Analysis of C4 and the C4 binding protein in the MRL/lpr mouse

The complement system is an important mediator of tissue injury in systemic lupus erythematosus (SLE) and other immune complex diseases. SLE is characterized by systemic complement activation, autoantibody production, the formation of circulating immune complexes, and the generation of autoreactive lymphocytes associated with multisystem injury, including nephritis, arthritis, serositis, dermatitis, and blood dyscrasias. Lupus nephritis is mediated in part by local deposition of circulating immune complexes and complement activation products. The relationship of complement to the pathogenesis of SLE is a complex one. Genetic deficiencies in the early components of the classical complement pathway (C1 inhibitor, C1q/r/s, C2, or C4) are some of the strongest risk factors for the development of SLE [1]. This is thought to be due to the role of the early classical pathway of complement activation in the clearance of immune complexes and apoptotic cells. Systemic complement activation, however, marked by depression of serum C3 and C4 levels and peripheral deposition of these proteins, is associated with increased disease activity [2, 3].

The complement system can be activated by three pathways: the classical pathway and the lectin pathway both require the fourth component of complement (C4), while the alternative pathway is independent of C4. All three pathways activate C3 by forming an enzyme, the C3 convertase, which cleaves C3 generating the C3a anaphylatoxin and the activation product C3b. The product C3b mediates a number of cellular reactions leading to proliferation and cell activation, release of proinflamatory cytokines, increased vascular permeability, cell recruitment, apoptosis, and, ultimately, parenchymal damage [4].

C4 binding protein (C4bp) negatively regulates activation of the classical pathway and the lectin pathway [5‐7]. Functionally, C4bp limits complement activation by blocking the formation of and promoting the decay of the classical pathway C3 convertase. It acts via three mechanisms: preventing the formation of the C3 convertase by binding to C4b; accelerating the natural decay of the classical pathway C3 convertase; and as a cofactor for the serine proteinase factor I in the proteolytic inactivation of C4b, which prevents the formation of the C3 convertase. Deficiency of C4bp would be expected to result in increased cleavage of C3 and in increased complement activity in response to classical pathway or lectin pathway activation by immune complex formation, bacterial infections, apoptosis, and other triggering mechanisms.

C4bp is present in human serum at concentrations of approximately 200 mg/l [8]. Human C4bp is synthesized primarily in the liver, and to a lesser degree by activated monocytes [9]. It is an acute phase reactant [10, 11], with expression upregulated by proinflammatory cytokines [9, 11]. In addition, C4bp protein levels have been shown to be upregulated in SLE [10]. Only one patient with C4bp deficiency has been described [12]. She had levels that were 15–29% of normal with repeated testing by radioimmunodiffusion. The patient presented at age 33 years with recurrent oral and genital ulcers, angioedema, malar rash, photosensitivity, dysuria, undetectable antinuclear antibodies, and normal C1 inhibitor levels. Biopsy of her skin lesions revealed arteriolar vasculitis with perivascular monocytic infiltrates, and increased C3 and IgM staining. The patient was diagnosed with atypical Behcet's disease and was treated with solumedrol and cyclophosphamide. Genotyping was not reported, but her father and her sister were reported to have similarly low serum C4bp levels [13]. There have been no reported cases of C4bp deficiency in patients with SLE.

C4bp belongs to a gene family of structurally related proteins designated the regulators of complement activation. There are three isoforms of C4bp in humans [6]. The predominant form is a 570 kDa glycoprotein composed of seven α chains covalently bound to each other and to one β chain. Other isoforms contain either seven α chains without a β chain or six α chains with one β chain. The α chain is composed of eight complement control protein domains, and the N-terminal three complement control proteins bind C4b [14]. The C-terminus contains a separate domain critical for multimerization. The β chain contains three complement control protein domains.

Human C4bp has been shown to bind other compounds including protein S (β chain), C-reactive protein, serum amyloid protein, soluble CD40 ligand, CD40 (α chain), heparin (α chain), low-density lipoprotein receptor protein (α chain), and several bacterial peptides (α chain) [6, 15‐21]. Isoforms containing the β chain can also bind to negatively charged phospholipids on the surface of apoptotic cells in a protein-S-dependent manner [22]. All isoforms regulate complement in an equivalent manner, and no binding partner has been shown to modulate C4bp complement regulatory activity.

The structure of murine C4bp differs from its human ortholog. The mouse protein lacks the β chain [23] and the murine α chain lacks two complement control protein domains and four cysteines present in human C4bp[24]. C4bp protein circulates in mouse serum as a multimer of noncovalently linked α chains [25]. Protein levels are elevated in serum during the acute phase response [26], and males have higher serum levels than females (160 mg/l versus 60 mg/l) due to an effect of testosterone [27, 28]. Expression of the murine C4bp α-chain mRNA has only been reported in the liver and in the epididymis [24, 29, 30]. As shown with human C4bp the mouse C4bp binds both mouse C4b in vivo and in vitro [25, 27], and mouse C4 is unable to form a functional C3 convertase when bound to C4bp [27]. We recently reported the phenotype of the C4bp knockout mouse [31]. Serum from the mice had depressed C4 levels and increased hemolytic activity using antibody-coated sheep erythrocytes.

There are several potential mechanisms by which C4bp deficiency may modify disease progression in SLE. Reduced classical pathway regulation could enhance the ability to clear apoptotic cells, thereby reducing the supply of autoantigens. Similarly, an unregulated C3 convertase could generate more C3 for opsonization and clearance of immune complexes, thus limiting accumulation of these complexes in the kidney and other organs. Alternatively, local classical pathway dysregulation in the kidney could lead to increased inflammation and exacerbation of tissue damage.

To study the role of C4bp in SLE, we used a C4bp knockout mouse in an established experimental model. The MRL/lpr mouse is a spontaneous disease model for complement-associated inflammatory kidney disease, similar to lupus nephritis [32]. The lpr mutation, a retroviral transposon insertion in the FAS gene, results in loss of FAS function and thus a defect in FAS-mediated apoptosis [33]. When present on the MRL genetic background, the loss of FAS-mediated apoptosis results in massive lymphoproliferation with expansion of the B220⁺CD3⁺CD4^-CD8^- cell population and the generation of autoreactive T cells [34]. The ensuing autoimmune disease is characterized by lymphadenopathy, complement activation, severe immune complex renal disease, and 50% lethality by 20–24 weeks of life [35]. We report here that C4bp deficiency does not modify disease severity in MRL/lpr mice.

We report the phenotype of C4bp-deficient MRL mice. Given that the MRL mouse has long been held as a murine model of the immune complex renal injury seen in patients with lupus nephritis, it was surprising that mice lacking the critical regulator of the classical pathway of complement activation had no differences in mortality or morbidity compared with C4bp-sufficient littermate control mice. There were no significant differences in the severity of renal injury between strains with respect to the glomerular deposition of complement proteins or immunoglobulins. Similarly there were no differences in either the degree of glomerular proliferation, of periglomerular inflammation, or of tubulointerstitial disease. In addition there was no evidence of increased complement activation either locally within the kidney or systemically in KO MRL mice at any age. C4bp, and thus negative regulation of the classical pathway of complement activation, therefore appears to play a minimal role in modulating disease severity in the MRL mouse.

There are several possible explanations for the lack of phenotypic differences between the C4bp-deficient mice and the control mice. First, it is possible that the classical pathway is maximally activated in the setting of autoimmunity in the MRL mouse, and that genetic targeting of C4bp does not increase the classical pathway hemolytic activity as it does in nonautoimmune mice (Soimo and Wetsel, manuscript in preparation). To investigate this possibility, serum C4 levels were measured by ELISA at various ages. The data indicate that, similar to humans, serum C4 levels rise early in life from 3 weeks to 8 weeks of age. After 8 weeks, the levels remain constant until at least 20 weeks, when kidney disease becomes evident. Interestingly, in comparison with CTRL MRL mice, CTRL B6 mice had significantly higher hemolytic activity and serum C4 levels. It is unlikely that these findings are due to complement consumption mediated by either tissue deposition or circulating immune complexes in the MRL mouse, as at 8 weeks of age, prior to the onset of overt injury, serum C3 levels were similar between the two strains (data not shown). It would therefore appear that, with respect to CTRL B6 mice, CTRL MRL mice have C4 deficiency marked by functional reductions in classical pathway hemolytic activity.

Two C4 genes map to the H-2 region of mouse chromosome 17. The MRL strain is H-2^k and encodes only one C4^k allele, which is aberrantly spliced in hepatocytes [41]. An intronic insertion encodes an alternative 5' splice site, resulting in an inframe stop codon in the mRNA and a truncated C4 protein that is not secreted [42]. Nonhepatic tissues do not utilize this splice site, and they express a full-length mRNA and a wildtype protein [43, 44]. As the majority of C4 in the serum is derived from the liver, mice with the H-2^k haplotype express 10-fold to 20-fold lower amounts of C4. C57BL/6 mice are H-2^b and encode C4 and a related protein Slp (sex-limited protein). The C4^b allele lacks the intronic insertion in C4^k and is expressed at higher levels, as we have confirmed. In addition, Slp is expressed in high levels in male mice. These differences are likely to explain the decreased hemolytic activity in MRL serum compared with C57BL/6 serum. MRL mice and BXSB mice, both mouse models for lupus-like disease, have previously been described to have lower C4 levels than B6 mice [45]. The H-2 region maps to qualitative trait loci Sle4 and Lbw1, both identified by genetic mapping in mouse models of SLE [46, 47]. It therefore seems more probable that the development of autoimmunity in MRL mice is in part related to a functional deficiency in C4, similar to that seen in humans with deficiencies in early classical pathway components such as C1q, C2, and C4.

As the local, nonhepatic, synthesis of C4 is normal in mice with the H-2^k allele, a second explanation for the lack of phenotype in C4bp KO MRL mice is that the classical pathway plays only a minor role in local complement-dependent injury in the MRL mouse. C3 deposition in the kidney was much more intense than C4 and C1q deposition, and this may be reflective of a larger role for the alternative pathway in cleavage and deposition of C3. The alternative pathway requires factor B to form the C3 convertase, and in the MRL background factor B knockout mice have less proteinuria, decreased renal pathology scores, less glomerular IgG staining, and less renal vasculitis [48]. Recent studies in a pure immune complex model of renal injury additionally conclusively demonstrated that the renal injury seen in this model was alternative pathway dependent [49]. Alternatively, it is possible that the lack of differences in local complement deposition and subsequent renal injury may also be reflective of the relative contribution of fluid phase regulatory proteins, such as C4bp, versus regulatory proteins expressed on the cell surface, such as MCP and DAF. Our data combined with those reported in the factor-B-deficient MRL mouse, however, strongly support the hypothesis that the principal pathway that drives complement-dependent renal injury in the MRL mouse is the alternative pathway.

In addition to the primary role of C4BP in negatively regulating classical pathway activation, C4bp has been proposed, either directly or indirectly, to modulate a variety of biologic processes including hemostasis, B-cell activation, and immune complex clearance. With respect to hemostasis, murine C4bp lacks the β chain present in human C4bp, and thus is unable to bind protein S. C4bp therefore plays no role in the mouse system in regulating the coagulation cascade. It has recently been reported that the α chain of C4bp has a functional role in mediating B-cell proliferation and class switching via its interactions with the CD40-CD40 ligand system. While this interaction was not directly examined in the current report, there were no differences in either absolute B-cell number, serum levels of autoantibodies, or subclasses of antidouble-stranded DNA antibodies between C4bp-sufficient mice or C4bp-deficient mice. In the context of the MRL mouse, therefore, it appears that C4BP plays no role in the regulation of B-cell responses. As an intact classical pathway is required for proper clearance of immune complexes and apoptotic bodies, C4bp as a negative regulator of the classical pathway should impact clearance of immune complex by limiting the activity of the classical pathway C3 convertase, and subsequent generation of C3b needed for solubilization of immune complexes. We were unable, however, to demonstrate any difference in circulating immune complex between the C4bp-sufficient mice and C4bp-deficient mice. Although it is possible that the reduced levels of C4 in the MRL strain limit the intrinsic capacity of the classical pathway to generate C3b, there are data to suggest that amplification of C3b generation via the alternative pathway is required for immune complex clearance [50]. This is believed to be due to the inefficiency of C3b binding to the immune complex: only 10% of generated C3b binds to the complex. Loss of the negative regulator the classical pathway therefore appears to have minimal impact on immune complex processing when the Alternative Pathway is intact. Further study of immune complex and apoptotic cell clearance in C4BP and factor B knockout mice in a C4-sufficient genetic background could confirm the relative importance of these two pathways in immune complex clearance.

One notable finding in C4bp knockout mice kidneys was their small perivascular infiltrates compared with very large infiltrates seen in control mice. This finding was tissue specific, as there were no differences in perivascular infiltrates in other tissues. The biology of C4 and its cleavage products in the mouse is unclear due to a paucity of reagents available for this animal. It is possible that local production of C4 in the kidney is more responsible for leukocyte accumulation in this than in other organs. C4bp may be required for optimal cell recruitment, perhaps due to binding of a chemotactic product of C4b cleavage. Alternatively, C4bp may modulate kidney endothelial cell function in a complement-independent manner. Nonetheless, differences in perivascular leukocyte accumulation in renal vessels did not correlate with other histologic parameters, with kidney function, or with survival.

In summary, the current studies in C4bp-deficient mice fail to demonstrate any significant impact on survival or disease severity in the MRL mouse model of lupus nephritis. Furthermore, this lack of impact on disease phenotype appears to be due to a relative deficiency of C4 in the MRL mouse strain that results in a functional reduction in the classical pathway hemolytic activity. Given previous data showing that renal injury in the MRL mouse is independent of Fc receptors [51], our studies showing the functional deficiency of C4 in these mice, and that the loss of the major negative regulator of the classical complement pathway fails to impact disease severity, the use of the MRL mouse as a prototypical model of immune complex renal injury may need to be reconsidered. Alternatively, new mechanisms for immune-complex-mediated renal injury need to be considered.