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Keywords:

  • ANCA-associated vasculitis;
  • Drug induced;
  • Propylthiouracil;
  • Hyperthyroidism

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Objective

To test whether antineutrophil cytoplasmic antibodies (ANCA) and ANCA-associated vasculitis (AAV) are not only induced during treatment with antithyroid drugs, but can also become evident when medication has been ceased, possibly after years.

Methods

Patients who visited our hospital for the treatment of hyperthyroidism were included (n = 207). Treatment consisted of antithyroid medications, radioactive iodide, thyroidectomy, or a combination of these treatment options. Patients were retested 3–6 years later to evaluate long-term effects of antithyroid drugs. Patients were tested for the presence of ANCA and, if positive, evaluated for the presence of AAV.

Results

Of 209 patients with hyperthyroidism, 12 patients (6%) were positive for myeloperoxidase- (MPO-), proteinase 3-, or human leukocyte elastase-ANCA. Seventy-seven of 209 patients were retested; 1 patient who had not been treated with antithyroid drugs had developed MPO-ANCA. In 3 of 6 patients previously positive, ANCA could still be detected. The presence of ANCA was highly associated with treatment with antithyroid drugs (odds ratio 11.8 [95% confidence interval 1.5–93.3]). Of 13 patients with a positive ANCA result on enzyme-linked immunosorbent assay, AAV with glomerulonephritis was diagnosed in 4 (31%).

Conclusion

The presence of ANCA with or without vasculitis is associated with previous treatment with antithyroid drugs, possibly after years.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Antineutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3) or myeloperoxidase (MPO) are strongly associated with small-vessel vasculitides, such as Wegener's granulomatosis and microscopic polyangiitis (1–3). There is evidence that ANCA play a pathophysiologic role in the induction of these vasculitides. First, elevations in ANCA titers appear to correlate with risk of future disease flares, although they do not reliably predict the timing of such flares (4, 5). Second, it has been demonstrated in vitro that ANCA induce neutrophil activation, i.e., degranulation and production of a respiratory burst (6). In addition, ANCA activate other cells in vitro, such as monocytes and endothelial cells (7). Finally, in vivo experimental data also suggest an important role for ANCA in the pathophysiology of vasculitis (8, 9). Certain drugs have been linked to the induction of ANCA and the onset of ANCA-associated vasculitis (AAV) (10). There are several case reports implicating hydralazine (11–14) and propylthiouracil (PTU) (15–19) in the induction of AAV. Occasionally, other drugs are mentioned, including other antithyroid drugs, such as carbimazole (17) and methimazole (20, 21).

ANCA in drug-induced AAV are most frequently directed against MPO (11–16, 18–22); however, cases in which patients were PR3-ANCA positive are also observed (12, 15, 18, 20, 22). ANCA in these patients may also be directed against human leukocyte elastase (HLE) or lactoferrin, and the presence of ANCA directed to 2 or more antigens may even be an indication of drug-induced disease (15, 23). Clinically, drug-induced AAV can be substantially milder than idiopathic vasculitis, and drug-induced AAV may resolve in many cases with discontinuation of the offending agent. However, severe disease courses may be observed and treatment with immunosuppressive therapy can be necessary.

To our knowledge, the time span between medication use and induction of ANCA and AAV has not been studied. Furthermore, whether ANCA are induced by hyperthyroidism or by antithyroid medication is still uncertain. We hypothesized that ANCA and AAV may not only be induced during treatment with antithyroid drugs, but can also become evident when medication has been ceased, possibly after years. To test this hypothesis, we evaluated patients with hyperthyroidism receiving various antithyroid treatment modalities for the presence of ANCA. Additionally, we evaluated induction of ANCA after long-term followup by retesting patients >3 years after previous testing for ANCA.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Patients.

All consecutive patients diagnosed with hyperthyroidism (both new diagnosis and relapse) between January 1994 and July 1996 and treated in University Hospital Groningen were included (n = 131). Treatment consisted of antithyroid medications (carbimazole, methimazole, PTU), radioactive iodide, thyroidectomy, or a combination of these treatment options. Additionally, patients who were diagnosed with hyperthyroidism, visited the outpatient clinic of University Hospital Groningen in 1999–2000, and had used antithyroid drugs (carbimazole, methimazole, PTU) during the course of their disease for at least 3 months were included (n = 76). Causes of hyperthyroidism were based on established clinical, laboratory, scintigraphic, and ultrasonographic findings and are presented in Table 1. Patients who were tested for the presence of ANCA between January 1994 and July 1996 were contacted for consent on retesting in 1999–2000. Informed consent was obtained from each patient. When patients were found positive for ANCA (see below), they were invited for evaluation, including physical and laboratory examination for signs of vasculitic disease according to our protocol (24).

Table 1. Causes of hyperthyroidism in groups A and B*
 Group A n (%)Group B n (%)
  • *

    Group A = Patients diagnosed with hyperthyroidism between January 1994 and July 1996; Group B = Patients diagnosed with hyperthyroidism between 1999 and 2000.

Graves and other autoimmune86 (66)58 (76)
Multinodular struma25 (19)14 (18)
Toxic adenoma3 (2)1 (1)
Unknown17 (13)3 (4)
Total13176

Sera.

Serum samples for ANCA testing were collected and stored at −20°C. Samples were coded and realiquoted in identical tubes. Laboratory personnel were blinded to patient identity and medication use.

Testing for ANCA by indirect immunofluorescence (IIF).

Sera were tested for the presence of ANCA by IIF as described previously (25). Briefly, slides coated with human granulocytes were incubated with patient sera in 2 dilutions (1:20 and 1:40) or with positive controls (for perinuclear [p] and classic [c] ANCA) or with a negative control (normal human serum). After addition of conjugate (fluorescein isothiocyanate-labeled rabbit F[ab']2 anti-human IgG; Dako, Glostrup, Denmark), slides were read independently by 2 experienced readers. Fluorescence patterns were scored as pANCA, cANCA, atypical, or negative. When readings were discordant, slides were read independently by a third reader without knowledge of prior fluorescence results. The IIF score was then decided by the majority.

Testing for ANCA by enzyme-linked immunosorbent assay (ELISA).

Each sample was tested by capture ELISA to detect PR3, MPO and HLE ANCA as previously described (1).

Final combined interpretation.

Results of IIF and ELISA were combined for final interpretation. Sera were considered positive for ANCA when capture ELISA was positive for MPO, PR3, or HLE ANCA. IIF staining showing cANCA, pANCA, or atypical ANCA was helpful, but not necessary, for ANCA positivity. Sera were considered negative when IIF was positive without a positive ELISA result or when the results on both assays were negative.

Testing for thyroid microsomal antibodies.

Titers of thyroid microsomal antibodies were determined by indirect haemagglutination using a commercial diagnostics kit according to the manufacturer's instructions (Murex Biotech, Dartford, UK). Thyroid peroxidase (TPO) has been determined as the thyroid microsomal antigen (26). Sera were considered positive when agglutination was present in at least a 1:100 dilution. All patients who were tested in 1999–2000 for the presence of ANCA were tested for anti-TPO antibodies.

Statistical analysis.

Comparisons between groups were analyzed by Fisher's exact test for categorical variables. All tests used a 2-tailed significance level of 0.05. We derived 95% confidence intervals (95% CIs) using exact methods for proportions. Analysis was performed using the GraphPad Instat software package version 2.04a (GraphPad Software Inc., San Diego, CA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cross-sectional evaluation of ANCA positivity in patients with hyperthyroidism.

Between January 1994 and July 1996, 131 patients with hyperthyroidism were tested for the presence of ANCA. An additional 76 patients were tested between 1999 and 2000. Patient characteristics are presented in Table 2.

Table 2. Patient characteristics
 All patients n = 207Patients receiving antithyroid drugs n = 107Patients not receiving antithyroid drugs n = 100Patients with followup sample n = 77Patients without followup sample n = 54
Age, mean ± SD, years49.8 ± 15.148.5 ± 15.251.2 ± 15.049.1 ± 14.751.8 ± 15.3
Female, n (%)165 (80)84 (79)83 (83)65 (84)44 (81)
Antithyroid drug use, n (%)107 (52)  56 (77)43 (80)

On immunofluorescence, cANCA was present in 2 serum samples (1.0%) and pANCA was present in 19 serum samples (9.1%). Atypical ANCA was present in 23 serum samples (11.1%). All other serum samples produced negative staining.

By ELISA, 12 of 131 patients (9.1%) reacted positive in 1 or more of the assays. Four patients (3.1%) were positive for MPO only and 4 patients were positive for HLE only. Additionally, 2 patients were positive for both PR3 and HLE (1.5%), 1 patient (0.8%) for both MPO and HLE, and 1 patient for MPO, PR3, and HLE. Ten patients who were positive by ELISA were also positive in IIF, with pANCA (n = 6) or atypical (n = 4) staining.

Long-term followup of patients with hyperthyroidism.

During followup, 9 of 131 patients previously tested had died (of nonvasculitic causes). Forty-five patients refused to be retested. These patients did not differ from patients who were retested with respect to age, sex, and antithyroid drug use (Table 2). Seventy-seven patients (6 of whom were previously positive for ANCA on ELISA) were retested in 2000. Median time between first and followup sample was 4.8 years (range 3.3–6.0 years). We found 5 of 6 patients still positive for ANCA. One patient, who was previously positive for PR3 and HLE ANCA, was now found negative. One of 71 patients previously negative for ANCA became positive for MPO ANCA. The remaining 70 patients were persistently ANCA negative.

The presence of ANCA is associated with previous treatment with antithyroid drugs.

Overall, 107 patients were or had been treated with ≥1 antithyroid drug (27 with carbimazole, 69 with methimazole, and 38 with PTU); 162 patients were treated with radioactive iodide, and 1 patient had surgery. Eleven (10.3%) patients treated with antithyroid drugs were positive for MPO, PR3, or HLE ANCA, whereas 2 patients who had not been treated with antithyroid drugs (2%) were positive for ANCA. Treatment with antithyroid drugs was highly associated with the presence of ANCA (odds ratio 11.8, 95% CI 1.5–93.3, P = 0.005). However, when patients positive for only HLE ANCA were excluded, there was no significant association between antithyroid drug use and ANCA positivity (P = 0.17). ANCA positivity was not related to any individual antithyroid drug (data not shown). There was no relationship between time on/off antithyroid drugs and the presence of ANCA, nor the time interval between drug initiation and the onset of vasculitic symptoms. Fifty-three percent of patients who were ANCA negative had anti-TPO antibodies compared with 67% of patients who were ANCA positive (P = 0.69). Forty-one percent of patients who were ANCA negative had anticolloid antibodies compared with 25% of patients who were ANCA positive (P = 0.65). Additionally, there was no association between cause of hyperthyroidism and the presence of ANCA (P = 1.0).

Clinical evaluation of ANCA-positive patients.

In 13 patients who tested positive for ANCA, 4 (2 MPO ANCA; 1 MPO and HLE ANCA; and 1 MPO, PR3, and HLE ANCA), all of whom had been or were treated with antithyroid drugs, had signs and symptoms of vasculitis and were treated in our hospital (Table 3). During followup, 2 patients with vasculitis continually tested positive for ANCA on IIF and ELISA, despite treatment with cyclophosphamide and steroids and discontinuation of their antithyroid medication. In the other 2 patients with vasculitis, ANCA titers normalized after treatment with cyclophosphamide and steroids and discontinuation of antithyroid drugs. In the remaining 9 patients, 1 patient with MPO ANCA had impaired renal function and proteinuria (Table 3). His renal biopsy showed changes typical of hypertension in addition to focal segmental glomerulosclerosis of uncertain origin; necrotizing and crescentic glomerulonephritis was not observed. He had discontinued his antithyroid drugs prior to ANCA testing. Seven other patients who had tested positive for ANCA did not show signs of vasculitis. In the ninth patient positive for ANCA, followup on clinical signs was not possible. Two of these 8 ANCA-positive patients without signs of vasculitis were retested in the followup study. The first was again found positive for HLE ANCA, without evidence of vasculitis and without the present use of antithyroid drugs. The second patient (previously PR3 and HLE ANCA positive) was now found negative, without being treated with any immunosuppressive drug, such as cyclophosphamide or steroids.

Table 3. Clinical features of ANCA-positive patients*
Sex/age, yearsDiagnosisDrug use, duration (interval)First ANCA resultSecond ANCA resultClinical result
  • *

    ANCA = antineutrophil cytoplasmic antibodies; interval = time interval between drug initiation and presence of vasculitic manifestations; MMI = methimazole; IIF = indirect immunofluorescence; ELISA = enzyme-linked immunosorbent assay; MPO = myeloperoxidase; CY = cyclophosphamide; CS = corticosteroids; NCGN = necrotizing and crescentic glomerulonephritis; PTU = propylthiouracil; PR3 = proteinase 3; HLE = human leukocyte elastase; FU = followup; CM = carbimazole.

F/68GravesMMI, 5 months (5 months)IIF: pANCA ELISA: MPONegativePleural effusion, renal functional deterioration but no active urine sediment [RIGHTWARDS ARROW] CY + CS
F/49GravesMMI, 8 months (7 years)IIF: pANCA ELISA: MPONegativeNCGN in renal biopsy [RIGHTWARDS ARROW] CY + CS
F/43GravesPTU, 21 months (23 months)IIF: pANCA ELISA: MPO, PR3, HLEIIF: pANCA ELISA: MPO, PR3, HLENCGN in renal biopsy, pulmonary infiltrates [RIGHTWARDS ARROW] CY + CS
F/51GravesPTU, 4 years (4 years)IIF: pANCA ELISA: MPO, HLEIIF: pANCA ELISA: MPO, HLENCGN in renal biopsy [RIGHTWARDS ARROW] CY + CS
M/57UnknownPTU, unknownIIF: atypical ELISA: HLENo FUNo signs of vasculitis
F/35GravesNoneIIF: atypical ELISA: PR3, HLENo FUNo signs of vasculitis
F/63UnknownMMI, unknownIIF: atypical ELISA: PR3, HLENegativeNo signs of vasculitis
F/56UnknownPTU, 6 yearsIIF: atypical ELISA: HLEIIF: pANCA ELISA: HLENo signs of vasculitis
F/88MNSNoneIIF: atypical ELISA: negativeIIF: pANCA ELISA: MPODied of pneumonia and septic shock
F/31MNSPTU, 6 monthsIIF: pANCA ELISA: HLENo FUNo signs of vasculitis
F/38GravesMMI/PTU, 2 yearsIIF: negative ELISA: HLENo FUNo signs of vasculitis
M/40GravesCM/MMI, 2 yearsIIF: negative ELISA: MPONo FUNo signs of vasculitis; glomerulosclerosis in biopsy
F/26GravesCM, unknownIIF: pANCA ELISA: MPONo FUNo followup on clinical signs

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In the current study, we investigated the presence of ANCA in patients diagnosed with hyperthyroidism receiving various treatment modalities. In our cross-sectional evaluation, we found 12 patients (5.7%) positive for MPO, PR3, or HLE ANCA on first testing. Furthermore during followup, 1 patient became ANCA positive. ANCA positivity was significantly related to the use of antithyroid drugs. Clinical signs were present in 2 of 6 ANCA-positive patients who had used PTU, in 2 patients using other antithyroid drugs, and in none of the patients who were not treated with antithyroid drugs.

The presence of ANCA, and of ANCA-associated vasculitis, in patients using antithyroid drugs for treatment of hyperthyroidism have been recurrently reported (15–23, 27). Reported prevalence of ANCA in patients treated with antithyroid drugs varies from 4% to 46%; prevalence of AAV is lower (0–1.4%) (22, 27–29). In our population, ANCA and AAV were present in 11% and 4%, respectively, of patients treated with antithyroid drugs. Prevalence of primary systemic vasculitis (Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, polyarteritis nodosa) has been estimated at 144.5/million (0.01%) in a population comparable with the general population in Groningen (30). Therefore, a prevalence of AAV of 4% in our patients treated with antithyroid drugs clearly suggests a relationship between these drugs and the presence of AAV.

Although ANCA are present relatively often in patients treated with antithyroid drugs, the prevalence of AAV is much lower (22, 27–29). In our study, 7 of 11 patients treated with antithyroid drugs who were positive for MPO, PR3, or HLE ANCA did not have, or develop, vasculitis (64%). This false-positive rate is much higher than could be expected from our earlier studies in which we found false-positive results in <1% of patients with related disorders or healthy controls (25, 31). Therefore, the use of PTU, carbimazole, and methimazole seems an important cause for positive ANCA results not related to small-vessel vasculitis.

The presence of ANCA without vasculitis indicates that ANCA alone are not enough for the induction of vasculitis. Indeed, in patients with clinically quiescent AAV, high ANCA titers can persist without reactivation of vasculitis (5, 32–34). It has been suggested that the characteristics of ANCA present in active disease may differ from those present in disease remission and in sera from patients without vasculitis, possibly in the mechanism of neutrophil activation (7, 35).

The mechanism by which PTU, and other antithyroid drugs, may induce ANCA and AAV remains to be elucidated, although some clues do exist. A study by Jiang et al showed that propylthiouracil, among other drugs, was highly cytotoxic in the presence of activated neutrophils, at concentrations that are therapeutically relevant (36). Oxidation of PTU by activated neutrophils provides a way to generate reactive drug metabolites, and the finding of MPO ANCA in patients with drug-induced lupus and vasculitis is consistent with an autoimmune reaction initiated by drug bioactivation mediated by neutrophil-derived MPO (37).

It is possible that AAV in the patients in our study was not induced by antithyroid drugs, but merely is associated with hyperthyroidism. Unfortunately, we did not have ANCA titers prior to the initiation of treatment in these patients. In a previous study on the induction of ANCA by minocycline, sulfasalazine, and penicillamine, we showed that when ANCA were present in patients after treatment with these drugs, they had already been present prior to treatment start (38). The presence of MPO ANCA may also be explained by the presence of anti-TPO antibodies that given the partial homology between both proteins, may crossreact with MPO (39), although this has been disputed by others (40). In our patients, the presence of MPO ANCA was not related to anti-TPO antibodies. Moreover, crossreactivity to TPO cannot explain the presence of HLE or PR3 ANCA in our patients.

In conclusion, we showed that the presence of ANCA, and associated vasculitis, is not uncommon in patients treated with antithyroid drugs. The presence of ANCA and AAV extends beyond antithyroid treatment duration. This risk of using antithyroid drugs should be included in the decision to use these drugs for the treatment of hyperthyroidism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors would like to thank Ms. Ilby Bouwman (University Hospital Groningen) for performing the ANCA assays, Ms. Kim Savelkouls (University Hospital Maastricht) for performing the anti-TPO assays, and Mr. Bart Meijer, MD for the retrieval of antithyroid antibody results.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES