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Frequency of Antineutrophil Cytoplasmic Antibody in Graves’ Disease Patients Treated with Methimazole

Rheumatology Section (M.G., M.V.-R., A.O.), Endocrinology Service (I.S., J.L.R.), Epidemiology Unit (J.R.), and Immunology Unit, Laboratori d’Immunobiologia per a la Recerca i Aplicacions Diagnòstiques (M.J.), Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain

Address all correspondence and requests for reprints to: Mònica Gumà, Immunology, Universitat Pompeu Fabra, C/Dr Aiguader 80, 08003 Barcelona, Spain. E-mail: monica.guma{at}cexs.upf.es.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Retrospective studies have shown antineutrophil cytoplasmic antibody (ANCA) positivity in patients treated for Graves’ hyperthyroidism; ANCA has been attributed to either antithyroid drugs or to the disease itself.

The aim of this study was to determine ANCA in Graves’ disease patients at diagnosis and after treatment with methimazole and to evaluate the relationship between ANCA and hyperthyroidism evolution.

Thirty patients recently diagnosed with Graves’ hyperthyroidism were prospectively studied. ANCA were determined by indirect immunofluorescence. ANCA autoantibodies against specific antigens (proteinase 3, myeloperoxidase, bactericidal/permeability-increasing protein (BPI), cathepsin, lysozyme, elastase, and lactoferrin) were detected by ELISA. The median observation period was 22 months. Kaplan-Meier analysis was performed to identify ANCA as an outcome variable.

Twenty patients (67%) were ANCA positive before the onset of treatment, and four (19%) remained positive after 1 yr of antithyroid drug treatment. No differences were observed in any clinical or analytical features between patients with or without positive ANCA. Before treatment, BPI-positive patients required radioiodine treatment or presented relapse more rapidly than BPI-negative patients (log-rank test P < 0.0002).

Patients with Graves’ hyperthyroidism show positive ANCA before medical treatment, which points to a relationship with the autoimmune disease itself. Our results suggest that BPI-positive patients tend to relapse with antithyroid medication.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
ANTIBODIES AGAINST NEUTROPHIL cytoplasmic antigens (ANCA) have been described as sensitive and specific markers for systemic vasculitis and crescentic glomerulonephritis (1). ANCA can be identified by indirect immunofluorescence (IIF) and ELISA techniques. Two classical patterns have been described: a cytoplasmic fluorescence pattern (c-ANCA) and a perinuclear pattern (p-ANCA). By ELISA, c-ANCA usually reacts with proteinase 3 (PR3), whereas p-ANCA reacts not only with myeloperoxidase (MPO) but also with bactericidal/permeability-increasing protein (BPI), cathepsin, lactoferrin, and other myeloid proteins that are found within azurophilic or primary granules of polymorphonuclear leukocytes. These latter antigens sometimes produce a positive staining pattern, different from those of c-ANCA and p-ANCA, named atypical ANCA (x-ANCA; Ref.2). Most laboratories usually test for MPO and PR3 because they have been related to vasculitis. This explains why some patients are ANCA positive by IIF but negative by ELISA. It is important to identify these patients because they could be positive for another antigen, which might have a clinical significance.

Circulating ANCA in serum are not confined to patients with systemic vasculitis, but can be found in other disorders such as connective tissue diseases and inflammatory bowel disease (3, 4, 5); however, their role and clinical significance remain unclear.

In Graves-Basedow (GB) disease, the presence of ANCA has been reported in two situations. First, MPO-ANCA-related vasculitis and nephritis have been described in patients with GB disease treated with propylthiouracil (6, 7, 8). Therefore, propylthiouracil therapy has been associated with the appearance of MPO-ANCA (9, 10, 11). However, few reports have described ANCA-associated vasculitis due to methimazole (MMI; Refs.12, 13, 14). Afeltra et al. (15) recently reported the presence of x-ANCA in untreated GB disease patients, in some of whom antilactoferrin antibodies were observed.

MMI is the drug of choice in the treatment of GB disease in continental Europe. The aim of this work was to evaluate the prevalence of ANCA and their specificities in patients with GB disease before and after MMI therapy. The correlation between ANCA positivity, clinical manifestations, and thyroid autoantibody titration was also assessed. Finally, the relationship between ANCA positivity and their specificities and the evolution of hyperthyroidism was investigated.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

Patients with untreated GB disease-induced hyperthyroidism were requested to participate in the study. All were recruited at the outpatient Endocrinology Clinic between January and December 2000. Thirty consecutive patients (5 men and 25 women; mean age, 35.9 ± 9.7 yr; range, 23–56 yr) agreed to participate and were studied prospectively. The diagnosis of GB disease was based on classical criteria: hyperthyroidism with or without clinical ophthalmopathy, a diffuse homogeneous goiter on palpation and on scintiscan, free T₄ (FT₄) and TSH measurements, and the presence of TSH receptor (TSH-R) antibodies (TRAb). All patients received MMI (Tirodril, Estedi, Barcelona, Spain) at an initial dose of 30–45 mg/d, which was adjusted according to thyroid function tests until a euthyroid state was achieved. The dose was progressively tapered until withdrawal after 12 months. Posttreatment follow-up was made at 3-month intervals or at the time of clinical hyperthyroidism relapse, if it occurred. Relapse after MMI withdrawal was defined as recurrence or persistence of abnormal thyroid hormones requiring further treatment.

FT₄, TSH, blood test, urine analysis, and clinical signs and symptoms were checked at intervals of 1–2 months. ANCA and antinuclear antibodies (ANA) were measured by IIF at diagnosis and at 1, 6, and 12 months after MMI therapy was started. The presence of autoantibodies against thyroid peroxidase (TPO), thyroglobulin (TG), and TSH-R (TRAb) was also examined. Sera at the time of diagnosis and the last obtained were assayed with ELISA for ANCA specificities.

Full ethical approval was obtained from the Ethics Committee, and written informed consent was obtained from all participants.

Hormone evaluation

Blood samples were drawn at each visit for measurement of FT₄ and TSH concentrations by the commercially available kits Immulite FT₄ Chemiluminescent Enzyme Immunoassay (Diagnostic Products Corp., Los Angeles, CA) and Immulite Third Generation THS Chemiluminescent Enzyme Immunoassay (Diagnostic Products Corp.). Reference ranges were: FT₄, 8.9–29.67 pmol/liter; and TSH, 0.4–4 mIU/liter.

IIF assay for ANCA

IIF assay for ANCA was performed by a standard method delineated at the First International Workshop on ANCA (16). Slides were read by two independent observers. Serum samples positive for ANCA at the screening dilution of 1:20 were subsequently tested at 2-fold dilution to determine a titer. Fluorescence patterns were classified as classic cytoplasmic (c-ANCA) signal when diffuse granular cytoplasmic staining with central accentuation was seen, perinuclear (p-ANCA) when a pattern was observed around the nucleus, or atypical (positive fluorescence different from that of c-ANCA or p-ANCA) when distinct, homogeneous, nongranular cytoplasmic staining was seen. All samples with perinuclear or combined perinuclear and nuclear staining patterns were additionally tested on formaldehyde-fixed granulocytes (The Binding Site, Birmingham, UK).

ELISA for ANCA specificities

ANCAcombi, a commercial semiquantitative indirect ELISA kit (Orgentec, Mainz, Germany), was used for simultaneous differential and qualitative detection of IgG autoantibodies against the following antigens of neutrophil cytoplasm: PR3, MPO, BPI, elastase, lactoferrin, cathepsin-G, and lysozyme, vertically coated in eight wells (the first well contained a positive control, the other seven were coated with the individual ANCA antigens). The antigen-antibody interaction was detected by an antihuman IgG conjugated with horseradish peroxidase and 3.3',5'5'-tetramethyl-benzidine as a colorimetric substrate. Results are expressed in relation to the OD of a cut-off serum, with a ratio of at least 1.0 being considered positive.

ANA detection

ANA were detected by an IIF on Hep-2 (human epithelioma type 2) cells using a Kallestad HEp-2 slide (Bio-Rad SA, Marnes-la Coquette, France). Serum samples positive for ANA at the screening dilution of 1:40 were subsequently tested at 2-fold dilution to determine a titer. The fluorescence patterns were assessed and recorded as homogenous, rimmed, fine or coarse speckled, nucleolar, or centromeric. Antibodies to double-stranded DNA (dsDNA) were measured by IIF on Kallestad Crithidia luciliae slides (Bio-Rad SA). Samples positive for anti-dsDNA at the screening dilution of 1:10 were subsequently tested at 2-fold dilution to determine the titer.

Anti-extractable nuclear antigens, including anti-SS-A, anti-SS-B, anti-Scl70, anti-Sm, and anti-U1RNP, were detected by indirect ELISA using specific commercial kits (Orgentec). The normal range is up to 25 U/ml for all of these autoantigens.

Antithyroid antibodies

Anti-TPO and anti-TG antibodies were determined by ELISA using commercial kits (Orgentec). The normal ranges are as follows: anti-TPO, up to 100 IU/ml; and anti-TG, up to 150 IU/ml. TRAb was detected using the Medizym T.R.A. (Medipan Diagnostica GMBH, Selchow, Germany), a competitive enzyme immunoassay. Normal range is up to 9 U/liter (equivalent to 2 IU/liter).

Statistical analysis

Quantitative variables are expressed as mean ± SD (range) and qualitative variables as percentages. Fisher’s exact test (frequencies) and nonparametric Mann-Whitney U test (means) were used to establish the statistical significance of the differences observed. Possible correlations were determined by Spearman’s rank correlation coefficient.

To evaluate the relationship between sex, TRAb, BPI, and cathepsin and hyperthyroidism evolution, the trial was planned with radioiodine treatment, when relapse occurred, as the primary end point. Therefore, the dependent variable was time (days) elapsed from diagnosis to when the radioiodine treatment was indicated in patients who required it. The remaining time was censored at the date of the last visit or at the date when statistical analysis began (i.e. March 1, 2002). Median follow-up for the overall population was 22 months (1–27 months). Progression time from diagnosis to failure of the medical treatment was estimated by the Kaplan-Meier method (17); a log-rank test (18) was used to compare Kaplan-Meier curves. The critical value necessary to establish that the group with positive BPI presented worse evolution compared with the group with negative BPI was 3.84, which corresponds to a two-tailed P value of 0.05.

Data were analyzed using the statistical software package SAS, version 8.2 (SAS Institute, Inc., Cary, NC). Differences were considered significant at P value no greater than 0.05.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Clinical, analytical, and, immunological manifestations at diagnosis

Clinical, analytical, and antithyroid autoantibody data are summarized in Table 1. Prevalence of TRAb, anti-TPO, and anti-TG antibodies was 84%, 57%, and 17%, respectively.

ANA and ANCA were measured 12 months after the start of MMI therapy in 21 patients who remained on drug treatment. The sera of nine patients were not included because three required surgery because of goiter size, two required radioiodine treatment, two were treated with dexamethasone for their Graves’ ophthalmopathy, and in two samples were not obtained.

ANA. Neither the pattern nor titers of ANA changed between determinations.

ANCA. Four of 21 patients (19%) were identified by IIF as positive for ANCA (2 p-ANCA and 2 x-ANCA). Six patients became negative by IIF after 3–6 months of MMI treatment, and only one became positive after treatment with MMI (x-ANCA pattern). By ELISA, 3 of 21 (14%) were positive (1 BPI and 2 cathepsin); thus, ANCA seroconversion from positive to negative affected eight patients, and none became positive by ELISA. Negative seroconversion was statistically significant by both IIF and ELISA. All MPO-positive sera became negative after 1 yr of treatment. These results are summarized in Table 3. No patient developed vasculitic disorders during this period.

No differences were found between any clinical features and positivity of ANA, ANCA, or different specificities of ANCA (BPI, cathepsin, MPO) in patients before MMI treatment. Regarding antithyroid autoantibodies, no statistically significant differences were observed between antithyroid autoantibodies or TRab and ANCA specificities.

ANCA and hyperthyroidism outcome

Twenty-six patients who remained under medical treatment were analyzed by Kaplan-Meier survival analysis. One patient was excluded because the observation period was less than 2 months, and three others were excluded for receiving surgical therapy. Medical treatment was switched to radioiodine in five patients during this period only if T₄ levels remained high after 4 months of increasing MMI doses up to 60 mg/d. In 21 patients, MMI was discontinued after 13.2 months (range, 11–17 months). Three of the 21 relapsed and required radioisotope therapy after MMI withdrawal.

Survival analysis was performed to identify outcome variables: sex, BPI, TRAb, and cathepsin. In BPI-negative patients, 11% (2 of 18) required radioiodine treatment in contrast to 75% (6 of 8) of BPI-positive patients. Patients BPI-positive at diagnosis required radioiodine treatment or presented relapse more rapidly than BPI-negative patients (log-rank test P < 0.0002; Fig. 1). No other variables were associated with prognosis.

View larger version (13K): [in this window] [in a new window]   Figure 1. Actuarial radioiodine free period in patients with Graves’ disease according to BPI. In Kaplan-Meier survival analysis, 26 patients who remained under medical treatment were analyzed. In BPI-negative patients, 11% (2 of 18) required radioiodine treatment, in contrast to 75% (6 of 8) of BPI-positive patients. BPI-positive patients at diagnosis required radioiodine treatment or presented relapse more rapidly than BPI-negative patients (log-rank test, P < 0.0002).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

It is worth emphasizing that most of our ANCA-positive patients became ANCA negative after 3–6 months of MMI therapy. In a recent work, we reported that 60% of GB disease patients showed ANCA positivity 2 months after starting drug treatment (20). In the present study, we confirm that GB patients were positive for ANCA before any treatment and became negative several months after therapy onset. These data are in agreement with some works that found neither MPO-ANCA-positive titers nor vasculitic disorders in patients treated with MMI (10, 11).

In the present study, a discrepancy was found between ANCA positivity determined by IIF and ELISA in 10 cases (33%); such a discrepancy has previously been reported (21). Some IIF-positive sera proved negative when tested by ELISA, which may occur because autoantibodies may be directed against antigenic or epitopic targets not screened by the method, or may be caused by the instability of the antigen at room temperature. Some of these sera were also positive for ANA. Patient samples for ANCA testing are frequently positive for ANA that can mimic p-ANCA staining patterns on ethanol-fixed neutrophils. This possibility could account for sera being ANCA-positive by IIF and negative by ELISA. To distinguish true p-ANCA staining patterns from other non-ANCA artifacts, all samples with perinuclear or combined perinuclear and nuclear staining pattern were tested on formaldehyde-fixed granulocytes.

On the other hand, some of the sera positive by ELISA were negative when tested by IIF. Factors that may influence the IIF test results, such as leukocyte preparation, fluorescence microscopy, antigen instability in ethanol-fixed neutrophils, or the investigator’s experience in staining patterns are difficult to standardize (21, 22), although all of these parameters are broadly evaluated in the immunology laboratory using internal and external controls.

Regarding ANA positivity, ANA were present in 77% and anti-dsDNA in 10% of Graves’ disease patients. The prevalence of ANA positivity reported by other authors ranges from 20–70% (23, 24, 25) and anti-dsDNA from 13–16% (25, 26). Neither the pattern of ANA nor their titers changed after 1 yr of treatment, in contrast to ANCA titers. ANA (especially those with diffuse pattern) seem to be nondisease-specific antibodies. The abnormal immune condition may influence some pathways of autoantibody production, particularly ANA (due to lack of control in B-cell autoreactivity), with the intrathyroidal germinal center-like structures constituting an important element for high autoreactive production (27). Furthermore, in our patients, no correlation was found between the ANA titer and presence of TRAb, anti-TPO, or anti-TG antibodies. Thus, positive ANA are probably not related to the etiology of autoimmune thyroid diseases.

In contrast, ANCA titers decreased and became negative in the majority of patients. An intriguing question is why ANCA are positive in GB disease patients. TRAb stimulate thyroid function through the TSH-R. However, TSH-R is expressed in a number of extrathyroid cells such as mono- and polymorphonuclear cells of peripheral blood (28). Little is known about interactions of polymorphonuclear granulocytes with TSH or anti-TSH-R, but some studies suggested that polymorphonuclear TSH-R are not mute receptors (29, 30). Thus, granulocytes, after being activated by anti-TSH-R autoantibodies, could degranulate and expose intracytoplasmic antigens to a noncontrol aberrant antigenic presentation. From these antigens, BPI could induce cross-presentation through its own capability of increasing membrane permeability.

On the other hand, antithyroid drugs appear to interfere with immunological abnormalities in Graves’ hyperthyroidism, thereby decreasing the titers of antithyroid antibodies in most patients (31). The mechanism of action of the immunosuppressive effects of antithyroid drugs has remained a matter of controversy (32, 33), but might offer a possible explanation for ANCA disappearance with their treatment. However, antithyroid drugs, particularly propylthiouracil, may produce vasculitic manifestations. The pathogenesis of propylthiouracil-induced vasculitis is unknown; however, some authors support the concept that during its treatment, if any infection activates neutrophils, a large quantity of MPO is released from the neutrophils and could transform the drugs into free radicals, resulting in injury to the vessel walls (6).

The second part of this study addressed the association between positive ANCA and hyperthyroidism evolution. BPI-ANCA-positive patients at diagnosis tended to require radioiodine treatment more frequently than BPI-negative patients. BPI-ANCA was originally detected in ANCA-positive sera of patients with vasculitis who were negative by ELISA to PR3 and MPO-ANCA (34). Recently, BPI-ANCA has been described and correlated with activity or evolution of some diseases. In systemic lupus erythematosus, BPI-ANCA levels correlated with disease activity rather than specific organ involvement (35, 36). Antibodies to BPI protein and cathepsin G were both associated with the presence of cirrhosis in patients with primary sclerosing cholangitis (37). BPI is a significant minority target antigen for ANCA in inflammatory bowel disease and appears to be related to colonic Crohn’s disease and activity in ulcerative colitis (4). Finally, anti-BPI levels, especially IgA isotype in patients with cystic fibrosis, are inversely correlated with the decline in pulmonary function (38).

In summary, GB hyperthyroid patients presented ANCA positivity before diagnosis, before any drug treatment was initiated. With MMI, ANCA titers decreased in the majority of these patients, thereby suggesting a possible immunomodulatory effect of the drug. Further studies with a large number of patients and long-term follow-up are required to determine whether BPI-ANCA positivity at diagnosis or the reappearance of ANCA levels after MMI withdrawal could predict GB hyperthyroidism relapse.

	Acknowledgments

 
We thank Carmen López and Olga Bernaus for their assistance as technicians in conserving the sera and measuring antithyroid and antineutrophil autoantibodies, and Christine O’Hara for helpful manuscript correction.

	Footnotes

 
This work was supported by a grant from the Catalan Society for Rheumatology.

Abbreviations: ANA, Antinuclear antibodies; ANCA, antineutrophil cytoplasmic antibody or antibodies; BPI, bactericidal/permeability-increasing protein; c-ANCA, cytoplasmic fluorescence staining pattern ANCA; dsDNA, double-stranded DNA; FT₄, free T₄; GB, Graves-Basedow; IIF, indirect immunofluorescence; MMI, methimazole; MPO, myeloperoxidase; p-ANCA, perinuclear staining pattern ANCA; PR3, proteinase 3; TG, thyroglobulin; TPO, thyroid peroxidase; TRAb, TSH receptor antibody; TSH-R, TSH receptor; x-ANCA, atypical ANCA.

Received August 30, 2002.

Accepted February 5, 2003.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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