Herein we describe an 18-year-old male who presented with life-threatening sepsis and meningitis secondary to Neisseria meningitidis and whose serum contained no detectable total hemolytic complement or C3 in standard clinical laboratory tests. Genomic sequencing demonstrated an intact C3 gene. Western blotting of the patient’s serum established the presence of C3 degradation fragments, compatible with accelerated C3 consumption. This possibility was confirmed by mixing experiments in which a factor in the patient’s serum cleaved native C3 in NHS. The proteolytic activity was identified as a classical pathway C3 convertase (C4bC2a) stabilized by an autoantibody. Thus, the normally short-lived (min) enzyme complex was altered to one with a half-life of hrs. This autoantibody prevented spontaneous and regulatory protein mediated decay and, further, stabilized the C5 convertase.
C3-NeF, an autoantibody to the alternative pathway C3 convertase (C3bBb), was initially described in the 1970’s [17
], and recently reviewed in depth by Paixão-Cavalcante et al [20
]. It stabilized this enzyme complex and was associated with type II membranoproliferative glomerulonephritis (MPGNII; Dense Deposit Disease), systemic lupus erythematosus (SLE), and occasionally partial lipodystrophy [19
]. There have also been four reports of an analogous C4-NeF, an autoantibody that stabilizes the CP C3-convertase [8
]. In one of these reports [9
], 16 patients with lupus nephritis were screened and two were identified to have stabilizing activity for the CP C3 convertase. About the same time, another article identified a similar stabilizing activity in one patient with post-infectious glomerulonephritis [21
]. In 1989, a study identified both C3-NeF and C4-NeF in two patients with hypocomplementemic MPGN [8
]. The last report in the literature on C4-NeF was by Ohi and Yasugi in 1994 who screened sera from 100 patients with non-SLE hypocomplementemic (C3 < 40% of normal) membranoproliferative glomerulonephritis for C3- and C4-NeFs [10
]. Thirty-one were positive for a nephritic factor, nine of them had only C4-NeF, and ten were positive for both C3- and C4-NeFs. The complement levels in the MPGN patients with only C4-NeF and the patient described herein are similar in that C3 was very low and C5 levels were about 50% of normal while C4 and Factor B were normal. However, in contrast to the previously reported cases [8
], our patient presented with sepsis and meningitis and had normal renal function that, despite ongoing C3 consumption, has remained so during a two-year follow up period.
Also notable in our patient is a lack of prior infections and absence over a two year follow up of additional infectious episodes. Given that C3 deficiency and the resultant lack of opsonic and bactericidal activity is a well-documented risk factor for pyogenic infections, it is likely that the development of the C4-NeF and the expansion of the corresponding B cell clones only recently predated the patient’s infection. Also, the patient has responded poorly to vaccination, presumably due to his C3 deficient state. The meningococcal vaccine was administered three years prior to his initial infection, but failed to provide protection against his serogroup Y infecting strain. Since that time, the patient has received yearly meningococcal (conjugated polysaccharide), pneumococcal (polysaccharide) and Haemophilus influenzae vaccines but continues to have undetectable or low antibody titers toward these antigens.
This patient’s C5 level was reduced by ~50%. Two explanations for this reduction have been considered. The first is that the stabilized CP C3 convertase cleaves C5 directly, a reaction reported to occur with about 10% the efficiency of C3 cleavage [22
]. The second possibility is enhanced C5 cleavage by a stabilized C5 convertase (C4b2a3b). We provide evidence for the latter in in vitro
experiments (), but this may be less likely in vivo
because the patient’s serum contains essentially no C3, which would be necessary to assemble the C5 convertase. These two explanations are not mutually exclusive.
The quantity of antibody required to stabilize the C5 convertase was considerably greater (10-fold) than what was needed to stabilize the C3 convertase, suggesting that either the binding of C3b to the C4b2a complex alters the neoepitopes or, alternatively, that only ~10% of the polyclonal antibody population can stabilize the C5 convertase. Additionally, stabilization of the C5 convertase by purified IgG was observed only if it was isolated using a Protein G column. IgG purified over a Protein A column did not stabilize the C5 convertase. In contrast, IgG purified from either Protein G or Protein A stabilized the C3 convertase to the same degree. Protein G binds to all isotypes of human IgG whereas Protein A binds poorly to IgG3. The discrepancy in the stabilization behavior of these two preparations supports the idea that this is a polyclonal response and that a subset of the autoantibodies containing IgG3 is specifically required for C5 convertase stabilization.
While there is a commercially available test for C3-NeF (National Jewish Center, Denver, CO), there is no such test for C4-NeF. However, the current test that is advertised for C3-NeF may give a low titer positive result if C4-NeF is present (Patricia Giclas, personal communication), as it did in our patient. This is because the basis of the test is the cleavage of native C3 by a factor in the unknown sample. Similar to the serum mixing experiments presented herein, the commercial test is performed by mixing NHS with the unknown sample and then monitoring for C3 cleavage products by electrophoresis. Thus, the presence of either C3-NeF or C4-NeF could result in cleavage of C3 in this assay. Therefore, if a patient has an undetectable, or very low, THC and undetectable C3, further testing should include C4 and Factor B (). In a patient with C4-NeF, Factor B and C4 levels will be normal. In contrast, a patient with C3-NeF will have reduced Factor B and normal C4 levels. Also, a patient does not necessarily need to have renal disease, as illustrated by our case. The proof that this is a C4-NeF then requires performing experiments as outlined in this report including mixing studies () and stabilization studies of the C4b2a enzyme (). Thus, having defined the nature of the autoantibody in our patient’s serum, we could now reinterpret the positive result in the C3-NeF assay performed in the clinical laboratory as resulting from C4-NeF activity. In view of the lack of reports over the past nearly 20 years of a C4-NeF, we suspect the diagnosis has not been considered or, if so, not addressed with appropriate tests. This report serves as a reminder of this autoimmune disease and extends its clinical spectrum.
Laboratory Results Suggesting the Presence of a Nephritic Factora
Structural and functional analyses of the classical pathway C3 and C5 convertases have been hampered by the highly transient nature of these bi- and tri-molecular enzymatic complexes. This patient’s autoantibody indefinitely stabilized the classical pathway C3 convertase. It also stabilized the C5 convertase, especially if added before C3b associates with C4b2a (); additionally, a subset of the IgG autoantibody stabilizes the convertase even if added to the pre-formed C5 convertase. Further studies of the convertases utilizing this patient’s IgG may lend additional insight into the assembly and stabilizing forces for these complexes.
Figure 8 Two models of convertase stabilization by the patient’s autoantibody. A. The patient’s IgG binds to and stabilizes the classical pathway C3 convertase. This convertase cleaves C3 to C3b and a fraction of this C3b then forms the C5 convertase. (more ...)
In summary, we describe a unique case of C4-NeF presenting with a meningococcal infection and C3 deficiency but without renal disease. We outline an approach to making a definitive laboratory diagnosis of this autoantibody-induced syndrome.