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PubMed2005

Reduced uptake of apoptotic cells by macrophages in systemic lupus erythematosus: correlates with decreased serum levels of complement.

BACKGROUND: Defects in phagocytosis of apoptotic cells have a role in the pathogenesis of autoimmune diseases. Decrease of phagocytosis of apoptotic cells occurs in systemic lupus erythematosus (SLE). Factors underlying this decrease are, presently, unknown. OBJECTIVE: To analyse the expression of relevant membrane receptors of monocyte derived macrophages (MDM) from patients with SLE and assess their ability to phagocytose apoptotic cells in comparison with MDM from healthy controls. Additionally, to compare phagocytosis in the presence of SLE sera with that in normal human serum (NHS). METHODS: Human peripheral blood monocytes were isolated from patients and controls, and cultured for 7 days to obtain MDM. Membrane expression of CD14, CD18, CD36, and CD51/61 was measured. MDM were incubated with apoptotic Jurkat cells in the presence of NHS or serum from patients with active or inactive disease. RESULTS: No differences in phagocytosis capacity were found between MDM from patients and controls. Membrane expression of the respective receptors was comparable in patients and controls. However, when MDM from controls were incubated with apoptotic cells in patient serum, phagocytosis was significantly decreased in comparison with incubation in NHS. This effect depended on the patients' disease activity and could be reversed by addition of NHS. Reduced uptake of apoptotic cells was associated with decreased levels of complement C1q, C4, and C3, but not with levels of complement factor B. CONCLUSIONS: Reduced uptake of apoptotic cells by MDM from patients with SLE is not an intrinsic defect but is serum dependent and associated with decreased levels of C1q, C4, and C3.

Adult↗
PubMed1986

Tropical pyomyositis: is it an immunodeficiency disease?

Serum immunoglobulin (IgG, IgM, IgA) and complement (C3, C4) levels were measured by radial immunodiffusion technique in a group of 16 Nigerian patients with tropical pyomyositis (TP). Sixteen healthy Nigerians, age- and sex-matched with the patients, were also included in the study as control subjects (CS). A significantly low level of circulating IgM (P less than 0.01) and elevation of the serum levels of IgG (P less than 0.01) and IgA (P less than 0.01) were observed in TP compared to CS. Mean serum complement (C3, C4) levels were found to be similar in patients and controls. Microbiological culture tests showed that 14 pus specimens (88%) and 3 blood specimens (19%) from the patients were positive for Staphylococcus aureus. It was therefore proposed that IgM antibody deficiency (primary or acquired) against S. aureus may be the cause of tropical pyomyositis in Nigerians.

Adolescent↗
PubMed1990

[Components of human complement system. Testing and isolation of C4].

A modified reagent for testing the hemolytic activity of human complement component C4 has been obtained. Reagent R4 was obtained by treatment of human blood serum pools with 0.075 M solution of hydrazine hydrate. This reagent was found to be rich in the serum fraction obtained by chromatography on DEAE-cellulose DE-52 and containing an active complement C3 component. To test the sensitivity and specificity of the reagent, component C4 was subjected to purification. This procedure resulted in a hemolytically active, electrophoretically and immunoelectrophoretically homogeneous component C4. DEAE-cellulose DE-52, DEAE-Sephacel, Ultragel AcA-34 and, again, DEAE-Sephacel were used consecutively as purification agents. The activity yield of component C4 with regard to the initial serum level was 20%.

Chromatography, Gel↗
PubMed1982

Complement consumption gonococcal peptidoglycan.

Purified peptidoglycan (PG) obtained from Neisseria gonorrhoeae was tested for the ability to consume complement in normal human sera. Sonicated PG (S-PG), a heterogeneous mixture of soluble fragments (molecular weight, greater than 10(6)), as well as intact (insoluble) PG, reduced the level of whole hemolytic complement in a pool of four human sera. The minimal concentration of S-PG required for this activity was approximately 500 micrograms of S-PG per ml of serum. Complete lysozyme digestion of S-PG, yielding PG fragments of less than 10(4) molecular weight, eliminated complement-consuming activity. S-PG-mediated complement consumption resulted in depletion of the individual complement components C4 and C3. Consumption of complement did not occur when C4-deficient human serum or normal human sera treated with Mg2+-(ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid to specifically impair classical complement pathway activity were used. The addition of rabbit anti-PG antibody greatly enhanced gonococcal PG-mediated complement consumption. Together, the data suggested that gonococcal PG-mediated complement consumption occurred via the classical complement pathway, was dependent on the presence of anti-PG antibody, and required glycosidically linked polymers of PG. Individual human sera varied widely in the extent of gonococcal PG-mediated reduction of complement levels, presumably a reflection of either different amounts of natural antibody to gonococcal PG, different levels of human PG hydrolase(s) capable of degrading PG to inactive fragments, or both.

Antibodies, Bacterial↗
PubMed1985

Localization of C4 genes within the HLA complex by molecular genotyping.

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.

Chromosome Mapping↗
PubMed1988

Sulfation of tyrosine residues does not influence secretion of alpha 2-antiplasmin or C4.

Sulfation of tyrosine residues is a biosynthetic modification of many secretory proteins. The function of this modification is not known, but it has been proposed that tyrosine sulfate residues may act as sorting signals to direct proteins along the secretory pathway. We tested this hypothesis by examining the effect of sulfation inhibitors on the kinetics of secretion of proteins by HepG2 cells. The inhibitors induced no change in the rate of secretion of alpha 2-antiplasmin and C4 (fourth component of complement), both of which contain tyrosine sulfate residues. Sulfation of tyrosine residues does not contribute to secretion of these proteins.

Cell Line↗
PubMed1987

The classical and alternate pathways of complement in oral contraceptive users.

Whole complement (CH50), C3, C4, the alternate pathway activity of complement (APH50) and factor B were measured in 159 women currently taking oral contraceptives and in 186 women who were not taking the pill. The mean levels of all components of the complement, except APH50, were found to be elevated in current users compared to non-users. The elevation in serum levels was seen in the first year after initiation of oral contraceptive use. Thereafter, levels changed little with duration of use. Among women who stopped using oral contraceptives, complement levels were similar to those of women who had never used the pill. Results appeared to be similar irrespective of the type of progestagen included in the oral contraceptive. These data provide support for the view that oral contraceptives have no adverse effect on the classical and the alternate pathways of complement.

Adolescent↗
PubMed1984

Significance of sequential changes in serum complement levels during acute anaphylactoid reactions.

Reduced levels of complement fractions C3 and C4 and total hemolytic complement (CH50) are usually attributed to classical complement pathway activation. However, studies in 26 patients undergoing severe anaphylactoid reactions during general anesthesia suggest that these changes may equally reflect plasma dilution and protein redistribution. Twelve patients showed a dramatic fall in complement complexes which tended to resolve after 24 h; however, this fall was not usually associated with the detection of C3 breakdown products or antigen-antibody complexes. Measurement of complement levels is not a reliable index of activation or cleavage when there are dynamic shifts in plasma volume.

Acute Disease↗
PubMed1995

In vitro evaluation of the efficacy and biocompatibility of new, synthetic ABO immunoabsorbents.

Synthetic ABO immunoabsorbents (known as Synsorbs) were in use for several years to specifically eliminate ABO antibodies from the patient's circulation before ABO-incompatible organ or bone marrow transplantation. Because Synsorbs are no longer available, we have developed new ABO immunoabsorbents. These substances, termed BioSorbents A and B, respectively, consist of synthetic A or B trisaccharides covalently coupled to macroporous glass beads via polyacrylamide. Here we report the evaluation of BioSorbents in regard to efficacy, specificity, and biocompatibility. Using a closed-circuit in vitro system, representing a 1:10-1:20 scale as compared with the immunoabsorption procedure with an adult patient, blood group O plasma was run through columns filled with ethylene oxide-sterilized BioSorbent. Hemagglutination was reduced by 4 titer steps after absorption, and anti-A and/or anti-B IgM/G/A, as measured by ABO ELISA, dropped by 85% or more, while no nonspecific absorption of immunoglobulins occurred. No significant changes could be observed for complement (C3, C4, and total hemolytic complement of the classical pathway) or for coagulation parameters (fibrinogen, prothrombin time, activated partial thromboplastin time). As monitored by immunoblotting, neither factor XII nor high molecular weight kininogen was cleaved. In addition, a monocyte phagocytosis inhibition test provided evidence that no significant aggregation of IgG had occurred during absorption. We conclude that BioSorbents A and B are efficient, specific, and biocompatible with human plasma.

ABO Blood-Group System↗
PubMed2005

Complement activation in acute coronary syndromes.

The complement system is part of the host defence response. However, considerable evidence suggests that complement plays an important role in the pathophysiology of ischemic heart disease. The aim of this study was to evaluate complement activation in patients with all forms of acute coronary syndromes (ACS) and to examine the relationship between the degree of complement activation and myocardial injury. The study population included 152 subjects (26 females): 82 with ACS (35 acute myocardial infarction (AMI), 22 non-Q wave MI (NQMI), 25 unstable angina (UAP)) (Group A), 35 stable angina (SA) (Group B), and 35 healty control subjects (Group C). Complement 3 (C3), Complement 4 (C4), C-reactive protein (CRP), troponin I (TnI) as well as creatine kinase MB (CK-MB) were evaluated. Patients' blood samples were taken on admission (day 1) and after 2, 3 and 7 days in group A. However, only one measurement was performed in the groups B and C. Plasma C3 and C4 peak levels were significantly higher in patients with AMI (141+/-29 and 35+/-11 mg/dl) and NQMI (136+/-13 and 35+/-7 mg/dl) than in patients with SA (128+/-14 and 27+/-10 mg/dl) and the control subjects (114+/-22 and 22+/-7 mg/dl) (p<0.03). Also, C3 and C4 serum levels in patients with SA and UAP (126+/-16 and 31+/-7 mg/dl) were significantly higher than those in control subjects (p<0.01, p<0.03, respectively). At 1-week follow-up, there were no significant differences between the plasma levels of C3 and C4 in patients with UAP (p>0.05). However, plasma levels of C3 and C4 were significantly different between days in patients with AMI and NQMI (p<0.0001). Plasma C3 and C4 levels in ACS showed a relationship with peak CK-MB and Tn I levels (p<0.01). Plasma CRP level in ACS showed positive correlation with C3 (p<0.01) and C4 (p<0.001). In this study, we determined that plasma C3 and C4 levels were elevated in ACS and SA. Although C3 and C4 were higher in ACS and SA, the systemic levels of inflammatory markers in patients with SA and UAP were lower than those found in the AMI and NQMI groups. The relationship between C3, C4 levels and ACS further suggests that the complement activation is related to necrosis within the myocardium.

Acute Disease↗
PubMed1983

Interaction of desialated guinea pig erythrocytes with the classical and alternative pathways of guinea pig complement in vivo and in vitro.

We examined the fate of desialated autologous erythrocytes injected intravenously into guinea pigs (GP). Desialated GP erythrocytes (E) were lysed directly or cleared by the reticuloendothelial system in normal GP (NIH-GP) and cleared by the reticuloendothelial system in GP genetically deficient in the classical complement pathway component C4 (C4D-GP), which activate complement only via the alternative pathway. Desialated E were also cleared in cobra venom factor-treated GP (CVF-GP), which had less than 1% of normal C3 levels, but were not cleared at all in C4D-CVF-GP. Preinjection of asialoorosomucoid (ASOR) and ovalbumin (OVA) had no effect on the rate of E clearance. These in vivo studies indicated that complement activation is essential for clearance of desialated E and that clearance is unaffected by blockade of galactose or mannose receptors. Inhibition of complement-mediated clearance required blockade of both classical and alternative complement pathways. In vitro studies showed that lysis of desialated E could occur in NIH-GP serum (GPS) but not in C4D-GPS. Surprisingly, CVF-GPS also caused lysis of desialated E. Lysis was dependent on both natural antibody to desialated E and classical pathway activation; natural antibody was of both the IgG and IgM classes. C3 uptake studies demonstrated that almost 10 times as many C3 molecules/E were deposited by NIH-GPS as by C4D-GPS or CVF-GPS onto desialated E. Approximately equal numbers of C3 molecules were deposited by CVF-GPS, which did lyse desialated E, and by C4D-GPS, which did not. We suggest that the molecular mechanism of in vivo clearance and in vitro lysis of desialated E by CVF-GP is via classical pathway deposition of C3b into sites on the erythrocyte surface protected from inactivation by H (beta 1H) and I (C4b/3b inactivator). Deposition of C3b into these sites by alternative pathway activation is sufficient to cause clearance but not lysis of desialated E. CVF-GPS may not represent an adequate reagent for testing the complement dependence of various biologic phenomena, particularly if the question involves surfaces that can provide protected sites for C3b molecules.

Animals↗
PubMed1991

[Value of biochemical and immunological studies in the diagnosis of silicosis].

A series of biochemical and immunological investigations was performed in a group of 33 silicosis patients and in 34 controls. The results obtained suggest future prospects of utilizing the following tests as silicotic-fibrosis markers: alpha 2-globulin and ceruloplasmin levels, activity of certain lysosomal enzymes (beta-galactosidase and beta-acetylglucosaminidase), IgM and IgG immunoglobulins, elastases, and the C4 component of the complement.

Acetylglucosaminidase↗
PubMed1986

[Immunological aspects of pulse therapy in systemic lupus erythematosus].

Twenty SLE patients were examined against a background of pulse-therapy with methylprednisolone. The analysis showed that there was a decrease in the CIC concentration, antibodies to native DNA and an increase in the level of C3c and C4 components of the complement against a background of pulse-therapy. A more rapid time course of the CIC level was noted shortly after pulse-therapy as compared to changes in other immunological indices. A dynamic study of immunological indices in SLE against a background of pulse-therapy was appropriate for a clinical assessment and a study of the mechanisms responsible for the therapeutic efficacy of this method.

Adolescent↗
PubMed1981

Acute non-A, non-B hepatitis with serum sickness-like syndrome and aplastic anemia.

We treated a patient in whom acute non-A, non-B hepatitis was complicated by a serum sickness-like syndrome (rash, arthralgias). Serum from this patient demonstrated a fall in C3, C4, and total hemolytic complement levels during the syndrome, in association with circulating IgM immune complexes. Fatal aplastic anemia developed later in the patient. This case illustrates that non-A, non-B hepatitis may be complicated by immune complex features that have previously been emphasized to occur with acute hepatitis B. Additional evidence supporting the occurrence of this clinical picture in non-B forms off viral hepatitis is presented.

Acute Disease↗
PubMed1986

Extractable nuclear antigen (ENA) autoantibodies in SLE: an immunogenetic relationship with HLA, C4 and Bf alleles.

Sera from 36 subjects with systemic lupus erythematosus (SLE) were examined for extractable nuclear antigen (ENA) autoantibodies by immunoassay with the ribonucleoprotein (RNP) subset being determined by immunodiffusion. The prevalence of the genetic markers of HLA, the fourth complement component (C4) and properdin factor (Bf), which are all coded for within the major histocompatibility complex on chromosome 6 were analysed in relation to various parameters of these autoantibodies. The following associations were observed: The lowest ENA antibody titres of the RNP negative group were associated with HLA A9 (P less than 0.05), while the lowest RNA-ase sensitive ENA (RSE) subset antibody levels were associated with HLA Dr 1 (P less than 0.05). For the complement markers, C4 AQo was associated with the lowest affinity ENA antibodies (P less than 0.05), while the BF F allele and Fs phenotype had lower RSE antibody levels than did the S allele (P less than 0.05) and the SS phenotype (P less than 0.05) respectively. This study demonstrated diverse association between various MHC markers and ENA antibody parameters, indicating that there are distinctive immunogenetic influences over ENA autoantibodies in SLE.

Adolescent↗
PubMed1984

Complement and its fractions (C3-C4) pattern in subjects with neoplasia.

The total complement (CH50) and its fractions C3 and C4 have been assayed for up to two years after surgery in subjects with breast, gastric and colon-rectum carcinomas. In all three types of pathology a constant pattern has been observed. Before surgery the CH50 stayed below the normal range but was increased following surgery. After a month it was again within the normal range and subsequently, according to the clinical evolution of the disease, it remained normal in those patients without relapse or any apparent metastasis, whereas in those patients who displayed metastasis and/or approached the terminal phase it fell below the normal range. The C3 fraction followed the CH50 pattern whereas the C4 did not show any variation correlated with the stages of the disease.

Breast Neoplasms↗
PubMed1991

Synthesis of complement components C2 and C4 by human monocyte-derived macrophages during in vitro differentiation in serum-free culture conditions.

In order to analyze the kinetics of complement component synthesis by human monocytes/macrophages, we have developed a system of defined culture conditions in the absence of serum. Moreover, the use of the polymerase chain reaction (PCR) provides a high sensitivity for the detection of mRNAs and the study of the regulation of complement component synthesis by these cells. Human blood monocytes were collected and purified by cytapheresis and elutriation, and then cultured in nonadherent cell culture bags for up to 3 weeks. Cells were grown in Iscove's modified Dulbecco's medium supplemented with alpha-phosphatidylcholine, transferrin, insulin, glutamine and antibiotics. The phenotypic and functional properties of macrophages differentiated under these serum-free culture conditions have been previously analyzed [1]. Secretion of complement component was measured by a hemolytic assay. mRNA was prepared using guanidine isothiocyanate extraction followed by cesium gradient ultra-centrifugation. cDNA was obtained by reverse transcription, then amplified by PCR. Fresh monocytes did not display any secretion of C2 as measured by hemolytic assay. However, C2 secretion was detected on and after the 3rd day of serum-free cultured unstimulated monocytes. The rate of C2 production increased along with macrophage differentiation up to the 3rd week of culture. We were not able to detect any functional C4 secretion. In preliminary studies, C2 and C4 mRNAs were detected in macrophages and in unstimulated fresh monocytes. These preliminary studies show that the serum-free culture conditions we have developed allow very satisfactory survival and differentiation of human monocytes, and provide optimal conditions for the study of their secretory activity.

Cell Differentiation↗