Anders G. Sjöholm

Protein SIC, a Novel Extracellular Protein of Streptococcus pyogenes Interfering with Complement Function

The human pathogen Streptococcus pyogenes possesses a chromosomal region, the mga regulon, that contains co-regulated genes important to the virulence of these bacteria. A novel gene located in the mga regulon of a S. pyogenes strain of serotype M1 was cloned and sequenced. It translates into a protein of 305 amino acid residues, including a signal sequence of 32 amino acids and a central region consisting of three tandem repeats. The sequence represents a novel structure with no significant homology to any previously published sequence. The protein was purified from the streptococcal culture media where it is present in substantial amounts. Affinity chromatography of human plasma on Sepharose coupled with the protein specifically adsorbed two plasma proteins which were identified as clusterin and histidine-rich glycoprotein (HRG). The interactions between the streptococcal protein and the plasma proteins were further characterized using purified clusterin and HRG. Inhibition experiments indicated that they have affinity for overlapping or closely located sites in the streptococcal protein. Both clusterin and HRG are regulators of the membrane attack complex (C5b-C9) of complement. When the streptococcal protein was added to serum, complement-mediated lysis of sensitized sheep erythrocytes and guinea pig erythrocytes was inhibited. In addition, the streptococcal protein was incorporated into C5b-C9 in serum, indicating the location of its action. The name, protein SIC, streptococcal inhibitor of complement-mediated lysis, is therefore suggested for this novel protein. The occurrence of protein SIC and its gene was investigated in a collection of S. pyogenes strains comprising 55 different M serotypes. Only M1 and M57 strains were positive in this screening, indicating that protein SIC could be a virulence determinant. Thus, during recent years, the M1 serotype has been connected with a world-wide increase of severe and toxic S. pyogenes infections.

Autoantibodies against neutrophil cytoplasm components in systemic lupus erythematosus and in hydralazine‐induced lupus

Anti‐neutrophil cytoplasm antibody (ANCA) has been shown to be no marker of systemic lupus erythematosus (SLE) including lupus nephritis or of progressive systemic sclerosis (PSS). Antibodies against myeloperoxidase (anti‐MPO) and elastase, two granulocyte lysosomal enzymes, were found in patients with SLE but not in those with PSS, except for one patient who had anti‐MPO. Anti‐MPO was present in 21% of patients with SLE, and at low concentrations in about 80% of these cases. Anti‐elastase was found in four patients with SLE. In another group of six patients with a SLE‐like syndrome induced by anti‐hypertensive treatment with the anti‐hypertensive hydralazine, anti‐MPO antibodies occurred in all six, and anti‐elastase antibodies in five. Monitored during a 2‐year follow‐up period, anti‐MPO antibodies were found to persist, whereas anti‐elastase antibodies were rapidly eliminated, after withdrawal of the drug.

Hereditary C2 Deficiency in Sweden: Frequent Occurrence of Invasive Infection, Atherosclerosis, and Rheumatic Disease.

Abstract: Although frequently asymptomatic, homozygous C2 deficiency (C2D) is known to be associated with severe infections and rheumatic disease. We describe the clinical findings in 40 persons with C2D from 33 families identified in Sweden over 25 years. Medical records covering 96% of the accumulated person-years were reviewed, giving a mean observation time of 39 years (range, 1-77 yr). Severe infection was the predominant clinical manifestation in the cohort: 23 patients had a past history of invasive infections, mainly septicemia or meningitis caused by Streptococcus pneumoniae, and 12 patients had repeated infections of this kind. Nineteen patients had at least 1 episode of pneumonia, and recurrent pneumonia was documented in 10 patients. Repeated infections occurred mainly during infancy and childhood. Systemic lupus erythematosus was found in 10 patients. Another 7 patients had undifferentiated connective tissue disease (n = 4) or vasculitis (n = 3). We found no correlation between susceptibility to invasive infection and rheumatologic disease. Cardiovascular disease occurred at a high rate, with a total of 10 acute myocardial infarctions and 5 cerebrovascular episodes in 6 patients. Causes of death among the C2D patients were infection (n = 5), acute myocardial infarction (n = 3), and cancer (n = 1). We suggest that severe infection may be the principal clinical manifestation of C2D. We also provide novel evidence for a possible role of C2D in the development of atherosclerosis consistent with findings in mannan-binding deficiency and experimental C3 deficiency. In addition, we confirm the well-known association between C2D and systemic lupus erythematosus. Abbreviations: ACR = American College of Rheumatology, AMI = acute myocardial infarction, ANA = antinuclear antibodies, C2D = homozygous C2 deficiency, MASP = MBL-associated serine protease, MBL = mannan-binding lectin, MHC = major histocompatibility complex, PCR = polymerase chain reaction, SLE = systemic lupus erythematosus.

EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis.

ABSTRACT The human pathogen Streptococcus pyogenes primarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival of S. pyogenes in human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests that S. pyogenes expresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system.

Complement components, complement activation, and acute phase response in systemic lupus erythematosus.

The investigation concerned 33 systemic lupus erythematosus (SLE) patients assigned to three groups representing mild SLE, more severe extra renal SLE, and SLE with significant renal involvement. In patients with extrarenal disease, the inflammatory plasma protein response was often pronounced during exacerbation, as evidenced by markedly increased concentrations of C-reactive protein (CRP), alpha 1-antichymotrypsin, alpha 1-antitrypsin, and orosomucoid. CRP responses were rare in patients with renal involvement, despite the increased concentrations of other acute-phase reactants in some of these patients. Superimposed bacterial infections were not clearly distinguished by raised CRP concentrations. The classical pathway of complement was activated in all patients during exacerbation, as indicated by increased concentrations of C1r-C1s-C1 inactivator complexes and C2a fragments. C1, C2, and probably also C3 activation varied according to the amounts of circulating C1q-binding immune complexes, as measured by solid-phase assay. Manifest hypocomplementemia was usually associated with glomerulonephritis. Participation of complement components in the inflammatory plasma protein response apparently counteracted the development of hypocomplementemia in many patients with extrarenal SLE. Circulating C3d was detected in all patients during exacerbation of renal disease and in most patients with severe extrarenal manifestations. Inverse relationships were found between immunochemical C2 concentrations and the percentage of cleaved C2 and between C3 and C3d. There was no appreciable consumption of factors B and D and properdin of the alternative pathway in the patients. High concentrations of factor D, a low molecular weight protein, were exclusively found in patients with renal involvement and could be ascribed to retention due to reduced glomerular filtration.

Deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship.

In cystic fibrosis (CF) prognosis concerning lung damage development is highly variable and difficult to predict. Mannan‐binding lectin (MBL) deficiency has been reported to be associated with poor outcome in CF lung disease. MBL is a recognition molecule of the MBL pathway of the complement system and is encoded by a gene characterized by a high degree of polymorphism. Some genotypes result in low serum concentrations of MBL. MBL‐associated serine protease 2 (MASP‐2) is another protein belonging to the MBL pathway. A mutation resulting in low levels of MASP‐2 in serum has been described recently. In the present study, 112 CF patients aged 4–54 years were investigated for MBL and MASP‐2 genotypes, serum levels of MBL and MASP‐2 and the MBL pathway function in serum. No correlation to reduced lung function or need for lung transplantation was seen, either for MBL deficiency, MASP‐2 gene mutation or reduced MBL pathway function. However, in the 27 patients colonized with Staphylococcus aureus, MBL‐deficient genotypes were associated with decreased lung function. As expected, MBL pathway function in serum was reduced both in MBL‐deficient patients and in patients carrying a mutant MASP‐2 allele. An unexpected finding was that CF patients had higher serum levels of MBL than healthy controls when corrected for MBL genotype. In conclusion, MBL pathway function was affected both by MBL and by MASP‐2 genotypes. However, MBL or MASP‐2 levels in serum did not affect the clinical outcome in the cohort of CF patients studied.

Dysfunctional properdin in a Dutch family with meningococcal disease.

DEVELOPMENT of systemic infections with Neisseria meningitidis can usually be ascribed to the absence of protective IgM or IgG antibodies, or to the presence of IgA antibodies that block serum bact...

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 mem‐branolytic 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.

Immunoglobulin g (IgG) anti-tissue transglutaminase antibodies used as markers for IgA-deficient celiac disease patients

ABSTRACT The role of immunoglobulin A (IgA) anti-tissue transglutaminase antibodies (IgA-tTG) as predictors of untreated celiac disease (CoD) is well documented, and the presence and levels of these antibodies are most accurately monitored with native or recombinant human antigens. However, IgA-deficient CoD patients are not identified by IgA serology, and conflicting results concerning the diagnostic validity of IgG antibodies against gliadin (IgG-AGA), endomysium (IgG-EmA), and tTG (IgG-tTG) have been reported. The aim of the present study was to evaluate the utility of IgG-tTG for the detection of CoD in IgA-deficient patients. Samples from 115 IgA-deficient and 200 IgA-sufficient subjects were collected and tested for the presence of IgA and IgG antibodies against tTG, EmA, and AGA. Antibodies against tTG were measured by an enzyme-linked immunosorbent assay based on recombinant human tTG, and antibodies against EmA were determined by immunofluorescence. The values for IgG-tTG showed a higher correlation (correlation coefficient [r] = 0.91) with those for IgG-EmA for the IgA-deficient subjects than for the IgA-sufficient subjects (r = 0.88). The overall concordance of the positive and negative results between IgG-tTG and IgG-EmA was 97%, and the IgG-tTG assay discriminated between IgG-EmA-positive and -negative subjects with IgA deficiency at a rate of 100%. Elevated levels of IgG-tTG and IgG-EmA were measured in 70% of the IgA-sufficient subjects. IgG-tTG detection with recombinant human tTG is a good alternative to IgG-EmA detection, and the addition of IgG-tTG assessment to present screening methods may improve the ability to identify IgA-deficient subjects with CoD.

Autoantibodies against C1q : view on clinical relevance and pathogenic roles

C. E. H. SIEGERT, M. D. KAZATCHKINE*, A. SJO¨HOLM§, R. WU¨RZNER†, M. LOOS‡ & M. R. DAHADepartment of Nephrology, Leiden University Medical Centre, Leiden, The Netherlands, *Institut National de la Sante´et de laRecherche Medicale, Hopital Broussais, Paris, France, † Institut fu¨r Hygiene, Leopold-Franzens-Universita¨t, Innsbruck, Austria,‡ Institute for Medical Microbiology and Hygiene, J-Gutenberg University, Mainz, Germany, and§Clinical Microbiology Laboratory,Lund University Hospital, Lund, Sweden(Accepted for publication 6 January 1999)INTRODUCTIONImmune responses to autoantigens are quite common andautoimmunity is considered to be a physiological part of theimmune system. Autoantibodies are an inherent property of theantibody repertoire of many healthy individuals and are thereforereferred to as natural autoantibodies [1,2]. The numerous kinds ofautoantibodies fall into two main categories, depending on whetherorgan-specific or non-organ-specific autoantigens are involved.Non-organ-specific antigens mostly occur in nucleated cells,such as DNA, or are found among circulating plasma proteins,such as coagulant proteins and the Fc portion of IgG.Natural autoantibodies have been proposed to be involved inthe clearance of degradation products that are formed during cellmetabolism. In this respect natural autoantibodies are also referredto as housekeeping antibodies [3]. Autoantibodies may also beassociated with disease states but do not necessarily play a role inthe pathogenesis of such diseases. The presence of autoantibodiesmay for example be secondary to the production of tissue damage,or independent pathogenic factors may directly induce both thedisease and the presence of autoantibodies.In 1984 autoantibodies to C1q (C1qAb) were reported to bepresent in serum of patients with systemic lupus erythematosus(SLE) [4]. The recognition that C1q may serve as a non-organ-specific autoantigen has attracted a growing number ofinvestigators. This study discusses the knowledge of C1q asautoantigen by reviewing the epidemiology, disease associations,and pathophysiology of C1qAb.ROLE OF C1q IN IMMUNE COMPLEXCLEARANCEActivation of the complement system is the first step in theprevention of damage by immune complexes. Initiation of com-plement activation occurs through three pathways: the classical,the alternative, and the lectin pathway. The classical pathway ofthe complement system is considered to be the most importantpathway in immune complex clearance. This pathway may beactivated by IgM- and IgG-containing immune complexes afterbinding of C1q [5]. C1q is a subcomponent of the first component(C1) of the classical pathway. It is a large highly cationicglycoprotein with a molecular weight of 410kD. C1q consists ofsix copies each of three polypeptide chains, A, B, and C. The A, B,and C chains are rich in hydroxylated amino acids and are linkedtogether by disulphide bonds into dimers [6] Together these dimersform a triple helix structure which resembles collagen. Towardsthe N-terminal end of C1q the triple helices lay parallel to eachother and towards the C-terminal end they diverge. The N-terminalend is called the collagen-like region which is linked by theconnecting strands to the C-terminal end, which is called theglobular heads region. The macromolecular structure of C1q issaid to resemble a bunch of tulips [5–7].The function of C1q is directly related to its structure. Bindingof Fc regions of immunoglobulins to the globular head portions ofC1q induces distortion of the connecting strand which changes theconformation of the collagen-like region [8,9]. The dynamicequilibrium between C1q and the other subcomponents of C1,C1r and C1s, subsequently shifts and induces further activation ofthe cascade of proteins composing the classical pathway. Thisresults in the prevention of lattice formation of immune complexesand ensures their clearance from the circulation by the mono-nuclear phagocyte system [10]. Although the recognition protein ofthe lectin pathway, mannose-binding lectin, is structurally relatedto C1q, it is not known to be involved in immune complexclearance mechanisms [11]. To summarize, activation of C1 bybinding of immune complexes to C1q is a prerequisite for immunecomplex clearance.HISTORY OF C1qAbSince SLE is considered to be the prototype of immune complexdiseases in man, a large variety of immune complex assays hasbeen employed to investigate possible pathogenic roles of circulat-ing immune complexes and to relate their titres to the presence ofmanifestations of the disease. The solid-phase C1q binding assay isone of the most frequently used assays for both purposes [12]. Thisradioimmunoassay is based on the binding of immune complexesto C1q, which is fixed to a solid phase. Studies in the early 1970s