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IgA complement activation

IgA complement activation


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Recently, I have been reading Janeway's immunobiology and had a question on immunoglobin A. I read that IgA activates the complement pathway using the Fab fragment of the IgA. How does IgA do that? I can't seem to find an information on that in the book or online.


I found some reports on it (like reference 1) but there is an oddly little amount of publications on this topic. then I found this review in Mucosal Immunology (reference 2, interesting to read) which doubts this activation. It says:

Interaction with complement

IgA lacks the residues identified in the Fc regions of IgG or IgM that bind to C1q, and consequently IgA does not activate the classical complement pathway. Although several papers have reported activation of the alternate pathway by heat-aggregated, denatured, or recombinantly generated IgA, this seems to be essentially artifactual, and intact native IgA antibodies complexed with antigen inhibit complement activation induced by IgG or IgM antibodies. This effect is also replicated by Fabα fragments generated by cleavage of IgA1 antibodies with IgA1 protease. It is telling that mixed aggregates of heat-denatured IgG and IgA activate the alternate pathway in proportion to the content of IgG, and that C3b becomes covalently linked to the IgG heavy chains, not to IgA. Intriguing reports that IgA antibodies promote complement-dependent lysis or opsonization of encapsulated bacteria probably also arise from facilitation of alternate pathway activation by bacterial polysaccharides

It names three papers to underline this (which are number 45-47 in the reference list of the article), which can be found as references 3-5. So the question here is not only how the mechanism looks like, but also if this is real or an artefact.

References:

  1. Activation of complement by human serum IgA, secretory IgA and IgA1 fragments.
  2. Structure and function relationships in IgA
  3. Anti-inflammatory activity of human IgA antibodies and their Fabα fragments: inhibition of IgG-mediated complement activation
  4. IgA blocks IgM and IgG-initiated immune lysis by separate molecular mechanisms.
  5. Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B.

Roos et al (Journal of Immunology, 2001, reference below) suggests that IgA binds MBL, and activates complement by facilitating the lectin binding pathway for complement activation rather than being a major player in the alternate pathway.

Another group of researchers (Daha et al, in Nephrology Dialysis Transplantation, 2006) discusses a potential fourth method for complement activation, as well as describing IgA nephropathy as a result of both alternate- and lectin-activated complement activity. (second reference)

http://www.jimmunol.org/content/167/5/2861.full

http://ndt.oxfordjournals.org/content/21/12/3374.full.pdf


Immunoglobulin A (IgA) nephropathy (IgAN) is an important cause of chronic and end-stage kidney disease. IgAN pathogenesis is incompletely understood. In particular, we cannot adequately explain the heterogeneity in clinical and histologic features and severities that characterizes IgAN. This limits patient stratification to appropriate and effective treatments and the development of disease-targeted therapies. Studies of the role of the alternative, lectin, and terminal complement pathways in IgAN have enhanced our understanding of disease pathogenesis and inform the development of novel diagnostic and therapeutic strategies. For example, recent genetic, serologic, and immunohistologic evidence suggests that imbalances between the main alternative complement pathway regulator protein (factor H) and competitor proteins that deregulate complement activity (factor H–related proteins 1 and 5, FHR1, and FHR5) associate with IgAN severity: a relative abundance of FHR1 and FHR5 amplifies complement-dependent inflammation and exacerbates kidney injury. Ongoing characterization of the mechanisms by which complement activity contributes to IgAN pathogenesis will facilitate the development of complement-based diagnostic techniques, biomarkers of disease activity and severity, and novel targeted therapies.

Financial Disclosure: N.R.M.-T. and M.M.O. have no disclosures to declare.


Introduction

Genetic sequence analysis and functional comparisons have shown that immunoglobulin A (IgA) is present in all mammals (placental, marsupials, and monotremes) and birds. Mammals, except for rabbits and certain primates, have a single gene encoding the α heavy-chain constant region that defines the IgA antibody class. Rabbits (Lagomorphs) possess 13 genes, whereas humans, chimpanzees, gorillas, and gibbons have 2 genes encoding distinct IgA subclasses, termed IgA1 and IgA2. 1, 2 Orangutans, possessing only a single IgA that resembles IgA1, have presumably lost their IgA2. IgA1 molecules are marked out by the length of their hinge regions, flexible stretches of polypeptide at the antibody's core that separate the regions responsible for antigen binding and effector capability. The elongated hinge regions of IgA1 are lacking both in IgA2 molecules and in the single IgAs present in the majority of mammals. This striking feature seems to have evolved relatively recently through an insertion event, and represents one of the important distinctions that exist between IgA molecules from different species. Here, the focus will be on human IgA.

Like all Igs, IgA molecules are made up of pairings of two identical heavy chains (α-chains in the case of IgA) and two identical light chains. In humans, the IgA in serum is chiefly monomeric, comprising ∼ 90% IgA1 and 10% IgA2 (Figure 1a and b). Further heterogeneity arises because both subclasses can form dimers. These are stabilized through disulfide linkages between the carboxy-terminal 18 amino-acid extension (tailpiece) of one of the heavy chains of each monomer and a 15-kDa joining or J chain (Figure 1d). Secretory IgA (S-IgA), the predominant Ig in milk, colostrum, tears, saliva, and the secretions that bathe mucosal surfaces such as our respiratory, gastrointestinal, and genitourinary tracts, is chiefly derived from local synthesis and is mainly dimeric (Figure 1e), although low levels of some larger polymers, particularly tetramers, are also present. The subclass proportions vary with mucosal site, but typically range from 80 to 90% IgA1 in nasal and male genital secretions, through 60% IgA1 in saliva, to 60% IgA2 in colonic and female genital secretions.

Human IgA structure. Schematic diagrams of a, IgA1 b, IgA2 d, dimeric IgA1 e, secretory IgA1 and f, polymeric immunoglobulin receptor. IgA heavy-chain domains are shown in pink, light-chain domains in light blue, J chain in yellow, and pIgR domains (D1–D5) in dark blue. N- and O-linked oligosaccharides are shown in red and green, respectively. In panel b, the IgA2m(1) allotype is depicted. In panel f, the arrow indicates the cleavage point to yield secretory component. In c and g, molecular models of IgA1, IgA2m(1), and secretory component based on X-ray and neutron scattering are shown (accession numbers 1IGA, 1R70, and 2OCW, respectively). The inset in panel g shows the X-ray crystal structure for domain 1 of pIgR (accession number 1XED). The loops implicated in binding to dIgA are shown in green.


Complement activation

There are 3 pathways of complement activation (see figure Complement activation pathways):

Complement activation pathways

The classical, lectin, and alternative pathways converge into a final common pathway when C3 convertase (C3 con) cleaves C3 into C3a and C3b. Ab = antibody Ag = antigen C1-INH = C1 inhibitor MAC = membrane attack complex MASP = MBL-associated serine protease MBL = mannose-binding lectin. Overbar indicates activation.

Classical pathway components are labeled with a C and a number (eg, C1, C3), based on the order in which they were identified. Alternative pathway components are often lettered (eg, factor B, factor D) or named (eg, properdin).

Classical pathway activation is either

Antibody-dependent, occurring when C1 interacts with antigen-IgM or aggregated antigen-IgG complexes

Antibody-independent, occurring when polyanions (eg, heparin , protamine , DNA and RNA from apoptotic cells), gram-negative bacteria, or bound C-reactive protein reacts directly with C1

This pathway is regulated by C1 inhibitor (C1-INH). Hereditary angioedema is due to a genetic deficiency of C1-INH.

Lectin pathway activation is antibody-independent it occurs when mannose-binding lectin (MBL), a serum protein, binds to mannose, fucose, or N-acetylglucosamine groups on bacterial cell walls, yeast walls, or viruses. This pathway otherwise resembles the classical pathway structurally and functionally.

Alternate pathway activation occurs when components of microbial cell surfaces (eg, yeast walls, bacterial cell wall lipopolysaccharide [endotoxin]) or immunoglobulin (eg, nephritic factor, aggregated IgA) cleave small amounts of C3. This pathway is regulated by properdin, factor H, and decay-accelerating factor (CD55).

The 3 activation pathways converge into a final common pathway when C3 convertase cleaves C3 into C3a and C3b (see figure Complement activation pathways). C3 cleavage may result in formation of the membrane attack complex (MAC), the cytotoxic component of the complement system. MAC causes lysis of foreign cells.

Factor I, with cofactors including membrane cofactor protein (CD46), inactivates C3b and C4b.


Abstract

IgA nephropathy (IgAN) is common and often progresses to end stage renal disease. IgAN encompasses a wide range of histology and clinical features. IgAN pathogenesis is incompletely understood the current multi-hit hypothesis of IgAN pathogenesis does not explain the range of glomerular inflammation and renal injury associated with mesangial IgA deposition. Although associations between IgAN and glomerular and circulating markers of complement activation are established, the mechanism of complement activation and contribution to glomerular inflammation and injury are not defined. Recent identification of specific complement pathways and proteins in severe IgAN cases had advanced our understanding of complement in IgAN pathogenesis. In particular, a growing body of evidence implicates the complement factor H related proteins 1 and 5 and lectin pathway as pathogenic in a subset of patients with severe disease. These data suggest complement deregulation and activity may be dominant drivers of renal injury in IgAN. Thereby, markers of complement activation may identify IgAN patients likely to progress to significant renal impairment and complement inhibition may emerge as an effective method of preventing and reducing glomerular injury in IgAN.


Trends

Three key immunologic molecules are found in the circulating immune complex (CIC): Gd-IgA1, IgG antibody anti-Gd-IgA1, and soluble CD89. At diagnosis, soluble CD89 and IgG anti-Gd-IgA1 antibodies in IgAN patient serum are strong candidate markers of disease activity and severity. They might also predict the risk for disease progression and recurrence after renal transplantation.

CD89 has been implicated in IgAN pathophysiology. It participates in humoral aggression of the kidney through CICs containing CD89. In addition, kidney infiltration by circulating monocytes can result from activation of ITAM-bearing CD89 bound to Gd-IgA1 complexes.

Glomerular damage can be mediated through fixation of CIC upon transferrin receptor (CD71) protein overexpression in the renal mesangium. The presence of CICs is amplified by surface expression of mesangial tranglutaminase 2 leading to mesangial cell activation and disturbing mesangial–podocyte interactions. This can lead to rupture of the glomerular barrier homeostasis with proteinuria and hematuria.

Local complement activation via C3a and C5a anaphylatoxins has more impact on glomerular inflammation than systemic activation in IgAN pathogenesis. Inhibition of C3a/C3aR or C5a/C5aR interactions or membrane attack complex formation may be useful in aggressive IgAN with an unsatisfactory response to traditional immunosuppression treatment.

Risk loci for IgAN have been recently linked to genes involved in immunity against intestinal pathogens. Indeed, the geographical breakdown of risk loci suggests an environmental influence of host–pathogen interactions through intestinal microbiota.

Immune responses against particular food and pathogen antigens can contribute to CIC formation, with exacerbated nephritogenic properties and hematuria mucosal infection can frequently trigger episodes of glomerulonephritis in IgAN.

Immunoglobulin IgA nephropathy (IgAN) is the leading form of primary glomerulonephritis associated with end-stage renal failure, requiring either dialysis or renal transplantation. Microscopic hematuria and proteinuria are the most common presentations, and mesangial cell proliferation with IgA deposition are found in renal biopsies. There is growing evidence that IgAN is an immune complex (IC)-mediated disease. To date, three key molecules have been implicated in IC formation, correlating with disease progression/recurrence after transplantation: galactose-deficient IgA1 (Gd-IgA1), IgG anti-Gd-IgA1 antibodies, and soluble CD89 (an Fc receptor for IgA). This review examines recent data on the role of these molecular players in IgAN. Understanding these factors is essential because such knowledge could lead to improved strategies for the future management of patients with IgAN.


Complement activation in experimental IgA nephropathy: an antigen-mediated process

Complement activation associated with immune complex glomerular deposition plays an important role in renal injury. In the present studies we performed three series of experiments to identify how IgA immune complexes activate complement. The first series of experiments was designed to determine whether the presence of an antigen within a glomerular IgA immune deposit is required for complement activation. In these experiments, large-sized covalently cross-linked IgA oligomers (X-IgA) were prepared with purified IgA anti-dinitrophenyl (DNP) and a bivalent affinity-labeling antigen, bis-2,4-DNP-pimelic acid ester. These X-IgA oligomers have free antigen-binding sites that will bind DNP-conjugated antigens. Two groups of mice were treated with either X-IgA or X-IgA followed, after two hours, by an antigen DNP-Ficoll. Immunofluorescent examination of renal tissues, obtained six hours after the initial injection, revealed an equal intensity of IgA glomerular deposits in both groups of mice. Glomerular C3 deposits were only detectable in the renal tissues of mice that had DNP-Ficoll bound to X-IgA. In the second series of experiments, a pair of preformed IgA immune complexes, differing only in one antigenic structural feature (DNP), were used to examine the role of the antigen in inducing glomerular C3 deposits in two groups of mice. These pre-formed immune complexes were prepared with IgA anti-phosphorylcholine (PC) and either PC-conjugated to bovine serum albumin (PC-BSA) or PC-BSA which was further modified with DNP (PC/DNP-BSA). Although the IgA immunofluorescent intensity and pattern in the glomerular deposits were equivalent for both groups, intense C3 deposits were exclusively associated with the PC/DNP-BSA-containing immune complexes. Analysis of the relative conversion of normal human serum C3 to inactive C3b (iC3b) by X-IgA, various antigens and their respective IgA immune complexes was highly dependent on the nature of the antigen.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction

Immunoglobulins are involved in the control and clearance of infectious diseases including viral (e.g. HIV), bacterial (e.g. Mycobacterium tuberculosis, N. meningitidis) and parasitic pathogens (e.g. Plasmodium spp., Leishmania spp.) via various different mechanisms such as neutralization, and fragment crystallizable (Fc) effector functions including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis and complement activation. 1 Immunoglobulin (Ig) G has been extensively studied and this is highlighted by the dozens of IgG monoclonal antibodies (mAbs) approved for therapeutic use by the US Food and Drug Administration. 2 Recently, there has been a growing appreciation for other antibody isotypes including IgA as mAb therapeutics for cancer treatment and some viral and bacterial pathogens. 3-5 IgA can neutralize invading pathogens and induce a range of Fc effector functions to control and clear various bacterial (e.g. N. meningitidis and Streptococcus pneumoniae) and viral infections (e.g. rotavirus and HIV). 4, 6-10 Furthermore, IgA maintains homeostasis of inflammation at mucosal surfaces and in the blood and tissues. 11 Mucosal IgA is important for first-line defense from invading pathogens at mucosal surfaces. However, the role of serum IgA and associated Fc functions in infectious disease is incomplete and understudied. Here we will discuss serum IgA Fc effector functions in the context of control and elimination of invasive pathogens.


Modeling the Contribution of Complement

Collectively, the library of ADCA-modifying Fc mutations is further supported by a suite of in vitro assays with diverse readouts, and animal models useful for evaluation of the contribution of complement activation to pathology or protection driven by antibodies. The detection of complement-fixation products has provided clinically useful biomarkers for numerous autoimmune diseases 24, 113 and cancer progression, 114 as well as in the prognosis of tissue/organ transplant tolerability and post-transplant monitoring. 27, 115, 116 While detection of complement activation end products on cells and tissues can be correlated with certain autoimmune disease states, preclinical characterization of drug candidates AMCA requires recapitulating the context of natural systems in which interventional AMCA would take place.

Given the physiological complexity and highly interconnected nature of the complement system, it would appear to be difficult to rely on in vitro experimentation as a model of in vivo outcomes. To best approximate their biological relevance, the design of in vitro experiments must take into consideration a number of factors. For example, there are individual- and tissue-specific differences in the concentration of complement components. To this end, reconstitution of the classical and alternative pathway by a defined mixture of the individual components might be useful in the standardization of complement assays, and may also reduce artifacts originating from other serum components that vary between serum sources. 117

Recapitulating native contexts is likely to produce more relevant in vitro results, but such target presentations and assay readouts are often less amenable to high-throughput screening. A common means to observe complement activation is through the use of C3 fragment-specific monoclonal detection reagents, 118 which are adaptable to bead-based antigen-multiplexed antibody screening. 119 The throughput of these methods come at the expense of capturing the functional consequences of complement activation, whether opsonophagocytosis, immune adherence and trafficking, lysis or others, which can be characterized through lower-throughput specialized assays. 120 Even specialized assessment of complement functions in vitro may fail to fully capture the clinical context, as they are often performed on laboratory time scales in the absence of effector cells, and using susceptible or sensitized targets. In vivo models can substantially increase the clinical relevance when characterizing the varied outcomes of AMCA, or lack thereof.

Animal models have been extensively used to parse the mechanisms of action of pathogenic or protective antibodies with a diversity of genetic knockouts, 121, 122 human cascade component knock-ins 106 and complement-modifying interventions including C5 and/or C3 depletion using cobra venom factors. 123 While animal models have demonstrated an indispensable role here, species-specific and human allelic differences in each of the many complement factors can also influence outcomes. Once a relevant difference is identified, models can be generated to more closely reflect native interactions. For example, a humanized C1q mouse, in which mouse C1qA, C1qB and C1qC genes were replaced with chimeric versions containing human globular head domains, has been generated. 106

One certainty remains, determining when, where and how complement matters in disease and antibody-based interventions is complicated. If carefully designed, experiments modulating AMCA in vitro and in vivo can provide unique insights that lead to the identification of druggable targets or assist in clinical translation of antibody drugs.


Activation of the alternative pathway of complement by human serum IgA

In order to study the activation of complement by soluble aggregates of human polyclonal serum IgA, lysis of sheep erythrocytes (E) coated with several IgA preparations was used as a model. A complement nonactivating monoclonal mouse IgG1 against IgA was used to coat the cells. IgA, isolated from normal human serum, was aggregated by either N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), glutaraldehyde, carbodiimide or heating. Depending on the size of the aggregates, and on the method of aggregation, E coated with aggregated IgA (Eγ1.AIgA) could be lysed. The alternative pathway of complement appeared to mediate the lysis because the latter was observed in the presence of EGTA containing 5 mM Mg 2+ (MgEGTA) and properdin (P) was deposited on the cells. Furthermore, no lysis was observed in C3-deficient serum. In the absence of AIgA the cells were not lysed, and no P deposition was observed. In another set of experiments Eγ1.AIgA were first reacted with purified C3, B, D and P for 30 min at 30°C, and subsequently in rat serum EDTA at 37°C. Lysis occurred when Eγ1.AIgA were prepared using SPDP-, glutaraldehyde- or carbodiimide-AIgA. Incubation of 100 μg/ml SPDP-AIgA with normal human serum for 30 min at 37°C in the presence or absence of MgEGTA also induced consumption of total complement. The other soluble AIgA preparations were less effective in activating complement. These results suggest that polymeric serum IgA is capable of activating the alternative pathway of complement.