Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.

Complement deficiency and disease: an update.

Complement deficiencies are probably vastly under-diagnosed within clinical medicine. Judging from a Swedish study of C2 deficiency, a deficiency with an estimated prevalence of about 1/20,000 in Western countries, less than 10% of the deficiencies of the classical and alternative pathways and the late complement components are identified in Sweden. C1 inhibitor deficiency and deficiencies of MBL and MASP-2 were not included in the assessment. The introduction of new screening methods should facilitate detection of complement deficiencies in clinical practice. In our study of C2 deficiency (n=40), 57% of the patients had a history of invasive infection with encapsulated bacteria, mainly Streptococcus pneumoniae. This emphasizes the importance of the classical and/or the lectin pathway in defence against severe infection. Rheumatological disease, mainly systemic lupus erythematosus was present in 43% of the patients. In addition, a significant association was found between C2 deficiency and atherosclerosis. Complement-dependent disease mechanisms are discussed together with the potential importance of non-complement genes for disease expression in complement deficiencies. Analysis of larger patient groups is required in order to establish guidelines for investigation and treatment of patients with complement deficiency.

Factor H binds to washed human platelets.

BACKGROUND: Factor H regulates the alternative pathway of complement. The protein has three heparin-binding sites, is synthesized primarily in the liver and copurifies from platelets with thrombospondin-1. Factor H mutations at the C-terminus are associated with atypical hemolytic uremic syndrome, a condition in which platelets are consumed. Objectives The aim of this study was to investigate if factor H interacts with platelets. METHODS: Binding of factor H, recombinant C- or N-terminus constructs and a C-terminus mutant to washed (plasma and complement-free) platelets was analyzed by flow cytometry. Binding of factor H and constructs to thrombospondin-1 was measured by surface plasmon resonance. RESULTS: Factor H bound to platelets in a dose-dependent manner. The major binding site was localized to the C-terminus. The interaction was partially blocked by heparin. Inhibition with anti-GPIIb/IIIa, or with fibrinogen, suggested that the platelet GPIIb/IIIa receptor is involved in factor H binding. Factor H binds to thrombospondin-1. Addition of thrombospondin-1 increased factor H binding to platelets. Factor H mutated at the C-terminus also bound to platelets, albeit to a significantly lesser degree. CONCLUSIONS: This study reports a novel property of factor H, i.e. binding to platelets, either directly via the GPIIb/IIIa receptor or indirectly via thrombospondin-1, in the absence of complement. Binding to platelets was mostly mediated by the C-terminal region of factor H and factor H mutated at the C-terminus exhibited reduced binding.

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Complement activation by Proteus mirabilis negatively charged lipopolysaccharides.

Proteus mirabilis strains are human pathogens responsible for urinary tract infections and bacteremias and may be involved in rheumatoid arthritis. Lipopolysaccharide (LPS, bacterial endotoxin), the major component of the cell wall, is one of the virulence factors of Proteus. In the presented studies, we have investigated complement activation by LPSs isolated from P. mirabilis O10, O23, O30, and O43 strains, which differ in the number of negative COO- groups on their polysaccharide components. Four P. mirabilis strains studied were sensitive to complement-mediated killing, despite complement binding by their LPSs. The optimal complement binding by LPSs was detected in serum with functional assays for both the classical and alternative pathways. Complement activation in 80% serum by the smooth, uronic acid, and hexosamine containing P. mirabilis LPSs was not critically determined by the structure of their O-chain polysaccharides. One of four LPSs used as a model, P. mirabilis O10 LPS, fragmented C3 in an LPS dose- and time-dependent manner. It was detected by crossed-immunoelectrophoresis and capture ELISA with anti-C3c antibodies. The lower complement activation by 023 LPS correlates with its reduced C3 fragmentation, compared with three other Proteus LPSs studied. Rabbit anti-O antibodies enhanced the complement binding and factor C3 fragmentation by O10, O23, O30, and O43 P. mirabilis LPSs.

New procedure for the detection of complement deficiency by ELISA. Analysis of activation pathways and circumvention of rheumatoid factor influence.

A procedure using enzyme-linked immunosorbent assays for the assessment of complement function has been evaluated. The sera investigated were incubated in microtiter plates with solid-phase complement activators. Human polyclonal IgG or monoclonal IgM were used for classical activation pathway assays and Salmonella typhosa lipopolysaccharide (LPS) for alternative activation pathway assays. The analysis focussed on deposition of C9 and properdin as detected with enzyme-conjugated antibodies. In an attempt to avoid spurious results due to rheumatoid factors in patient sera, monoclonal mouse and chicken antibodies were unsuccessfully tested as indicator reagents in the assay with solid-phase IgG. However, the use of solid-phase IgM as an activator completely circumvented the influence of rheumatoid factors. With solid-phase IgG or IgM, properdin deposition occurred in the absence of factor D. A combination of assays is suggested for diagnostic purposes: IgM-coated plates with detection of bound C9 and properdin for the classical pathway and LPS-coated plates with detection of bound properdin for the alternative pathway. The procedure distinguished between defects of the classical activation pathway (C1, C4, C2), the alternative activation pathway (C3, factor B, factor D, properdin) and the terminal components (C5-C9). This analytical approach may be useful for detection of inherited complement deficiency and the assessment of complement function in acquired complement deficiency states.

Trimer and tetramer complexes containing C1 esterase inhibitor, C1r and C1s, in serum and synovial fluid of patients with rheumatic disease.

During activation, the first component of complement C1q (C1r-C1s)2 is dissociated in conjunction with the formation of complexes containing C1 esterase inhibitor (C1-INH). Trimer complexes, with zymogen C1s associated with a firm C1-INH-C1r complex (C1-INH-C1r-C1s) can be distinguished from tetramer complexes C1-INH-C1r-C1s-C1-INH) in which C1-INH is firmly bound to both proteases. In the present study a two-stage electroimmunoassay was developed for the specific measurement of C1-INH-C1r-C1s. In the first step, C1-INH and its complexes were immunoprecipitated with anti-C1-INH during electrophoresis in the presence of Ca2+. In the second step, C1s contained in C1-INH-C1r-C1s was dissociated in the presence of EDTA and was measured by immunoprecipitation with anti-C1s. C1-INH-C1r-C1s were consistently found in normal sera. Normal sera did not contain C1-INH-C1r-C1s-C1-INH as assessed with a previously described ELISA procedure. Sera and synovial fluids from two groups of patients with inflammatory arthritis were investigated. In rheumatoid arthritis patients (n = 15) C1-INH-C1r-C1s complexes were usually found at high concentration both in serum and synovial fluid. C1-INH-C1r-C1s-C1-INH complexes were also present with values that were higher in synovial fluid than in serum, in accord with previous findings of classical pathway activation in the inflamed joints of the patients. Patients with spondylarthritic syndromes (n = 7) had serum and synovial fluid C1-INH-C1r-C1s concentrations that were comparable to those of the rheumatoid arthritis patients. If at all present, C1-INH-C1r-C1s-C1-INH were detected in trace amounts. Thus, C1 activation in patients with spondylarthritic syndromes appeared to be efficiently controlled at the C1r level. Distinguishing between C1-INH-C1r-C1s and C1-INH-C1r-C1s-C1-INH may prove of value in further studies of the activation and control of C1 in disease.

C1 activation and dissociation in disease.

The composition of complexes containing C1 inactivator (C1 IA), C1r and C1s was investigated in normal serum after activation of C1 under various conditions. Analyses were performed with PAGE of eluates from Sepharose beads coated with F(ab')2 fragments of anti C1s followed by immunoblotting with anti C1 IA, anti C1s or anti C1r. Eluates obtained from serum treated with aggregated IgG (AGG) contained C1 IA in complex with C1r and C1s with both subcomponents in activated form. Eluates from serum incubated at 37 degrees C for 1, 2 or 3 days without activators showed C1 IA complexed with activated C1r and with C1s in proenzyme state associated to the complex. On analysis of serum, treated as mentioned above, by a variant of the electroimmunoassay using an intermediate gel containing anti-C1 IA and with anti-C1s in the anodal gel the two types of C1r--C1s--C1 IA complexes could be distinguished. Investigation of fresh sera and synovial fluids from patients with rheumatoid arthritis in this assay showed complexes containing C1 IA and C1r-C1s in activated form in the synovial fluids, while C1 IA-activated C1r-proenzyme C1s complexes were found in the corresponding sera.

Influence of factor D concentrations on fluid phase C3 activation, lysis of rabbit erythrocytes and solubilization of immune complexes.

Factor D depleted serum did not support hemolysis of rabbit erythrocytes and solubilization of performed immune complexes. Fluid phase C3 cleavage increased in a dose dependent manner when D protein was added to normal or to factor D depleted serum. During short incubation factor D concentrations were correlated with the capacity of serum to solubilize immune complexes and to lyse rabbit erythrocytes. With prolonged incubation, the hemolytic activity decreased in a factor D dose dependent manner. This was probably due to fluid phase breakdown of C3 and factor B in the presence of high factor D concentrations. Hypocomplementemic sera from patients with active systemic lupus erythematosus (SLE) did not support solubilization of bovine serum albumin (BSA) anti-BSA complexes when factor D was added in excess. Patients with polycystic kidney disease with reduced renal function and high factor D concentrations showed increased concentrations of circulating C3d/dg fragments. The possibility was considered that high factor D concentrations in uremia might promote fluid phase C3 degradation and thereby limit the in vivo efficiency of alternative pathway activation on target surfaces.

Characterization of non-expressed C4 genes in a case of complete C4 deficiency: identification of a novel point mutation leading to a premature stop codon.

The genetic basis of complete C4 deficiency in a patient with SLE was investigated. Previous studies have demonstrated that this patient has two different major histocompatibility complex (MHC) haplotypes that each contain a major deletion and a non-expressed C4 gene. In the present study, non-expression of the C4 genes was explained by the finding of two distinct C4 gene mutations. A previously described two base pair insertion in exon 29 of the C4 gene was detected in the paternal MHC haplotype [HLA-A2, B40, SC00, DR6]. The maternal haplotype [HLA-A30, B18, F1C00, DR3] carried a C4 gene with a one base pair deletion in exon 20 generating a premature stop codon. This mutation was neither found in 10 individuals with known non-expressed C4 genes nor in 9 individuals homozygous for the complotype F1C30. The isotype and allotype specific regions of the patient's C4 genes were sequenced, and both contained C4A3a sequence. In conclusion, two different MHC haplotypes resembling the extended haplotypes [HLA-A2, B40, SC02, DR6] and [HLA-A30, B18, F1C30, DR3] both contained a non-expressed C4A gene that was due to either of two distinct mutations, demonstrating the heterogeneous genetic background of C4 deficiency.

Complement activation in patients with primary Sjögren's syndrome: an indicator of systemic disease.

Sixteen patients with primary Sjögren's syndrome, verified according to the Copenhagen criteria, were investigated for evidence of complement activation. Thirteen of the patients had intact functional activity of both the classical and alternative pathways, with normal concentrations of the complement proteins C1q, C1s, C3, C4 and the complement protein fragments C2a and C3d in the circulation. In contrast, three patients showed clear evidence of complement activation. Further investigation of these patients revealed manifestations of glomerulonephritis, vasculitis and primary biliary cirrhosis. Six months later, one patient developed a malignant non-Hodgkin lymphoma. We conclude that complement activation is generally not associated with primary Sjögren's syndrome. Evidence of complement activation in patients considered to have primary Sjögren's syndrome should raise the suspicion of concomitant systemic disease and/or extraglandular activity.

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.

Granulocyte functions and Neisseria meningitidis: influence of properdin-deficient serum.

Granulocyte-mediated reactions such as opsonization, chemotaxis, and release of granulocyte myeloperoxidase and lactoferrin were studied in properdin-deficient and normal human serum incubated with serogroup A and W-135 meningococci. There were no differences between the sera when serogroup A meningococci were studied. Opsonic and chemotactic activity were impaired against serogroup W-135 meningococci in properdin-deficient serum. Restitution with properdin restored both activities. We found similar release of myeloperoxidase and lactoferrin from granulocytes challenged with serogroup A or W-135 meningococci in either sera. These findings are in accordance with the clinical observations of meningococcal infections caused by serogroup W-135 in properdin-deficient patients as well as the absence of infections caused by serogroup A meningococci.

Normal human serum depleted of C1q, factor D and properdin: its use in studies of complement activation.

Normal human sera were depleted of C1q, factor D (D) and properdin (P) by a simple and reproducible procedure providing reagents for analysis of complement-dependent functions. Classical pathway activity was restored with purified C1q, and alternative pathway activity with purified D and P. Since both activation pathways were abolished, antibodies and other components could be removed without loss of complement activity during immunoabsorption procedures. Synergism between the two pathways during haemolysis of rabbit erythrocytes was clearly demonstrated, and was also found on analysis of C3 cleavage in serum incubated with other alternative pathway activators such as zymosan and inulin. Experiments with a Neisseria meningitidis serogroup W-135 strain isolated from a patient with inherited P deficiency showed that both pathways were capable of supporting antibody-dependent killing of the bacteria in serum. The alternative pathway was possibly more efficient than the classical pathway in the assay system. In C1q,D,P-depleted serum with high concentrations of anticapsular IgG antibodies, the addition of D alone resulted in efficient alternative pathway-mediated killing. The alternative pathway was equally efficient in a C1q,D,P-depleted serum with low concentrations of anticapsular antibody, but in this case the reaction required both D and P.

Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins.

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components. 55 kDa and 43-40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.

Kinetic analysis of immune complex solubilization: complement function in relation to disease activity in SLE.

Solubilization of preformed bovine serum albumin (BSA) rabbit anti-BSA complexes in serum with kinetic analysis, haemolytic complement function, complement proteins C1q, C4, C3 and complexes containing C1 inhibitor (C1 INH-C1r-C1s-C1 INH) were serially investigated in relation to disease activity in 25 patients with systemic lupus erythematosus (SLE). Clinical assessment of disease activity was expressed using a validated global index (SLEDAI). Markedly decreased capacity to solubilize immune complexes in serum was mainly found in sever disease. By serial analysis, evidence of fairly persistently impaired classical pathway function was found in most of the patients. In partial contrast, impaired alternative pathway function was more clearly associated with active severe disease. Immune complex solubilization during short incubation (5-10 minutes) correlated with classical and alternative pathway-mediated haemolysis. Solubilization during long incubation (40 minutes) was correlated with haemolytic alternative pathway function. In some patients gradual impairment of solubilization during short incubation, and reduced classical pathway haemolytic activity were detectable 2-4 months before clinical manifestations prompted therapeutical intervention. SLEDAI was negatively correlated with solubilization during prolonged incubation (40 minutes) and with haemolytic alternative pathway function, further emphasizing involvement of the alternative pathway in severe disease. The findings emphasize the importance of impaired complement function due to complement activation in SLE. Assays for immune complex solubilization or other complement functions appear to be useful for monitoring disease activity in SLE.

Complement activating rheumatoid factors in rheumatoid arthritis studied by haemolysis in gel: relation to antibody class and response to treatment with podophyllotoxin derivatives.

Complement (C) activating rheumatoid factors (RF) were measured in 16 patients with rheumatoid arthritis (RA) by a simple haemolysis in gel (HIG) assay. IgM-RF, but not IgG-RF or IgA-RF, measured by an enzyme linked immuno-sorbent assay, were closely correlated with C activating RF (r = 0.86). In neither assay did the concentrations of RF appear to be directly related to C1 activation as expressed by serum concentrations of C1r-C1s-C1 inactivator complexes or of C4. In the sera, C1q binding substances, measured by C1q binding assay, were markedly correlated with C activating RF (r = 0.88), whereas C1q binding substances detected by the C1q deviation test were not. Treatment with podophyllotoxin derivatives for 6 months clearly reduced patients' RF concentrations. The decrease of IgG-RF, IgA-RF and IgM-RF was significantly more pronounced than that of the IgG, IgA and IgM concentrations, respectively.

Prospective analysis of C1 dissociation and complement activation in patients with systemic lupus erythematosus.

OBJECTIVE: To evaluate the results of complement analysis for assessment of disease activity and severity, and prediction of flares in systemic lupus erythematosus (SLE). METHODS: Patients with mild extra-renal flares, severe extra-renal flares or flares of lupus glomerulonephritis were followed for eight months, with investigations being performed every second month. Findings in initial samples four months before the flares were compared with findings in a control group with stable disease. C-reactive protein, and circulating C1q, C4 and C3 were determined together with two types of complexes containing C1 inhibitor (C1 INH), C1 INH-C1r-C1s and C1 INH-C1r-C1s-C1 INH, and the C3 breakdown product C3d. RESULTS: Enhanced formation of C1 INH-C1r-C1s appeared to be a marker of low specificity and was mainly seen in patients with extra-renal disease. Concentrations of C1 INH-C1r-C1s-C1 INH, C3d, C1q and C3 clearly varied according to disease activity in patients with severe disease. Interestingly, high C1 INH-C1r-C1s-C1 INH values were found four months before the flares in all but one patient with lupus glomerulonephritis. Assessment of the relative predictivity for a subsequent flare indicated low C1q to be the most reliable marker, the predictivity of the complexes being: low C1q > high C1 INH-C1r-C1s-C1 INH > low C3 > high C3d > low C4. CONCLUSION: The importance of C1q and C1-related events in SLE may be underestimated. In addition, our results demonstrate the relevance of serial complement analysis for the assessment of disease activity and severity.

C4 phenotypes in IgA nephropathy: disease progression associated with C4A deficiency but not with C4 isotype concentrations.

IgA nephropathy (IgAN) is a common glomerular disease and is thought to have an immunological origin which may involve complement-mediated pathogenic mechanisms. We performed C4 phenotyping and C4 isotype quantification in 93 IgAN patients in Southern Sweden. Phenotype frequencies did not deviate from those of healthy controls. However, three patients had homozygous C4A deficiency and these all belonged to a group of fifteen patients with end-stage renal failure (p < 0.0035). Progression to end-stage renal failure did not appear to be faster than in other IgAN patients. Both C4A and C4B concentrations were raised in the IgAN patients, but the C4A/C4B concentration ratios did not deviate from those of healthy controls. This indicated that heterozygosity for C4A or C4B deficiency or other reasons for the relatively low concentrations of the protein were not associated with disease susceptibility. There was no correlation between low C4A/C4B ratio and poor prognosis. In conclusion, the findings suggested that homozygous C4A deficiency predisposes to development of end-stage renal failure. The question if this is due to complement dysfunction or to linked genetic factors remains to be elucidated.

Complement pathways and meningococcal disease : diagnostic aspects.

Complement is an immunological effector system that bridges innate and acquired immunity in several ways. There is a striking association between susceptibility to meningococcal disease and various forms of complement deficiency (1,2). In defense against bacterial infection, the most important function of complement is probably to serve as a mediator of antibody-dependent immunity. Specific antibodies can trigger activation of the classical and the alternative pathways of complement activation (3-5). It is well known that antibody-independent mechanisms interfere with alternative pathway activation on the bacterial surface (6,7). The newly discovered mannan-binding lectin (MBL) pathway of complement activation appears to be protective against many types of infection (8) and adds previously unsuspected aspects of innate immunity to complement-mediated defense. Interestingly, immune responses are influenced by complement (9), and it could be that acquisition of protective antibodies is impaired in some types of complement deficiency. A further aspect of interactions between Neisseria and complement is the potential role of membrane-bound complement regulators as cellular receptors for the microbes (7).