C1q and Systemic Lupus Erythematosus

Immunobiol., vol. 199, pp. 265-285 (1998)

C1q Deficiencies and C1q in Autoimmunity Department of Medicine, Rheumatology Section, Imperial College School of Medicine, London, U.K.

C1q and Systemic Lupus Erythematosus MARK

J. WALPORT, KEVIN A. DAVIES, and MARINA BOTTO

Abstract In this chapter we review the association between SLE and C1q. In the first part of the chapter we discuss the clinical associations of C1q deficiency, and tabulate the available information in the literature relating to C1q deficiency and autoimmune disease. Other clinical associations of C1q deficiency are then considered, and we mention briefly the association between other genetically determined complement deficiencies and lupus. In the review we explore the relationship between C1q consumption and lupus and we discuss the occurrence of low molecular weight (7S) C1q in lupus, which raises the possibility that increased C1q turnover in the disease may result in unbalanced chain synthesis of the molecule. Anti-C1q antibodies are also strongly associated with severe SLE affecting the kidney, and with hypocomplementaemic urticarial vasculitis, and these associations are also examined. We address the question of how C1q deficiency may cause SLE, discussing the possibility that this may be due to abnormalities of immune complex processing, which have been well characterised in a umber of different human models. There is clear evidence that immune complex processing is abnormal in patients with hypocomplementaemia, and this is compatible with the hypothesis that ineffective immune complex clearance could cause tissue injury, and this may in turn stimulate an autoantibody response. We have also considered the possibility that C1qClq receptor interactions are critical in the regulation of apoptos is, and we explore the hypothesis that dysregulation of apoptosis could explain important features in the development of autoimmune disease associated with C1q deficiency. An abnormally high rate of apoptosis, or defective clearance of apoptotic cells, could promote the accumulation of abnormal cellular products that might drive an autoimmune response. Anti-C1q antibodies have been described in a number of murine models of lupus, and these are also briefly discussed. We focus on the recently developed C1q «knockout» mice, which have been developed in our laboratory. Amongst the C1q deficient mice of a mixed genetic

Abbreviations: SLE = systemic lupus erythematosus; CNS = central nervous system; ANA = antinuclear antibodies; ENA = antibodies to extractable nuclear antigens; anti-RNP = antibodies to the extractable nuclear antigens Ul ribonucleoprotein; anti-dsDNA = antibodies anti double strand DNA; anti-SM = antibodies to the extractable nuclear antigen SM; anti-Ro = antibodies to the extractable nuclear antigen Ro; CMV retinitis = cytomegalovirus retinitis; HBsAg = Hepatitis B surface antigen; CRI = complement receptor 1; VnR = vitronectin receptor; anti-Clq CLR = antibodies to the collagen-like region of Clq; NZBINZW = New Zealand Black/New Zealand White; FFP = fresh frozen plasma; RF = rheumatoid factor; MPGN = mesangioproliferative glomerulonephritis; ACA = anti-cardiolipin antibodies; IVIG = intravenous IgG; Wkly = weakly; ELISA = enzyme linked immunosorbent assay; LBT = Lupus band test °1998 by Gustav Fischer Verlag

266 . Mo]' WALPORT, Ko Ao DAVIES and M. BOTTO background high titres of antinuclear antibodies were detected in approximately half the animals, and around 25% of the mice, aged eight months had evidence of a glomerulonephritis with immune deposits. Large numbers of apoptotic bodies were also present in diseased glomeruli, and this supports the hypothesis that Clq may have a critical role to play in the physiological clearance of apoptotic cells. .

Introduction There are three extremely strong links between C 1q, the first molecule in the activation pathway of the classical pathway of complement, and the autoimmune disease, systemic lupus erythematosus (SLE). At first sight, these links are apparently paradoxical. Hereditary deficiency of C1q is a powerful susceptibility factor for the development of SLE. In complete contrast, SLE itself causes consumption of C1q in the vast majority of patients with SLE, who are C1q sufficient. The final twist is that autoantibodies are found in approximately 33% of SLE patients, which bind with high affinity to a neo-epitope expressed in C1q, that is free of the C1r-C1s tetramer. This review will be devoted to describing these links between C1q and SLE, and developing a testable hypothesis that can explain the three associations.

C1q deficiency Clinical associations of C 1q deficiency

The clinical features of all of the reported patients with C1q deficiency are described in Table 1. The molecular genetics of C1q deficiency are described in detail by F. PETRY in this volume. The vast majority, 38 out of 41, humans with homozygous C1q deficiency have developed a clinical syndrome closely related to SLE, with rash in 36, glomerulonephritis in 16 and CNS disease in 7. Disease has been described equally in males and females, and is typically of early onset, with a median age of 6 (range 6 months to 42 years). There is a similar spectrum of autoantibodies to that described in other patients with SLE, though the antibody prevalence is lower, with 24 of 34 patients in whom a result is reported having anti-nuclear antibodies, and 15 of 24 having antibodies to one of the extractable nuclear antigens (Sm, RNP, Ro and / or La). Anti-dsDNA antibodies appear to be uncommon in patients with C1q deficiency and have only been reported to be positive in 5 out of 24 patients. Other clinical associations of C 1q deficiency

There is a substantial body of evidence that the complement system plays an important role in host defence against infections. Amongst the 41 C1q deficient patients described in table 1, 13 suffered from recurrent bacterial infections including otitis media, meningitis and pneumonia. Four of them died with septicaemia in early childhood. In addition to the common infections caused by

Onset, sex, race

Clinical features

ANA, DNA -ve, RF 1:640; immunofluorescence renal biopsy +ve IgA, IgM, C3, C3PA ANA 1180, RF 11640; DNA-ve immunofluorescence renal biopsy +ve IgG, IgM, C3, C3PA

9, M, Spanish rash, hair loss, MPGN (Canary Islands) Rothmund-Thomson syndrome (poikiloderma congenitale) bilat capsular posterior cataract

Rothmund-Thomson syndrome bilat capsular posterior cataract rash, haematuria, mesangioproliferative nephritis

Rothmund-Thomson syndrome bilat capsular posterior cataract rash, haematuria, mesangioproliferative nephritis

10, M, Turkish

8, F

5,M

29, M, Japanese

4a.

4b.

4c.

5.

Discoid lupus

deformed finger and toenail with monilia hyperkeratotic desquamative rash, fits, mouth monilia and aphthae, otitis media, died septicaemia age 10

ANA, ENA, DNA -ve

FFP infusions - rash improved at 10 days, worse by 1 month; 0.4 pg/ml C1q detected; brother died aet 10 «SLE»

Notes

parents consanguineous; response to plasmapheresis and FFP 10 days; genetic analysis of the family: C to T transition at posn 186 of the A chain, Gin to stop codon, no DNA available from the propositus

ANA 1130, anti-smooth muscle 1130 Latex 1180; DNA-ve immunofluorescence renal biopsy +ve IgG, IgA, IgM, C3, C3PA

ANA, Latex, ENA, DNA, LE -ve; anti-smooth muscle +ve; anti-HBsAg +ve; IgG and C3 in skin biopsy; necropsy: mesangioproliferative glomerulonephritis

Latex +ve, RNP +ve; ANA,DNA-ve

3.

SLE, glomerulonephritis died aged 6

5, F, Japanese

2.

ENA +ve (RNAse resistant); ANA +ve 1120, DNA, LE -ve; immunofluorescence lesional skin: -ve Ig and complement

37, M, Japanese

discoid rash, erythema multiforme soles and palms

Laboratory tests

1.

Absent or extremely low C1q concentration

Family

Table 1. C1q-deficiency and lupus-like illness: 41 patients from 23 families are summarised (modified from BOWNESS et al.) (19).

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(8)

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ANA, LE cell-ve; kidney biopsy mild mesangial proliferation; immunofluorescence renal: capillary IgG, mesangial IgM, C3; skin: IgM, IgG, C3, C5

light sensitive cutaneous vasculitis since age 1, mild alopecia, Raynaud's apthous ulceration, hyperkeratotic and atrophic skin

1, F, Greek caucasoid

7.

13 grand mal seizure, 15 meningitis polyarthritis, fever, extensive rash, alopecia, oral thrush, skin biopsy hyperkeratosis with basal vacuolation, leucopenia, thrombocytopenia

rash: crusted lesions on erythematous base; fatal meningitis age 9

malar rash, facial swelling, stomatitis extensive macular eruption with erythema and desquamation, died sepsi age 6

facial and truncal edema, haematuria

13,F, Saudi Arabian

7, F, Turkish

4, F, Turkish

6, F,Turkish

9.

10.

11a.

11b.

butterfly rash, cut vasculitis palms and toes, fever

3, M, Yugoslav cauc.

sb.

purulent otitis media, febrile then non-febrile convulsions, rash on palms and soles, photosensitivity and buttefly rash,mesangioproliferative glomerulonephritis, died age?

1, M, Yugoslav cauc.

Sa.

ANA, anti-mitocondrial -ve

ANA+ve; dsDNA, ENA -ve; LE cells, cryoglobulin -ve; immunofluorescence skin: IgG, IgM, C3 in vessel walls

ANA+ve; dsDNA, ENA -ve; LE cells, RF -ve C3 at dermo-epidermal junction

ANA 1/16 DNA,ENA-ve lupus band test +ve for IgG, IgM, C3

+ve LBT IgM, ANA 1/ SO speckled

C to T transition at posn lS6 of the A chain; Gin to stop codon

Abnormal immune response to T-cell dependent Ag

ANA 1:S0 speckled, DNA 94% (<30%), anti-Sm, anti-RNP, anti-Ro +ve; deletion of C at codon 43 RA latex 1:40, IgG2 deficiency; in C-chain plasma exchange + FFP: transient improvement

(7)

(15,16)

(15)

(14)

(12,13)

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(10)

ANA, LE, Latex -ve

itchy spreading rash on upper limbs and shoulder, leukopenia, discoid

21, M, Japanese

Ref.

6.

Notes

Laboratory tests

Clinical features

Onset, sex, race

Family

Table 1. (Continued).

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widespread rash, alopecia, photosensitivity onychomycosis, fits, cerebral atrophy basal ganglion calcification, CMV retinitis died age 28

erythematosus, desquamated skin lesions, aphthae,otitis media, broncopneumonia, died renal failure age 9

asymptomatic,age 22

Arthralgia, photosensitive rash, mouth monilia and aphthae, deformed finger and toenail with monilia

photosensitive rash,two episodes of macroscopic haematuna, IgA nephropathy associated with mesangioproliferative glomerulonephritis

?, F, Slovakian

9, F, Caucasoid

5, M, Turkish

F, Turkish

3, F, Turkish

15, F,Turkish

12c.

13.

14a.

14b.

15a.

15b.

SLE-like syndrome and recurrent bacterial infections

malar rash, diffuse discoid lupus, arthritis, pericarditis, photosensitivity, vasculitis, alopecia, recurrent bacterial infections

?, M, Slovakian

12b.

malar rash, arthritis, photosensitivity necrotising vasculitis, recurrent bacterial infections

?, M, Slovakian

12a.

ANA,ENA, DNA, RF -ve; ACA IgG weakly +ve, ACA IgM -ve; biopsy: mesangial deposits of IgA, IgM, C3, C5b-9

ANA, DNA -ve; RF +ve (1180), anti-Ro+ve, ACA IgG +ve high titre, ACAIgM -ve; mesangioproliferative glomerulonephritis

ANA, Latex, DNA, ENA, LE cells, cryoglobulin -ve, kidney histopathology; consistent with SLE DNA +ve with progressive renal failure

C to T transition of the A chain, GIn to stop codon; normal immune response to Hepatitis B vaccine

C to T transition of the A chain, GIn to stop codon; normal immune response to Hepatitis B vaccine treated with FFP and IVIG: no response

C to T transition of the A chain, GIn to stop codon

parents unrelated no DNA available no response to FFP

ANA 1:2560; anti-RNP, -Sm, C to T transition at posn 41 Ro +ve; Latex 1:160; dsDNA -ve; of C chain; arg to stop codon immunofluorescence skin: IgG, IgM, C3 at epidermal basement membrane

C to T transition at posn 6 of the A chain; GIn to stop codon

C to T transition at posn 6 of the A chain; GIn to stop codon

C to T transition at posn 6 of the A chain; GIn to stop codon

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(20)

(13,19)

(17,18)

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Onset, sex, race

Clinical features

rash aet 3, widespread discoid lupus, age 15 thrombocytopenia (45 x 109/1), growth retardation

subacute cutaneous lupus erythematosus ANA 1:160; anti-Ro +ve; arthralgia DNA -ve

M, Moroccan

3, M, Moroccan

16, F, Moroccan

23, M, Moroccan subacute cutaneous lupus erythematosus ANA 1:320; anti-Sm+ve

7, F, Dutch caucasoid

4, F, Dutch caucasoid

17b.

17c.

17d

18a.

18b.

nephritis aet 4, 23 discoid rash, arthralgia, hair loss, fever, butterfly rash, aphthae, In +

7 glomerulonephritis, 20 fever butterfly rash, apthae, hair loss myositis, grand mal seizure, somnolence, died sepsis age 20

healthy age 42

ANA 1120000, leukopenia, anti-RNP, RF +ve; LE, DNA -ve

ANA, anti-RNP, Latex +ve; DNA, LE -ve, immunofluorescence: skin: IgG, C3+ve, kidney: granular IgG, IgM, IgA, C3

ANA +ve; dsDNA weak +ve; anti-Ro+ve; skin biopsy: lymphocytic infiltration, of the upper dermis with exocytosis, epidermal atrophy; immunofluorescence: IgM, IgG, no C3

ANA+ve; DNA-ve initially, no subsequent tests done

17a.

vasculitis with oral and facial lesions died age 2.5

6mths, F, Pakistani

16c.

Ref.

Gly to Asp at posn 15 of the B chain (28)

(25-27)

(22-24)

Gly to Asp at posn 15 of the B chain

Gly to Asp at posn 15 of the B chain

parents consaguineous

Ron Thompson, personal communication

mucous and skin lesions improved on thalidomide

vasculitic rash digits, face and trunk, oral ulcers, pneumonia, died age 7

18mths, Pakistani

16b.

ANA, Latex +ve; dsDNAwk+ve

fever, mesangiocapillary nephritis facial discoid, died age 8

4, M, Pakistani

parents consanguineous point mutation resulting in premature stop codon B chain

Notes

ANA, Latex, DNA +ve; immunofluorescence renal biopsy: IgG, IgM, C3, C5, no Clq or C4

Laboratory tests

16a.

Dysfunctional Clq detected

Family

Table 1. (Continued).

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42, M, Dutch caucasoid

membranous nephritis ANA 1/80; immunofluorescence kidney: IgG, IgM, IgA +ve no C3 or Clq

6, F, German

photosensitivity rash, facial erythema ANA +ve high titre; arthritis, Libman-Sacks endocarditis; anti-Sm, dsDNA +ve fits and psychosis, peritonitis, mesangiocapillary glomerulonephritis pneumonitis, generalized skin vasculitis, died age 29

150 kD Clq molecule found comprising single structural (18,29) subunit containing two A chain B chain dimers and a C-C chain dimer. G to A at posn 6 of the C chain, Gly to Arg

4, F, Indian

5,F, Saudi Arabian

20b.

21a.

severe mucocutaneous lesions,alopecia photosensitivity, renal disease, cerebral atrophy

discoid rash, photosensitivity

photosensitivity rash, facial erythema parotitis;

ANA, Ro, La, Sm +ve

ANA 1/80, ENA wkly +ve

ANA +ve; anti-Ro +ve anti-cardiolipin +ve

G to A at posn 6 of the C chain, Gly to Arg; sibling died age 12, ?SLE

(30)

G to A at posn 6 of the C chain, Gly to Arg; parents consaguineous (13)

rash, photosensitivity, aphthae, discoid, syncope and peripheral numbness, basal ganglion and temporal lobe calcification

10, F, Japanese

23.

ANA 1/160; latex 1/320 anti-RNP, -Sm, -Ro +ve; immunofluorescence -ve at dermo-epidermal junction

Latex 1 16400; ANA speckled; ENA 1/ 2000000 RNase resistant; DNA -ve; lupus band test +ve

ANA, Ro +ve

sister died age 7 mucocutaneous candidiasis; parents consanguineous; 5% normal Clq conc. in patient; FFP transiently helped rash

Clq = 44 ]Jg/ml (n = 160±50)

G to A at posn 6 of the C chain

G to A at posn 6 of the C chain

(3)

(31)

Footnote: Material from three further patients with Clq deficiency and SLE, two of Swiss origin and one of Mexican origin, were studied by McAdam et al but the clinical histories of these patients have not been fully reported. .

photosensitivity rash, arthritis butterflr rash, vasculitis palm and soles; monilia stomatitis, staphylococcal meningitis, pneumococcal sepsis

18mths, F, ?

22.

Low levels of Clq detected

M, Saudi Arabian asymptomatic age 5

21c.

discoid lupus, photosensitivity,

14, M, Saudi Arabian

21b.

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20a.

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272 . M. J. WALPORT, K. A. DAVIES and M. BOTTO

encapsulated bacteria, 9 C1q deficient patients developed diffuse monilia and aphthous lesions in the mouth and toenail deformity secondary to moniliasis. These repeated and severe infections emphasize the physiological role of the classical pathway, especially in early childhood, when protective antibodies and anamnestic response have not yet developed. Other complement deficiencies and SLE The published data on the association of complement deficiencies and SLE shows that there is a hierarchy of susceptibility and severity of disease according to the position of the missing protein in the activation cascade of the classical pathway of complement (32). As we have seen C1q deficiency is associated with a very high prevalence of severe disease. In contrast, SLE of moderate severity is found in approximately 75% of subjects with homozygous C4 deficiency, and in less than 33% of subjects with homozygous C2 deficiency with a spectrum of severity similar to that seen in SLE without homozygous complement deficiency. C3 deficiency is associated with mesangiocapillary glomerulonephritis in 8 of 22 patients and with post-infectious rashes in 5 of 22 (33-35). Lupus-like illness has only been reported in 5 subjects with C3 deficiecy. There is a small number of patients with SLE and homozygous deficiencies of membrane attack complex proteins of the complement system. However, this association may well be due to a certain artefact and appears to be extremely rare. A population survey of blood donors in Japan identified 138 subjects out of 145,640 with homozygous deficiencies of membrane attack complex proteins, mainly of C9. No classical pathway complement deficiencies were identified (36). In contrast 5 Japanese patients with C1q deficiency and SLE have been identified and only 1 with SLE and deficiency of C9 (37). These findings would be compatible with the classical pathway association with SLE being causal, and the C9 association being due to ascertainment artefact.

C1q and anti-C 1q antibodies in SLE C1q consumption

C1q levels are low in patients with active SLE. Similar to other measures of complement activation, there is a correlation between low levels of C1q and disease activity. There is evidence of hypercatabolism of C1q from turn-over studies in vivo in humans, though data from only a single patient with SLE has been published (38). Reduction of C1q levels was closely correlated with reduction in levels of other classical pathway complement proteins, C1r and C1s (39), C4 (40), C2 and C3 (41). These findings are all compatible with the hypothesis that C1q is reduced because of increased turnover secondary to classical pathway activation of complement occurring in the context of active disease. More recent data show quite marked correlations between reduced levels of C1q and the presence of anti-C1q autoantibodies. This raises the possibility that

Clq and SLE . 273

at least some of the classical pathway complement activation seen in 5LE is caused by the presence of these autoantibodies, or alternatively that these antibodies develop in response to chronic classical pathway activation in 5LE. These possibilities are discussed below. 7S (1 q and SLE

A finding in relation to the expression of Clq in 5LE which has not been fully explained is the description of 75 Clq in sera from some patients with 5LE (42). This material resembles the non-functional low molecular weight Clq variants, found in some patients with complement deficiencies, described in this issue by F. PETRY. It may be that, in the context of increased Clq turnover in 5LE, unbalanced chain synthesis of Clq occurs, leading to the expression of detectable amounts of 75 Clq. Anti-( 1q antibodies, complement levels and disease activity in SLE

A number of different groups have established that autoantibodies to the collagenous region of the complement component Clq (Clq) (CLR) occur in patients with 5LE. This autoantibody is also found in patients with hypocomplementaemic urticarial vasculitis, and mesangiocapillary glomerulonephritis. (43-46). Between 20 and 40% of patients with 5LE are reported to have antibodies to Clq (CLR), and there is a strong association with proliferative glomerulonephritis (43). There is evidence that rises in anti-Clq antibody levels may predate renal flares in lupus (47). Levels of anti-Clq CLR in patients with lupus correlate inversely with the levels of C3, C4 and Clq (48).

Mechanisms of association There are two essential questions about the association of Clq with 5LE. The first is - how does Clq deficiency cause 5LE? The second is - do Clq or antiClq antibodies participate in the pathogenesis of the disease by causing or amplifying tissue injury? We will first consider the role of Clq, or more pertinently, the lack of it, in the causation of disease, and then turn to the second question. How does (1 q deficiency cause SLE?

The observations of the link between homozygous complement deficiency and 5LE imply that there is a physiological activity of the early part of the classical pathway of complement activation that protects humans against the development of 5LE. The hierarchy of severity and susceptibility of the association, discussed above, implies that Clq provides the most protection, followed by Clr and Cls, C4 and C2. The challenge is to identify the relevant physiological activity and, for this, there are two strong candidates and one weaker one. The two strong candidates are the role of the classical pathway of complement in the

274 . M. J. WALPORT, K. A. DAVIES and M. BOTTO processing of immune complexes and a possible role of C1q in the clearance of apoptotic cells, for which there is, at this stage, only suggestive evidence. The third possibility is the role of C1q and the classical pathway in host defence against infection. The hypothesis that complement deficiency causes SLE because of impaired host defence against a causal infectious agent has no experimental support, but because of the key role of complement in innate immunity, can not be dismissed simply. It seems very unlikely for two reasons that a bacterial trigger is involved. The first of these is that other immunodeficiencies leading to increased bacterial infecion are not associated with increased incidence of SLE. The second is that host defence against bacteria is mediated in the complement system predominantly by C3 as opsonin (and to a lesser extent C4). In view of this, it would be predicted that C3 deficiency should be as strongly associated with SLE as C1q deficiency, if defective host defence against bacteria was the explanation for the link of complement deficiency with SLE. Could a viral trigger be involved? This is a much harder possibility to exclude. Although complement does not play an important role in host defence against the majority of viral infections, there are some significant exceptions, such as complement-mediated enhancement of viral uptake which is recognised as a pathogenic factor in the invasion of certain flaviviruses and of HIV (49,50). It is impossible to reject the hypothesis that complement deficiency leads to the development of SLE by allowing infection by a pathogenic virus that triggers disease - but there are other hypotheses that we favour and we consider these in the following sections. Complement and the processing of immune complexes

It has been known since the experiments of Heidelberger in the 1940s that complement interacts with immune complexes (51). Current knowledge on the role of complement in immune complex processing is reviewed in (52). In summary the interactions of complement with immune complexes may be considered under two headings, firstly the role of complement in the processing and clearance of immune complexes and secondly, interactions of complement with immune complexes in tissues which may provoke inflammatory injury. The role of complement in the processing of immune complexes promotes the disruption of the complex lattice, by non-covalent interactions with C1q and by the covalent binding of C3 and C4 within the immune complexes (53). Each of these interactions between complement proteins and immune complexes is thought to promote the disruption of the non-covalent bonds between antigen and antibody. The second role of complement in the processing of immune complexes is to promote receptor-mediated transport and clearance of immune complexes, reviewed in (54). The role of complement in promoting tissue injury when bound to immune complexes in tissues has become controversial recently and is considered below. The hypothesis has been advanced that defects in the complement-mediated processing of immune complexes provide the explanation for the link between complement deficiency and the development of SLE. In the absence of comple-

Clq and SLE . 275

ment, immune complexes may escape clearance by the mononuclear phagocytic system and end up in tissues where they trigger an inflammatory response, with the release of autoantigens, leading to development of an autoimmune response. In support of this hypothesis is abundant evidence showing abnormalities of immune complex processing in the presence of hypocomplementaemia. In humans, the clearance in vivo of large, complement-fixing soluble immune complexes has been studied by intravenous injection of a number of different model immune complexes, including IgG aggregates (55), tetanus toxoid-containing complexes (56), and anti-Hepatitis B surface antigen (HBsAg) / HBsAg complexes (57). These studies showed relatively consistent results and the major findings were as follows. Within the circulation immune complexes bound rapidly to erythrocyte complement receptor type 1 (CR1) and binding was correlated with CR1 numbers and with levels of complement activity. The major sites of clearance from the circulation were the liver and spleen, confirming earlier studies of immune complex clearance in rabbits and rodents. Against prior expectations, the initial rate of clearance from the circulation of immune complexes by the liver was faster in hypocomplementaemic subjects compared with controls. However, in hypocomplementaemic individuals immune complexes were retained less efficiently by the liver and were slowly released back into the circulation. The explanation for these findings has not been fully established. One hypothesis is that, in the absence of normal complement activation, immune complexes remained larger and were retained very efficiently by hepatic Fc receptor-bearing cells, accounting for the accelerated uptake. The release of immune complexes back into the circulation may be accounted for by less efficient internalization of immune complexes bound by Fc receptors alone, in comparison with immune complexes ligating both Fc and complement receptors, which may promote more efficient internalization of immune complexes, followed by immune complex catabolism. These hypotheses are, as yet untested. A further abnormality of immune complex processing associated with hypocomplementaemia was reduced uptake by the spleen, which in a patient with C2 deficiency was absent and restored to normal following the infusion of fresh frozen plasma (57). The explanation for the contrasting findings in respect of immune complex uptake by the spleen and liver in hypocomplementaemia may be related to the mode of delivery of complexes to the fixed mononuclear phagocytic system. In the spleen, the anatomical organisation of the organ specifically favours the uptake of particles (58). The haematocrit in the spleen is relatively high compared with major vessels, and splenic macrophages have been shown to play an important role in the processing of IgG-coated red cells (59). It might therefore be expected that immune complexes bound to red cell CR1 would be selectively processed in the spleen, whereas complexes presented in the fluid phase would localise in the liver. Taken together, these findings show that immune complex processing is abnormal in patients with hypocomplementaemia and are compatible with the hypothesis that ineffective immune complex clearance could cause tissue injury and this, in turn, could stimulate an autoantibody response.

276 . M. J. WALPORT, K. A. DAVIES and M. BOTTO C 1q and apoptosis

Programmed death via apoptosis is the physiological mode of cell death. Apoptotic cells are characterised by cell shrinkage, collapse of the nucleus and cytoplasmic blebbing. Plasma membranes undergo anionic phospholipid exposure and modification of carbohydrate moieties. The resulting cellular fragments, or apoptotic bodies, are subjected to rapid receptor-mediated ingestion by macrophages or resident tissue phagocytes without the inflammatory changes which accompany necrosis. Some of the receptor pathways in the processing of apoptotic cells have been elucidated, for example the phagocytosis of human apoptotic neutrophils by human monocyte-derived macrophages involves a charge-sensitive recognition that is mediated by the macrophage aV~3 integrin, the vitronectin receptor (VnR) (60). In addition there have been reports that murine inflammatory macrophages can recognise apoptotic lymphocytes, thymocytes and neutrophils by means of a mechanism inhibitable by phosphatidylserine. This phospholipid, which is normally sequestered in the inner surface of the cell membrane, became translocated to the external leaflet of the plasma membrane when cells undergo death by apoptotis and the involvement of a macrophage phosphatidylserine receptor has been suggested (61). There appear to be several mechanisms of uptake of apoptotic cells and it seems likely that different pathways of clearance may operate in different tissues. In this context, KORB and AHEARN (62) have recently demonstrated that when human keratinocytes become apoptotic they develop the capacity to directly bind to Clq in the absence of antibodies. This suggests the hypothesis that C1q may playa role in receptor-mediated clearance of apoptotic cells. If this is confirmed, this would represent a newly-described function of the complement system and may provide an important clue to the aetiology of SLE associated with complement deficiency. In addition, in the last few years it has become apparent from a number of observations (63-65) that cells undergoing death by apoptosis generate discrete subcellular structure that are called surface blebs, which contain either nuclear or cytoplasmatic constituents, many of which are targeted by autoantibodies in patients with SLE. One hypothesis that follows from these findings is that, in situations where the uptake of apoptotic cells is abnormal, as might occur in C1q deficiency, ineffectively-cleared apoptotic cells may become a source of autoantigens that stimulate the immune response. This hypothesis might also explain the paradoxical finding that, on the one hand, C1q deficiency causes SLE, whilst, on the other, a third of patients with SLE have anti-C1q antibodies. If apoptotic cells provide the autoantigens that drive the autoimmune response, maybe proteins that participate in the clearance of apoptotic cells might themselves stimulate an autoimmune response. This raises a number of challenging experimental questions. What is the nature of the molecular recognition pathway of apoptotic cells by C1q? Do anti-C1q antibodies themselves participate in the pathogenesis of disease by interfering with the C1q-mediated processing of apoptotic cells or by causing secondary C1q deficiency?

Clq and SLE . 277

Dysregulation of apoptosis is emerging as a new hypothesis to explain important features in the development of autoimmune disease associated with C1q deficiency. An inappropriately high rate of apoptosis or a defect in the clearance of apoptotic cells may promote the accumulation of abnormal cells that might drive an autoimmune response. How do C 1q and anti-C 1q antibodies participate in the pathogenesis of SLE? Anti-C 1q antibodies and SLE

C1q was historically used as a solid phase ligand in assays which were designed to measure immune complexes in serum (66). Some studies showed that there was a good correlation between «immune complex levels» and disease activity in patients with lupus (67). Inconsistencies in results were often attributed to differences in the apparent immunochemical and physical characteristics of the immune complexes (68,69). It had long been known that monomeric IgG 7S precipitins from SLE serum could behave like an immune complex and bind to C1q (70), though it is only comparatively recently that the autoantibody nature of this immunoglobulin has been elucidated. It was initially postulated that small immune complexes were responsible for these observations (71). However it was subsequently demonstrated that F(ab')2 fragments of IgG from SLE serum retained their ability to bind C1q (72). Similarly, high salt concentrations, or heat-inactivation of C1q were shown to be ineffective at abrogating the binding of IgG in many SLE patients (73). It was then shown by Antes and colleagues that some patients with SLE had antibodies which were directed to epitopes on the collagen-like region (CLR C1q), in studies in which they demonstrated that intact IgG from patients precipitated radiolabelled Clq CLR in the presence of polyethylene glycol (74). It has since been demonstrated that autoantibodies to Clq(CLR) only bind the molecule in the solid phase. ANTES and colleagues showed that anti-C1q antibodies are directed against neo-epitopes which become exposed once the molecule is bound in the solid phase (74). Earlier studies with monoclonal antibodies (75,76) also supported this hypothesis. It is now clear that anti-C1q CLR may interfere with solid phase Clq binding assays designed to detect circulating immune complexes, and these assays are now rarely used. A number of assay systems have since been developed that are specifically designed for the detection of anti-C1q antibodies. These are generally ELISA-based systems with the CLR of Clq, or the intact molecule immobilised on the plate. In the latter case, serum samples are incubated in the presence of 1M salt. Since the development of these tests anti-Clq antibodies have been detected in a number of different diseases - SLE, membranous glomerulonephritis, rheumatoid vasculitis, and hypocomplementaemic urticarial vasculitis (43-46). As discussed above these antibodies are strongly associated with hypocomplementaemia, and levels vary in many patients with disease activity. They are particularly associated with glomerulonephritis in lupus.

278 . M. J. WALPORT, K. A. DAVIES and M. BOTTO C 1q, anti-C 1q antibodies and tissue injury - immune complex hypothesis

We have seen how C1q deficiency may contribute to the pathogenesis of SLE. It has been thought for a long time that the fixation of C1q by immune complexes located in the tissues of patients may contribute to inflammatory injury through the products of activation of complement. However, it is clear that both complement - and Fc receptor-mediated mechanisms are implicated in both the clearance of immune complexes from the circulation, and in the development of tissue injury consequent upon the localisation of complexes in the tissues. The role of Fc receptors in immune complex clearance is discussed briefly below. There is very recent experimental evidence from animal studies that leukocyte Fc-gamma receptors have a critical role to play in the development of immune complexmediated inflammation. Clynes and colleagues demonstated that Fc-gamma chain-deficient NZB/NZW mice generated and deposited immune complexes in the kidney, and activated complement, but were protected from developing severe nephritis, compared with wild type controls (77). It is clearly possible that anti-C1q antibodies have a role to play in this process, and may provide a key link between complement and Fc-mediated inflammation. The localisation of potentially pathogenic immune complexes at an inflammatory site may result both in the direct activation of leukocytes via Fc receptors, and the binding of C1q, with subsequent complement activation. As discussed previously, exposure of neo-epitopes on the C1q molecule, consequent upon complement activation, may stimulate anti-C1q antibody production and these antibodies may bind locally, further enhancing Fc-mediatd inflammatory mechanisms. Is there any direct evidence supporting pathological Fc gamma receptor function in lupus, or linking anti-C1q antibodies with abnormalities of Fc receptor function? Much experimental effort has been -devoted to addressing whether there are fundamental abnormalities of mononuclear phagocytic system function in patients with lupus. There is recent evidence that defects in Fc receptor-mediated functions may be important (78-81). For example, a polymorphic variant of FcyRIIA which results in defective internalisation and processing of immune complexes by leukocytes is associated in the African-American population with severe lupus, particularly affecting the kidney (82). This receptor specifically binds IgG2, and SALKON and colleagues have recently demonstrated a more specific association between anti-C1q antibodies and the polymorphic variant of this Fc receptor (R 131) which exhibits defective antibody binding, in patients with lupus (83). At present the pathogenic role of anti-C1q CLR remains unclear, but the strong association of these autoantibodies with hypocomplementaemia and severe disease in lupus, particularly affecting the kidney, suggests that the antibodies may have a key role to play in mediating inflammation in the disease. This could be as a result of formation of C1q anti-C1q immune complexes, with the perpetuation of classical pathway complement activation. For example, it has been shown that anti-C1q CLR are deposited and concentrated in glomeruli from patients with SLE (84). Other possible roles for anti-C1q antibodies in the pathogenesis of SLE have been considered above.

Clq and SLE . 279

Murine models Anti-C 1q antibodies in mice

C 1q binding IgG in MRL/lpr sera was first described in the mid-1980's using a solid phase assay with human C1q as the solid phase ligand (85). Assays of this type using C1q in the solid phase were frequently used for measuring circulating immune complexes, reflecting Fc binding of IgG in the complexes to the globular portion of the C1q molecule. As discussed above, it was the adaptation of such assays by the addition of 1M salt to test sera, or by specifically employing purified collagenous fractions of C1q as a solid phase ligand, which facilitated a demonstration of anti-C1q (CLR) in humans. Similar approaches have been used to evaluate the recurrence of anti-C1q CLR in mouse models of lupus. TRINDER and colleagues demonstrated anti-C1q (CLR) in MRL/lpr mice using an assay based on the use of murine C1q as a solid phase ligand (86). More recently our own group has demonstrated the presence of autoantibodies to C1q both in MRL/lpr mice and in two other lupus prone mice strains - NZB/W and BXSB (87). We demonstrated high binding activity in our assay system using human C1q as a solid phase ligand in the presence of 1M NaCl. The addition of salt produced binding in most cases by only 30-50%. We were also able to demonstrate specific binding to both human C1q (CLR alone) and purified mouse C1q. These findings in three different murine models of lupus corroborate observations made in humans, and suggest that anti-C1q CLR may have an important pathogenic role to play in the development of SLE. C1q knock-out mice

Although the clinical observations in humans demonstrate a very strong association between C1q deficiency and SLE, in order to perfom the experiments that will provide an explanation for this link it has become necessary to turn to animals models. There are a number of spontaneous models of inherited deficiency of the classical pathway proteins, but no C1q deficiency has ever been described in animals. In the last few years, thanks to the advent of the gene-targeting technique, a number of mouse strains have been developed with classical and alternative pathway complement deficiencies. The gene for C3 (88), C4 (89), CR1/2 (90,91), and factor B (92), have been successfully disrupted in mice, but no spontaneous phenotype has been observed in any of these mice. We have recently engineered a strain of C1q-deficient mice by targeted disruption of the first exon of the C1q A-chain gene. Homozygous C1q-deficient (C1qa-I -) mice had no circulating C1q protein detectable by antigenic (ELISA and Western Blot) or functional assays. The C1qa-l - mice are fully viable and fertile. Amongst the C1q-deficient mice of a mixed genetic background (129/01a x C57BL/6) high titres of antinuclear antibodies were detected in approximately 50% of the animals Furthermore 25% of the mice, aged eight months, had on histological examination, evidence of glomerulonephritis with immune deposits.

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A. DAVIES and M. BOTTO

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Dr. KEVIN A. DAVIES, Senior Lecturer, Rheumatology Section, Imperial College School of Medicine at Hammersmith Campus, Du Cane Road, London, UK, W12 OHS. Tel.: 0181/ 3833276, Fax: 0181/743 3109; e-mail: [email protected]