Methods: Plasma samples 0. The 55 patients consisted primarily of adult but also of pediatric patients with ITP, undergoing various treatment regimens. The most common included IVIG, rituximab, and especially thrombopoietic agents eltrombopag, romiplostim. The complement activating capacity CAC of patient plasma was evaluated with a previously described in vitro assay Peerschke et al. CAC represents assay optical density readings normalized to reference normal plasma pool.
Patient CAC values were correlated with platelet count. The ability of TNT to block in vitro complement activation was assessed relative to an isotype matched control. There was a non-significant trend for higher C4d levels on platelets and lower AIPF absolute immature platelet fraction, equivalent to platelet reticulocytes. Conclusions: The heterogeneity of patient responses to different treatment modalities in ITP support the concept of different immune mechanisms contributing to thrombocytopenia.
Our data demonstrate classical complement pathway activation in a subgroup of patients with ITP, and further present the first evidence of CP complement inhibition by a novel C1s inhibitor in this setting. The ability of TNT to more completely inhibit C3 activation and C5b-9 assembly downstream of C4 in the in vitro assay system is consistent with direct activation and inhibition of complement at the platelet surface. Thus, TNT may mitigate enhanced platelet clearance by RES via inhibition of complement mediated platelet opsonization by C3b and platelet lysis by C5b Panicker: True North Therapeutics: Employment.
Sign In or Create an Account. Sign In. Skip Nav Destination Content Menu. This C3 convertase molecule is distinct from that within the alternative pathway, but it is from this point onwards that parallels can be drawn between the two cascades. The host proteins that serve key regulatory functions within the alternative pathway DAF, CR1 factor I, CD59 also serve similar functions within the classical pathway.
However, in contrast to the alternative pathway the activation step in the classical pathway requires specific antibody—antigen interactions.
In this context the C1 protein can only become catalytically active when it is bound to at least two adjacent Fc domains. In the case of the IgG and IgM molecules the Fc domains will only align adjacent to each other when the corresponding Fab domains bind antigen. Further, when C1 is free in the circulation it is bound to a protein termed C1 inhibitor C1-INH which prevents any possible activation of C1 in the absence of antibody.
The functions of the classical complement pathway are similar to those described for the alternative pathway, i. The terminal phase is similar for the classical, lectin, and alternative pathways. C5a, and the other anaphylatoxin C3a that generates from C3 cleavage, are potent bioactive molecules that can act on a wide variety of cell types expressing their high-affinity binding transmembrane receptors C5aR and C5L2 19 , 21 for C5a and C3aR for C3a.
C5a and C3a are potent inflammatory mediators targeting a broad spectrum of immune and nonimmune cells. By interacting with their G-protein—coupled receptors C5aR and C3aR, C5a and C3a regulate vasodilatation, increase the permeability of small blood vessels, and induce contraction of smooth muscles. In eosinophils, C5a and C3a regulate the production of eosinophil cationic protein, their adhesion to endothelial cells, and their migration.
C5a is a powerful chemoattractant for macrophages, neutrophils, activated B and T cells, basophils, and mast cells, 19 , 25 the latter of which also migrate toward a C3a gradient Table 1.
On the other hand, the other C5a receptor, C5L2, may function as a decoy receptor regulating the extracellular bioavailability of C5a, and so limiting the proinflammatory response to C5a. It should be noted that once plasma C5a and C3a are released, plasma carboxypeptidases rapidly metabolize them, by cleaving the C-terminal arginine to less-potent forms: C5adesArg and C3adesArg.
In contrast to the early steps of complement activation, assembly of the cytolytic C5b-9 Fig. It starts with the interaction of C5b, released from C5 convertase cleavage of C5, with C6. Binding of C7 to C5b6 leads to the formation of a more stable trimeric complex, C5b-7, which exposes transient lipid binding sites, allowing its association with the cell membrane.
Interaction of C8 with C5b-7 results in the assembly of the tetrameric complex C5b-8, which promotes binding and polymerization of 10 to 16 molecules of C9. The final step of this process is the insertion of C5b-9 MAC complex into the cell plasma membrane, which causes cell death Table 1. For a long time, the damaging effect of MAC has been associated almost exclusively to its cytolytic activity.
Although this may be true for some cell targets under special conditions, as is the case of erythrocytes sensitized by antibodies of well-defined classes and subclasses and in sufficient numbers, the MAC does not appear, in general, to be very efficient, particularly on nucleated cells.
Nucleated cells are able to withstand MAC insertion but they sustain sublethal injury, which leads to a cascade of cellular events as discussed in the article by Tamoko and Cybulsky. Endothelial cells represent a potential target of MAC, which exerts a number of noncytolytic effects.
This favors the shedding of vesicles that support the formation of a prothrombinase complex, and contributes to modify the vascular tone, promoting the release of the vasodilator prostaglandin I2 and the vasoconstrictor thromboxane A2 from endothelial cells. The powerful effector functions of complement have the potential to harm the host.
The activation of CP and LP is largely dependent on foreign material, but under certain situations eg, tissue ischemia and reperfusion , both pathways can be activated and cause autologous injury.
More relevant to complement-related human diseases, deposition of C3b via AP activation and amplification is nondiscriminatory and, if not properly regulated, can damage host cells rapidly. Thus, it is essential that activity be down-regulated on host cells while efficient activation is permitted on foreign targets. Human beings and other mammals have developed a variety of both plasmatic and membrane-bound inhibitory proteins to regulate the location and activity of complement Table 2.
Abbreviation: TAFI, thrombin activatable fibrinolysis inhibitor. Deficiency of C1INH results in episodic angioedema. C1INH, in addition to its role as a complement inhibitor, is the major inhibitor of factor XIIa and kallikrein of the contact system.
The lack of inhibition of these enzymes by C1INH results in inappropriate bradykinin generation. This in turn mediates the increased vascular permeability characteristic of angioedema. Mechanisms of complement regulation.
C C1 complex inactivation. If one of them is missing, or totally dysfunctional, AP activation in plasma is vigorous and leads to secondary complement deficiency via overconsumption of C3 and other complement components. CFI is a plasma serine protease that is able to permanently inactivate C3b to iC3b by proteolysis, but needs a cofactor.
The major source of CFH is the liver, but extrahepatic synthesis has been shown in a variety of tissues including endothelial cells, 40 glomerular mesangial cells, 41 and, in rodents, podocytes. In addition to its regulatory activity in plasma, CFH is practically the only molecule that is involved in down-regulating AP activation on host structures that lack other surface-bound regulators such as basement membranes in kidney glomeruli.
Deletion mutagenesis studies have shown that regulation of fluid-phase C3 activation requires the N-terminal five short consensus repeat domains 46 , 47 in which both the cofactor and decay-accelerating activities of CFH reside. In contrast, targeting of the protein to cell surfaces surface recognition function is mediated by C-terminal domains.
Of interest, C4b-binding protein also can favor C3b inactivation, although to a lesser degree Fig. DAF also decreases the stability of the C3 convertase of the classic and lectin pathways, C4bC2a, by accelerating its dissociation to C4b and C2a. In addition, the terminal MAC is regulated closely both in fluid phase and on cell membranes Table 2 , Fig.
Vitronectin, also known as protein S, is one of these regulators, which binds preferentially to C5b-7 and interacts with C9 to form SC5b-9, inhibiting its polymerization. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to compete efficiently with CFH for ligand binding.
CD59 is the key regulator of the terminal pathway Table 2 , Fig. It inhibits C9 association with C5b-8 to prevent C5b-9 formation. Somatic mutations in the phosphatidylinositol glycan complementation class A gene in red blood cells 3 result in a rare clonal disorder called paroxysmal nocturnal hemoglobinuria.
As a result of defective phosphatidylinositol glycan complementation class A function, affected red cells lack all glycosyl phosphatidylinositol—linked membrane proteins to autologous complement-mediated lysis with consequent hemolytic anemia.
Immune cells, including T cells and antigen-presenting cells, produce complement products that have implications for organ transplantation and autoimmune diseases 60 , 61 see the article by Cravedi and Heeger.
Cognate T-cell——antigen-presenting cell interactions that result in T-cell activation are associated with the up-regulation and release of the AP complement components C3, CFB, and CFD by both cell types.
More recent studies have shown that natural regulatory T cells express C3aR and C5aR and that signaling through these receptors inhibits natural regulatory T-cell function. Complement-dependent effects on alloreactive T-cell immunity regulate the phenotypic expression of immune-mediated injury in animal models.
Further confirming a key role for C5a—C5aR interactions as pathogenic in transplant rejection are data indicating that C5aR blockade prolongs kidney transplant survival in rodents. In addition, mice deficient in either or both C5aR and C3aR develop less autoimmunity and are resistant to experimental allergic encephalomyelitis.
Because of its highly specialized function, the kidney is subject to significant stress from exogenous factors eg, pathogens, toxins, and cytokines filtered from the bloodstream. Consequently, renal function is dependent on a finely calibrated immune response including proper complement activation and regulation.
Although traditional thinking focuses on liver-derived serum complement as a principal pathogenic mediator of kidney injury, it is now abundantly clear that complement components are produced by parenchymal kidney cells as well.
A comprehensive range of complement genes are expressed in the kidney. C1q, which is synthesized mainly in mononuclear phagocytes and epithelial cells, has been suggested to play an important role in the clearance of immune complexes and apoptotic bodies in the kidney.
At variance with the glomerulus, tubules more strongly express the main components of complement synthesized in the liver C2, C4, C3, and factor B. This area of the kidney has adapted to make its own complement proteins, possibly because of exposure to microorganisms ascending through the urinary tract.
Thus, clustering of the components of the activation pathways in the tubule may act as a front-line defense against pathogens. Four types of resident kidney cells are capable of synthesizing complement proteins when grown in tissue culture: glomerular epithelial cells, glomerular endothelial cells, mesangial cells, and proximal tubular cells. This discrepancy between in vivo and in vitro expression may be owing to rapid loss of ability to synthesize C4 in vitro, as noticed in monocyte studies.
However, many stimulators, including cytokines, growth factors, immune complexes, serum proteins, and human cytomegalovirus, can increase the expression of C3, C4, and factor B by cultured cells. Numerous factors have been shown to regulate the production of complement by renal cells.
For example, interleukin-1 may induce a two-fold to three-fold increase in the renal expression of C2 in mice, but has no effect on hepatic synthesis of C2. The transmembrane receptors for C3a and C5a C3aR and C5aR , and the integral membrane CRs for C3b, iC3b, and C3dg, are expressed outside the kidney, particularly in cells of hematopoietic and immune lineage. There is mounting evidence that intrinsic glomerular and tubular cell C3aR and C5aR expression and activation also can affect renal injury.
These can be engaged by C3 and C5 activation products derived from systemic and local pools in renal injury. The deposition of the MAC complex, C5b-9 on glomeruli and tubules of diseased kidney, has led to investigation of the activity of the complex on renal cells.
In mesangial and tubular cells, the complex has a dual effect. On one hand, C5b-9 stimulates both cell types to release cytokines and collagen, which cause tissue fibrosis, 79 and on the other hand it is able to induce or enhance apoptosis.
In addition to the complement activation pathway components, a number of complement regulatory proteins, particularly membrane-bound proteins, are synthesized within the kidney. The distribution of these inhibitory proteins in normal and diseased kidneys has been studied comprehensively.
DAF is found mostly on podocytes, endothelial cells, and in the juxtaglomerular apparatus. MCP is expressed throughout human renal tissues, but rodents do not normally express MCP beside spermatozoa. The rodent-specific complement regulator Crry is expressed ubiquitously in the kidney and is considered a functional homolog of human MCP. The renal tubule is relatively deficient in complement regulators, helping to explain the vulnerability of the renal tubule to complement attack.
In addition, we found that CFH binds to heparan sulfate residues on human proximal tubular cell surface through its C-terminal domain. The expression of regulatory molecules is up-regulated during the activation of complement, 66 as occurs in a number of forms of renal disease.
The kidney therefore has a set of positive and negative internal controls with the ability to influence complement activation on the renal structures. A brief overview of the involvement of complement in kidney diseases that is discussed in detail in other articles in this issue is given below Table 3.
One of the fundamental protective actions of complement is the clearance of invasive pathogens and the removal of immune complexes and cell debris. This is evident in individuals with inherited C3 deficiency who develop recurrent bacterial infections and immune complex glomerulonephritis. Many studies have shown that the complement system affects anti-GBM glomerulonephritis in human beings by amplifying antibody-mediated injury through CP and enhancing the inflammatory response through C5 activation.
Complement has been shown to play a key role in experimental ANCA -associated glomerulonephritis. Membranous nephropathy MN is another form of immune-mediated glomerular injury in which immune complexes of immunoglobulins and complement accumulate in a granular pattern along the outer side of the GBM, leading to glomerular visceral epithelial cell detachment 90 see the articles by Beck et al and Takano and Cybulsky.
Seminal studies have identified several autologous antigens that are targets of antibody response in MN. In most patients with the idiopathic autoimmune form of MN, the autoantibodies are of the IgG4 and IgG1 type and are directed to the phospholipase A2-receptor. The role of complement has been established clearly in experimental MN.
Indeed, complement activation and local formation of C5b-9 is a prerequisite for the development of glomerular injury in rats with passive Heymann nephritis, an experimental model of MN, as documented by studies showing that complement inhibition with recombinant CR1 led to a reduction in proteinuria in this model. In human secondary MN, especially the forms associated with lupus, most studies found abundant C1q, whereas the role of the classic pathway in primary MN is uncertain because renal C1q deposits rarely are found.
On the other hand, C3, C4, CFB, MBL, and C5b-9 typically are present and co-deposited with IgG, suggesting that the lectin and the alternative pathways of complement activation could play a role see articles in this issue by Ma et al and Takano and Cybulsky. Inappropriate activation of the AP of complement system as a result of inefficient inhibition may lead to prolonged tissue damage and glomerular diseases. This mechanism recently was shown to underlie the Escherichia coli —associated and atypical forms of hemolytic uremic syndrome aHUS.
Similar to HUS, idiopathic forms of membranoproliferative glomerulonephritis MPGN are associated commonly with defective complement regulation. A recent immunofluorescence-based classification approach distinguishes those forms of MPGN with isolated C3 deposits, known as C3 glomerulopathies, and characterized by defective control of the AP of complement from MPGN type I with deposits of immunoglobulin and complement and characterized by activation of the CP by antigen-antibody immune complexes.
Progressive glomerular diseases invariably are accompanied by tubulointerstitial damage, the extent of which is linked closely to an adverse renal outcome. The various proteins of the complement system are among the proteins appearing in the urine in nonselective glomerular proteinuria, leading to intratubular deposition of C3 and of C5b-9, and their activation also has been proposed to contribute to tubulointerstitial damage 98 via cytotoxic, proinflammatory, and fibrogenic effects.
Abnormal C3 and C5b-9 staining in proximal tubular cells and along the brush border is a long known feature in chronic proteinuric diseases. In mice with protein overload—induced proteinuria, complement appeared to be an important effector of interstitial mononuclear cell infiltration and fibrogenesis, as shown by significant attenuation of injury in C3-deficient mice.
Renal tubular cells synthesize C3 and other complement factors and exposure of cultured proximal tubular cells to total serum proteins up-regulated C3 messenger RNA expression and protein biosynthesis. Therefore, both excess ultrafiltration and protein-overload—induced proximal tubular cell synthesis of complement components could underlie complement-mediated injury in chronic proteinuric renal diseases. For determination of the injurious role of plasma-derived C3, as opposed to tubular cell—derived C3, C3-deficient kidneys were transplanted into wild-type mice 99 before inducing protein-overload proteinuria.
Protein overload led to the development of glomerular injury, accumulation of C3 in podocytes and proximal tubules, and tubulointerstitial changes. Conversely, when wild-type kidneys were transplanted into C3-deficient mice, protein overload led to a milder disease and abnormal C3 deposition was not observed. All of the earlier-mentioned studies have been performed in rodents. Limited information is currently available from human beings and larger animals.
The involvement of the complement system in renal diseases is complex. There are multiple mechanisms of complement activation, and activation generates multiple biologically active fragments.
Complement is involved in the resolution of injury as well; part of the complement activation seen in glomerulonephritis is the normal reaction to remove immune complexes and damaged cells from the glomerulus.
Although these conditions are rare in the population, their studies have provided important insight to the pathogenesis of complement-mediated renal tissue injury as well as new understanding of mechanisms of action of complement regulatory proteins. These advances also have fueled many efforts to develop new targeted complement inhibitors and it is likely that in the near future complement-inhibitory drugs will be used in patients with more common types of renal diseases.
Conflict of interest statement: none. National Center for Biotechnology Information , U. Sponsored Document from. Semin Nephrol. Author information Copyright and License information Disclaimer. Marina Noris: ti. This article has been cited by other articles in PMC. Summary Complement is an important component of the innate immune system that is crucial for defense from microbial infections and for clearance of immune complexes and injured cells.
Keywords: Complement, kidney, complement regulators, innate immunity, adaptive immunity, kidney diseases. Open in a separate window. Figure 1. The Three Complement Activation Pathways Three main pathways can activate the complement system: classical, lectin, and alternative Fig. Figure 2. Complement Regulators The powerful effector functions of complement have the potential to harm the host.
Figure 3. Complement and Adaptive Immunity Immune cells, including T cells and antigen-presenting cells, produce complement products that have implications for organ transplantation and autoimmune diseases 60 , 61 see the article by Cravedi and Heeger. Complement in the Kidney Because of its highly specialized function, the kidney is subject to significant stress from exogenous factors eg, pathogens, toxins, and cytokines filtered from the bloodstream.
Complement in Kidney Diseases Glomerulopathies One of the fundamental protective actions of complement is the clearance of invasive pathogens and the removal of immune complexes and cell debris. Tubulointerstitial Injury in Progressive Kidney Diseases Progressive glomerular diseases invariably are accompanied by tubulointerstitial damage, the extent of which is linked closely to an adverse renal outcome. Conclusions The involvement of the complement system in renal diseases is complex.
References 1. Walport M. First of two parts.
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