component of serum) by Buchner and Bordet. It has been established that complement is not solely produced systemically but may have origin in different tissues where it can influence organ specific functions that may affect the outcome of transplanted organs. This review looks at the role of complement in particular to kidney transplantation. We look at current literature to determine whether blockade of the peripheral or central compartments of complement production may prevent ischaemic reperfusion injury or rejection in the transplanted organ. We also review new therapeutics that have been developed to inhibit components of the complement cascade with varying degrees of success leading to an increase in our understanding of the multiple triggers of this complex system. In addition, we consider whether biomarkers in this field are effective markers of disease or treatment.

Thrombalexins: Cell-Localized Inhibition of Thrombin and Its Effects in a Model of High-Risk Renal Transplantation.

Allograft transplantation into sensitized recipients with antidonor antibodies results in accelerated antibody-mediated rejection (AMR), complement activation, and graft thrombosis. We have developed a membrane-localizing technology of wide applicability that enables therapeutic agents, including anticoagulants, to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLNs). Using a rat model of hyperacute rejection, we investigated the potential of one such inhibitor (thrombalexin-1 [TLN-1]) to prevent acute antibody-mediated thrombosis in the donor organ. TLN-1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.

Coexpression of donor peptide/recipient MHC complex and intact donor MHC: evidence for a link between the direct and indirect pathways.

T lymphocytes recognize foreign antigens presented by donor or recipient cells through the direct and indirect pathways respectively. This raises the question of how directly and indirectly activated T cells interact. A 4-cell model involving the interaction of CD4(+) and CD8(+) T cells recognizing major histocompatibility complex (MHC) class II on recipient antigen presenting cell (APC), and MHC class I on donor APC, has been proposed. However, this would require complex co-ordination between all the participating cell types. The semidirect pathway of alloantigen presentation suggests a simpler mechanism. Although exchange of MHC class II molecules between donor and recipient cells has been described, coexpression of recipient MHC molecules presenting donor derived allopeptides (indirect presentation) and donor MHC (direct presentation) on the same cell, a key requirement for the semidirect alloantigen presentation pathway, has not been demonstrated. We have used a mouse transplantation model to demonstrate the presence of cells expressing both donor MHC class I/II molecules, and a donor MHC class II peptide in the context of a recipient MHC class II molecule. This would allow indirectly activated CD4(+) T cells to regulate directly activated CD4(+) T cells, or to help directly activated CD8(+) T cells, thus providing physical evidence for the semidirect pathway.

SPECT/CT lymphoscintigraphy of heterotopic cardiac grafts reveals novel sites of lymphatic drainage and T cell priming.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients' during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

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.

Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation.

Experimental unilateral ureteral obstruction (UUO) is widely used to study renal fibrosis; however, renal injury can only be scored semiobjectively by histology. We sought to improve the UUO model by reimplanting the obstructed ureter followed by removal of the contralateral kidney, thus allowing longitudinal measurements of renal function. Mice underwent UUO for different lengths of time before ureteral reimplantation and contralateral nephrectomy. Measurement of blood urea nitrogen (BUN) allows objective evaluation of residual renal function. Seven weeks after reimplantation and contralateral nephrectomy, mean BUN levels were increased with longer duration of UUO. Interstitial expansion, fibrosis, and T-cell and macrophage infiltration were similar in kidneys harvested after 10 days of UUO or following 10 weeks of ureter reimplantation, suggesting that the inflammatory process persisted despite relief of obstruction. Urinary protein excretion after reimplantation was significantly increased compared to control animals. Our study shows that functional assessment of the formerly obstructed kidney can be made after reimplantation and may provide a useful model to test therapeutic strategies for reversing renal fibrosis and preserving or restoring renal function.

An antibody combination that targets activated T cells extends graft survival in sensitized recipients.

Memory T cells are the very essence of adaptive immunity with their rapid and efficient response to antigen rechallenge and long-term persistence. However, it is becoming increasingly evident that when primed with self or transplanted tissue, these cells play a key role in causing and perpetuating tissue damage. Furthermore, current treatments, which efficiently control the naive response, have limited effects on primed T cells. We have used a treatment based on a combination of antibodies specific for molecules expressed by activated T lymphocytes to selectively remove these cells. This approach, which we termed multi-hit therapy, leads to cumulative binding of antibodies to the target T cells and a striking prolongation of skin graft survival in presensitized recipients in a stringent skin transplant model. The findings are consistent with the depletion of graft-specific CD4+ and CD8+ T cells, although other modes of action, such as T-cell regulation and altered migration could play a role. In conclusion, our therapeutic strategy controls primed T cells which are a major driving force in the pathology of many autoimmune diseases and in transplant rejection.

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.

Human blood group isoantigen expression on normal and malignant gastric epithelium studied with anti-A and anti-B monoclonal antibodies.

Variation in human blood group isoantigen expression on normal and malignant gastric epithelium was demonstrated with monoclonal antibodies to blood groups A and B in an indirect immunoperoxidase technique. The expected isoantigen expression was demonstrated on endoscopic biopsy specimens of normal gastric mucosa from 11 patients. Of 17 patients with gastric carcinoma (blood group A, 15; blood group AB, 2), complete loss of isoantigen expression was noted in 6 (35%). In these 6 patients, blood group isoantigen remained both in the adjacent uninvolved mucosa and at the margin of resection. The loss of isoantigen did not appear to be related to the degree of differentiation within the tumor, to the secretor status of the patient, or to the blood subgroup. Lymph node metastases reflected the isoantigen status of the primary tumor, being positive in 5 of 6 expression in all 17 patients or in an additional 15 patients studied with blood group O. These findings were discussed in the light of previously reported work on the localization of blood group isoantigens on malignant and nonmalignant gastric epithelium with the use of conventional antisera and a variety of immunohistologic techniques.

Strategies for targeting complement inhibitors in ischaemia/reperfusion injury.

A transplanted organ suffers inherently from an ischaemic insult and subsequent reperfusion injury. The severity of such early events is thought to influence the success of the transplant procedure, not only in the immediate post-transplant period, but also to predispose the graft to both acute and chronic rejection. In this paper, we review the influence of the complement system upon ischaemia,reperfusion injury. The recognition of the involvement of complement has led to novel strategies to try to modulate ischaemia/reperfusion injury, some of which we have summarized. Finally, we note our own strategy to target complement inhibition in ischaemic tissues.

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.

Molecular analysis of C3 allotypes related to transplant outcome in human renal allografts.

The third component of complement (C3) exists in two main allotypic forms, C3S and C3F, which can be distinguished at the molecular level using a variation of the polymerase chain reaction. An increased frequency of the C3F allele has been noted in a number of autoimmune and inflammatory conditions affecting the kidney, including systemic vasculitis, IgA nephropathy, and type II mesangiocapillary nephritis. Recently, in an unrelated study, we found (with small numbers) an increased incidence of graft loss associated with the presence of the C3F allele. To further assess this, we analyzed the S/F polymorphism in 183 donor-recipient pairs of patients undergoing renal transplantation. Forty-one of 183 grafts were lost, but graft loss was not associated with the C3F allele over 14-month follow-up. However, the presence of the C3F allele predicted an increased risk of graft dysfunction (defined as serum creatinine > 150 mumol/L): 61/105 versus 36/78, with a relative risk of 1.4 (P < 0.05). The C3F allele predisposed toward graft dysfunction when present in either donor or recipient. The presence of two C3F alleles gave a relative risk for graft dysfunction of 1.8, suggesting a dose-dependent effect, although numbers were small. The presence of the C3F allele was not significantly correlated with the number of rejection episodes, serum creatinine, or duration of primary nonfunction. These findings suggest that C3F may be a susceptibility allele for allograft injury. Possible mechanisms for this association are discussed.

Rhesus immunization after renal transplantation.

Previous reports, before the advent of cyclosporine, suggest that the small amount of blood transplanted with a kidney can result in rhesus D (RhD) antibody production. We looked for retrospective and current evidence of primary RhD antibody production following renal transplantation in RhD-negative recipients of an RhD-positive kidney. Of 42 patients, all on triple immunosuppressive therapy, 2 (5%) were found to have an RhD antibody identified for the first time after transplantation. As the number of pregnancies in transplant recipients increases, the small risk of primary immunization and subsequent risk of hemolytic disease of the newborn will become more important. Therefore, we recommend that all RhD-negative women of child-bearing age receiving an RhD-positive solid organ transplant are given a prophylactic dose of 500 IU of anti-D immunoglobulin intramuscularly at the time of transplantation.

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.

Up-regulation of type 1 plasminogen activator inhibitor messenger RNA with thrombotic changes in renal grafts.

Small vessel thrombosis is a prominent feature in kidneys undergoing vascular rejection. Type I and type 2 plasminogen activator inhibitors (PAI-1 and PAI-2, respectively) are known to mediate thrombosis. To examine the potential role of PAI-1 and PAI-2 in the mediation of vascular injury, the relationship and the time course of gene expression of PAI-1 and PAI-2 with the thrombotic changes in renal grafts were investigated in an unmodified rejection model in rats. Orthotopic renal transplantation was performed from Lewis to dark agouti (DA) rats and from DA to DA isografts; untreated normal rat kidneys were used as controls. The rats were killed on days 1-9 posttransplantation (n=18 in each allograft and isograft group). The grafts were analyzed by histopathology, in situ mRNA hybridization and Northern blot methods. The results show that PAM mRNA was first detected at day 4, when the thrombotic changes in the grafts were first seen, and that this relationship persisted during the time course observed to day 9. There was no detectable PAI-1 mRNA in the control groups and no PAI-2 in either group. In situ hybridization showed that PAI-1 positive cells were predominantly located in the cortical interstitium, consistent with the distribution of interstitial microthrombi. These results provide experimental evidence that the thrombotic changes in rejecting allografts are associated with the up-regulation of PAI-1 in the donor tissue, whereas PAI-2, from our results, does not seem to influence these changes. The data are consistent with a role for PAI-1 in the pathogenesis of vascular rejection.

Functional and biochemical subtypes of the haplotype HLA-DR3 in patients with celiac disease or idiopathic membranous nephropathy.

HLA class II beta-chain polymorphism was investigated in the haplotype HLA-DR3 to determine if patients with HLA-DR3-associated diseases express normal or variant class II polymorphisms. Analysis was carried out by two-dimensional gel electrophoresis of immunoprecipitated HLA class II molecules, DNA hybridization with DR beta and DQ beta gene probes on Taq 1, Bam H1, or Rsa 1 digests, and mixed lymphocyte culture. Two subtypes of HLA-DR3 were identified in normal homozygous DR3 individuals on the basis of polymorphism in one of two DR beta chains detected, corresponding to differences in DR beta restriction fragment patterns. These polymorphisms exhibited significant linkage disequilibrium with the A1,B8,DR3 and B18,DR3 haplotypes, respectively. In proliferative experiments, cells with the B18,DR3-associated polymorphism strongly stimulated cells from donors with the B8,DR3-related polymorphism, suggesting that a T-cell epitope recognized by B8,DR3 cells lies on the B18,DR3-associated DR beta chain. In seven HLA-DR3 homozygous patients with celiac disease and three HLA-DR3-homozygous patients with idiopathic membranous nephropathy, only the normal patterns of HLA class II molecules were displayed, the B8,DR3 type occurring in all patients and the B18,DR3 type in one patient. These data suggest that celiac disease and idiopathic membranous nephropathy are not related to disease-specific HLA-DR beta or -DQ beta gene variants within the DR3 population that are revealed by these methods.

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.

Analysis of activated T cell infiltrates in rat renal allografts by gamma camera imaging after injection of 123iodine-interleukin 2.

Activated T cells bearing receptors for interleukin 2 (IL-2) play an important role in immunity and in immunopathological processes such as allograft rejection. In order to investigate the presence of activated T cells in lymphocytic infiltrates in transplanted kidneys, we investigated the uptake and retention of radioactivity after an intravenous injection of radioiodinated IL-2 in experimentally transplanted rats. IL-2 was enzyme radiolabelled with 123iodine using a glucose oxidase/lactoperoxidase method and shown to retain specific binding on an IL-2 receptor positive cell line, C58E6. To examine the kinetics of 123iodine-interleukin 2 (123I-IL-2) uptake in vivo, animals that had been transplanted five days previously with allogeneic or syngeneic grafts were injected with 123I-IL-2 and then imaged using an external gamma camera. Radioactivity was measured at time points up to 240 min after intravenous injection of 123I-IL-2. Four groups of animals were examined: allogeneic grafts (n = 7); syngeneic grafts (n = 6); ischaemic native kidneys (n = 5) all following injection with 123I-IL-2; and allogeneic transplants (n = 5) after injection of 123I-lactalbumin, an irrelevant molecule of similar molecular weight to IL-2. The peak radioactivity after injection was measured and the amount of radioactivity retained in the graft at increasing time intervals after injection was expressed as a function of initial peak radioactivity. At four hours after injection of 123I-IL-2, mean retention of activity by rejecting grafts was 77(+/- 2.68)% of peak activity, compared to 45(+/- 6.38)% in syngeneic controls (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Allograft immune response with sCR1 intervention.

The deposition of complement (C) components on tissues of transplanted organs may induce many proinflammatory responses. The role of such C activation in allograft rejection is uncertain. We addressed this question by inhibiting C at the level of the C3 and C5 convertases, preventing C activation and progression of its cascade, using recombinant human soluble complement receptor 1 (sCR1) in an unsensitized rat renal allograft model. Fully MHC disparate Lewis to DA rat renal allograft recipients given 25 mg/kg sCR1 daily, with saline-treated allograft recipients as controls (n = 15 in each group), were sacrificed from day 1 to day 5 post-transplant, and examined histopathologically, and for the deposition of C3 and C5b-9 membrane attack complex (MAC), and for the presence of leucocyte antigen markers. Treated animals demonstrated a reduction in vascular injury and cellular infiltration, coincident with reduced C deposition. Flow cytometric analysis of leucocyte subpopulations in the spleen showed a reduction in activated (CD25 positive) B and T cells in treated animals, compared to saline treated controls. The results suggest that C inhibition with sCR1, in an unsensitized model of allograft rejection, was able to suppress the vascular and cell mediated components of tissue injury. The data support not only a role for C in antibody and possibly cell mediated cytotoxicity in the graft, but also suggest a role in the primary immune response leading to both T cell and B cell activation.

Molecular mapping of the HLA class II region in HLA-DR3 associated idiopathic membranous nephropathy.

Susceptibility to IMN is associated, in European Caucasoids, with the extended HLA haplotype in A1, B8, and DR3. It is unclear from previous investigations of HLA class II genes whether the association with A1, B8, DR3 is due to an HLA-DR or -DQ locus, or both, or to another locus linked to HLA class II. To examine genetic polymorphism over a more extensive area of DNA than previously, we carried out long range mapping of the HLA class II region of A1, B8, DR3 patients and healthy controls to discover if new markers of disease could be identified at this level of organization. Large fragments of genomic DNA were cut using enzymes with infrequent restriction sites, and were separated by pulsed field gel electrophoresis (PFGE) and analyzed using a series of probes which cover the HLA class II region. In several different DR3 haplotypes examined, the overall content of DNA and organization of the class II region were similar. However, both patient and control B8, DR3 haplotypes contained an extra Pvul site in the DRB region, compared to the disease-neutral B18, DR3 haplotype. Further, the DP region of the patient B8, DR3 haplotypes contained an additional partial BssHII cutting site which was not identified in the control B8, DR3 haplotypes. This structural heterogeneity in the vicinity of DP could have implications for genetic susceptibility to IMN and for linkage disequilibrium.