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. 2002 Jan;127(1):92–98. doi: 10.1046/j.1365-2249.2002.01720.x

Drug-induced neutropenia associated with anti-neutrophil cytoplasmic antibodies (ANCA):possible involvement of complement in granulocyte cytotoxicity

T Akamizu *, S Ozaki *, H Hiratani *, H Uesugi *, J Sobajima *, Y Hataya *, N Kanamoto *, M Saijo *, Y Hattori *, K Moriyama *, K Ohmori *, K Nakao *
PMCID: PMC1906299  PMID: 11882038

Abstract

Although antineutrophil antibodies are thought to be involved in drug-induced neutropenia, neither the precise mechanisms nor the particular antigens on the neutrophil surface have yet been clarified. Recently, we examined a patient with Graves’ disease who developed antineutrophil cytoplasmic antibodies (ANCA) after propylthiouracil treatment and exhibited neutropenia. Because several target antigens of ANCA are expressed on the surface of neutrophils, it was suggested that ANCA might contribute to neutropenia. The patient’s serum bound specifically to neutrophils and HL-60 cells differentiated into granulocytes, and lysed the HL-60 cells via a complement-mediated mechanism. Furthermore, two representative ANCA antigens, proteinase 3 and myeloperoxidase, significantly inhibited both the binding and cytotoxicity of the serum. Finally, tumour necrosis factor-α, which is known to up-regulate cell surface expression of several ANCA antigens, enhanced both the binding and cytotoxicity of the serum. These findings suggest that ANCA induced by propylthiouracil contributed to leucopenia through a complement-mediated mechanism.

Keywords: neutropenia, Graves’ disease, propylthiouracil, ANCA, autoantibody

INTRODUCTION

Drug-induced neutropenia is caused by a variety of mechanisms, including direct toxic effects and immunological reactions. As the immunological mechanisms, opsonizing neutrophils, neutrophil cytotoxicity and neutrophil agglutination by antineutrophil antibodies have been documented [1–4]. Due to the technical difficulties of detecting antibodies to neutrophils, however, the precise role and mechanisms of the antibodies in drug-associated neutropenia as well as the self-antigens have not yet been clarified.

There have been recent studies of antineutrophil cytoplasmic antibodies (ANCA)-positive vasculitis associated with antithyroid drugs, especially propylthiouracil (PTU) [5–7]. Two different ANCA types have been described using indirect-staining immunofluorescence (IIF):cytoplasmic staining (C-ANCA) and perinuclear staining (P-ANCA). Several ANCA antigens, such as proteinase 3 (PR3) and cathepsin G, are known to be translocated from the intracellular region to the plasma membrane and to be accessible to ANCA [8–11]. Although several studies have postulated a direct pathogenic effect of ANCA on vascular endothelial cells [12–14], only a few have documented the effects on neutrophils [15,16]. We examined a patient with Graves’ disease who developed both P- and C-ANCA after PTU treatment and exhibited leucopenia. In the present study we investigated if ANCA plays a pathogenic role in neutropenia, and found that ANCA killed differentiated HL-60 cells by a complement-dependent cytotoxicity.

PATIENT AND METHODS

Patient

A 45-year-old Japanese woman, suffering from Graves’ disease, had been administered 300 mg PTU daily since 31 May 1991 (Fig. 1). She was admitted to hospital on 22 July 1997 due to neutropenia (WBC; 1700 μl, neutrophils; 18% (306 μl), lymphocytes; 78%, monocytes; 2%, eosinophils; 1%, basophils; 1%). Before PTU administration, her neutrophil counts were 2300–2600 μl. Her neutropenia was noted on 2 August 1993 (1065 μl) and 27 April 1994 (238 μl) under 100 mg administration per day of PTU, but she recovered spontaneously without cessation of PTU on both occasions. Since July 1995, chronic neutropenia (98–1460 μl) appeared under 50–75 mg per day of PTU and she was admitted for a radioisotope therapy to our hospital. After admission, PTU was discontinued and 131I] treatment was performed. Cessation of PTU resulted in the gradual increase of neutrophils. Serum at admission was positive in antinuclear antibody (homogeneous pattern), antithyroperoxidase antibody (7·8 U/ml, normal cut-off; <0·3 U/ml) and thyroid-stimulating antibody (1·0 μU/ml bovine TSH equivalent, normal cut-off; <0·3 μU/ml), but negative in antithyroglobulin antibody and TSH-binding inhibitor immunoglobulin. The complements levels were slightly low or lower normal (C3; 58·6 mg/dL (normal 66–153 mg/dL), C4; 20·9 mg/dL (normal 10–43 mg/dL), CH50; 35·1 U/ml (normal 28–51 U/ml)) despite the slight elevation of C-reactive protein (CRP) (0·3 mg/dL, normal cut-off; <0·2 mg/dL). Bone marrow aspiration at admission showed hypercellular marrow, compatible with neutrophil lysis in the peripheral blood. There were no symptoms or signs that suggested vasculitis, such as fever, skin eruption, mononeuritis and proteinuria. Informed consent for the present study was obtained from this patient.

Fig. 1.

Fig. 1

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Changes in neutrophil counts and ANCA activities during PTU treatment. C131I]-Tx; radioisotope treatment by using radioiodine, 131I].

Measurement of ANCA

P-ANCA and C-ANCA were identified by the method of IIF [17,18]. Anti-MPO and anti-PR3 antibodies were measured by the enzyme-linked immunoadsorbent assay (ELISA) and their normal ranges are <13 U/ml or <10 EU/ml [17,18], respectively.

Preparation of neutrophils and HL-60

Peripheral blood mononuclear cells were isolated from heparinized venous blood of a normal individual by centrifugation through a Ficoll Hypaque gradient, as described previously [17]. Subsequently, neutrophils were separated with 2% methylcellulose (Sigma, St Louis, MO, USA) from red blood cells. The HL-60 promyelocytic leukaemia cell line was a gift from Dr Kaoru Toyama (Kyoto University, Japan) and was incubated with dimethyl sulphoxide (DMSO) (Nakarai tesque, Kyoto, Japan) for at least 12 h to induce maximum differentiation to granulocytes [19].

Flow cytometry

Cytofluorographic analysis was performed by a FACScan flowcytometer (Becton Dickinson, Mountain View, CA, USA) using mouse fluorescein isothiocyanate (FITC)-conjugated antihuman IgG antibody (Amrad Biotech, Boronia, Australia). Approximately 106 neutorophils or HL-60 cells incubated with 1 ng/ml of tumour necrosis factor (TNF)-α (Boehringer-Mannheim, Germany). After stained with FITC-antihuman Ig, cells were gated by forward and side scatter. Dead cells were excluded by using propidium iodide or 7-amino-actinomycin D staining and appropriate gating. Dilutions, washings and incubations were performed in phosphate-buffered saline (PBS) at 4 days. Cell surface expression of PR-3 and MPO in HL-60 was tested using monoclonal anti-PR3 (RDI-TRK4P44–1B10; Research Diagnostics, Inc., Flanders, NJ, USA) and anti-MPO (Mob206, MPO-7 clone; DBS, Pleasanton, CA, USA) antibodies. The antibodies diluted from 1:10 to 1:100 were used for cytofluorographic analysis. FITC-conjugated antimouse IgG antibody was used for the second antibody (Amrad Biotech).

Cytotoxicity test

A chromium release assay using HL-60 cells was performed by the method described previously [20]. Cells were labelled with (Na)251CrO4 as 100 μCi/105 cells and used for target cells. For antibody-dependent cell-mediated cytotoxicity (ADCC), target cells were preincubated with 5 μl of the patient’s serum and then various numbers of effector cells, which were mononuclear cell fractions isolated from human peripheral blood, were added. For the cytotoxicity test, 5 × 105 neutorophils or HL-60 cells/ml were incubated with 5 μl of the patient’s serum in the presence or absence of 4 μl of twice-diluted rabbit complement (Cedarlane Laboratories, Hornby, Canada). Percentage specific cytotoxicity was calculated using the following formula:100× (experimental cpm – background release cpm)/(total release cpm – background release cpm), where the total release value was measured by suspending the labelled target cells in water and freeze-thawing three-times. Rabbit antiserum directed against HLA ABC (Biotest, Dreieich, Germany) was used as a positive control. The assays were performed in triplicate. Statistical analysis was performed by using the Mann–Whitney U-test and multiple analysis of variance (manova).

The dye exclusion microcytotoxicity test was performed as described previously [21]. Target cells were incubated with serum and complement as in the chromium release assay. The incidence of cell death was measured from 0·1% nigrosin uptake (Nigrosin B, Nakarai tesque, Kyoto, Japan).

Agents

MPO and PR3 were purchased from Athens Research Technology, Inc., Athens, GA, USA. Both were intact proteins purified from leucocytes.

RESULTS

ANCA activity

The patient’s serum was negative in ANCA in IIF before PTU administration (31 May 1991), and it became positive in both P-ANCA and C-ANCA after administration, 27 April 1994 and 22 July 1997. As shown in Fig. 2, the latter serum stained ethanol-fixed neutrophils in a perinuclear/cytoplasmic pattern (Fig. 2a) and formalin-fixed neutrophils in a cytoplasmic pattern (Fig. 2b), indicating that the serum was positive in both P- and C-ANCA. The former serum did not stain either neutrophils (Fig. 2c,d). In ELISA, the serum possessed positive anti-PR3 antibody (75EU/ml) and anti-MPO antibody (302 U/ml) at admission, while both were negative on 31 May 1991 (Fig. 1). These results suggested that ANCA were induced by PTU and that the appearance of ANCA coincided with neutropenia. In fact, the cessation of PTU resulted in a gradual increase of neutrophils and disappearance of anti-PR3 and anti-MPO antibody activities (Fig. 1).

Fig. 2.

Fig. 2

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Detection of ANCA by IIF using the patient’s serum during (4/27/94) and before (5/31/91) PTU administration. Human granulocytes were incubated with 1 μl of the patient’s serum (a, b:4/27/94, c, d:5/31/91) after ethanol (a, c) or formalin fixation (b, d) and stained with FITC-conjugated antihuman IgG antibody. Arrows show eosinophils, and the other stained cells are neutrophils.

Binding of the patient’s sera to neutrophils

In FACS analysis, the patient’s serum at the time of neutropenia bound to neutrophils, while her serum before PTU administration showed no binding (Fig. 3a). The binding of the serum was significantly enhanced in the presence of tumour necrosis factor (TNF)-α, suggesting strongly that the cytokine enhanced the translocation of self-antigens to the surface of neutrophils (Fig. 3b). The binding activity of her sera disappeared after cessation of PTU (data not shown).

Fig. 3.

Fig. 3

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Binding of the patient’s sera to surface of neutrophils (a), the effect of TNF-α on the binding (b) and absorption of binding the of patient’s sera to neutrophils by PR3 and MPO (c) using flow cytometry. (a) Cells were stained using a 1:50 dilution of the patient’s serum (bolder solid line, during PTU administration (7/22/97); fainter dotted line, before PTU administration (5/31/91)) or control human serum (fainter solid line) and FITC-conjugated antimouse Ig antibody, and analysed by a flow cytometer. (b) Approximately 106 cells were preincubated with 1 ng/ml of TNF-α (Boehringer-Mannheim, Germany) for 2 h at 37ºC. (c) Neutrophils treated with TNF-α were preincubated in the presence (fainter dotted line) or absence (bolder solid line) of both 10–6m PR3 and MPO for 1 h at room temperature and stained using the patient’s serum during PTU administration followed by FITC-conjugated antihuman IgG. As a negative control, the dilution buffer (PBS) was added instead of the peptides. Fainter solid line shows the staining by the patient’s serum before PTU administration. (a) and (b) were performed at the same time, while (c) was performed separately.

In order to clarify the involved antigens, we performed the binding inhibition test. As shown in Fig. 3c, the binding was significantly absorbed by adding a mixture of PR3 and MPO but not by the buffer only, suggesting that anti-PR3 or anti-MPO antibody contributed to the binding. We could not detect their individual inhibitory effects due to the limited amount of the patient’s serum.

Cell surface expressions of PR-3 and MPO in HL-60 cells were tested by staining with monoclonal anti-PR3 and -MPO antibodies. Anti-PR3 antibody stained the cells as shown in Fig. 4, while anti-MPO antibody did not at any dilutions (data not shown). The binding of anti-PR3 antibody was not significantly absorbed by adding 10–6M PR3 protein (Fig. 4).

Fig. 4.

Fig. 4

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Binding of monoclonal anti-PR3 antibody (1:100 dilution) to surface of HL-60 and absorption of the binding by PR3 using flow cytometry. HL-60 cells treated with TNF-α were preincubated in the presence (fainter dotted line) or absence (bolder solid line) of both 10–6 m PR3, as in Fig. 3c. Fainter solid line shows the binding of control mouse IgG.

Cytotoxicity tests

To see the mechanism of neutropenia, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent microcytotoxicity tests were performed. The patient’s serum at admission showed no significant cytotoxicities at any (effector cell)/(target cell) ratio tested in the ADCC test (data not shown). In contrast, target cells were lysed in the presence of complement and the serum without effector cells (Fig. 5a). These results strongly suggested that the ANCA-positive serum killed neutrophils via a complement-dependent mechanism, but not by ADCC, although we cannot entirely exclude other mechanisms. A dye exclusion microcytotoxicity test supported cytotoxicity via a complement-dependent mechanism (data not shown). The finding that the patientís serum with positive ANCA exhibited lower normal complement levels may support the complement-mediated cytotoxicy.

Fig. 5.

Fig. 5

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Effects of TNF-α (a), and PR3 and MPO (b) on the complement-mediated cytotoxicity test. (a) 5 × 105/ml HL-60 cells preincubated in the presence or absence of TNF-α were subjected to the complement-mediated cytotoxicity test:the cells were incubated with 5 μl of the patient’s serum in the presence or absence of rabbit complement. Control human serum (Cont) and antiserum directed against HLA class ABC (HLA) were used as negative and positive controls, respectively. The bars represent the mean ± s.d. of triplicate assay samples. Asterisks mean statistical significance of <0·05. (Mann-Whitney U-test) (b) Variously diluted patientís sera during PTU administration (7/22/97) were preincubated with 10–6m PR3 and MPO (open circle) or buffer (closed circle). They were subjected to the complement-mediated cytotoxicity test using HL-60 cells treated with TNF-α. Rabbit antiserum directed against HLA-ABC (HLA) were used as positive controls (closed square). The symbols are the same as in (b) (manova).

To clarify further the role of ANCA in the cytotoxicity, we treated the target cells with TNF-α to enhance the surface expression of ANCA antigens and tested its effects on the cytotoxicity. As shown in Fig. 5a, percentage specific cytotoxicity significantly increased after preincubation with TNF-α. Taken together with the findings of the binding assay (Fig. 3b), target antigens of cytotoxic antineutrophil antibodies in the patientís serum appeared to be translocated to the cell surface of the neutrophils by TNF-α. To address this point, we examined the inhibitory effect of PR3 and MPO on the cytotoxicity. As shown in Fig. 5b, preincubation of the patientís serum with these agents significantly absorbed the cytotoxicity activity of optimally diluted serum. This result indicates that ANCA, at least in part, contributed to the lysis of the neutrophils via the interaction with their target antigens on the surface of the neutrophils.

DISCUSSION

Here we report a patient who exhibited neutropenia associated with ANCA. Both neutropenia and ANCA appeared during PTU treatment and disappeared after cessation of the drug. Her ANCA-positive serum, which possessed anti-PR3 and anti-MPO antibodies, bound to neutrophils and the binding was inhibited in the presence of PR3 and MPO. Cytotoxicity tests demonstrated evidence that her antineutrophil antibodies lysed the neutrophils via a complement-dependent mechanism, but not by ADCC. The killing activities were augmented by TNF-α and inhibited by a mixture of PR3 and MPO. Since all experiments were performed using serum rather than purified IgG due to limited availability of the patient sera, it cannot be excluded completely that the observed effects could be caused by anything in the serum. These results, however, strongly suggested that ANCA, at least partly, contributed to the cytotoxicity.

PTU, as well as methylmercaptoimidazole (MMI), is widely used for treatment of Graves’ disease. These drugs directly induce neutropenia as do other drugs, such as penicillins and antimicrobial agents. Approximately 0·5–1% of patients treated develop agranulocytosis and a slightly greater percentage of patients may develop a mild to moderate neutropenia [22]. Similar to neutropenia associated with other drugs, the pathogenic mechanisms remain obscure. There are only a couple of studies concerning the mechanism of PTU-induced neutropenia [4,23]. In spite of the possible contribution of antineutrophil antibodies, none of the studies reported on the particular antigens on the surface of the neutrophils. Although Graves’ disease causes frequently neutropenia and harbours antineutrophil antibody [24,25], this will not be true of this patient because neutropenia was not present before PTU administration or after the cessation.

Increasing numbers of case reports have identified associated diseases with ANCA reactivity, which are presumed to be induced by certain drugs, including PTU-induced vasculitis. There are more than 24 previously reported cases of ANCA positive vasculitis in association with PTU [7], although none of them clearly showed neutropenia. The mechanism how PTU induces ANCA is not clearly understood. PTU has been shown to accumulate in neutrophils [26] and bind to MPO, changing its structure [27]. This alteration in configuration may allow initiation of autoantibody formation in susceptible individuals.

There have been only a few studies on neutropenia induced by ANCA [28,29], while a direct pathogenic effect of ANCA on vascular endothelial cells in vasculitis is postulated [12–14]. Since ANCA antigens such as PR3 and cathepsin G can be expressed on the surface of neutrophils as well as endothelial cells and monocytes [8–11], similar effects on neutrophils in drug-induced neutropenia should be considered. More recently, neutrophil apoptosis, as well as activation of neutrophils, was reported to be associated with the translocation of cytoplasmic proteins to the cell surface [15,16]. The rarity of studies about ANCA-related neutropenia may be partly because ANCA are measured only in limited disorders which do not necessarily manifest neutropenia. Alternatively, ANCA may not be a major cytotoxic antibody in ordinary drug-induced neutropenia.

In addition, neutropenia might induce ANCA. Cytokines, such as TNF-α, not only increase the expression of ANCA antigens, but also cause them to translocate from the intracellular region to the plasma membrane of neutrophils [8–11,30]. Bacterial infections facilitated by neutropenia may cause the production of various cytokines and result in induction of ANCA. Although in this situation ANCA production is merely a result of neutropenia, a vicious cycle may arise for neutropenia. The more antigens neutrophils express on their surface, the more molecules of ANCA bind to and damage them. In fact, expression of cell surface antigens of neutrophils and their lysis by the patientís serum were both augmented by TNF-α (Figs 3b and 5a). Furthermore, the binding and cytotoxicity activities were specific to PR3 and MPO (Figs 3c and 5b). These results suggest that ANCA, at least in part, contributes to the lysis of neutrophils.

Although cell surface expression of PR3 or MPO has been shown in neutrophils [8–11,31], that in HL-60 cells is controversial. Most studies showed PR3 or MPO expression only in the cytoplasm of HL-60 [32]. However, even in neutrophils, there was discrepancy; some studies showed the cell surface expression, whereas others did not [9]. This discrepancy might be explained by methodological differences (e.g. the sensitivity of the staining procedure) and/or very limited kinds of monoclonal anti-PR3 and -MPO antibodies (e.g. only one monoclonal anti-MPO antibody and one source of monoclonal anti-PR-3 antibodies are commercially available). Although we found binding of monoclonal anti-PR3 antibody to the surface of HL-60, the binding was not absorbed by PR3 protein and we could not find the binding of monoclonal anti-MPO antibody. Further studies are necessary to clarify these problems.

The antineutrophil antibody of the patient’s serum appeared to lyse neutrophils by the mechanism of complement-mediated cytotoxicity, not ADCC, although we cannot entirely exclude other mechanisms such as leucoagglutins, opsonization of neutrophils or immune complexes of drug and antibody. It should also be kept in mind that HL-60 cells might not substitute directly for mature neutrophils in these experiments. However, the finding that the patientís ANCA-positive serum exhibited slightly low or lower normal complement levels in spite of the slightly elevated CRP may support the complement-mediated cytotoxicy. Complement-mediated cytotoxicy and ADCC were postulated as mechanisms of drug-induced neutropenia [1–4], but neither has been clearly proven. Although both IgG and IgM can mediate the complement-dependent cytotoxicy and be involved in drug-induced neutropenia, complement fixation activities vary among Ig class or IgG subclass (IgM = IgG3 > IgG1 > IgG2). The effects of ANCA on neutrophil function may also change by IgG subclass [33]. Because we presently tested the IgG-class ANCA alone, the issue of Ig class and subclass remains to be elucidated.

ANCA-associated neutropenia was illustrated in the present study. ANCA are known to be induced by association with a variety of drugs and are linked to various autoimmune clinical sequelae [14]. Autoimmune drug-induced neutropenia might be one of the sequelae. To clarify this, it will be essential not only to survey ANCA-positive patients extensively, but also to determine the biological and pathological roles of ANCA in neutrophil intensively.

Acknowledgments

This work was partly supported by a grant in aid from the Mochida Memorial Foundation for Medical and Pharmaceutical Research 1999 to TA. We thank Miss Maki Kochi for excellent secretarial assistance.

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