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The significance of serial measurement of serum anti-native DNA antibodies and complement C3 and C4 components in the management of patients with systemic lupus erythematosus.

Serial measurements were made over a period of three years of serum DNA-binding capacity and complement C3 and C4 levels in parallel with documentation of clinical features of disease activity in Glasgow patients with SLE. Raised DNA-binding levels were noted in 27 of the 32 patients over this time period. High levels (over 80%) were found in patients with both renal and non-renal disease. In some patients increases in DNA-binding capacity and decreases in C3 levels were associated with changes in disease activity. This pattern was commonest in patients with renal SLE. In others DNA-binding capacity was elevated without any relationship to SLE disease activity. A fall in serum C3 levels was usually significant while serum C4 levels frequently fell without any change in clinical features of disease activity. Very low serum C4 levels (below 15 mg/dl), however, were usually of clinical relevance. The uses of immunological measurements in the differential diagnosis of a major illness in a patient with SLE are discussed. The finding of such an illness in a patient with normal serum DNA-binding levels made it unlikely that the illness was due to an exacerbation of the SLE and more likely that an alternative cause such as supervening bacteraemia was responsible.

Antibodies↗

Extrahepatic secreted complement C3 contributes to circulating C3 levels in humans.

The majority of complement protein C3 is synthesized by the liver, but many other cell types produce small amounts of functionally active C3. The overall contribution of such extrahepatic C3 production to the total circulating C3 level is unknown. Bone marrow and extrahepatic C3 productions were quantified in bone marrow transplant (BMT) and liver transplant (LT) recipients, respectively, where a mismatch for the C3 allotypes distinguished by the mAb HAV 4-1 had occurred. In the BMT group, donor-derived C3 was detected by ELISA and immunoblotting techniques. It contributed to 0.1 to 2.6% of the total circulating C3 during the immediate post-BMT period in response to inflammatory stimuli. Cell culture and immunostaining techniques demonstrated that monocytes were the source of the C3. By 6 wk following BMT, donor-derived C3 levels had decreased to below the detection limit of the assays. By contrast in the LT group, total extrahepatic C3 levels were higher (3.1-5.7% of the total circulating C3) and remained stable for up to 1 yr post-LT. This study demonstrates that extrahepatic derived C3 forms a larger proportion of the circulating C3 levels than was considered previously and that in the resting state, most of this extrahepatically derived C3 comes from nonmyeloid sources. In addition, monocytes, which in the resting state contribute negligible amounts of C3, have the potential when stimulated to contribute significantly to the total systemic C3 pool. These findings highlight the importance of locally secreted complement proteins.

Antibodies, Monoclonal↗

Complement C3 and immunoglobulin in inflammatory acne vulgaris.

In patients with moderate to severe inflammatory acne, complement (C3) was detected by immunofluorescence in sixteen early inflammatory acne lesions but in only one of thirteen biopsies of non-inflamed skin from acne sites. C3 deposition occurred particularly in the walls of small dermal blood vessels and at the dermo-epidermal junction. IgM was identified in vessel walls in four of sixteen early lesions. In eight late inflammatory lesions C3 deposition was much less prominent and was present in vessel walls in only two. None of the late lesions showed vascular deposition of IgM. The observations indicate that complement activation occurs in inflammatory acne and it is suggested that this may play a pathogenic role in the inflammation.

Acne Vulgaris↗

Estimation of postmortem interval based on the third component of complement (C3) cleavage.

To estimate postmortem interval (PMI), the spontaneous conversion of the native third component of complement (C3) to its derived fragments in whole blood was studied by crossed immunoelectrophoresis. C3 cleavages in vitro at different temperatures showed that the incubation of whole blood at a higher temperature led to a faster conversion of beta 1C (native C3) to beta 1A (C3c). In cadaveric blood, we found a significant positive correlation between percentage of C3 cleavage and PMI. From these results, it is possible to estimate PMI from the ratios of C3 cleavage.

Body Temperature↗

Inherited complement C3 deficiency: reduced C3 mRNA and protein levels in a Laotian kindred.

To determine the molecular basis of complement C3 deficiency in a Laotian kindred, the homozygous C3-deficient male propositus was studied. By ELISA, this individual's serum was determined to contain approximately 4 microg/ml C3 (0.3% of normal). In accord with this result, anti-C3 immunoprecipitation of [35S]-methionine-labeled fibroblasts from this C3D individual revealed pro-C3 of normal size (180,000 Mr), but in significantly reduced amounts (approximately 1% of normal fibroblasts), that was processed and secreted with normal-size alpha- and beta-chains. In addition, C3-specific mRNA of normal size (5.2 kb) but in reduced quantity (approximately 1% of normal) was detected in this individual's fibroblasts by Northern analysis. The nucleotide sequence of the transcriptional initiation site, the promoter, and the IL-1beta/IL-6 cis-regulatory elements of the C3-deficient gene are normal in this C3-deficient individual, indicating that the low C3 mRNA and protein levels are not caused by reduced C3 transcription that is the result of a cis-mutation. Moreover, cDNA sequencing studies revealed no defect in the C3-deficient mRNA, including the areas mutated in four previously characterized C3-deficient patients. These data indicate that (1) C3 protein deficiency in this Laotian patient results from reduced levels of C3-specific mRNA, (2) the small amount of expressed C3 protein is processed and secreted normally from the deficient cells, and (3) the molecular genetic defect(s), although not yet delineated, is different from those described in other C3-deficient individuals, thereby providing additional evidence for numerous mutations that cause inherited C3 deficiency in humans.

Cells, Cultured↗

Characterization of complement C3 as a glycyrrhizin (GL)-binding protein and the phosphorylation of C3alpha by CK-2, which is potently inhibited by GL and glycyrrhetinic acid in vitro.

The physiological interaction between glycyrrhizin (GL) and serum complement C3, and the inhibitory effects of GL, glycyrrhetinic acid (GA), and a GA derivative (oGA) on the phosphorylation of C3 by casein kinase 2 (CK-2), were investigated in vitro. C3 was found to be a GL-binding protein (gbP), because (i) of its high affinity for a GL-affinity HPLC column; and (ii) both GL and GA induce conformational changes in C3. At least four trypsin-resistant fragments (p30, p25, p18, and p15) were detected when the (32)P-labeled C3alpha was digested with trypsin in the presence of 100 micro M GA. Two of these (p25 and p15) were immuno-precipitated with anti-C3a serum. Furthermore, it was found that C3a contains GL-binding domains, because (i) C3a (anaphylatoxin) could be selectively purified from the synovial fluids of patients with rheumatoid arthritis by GL-affinity column chromatography (HPLC); and (ii) purified human C3a has a high affinity for a GL-affinity column. In addition, C3alpha (p115) of C3 was effectively phosphorylated by CK-2 in the presence of poly-Arg (a CK-2 activator) in vitro. This phosphorylation was completely inhibited by 10 micro M oGA, 30 micro M GA, or 100 micro M GL. Taken together, these results suggest that the GL-induced inhibition of the physiological activities of C3a and C3alpha may be involved in the anti-inflammatory effect of GL in vivo.

Arthritis↗

A model system for the study of the assembly and regulation of human complement C3 convertase (classical pathway).

The formation of classical C3 convertase of complement and its regulation by C4b-binding protein (C4bp) were studied using two different approaches: (a) the analysis was first carried out in fluid phase; a soluble stabilized C3 proconvertase could be assembled from C4b (or C4b-like C4) and iodine-treated C2 in the presence of Ni2+ ions. Upon activation of this complex by C1s, a C3 convertase C4b(C4b-like C4)-C2a was generated which was able to cleave purified C3. C4bp dissociated both C3 proconvertase and C3 convertase, but its effect was more important on C3 convertase. (b) A model system of phospholipid vesicles has been developed to study the assembly of the C3 convertase on a membrane. Among different phospholipid mixtures tested, P-glycerol/P-choline vesicles were found most effective for C4b binding. Optimal conditions were determined for C4b fixation on these vesicles; bound C4b participated in the formation of a functional membrane-associated C3 convertase. C4bp was found to bind to phospholipid vesicles with a higher affinity than C4b; it was able to dissociate the vesicle-associated C3 convertase.

Buffers↗

[Partial lipodystrophy with C3 complement deficiency in 8 years old girl].

Lipodystrophy is a rare, heterogenic disorder, leading to the loss of adipose tissue. Several main types of the disease are distinguished according to age of onset and localization of fat atrophy. The authors present the case of 8 years old girl with partial lipodystrophy, C3 complement deficiency and autoimmunologic disorder.

Brain↗

Changes in conglutinin, immunoconglutinin, complement C3 and fibronectin concentrations in cattle acutely infected with Babesia bovis.

Further pathophysiological studies of the hypotensive shock syndrome associated with acute Babesia bovis infection in cattle have demonstrated changes in the plasma concentrations of conglutinin, complement C3, fibronectin and immunoconglutinin. Decreases in the first three components are presumably due to their contribution towards intravascular sludging of infected erythrocytes within the microvascular system during the acute phase of infection. In contrast, the increase in fibronectin and immunoconglutinin in the recovery phase suggests the involvement of these proteins in the reticulo-endothelial clearance of infected or damaged erythrocytes, immune complexes and cryofibrinogen.

Acute Disease↗

Acute induction of interleukin-6 and biphasic changes of serum complement C3 by carrageenan in mice.

Carrageenan has been widely used as an irritant to evoke inflammation in animals or to selectively deplete macrophages in vivo. Although precise understanding of carrageenan activity is a prerequisite for the experimental use of this polysaccharide, the effects of this agent on host-biological systems are still poorly understood. We investigated the effect of carrageenan on serum concentrations of complement C3 and interleukin (IL)-6, a potent complement-inducing factor. Intraperitoneal administration of carrageenan (4 mg) in mice resulted in an initial fall in serum C3 (70% of control, P < 0.05) between 3 and 6 h, but was followed by a significant rise (180% of the control, P < 0.05) at 24h. Prior to the rise in complement C3, a sharp peak of serum IL-6 was observed at 6h after carrageenan treatment. These results indicate potential of carrageenan to enhance host complement systems, which may be associated with, at least in part, an acute induction of IL-6.

Animals↗

Pathways of complement (C3) activation in rabbits infected with Trypanosoma evansi.

Immunoelectrophoresis was used to evaluate the pathways of complement (C3) activation with sera from rabbits infected with Trypanosoma evansi. C3 activation via the classical pathway was of a higher magnitude than via the alternative pathway using both the intact parasite and soluble parasite antigens. This observation indicates that although the two pathways may be activated during infection, the activation of the classical pathway may be dominant in a host's response to a T. evansi infection during both the early and late stages of the disease.

Animals↗

Adiponectin, acylation stimulating protein and complement C3 are altered in obesity in very young children.

OBJECTIVE: Childhood obesity is increasing worldwide. This, in turn, is associated with chronic disease risk factors including hyperlipidaemia. The aim of the present study was to examine adiponectin, acylation-stimulating protein (ASP) and its precursor, complement C3, in very young obese and nonobese children and the corresponding associations with plasma lipid and lipoproteins. All three adipose tissue secreted factors are involved in fat metabolism, and little is known of the levels in very young children. DESIGN AND METHODS: A total of 124 healthy children from 2 to 6 years old were evaluated for weight, height, plasma lipids and adipokines. Based on percentage ideal body weight (%IBW), 60 children were nonobese and 64 were overweight/obese. RESULTS: ASP and C3 were significantly increased (P = 0.0002 and P < 0.0001, respectively) in obese vs. nonobese, and this pattern held true when separated in three age groups: toddler (2-3 years), preschool (4-5 years) and primary school (6 years). By contrast, adiponectin was significantly decreased (P = 0.04). When separated based on a positive family history of obesity, ASP was increased (P = 0.005). Other than a small (23.4%) increase in plasma triglyceride, all other lipids [cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and nonesterified fatty acids] were normal. ASP, C3 and adiponectin were strongly correlated with %IBW (r = 0.515, P < 0.0001; r = 0.383, P < 0.0001; r = -0.211, P = 0.03, respectively). CONCLUSIONS: Changes in plasma adipokines are seen in very young obese children in the absence of lipid changes. These changes in ASP, C3 and adiponectin in very young obese children may predispose towards enhanced fat storage (ASP) and decreased fat oxidation (adiponectin) further driving the obesity profile.

Adiponectin↗

Immunoglobulin (IgG and IgA) and complement (C3) concentrations in uterine secretion following an intrauterine challenge of Streptococcus zooepidemicus in mares susceptible to versus resistant to chronic uterine infection.

The validity of measuring concentrations of immunoglobulins in undiluted uterine secretions was established. The concentrations of IgG, IgA, and cleavage factor C3 of the complement system in uterine secretions were compared in mares with different resistance to chronic uterine infection (CUI). The uteri of mares susceptible (n = 6) and resistant to CUI (n = 5) were inoculated with 5 x 10(6) Streptococcus zooepidemicus when the mares were in estrus. Uterine secretions were sampled, and sampling was immediately followed by a uterine lavage at 5 and 24 h after bacterial inoculation. During a subsequent estrus, bacterial inoculation of the uterus was repeated, and samples were taken from the mares 12 and 36 h later. The fraction of uterine secretion in each uterine washing was determined by dividing the protein concentration of the undiluted uterine secretion by the protein concentration of the corresponding uterine washing. There was a significant correlation between measured concentrations of immunoglobulins in uterine secretions and calculated concentrations in the uterine washings (p < 0.05). Concentrations of IgG and C3 in uterine secretions declined similarly in both susceptible and resistant mares during the first 24 h after bacterial inoculation (p < 0.04). In contrast to the susceptible group, which showed a continuous decline at 36 h, resistant mares demonstrated an increased concentration of both IgG and C3 at this time. Concentrations of IgA did not differ between susceptible and resistant mares. It was concluded that the described method of sampling uterine secretions was useful for analyzing IgG and IgA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC).

C3(H2O), an inactive form of C3 present to a variable extent in most C3 preparations, has been isolated in 40 min from previously purified C3 using FPLC ion exchange chromatography on a Mono Q column. As many as six peaks were obtained from some C3 preparations, corresponding to different molecular forms of the protein. One of these peaks consisted of a molecular form of C3 with intact alpha and beta chains, a free sulfhydryl group but no hemolytic activity and was identified as C3(H2O). C3(H2O) eluted as a homogeneous peak well resolved from native C3, C3b, high molecular weight aggregates and small degradation fragments. The same C3(H2O) peak was generated from native C3 by repeated freeze-thaw cycles or NH2OH treatment. C3(H2O) alpha chain appeared as a doublet about 2 kDa heavier than native C3 alpha chain in low cross-linked gels. Two forms of C3b could be separated on the Mono S column, both able to form the C3 convertase. The present report describes a very fast method to resolve and isolate to homogeneity C3(H2O) and native C3 from C3 preparations. Both molecular forms of C3 are very suitable for studies of the initial and amplification C3 convertases of the alternative pathway of complement.

Chromatography, High Pressure Liquid↗

Complement C3 proteins in psoriasis.

A new polymorphism of the complement factor C3 in human plasma was demonstrated by isotachophoresis in agarose gels followed by immunodetection with rabbit anti-human C3c and C3d immunoglobulins. Four bands were detected in the immunoprint of freshly drawn EDTA-plasma, which were C3s1, C3s2, C3f1 and C3f2. At least four additional C3 components in Mg2+ -zymosan activated plasma were present, which were C3b1 to C3b4. The different forms of C3 in frozen and thawed heparin-plasma from 20 patients with psoriasis and 20 healthy individuals were studied from the immunoprint. The total content of C3 components was 29% greater in the patients with psoriasis than controls. The major difference was in the C3b components which were increased by 46%. In psoriatic patients, the two slow C3 components C3s1 and C3s2 were increased by 24 and 56% respectively, when compared with controls. The two fast C3 components C3f1 and C3f2 were decreased to 29 and 37%. The results suggest a direct involvement of the complement factor C3 in psoriasis.

Adult↗

Platelet-bound complement (C3) in immune thrombocytopenia.

The fixation of complement to the circulating platelet in immune thrombocytopenia was detected by measurement of one of the complement components, C3, on the surface of platelets from patients with idiopathic thrombocytopenic purpura (ITP) and systemic lupus erythematosus (SLE) using the anti-C3 consumption assay. The surface IgG was determined simultaneously using the previously described anti-IgG consumption assay. Washed platelets from normal controls had 3.5 fg (10(-15) g) of C3, or about 11,000 molecules, per platelet, an amount comparable to the IgG (4.1 FG, or 15,000 molecules, per platelet). For most patients with ITP both C3 and IgG were increased on the platelet surface, although for 5 of 16 patients only IgG was increased. Two patients with SLE and thrombocytopenia had an increase in both C3 and Ig, six patients with SLE who were not thrombocytopenic had normal amounts of membrane-bound C3 and IgG. In 5 patients, 3 with ITP and 2 with collagen vascular disease, both surface immunoproteins decreased with successful treatment of the thrombocytopenia.

Blood Platelets↗

Complement (C3)-receptor-mediated phagocytosis of agarose beads by mouse macrophages. II. Extracellular activation of macrophage-derived complement on agarose via the alternative pathway.

Phagocytosis of agarose beads by macrophages cultured under serum-free conditions was studied. 48 h was needed before a plateau in the uptake was reached. The ingested agarose beads were coated extracellularly with macrophage-derived protein before attachment and ingestion of the beads. Intracellularly, the agarose-linked protein was removed from the agarose. If the ingested agarose beads were extracted from the macrophages within 24 h after the plateau in the uptake was reached, a fraction of the beads could attach to new macrophages, demonstrating modification of the agarose beads by opsonin(s). Because of binding of anti-human C3c antibodies to beads extracted from the macrophages after 24 h of phagocytosis and the trypsin sensitivity of the protein on the agarose, we conclude that the main opsonin on the agarose beads is C3bi. Requirements for the stimulatory effect of agarose on macrophages are summarized.

Animals↗