Inherited structural polymorphism in human C2: evidence for genetic linkage between C2 and Bf.

Structural variation in the second component of human complement was identified in about 4% of serum samples from random unrelated individuals of all the major races. Three forms of C2 have been identified by isoelectric focusing in polyacrylamide gel and development of patterns in agarose gel containing antibody-sensitized sheep red cells and C2-deficient serum: C2 C (common), C2 A (acidic), and C2 B (basic). The C2 variants were shown to be inherited as autosomal codominant traits, and suggestive evidence for close linkage between C2 and Bf was obtained.

Genetic polymorphism in human glycine-rich beta-glycoprotein.

Extensive polymorphism of glycine-rich beta-glycoprotein (GBG) was found in human sera. In all instances, GBG consisted of at least five components on electrophoresis. Patterns were such that they provided evidence for four alleles (at a locus designated Gb) which were expressed as autosomal codominant traits. Gb(S) and Gb(F) were found in all populations but with different frequencies, Gb(F1) was found in Negroes, and Gb(S1) was found in Caucasians. From electrophoretic studies of GBG, evidence was obtained that suggested that the GBG molecule was a tetramer consisting of A and B subunits in a proportion of about 1.6:1. The genetically controlled differences in GBG embodied in the Gb system indicated the presence of a third moiety of the molecule (C), possibly a polypeptide subunit. Electrophoretic studies of fragments from defined types of GBG suggested that GBG cleavage induced by complement or properdin activation in serum occurred through this C moiety, since two variants were detectable in one fragment and two were found in the other fragment. On comparison of fetal-maternal Gb types, approximately one-half the pairs showed differences. This indicated that GBG did not cross the placental barrier.

The relationship of glycine-rich -glycoprotein to factor B in the properdin system and to the cobra factor-binding protein of huan serum.

Factor B activity of the properdin system was found to be identical with purified glycine-rich beta-glycoprotein (GBG) but was distinct from the normal human serum protein capable of forming a C3-inactivating complex with a protein from cobra venom (CoF). Factor B activity coincided with electrophoretically separated GBG genetic variants, whereas the CoF-binding protein did not. GBGase destroyed factor B as it cleaved GBG but did not destroy the C3-inactivating activity of the CoF-binding protein. During incubation of serum with CoF, GBG did not change in molecular size, nor was there any coincidence in the immunoelectrophoretic mobilities of CoF and GBG. It was not possible to precipitate labeled CoF incubated with serum by anti-GBG, nor labeled GBG from serum incubated with CoF by anti-CoF. The CoF-binding capacity of serum was 2 mg/100 ml or less or under 6.5% of the serum concentration of GBG. When labeled CoF was added to serum below the binding capacity, complete complexation of the CoF was demonstrated, whereas CoF was largely uncomplexed when CoF was added in amounts equimolar to GBG.

Acute phase reactants ceruloplasmin and haptoglobin and their relationship to plasma prostaglandins in rabbits bearing the VS2 carcinoma.

Results of previous studies have shown that the VX2 carcinoma in rabbits synthesizes large amounts of prostaglandin E2 (PGE2). PGE2 secreted by the tumor is rapidly metabolized and can be measured in plasma as the metabolite 13,14-dihydro-15-keto-PGE2 (PGE2-M). We have previously proposed that the hypercalcemia that occurs in rabbits bearing the VX2 carcinoma is due to excessive secretion of PGE2 by the tumor and its subsequent action on the skeleton as a bone resorption-stimulating factor. In the course of these studies, we noted that the plasma of rabbits bearing the VS2 carcinoma became blue about 1 wk after tumor implantation. The intensity of the color increased markedly thereafter. We therefore measured ceruloplasmin in plasma by both chemical and immunological assay methods. Plasma ceruloplasmin and PGE2-M rose in parallel (within 7-10 days) and preceded by 7-10 days the development of hypercalcemia. 2 wk after tumor implantation, plasma PGE2-M and ceruloplasmin had risen about 20- and 6-fold, respectively, while the rise in plasma calcium was just beginning. Indomethacin, an inhibitor of prostaglandin synthesis, given from the time of tumor implantation prevented completely the hypercalcemia and largely inhibited the rise in ceruloplasmin. When given after hyperprostaglandinemia had developed, indomethacin produced a fall in both PGE2-M and ceruloplasmin. A rise in plasma haptoglobin concentrations similar to that seen for ceruloplasmin was also observed. No changes in plasma albumin concentrations occurred. We conclude that the acute phase reactants ceruloplasmin and haptoglobin rise rapidly in the plasma of rabbits bearing the VX2 carcinoma, and that this increase is related to arachidonic acid metabolism in these animals. It is possible that arachidonic acid metabolites also play a role in the elevations of these two plasma proteins observed in certain patients with malignant tumors.

Serum-dependent phagocytosis of paraffin oil emulsified with bacterial lipopolysaccharide.

Paraffin oil containing oil red O and emulsified with lipopolysaccharide obtained from Escherichia coli was ingested rapidly by guinea pig polymorphonuclear leukocytes or human peripheral blood granulocytes and monocytes after opsonization by fresh homologous serum. The initial rate of engulfment of the particles was spectrophotometrically assayed by determination of cell-associated oil red O and reflected the opsonic activity of the serum. This activity was resistant to dialysis but labile to heat, hydrazine, and zymosan, required divalent cations, and was maximal in the presence of Ca(++) and Mg(++). It was associated with the fixation of [(125)I]C3 to the lipopolysaccharide particles. Genetically C3-deficient serum had no opsonic activity, and this activity was restored by the addition of purified C3. Normal and C4-deficient guinea pig serum and normal, C2-, and C4-deficient human sera were equally effective in opsonizing lipopolysaccharide particles and lipopolysaccharide particles sensitized with heat-inactivated lipopolysaccharide immune serum. Cord serum deficient in glycine-rich beta-glycoprotein (GBG) (properdin factor B) had diminished opsonic activity which was improved by addition of purified GBG. Thus, C3 fixation to lipopolysaccharide particles occurs by means of the properdin system, and the opsonization and ingestion of lipopolysaccharide particles constitutes a quantitative functional assay of this pathway.

The opsonic fragment of the third component of human complement (C3).

Human peripheral blood phagocytes ingest Escherichia coli 026:B6 lipopolysaccharide (LPS)-coated paraffin oil droplets containing Oil red O only if fresh serum deposits C3 on the surfaces of the particles (opsonizes them), by reactions involving the properdin system. The rate of binding of purified [125-I]C3 in serum to LPS-coated particles correlated precisely with the rate of acquisition of ingestibility assayed spectrophotometrically. Once opsonized, LPS-coated particles remained fully ingestible and retained fixed [125-I]C3 radioactivity even after exposure to extremes of temperature, pH, ionic strength, phospholipases, urea or guanidine, some nonionic and ionic detergents, and organic solvents. Trypsin, human conglutinogen-activating factor, another heat-stable activity found in human serum, and sodium dodecyl sulfate removed radioactivity and diminished ingestibility of the opsonized particles. Alkylation, reduction plus alkylation and F(ab')2 from anti-C3 blocked ingestibility but did not alter particle-bound radioactivitymelectrophoretic and tryptic peptide autoradiographic analysis of dodecyl sulfate eluates of opsonized particles, cleansed of many contaminating proteins by boiling with 2 M NaCl (yet still opsonized), revealed that the polypeptide with C3-derived radioactivity had a mol wt of approximately 140,000 and was composed of 70,000 mol wt subunits linked by disulfide bonds. Immunochemical analysis and comparison of the peptide structure of the eluate with that of C3 indicated that the opsonic fragment is not the fragment defined as C3b but a smaller derivative of C3.

The immune response to hepatitis B vaccine in humans: inheritance patterns in families.

We have recently shown that the human antibody response to the hepatitis B virus surface antigen (HBsAg) vaccine is major histocompatibility complex (MHC) associated. In studies of nonresponders to the vaccine, we found an increased incidence of individuals homozygous for human histocompatibility leukocyte antigen (HLA) proteins associated with the extended (conserved) haplotype [HLA-B8,SC01,DR3]. In later prospective vaccination trials, we showed that none of five individuals homozygous for this haplotype developed more than 1,300 radioimmunoassay (RIA) units of antibody (mean, 467 RIA units), while all heterozygotes made at least 2,500 RIA units (mean antibody level, 15,608 units). Our results suggested that [HLA-B8,SC01,DR3] lacks an immune response gene for HBsAg, and that response is inherited in a dominant fashion. To provide further evidence for this hypothesis, we have now analyzed the results of HBsAg immunization in families. 43 members of 10 families were immunized with the hepatitis B vaccine, including seven families where at least one member bore the haplotype [HLA-B8,SC01,DR3], and three families where one member had already received, but failed to respond to, the vaccine. In two of these three families, the presence of [HLA-B8,SC01,DR3] was subsequently found. Of nine MHC-identical sibling pairs in the study, both members of eight pairs had similar antibody responses (five nonresponder and three responder pairs). In all families with such sibling pairs, including the discordant pair, rank-ordering members by antibody level demonstrated that no relative's value came between the sibling pair values. Furthermore, of nine [HLA-B8,SC01,DR3]-haplotype-homozygous individuals, six were nonresponders, and two others had only low-normal responses. [HLA-B8,SC01,DR3]-heterozygous family members always had higher levels of antibody than their homozygous relatives. Linkage analysis of nonresponse to HLA haplotypes revealed a maximum likelihood LOD (logarithm of the odds) score of 6.3 at a recombination fraction of 0.1. The MHC association with lack of antibody response to HBsAg was not seen with tetanus immunization, where 1 of 20 HBsAg responders and 1 of 21 poor or nonresponders had tetanus titers of less than 1:512; both tetanus nonresponders were [HLA-B8,SC01,DR3] heterozygotes. Our results indicate that: (a) response to the HBsAg vaccine is MHC linked, and inherited in a dominant fashion; (b) an abnormal or missing immune response (Ir) gene for HBsAg is a characteristic of most examples of the extended haplotype [HLA-B8,SC01,DR3]; and (c) other haplotypes also have abnormal or missing Ir genes for HBsAg.

Polymorphic Hh genes in the HLA-B(C) region control natural killer cell frequency and activity.

We demonstrated earlier that individuals homozygous for conserved major histocompatibility complex (MHC)-extended haplotypes have low natural killer (NK) activity as measured by cytolysis of the K562 tumor cell lines. In the present study, we investigated the segregation and MHC linkage of NK activity in families in which MHC haplotypes of human histocompatibility leukocyte antigens (HLA)-A, -C, and -B, complotype, and DR specificities are known. In two informative families, low activity was inherited as a recessive trait linked to the MHC. By using individuals homozygous for specific fragments of extended haplotypes or for HLA-B alleles, we found that the HLA-C and -B and not the complotype or HLA-DR region contains genes controlling NK activity. The majority of the unrelated individuals with low NK activity were homozygous or doubly heterozygous for HLA-B7 (Cw7), B8 (Cw7), B44 (Cw5), B18, or B57 (Cw6). Thus, these alleles form one complementation group designated NKB1. Another less frequent group, NKB2, was also identified, and consisted of individuals homozygous for B35 (Cw4). NK activity was correlated with the number of circulating NK (CD16+ CD56+) cells. Individuals homozygous for the NKB complementation groups have fewer circulating NK cells than individuals heterozygous for these alleles and alleles of other complementation groups, possibly explaining the low activity of cells in these subjects. These findings suggest that during the maturation of NK cells there is NK cellular deletion in donors homozygous for NKB genes resulting in low NK cell numbers and activity.

The enhancement of bacterial phagocytosis by serum. The role of complement components and two cofactors.

The role of serum factors in the phagocytosis of pneumococci was studied employing a spectrophotometric assay which measures reduced nitro blue tetrazolium (NBT) dye. Dye reduction occurs within the phagocyte shortly after bacterial ingestion as measured by the phagocytic index technique and by the uptake of (125)I-pneumococci. Bacteria prepared with gammaG antibody were not phagocytosed unless a small volume of fresh normal serum was added. Using fresh sera deficient in single complement components, it was demonstrated that the first four components are necessary for optimal bacterial phagocytosis. When highly purified complement components were added to the antibody-coated pneumococci, enhancement of phagocytosis was achieved only with the sequential addition of C1, C4, C2, and C3. Evidence has been presented that human C3 bound to an immune complex exhibits peptidase activity and that this activity is essential for phagocytosis. A heat-labile, dialyzable serum cofactor which enhances C3 peptidase activity enhanced the phagocytosis of pneumococci prepared with purified complement components. A second phagocytosis-promoting cofactor, which is not a complement component, was found to be a heat-labile, 5-6S, beta pseudoglobulin. This protein may stabilize C3 peptidase activity or inhibit enzymatic inactivation of C3.

Linkage of pemphigus vulgaris antibody to the major histocompatibility complex in healthy relatives of patients.

Pemphigus vulgaris (PV) is an autoimmune disease caused by high concentrations of antibody to an epidermal cadherin. The disease is associated with two kinds of HLA-DR4, DQ8 haplotypes dominantly distributed among Jewish patients, and these plus DR6, DQ5 haplotypes in non-Jewish patients. Low levels of the PV antibody were found in 48% of a total of 120 asymptomatic parents, children, and siblings of 31 patients, thus exhibiting dominant inheritance. The inheritance of these low levels of antibody in asymptomatic relatives was linked to the major histocompatibility complex with a highly significant logarithm of the odds score of 9.07, almost always to a DR4 or DR6 haplotype of the patient. Disease appears to occur in susceptible individuals with low levels of antibody when a second factor, either environmental or genetic, induces high levels, sufficient to produce blisters.

Major histocompatibility complex susceptibility genes for dermatitis herpetiformis compared with those for gluten-sensitive enteropathy.

Dermatitis herpetiformis (DH) shares some clinical features and major histocompatibility complex (MHC) markers with gluten-sensitive enteropathy (GSE). We compared MHC haplotypes in 27 patients with DH, 35 patients with GSE, and normal controls. As in GSE, the frequencies of two extended haplotypes, [HLA-B8, SC01, DR3] and [HLA-B44, FC31, DR7], were increased in patients with DH. Distributions of fragments of extended haplotypes, consisting of some but not all of the elements of complete extended haplotypes, were analyzed to attempt to localize a susceptibility gene. Besides complete extended susceptibility haplotypes, (DR3, DQ2) and (DR7, DQ2) fragments were most common in GSE. In contrast, DH showed only a few such fragments but many instances of the fragment (SC01). The differences in distribution of these fragments in the two diseases were highly significant (P < 0.002). HLA-DQ2 and DR3 had the highest odds ratios for GSE, but the highest odds ratio for DH was for the complotype SC01. These findings suggest that the MHC susceptibility gene for DH is between class II and complotype regions, closest to the complotype, whereas that for GSE is in the class II region.

The cellular basis for lack of antibody response to hepatitis B vaccine in humans.

We had previously obtained evidence that among normal subjects the humoral antibody response to hepatitis B surface antigen (HBsAg) was bimodally distributed with about 14% of subjects producing less than 1,000 estimated radioimmunoassay RIA units. From the study of major histocompatibility complex (MHC) markers in the very poor responders who produced less than 36 estimated RIA units of antibody, it appeared that there was an excess of homozygotes for two extended haplotypes [HLA-B8, SC01, DR3] and [HLA-B44, FC31, DR7]. This finding suggested that a poor response was inherited as a recessive trait requiring nonresponse genes for HBsAg on both MHC haplotypes and was strengthened by finding a much lower antibody response among prospectively immunized homozygotes for [HLA-B8, SC01, DR3] compared with heterozygotes. In the present study, we have analyzed the cellular basis for nonresponse to this antigen by examining antigen-specific proliferation of T cells from responders and nonresponders in the presence and absence of autologous CD8+ (suppressor) cells. Peripheral blood cells from nonresponders to HBsAg failed to undergo a proliferative response to recombinant HBsAg in vitro, whereas cells from responders proliferated vigorously. This failure of cells from nonresponders to proliferate was not reversed in cell mixtures containing CD4+ and antigen-presenting cells devoid of CD8+ cells. There was no difference between responders and nonresponders with respect to the number of circulating T cells or their subsets, or the proliferative response to mitogens such as pokeweed or phytohemagglutinin or another antigen, tetanus toxoid. Our results indicate that our HBsAg nonresponding subjects have a very specific failure in antigen presentation or the stimulation of T helper cells, or both. Our evidence is against specific immune suppression as the basis for their nonresponsiveness. The failure of antigen presentation or T cell help is consistent with recessive inheritance of nonresponsiveness and suggests that response is dominantly inherited.

Cobra venom factor: evidence for its being altered cobra C3 (the third component of complement).

Evidence is presented that cobra venom factor, the anticomplementary protein in Naja naja venom, is modified cobra C3 (the third component of complement). Antiserum to the cobra venom factor cross reacts with human C3. A protein in cobra serum reacts strongly with antiserum to the venom factor and the former protein, like human C3, is converted by incubation of cobra serum with endotoxin, hydrazine, or simple storage at 37 degrees C. Incubation of cobra venom factor with cobra serum destroys the C3 cleaving activity of the venom factor in human serum, whereas human C3b inactivator is ineffective. Thus, the cobra venom factor appears to be a form of C3 (perhaps C3b); its potent action in human serum probably derives from its lack of sensitivity to human C3b inactivator.

C'3 synthesis in the human fetus and lack of transplacental passage.

Allotypic differences in the third component of complement between mothers and their newborns provided evidence for synthesis of this complement component by the fetus. There was no indication that this protein traversed the placenta. The known low level of C'3 in the neonate was confirmed, and the maternal concentration was found to be significantly elevated.

Beta-1C-globulin: metabolism in glomerulonephritis.

The metabolism of beta(1C)-globulin labeled with iodine-131 was studied in six normal individuals and in three individuals with glomerulonephritis who exhibited markedly reduced serum concentrations of this protein. Fractional of serum beta(1C)-globulin in glomerulonephritis appears to be chiefly secondary to decreased synthesis.

Human C'3: evidence for the liver as the primary site of synthesis.

The liver is the primary, if not sole, site of synthesis of the third component of human complement, as shown by a change in the recipient from C'3 FS(0.6) to C'3 SS, the donor type, following homotransplantation of the liver.

C1 inhibitor: evidence for decreased hepatic synthesis in hereditary angioneurotic edema.

Although the Cl inhibitor was detected in 5 to 10 percent of normal hepatic parenchymal cells by means of the immunofluorescent technique, none was seen in liver biopsies from two individuals with hereditary angioneurotic edema having low concentrations of Cl inhibitor in the serum. In contrast, the percentages of cells which reacted with fluorescent antiserums to C4 and transferrin were normal. These data suggest that in most subjects with hereditary angioneurotic edema, there is decreased synthesis of the C1 inhibitor but normal synthesis of C4, and that the disease results from this biosynthetic error.

Synthesis of human plasminogen by the liver.

Genetic types of plasminogen were determined from a donor and a recipient before and after hepatic homotransplantation. Examination of the plasminogen types demonstrated that the liver is the principal site of synthesis of human plasminogen.

Alper CA, Rosen FS: Studies of the in vivo behavior of human C'3 in normal subjects and patients.

The metabolic behavior of C'3 labeled with radioactive iodine was investigated in 10 normal subjects and in 20 patients with diseases in which complement is thought to play a pathophysiological role. The mean fractional catabolic rate of C'3 in normal subjects was 2.3 +/- 1.0% of the plasma pool per hr, whereas the fractional catabolic rate of C'3(i), the inactive conversion product of C'3 produced by complement activation, was at least five times as great. Increased catabolic rates were found in some patients with acute glomerulonephritis, systemic lupus erythematosus, idiopathic nephrotic syndrome of childhood, and progressive glomerulonephritis. Depressed synthesis was found in each of four studies of patients with progressive glomerulonephritis and seemed to be the major factor in the lowering of plasma C'3 concentrations regularly observed in patients with this disease. Of three patients with acute glomerulonephritis, synthesis rates of C'3 were markedly depressed in one subject, at the lower limit of normal in another, and entirely normal in the third. Increased extravascular: plasma pool ratios were observed in the studies of C'3(i) metabolism in a normal subject, and of C'3 metabolism in two of three patients with acute glomerulonephritis, in one of four patients with systemic lupus erythematosus, and in one patient with idiopathic nephrotic syndrome. The increased pool ratios are possibly compatible with tissue attachment of part of the injected C'3 or its conversion products. No important abnormalities of metabolism were found in patients with acquired hemolytic anemia, paroxysmal nocturnal hemoglobinuria, hereditary angioneurotic edema, or rheumatoid arthritis.By means of antigen-antibody crossed electrophoresis, C'3(i) could be demonstrated in the fresh plasma of three of eight patients who had acute glomerulonephritis. This finding was used as evidence for in vivo complement activation in this disease. Since C'3(i) was demonstrated only in plasma from patients with very low plasma concentrations whose onset of symptoms was very recent, there may be two phases in the metabolism of C'3: early complement activation with resultant increased catabolism and later depressed synthesis, both of which lead to lowered serum concentrations.

Genetic polymorphism of the third component of human complement (C'3).

Polymorphism of human C'3 has been defined by prolonged agarose electrophoresis of fresh serum. At least four, and probably five, alleles have been identified by the electrophoretic mobilities of gene products. Inheritance of three alleles, F(1) F, and S, is consistent with the autosomal condominant type. The inheritance of S(1) is probably codominant and that of F(0.8) is not known. Of the 15 phenotypes predicted by these alleles, eight have been observed. The allotypes appear to differ in net surface charge at pH 8.6, but show no obvious differences in complement activity, in molecular size, or in binding of Ca(++). The concentrations of the two gene products in serum from all known heterozygotes are approximately equal. The S gene is most common in the three major races of man. The F gene is relatively common in Caucasians, less common in American Negroes, and relatively uncommon in Orientals.