Second Component Of Complement Research Articles

Hereditary Deficiency of the Second Component of Complement: Early Diagnosis and 21-Year Follow-Up of a Family

Complement deficiencies are rare and often underdiagnosed primary immunodeficiencies that may be associated with invasive bacterial diseases. Serious infections with encapsulated organisms (mainly Streptococcus pneumoniae, but also Neisseria meningitides and Haemophilus influenzae type B) are frequent in patients with a deficiency of the second component of complement (C2), but no data are available on long-term follow-up. This study aimed to evaluate the long-term clinical outcome and the importance of an early diagnosis and subsequent infection prophylaxis in C2 deficiency. Here, we report the 21-year follow-up of a whole family which was tested for complement parameters, genetic analysis and biochemical measurements, due to recurrent pneumococcal meningitis in the elder brother. The two sons were diagnosed with homozygous type 1 C2 deficiency, while their parents were heterozygous with normal complement parameters. For the two brothers, a recommended vaccination program and antibiotic prophylaxis were prescribed. During the long-term follow-up, no severe/invasive infections were observed in either patient. At the age of 16, the younger brother developed progressive hypogammaglobulinemia of all three classes, IgA, IgM and IgG. A next generation sequencing panel excluded the presence of gene defects related to primary antibody deficiencies. Our data show that early diagnosis, use of vaccinations and antibiotic prophylaxis may allow a normal life in hereditary C2 deficiency, which can be characterized using functional and genetic methods. Moreover, a periodical check of immunoglobulin serum levels could be useful to detect a possible hypogammaglobulinemia.

Bradykinin and the Pathogenesis of Hereditary Angioedema

Bradykinin and the Pathogenesis of Hereditary Angioedema

The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation

The second component of complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human complement. The formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 A resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b-C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.

Unraveling the Genetic Predisposition to Systemic Lupus Erythematosus

It has long been suggested that patients who develop systemic lupus erythematosus (SLE) have a genetically determined predisposition. Researchers have noted that deficiency of the second component of complement is associated with SLE and with subacute cutaneous lupus erythematosus (SCLE). SLE is, perhaps, not just one disease; it has manifestations in many organs, and prognosis and the mechanisms by which inflammation develops differ depending on the organ systems involved. Four recent reports and two excellent editorials published in the New England …

The complement inhibitor, CRIT, undergoes clathrin-dependent endocytosis

Complement C2 receptor inhibitor trispanning (CRIT) is a receptor for the second component of complement and is found in various tissues and hemopoietic cells. On binding to CRIT, C2 cannot be activated to potentially form a variant-C3 convertase as it is rendered non-cleavable by C1s. CRIT thus limits the amount of C3 convertase formed on the cell surface. In this study we have shown, using flow cytometry and immunofluorescence microscopy, that human CRIT undergoes endocytosis from the plasma membrane. The endocytosis, possibly ligand mediated, occurs via clathrin-coated pits as it can be inhibited by prior incubation of cells in hypertonic medium or with chlorpromazine, at 37 degrees C. However, inhibition of endocytosis was not possible after treatment with nystatin, or filipin, inhibitors of caveolae/raft-dependent endocytosis. In the presence of C2 alone, CRIT associates with the adapter protein, beta-arrestin-2, and whether in association with C2 or not, then appears in the perinuclear region, but does not appear to be translocated into the nucleus. Apart from the C3aR and C5aR that internalize the anaphylatoxic peptides, this is the first report of the internalization via the clathrin pathway of a receptor for a complement serum protein.

Impaired IgG responses in a child with homozygous C2 deficiency and recurrent pneumococcal septicaemia

Homozygous deficiency of the second component of complement (C2) is the most common inherited deficiency of complement (1). Although C2 deficiency has been detected in asymptomatic individuals, patients usually present with either autoimmune disease or recurrent pyogenic infection, particularly due to encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis. Interestingly, infection is the most common mode of presentation of C2 deficiency in young children (1). An association between C2 deficiency and IgG subclass deficiency has also been previously described (2–4). We now report a female child with C2 deficiency that presented at the age of 3 mo with recurrent pneumococcal septicaemia. Although IgG subclass levels were normal, specific IgG responses to vaccination against S. pneumoniae and H. influenzae were significantly impaired.

Local bradykinin generation in hereditary angioedema

Genetic deficiency of C1 inhibitor results in recurrent angioedema attacks consisting of localized swelling of the subcutaneous and mucous tissues. Ineffectiveness of histamine H1 receptor antagonists in controlling these attacks excludes histamine as a mediator, whereas several lines of evidence indicate that the kinin family is involved. The possibility that bradykinin itself or a kininlike peptide derived from the second component of complement is responsible for angioedema has been largely debated,1,2 and recently a predominant role of bradykinin in the process has begun to be accepted.

A Study of Membranoproliferative Glomerulonephritis in Iran

The aim of this study was to review the morphologic patterns of membranoproliferative glomerulonephritis (MPGN) in 100 Iranian patients using light microscopy (LM) and electron microscopy (EM), and to compare the treatment and outcome in 13 patients with two biopsies. A retrospective study of 713 kidney biopsies of Iranian patients received between 1981 to 1994 was carried out. Of the 713 kidney biopsies, MPGN (n=106) and membranous glomerulopathy (n=112) made up the highest numbers of cases. Among 100 MPGN patients, 55 (55%) were MPGN type I, 10 were type II (10%), and 35 type III (35%). Eighty-three (83%) had nephrotic proteinuria, 39 (39%) had hematuria, and 52 (52%) were hypertensive. Complement levels were estimated in 58, with low C3 in 10. The glomerular involvement was irregular, with focal hypercellularity in 47 patients (47%), widely patent capillaries in 50 (50%), arteriosclerosis in 48 (48%), and with hyaline change in 25 (25%). Follow-up data (22-130 months) was available in 61 (61%) patients: 6 (10%) died after 14-56 months, 27 (44%) were on maintenance hemodialysis for 15-110 months, and three received transplants. Thirteen patients had detailed follow-up and a second biopsy after 24-120 months. All 13 presented with edema and nephrotic range proteinuria, with hematuria and hypertension in five and azotemia in four. Seven of the 13 patients received initial steroids, followed by antiplatelet or antihypertensive drugs. Four (type III) patients received antiplatelet and antihypertension drugs, and two (type III) received only antihypertensive drugs. In the first biopsy, glomerular changes by light microscopy were non-uniform in 7 of 10 (70%) type III MPGN cases. Vascular changes were absent or mild in 11, and moderate in two. In the second biopsies, 10 showed decrease in cellularity, with many open capillaries, persistence of deposits by EM in all, and progression of vascular sclerosis in eight, and tubulointerstitial changes in 10. Among the 13, six were clinically stable, another six received dialysis followed by transplant in three, and one had relapses with episodes of cryoglobulinemia. Three patients died. There is a high incidence of MPGN in Iranian patients, with a substantial number of type III MPGN cases. Second biopsies showed decreased cellularity, but increase in chronic tubulointerstitial and vascular cases. Steroids did not appear to benefit the outcome in types I and III MPGN patients compared to patients who received antihypertensive and antiplatelet treatment without steroids.

Cloning and nucleotide sequence of retroposons specific to hominoid primates derived from an endogenous retrovirus (HERV-K).

595 ENDOGENOUS RETROVIRUSES comprise approximately 1% of the human genome.1 Although most human endogenous retroviruses (HERVs) are defective, one class, HERV-K (homologous to mouse mammary tumor virus [MMTV]), encodes viral proteins and particles3 and is highly expressed in germ cell tumors. Retroposons (a retroelementso and a retrovirus-like elements,o which multiply in the genome by retrotransposition) are distinguished from retroviruses by the absence of one or both long terminal repeats (LTRs). These repetitive elements are classified into two groupsÐshort (up to 600 bp) interspersed nuclear elements (SINEs) and long (about 6 kg) interspersed nuclear elements (LINEs)Ð that include a number of families that have diverged in the course of mammalian evolution.4 One class of SINEs, the Alu sequences, is specific to primates. Another class, termed SINE-R (for retrovirus), which is derived from the HERV-K sequence, has apparently undergone modification in the hominoid lineage.5 Such elements are a potential agency of genome change.1,6,7 The human-specific retroposon SINE-R.C2, a member of the SINE-R retroposon family (present at 4000 to 5000 copies per haploid human genome), was discovered in an investigation of the variable number of tandem repeats (VNTR) locus within intron 3 of the gene for C2, the second component of complement, located in class III of the major histocompatibility complex on the short arm of human chromosome 6.8,9 Other members of the SINE-R family in the human genome, SINER11, -14, and -19, were isolated by colony blot hybridization with an HERV-K10 probe. By BLAST search, schizophrenic brain S11 cDNA (GenBank, accession No. AA772777) was also found to have high homology with the 39 LTR of HERV-K10. Therefore, specific polymerase chain reaction (PCR) primers, JS902 and JS903, were designed from S11 cDNA. Using these primers, the sequences HS307 (GenBank, accession No. AB016781) and HS408 (GenBank, accession No. AB016782), which have a high degree of homology to the SINE-R.C2, -R11, -R14, and -R19 retroposons, were identified in an investigation of the Xq21.3 region that has been tentatively linked to schizophrenia and schizo-affective disorder by Laval et al.10 and Kim et al. Each of these retroposons derives from the 3 9 long terminal repeat and the small upstream flanking regions of the human endogenous retrovirus HERV-K10,5 the entire sequence of which has been reported as a candidate autoimmune gene in type I diabetes.11 The HERV-K10 (present at 50 copies per haploid human genome) has been found to be present in all primates except for New World monkeys, whereas SINE-R.C2 is present only in Homo sapiens. Other members of the SINER family have been found to be present in the human, chimpanzee, and gorilla genomes by Southern blot analysis.12 To obtain sequence information relating to SINE-R-type retroposons in hominoid primates, genomic DNAs from the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and agile gibbon (Hylobates agilis) were amplified by PCR using primers JS902 (5 9 -GAGA ATGGGCCATGATGAC-3 9 , bases 4±22) and JS903 (59 GATCATTCTTGGATGTTTCT-3 9 , bases 466±485) derived from schizophrenic brain S11 cDNA (GenBank, accession No. AA772777). The PCR conditions followed were those of Kim et al. with an annealing temperature of 56°C. PCR products were separated on a 1.8% agarose gel, purified with the QIAEX II gel extraction kit (Qiagen, Chatsworth, CA) and cloned into the T-khs307 vector.14 Plasmid DNA was isolated by the alkali lysis method, using the High Pure plasmid isolation kit (Boehringer Mannheim, Indianapolis, IN). Individual plasmid DNAs were screened for inserts by PCR and positive samples were subjected to sequence analyses on both strands with T7 and M13 reverse primers using an automated DNA sequencer (model 373A; Applied Biosystems, Foster City, CA) and the DyeDeoxy terminator kit. Sequence analyses were done with the aid of GAP and PILEUP from the GCG program. The neighbor-joining phylogenetic analysis was performed with the CLUSTAL W program.15 We searched for sequence homology using the BLAST network server.16 Using genomic DNA from hominoid primates, SINE-R-type retroposons were amplified and nucleotide sequences deter-

Pathogenetic and Clinical Aspects of C1 Inhibitor Deficiency

People deficient in C1-INH present recurrent angioedema localized to subcutaneous or mucous tissues. The defect can be caused by impaired synthesis, due to a genetic defect (hereditary angioedema), or by increased catabolism (acquired angioedema). In our experience the majority of patients with acquired angioedema (16 of 18) have autoantibodies to C1-INH in their serum. These autoantibodies bind to C1-INH with different and generally low affinity. The vasopermeability mediator responsible for attacks is still undefined: bradykinin (derived from cleavage of high molecular weight kininogen) and a kinin-like peptide (derived from the second component of complement) still remain the two primary candidates. We examined the systems controlled by C1-INH (complement, contact system, fibrinolysis and coagulation) and found that all of them are activated during angioedema attacks. Activation of the coagulation leads to generation of thrombin whose vasoactive effect can thus influence edema formation. Treatment of severe angioedema attacks is satisfactorily performed with C1-INH plasma concentrate although patients with an acquired defect frequently need very high doses. Attenuated androgens effectively prevent attacks in hereditary angioedema, but their safety, on the very long-term, needs to be further assessed. Acquired angioedema generally fail to respond to these drugs, but can be treated prophylactically with antifibrinolytic agents.

P1 and cosmid clones define the organization of 280 kb of the mouse H-2 complex containing the Cps-1 and Hsp70 loci

A 280 kilobase (kb) contig was isolated from mouse genomic P1 and cosmid libraries, using as probes human cDNA and genomic DNA fragments that map in the interval between the second component of complement and tumor necrosis factor genes of the HLA complex. The clone contig demonstrates synteny of eleven mouse genes that are homologous to genes initially mapped within the human major histocompatibility complex. These include the mouse homologs of BAT2 (HLA-B-associated transcript 2) through BAT9 and also three HSP70-related genes. Five P1 clones form a contig of 240 kb that spans from BAT9 through BAT3. Twelve cosmid clones are arranged in three contigs that confirm most of the structure of the P1 contig and link the mouse BAT3 homolog to the BAT2 homolog approximately 15 kb farther telomeric. Polymorphic DNA markers within the cloned region were used to map the cleft palate susceptibility-1 (Cps-1) locus to the interval between Hsp70.1 and BAT6 (valyl-tRNA synthetase). This refines the location of the Cps-1 locus to a 45 kb region contained in the H2-124 P1 insert.

The Complement System of Carp Cyprinus carpio-VI. Isolation of the Second Component of Complement (C2) from Carp Serum.

The second component (C2) of carp complement was isolated from carp serum by a six-step purification procedure: 1) affinity chromatography on Blue-Cellulofine, 2) affinity chromatography on heparin-agarose, 3) gel filtration on Sephadex G-200, 4) TSKgel DEAE-5PW chromatography (HPLC), 5) TSKgel Heparin-5PW chromatography (HPLC), and 6) TSKgel G3000SWXL chro-matography (HPLC). Hemolytic activity of carp C2 in column eluates was measured using EAC14 (sheep erythrocytes sensitized with carp antibody, C1 and C4) and heat-inactivated carp serum (deficient in C1 and C2) as reagents for detecting C2. About 0.4mg of carp C2 was obtained from 100ml of serum. The final recovery was 9.4%, representing a 799-fold purification. Carp C2 was composed of a single polypeptide chain with molecular weight of 108, 000 and was susceptible to heat-inactivation at 45°C. These physicochemi-cal properties of carp C2 closely resemble those reported for mammalian C2.

Defective activation of the alternative pathway of complement in patients with homozygous C2 deficiency: studies in two unrelated families

Selective homozygous deficiency of the second component of complement, C2, with increased susceptibility to infection was detected in five children of two unrelated families. Because the haemolytic activity of the alternative complement pathway (AP) was in the low normal range, we evaluated the AP activation pattern. Serum levels of factor B measured immunochemically and the haemolytic function of factor B were low normal. Levels of C3d were not increased. Activation products of factor B were undetectable indicating the absence of in vivo activation of AP. Activation of C3 in vitro by activators of the AP (zymosan A and lipopolysaccharide) was profoundly deficient in homozygous C2 deficiency while heterozygous carriers exhibited intermediate values. There was no correlation between serum levels of factor B and in vitro C3 activation. We conclude that defective AP activation may contribute to increased susceptibility to bacterial infections in some patients with homozygous C2 deficiency.

In Vitro Induction of IgG1 and IgG2 Secretion by B Cells of Children Who Developed Invasive Haemophilus Disease despite Vaccination

Lymphocytes from seven patients who developed Haemophilus influenzae type b (Hib) disease after being vaccinated with Hib polysaccharide vaccine and from one patient who developed disease after conjugate vaccination were investigated for the ability to secrete IgG1 and IgG2, in vitro, in response to a combination of pokeweed and Staphylococcus aureus Cowan I mitogens. The eight patients were selected because they had low anticapsular antibody responses to Hib disease. Only one of the eight children had a history of previous severe, recurrent infections. This child (in whom a polysaccharide vaccine failed) had a deficiency of the second component of complement and also had a subnormal serum concentration of IgG4. Only one of the eight children, an otherwise healthy 54-mo-old with normal serum Ig concentrations, had subnormal mitogen-induced B-cell secretion of IgG1 and/or IgG2. When this child's lymphocytes were separated into T- and B-cell fractions and cocultivated with the respective fractions of the father's lymphocytes, the child appeared to have an intrinsic B-cell defect and normal T cells. There were no significant differences (p greater than 0.3) in the respective geometric means of the in vitro secretion of IgG1 or IgG2 of the B cells from the children with polysaccharide vaccine failure and those of 14 healthy controls of similar ages as the patients (IgG1, 1524 versus 3497 ng/mL per 10(5) lymphocytes; IgG2, 79 versus 89 ng/mL per 10(5) lymphocytes). Thus, despite the presence of impaired serum anticapsular antibody responses to Hib disease, most children who develop Hib disease after Hib polysaccharide vaccination have normal in vitro B-cell secretion of IgG1 and IgG2 in response to mitogens.

Disseminated gonococcal infection associated with deficiency of the second component of complement

A case of C2 deficiency presenting with disseminated gonococcal infection is described. The predisposition of C2-deficient individuals to infection in addition to the commoner problem of immune complex diseases is noted. Attention is drawn to the absence of documented cases of gonococcal infection associated with C2 deficiency. No other homozygous C2 deficient family members were identified. Lifelong penicillin prophylaxis was recommended for the patient.

Interferon‐mediated transcriptional and post‐transcriptional modulation of complement gene expression in human monocytes

The addition of lymphoblastoid interferon alpha, fibroblast interferon beta and recombinant interferon gamma to in vitro monocyte cultures produced dose-dependent increases in transcription rates of the genes encoding the second component of complement (C2), factor B (B) and C1 inhibitor, and the abundance of their respective mRNA. Interferon gamma was the most effective at stimulating transcription of the C1-inhibitor gene whereas interferons alpha and beta were more effective at increasing the transcription of the C2 and B genes. Transcription of the C3 gene was reduced by interferon gamma. None of these cytokines altered the level of transcription of the actin gene. Interferon-induced changes in the levels of transcription of the C2, B and C1-inhibitor genes occurred rapidly, with significant changes occurring within 30 min of exposure to these cytokines. Within 4 h of removal of the interferons from the culture fluid, the level of transcription of the C1-inhibitor, C2, B and C3 genes returned to control values, as did abundance of C2, B and C3 mRNA. However, the abundance of C1-inhibitor mRNA remained elevated in interferon-gamma-treated monocytes. Combinations of interferons produced less than additive effects on the stimulation of the transcription of C2, B and C1-inhibitor genes, whereas measurements of C1-inhibitor mRNA and B mRNA showed that interferon gamma acted synergistically with interferon gamma to increase the abundance of the mRNA. Their effects on C2 mRNA abundance were less than additive. The half-lives of C1-inhibitor, C2, B and C3 mRNA were not altered by interferon alpha, whereas interferon gamma shortened the half-life of C2 mRNA by approximately 50%, and prolonged the half-lives of B and C1-inhibitor mRNA approximately twofold and fivefold, respectively. The half-life of C3 mRNA was unaltered by either interferon. These results show that the large increase in C1-inhibitor synthesis which occurs in interferon-gamma-treated monocytes, is due to a combination of increased transcription and increased C1-inhibitor mRNA stability. They also suggest that the synergistic effects of interferon alpha together with interferon gamma on C1-inhibitor and factor B synthesis is also dependent upon increased transcription and increased mRNA stability.

Recurrent systemic bacterial infections in homozygous C2 deficiency

Homozygous deficiency of the second component of complement (C2) occurs in one in 10, 000 individuals. Its clinical manifestations range from essentially no symptoms to recurrent infections or evidence of collagen‐vascular disease. We present here a case report and a review of the literature focusing on recurrent systemic infections in C2‐deficient individuals. Thirteen of 20 patients vi'ith C2 deficiency and a history of invasive bacterial infections have had recurrent episodes of bacteremia or meningitis. Most of these patients were children, and Streptococcus pneumoniae was the most common pathogen reported. The use of prophylactic antibiotics. Haemophilus influenzae type b and pneumococcal vaccines, and prompt medical attention for any febrile illness should be encouraged in children with documented C2 deficiency. Although there are no studies to date to document their efficacy, the above measures may serve to diminish the frequency of recurrent systemic bacterial disease in these children.

Lysis of sensitized sheep erythrocytes in human sera deficient in the second component of complement.

Analysis of C-dependent lysis of sensitized SRBC by C2-deficient sera (C2D) led to the characterization of a C2 bypass pathway. Lysis in the total hemolytic C assay by C2D sera was Ca2+-dependent and required a high concentration of hemolysin to sensitize E. Selective component depletion indicated a requirement for C1 and C4 of the classical pathway (CP) and proteins B, P, and probably D of the alternative pathway (AP). Total hemolytic C could be restored to normal in these C2D sera by utilizing heavily sensitized E or by the addition of a supranormal concentration of B. This system most closely resembles a pathway described by J. E. May and M. M. Frank which requires antibody, C1, and the AP but not C4 or C2. It differs in its requirement for C4. We hypothesize that this pathway represents vestiges of a more primitive C pathway. It becomes evident and possibly clinically important in the setting of C2 deficiency, by allowing C activation, other than the AP, and perhaps in normal individuals, by damaging microorganisms that have evolved means to inhibit early components of the CP.

Interferon-gamma is a major regulator of C1-inhibitor synthesis by human blood monocytes.

C1 inhibitor (C1INH) is the major control factor for the activation of the classical pathway of complement and for contact system activation. Hepatocytes and blood monocytes are known to synthesize this protease inhibitor. We studied the regulation of monocyte C1INH production by mediators that are generated during inflammatory responses. Purified blood monocytes spontaneously synthesized and secreted C1INH only after prolonged culture. In the presence of interferon (IFN)-gamma, C1INH was detectable within 24 hr and continued to be released at high levels throughout an 8-day culture period. Monocyte C1INH was newly synthesized and was functionally active as determined by forming stable complex with C1s. Other monocyte stimuli were either less potent (IFN-alpha, IFN-beta) or not capable of increasing C1INH release (lipopolysaccharide, interleukin 1, and tumor necrosis factor). The second component of complement, C2, was induced by IFN-gamma to a similar extent as C1INH. These findings demonstrate that IFN-gamma is a major regulator of monocyte C1INH production and may warrant consideration of IFN-gamma in the treatment of C1INH deficiency states.

Modulation of monocyte complement synthesis by interferons

Recombinant Escherichia coli-derived gamma-interferon has been shown to stimulate synthesis of the second component of complement (C2), factor B and C1 inhibitor, but to inhibit synthesis of the third component (C3). alpha- and beta-interferons stimulate synthesis of factor B and C3 inhibitor, inhibit C5 synthesis but do not alter synthesis of C2. alpha- and beta-interferons act synergistically with gamma-interferon to enhance both factor B and C1-inhibitor synthesis.