Linda S. Beischel

Differential expression of complement C3 and C4 in the human kidney

Complement activation is associated with a variety of immunologically-mediated renal diseases. Proximal tubular epithelial cells in situ constitutively express messenger RNA for C4 of the complement system. These same epithelial cells in culture have been reported to contain message for C3 and to secrete this protein when stimulated by IL-2. The present study compared the in situ localization of C3 and C4 message in parallel in a variety of renal biopsy and nephrectomy specimens. All adequate tissue samples (n = 23) had C4 mRNA throughout in the cortical tubular epithelium. Although C3 message was also expressed in tubular epithelial cells, there was much greater variation in its distribution. mRNA for C3 was not detected in histologically normal specimens (n = 4) either by in situ or Northern hybridization. Focal C3 message correlated with focal histologic abnormalities (e.g., focal glomerulosclerosis), while more generalized C3 signal occurred in specimens with more diffuse inflammatory processes (e.g., SLE). Infiltrating inflammatory cells and cells of the glomeruli were uniformly negative for C3 (and C4) message. Tubular C3 and C4 mRNA appeared to be translated, since selected specimens showed cytoplasmic staining by monoclonal antibodies to C3c and C4c. These observations are consistent with the hypothesis that local production of inflammatory mediators could induce C3 synthesis in the renal interstitium, with the possibility that subsequent complement activation could enhance the pathogenic process.

Uniparental isodisomy 6 associated with deficiency of the fourth component of complement.

We identified an extremely rare condition, isolated complete deficiency of the fourth component of complement, in a child with systemic lupus erythematosus. The genes for C4 are located within the major histocompatibility complex (MHC) on the short arm of chromosome 6. The patient expressed only paternal phenotypes for proteins encoded by the MHC (HLA and GLO), yet was 46XX with no detectable 6p deletion. Genomic DNA from patient, parents, and sibling was digested with restriction enzymes, and blots were probed for five chromosome 6 markers. At all loci, maternal and paternal RFLPs could be distinguished, and the patient showed only paternal bands. RFLP analysis of markers from four other chromosomes showed maternal and paternal contribution. The data are consistent with uniparental isodisomy 6 (inheritance of two identical chromosome 6 haplotypes from the father and none from the mother). Direct analysis of genetic material from both parents, as well as detection of multiple protein polymorphisms encoded on chromosome 6, clearly demonstrates this novel mechanism for the expression of a recessive genetic condition.

Major-Histocompatibility-Complex Extended Haplotypes in Membranoproliferative Glomerulonephritis

Membranoproliferative glomerulonephritis is often associated with evidence of immune derangement, especially hypocomplementemia. We studied genetic markers for membranoproliferative glomerulonephritis within the major histocompatibility complex in 34 patients and their families and in 29 normal families. We examined the frequencies of extended haplotypes (combinations of alleles that tend to occur together) in patients and controls. The extended haplotype HLA-B8,DR3,SC01,GLO2(glyoxalase I 2) was observed in 9 of 68 disease-associated haplotypes (13 percent), but in only 3 of 205 controls (1 percent) (relative risk, 14.79; P less than 0.001). An extended haplotype similar except for a different glyoxalase allotype (B8,DR3,SC01,GLO1) did not occur with increased frequency, nor did any other extended haplotypes. Patients with the extended haplotype B8,DR3,SC01,GLO2 had a higher incidence of renal insufficiency than those without it (P less than 0.01). The data support the hypothesis that a specific extended haplotype of the major histocompatibility complex is associated with susceptibility to membranoproliferative glomerulonephritis, and that patients with glomerulonephritis who have this extended haplotype have a poorer prognosis for kidney survival than those without the haplotype.

C4 isotype deficiency in IgA nephropathy

C4 and factor B typing were performed in 37 pediatric patients with primary IgA nephropathy. Null alleles for C4B occurred with a frequency of 26% in patients, as compared to 15% in healthy controls (NS). The phenotype of C4B deficiency (homozygous C4B null), however, was found in 16% of patients and 4% of controls (P<0.05). Comparison of observed C4B phenotypes with those predicted from the Hardy-Weinberg equilibrium also confirmed an excess of C4B deficiency (P<0.0005). In contrast, there was no evidence of distortion in the frequencies of the C4A null allele or phenotype, or of the factor B alleles. The data suggest that C4B deficiency may be one of multiple interacting factors contributing to the development of this glomerulopathy.

Functional consequences of the genetic polymorphism of the third component of complement

The third component of complement, the central protein of the complement cascade, occurs in two principal allotypes, C3S and C3F. An excess frequency of the F allotype has been implicated in a number of disease states, including some forms of glomerulonephritis. These associations have been explained by functional differences between C3S and C3F. We examined several complement functions, using purified preparations of C3S or C3F. The C3S allotype was 1.3 times more efficient than C3F in a hemolytic assay employing sensitized sheep erythrocytes; this difference was shown to arise from a slightly more efficient deposition of C3F on the cell surface. These differences are trivial and of much less magnitude than the functional differences between C4A and C4B. There were no differences between allotypes in their ability to be converted to inactive C3b (C3bi) by complement factors H and I or by CR1 and factor I. No significant differences were seen between the allotypes and their ability to support solubilization of preformed immune complexes.

POLYMORPHISM OF HUMAN C3: CLINICAL AND FUNCTIONAL CONSEQUENCES

Distortions in frequency distribution of the allotypes of human C3 (C3*S and C3*F) have been linked to disease. Explanations for these associations have invoked the possibility of functional differences between the allotypes. We report the first detailed studies of C3 allotype function and distribution in disease.Purified C3 of each type was prepared from plasma of homozygous donors. In a standard functional assay with sheep erythrocytes (EAs), the hemolytic efficiency of C3S was 1.3 times that of C3F. Correspondingly, C3S uptake on EAs via the classical C3 convertase (assessed by anti C3c binding) was 1.4 times that of C3F.Interaction of C3S and C3F with regulators was measured using C3b of each type incubated with purified I and increments of purified H or a source of CR1. No difference in the proteolytic formation of C3bi by each allotype was seen.Labeled BSA and anti-BSA complexes were solubilized In sera containing an excess of C3S or C3F, and uptake of complexes onto erythrocytes was measured. Uptake at 15 minutes was identical, and no kinetic differences were apparent.Distribution of C3S and C3F was examined in groups of patients with diseases in which C3 is thought to be involved; SLE.(30), MPGN (33), and IgA nephropathy (31). No significant differences from the distribution of allotypes in 100 healthy controls (C3S =.8; C3F = .2) were evident.We thus found no association between C3 genetic polymorphism and diseases in which C3 is relevant. More importantly, we identified no important functional differences which could explain such associations, other than trivial differences in affinity for EAs.

1036 THE RELATIONSHIP BETWEEN THE COMPONENT AND REGULATORY PROTEINS OF THE CLASSICAL PATHWAY C3 CONVERTASE

The classical pathway C3 convertase (C4b2a) is regulated by the action of two components. C4 binding protein (C4bp) accelerates its intrinsic decay and acts as a cofactor with the C3 inactivator (I) in the cleavage of C4b. We examined the possibility that an equilibrium existed between the serum concentration of the components (“C”[C4 and C2]) and regulators (“R”[C4bp and I]) of the classical C3 convertase.Complete measurement of serum complement components was made in 184 normals. The r value of C vs R (each expressed as 7, normal) was +0.64; the 95% confidence interval was defined as the normal classical convertase C vs R relationship (CCC vs R).A normal CCC vs R was found in 23/25 patients with heterozygous genetic deficiency of C2, C4, I, or C4bp and 11/13 newborns with low complement levels in cord sera. These data suggest that hypocomplementemia in the absence of complement consumption would not disturb the CCC vs R. In situations of classical pathway mediated complement consumption, the CCC vs R was usually abnormal (19/23 SLE, 2/2 bacteremla, 8/8 HANE, and 14/24 MPGN I). Patients with MPGN II with evidence of alternative pathway activation, however, generally had normal values of CCC vs. R (10/13).Simultaneous examination of C4bp and I along with C4 and C2 may help to document the contribution of classical pathway activation to hypocomplementemia.