Alternative pathway of complement.
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Animals↗
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Evasion of alternative complement pathway by Trypanosoma cruzi results from inefficient binding of factor B.
During its differentiation in the insect vector to a stage infective for the mammalian host, Trypanosoma cruzi becomes resistant to lysis by the alternative pathway of complement. To elucidate the mechanism of complement evasion, we studied control of complement activation on the surface of the noninfective epimastigote and the infective culture-derived metacyclic trypomastigote stages (CMT) of T. cruzi. It was found that the predominant form of complement component C3 on epimastigotes is C3b, whereas the majority of C3 on CMT is in the form of the hemolytically inactive fragment iC3b, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Our results also showed that C3 binds by a covalent ester linkage to surface molecules of different molecular weight in the epimastigote stage and CMT. Binding studies with purified complement components indicated that CMT do not support efficient formation of an alternative pathway C3 convertase. C3b on the parasite surface fails to bind the amplification component, factor B, rather than showing enhanced binding of the control component, factor H. These results identify the biochemical basis for evasion of complement-mediated killing in T. cruzi and reveal a mechanism for developmental regulation of complement activation.
Animals↗
Inherited complement deficiency states: implications for immunity and immunological disease.
The study of complement deficiency states and their influence on immune function has generated new insights and still provides a challenge to continued investigation. The association of classical pathway deficiencies (C1, C4, C2 or C3) with immunological diseases such as SLE and glomerulonephritis has contributed to current knowledge concerning complement-dependent immune complex handling and elimination. Susceptibility to systemic infection with encapsulated bacteria is encountered in most forms of inherited complement deficiency. Recurrent neisserial infection is the only clinical manifestation clearly associated with defects of the membranolytic sequence C5-C9, while deficiency of properdin, a component of the alternative activation pathway, appears to predispose to nonrecurrent meningococcal disease. Inherited complement deficiency is rare, but the perspective is widened by the more common occurence of acquired defects in immunological diseases, and the apparent requirement for efficient complement recruitment in host defense. Another aspect is the possibility that complement deficiency might alleviate or prevent inflammatory symptoms. Notably, complement deficiency has not been reported in classical rheumatoid arthritis. Considerations of this kind would be refuted or modified by findings of complement deficiency in single patients.
Animals↗
Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.
Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with cryptococci opsonized with the reconstituted pathway compared with that observed when yeast cells were opsonized with normal serum.
Complement Activation↗
Adipsin and an endogenous pathway of complement from adipose cells.
The alternative complement pathway is best known for its role in humoral suppression of infectious agents. We have previously shown that adipose cells synthesize adipsin, the mouse homolog of human complement factor D, and that the synthesis of this protein is reduced in several rodent models of obesity. We show here that adipose cells and adipose tissue also synthesize two other essential components of the alternative pathway of complement, factors C3 and B, and activate the proximal portion of this pathway. This activation occurs in the absence of infectious agents and without triggering the terminal, lytic part of this pathway. We demonstrate the production in vitro of several polypeptides characteristic of complement activation that are known to have potent biological activities, including the anaphylatoxin C3a. Cultured adipocytes require stimulation with cytokines to activate complement, while explanted adipose tissue has no such requirement. The adipose tissue from obese mice is deficient in this localized activation of the alternative pathway. These results indicate that complement activation occurs in a localized site, adipose tissue, in normal mice and is impaired in a state of metabolic dysfunction. This suggests a novel function for the proximal portion of this complement pathway related to adipose cell biology or energy balance.
3T3 Cells↗
Human factor D of the alternative complement pathway. Physicochemical characteristics and N-terminal amino acid sequence.
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Amino Acid Sequence↗
Serum levels of the soluble interleukin-2 receptor are dependent on the kidney function.
We have investigated the influence of kidney function on soluble interleukin-2 receptor (S-IL-2R) serum levels. S-IL-2R is a glycoprotein with a molecular weight of 45 kDa. The serum concentration of S-IL-2R was positively correlated to serum creatinine (r = 0.35) and complement factor D (r = 0.30), a low molecular weight protein, and the reciprocal value of S-IL-2R was negatively correlated to 51Cr-EDTA clearance (r = -0.35). When measuring S-IL-2R and factor D in a wide range of serum creatinine (140-1,380 mumol/l), a high positive correlation was found between factor D and serum creatinine. S-IL-2R and serum creatinine had r = 0.92 and r = 0.79, p = 0.0001, respectively. The correlation coefficient between factor D and S-IL-2R was 0.86. S-IL-2R is used as a marker for an increased activity of the immune system and in a variety of lymphoproliferative disorders. However, it is important to keep in mind that a reduced kidney function may contribute to the increased S-IL-2R levels seen in different immune diseases. Therefore, renal function should be taken into account in the interpretation of elevated plasma concentrations of S-IL-2R.
Complement Factor D↗
Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes.
The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.
Adipose Tissue↗
Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA.
The mouse adipsin gene encodes a serine protease with complement factor D activity that is expressed during adipocyte differentiation and is deficient in several animal models of obesity. We have investigated the regulation of adipsin expression by transfecting preadipocytes and adipocytes with plasmids containing the 5'-flanking region of the adipsin gene linked to a reporter gene. Constructions containing a -950 to +35 segment of the adipsin promoter were preferentially expressed in adipose cells. Deletion experiments identified a region from -114 to -38 which contains a large inverted repeat sequence and negatively regulated gene expression in preadipocytes and positively regulated expression in fat cells. Exonuclease III protection and gel retardation assays indicated that this region of duplex DNA had multiple binding sites for nuclear factors, several of which were preadipose specific. In addition, we also identified two distinct factors that bound symmetrically and sequence specifically to the inverted repeat sequences only when they were in single-stranded form; one of these factors was induced during adipocyte differentiation. These results suggest that the control of the adipsin promoter in differentiation may involve an interplay of multiple regulated DNA-binding proteins, including two that have preferential affinity for single-stranded DNA.
Adipose Tissue↗
Differentiation dependent biphasic regulation of adipsin gene expression by insulin and insulin-like growth factor-1 in 3T3-F442A adipocytes.
Adipsin is a serine protease with complement factor D activity that is synthesized by adipocytes and secreted into the blood stream. Expression of adipsin is deficient in models of genetic (ob/ob, db/db) and acquired (monosodium glutamate-lesioned) obesity, but the cellular mechanisms responsible for this deficiency are unknown. Because hyperinsulinemia is frequently associated with obesity, we evaluated the effects of this hormone and insulin-like growth factor 1 (IGF-1) on adipsin secretion and adipsin messenger RNA (mRNA) levels in 3T3-F442A adipocytes. In the present study, we report that in fully differentiated adipocytes (after 11 days post confluence), insulin exposure progressively decreases adipsin secretion by 40%, 67%, and 78% after 2, 4, and 6 days of treatment. The inhibition of adipsin secretion by insulin is the result of a corresponding decrease in adipsin mRNA and is specific since two other differentiation-dependent fat cell mRNAs encoding aP2 (a fatty acid binding protein) and glycerophosphate dehydrogenase (GPD), are unaffected. Insulin suppresses adipsin gene expression via high affinity insulin receptors, because physiological levels of insulin produce this effect, and dose-response curves for insulin stimulation of 2-deoxyglucose uptake and glucose utilization are similar to insulin's effect on adipsin. In contrast, insulin when present during days 1-8 post confluence (during differentiation) markedly increases adipsin secretion and adipsin mRNA levels. This stimulation is due to the ability of insulin to accelerate differentiation as evidenced by corresponding increases in aP2 and GPD mRNAs as well. Insulin and IGF-1 are equipotent in this effect, suggesting that both insulin and IGF-1 receptors can mediate this response. In summary, during the differentiation of 3T3-F442A adipocytes, insulin stimulates adipsin gene expression by accelerating differentiation. As the cells become mature adipocytes, they acquire some differentiation-dependent factor, which couples insulin receptor stimulation to inhibition of adipsin gene expression. This model should aid our search for the molecular links between insulin receptor stimulation and altered gene expression.
Adipose Tissue↗
Activation of factor B and C3 in guinea pig serum by zymosan or by activated properdin depends on the presence of factor D.
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Animals↗
Initiation of the alternative pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway from six isolated proteins.
An intact alternative pathway of complement activation was assembled from six isolated proteins present at their respective physiological concentrations (C3, 1200 microgram/ml: factor B, 200 microgram/ml; factor D, 2 microgram/ml; beta1H, 560 microgram/ml; C3b inactivator, 34 microgram/ml; and native properdin, 20 microgram/ml). Initiation of the pathway required the presence of five of these proteins not including properdin. The initial C3 convertase of the system was shown to be a fluid-phase rather than a surface-bound enzyme. The ability of the pathway to discriminate between activator and nonactivator was found to reside in the bound C3b molecule. When bound to the surface of an activator through its labile binding site, C3b interacts with surface structures of the activator through another site on the molecule. This interaction results in diminished beta1H binding to C3b and thereby allows the bound C3b molecule to escape control and participate in C3 convertase formation. Thus, initiation of the alternative pathway is a two-step process, the first being non-specific and the second being discriminatory.
Binding Sites↗
Fluid-phase activation of the alternative pathway of complement by excess factor D in regularly dialyzed patients.
We examined the effect of excess factor D on the alternative pathway of complement (APC). First, we demonstrated that the production of C3a is accelerated in the fluid-phase with the addition of purified factor D. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions showed that the serum iC3b level was elevated when incubated with excess factor D. Secondly, we demonstrated, by measuring the C5a-des-Arg level, that the generation of C5a was promoted in the fluid-phase with the addition of purified factor D. We then studied whether activation of APC is elevated in the blood of patients on maintenance hemodialysis whose sera contained a high concentration of factor D. First, we detected, by fluorescence activated cell sorter analysis, greater amounts of C3d on erythrocytes from the patients (mean fluorescence intensity +/- SD: 7.7 +/- 1.7 arbitrary units) than those from healthy individuals (5.4 +/- 0.5 arbitrary units; p less than 0.001). Secondly, serum C3 level was significantly lower (p less than 0.001) in patients (mean +/- SD: 63.3 +/- 8.2 mg/dl) than in healthy individuals (84.8 +/- 9.5 mg/dl), whereas there was no difference in serum C4 level between patients (32.4 +/- 6.9 mg/dl) and healthy individuals (33.0 +/- 7.4 mg/dl). Serum C5 level was almost the same in patients (10.5 +/- 1.5 mg/dl) and in healthy individuals (11.2 +/- 1.3 mg/dl). These results provide supportive evidence of elevated APC activation in patients with high serum factor D.
Blood Circulation↗
Growth-supporting activity of fragment Ba of the human alternative complement pathway for activated murine B lymphocytes.
We have investigated the effects of cleavage of factor B by its activating enzyme, factor D, as well as its activation fragments Bb and Ba, on the growth of mouse spleen B lymphocytes preactivated by LPS. Neither factor B nor factor D show any growth-supporting activity when tested alone. The coaddition of factor B and factor D to serum-free cultures of LPS-preactivated B cell blasts increased the proliferation of the responding cells up to the level obtained by restimulation with LPS. Such growth-supporting activity was shown to be mediated by Ba, whereas Bb did not show any significant effect. Furthermore, this effect was not restricted to the LPS-preactivated B cell blasts; in fact, Ba also supported the growth of in vivo, activated B cell blasts of unprimed mice of the LPS-nonresponder C3H/HeJ strain. In contrast, Ba did not maintain growth of Con A-activated T cells or TCGF-dependent CTL cells. Taken together, these results describe the first biological activity of human Ba as a B cell stimulatory factor.
Animals↗
A reexamination of the role of magnesium in the human alternative pathway of complement.
The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.
Centrifugation, Density Gradient↗
Insoluble polyanions as activators of both pathways of complement.
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Complement C3↗
Complement activation in asymptomatic patients with sickle cell anaemia.
Previous reports have suggested that a defect in serum complement may contribute to the increased susceptibility to infection shown by patients with sickle cell anaemia (SCA). In order to define the nature of any complement abnormality in SCA, we investigated the complement system in eighty-seven patients during asymptomatic periods, and analysed factor B turnover in a small sample. In these patients geometric mean serum concentrations of functionally active factor B and factor D, and of C3 and C4 protein (expressed as a percentage of normal reference serum) wer lower than in controls (78% vs 107%, P less than 0.001, 86% vs 103%, P less than 0.001, 91% vs 100%, P less than 0.01, 89% vs 105%, P less than 0.05 respectively). The ratio of the serum concentration of functionally active factor B to factor B protein was lower in patients than in controls (means 75% s.d. 16% vs mean 93%, s.d. 22% P less than 0.001), indicating a functional deficiency of factor B protein. In addition, the fractional catabolic rate of radiolabelled factor B was markedly increased in four out of seven asymptomatic patients studied, and was inversely related to the functional factor B concentration in serum (r = -0.59, P less than 0.05); factor B synthesis was uniformly increased. Complement activation was not related to the presence of circulating C1q binding material. We conclude that complement activation, rather than defective synthesis as previously suggested, contributes to the abnormalities in complement componenet concentration and function in asymptomatic subjects with sickle cell anaemia.
Adolescent↗
Regulation of complement functional efficiency by histidine-rich glycoprotein.
The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.
Animals↗