M. Botto

Complement deficiency and autoimmunity.

There are three unexplained paradoxes that characterize the association between the complement system and systemic lupus erythematosus (SLE). The first is that complement participates in the inflammatory lesions of SLE and yet inherited homozygous deficiency of certain complement proteins is powerfully associated with the development of SLE. The second is that C lq deficiency shows the strongest association with the development of SLE and yet, in patients with SLE who do not have Clq deficiency, autoantibodies to Clq commonly develop. The third paradox is that complement deficiency is associated with an impaired primary and secondary antibody response to T lymphocyte4ependent antigens, yet SLE is characterized by high levels of autoantibodies to many intracellular and cell surface antigens. In this report, we shall review the data related to these three paradoxes and some hypotheses to explain them.

Accelerated Nephrotoxic Nephritis Is Exacerbated in C1q-Deficient Mice

C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.

Overrepresentation of the FCγ receptor type IIA R131/R131 genotype in caucasoid systemic lupus erythematosus patients with autoantibodies to C1q and glomerulonephritis

OBJECTIVE To test the hypothesis that there is an association between the Fcgamma receptor type IIA (FcgammaRIIA)-H/R131 polymorphism and autoantibodies to the collagenous region (CLR) of C1q in patients with systemic lupus erythematosus (SLE). METHODS One hundred ninety-five Caucasoid lupus patients were studied. Anti-C1q(CLR) antibodies in serum were measured by enzyme-linked immunosorbent assay (ELISA) and FcgammaRIIA genotype analysis was performed by polymerase chain reaction. Immunoglobulin subclass of the autoantibodies was measured by ELISA. RESULTS Fifty-six patients were anti-C1q antibody positive, and Ig subclass analysis indicated a predominance of IgG2 anti-C1q antibodies. Analysis of the SLE population as a whole revealed no significant difference in the allele frequencies of R131 and H131 compared with controls. There was, however, a significantly increased frequency of the R131 allele both in the anti-C1q-positive subgroup of patients (chi2 = 7.66, P<0.01) and in the 71 patients with nephritis (chi2 = 7.76, P< 0.01), compared with controls. CONCLUSION FcgammaRIIA-R131 constitutes a heritable susceptibility factor for the development of SLE with manifestations in the kidney in Caucasoid patients. The close associations demonstrated between this FcgammaRII variant, antibodies to C1q(CLR), and glomerulonephritis may be due to a failure of clearance of the potentially pathogenic IgG2 autoantibody.

Hereditary Deficiency of C3 in Animals and Humans

Inherited deficiency of complement C3 has been described in guinea pigs, dogs and 20 humans. Homozygous deficiency of C3 is associated with recurrent pyogenic infections by encapsulated bacteria, especially H. influenzae, S. pneumoniae and N. meningitidis. In dogs and humans there is also an association with development of glomerulonephritis of the mesangiocapillary type. Some patients also develop transient erythematous rashes in association with pyogenic infections, with histology showing predominantly neutrophil infiltration and small vessel vasculitis. Studies of antibody responses, mainly in experimental animals have shown impaired primary and secondary responses to both thymus-dependent and -independent antigens at low immunizing doses, with a reduced switch from IgM to IgG production. The molecular basis of C3 deficiency has been established in two humans with C3 deficiency. In one it was due to a splice junction mutation and in another, to a partial gene deletion. These mutations are not compatible with the production of functional C3 in any tissue. Such patients with absolute C3 deficiency are a valid model for understanding the physiological role of C3 in vivo.

Identification of chromosome intervals from 129 and C57BL/6 mouse strains linked to the development of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease in which complex interactions between genes and environmental factors determine the disease phenotype. We have shown that genes from the non-autoimmune strains 129 and C57BL/6 (B6), commonly used for generating gene-targeted animals, can induce a lupus-like disease. Here, we conducted a genome-wide scan analysis of a cohort of (129 × B6)F2 C1q-deficient mice to identify loci outside the C1qa locus contributing to the autoimmune phenotype described in these mice. The results were then confirmed in a larger dataset obtained by combining the data from the C1q-deficient mice with data from previously reported wild-type mice. Both analyses showed that a 129-derived interval on distal chromosome 1 is strongly linked to autoantibody production. The B6 genome contributed to anti-nuclear autoantibody production with an interval on chromosome 3. Two regions were linked to glomerulonephritis: a 129 interval on proximal chromosome 7 and a B6 interval on chromosome 13. These findings demonstrate that interacting loci between 129 and B6 mice can cause the expression of an autoimmune phenotype in gene-targeted animals in the absence of any disrupted gene. They also indicate that some susceptibility genes can be inherited from the genome of non-autoimmune parental strains.

Shiga toxin-2 results in renal tubular injury but not thrombotic microangiopathy in heterozygous factor H-deficient mice.

Haemolytic uraemic syndrome (HUS) is characterized by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure because of thrombotic microangiopathy (TMA). It may be caused by infection with Shiga toxin‐producing enteropathic bacteria (Stx‐associated HUS) or with genetic defects in complement alternative pathway (CAP) regulation (atypical HUS). We hypothesized that defective complement regulation could increase host susceptibility to Stx‐associated HUS. Hence, we studied the response of mice with heterozygous deficiency of the major CAP regulator, factor H, to purified Stx‐2. Stx‐2 was administered together with lipopolysaccharide to wild‐type and Cfh+/− C57BL/6 animals. Forty‐eight hours after administration of the first Stx‐2 injection all animals developed significant uraemia. Renal histology demonstrated significant tubular apoptosis in the cortical and medullary areas which did not differ between wild‐type or Cfh+/− Stx‐2‐treated mice. Uraemia and renal tubular apoptosis did not develop in wild‐type or Cfh+/− animals treated with lipopolysaccharide alone. No light microscopic evidence of TMA or abnormal glomerular C3 staining was demonstrable in the Stx‐2 treated animals. In summary, Stx‐2 administration did not result in TMA in either Cfh+/− or wild‐type C57BL/6 mice. Furthermore, haploinsufficiency of factor H did not alter the development of Stx‐2‐induced renal tubular injury.

Murine glomerular mesangial cell uptake of apoptotic cells is inefficient and involves serum-mediated but complement-independent mechanisms

An increased number of apoptotic bodies have been detected in glomeruli of non‐nephritic kidneys of C1q‐deficient mice. In these mice an in vivo impaired uptake of apoptotic cells by peritoneal macrophages was also demonstrated. Here we investigated whether C1q plays a role in the in vitro clearance of apoptotic cells by glomerular mesangial cells. Phagocytosis was assessed using a novel flow cytometric assay that was validated by immunofluorescence studies. The uptake of apoptotic cells by mesangial cells, measured as percentage of mesangial cells ingesting apoptotic cells, was ∼25%, 10% and 10% for a T cell lymphoma line (RMA), thymocytes and neutrophils, respectively. The uptake reached a plateau phase after 3 h, was specific for apoptotic cells and was mediated by serum but not by complement components C1q or C3. The phagocytosis of apoptotic cells was significantly inhibited by Arg‐Gly‐Asp‐Ser (RGDS), a peptide capable of blocking the interaction of thrombospondin with CD36 or the vitronectin receptor. Pretreatment of the mesangial cells with dexamethasone (200 nm) but not with LPS increased the uptake markedly. These findings indicate that murine mesangial cells are capable of taking up syngeneic apoptotic cells, although much less efficiently than professional phagocytic cells. They also show that serum proteins other than complement components mediate the removal of apoptotic cells by murine mesangial cells in vitro.

A lupus-susceptibility C57BL/6 locus on chromosome 3 (Sle18) contributes to autoantibody production in 129 mice.

Epistatic interactions between the non-autoimmune strains 129 and C57BL/6 (B6), used for generating gene-targeted animals, can induce a lupus-like disease. Genome-wide scan analyses of testcross progeny between these two strains have identified several lupus susceptibility loci, with the strongest linkage to the production of autoantibodies (auto-Abs) displayed by an interval on chromosome 1 of 129 origin (Sle16). However, the contribution of B6 loci to the lupus phenotype remained unknown. We used a congenic approach to deduce the contribution to the autoimmune traits of the B6 genomic interval on chromosome 3 (Sle18), previously shown to be linked to antinuclear Ab production. This interval, when transferred on a 129 background (a strain termed 129.B6–Sle18), promoted auto-Ab production targeting a broad spectrum of autoantigens, expansion of activated CD4+T and B cells and mild glomerulonephritis. Surprisingly, these immunological and serological defects were accompanied by a significant increase in the percentage of regulatory T cells (Tregs; CD4+ Foxp3+). However, these cells, that expressed lower levels of Foxp3, had no impaired regulatory function when tested in vitro. These findings illustrate further the efficacy of congenic dissection for functional characterisation of individual lupus susceptibility loci and highlight the contribution of loci derived from non-autoimmune strains to the disease pathogenesis.

HLA class I expression on erythrocytes and platelets from patients with systemic lupus erythematosus, rheumatoid arthritis and from normal subjects

It has previously been shown, by a haemagglutination assay, that patients with systemic lupus erythematosus (SLE) express increased levels of HLA class I on erythrocytes compared with normal subjects and patients with rheumatoid arthritis (RA). A radioligand‐binding assay, using monoclonal antibody W6/32, was devised to quantify HLA class I expression on erythrocytes and platelets. An increased number of class I molecules was expressed on erythrocytes from 45 patients with SLE (mean = 354 molecules per cell, median = 255 molecules, range = 30–1270 molecules per cell), compared with cells from 46 normal subjects (mean = 132, median = 78, range = 40–550) and 31 RA patients (mean = 132, median = 89, range = 26–497). The presence of HLA‐B7 correlated with increased class I expression on erythrocytes from both normal subjects and patients with SLE. Levels of HLA class I in serum were measured. All subjects with HLA‐A9 (A23, 24) showed higher levels of serum class I than their A9‐negative counterparts, and there was no difference in levels between SLE patients and normal subjects. There were no correlations between class I levels in serum and on erythrocytes amongst SLE patients or normal subjects. Red cells were fractionated, according to their age in vivo, on Percoll gradients. Class I levels fell with increasing erythrocyte age in all individuals, but were higher in all fractions from SLE patients compared with age‐matched fractions from normal subjects. HLA‐B7‐positive erythrocytes also expressed higher class I levels in each Percoll fraction, compared with their HLA‐B7‐negative counterparts, suggesting that enhanced B7 expression is not due to greater structural stability of this class I allotype. These data are compatible with the hypothesis that class I is expressed as an intrinsic protein of erythrocyte membranes and that expression is increased amongst patients with SLE.

Identification and Characterization of a Lupus Suppressor 129 Locus on Chromosome 3

The 129-derived Sle16 is a susceptibility locus for systemic autoimmunity when present on the C57BL/6 (B6) background. Genetic analysis of a (129×B6)F2 cross identified a region from the B6 chromosome 3 (Sle18) with positive linkage to antinuclear Abs. In this study, we have generated a B6 congenic strain harboring the 129 allele of Sle18 and intercrossed this line with the lupus-prone B6.129-Sle16 strain. The presence of the 129-Sle18 allele in the B6.129-Sle16Sle18 double congenic mice suppressed the development of Sle16-mediated autoantibody production and ameliorated the renal pathology. The 129-Sle18 locus rectified the B cell abnormalities detected in the B6.129-Sle16 mice, such as the reduction in the percentage of marginal zone B and B1a cells and the increased number of germinal centers. The B6.129-Sle16Sle18 spleens still displayed an increased percentage of activated T and B cells. However, in the B6.129-Sle16Sle18 strain the percentage of naive T cells was equivalent to that in B6.129-Sle18 and B6 mice and these cells showed a reduced proliferative response to anti-CD3 stimulation compared with B6.129-Sle16 T cells. There was a significant increase in the percentage of CD4+FoxP3+regulatory T cells in all congenic strains. These cells had normal regulatory function when tested in vitro. Thus, 129-Sle18 represents a novel, non-MHC lupus-suppressor locus probably operating as a functional modifier of B cells that, in combination with other factors, leads to lupus resistance. Further characterization of this locus will help to uncover the immune mechanism(s) conferring protection against lupus.