Anti-neutrophil cytoplasm antibody IgG subclasses in Wegener's granulomatosis: a possible pathogenic role for the IgG4 subclass

A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.     

IgG subclass distribution of antibody responses to protein and polysaccharide mycobacterial antigens in leprosy and tuberculosis patients

Immunoenzymatic assays were developed for the measurement of antibodies against mycobacterial lipoarabinomannan (LAM), a cell-free proteic extract (CFX) of Mycobacterium leprae, and the 38-kD protein antigen of M. tuberculosis. Sera from 108 leprosy patients, belonging to all clinical–immunological forms of the spectrum, and 81 patients with localized or disseminated tuberculosis (TB) were tested for antibodies of the four IgG subclasses. Standard calibration curves were used to allow comparisons between results of different isotypes and specificities. Mean concentrations of total IgG antibodies were higher in the overall leprosy population than in TB patients. In leprosy, levels of anti-CFX increased from tuberculoid toward lepromatous forms, with a clear switch from IgG1 to IgG2 subclass predominance. A similar IgG1 to IgG2 conversion was observed in anti-LAM antibodies, although total levels of anti-LAM were similar in patients with tuberculoid and lepromatous forms. In TB, antibodies against polysaccharide and protein antigens were both predominantly of IgG1 subclass, whatever the patient's clinical status, although lower in disseminated forms, probably due to concomitant HIV infection. A hypergammaglobulinaemia was also found in most leprosy and TB patients. In TB this was due to increased IgG1 and IgG3, especially in HIV co-infected patients. Based on the current knowledge of the influence of T cell-secreted cytokines on human immunoglobulin isotype expression, these results do not fit with a putative role of Th1 (such as found in TB and tuberculoid leprosy (TT)) and Th2 (such as found in leprosy lepromatous (LL) leprosy) environment in the isotypy of antibody responses in mycobacterial infections. Nor do variations of isotypy according to pathological conditions seem to be related to the biochemical nature of antigens, since antibodies to LAM and protein antigens had comparable evolutions of their subclass distribution. Other factors are to be investigated in order to understand better the significance and possible roles of antibodies in mycobacterial diseases.     

IgG subclass distribution of autoantibodies to neutrophil cytoplasmic antigens in systemic vasculitis

The IgG subclass distribution for both ANCA-specific, and total IgG subclasses, of sera from 48 patients with active systemic vasculitis was determined by solid-phase assay using monoclonal anti-human subclass reagents. To control for varying performance of the subclass reagents, the assays were calibrated with sera affinity-depleted into subclass specific fractions, whose ANCA IgG subclass distribution was determined by a common reagent. In 10 patients whose ANCA persisted during clinical remission, the ANCA IgG subclass distributions between active and remission phases of disease were compared. The total IgG subclass distribution of sera from patients with active vasculitis was similar to that found in a normal population. The ANCA IgG subclass distribution of the same sera showed relative enrichment for IgG3 and depletion of IgG2 (P less than 0.05). When the active and clinical remission ANCA IgG subclass distributions were compared, ANCA IgG3 levels had fallen and ANCA IgG2 levels were increased during remission (P less than 0.01).     

IgG subclasses in human chronic schistosomiasis: over-production of schistosome-specific and non-specific IgG4

IgG subclasses were determined quantitatively in sera from 63 Egyptian men who were infected with Schistosoma mansoni. Total and antigen-specific IgG was measured pre- and post-treatment. Total IgG subclass antibodies were determined by immunoradiometric assay (IRMA) using monoclonal antibodies (MoAbs). The anti-worm and anti-egg specific S. mansoni IgG subclass antibodies were quantitatively measured by ELISA using specific MoAbs and standards obtained by affinity chromatography. Our data show that total IgG of the patients was elevated in the range of two to three times above normal. The magnitude of increase differed markedly among the four subclasses of IgG. The IgG1, IgG2 and IgG3 concentrations were approximately two to four times higher than normal, whereas the IgG4 concentrations was 20 times normal (9000 mg/l). IgG1 and IgG4 tended to dominate the IgG subclass distribution of anti-soluble worm antigen preparation (SWAP) antibodies followed by IgG2 and IgG3. On the other hand, IgG1 and IgG2 dominated the IgG subclass distribution of anti-soluble egg antigen (SEA) antibodies. As with IgG1, IgG2 and IgG3, most IgG4 was non-specific. The role of IgG subclasses in the pathogenesis of schistosomiasis is not clear. However, the high concentration of IgG4 might act as IgE blocking antibody, possibly as anti-idiotypes that may play a role in down-regulation of the immune system when it is challenged with an excess of antigen.     

IgG subclass distribution and relative functional affinity of anti-myeloperoxidase antibodies in systemic vasculitis at presentation and during follow-up.

Circulating IgG autoantibodies to myeloperoxidase (MPO) are associated with renal vasculitis and have been implicated in its pathogenesis. However, raised levels of these autoantibodies may persist during clinical remission. We tested whether this paradox could be explained by immunoglobulin subclass switching during disease evolution, since different subclasses have different immunological and biochemical properties. Sera with anti-myeloperoxidase (anti-MPO) activity from 33 patients with active disease and 20 anti-MPO positive follow-up sera were studied by an ELISA using a panel of anti-human IgG subclass monoclonal reagents previously calibrated on human myeloma proteins. Anti-MPO subclass distribution in initial sera was: IgG1, 31 (94%); IgG2, 10 (30%); IgG3, 24 (73%); and IgG4, 22 (67%). IgG3 anti-MPO decreased during follow-up (P less than 0.02), with no change in IgG1 and IgG4. Relative functional affinity of anti-MPO antibodies in purified IgG subclasses was studied by the diethylamine method. IgG3 fractions consistently had a greater affinity for MPO than the other subclasses. Sequential studies in four patients demonstrated an affinity maturation for IgG1 and IgG4 anti-MPO as IgG3 anti-MPO disappeared. We conclude that dynamic changes of subclass distribution and affinity may explain discrepancies between anti-MPO antibody titre and disease expression.     

Sensitive analytic ELISAs for subclass herpes virus IgG

The subclass distribution of antiviral antibodies to three herpes viruses was studied in a population of healthy blood donors. Subclassification by monoclonal antibodies led to the identification of certain viral IgG patterns. IgG1 appeared to be formed in response to almost all CMV, HSV-1 and VZV infections. A higher frequency of virus-specific IgG3 to CMV and HSV-1 suggested that these infections may be reactivated subclinically more often than VZV. The presence of CMV and VZV IgG4 showed a familial relationship, while IgG4 responses to HSV-1 were common. Persons with IgG4 as the only subclass-reactive antibody to CMV showed cell-related reactivities in a high frequency. Patients with leukemias, myelomas and Crohn's disease had a near-normal subclass pattern to the herpes viruses.     

The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity

Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns. The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass. The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E. coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV). Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E. coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.     

ELISA quantitation of IgG subclass antibodies to dietary antigens

The IgG subclasses of human antibodies against 2 dietary antigens, ovalbumin (OA) and beta-lactoglobulin (BLG), were studied by ELISA using monoclonal anti-human IgG subclass antibodies. Under the assay conditions used, the anti-IgG subclass antibodies were subclass specific. Quantitative estimates of the subclass antibodies were obtained by reference to a 'capture' assay using F(ab')2 anti-light chain antibody as ligand and IgG myelomas as standards. The validity of these estimates was supported by antibody quantitation using the Farr assay. In healthy adults with serum anti-OA or anti-BLG antibodies, anti-OA antibodies were found mainly in the IgG1 (9/11) and IgG4 (6/11) subclasses, whereas 5 sera showed high levels of IgG2 antibodies. In contrast, the IgG subclass distribution of anti-BLG antibodies was predominantly IgG4 (10/10).     

Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis

The triad of small vessel vasculitides (SVV) comprise Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CS). All three are associated with presence of circulating IgG antineutrophil cytoplasm antibodies (ANCA) which target autoantigens contained, primarily, within neutrophil azurophilic granules. The widely accepted model of pathogenesis suggests that ANCA activate cytokine-primed neutrophils within the microvasculature, leading to by-stander damage to endothelial cells, and rapid escalation of inflammation with recruitment of mononuclear cells. Activation may be initiated, in vitro, by the coligation of the PR3 or MPO antigen, translocated to the cell surface, and FcgammaRIIa/FcgammaRIIIb receptors. This suggests that the IgG subclass profile of ANCA and, possibly, its glycosylation status could influence the inflammatory mechanisms activated. The glycosylation status of total IgG isolated from the sera of patients with WG (13), MPA (6) and CSS (1) was determined by analysis of the released oligosaccharides. A deficit in IgG galactosylation is demonstrated for all patient samples, compared to controls. The mean percentage values for the agalactosylated (G0) oligosaccharides were 57% (SD +/- 9.71), 47% (SD +/- 4.25) and 28% (SD +/- 4.09) for WG, MPO and control samples, respectively. The G0 levels for polyclonal IgG isolated from the sera of both WG and MPA patients were significantly increased compared to controls (P < 0.0001). The major glycoform present therefore is agalactosylated (G0) IgG. In previous studies the G0 glycoform of IgG has been shown to bind and activate mannan binding lectin, and hence to activate the complement cascade, and to facilitate mannose receptor binding and the uptake of IgG complexes by macrophages and dendritic cells. Both of these activities could impact on the processing and presentation of self-antigens in autoimmune disease.     

ELISA detection of human IgG subclass antibodies to Streptococcus mutans

A sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to measure IgG subclass antibodies against whole cells of Streptococcus mutans and to a purified streptococcal antigen (SA I/II). Bacterial cells were bound to the solid phase using methyl glyoxal and mouse monoclonal antisera against IgG and each IgG subclass were used to detect antibodies. Natural antibodies to S. mutans were predominantly of the IgG1 and IgG2 subclasses, though IgG3 and IgG4 antibodies were detectable in most subjects, and were the majority response in a few subjects. Antibodies to SA I/II were predominantly of the IgG1 subclass with virtually no activity detectable in the IgG3 and IgG4 subclasses. Inhibition studies suggested some restriction of IgG subclass responses to bacterial antigens since SA I/II and c polysaccharide could inhibit binding of all subclasses to whole cells of S. mutans equally, whereas glucosyltransferase, lipoteichoic acid and dextran showed greatest inhibition of the IgG3 and IgG4 subclasses.     

Elevated serum IgG4 levels in cystic fibrosis patients

The quantitative measurement of the IgG subclass composition of the sera from sixteen patients with cystic fibrosis has revealed grossly elevated levels of IgG4 in seven patients. The possible significance of this observation is discussed in relation to recent reports of a high incidence of immediate-type hypersensitivity in such patients.     

IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections

The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections.     

An enzyme-linked immunosorbent assay for the determination of human IgG subclass antibodies directed against Branhamella catarrhalis

An ELISA procedure to determine the distribution of human IgG subclass antibodies directed against the gram-negative bacterium Branhamella catarrhalis has been developed using commercially available monoclonal anti-IgG subclass antibodies. Using whole bacteria as coating antigen the specificity of the assay was determined and showed minimal cross-reactivity with a range of other bacteria. Estimations of IgG1, IgG2, IgG3, IgG4 and total IgG antibodies directed against this antigen were performed. All normal adult sera tested had measurable antibody levels of specific IgG1, IgG2, IgG3 and total IgG. Specific IgG4 was undetectable in the majority of adult sera. These assays will be of value for investigation of both children and adults with suspected immunodeficiency and recurrent upper respiratory tract infection.     

Neutrophil cytoplasmic antibodies (p-ANCA) in ulcerative colitis.

AIMS--To study ulcerative colitis associated neutrophil cytoplasmic antibodies (p-ANCA) in respect of class and subclass distribution, antigen specificity, and (sub)cellular localisation of the antigen(s) to which these antibodies are directed. METHODS--p-ANCA positivity was determined using the standard indirect immunofluorescence test (IIFT). The immunoglobulin (Ig) subclass distribution of p-ANCA was investigated using monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4. Intracellular antigen localisation studies were performed on (fractionated) neutrophils using antigen-specific antibodies. RESULTS--In contrast to vasculitis associated ANCA, ulcerative colitis p-ANCA are mainly of IgG1 and IgG3 subclass and lack IgG4. Ulcerative colitis p-ANCA are myeloid specific. IIFT data indicate that the related antigen(s) seem(s) to be located not in the cytosol, but in the granules (most likely the azurophil granules) of the neutrophil. CONCLUSIONS--p-ANCA in ulcerative colitis have a different immunoglobulin subclass distribution than the ANCA of systemic necrotising vasculitis and necrotising and crescentic glomerulonephritis. This may point to differences in immune regulation between these diseases. Both cathepsin G and lactoferrin are recognised by a subpopulation of ulcerative colitis p-ANCA. In our series, eight out of 36 (22%) of ulcerative colitis associated p-ANCA react with lactoferrin and seven (19.5%) other sera with cathepsin G. None of them recognised both antigens. The main target antigen(s) of ulcerative colitis p-ANCA still remain(s) to be identified.     

Affinity Purification of IgG Subclasses and the Distribution of Thyroid Auto-Antibody Reactivity in Hashimoto''s Thyroiditis

To delineate accurately the IgG subclass distribution of thyroid auto-antibodies, sera from nine patients with Hashimoto's thyroiditis were fractionated into IgG subclasses by complete depletion of the other IgG subclasses on affinity columns. All IgG subclass fractions contained thyroglobulin and microsomal (or thyroid peroxidase) antibody activity, although when compared to the total serum concentrations of IgG subclasses, IgG4 antibodies were overrepresented. However, in contrast to recent studies, this particular subclass never predominated--IgG4 antibody levels being exceeded by those of the IgG1 and IgG2 subclasses; it seems likely that these differences relate to varying sensitivity for different subclasses in previously used assay methods. This pattern of subclass activity differed from that of tetanus toxoid antibodies, which were found in six subjects. There was no light chain restriction within any subclass, showing that the overproduction of IgG4 thyroid antibodies is not of monoclonal origin. The functional affinity of subclasses for both thyroid antigens varied between patients, but IgG2 subclass fractions showed the highest functional affinity in the majority of samples. We also found that IgG2 subclass thyroid antibodies were ineffective in eliciting antibody-dependent cell-mediated cytotoxicity, as distinct from the other three subclasses. Our results show that thyroid antibodies are less restricted in their IgG subclass distribution and patients are less heterogeneous than previously described. Moreover, IgG2 thyroid antibodies are quantitatively important and differ in relative functional affinity and effector function from IgG1 and IgG4 thyroid antibodies.     

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.     

The IgG4 subclass is associated with a low affinity antibody response to tetanus toxoid in man

The amount and relative affinity of antibodies to tetanus toxoid were measured, following immunization, in patients with chronic or recurrent acute chest infections and in healthy controls. The responding patients and controls produced similar amounts of antibody and, although antibody affinity was higher in the controls compared to the patients, the differences were not significant. Most individuals (65%) produced antibody of the IgG1 subclass with little or no IgG4 antibody, but in the remainder antibody was either predominantly IgG4 (29%) or equally distributed between the IgG1 and IgG4 subclasses (6%). Antibody affinity was significantly lower in both patients and controls producing IgG4 antibodies compared to those with a predominantly IgG1 response, and antibody affinity increased with the amount of IgG1 antibody present. These results provide preliminary evidence of an association between low antibody affinity and the IgG4 subclass in man.     

Humoral immune response in Epstein-Barr virus infections. I. Elevated serum concentration of the IgG1 subclass in infectious mononucleosis and nasopharyngeal carcinoma

Using radial immunodiffusion we measured IgG subclass concentrations and studied their distribution in serum samples from patients with infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC), two Epstein-Barr virus (EBV)-associated diseases, in comparison with two control groups [completely anti-EBV negative persons and subjects carrying antibodies to the viral capsid antigen (VCA)]. Antibody titres to VCA and to the early antigen (EA) were determined by indirect immunofluorescence and revealed characteristic patterns for the respective diagnostic groups. Nephelometric assays served for quantitating total protein, albumin, total IgG, IgA and IgM in all the sera. In the IM and NPC groups the concentration of IgG1 was significantly elevated by more than 50% whereas the other three subclasses remained unchanged as compared with the controls. Correspondingly, we found a significant increase of total IgG in IM and NPC. In IM, the only disease where VCA-specific IgM antibodies have been reported to occur, IgM levels were markedly elevated. Our data suggest that the IgG1 subclass plays an important role in the humoral immune response to EBV-determined antigens and that it is possibly involved in the control of virus infection.     

Anti-myeloperoxidase IgG subclass distribution and avidity in sera from patients with propylthiouracil-induced antineutrophil cytoplasmic antibodies associated vasculitis

OBJECTIVE: Propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The aim of this study was to compare the IgG subclass distribution and avidity of MPO-ANCA in sera from patients with primary ANCA-associated vasculitis (AASV) and PTU-induced vasculitis. METHODS: Nineteen patients with primary AASV with MPO-ANCA and thirteen patients with PTU-induced vasculitis were enrolled in the current study. Sera in both active phase and remission were collected. Anti-MPO IgG subclasses were detected by antigen specific ELISAs using specific monoclonal antibodies as second antibodies, and MPO-ANCA avidity was assessed by antigen-inhibition ELISAs. RESULTS: In primary AASV, all four anti-MPO IgG subclasses could be detected in active phase with IgG1 (100%), IgG2 (73.7%), IgG3 (63.2%) and IgG4 (94.7%), and in remission, IgG1 and IgG4 subclasses in most patients remained positive. However, in PTU-induced vasculitis, anti-MPO IgG3 subclass could not be detected, the anti-MPO IgG subclasses in active phase were IgG1 (100%), IgG2 (61.5%) and IgG4 (46.2%). Furthermore, five out of the six patients (88.8%) with PTU-induced vasculitis with positive IgG4 subclass in active phase turned to negative in remission, however, only eight out of the fourteen patients (57.1%) with primary AASV turned to negative. The median avidity constant of MPO-ANCA was 56 (8.96 to >140) x 10(7) mol/l for patients with primary AASV and 0.7 (<0.28 to >140) x 10(7) mol/l for patients with PTU-induced vasculitis respectively. Furthermore, the relative levels of MPO-ANCA avidity were associated with elevation of ESR in primary AASV and were associated with BVAS scores in patients with PTU-induced vasculitis, respectively. CONCLUSION: MPO-ANCA IgG subclass distribution and avidity were different between patients with primary AASV and PTU-induced vasculitis. It was suggested that the mechanism of ANCA production in PTU-induced vasculitis was different from that in primary AASV, and the avidity of MPO-ANCA might be associated with disease activity.     

Detection by enzyme-linked immunosorbent assay of specific immunoglobulin G isotypes in primary and established cytomegalovirus infections

An enzyme-linked immunosorbent assay using monoclonal antibodies was developed to study the subclass distribution of immunoglobulin G (IgG) to cytomegalovirus (CMV) in individuals from a number of clinical groups. Most CMV-seropositive individuals had IgG1 and IgG3. IgG2 and IgG4 were detected less frequently at very low levels of activity, mostly among mothers at delivery and renal patients. Most seroconversions were accompanied by an important increase of the IgG1 activity, whereas IgG3 appeared at lower levels; neither IgG2 nor IgG4 occurred. This suggests that these isotypes play a secondary role in the response to the CMV infection and that they may be considered markers of past infections. Anti-CMV IgG1 is the most efficiently transmitted through the placenta. Whether infected or not, newborns had the same subclass distribution and activity levels as their mothers. Isotype determination did not offer a decisive explanation of a number of discrepancies observed between CMV IgG enzyme-linked immunosorbent assay and complement fixation test results.