Conrad A. Farrar

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.

Fc-dependent depletion of activated T cells occurs through CD40L-specific antibody rather than costimulation blockade

Although the underlying mechanisms are not well understood, it is generally believed that antigen recognition by T cells in the absence of costimulation may alter the immune response, leading to anergy or tolerance. Further support for this concept comes from animal models of autoimmunity and transplantation, where treatments based on costimulation blockade, in particular CD40 ligand (CD40L)-specific antibodies, have been highly effective. We investigated the mechanisms of action of an antibody to CD40L and provide evidence that its effects are dependent on the constant (Fc) region. Prolongation of graft survival is dependent on both complement- and Fc receptor–mediated mechanisms in a major histocompatibility complex (MHC)-mismatched skin transplant model. These data suggest that antibodies to CD40L act through selective depletion of activated T cells, rather than exerting immune modulation by costimulation blockade as currently postulated. This finding opens new avenues for treatment of immune disorders based on selective targeting of activated T cells.

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)

Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2–deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti–MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

Complement Activation Regulates the Capacity of Proximal Tubular Epithelial Cell to Stimulate Alloreactive T Cell Response

Tissue expression of C3 is an unexpected regulator of the alloimmune response in mouse kidney transplantation. It is unclear, however, whether a direct or an indirect action of complement on the host immune response is involved. Also unknown is which of the complement effector products, cleaved C3, cleaved C5, or C5b-9, is responsible. Proximal tubular epithelial cells (PTEC) not only constitute a major target of the alloimmune response but also produce substantial amounts of C3. This study investigated the property of mouse PTEC to stimulate alloreactive T cells in a complement-dependent manner. The proliferative and cytokine responses of primed alloreactive T cells were measured after exposure to donor-specific PTEC that had been pretreated with normal mouse serum, heat-inactivated mouse serum, or complement- deficient (C3, C5, or C6) mouse sera to differentially deposit complement components. PTEC were able to stimulate alloreactive T cells in an antigen-specific manner. Complement activation leading to the deposition of cleaved C3 on PTEC enhanced the alloreactive T cell response. This complement-mediated stimulation of the T cell response was dependent on C3 but not on C5 or C6. The primary influence of tissue-bound complement was on CD4(+) T cells. Moreover, the effect of complement on alloreactive T cells was B7 dependent, shown by inhibition studies with CTLA4-Ig. These results suggest that donor epithelium-bound C3 can upregulate the alloimmune response. It is postulated that surface-bound C3 interacts with complement receptors on alloreactive T cells or on antigen presenting cells to increase allo-immune stimulation.

Collectin-11 detects stress-induced L-fucose pattern to trigger renal epithelial injury

Physiochemical stress induces tissue injury as a result of the detection of abnormal molecular patterns by sensory molecules of the innate immune system. Here, we have described how the recently discovered C-type lectin collectin-11 (CL-11, also known as CL-K1 and encoded by COLEC11) recognizes an abnormal pattern of L-fucose on postischemic renal tubule cells and activates a destructive inflammatory response. We found that intrarenal expression of CL-11 rapidly increases in the postischemic period and colocalizes with complement deposited along the basolateral surface of the proximal renal tubule in association with L-fucose, the potential binding ligand for CL-11. Mice with either generalized or kidney-specific deficiency of CL-11 were strongly protected against loss of renal function and tubule injury due to reduced complement deposition. Ex vivo renal tubule cells showed a marked capacity for CL-11 binding that was induced by cell stress under hypoxic or hypothermic conditions and prevented by specific removal of L-fucose. Further analysis revealed that cell-bound CL-11 required the lectin complement pathway-associated protease MASP-2 to trigger complement deposition. Given these results, we conclude that lectin complement pathway activation triggered by ligand-CL-11 interaction in postischemic tissue is a potent source of acute kidney injury and is amenable to sugar-specific blockade.

Deficiency of C5aR Prolongs Renal Allograft Survival

Interaction between C5a, a product of complement activation, and its receptor (C5aR) upregulates antigen-specific T cell responses by modulating the activation of antigen-presenting cells and T cells. Whether this C5a-C5aR interaction contributes to the immune responses that promote renal allograft rejection is unknown. Here, we found that deficiency of C5aR in both graft and recipient reduced allospecific T cell responses and prolonged renal allograft survival. In addition, lack of C5aR impaired the function of donor and recipient antigen-presenting cells and inhibited the response of recipient T cells to allostimulation. Furthermore, deficiency of C5aR in both graft and recipient reduced early inflammation in the grafts, with less cellular infiltration around the vessels and fewer F4/80 positive cells in the peritubular interstitium. These data demonstrate that C5aR is critical for a full adaptive immune response and mediates renal allograft rejection. Engagement of C5aR on dendritic cells and T cells modulates their function, enhancing allospecific T cell responses that lead to allograft rejection. Targeting C5a signaling may have therapeutic potential for T cell-mediated graft rejection.

Mannan-binding lectin-associated serine protease 2 is critical for the development of renal ischemia reperfusion injury and mediates tissue injury in the absence of complement C4

Mannan‐binding lectin‐associated serine protease 2 (MASP‐2) has been described as the essential enzyme for the lectin pathway (LP) of complement activation. Since there is strong published evidence indicating that complement activation via the LP critically contributes to ischemia reperfusion (IR) injury, we assessed the effect of MASP‐2 deficiency in an isogenic mouse model of renal transplantation. The experimental transplantation model used included nephrectomy of the remaining native kidney at d 5 post‐transplantation. While wild‐type (WT) kidneys grafted into WT recipients (n=7) developed acute renal failure (control group), WT grafts transplanted into MASP‐2‐deficient recipients (n=7) showed significantly better kidney function, less C3 deposition, and less IR injury. In the absence of donor or recipient complement C4 (n=7), the WT to WT phenotype was preserved, indicating that the MASP‐2‐mediated damage was independent of C4 activation. This C4‐bypass MASP‐2 activity was confirmed in mice deficient for both MASP‐2 and C4 (n=7), where the protection from postoperative acute renal failure was no greater than in mice with MASP‐2 deficiency alone. Our study highlights the role of LP activation in renal IR injury and indicates that injury occurs through MASP‐2‐dependent activation events independent of C4.—Asgari, E., Farrar, C. A., Lynch, N., Ali, Y. M., Roscher, S., Stover, C., Zhou, W., Schwaeble, W. J., Sacks, S. H. Mannan‐binding lectin‐associated serine protease 2 is critical for the development of renal ischemia reperfusion injury and mediates tissue injury in the absence of complement C4. FASEB J. 28, 3996‐4003 (2014). www.fasebj.org

Independent Pathways of P-Selectin and Complement-Mediated Renal Ischemia/Reperfusion Injury

Evidence from in vitro studies indicates that complement activation regulates the expression of P-selectin on endothelial cells. This suggests that in disorders such as ischemia/reperfusion injury, in which both complement and P-selectin have been shown to play a role, complement activation is a primary event and the effects of P-selectin are secondary. To test this hypothesis in vivo, we examined a mouse kidney model of ischemia/reperfusion injury. Surprisingly, the time course and extent of expression of P-selectin was unaltered in C3-deficient mice compared with wild-type mice, in which there was rapid but transient up-regulation of P-selectin on capillary walls and slower accumulation of complement split product on the tubular epithelium. In addition, treatment with anti-P-selectin antibody to reduce the neutrophil-mediated reperfusion damage was equally effective in the absence of C3. These data imply that complement and P-selectin-mediated pathways of renal reperfusion injury are mutually independent, a conclusion that is possibly explained by the differences in the location and time kinetics of complement activation and P-selectin expression. We conclude that in vivo interaction between complement and P-selectin is limited because of time and spatial considerations. Consequently, complement and P-selectin pose distinct targets for therapy.

The Innate Immune System and Transplantation

The sensitive and broadly reactive character of the innate immune system makes it liable to activation by stress factors other than infection. Thermal and metabolic stresses experienced during the transplantation procedure are sufficient to trigger the innate immune response and also augment adaptive immunity in the presence of foreign antigen on the donor organ. The resulting inflammatory and immune reactions combine to form a potent effector response that can lead to graft rejection. Here we examine the evidence that the complement and toll-like receptor systems are central to these pathways of injury and present a formidable barrier to transplantation. We review extensive information about the effector mechanisms that are mediated by these pathways, and bring together what is known about the damage-associated molecular patterns that initiate this sequence of events. Finally, we refer to two ongoing therapeutic trials that are evaluating the validity of these concepts in man.