Wuding Zhou

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.

Dendritic cell synthesis of C3 is required for full T cell activation and development of a Th1 phenotype

Previous studies have found that deficiency of complement component C3 is associated with reduced T cell responses in several disease models including viral infection, autoimmune disease, and transplantation. However, the underlying mechanism is unclear. In this study, we demonstrate that dendritic cells (DCs) are able to synthesize C3 and this synthesis is required for the capacity of DCs to stimulate alloreactive T cell responses in vitro and in vivo. Compared with C3-producing DCs, C3-nonproducing DCs exhibit reduced potency to stimulate an alloreactive T cell response, favor the polarization of CD4+ T cells toward Th2 phenotype, and have regulatory T cell-driving capacity. In addition, priming mice with C3-deficient DCs compared with wild-type DCs led to delayed skin allograft rejection. Our findings that nonproduction of C3 by DCs significantly reduced T cell stimulation and impaired allograft rejection provide a potentially important explanation of how C3-deficient mice develop reduced T cell responses and of how C3-deficient donor kidney is protected from T cell-mediated graft rejection.

Local extravascular pool of C3 is a determinant of postischemic acute renal failure

The third complement component (C3) is an acute phase protein that plays a central role in reperfusion injury in several organ models. To investigate the contribution of local synthesis of C3 and distinguish it from that of circulating complement mainly produced by hepatic synthesis, we employed a mouse renal isograft model. Our model demonstrated a close relationship between the extent of intrarenal expression of C3 and cold‐ischemia induced injury. Ischemic C3‐positive donor kidneys transplanted into C3‐positive or C3‐negative recipients developed widespread tissue damage and severe acute renal failure. In contrast, ischemic C3‐negative isografts exhibited only mild degrees of functional and structural disturbance, even when transplanted into normal C3‐positive recipients. Thus local synthesis of C3, mostly identified in the tubular epithelium, was essential for complement‐mediated reperfusion damage, whereas circulating C3 had a negligible effect. Our results suggest a two‐compartment model for the pathogenic function of C3, in which the extravascular compartment is the domain of local synthesis of C3, and where the role of circulating C3 is redundant. Our data cast new light on the mechanism of complement‐mediated tissue injury in nonimmunological disorders, and challenges the long‐standing dogma that circulating components are the main complement effectors of extravascular tissue damage.—Farrar, C. A., Zhou, W., Lin, T., Sacks, S. H. Local extravascular pool of C3 is a determinant of postischemic acute renal failure. FASEB J. 20, 217–226 (2006)

The role of complement in the early immune response to transplantation

The complement system is a key element of the innate immune system, and the production of complement components can be divided into central (hepatic) and peripheral compartments. Essential complement components such as C3 are produced in both of these compartments, but until recently the functional relevance of the peripheral synthesis of complement was unclear. Here, we review recent findings showing that local peripheral synthesis of complement in a transplanted organ is required for the immediate response of the donor organ to tissue stress and for priming alloreactive T cells that can mediate transplant rejection. We also discuss recent insights into the role of complement in antibody-mediated rejection, and we examine how new treatment strategies that take into account the separation of central and peripheral production of complement are expected to make a difference to transplant outcome.

Cyclic AMP plays a critical role in C3a-receptor mediated regulation of dendritic cells in antigen uptake and T cell stimulation

The biochemical basis for complement acting directly on antigen-presenting cells to enhance their function in T-cell stimulation has been unclear. Here we present evidence that engagement of C3a receptor (C3aR) on the surface of dendritic cells (DCs) leads to alterations in the level of intracellular cyclic adenosine monophosphate (cAMP), a potent negative regulator of inflammatory cytokines. C3aR activation-induced depression of cAMP was associated with enhanced capacity of DCs for antigen uptake and T-cell stimulation. Conversely, C3aR-deficient DCs showed elevation of cAMP and impaired properties for antigen uptake and immune stimulation. Similarities in the phenotype of C3-deficient and C3aR-deficient DCs suggest that local production of C3 with extracellular metabolism to C3a is an important driver of DC alterations in cAMP. The finding of a link between complement and adaptive immune stimulation through cAMP offers new insight into how innate and adaptive immunity combine to generate efficient effector and memory responses.

C3a and C5a Promote Renal Ischemia-Reperfusion Injury

Renal ischemia reperfusion injury triggers complement activation, but whether and how the small proinflammatory fragments C3a and C5a contribute to the pathogenesis of this injury remains to be elucidated. Using C3aR-, C5aR-, or C3aR/C5aR-deficient mice and models of renal ischemia-reperfusion injury, we found that deficiency of either or both of these receptors protected mice from injury, but the C3aR/C5aR- and C5aR-deficient mice were most protected. Protection from injury was associated with less cellular infiltration and lower mRNA levels of kidney injury molecule-1, proinflammatory mediators, and adhesion molecules in postischemic kidneys. Furthermore, chimera studies showed that the absence of C3aR and C5aR on renal tubular epithelial cells or circulating leukocytes attenuated renal ischemia-reperfusion injury. In vitro, C3a and C5a stimulation induced inflammatory mediators from both renal tubular epithelial cells and macrophages after hypoxia/reoxygenation. In conclusion, although both C3a and C5a contribute to renal ischemia-reperfusion injury, the pathogenic role of C5a in this injury predominates. These data also suggest that expression of C3aR and C5aR on both renal and circulating leukocytes contributes to the pathogenesis of renal ischemia-reperfusion injury.

Dendritic Cell Function in Allostimulation Is Modulated by C5aR Signaling

Regulation of T cell immunity by C5a has been suggested from recent studies. However, the underlying mechanisms, particularly the involved cells and biochemical basis, are not well defined. In this study, the direct modulation of dendritic cell (DC) activation and its function in T cell stimulation by C5a-C5aR interaction and the involved signaling pathways were investigated. We show that DCs from C5aR−/− mice and normal DCs treated with C5aR antagonist have less-activated phenotype characterized with increased IL-10 and decreased IL-12p70 production in response to LPS stimulation, lowered surface expression of MHC class II, B7.2, and consequently have reduced capacity to stimulate allospecific T cells. Conversely, C5a stimulation up-regulates DC activation and its function in allostimulation. Furthermore, stimulation of C5aR mediates the inhibition of cAMP production and protein kinase A activity and is involved in activation of PI3K/AKT and NF-κB signaling in DCs. These results demonstrate that C5a acts directly on C5aR expressed on DCs resulting in the cell activation and subsequently enhances its capacity for allospecific T cell stimulation. It also suggests that NF-κB signaling induced by down-regulation of cAMP/ protein kinase A pathway and up-regulation of PI3K/AKT pathway following C5a stimulation may contribute to up-regulation of DC function.

Nontransgenic Hyperexpression of a Complement Regulator in Donor Kidney Modulates Transplant Ischemia/Reperfusion Damage, Acute Rejection, and Chronic Nephropathy

Complement activation during ischemia and reperfusion contributes to the development of tissue injury with severe negative impact on outcomes in transplantation. To counter the effect of complement, we present a strategy to deliver a novel complement regulator stabilized on cell surfaces within donor organs. The membrane-bound complement regulator is able to inhibit complement activation when the donor organ is revascularized and exposed to host-circulating complement. Application of this construct to donor kidneys protected transplanted tissues from ischemia/reperfusion injury and reduced the deposition of activated complement and histological signs of damage under conditions in which a nontargeted control construct was ineffective. Treatment of donor organs in this way improved graft performance in the short and long term. An analysis of the immune response in allograft recipients showed that reducing graft damage at the time of transplantation through complement regulation also modulated the alloresponse. Additionally, the results of perfusion studies with human kidneys demonstrated the feasibility of targeting endothelial and epithelial surfaces with this construct, to allow investigation in clinical transplantation.

Therapeutic Strategy with a Membrane-Localizing Complement Regulator to Increase the Number of Usable Donor Organs after Prolonged Cold Storage

A shortage of donor organs and increasing dependence on marginal grafts with prolonged ischemic times have meant that new methods are needed to prevent postischemic damage. Herein is reported a new strategy aimed to protect donor kidney from complement-mediated postischemic damage and therefore increase the number of successful transplants. Rat donor kidneys were perfused with a membrane-localizing complement regulator derived from human complement receptor type 1 (APT070) and then subjected to prolonged periods of cold storage (at 4 degrees C). A relationship was found between the duration of cold ischemia and the extent of complement-mediated tubule damage and loss of graft function. After 16 h of cold storage, APT070-treated kidneys that were transplanted into syngeneic recipients showed a significant increase in the number of surviving grafts, compared with control-treated grafts (63.6 versus 26.3%). Surviving grafts also displayed less acute tubular injury and better preservation of renal function. These results not only enhance the understanding of the mechanism by which prolonged cold ischemia reduces immediate graft survival but also provide essential information about the effectiveness of membrane-localizing complement regulator with prolonged cold storage. This could lead to more effective strategies for improving the use of severely ischemic donor organs.

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.