The complement system and human autoimmune diseases

Abstract

Genetic deficiencies of early components of the classical complement activation pathway (especially C1q, r, s, and C4) are the strongest monogenic causal factors for the prototypic autoimmune disease systemic lupus erythematosus (SLE), but their prevalence is extremely rare. In contrast, isotype genetic deficiency of C4A and acquired deficiency of C1q by autoantibodies are frequent among patients with SLE. Here we review the genetic basis of complement deficiencies in autoimmune disease, discuss the complex genetic diversity seen in complement C4 and its association with autoimmune disease, provide guidance as to when clinicians should suspect and test for complement deficiencies, and outline the current understanding of the mechanisms relating complement deficiencies to autoimmunity. We focus primarily on SLE, as the role of complement in SLE is well-established, but will also discuss other informative diseases such as inflammatory arthritis and myositis.

Keywords: Antiphospholipid syndrome; Autoantibodies; Classical pathway; Complement; Gene copy number variations; Genetic and acquired deficiencies; Idiopathic inflammatory myopathies; Juvenile dermatomyositis; Polymorphisms; Systemic lupus erythematosus; juvenile idiopathic arthritis; rheumatoid arthritis; type I interferon induced gene expression.

Figure 1. Activation and regulation of the human complement system.

There are 3 known arms of the complement cascade, including the classical, alternative, and lectin pathways (10). The ligands and sequence of activation are shown for each pathway. Note that all three pathways converge to cleave complement C3, which in turn allows for the generation of C5 convertases and formation of the membrane attack complex (MAC) composed of C5b, C6, C7, C8, and C9 multimers. This complex serves to perforate the outer membrane of invading microbes or aberrant cells necessitating clearance and can result in lysis or sub-lytic permeabilization of the target. Incorporated in the system are tight regulations that prevent or abort inadvertent activations on self or host cell membrane by dissociation of multimolecular complexes and proteolytic degradation of anchor proteins such as C3b and C4b. Eculizumab is a drug or biologic that blocks activation of complement C5. Activation of zymogens and progression of pathways are shown in red and regulatory steps in green. A positive feedback loop (shown in blue) leading to auto-amplification is common for all three activation pathways.

Figure 2. Systemic lupus erythematous and homozygous deficiency of early components of the classical pathway of complement activation.

Severe cutaneous lesions are common clinical presentations in SLE patients with a complete complement deficiency. (A) A homozygous C1q-deficient male child with cutaneous infection (upper panel), and with discoid lupus erythematosus and scarring lesions on face when he was 22 years old (lower panel). (B) A male child with discoid lupus at 16 months of age with homozygous C1r-deficiency. (C) Complete C4-deficiency in a girl at age 3 with a butterfly rash and cheilitis. (D) A homozygous C2-deficient young woman with acute cutaneous lupus erythematosus. The upper panel shows the butterfly rash, and the lower panel shows photosensitive lesions on sun exposed area. (Source of photographs: (116, 249).)

Figure 3. C1 complex: structure and genetics.

(A) The structure of hexameric C1q in a complex with 2 subunits each of C1r and C1s (C1r₂s₂) is shown in a cartoon. The structural domains of each C1q subunit are outlined in the bottom panel, including the globular and collagen-like regions. Each C1q subunit is composed of C1qA, C1qB, and C1qC. Six of these subunits then assemble into a bouquet conformation as depicted. Note the N terminal cysteine residues, which are critical for disulfide bond formation between C1qA, B, and C. (B) The configuration of C1qA, C1qB, and C1qC on chromosome 1 is shown in the top panel with each gene structure represented diagrammatically below. Known mutations and their relative location in the various introns and exons of C1qA, C1qB, and C1qC are listed (–95). GenBank accession numbers for C1q genes, mRNA and proteins are shown.

Figure 4. C4 genetics.

(A) Genetic locations for constituents of the C3 convertases for classical and alternative pathways. (B) Segmental duplications with one to five modules of the RP-C4-CYP21-TNX (RCCX) in haplotypes are located in the class III region of the HLA. Panel B inset: Dichotomy of human C4 gene size with the long gene containing endogenous retrovirus HERV-K(C4) in the ninth intron and the short gene without the endogenous retrovirus. Note the otherwise complex gene structure, with 41 exons and intervening introns (133, 134, 137, 138, 250).

Figure 5. C4A and C4B genetic and functional differences.

(A) Gene sequence differences between C4A and C4B are shown with the corresponding amino acid differences noted above and below. (B) Graphical representation of C4A and C4B, including amino acid substitutions. Biochemical properties and bioreactivity for C4A and C4B are included (, , –156). Note that despite high homology, there are dozens of different allotypes of C4A and C4B, allowing for incredible diversity.

Figure 6. Interferon signature in juvenile dermatomyositis.

(A) A heat map showing a comparison of blood transcripts between patients with JDM and healthy subjects. Type 1 interferon stimulated gene expression was prominent among seven patients without HLA-DRB1*03 who were C4A proficient (DR3-ve, C4A+ve, left columns). T-cell related transcripts were markedly reduced (green), suggesting migration of T lymphocytes away from blood to tissues (possibly to muscles and skin). Those with HLA-DRB1*03 and C4A deficiency (DR3+ve, C4A-def) demonstrated moderate IFN-stimulated gene expression and the reduction of T cell specific transcripts was also less remarkable (middle columns). Healthy control results are shown in the right most columns. (B) These results were further confirmed by SYBR-green qPCR assays for IFN-stimulated gene expression (red) and reduced expression of T-cell related genes (blue) (77).

Figure 7. Copy number variation of C4 genes in healthy subjects and patients with systemic lupus erythematous.

(A) Comparisons of gene copy number (GCN) groups for total C4, C4A, and C4B between healthy American subjects and patients with systemic lupus erythematous (SLE). Both groups were of European ancestry. SLE patients show significantly higher frequencies for lower copy numbers of total C4 and C4A but not C4B. More than one-third of patients with SLE had only 0 or 1 copies of the C4A gene, compared with one-fifth in healthy White control subjects. Lower panels: (B) Comparisons of C4 GCN groups in healthy subjects of either European or East Asian (EA) descent. (C) Comparison of C4A GCN groups in SLE patients versus healthy subjects (132, 153). White subjects are of European American descent.

Figure 8. Complement, systemic lupus erythematous, and anti-phospholipid syndrome.

(A) Clinical differences in the frequency of reported hypertension, headache, and pericarditis between patients with more than two copies of C4B genes (red columns) and those with less than two copies of C4B genes (green columns) (170). Patients with hypertension consistently presented with higher C3 (panel B) and C4 (panel C) serum protein levels (red curves) than those without (blue curves). (D) Study design of patients with anti-phospholipid (aPL) antibodies. Subjects were divided into groups based on the presence or absence of thrombosis and systemic lupus erythematous (SLE). T₀= patients with thrombosis only. TS= patients with both thrombosis and SLE. S₀= patients with SLE only. NTS= patients with neither thrombosis nor SLE. (E) Remarkably, patients with aPL antibodies and thromboses had significantly higher serum C4 protein levels than those without thromboses. (F) As expected, patients who were diagnosed with SLE who also had aPL antibodies had lower levels of serum C4 than those who did not present with SLE (right bottom panel) (175).

Figure 9. Complement C4 gene copy number variation in the idiopathic inflammatory myopathies.

(A through E) Gene copy number (GCN) variation of total C4 (C4T), C4A, C4 long (C4L), C4B, and C4 short (C4S) between healthy control subjects (CTL) and patients with idiopathic inflammatory myopathy (IIM) and its major subgroups: dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), and juvenile dermatomyositis (JDM). Note that more than half (52.3%) of patients with IIM had C4T GCN<4 (panel F); C4A deficiency (C4A GCN<2) was present in 42.9% of the IIM patients (panel G) (77, 79).

Figure 10. Comparisons of plasma protein levels among patients with idiopathic inflammatory myopathies. Plasma complement levels were determined by single radial immunodiffusion assays.

(A and B) Serum C4 and C3 protein levels were measured in patients with inflammatory idiopathic myopathies (IIM) who also had myositis-specific (MSA) and myositis-associated (MAA) antibodies. Specific MSA including Jo-1 are shown, as are the MAA PM/Scl and Ro. (C) C4 protein levels were corrected for gene copy number (GCN) variation, and C4 protein yield per copy of C4 gene was plotted for patients with MSA, MAA, Jo-1, PM-Scl, and Ro antibodies. (D) C4 (left) and C3 (right) protein levels were compared between males and females (79).

Figure 11. Erythrocyte-bound C4d (E-C4d) and E-C3d in patients with juvenile dermatomyositis and healthy controls.

(A and B) A comparison of E-C4d and E-C3d in juvenile dermatomyositis (JDM) and controls. (C) A comparison of E-C4d in C4A-deficient (C4A gene copy number <2) and C4A-proficient (C4A GCN≥2) JDM patients. (D) A comparison of E-C4d in C4B-deficient (C4B GCN <2) and C4B-proficient (C4B GCN≥2) JDM patients. The median for each group is indicated by a horizontal bar, while the shorter bars represent interquartile ranges; the p-value for Mann Whitney test is indicated.