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Introduction

  1. Top of page
  2. Introduction
  3. Case Report
  4. Discussion
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

The complement system is a highly conservative defense mechanism belonging to the innate immune system and composed of more than 30 membrane-bound and soluble proteins (1). The complement system can be activated via 3 distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. Besides direct pathogen-associated molecular pattern recognition, the classical pathway is involved in antibody-mediated cytotoxicity, immune complex solubilization and elimination, adaptative immunity modulation, and clearance of the apoptotic bodies. The activation of the classical pathway sequentially involves the C1 complex, C4 that is cleaved by C1s into C4b and C4a, and C2, which binds C4b and is subsequently cleaved by C1s into C2a and C2b. C4b and C2a form the classical C3 convertase C4b2a, an enzymatic complex that elicits the release of anaphylatoxins, opsonins, and the recruitment of the membrane attack complex C5b–9. The C1 complex is composed of 3 subunits: C1q, C1r, and C1s molecules. C1q binding to immune complexes, apoptotic bodies, or pathogen-associated motives initiates C1r and C1s sequential activation. C1s is a 79.8 kDa serine protease, activated by C1r through proteolytic cleavage, whose function is to cleave sequentially C4 and C2, thereby enabling the formation of the C3 convertase (2). The gene encoding C1s is located on the short arm of chromosome 12 in the vicinity of the C1r locus. The complement system plays an ambivalent role in systemic lupus erythematosus (SLE) nephritis. On one hand, it appears to mediate tissue damage through activation of the classical pathway by immune complexes, and on the other hand, complete deficiencies in C1q, C1r, C2, or C4 have been associated with SLE. Excluding the present case, selective complete C1s deficiency has been reported in only 8 cases so far (3–7). One patient had SLE without renal involvement and two had SLE and renal disease (one had IgG and C1q deposition in the glomeruli and one had chronic renal failure associated with proteinuria and hematuria, but renal biopsy was not performed in this last case). One patient presented with a coma and convulsions and died 6 months thereafter without recovering consciousness. Finally, 3 additional subjects with complete C1s deficiency were asymptomatic at the ages of 10, 11, and 20 years (Table 1). We report here the first well-documented biopsy-proven lupus nephritis in a patient with a homozygous C1s nonsense mutation, a condition resulting in a complete impairment of the classical pathway but preserving the lectin pathway, and the response to rituximab therapy.

Table 1. Summary of the published cases of complete C1s deficiency*
Author, year (ref.)SexAge at onset, yearsSLEInfectionKidney diseaseC1s mutation(s)
  • *

    SLE = systemic lupus erythematosus; ANA = antinuclear antibody.

Inoue et al, 1998 (6)Male26YesNoChronic glomerulonephritisT376fsX407 homozygous
Amano et al, 2008 (3)Female7YesYesIgG and C1q deposition in glomeruliY204X homozygous
Amano et al, 2008 (3)Male13YesYesNoY204X homozygous
Amano et al, 2008 (3)MaleAsymptomatic at 20NoNoNoY204X homozygous
Amano et al, 2008 (3)MaleAsymptomatic at 10NoNoNoY204X homozygous
Abe et al, 2009 (7)Male6NoNoNoE597X + T376fsX407
Abe et al, 2009 (7)Female13No (high ANA titers)YesNoE597X + G630Q
Abe et al, 2009 (7)MaleAsymptomatic at 11NoNoNoE597X + G630Q
Dragon-Durey et al, 2001 (4), and present reportFemale2YesYesLupus glomerulonephritisR534X

Case Report

  1. Top of page
  2. Introduction
  3. Case Report
  4. Discussion
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

An 11-year-old girl was hospitalized because of nephrotic range proteinuria. Her initial history prior to the development of the nephropathy has already been reported (4). She was born at term and eutrophic from nonconsanguineous parents. At the age of 12 months, she presented with a bilateral malar rash after sun exposure, polyarthritis, oral aphthous ulcers, digital pulp vasculitis, and thyroiditis. The estimated glomerular filtration rate (GFR) and urine sediment were normal and proteinuria was absent. Antinuclear anti-SSA and anti-SSB were present at diagnosis, whereas no anti–double-stranded DNA (anti-dsDNA) antibodies could be detected. The CH50 level was 0% (normal range 70–130%), whereas C3 and C4 levels were elevated (1.74 gm/liter [normal range 0.6–1.10] and 0.58 gm/liter [normal range 0.12–0.32], respectively). Homozygous R534X mutation of the C1s gene confirmed the diagnosis of complete and selective C1s deficiency. She was treated with prednisone and L-thyroxine. Fifteen months later, she required third-generation cephalosporin for uncomplicated pneumococcal pneumonia. Apparition of an active autoimmune hepatitis with positive dsDNA at 22 months of evolution required an intensification of her treatment regimen, with introduction of azathioprine and hydroxychloroquine and an increase in the prednisone dose. On this regimen, liver enzymes normalized progressively. Six years later, systematic laboratory test surveys revealed the apparition of proteinuria and the patient was referred to a pediatric nephrology unit. Physical examination was unremarkable. Routine laboratory tests showed an estimated GFR of 135 ml/minute/1.73 m2 according to Schwartz's formula and nephrotic range albuminuria (2.16 gm/day). Microscopic hematuria and leukocyturia were present (erythrocytes 1,000/mm3 and leukocytes 1,000/mm3). A complete blood cell count revealed normocytic anemia (9.7 gm/dl) and lymphopenia (900/mm3). Native anti-DNA antibodies were highly elevated (Farr assay 80%). Anti-C1q antibodies were not detected by enzyme-linked immunosorbent assay. A kidney biopsy was performed, allowing the visualization of 14 glomeruli (Figure 1). By light microscopy study, all of the glomeruli showed active lesions with necrosis and global cellular crescent in 20% of glomeruli, global endocapillary proliferation in 50% of glomeruli, numerous “wire loop” aspects and, with Jones' silver staining, rupture of the glomerular basement membrane (GBM) but also abnormal GBM with diffuse spikes. No thrombotic microangiopathy lesions were observed. By immunofluorescence study, global granular deposits were observed in the mesangium and along the GBM with anti-sera against IgG, IgA, IgM, C3, and C1q. Granular IgG deposits were also observed in the interstitium and along the tubular basement membrane. According to the International Society of Nephrology/Renal Pathology Society 2003 classification, this renal biopsy was classified as class IV-global (activity 70%, chronicity 0%) and class V. C4d staining revealed glomerular parietal granular deposits contrasting with the thin linear staining observed in glomeruli from control subjects. Using TUNEL, apoptotic bodies were detected in the affected glomeruli (Figure 1).

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Figure 1. Pathologic examination of the initial kidney biopsy: periodic acid–Schiff staining (magnification × 200) disclosed interstitial cellular infiltrate and segmental glomerulonephritis (A). Masson's trichrome stain (magnification × 400) showed active necrotizing glomerulonephritis with parietal necrosis, epithelial crescent, endocapillary proliferation, and subendothelial deposits (B). Jones' silver staining (magnification × 600) revealed parietal abnormalities with double contour and spike-like projection of the glomerular basement membrane (C). Apoptotic bodies were detected by TUNEL in the case biopsy (D). Immunofluorescence staining revealed IgG (E), C3 (F), C1q (G), and C4d (H) deposition in the glomeruli.

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The patient was treated with 6 cyclophosphamide intravenous pulses 500 mg/1.73 m2 every other week, followed by mycophenolate mofetil 500 mg twice a day. Prednisone was initially raised to 1 mg/kg/day, and then progressively tapered. Angiotensin-converting enzyme inhibitors were introduced. By month 4, a repeat renal biopsy was performed showing class IV-segmental (activity 50%, chronicity 20%) and class V. Ten months later, a second repeat renal biopsy was performed showing no histologic improvement (class IV-segmental [activity 55%, chronicity 20%] and class V). The patient was treated with 4 weekly pulses of rituximab (375 mg/m2), achieving B cell depletion (Figure 2). Hematuria and proteinuria decreased progressively and 13 months after the initiation of rituximab, the patient felt well and had stable estimated GFR (147 ml/minute/1.73 m2). Proteinuria was in the physiologic range (0.04 gm/day), whereas albuminemia was 39.1 gm/liter. Urinary sediment was unremarkable, confirming complete renal remission.

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Figure 2. Evolution of biologic parameters and therapy after the first kidney biopsy. RTX = rituximab infusion; Cyc = cyclophosphamide infusion; MMF = mycophenolate mofetil.

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Discussion

  1. Top of page
  2. Introduction
  3. Case Report
  4. Discussion
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

C1q, C2, and C4 complete deficiencies have been associated with proliferative glomerulonephritis (8). The disease usually manifests early in life during childhood or early adulthood, and tends to occur earlier in C1q deficiency. In two-thirds of the cases, the disease is associated with dermatologic manifestations such as extended discoid lupus and photosensitivity. Although criteria for lupus are frequently present, a low titer of antinuclear antibodies and the absence of anti-dsDNA are commonly reported in patients with C1q, C2, and C4 deficiencies. The renal disease is usually mild with isolated proteinuria. Severe lesions such as crescent and “wire loop” aspects are rarely reported on renal biopsies (8).

The present report demonstrates efficient rituximab treatment in severe lupus nephritis associated with glomerular C4d deposition in a patient with complete C1s deficiency. This exceptional condition results in a complete and selective impairment of the complement classical pathway.

The occurrences of glomerulonephritis in patients with C1q, C2, or C4 deficiencies and in C1q−/− mice (9) led to the idea that a deficit in the classical pathway is responsible for the development of an autoimmune process. Three non–mutually exclusive hypotheses have been proposed to explain this autoimmune-prone phenotype: 1) a reduced solubility and clearance of immune complexes, 2) a deficiency in apoptotic body clearance, and 3) a defective clonal deletion of autoreactive B cells (10).

However, the physiologic consequences of C1q, C2, or C4 deficiencies are not restricted to classical pathway impairment. C1q is able to interact with specific receptors and has a classical pathway independent function that may be involved in the occurrence of glomerulonephritis in C1q-deficient patients (11). C2 and C4 deficiencies also impair the lectin pathway. In contrast, C1s is exclusively involved in the classical pathway. Of the 9 reported subjects with complete C1s deficiency, 4 developed SLE and, among them, 3 developed renal diseases (Table 1). Therefore, selective classical pathway impairment seems to be a major predisposing factor to SLE and lupus nephritis, confirming a specific role of the classical pathway in the maintenance of self-tolerance. The presence of apoptotic bodies within the patient's glomeruli may be related to acute cellular injury and does not demonstrate defective apoptotic body clearance in the patient.

If C1s deficiency represents a risk factor for SLE, the activation of the classical pathway by immune complexes has been advocated to participate in kidney damage in lupus nephritis. This observation demonstrates that severe lupus nephritis can occur in spite of complete classical pathway deficiency. Because this is only a single case report, we cannot conclude on the exact contribution of the classical pathway to renal injury in lupus nephritis. However, if such a role exists, it does not seem to be central because it is, at least in this observation, dispensable for the induction of severe glomerular lesions.

Parietal C4d staining was observed in the patient's glomeruli. C4d is an inactivation product of C4b that is formed through proteolytic cleavage by the serine protease factor I. C4d stays covalently bonded to the surface where C4b deposition has occurred. Interestingly, C4d can reflect an activation of the classical pathway but also an activation of the lectin pathway because mannose-binding lectin–associated serine protease 2, a protein functionally related to C1s in the lectin pathway, can also cleave C4, initiating the formation of the classical C3 convertase (12). In this context of complete C1s deficiency, C4d deposition can only occur through lectin pathway activation, suggesting that the lectin pathway is involved in the lesional process. Given that certain immunoglobulins such as multimeric IgA or specific IgG glycoforms can activate lectin pathway at least in vitro and that some data argue for a detrimental role of the lectin pathway in glomerular diseases (13), it is tempting to propose that an activation of the lectin pathway contributes to the antibody-mediated glomerular damage in this observation. In contrast to C2- and C4-deficient patients, C1q, C1s, and C1r patients retain the ability to form C4b2a complexes through the lectin pathway. This may explain the severity of renal disease in this patient in contrast to the usual presentation of glomerulonephritis associated with C2 deficiency. However, it should be stressed that although C4d deposits favored an involvement of the lectin pathway in the disease, the alternative pathway may also participate in the massive glomerular C3 deposition found in the patient's glomeruli. Further studies are needed to assess the role of complement activation and particularly of the lectin pathway in human lupus nephritis.

Interestingly, due to poor response to low-intensity cyclophosphamide treatment, the patient was treated with monoclonal anti-CD20 antibody. Rituximab efficacy in lupus nephritis has been reported in retrospective studies (14). However, the mechanisms of action of rituximab remain uncertain. Three main effects are thought to mediate B cell depletion: direct cytotoxicity through direct cross-linking of CD20 on B cells, complement-dependent cytotoxicity through the classical pathway, and antibody-dependent cellular cytotoxicity (ADCC), a mechanism that involves recognition of an antibody's Fcγ by Fcγ receptors on effector cells, mainly natural killer lymphocytes (15). In this C1s-deficient patient, rituximab treatment induced B cell depletion and complete renal remission, demonstrating that the classical pathway activation is dispensable for a rituximab therapeutic effect in lupus. Therefore, ADCC and direct toxicity may account for efficient B cell depletion and therapeutic effects of rituximab in SLE.

In addition to its rarity, this documented unique observation emphasizes the complex role of the complement system in the development of lupus nephritis, confirms that a selective impairment of the classical complement pathway predisposes to autoimmune nephritis, suggests that the lectin pathway may contribute to kidney injury in lupus nephritis, and demonstrates that the classical pathway is not required to achieve B cell depletion and clinical remission in severe lupus nephritis after rituximab therapy.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Introduction
  3. Case Report
  4. Discussion
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be submitted for publication. Dr. Bienaimé had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Bienaimé, Frémeaux-Bacchi.

Acquisition of data. Bienaimé, Quartier, Dragon-Durey, Frémeaux-Bacchi, Bader-Meunier, Patey, Salomen, Noël.

Analysis and interpretation of data. Bienaimé, Quartier, Frémeaux-Bacchi, Patey, Noël.

Acknowledgements

  1. Top of page
  2. Introduction
  3. Case Report
  4. Discussion
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES

The authors would like to thank Drs. Mordi Muorah and Helene Decaluwe for their careful rereading of this manuscript.

REFERENCES

  1. Top of page
  2. Introduction
  3. Case Report
  4. Discussion
  5. AUTHOR CONTRIBUTIONS
  6. Acknowledgements
  7. REFERENCES
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