Joshua M. Thurman

Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction

Immune mechanisms have been implicated in placental dysfunction in patients with recurrent miscarriages and intrauterine growth restriction (IUGR), but the mediators are undefined. Here we show that complement activation, particularly C5a, is a required intermediary event in the pathogenesis of placental and fetal injury in an antibody-independent mouse model of spontaneous miscarriage and IUGR, and that complement activation causes dysregulation of the angiogenic factors required for normal placental development. Pregnancies complicated by miscarriage or growth restriction were characterized by inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor 1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation in vivo blocked the increase in sVEGFR-1 and rescued pregnancies. In vitro stimulation of monocytes with products of the complement cascade directly triggered release of sVEGFR-1, which sequesters VEGF. These studies provide the first evidence linking the complement system to angiogenic factor imbalance associated with placental dysfunction, and identify a new effector of immune-triggered pregnancy complications.

The central role of the alternative complement pathway in human disease.

The complement system is increasingly recognized as important in the pathogenesis of tissue injury in vivo following immune, ischemic, or infectious insults. Within the complement system, three pathways are capable of initiating the processes that result in C3 activation: classical, alternative, and lectin. Although the roles that proinflammatory peptides and complexes generated during complement activation play in mediating disease processes have been studied extensively, the relative contributions of the three activating pathways is less well understood. Herein we examine recent evidence that the alternative complement pathway plays a key and, in most instances, obligate role in generating proinflammatory complement activation products in vivo. In addition, we discuss new concepts regarding the mechanisms by which the alternative pathway is activated in vivo, as recent clinical findings and experimental results have provided evidence that continuous active control of this pathway is necessary to prevent unintended targeting and injury to self tissues.

C3 glomerulopathy: consensus report

C3 glomerulopathy is a recently introduced pathological entity whose original definition was glomerular pathology characterized by C3 accumulation with absent or scanty immunoglobulin deposition. In August 2012, an invited group of experts (comprising the authors of this document) in renal pathology, nephrology, complement biology, and complement therapeutics met to discuss C3 glomerulopathy in the first C3 Glomerulopathy Meeting. The objectives were to reach a consensus on: the definition of C3 glomerulopathy, appropriate complement investigations that should be performed in these patients, and how complement therapeutics should be explored in the condition. This meeting report represents the current consensus view of the group.

Lack of a Functional Alternative Complement Pathway Ameliorates Ischemic Acute Renal Failure in Mice

Ischemia/reperfusion (I/R) injury of the kidney is a common cause of acute renal failure (ARF) and is associated with high morbidity and mortality in the intensive care unit. The mechanisms underlying I/R injury are complex. Studies have shown that complement activation contributes to the pathogenesis of I/R injury in the kidney, but the exact mechanisms of complement activation have not been defined. We hypothesized that complement activation in this setting occurs via the alternative pathway and that mice deficient in complement factor B, an essential component of the alternative pathway, would be protected from ischemic ARF. Wild-type mice suffered from a decline in renal function and had significant tubular injury, particularly in the outer medulla, after I/R. We found that factor B-deficient mice (fB−/−) developed substantially less functional and morphologic renal injury after I/R. Furthermore, control wild-type mice had an increase in tubulointerstitial complement C3 deposition and neutrophil infiltration in the outer medulla after I/R, whereas fB−/− mice demonstrated virtually no C3 deposition or neutrophil infiltration. Our results demonstrate that complement activation in the kidney after I/R occurs exclusively via the alternative pathway, and that selective inhibition of this pathway provides protection to the kidneys from ischemic ARF.

Acute Renal Failure after Bilateral Nephrectomy Is Associated with Cytokine-Mediated Pulmonary Injury

Clinical studies demonstrate that acute renal failure (ARF) is associated with increased mortality, which may be due to pulmonary complications. ARF may affect the lung via increased renal production or impaired clearance of mediators of lung injury, such as proinflammatory cytokines. Bilateral nephrectomy is a method to examine directly the deleterious systemic effects of absent renal clearance in ARF without the confounding effects that are associated with ischemia-reperfusion injury (e.g., ischemic ARF) or systemic toxicity (e.g., cisplatin-induced ARF). This study contrasts the effects of ischemic ARF and bilateral nephrectomy on serum cytokines and lung injury. It demonstrates that the acute absence of kidney function after both ischemic ARF and bilateral nephrectomy is associated with an increase in multiple serum cytokines, including IL-6 and IL-1beta, and that the cytokine profiles were distinct. Lung injury after ischemic ARF and bilateral nephrectomy was similar and was characterized by pulmonary vascular congestion and neutrophil infiltration. For investigation of the role of proinflammatory cytokines in pulmonary injury after ARF, the anti-inflammatory cytokine IL-10 was administered before bilateral nephrectomy. IL-10 treatment improved pulmonary architecture and was associated with a reduction in inflammatory markers, including bronchoalveolar lavage fluid total protein, pulmonary myeloperoxidase activity (a biochemical marker of neutrophils), and the chemokine macrophage inflammatory protein 2. These data demonstrate for the first time that the acute absence of kidney function results in pulmonary injury independent of renal ischemia and highlight the critical role of the kidney in the maintenance of serum cytokine balance and pulmonary homeostasis.

New Approaches to the Treatment of Dense Deposit Disease

The development of clinical treatment protocols usually relies on evidence-based guidelines that focus on randomized, controlled trials. For rare renal diseases, such stringent requirements can represent a significant challenge. Dense deposit disease (DDD; also known as membranoproliferative glomerulonephritis type II) is a prototypical rare disease. It affects only two to three people per million and leads to renal failure within 10 yr in 50% of affected children. On the basis of pathophysiology, this article presents a diagnostic and treatment algorithm for patients with DDD. Diagnostic tests should assess the alternative pathway of complement for abnormalities. Treatment options include aggressive BP control and reduction of proteinuria, and on the basis of pathophysiology, animal data, and human studies, plasma infusion or exchange, rituximab, sulodexide, and eculizumab are additional options. Criteria for treatment success should be prevention of progression as determined by maintenance or improvement in renal function. A secondary criterion should be normalization of activity levels of the alternative complement pathway as measured by C3/C3d ratios and C3NeF levels. Outcomes should be reported to a central repository that is now accessible to all clinicians. As the understanding of DDD increases, novel therapies should be integrated into existing protocols for DDD and evaluated using an open-label Bayesian study design.

Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.

Oxidative Stress Renders Retinal Pigment Epithelial Cells Susceptible to Complement-mediated Injury

Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H2O2 (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H2O2 reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H2O2 and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.

Alternative Complement Pathway Activation Is Essential for Inflammation and Joint Destruction in the Passive Transfer Model of Collagen-Induced Arthritis

Activation of each complement initiation pathway (classical, alternative, and lectin) can lead to the generation of bioactive fragments with resulting inflammation in target organs. The objective of the current study was to determine the role of specific complement activation pathways in the pathogenesis of experimental anti-type II collagen mAb-passive transfer arthritis. C57BL/6 mice were used that were genetically deficient in either the alternative pathway protein factor B (Bf−/−) or in the classical pathway component C4 (C4−/−). Clinical disease activity was markedly decreased in Bf−/− compared with wild-type (WT) mice (0.5 ± 0.22 (n = 6) in Bf−/− vs 8.83 ± 0.41 (n = 6) in WT mice (p < 0.0001)). Disease activity scores were not different between C4−/− and WT mice. Analyses of joints showed that C3 deposition, inflammation, pannus, cartilage, and bone damage scores were all significantly less in Bf−/− as compared with WT mice. There were significant decreases in mRNA levels of C3, C4, CR2, CR3, C3aR, and C5aR in the knees of Bf−/− as compared with C4−/− and WT mice with arthritis; mRNA levels for complement regulatory proteins did not differ between the three strains. These results indicate that the alternative pathway is absolutely required for the induction of arthritis following injection of anti-collagen Abs. The mechanisms by which these target organ-specific mAbs bypass the requirements for engagement of the classical pathway remain to be defined but do not appear to involve a lack of alternative pathway regulatory proteins.

Treatment with an Inhibitory Monoclonal Antibody to Mouse Factor B Protects Mice from Induction of Apoptosis and Renal Ischemia/Reperfusion Injury

Complement activation in the kidney after ischemia/reperfusion (I/R) seems to occur primarily via the alternative complement pathway. The ability of an inhibitory mAb to mouse factor B, a necessary component of the alternative pathway, to protect mice from ischemic acute renal failure was tested. Treatment with the mAb prevented the deposition of C3b on the tubular epithelium and the generation of systemic C3a after renal I/R. Treated mice had significantly lower increases in serum urea nitrogen and developed significantly less morphologic injury of the kidney after I/R. For gaining insight into potential mechanisms of protection, the activity of caspases within the kidney also was measured, and it was found that caspases-2, -3, and -9 increased in a complement-dependent manner after renal I/R. Apoptotic cells were detected by terminal deoxynucleotidyl transferase catalyzed labeling of DNA fragments, and mice in which the alternative pathway was inhibited demonstrated significantly less apoptosis than control mice. Thus, use of an inhibitory mAb to mouse factor B effectively prevented activation of complement in the kidney after I/R and protected the mice from necrotic and apoptotic injury of the tubules.