American Journal ofPathology, Vol. 137, No. 5, November 1990 Copyright © American Association of Pathologists

Ultrastructural Localization of Proteinase 3, the Target Antigen of Anti-cytoplasmic Antibodies Circulating in Wegener's Granulomatosis Elena Csernok,* Jens LUdemann,* Wolfgang L. Gross,* and Dorothy F. Baintont From the Department of Clinical Rheumatology, Medical University of Liubeck and Rheumaclinic Bad Bramstedt GmbH,* Federal Republic of Germany; and the Department of Pathology,t Universitj of California School of Medicine, San Francisco, Californzia

To investigate the distribution ofproteinase 3, the target antigen of anti-cytoplasmic antibodies (ACPA or C-ANCA), within the organelles of resting normal human polymorphonuclear leukocytes and monocytes, the authors used immunocytochemical techniques on thin frozen sections. To obtain valuable toolsfor immunolabeling, two murine monoclonal antibodies (MAbs) directed against the ACPA antigen were produced. In neutrophils, the authors observed immunogold labelfor the ACPA antigen, predominantly in myeloperoxidase-positive azurophil granules, and in smaller amounts on the plasma membrane. In monocytes, the ACPA antigen could be detected in small granules, which occasionally also contained myeloperoxidase, and rare labeling was found on the monocyte membrane. The finding that the ACPA antigen is expressed on the plasma membrane of neutrophils and monocytes, thereby becoming accessible to circulating autoantibodies, supports the supposition that ACPA are not only markers of disease activity, but also are involved in the pathogenesis of Wegener's granulomatosis. (AmjPathol 1990; 137:1113-1120)

Autoantibodies directed against a cytoplasmic antigen of human neutrophils and monocytes (ACPA or cytoplasmic pattern anti-neutrophil cytoplasm antibodies = C-ANCA) have been described as diagnostic markers of Wegener's granulomatosis (WG),1-4 a systemic necrotizing vasculitis

associated with granuloma formation. The ACPA (CANCA) immunofluorescence pattern is defined by diffuse, granular cytoplasmic staining with central accentuation of ethanol-fixed normal neutrophils and monocytes. The antigen associated with the C-ANCA has been shown to be a serine protease of 29 kd.5'6 Recently we have provided strong evidence that the target antigen of ACPA is proteinase 3,7 the third neutral serine proteinase of human polymorphonuclear leukocytes (PMN)."9 Furthermore it was shown that ACPA serum concentrations are closely related to disease activity,10-12 indicating that ACPA may play a pathogenetic role.11 This is further substantiated by the observation that ACPA are able to activate neutrophils in vitro, as evidenced by induction of a respiratory burst and degranulation.13 In this respect it is of particular interest to determine the subcellular localization of the ACPA antigen and, especially, to investigate whether the antigen is expressed in small amounts on the plasma

membrane. This would make the antigen accessible to interaction with the circulating autoantibodies, which might result in neutrophil activation, leading to necrotizing inflammation within the blood vessels. In previous studies, subcellular fractionation techniques based on nitrogen cavitation and Percoll density gradient fractionation were employed for subcellular localization of the ACPA target antigen. The results led to the conclusion that the antigen is associated with azurophil granules of human neutrophils, 4-17 and the same localization was also suggested for proteinase 3.89 But, as indicated by Borregaard and Rasmussen,16 immunoelectron microscopy should be applied to resolve whether the ACPA antigen is part of the azurophil granules or localized in a hitherto unidentified cell constituent that co-purifies with azurophil granules on subcellular fractionation. Supported by grants DK-10486 of the National Institutes of Health and 01 VM 8622 of the Bundesministerium fOr Forschung und Technologie and by the Verein zur Ffrderung der Erforschung rheumatischer Erkrankungen Bad Bramstedt e.V. Accepted for publication June 6, 1990. Address reprint requests to Elena Csernok, Rheumaklinik Bad Bramstedt, Oskar-Alexander Str. 26, 2357 Bad Bramstedt, FRG.

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Figure 1. Typical cytoplasmic staining of neutrophils in indirect immunofluorescence induced by WGM2, a monoclonal antibody directed against theACPA antigen (a) and an ACPA-positive serum from a WG patient (b) (X 700).

In this study we describe the production and characterization of two murine monoclonal antibodies (MAbs) directed against the ACPA antigen. These MAbs were used for ultrastructural localization of the ACPA target antigen with immunocytochemical methods on thin frozen sections of normal PMN and monocytes fixed with paraformaldehyde and/or glutaraldehyde. Using this technique we demonstrate that the antigen is located mainly within large myeloperoxidase-positive azurophil granules of human PMN and also in small cytoplasmic granules of monocytes. In addition, smaller amounts of the antigen are expressed on the plasma membrane of PMN and monocytes.

Materials and Methods Preparation of Monoclonal Antibodies Hybridoma technology was used to generate monoclonal antibodies directed against the ACPA target antigen. The

antigen preparation for immunization was obtained from human neutrophils during degranulation induced by phorbol myristate acetate, as described previously.18 Six-weekold female CD2F1 mice were immunized by subcutaneous injection of 0.05-mg crude antigen in Freund's complete adjuvant (Difco Laboratories, Detroit, Ml). One week after the first immunization, the mice received antigen in phosphate-buffered saline (PBS) 0.05 mg per day intraperitoneally. Five days later, the spleen cells were fused with murine myeloma X63-Ag8.653 cells at a ratio of four spleen cells per myeloma cell. A first selection of hybridomas was made by screening in an enzyme-linked immunosorbent assay (ELISA) with the degranulation supernatant on the solid phase. Thirty-eight culture supernatants were found positive, and they were further tested for reactivity in an ELISA using affinity-purified antigen.18 Two hybridomas (designated WGM1 and WGM2) showed the desired ACPA specificity. They were cloned by three cycles of limiting dilution at one cell per well and grown in serumfree medium (GIBCO BRL GmbH, Eggenstein, FRG) and

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eluted with 0.1 mol/l glycine/HCI, pH 3, neutralized immediately, and dialyzed against PBS.

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Immunocytochemical Techniques on Thin Frozen Sections A

30 20.1 14. 1

2

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Figure 2. Analysis of the monoclonal antibodies in immunoblotting. The target antigen of ACPA was separated in SDSPAGE under nonreducing conditions and probed in immunoblotting with a serum pool containiing ACPA (lane 2), and the murine MAbs WGM1 (lane 3) and WGM2 (lane 4). Relative molecular masses of the marker proteins separated under reducing conditions (lane 1) are shown on the Ieft in kilodaltons.

mouse peritoneum for further characterization of the antibodies. The immunoglobulin isotype was determined using isotyping kits (Serotec, Oxon, England and Nordic Immunology, Tilburg, The Netherlands). The antigen specificity was determined in an ELISA using affinity-purified antigen,18 in immunofluorescence'° and immunoblots.7 To test wether the MAbs recognize the same antigenic site, we used the ELISA additivity test described by Friguet et al'9

Preparation of IgG from an ACPA-positive Serum Thirteen milliliters of an ACPA-positive serum from a patient with WG were diluted 1:2 with 1.5 mol/l (molar) glycine/ NaOH containing 3 mol/l NaCI, pH 8.3 (running buffer), and applied to a 6-ml Protein A Sepharose column (Pharmacia/LKB Biotechnology, Uppsala, Sweden). After extensive washing with running buffer, the bound IgG was

Blood of normal donors was anticoagulated with heparin, and neutrophils and monocytes were sedimented in dextran and washed in Hank's buffered saline solution as described previously.2' Several variations of fixation were used as follows: 1) 8% paraformaldehyde in Nakane's buffer at 40C for 3 hours.; 2) 2% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mol/l phosphate buffer (pH 7.4) at 40C for 1 hour; and 3) 0.5% or 1.5% glutaraldehyde in 0.1 mol/l phosphate buffer (pH 7.4) at 4°C for 10 to 30 minutes. The cells then were transferred to 2.1 mol/l sucrose, frozen, and prepared for thin frozen sections as described previously.?' Sections were incubated as follows: 1) Monoclonal antibodies (serum-free culture supernatant) directed against the ACPA antigen were used in dilutions of 1/400 to 1/1000. The 1 0-nm immunogold probe goat anti-mouse gamma G immunoglobulin (IgG), IgM-gold (GAM-10) (Janssen Life Sciences Products, Olen, Belgium) was used at dilutions of 1/20 to 1/100; 2) Polyclonal antibodies to human myeloperoxidase (Calbiochem Corp., San Diego, CA) or to human lactoferrin (Davo Corp., Cappinteria, CA) were used at dilutions of 1/500 to 1/1000. The 5-nm immunogold probe was goat anti-rabbit Ig-gold (GAR-5) (Janssen Life Sciences Products). Double-labeling experiments were performed by incubating the ACPA MAbs with either of the polyclonal antibodies to myeloperoxidase or lactoferrin, followed by incubation with GAM-10 and GAR-5. As controls, nonimmune purified mouse IgG, IgM, or purified rabbit IgG were used.

Results

Characterization of Monoclonal Antibodies The two hybridomas reacting with the ACPA target antigen produced immunoglobulin of IgM (WGM1) or IgGl (WGM2) class, both with kappa light chains. Both MAbs reacted with affinity-purified ACPA antigen in ELISA and showed the typical diffuse granular staining of the cytoplasm, with some accentuation near the center of the cells, in indirect immunofluorescence on granulocytes and monocytes (Figure 1a). For comparison, immunofluorescence induced by an ACPA-positive serum from a WG patient is shown (Figure 1b). To provide further confirmation of the antigen specificity of the two MAbs, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

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Figure 3. Frozen thin sections of normal neutrophils incubated with antibodies followed by immunogold probes. The cells were incubated with WGM1, a monoclonal antibody directed against theACPA antigen and labeled with GAM- 10 (a). Note that most of the gold grains are in large extracted granules (*). Other organelles such as specific granules (sg) are not labeled. N, nucleus (X50, 000). A similar section has been incubated with a rabbit polyclonal antibody to myeloperoxidase and labeled with GAR-5 (b). The large granules, whose contents are partially solubilized, are labeled for myeloperoxidase, a marker of the azurophil (primary) granules (ag)

(X50, 000).

PAGE) was performed, followed by immunoblotting. Both MAbs reacted with the antigen separated under nonreducing conditions (Figure 2, lanes 3 and 4). The same band was also recognized by an ACPA-positive serum pool from WG patients (Figure 2, lane 2). The MAbs showed no reaction with the antigen separated under reducing conditions (data not shown). Moreover, we have tested by ELISA and immunoblotting the reactivity of our antibodies with purified proteinase 3, which was obtained by purifying with the same chromatographic techniques described by Kao et al.9 Both MAbs reacted with documented proteinase 3. Using the additivity index, we compared the epitope specificities of the two MAbs and found no significant addition in absorbance using an ELISA with affinity purified antigen for the two MAbs (additivity index = 6.2%). Therefore, the MAbs WGM1 and WGM2 recognize either the same antigenic site or overlapping regions.

Localization of the ACPA Antigen Our goal was to investigate the distribution of the ACPA target antigen within the organelles of resting normal hu-

man PMN and monocytes. Thin frozen sections were prepared and incubated with the monoclonal antibodies, using mainly the IgM antibody (WGM1) and immunogold to label the antigenic sites, because background staining was minimal and better than with IgG. As can be seen in Figure 3a, many (approximately 85%) of the large extracted granules contained immunogold grains. Rarely labeling was also seen on the plasma membrane (illustrated later). Other organelles, including the specific granule population, were negative. The IgG preparation of an ACPA-positive serum from a WG patient labeled mainly the same granules as the monoclonal antibodies, but more background labeling over the whole section was observed (not shown).

Localization of Myeloperoxidase Because the large extracted granules were believed to correspond to azurophil (or primary) granules, a known content of these granules, myeloperoxidase, was detected using polyclonal antibodies. As can be seen in Figure 3b,

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Figure 4. A portion of a human neutrophil that has been double immunolabeled with the monoclonal antibody WGM1 and GAM10 to detect the presence of the ACPA antigen and a polyclonal antibody to myeloperoxidase labeled with GAR-5. Both sizes of gold grains are present in azurophil granules (ag). Note the absence of labeling in the specific granules (sg). M, mitochondria; N, nucleus (X80,000).

the gold particles (GAR-5) labeled the large extracted azurophil granules. The specific granules and other organelles were not labeled.

Double Labeling of the ACPA antigen and Myeloperoxidase or Lactoferrin The results of double labeling the ACPA antigen with the MAb WGM1, followed by GAM-10 and myeloperoxidase with a polyclonal antiserum followed by GAR-5, are shown in Figure 4. The two sizes of gold particles colocalized in the large extracted granules, allowing us to conclude that the ACPA antigen is located in the azurophil (primary) granule population. Counts of 126 large extracted granules showed colocalization in 85% of the extracted granules, whereas 15% labeled with myeloperoxidase (MPO) alone. In addition, when the MAb WGM1 and an anti-lactoferrin antiserum were incubated, they did not colocalize (Figure 5a, b). Labeling for lactoferrin (GAR-5) was present in the matrix of many specific granules, whereas the ACPA an-

tigen (GAM-10) was present in azurophil granules, and in smaller amounts on the plasma membrane.

Double Labeling of the ACPA Antigen and Myeloperoxidase in Monocytes Monocytes also were examined in the double-labeling experiments. The ACPA antigen could be detected in small granules (Figure 6), which are found less frequently than the azurophil granules in PMN, and myeloperoxidase occasionally colocalized with the ACPA antigen (see inset, Figure 6). Rare plasma membrane labeling was also observed.

Discussion The present report describes the development and characterization of two monoclonal antibodies directed against the target antigen of anti-cytoplasmic antibodies associated with WG, which has recently been identified as

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Figure 5. Portions ofa normal human neutrophil that has been double labeled for lactoferrin with a polyclonal serum and the small gold grains (GAR-5) andfor the ACPA antigen with the monoclonal antibody WGM1 and the large gold grains (GAM- 10). The ACPA antigen labeled by large gold grains is localized in the larger, more extracted azurophil granules (ag), and lactoferritn is detected by the small gold grains in the specific granules (sg). Note that a small amount of label is present on the plasma membrane (arrows). N, nucleus. a: X 60, 000; b: X84, 000.

proteinase 37 These MAbs were used for immunolocalization of the ACPA antigen at the ultrastructural level of normal human PMN and monocytes. Both MAbs reacted similarly in different assays, including ELISA, immunoblofting, and immunofluorescence. Investigations of epitope specificities demonstrate that the two MAbs recognize the same antigenic site. In immunoblot analysis, both antibodies reacted with the ACPA antigen separated in SDS-PAGE under nonreducing conditions, but no reaction was observed with the reduced antigen. This observation suggest that the MAbs recognize a conformational epitope of the protein. The same findings were obtained using sera from patients with Wegener's granulomatosis that contained ACPA.7 In indirect immunofluorescence, both MAbs stained neutrophil and monocyte cytoplasm in the typical granular manner, whereas no immunostaining of the plasma membrane was observed. In our study, the ultrastructural localization of the ACPA antigen was done by an immunogold staining technique on thin frozen sections. The advantage of this method is that the integrity of the cell is preserved and the risk of artifacts is decreased.21 However, to preserve the antigenicity, we initially used paraformaldehyde fixation to lo-

calize the ACPA antigen, as many antigens are sensitive to glutaraldehyde fixation. We frequently found 'spillage' of the gold near the granules and poor localization. As we increased the concentration of glutaraldehyde to 1.5%, we were able to decrease the 'leakage' and localized the antigen mainly in azurophil granules. We believe that small molecular weight contents of the granules may be difficult to contain when incubated on thin frozen sections. For example, localization of defensins (molecular weight: 3500) required 0.5% glutaraldehyde.22 However, the MAbs directed against the ACPA antigen have proved to be a useful tool for ACPA-antigen detection at the ultrastructural level. Finally both subcellular fractionation techniques and immunoelectron microscopy are important methods for ultrastructural analyses and should be used complementary in localization studies of intragranular constituents. We have confirmed by immunoelectron microscopy that the ACPA antigen is located in azurophil granules of human PMN, as previously described using the subcellular fractionation technique,14'17 and moreover we could show that it is expressed in small amounts on the plasma membrane of normal blood PMN and monocytes. In addition, we have detected this antigen in small granules of monocytes, which occasionally also contained myeloperoxi-

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vg%,.

Figure 6. A portion of a normal human monocyte which has been double-labeled for the ACPA antigen and myeloperoxidase, as describedfor PMN in Figure 4. Granules (g) containing either myeloperoxidase (GAR-5) or the ACPA antigen (GAM- 10) can be detected in the monocyte cytoplasm. Occasionally colocalization of myeloperoxidase and the ACPA antigen can be seen (inset). Rare labeling for the ACPA antigen by the large gold grains is present in a clear vacuole, which is presumably associated with the plasma membrane (arrow)

(X52, 000; inset, X81, 000).

dase. As the antigen is not only found in neutrophils, but also in monocytes, we still prefer the more general acronym ACPA (anti-cytoplasmic antibodies) instead of the new nomenclature ANCA (anti-neutrophil cytoplasm antibodies). Recently, Calafat et al23 have shown similar results by demonstrating the localization of the ANCA-antigen in azurophil granules of normal human neutrophils and in MPO-positive granules of monocytes using immunoglobin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. But in contrast to our results, they did not see membrane labeling. Although partial degranulation is always possible with isolation procedures, it should be pointed out that even with intentional degranulation (Calafat et a123), the authors did not see membrane staining. We believe our frozen thin sections procedure is more sensitive then the Lowicryl K4M (Bio-Rad, Veenendal, The Netherlands) method used, and may explain this difference in findings.

Previous studies have shown that ACPA are directed against proteinase 3, the third neutral serine proteinase of human neutrophils,7 which seems to be identical to an antimicrobial protein described by Gabay et al.24 Therefore the current investigations implicate that proteinase 3 is not only located in the azurophil granules of human neutrophils, but also in small cytoplasmic granules of monocytes, and moreover, is expressed in small amounts on the plasma membrane of both cell types. Although proteinase 3 was already described in 1978 by Baggiolini et al,8 no further characterization was done until recently.9 Kao et a19 have demonstrated that proteinase 3 is only the second enzyme along with elastase purified from human neutrophils that causes experimental emphysema, indicating that it might be involved in the induction of pulmonary damage and possibly other destructive diseases. In a recent study the hypothesis was raised that the neutrophils might express on their surface small amounts of primary granule constituents, which thereby become

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available to interact with ACPA, resulting in neutrophil activation.13 We have confirmed by immunoelectron microscopy that the ACPA antigen is indeed expressed on the plasma membrane, and this finding supports the supposition that ACPA are a pathogenetic factor and that immunologic mechanisms are important in Wegener's granulomatosis.

References 1. van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es LA, van der Giessen M, van der Hem GK, The TH: Auto-antibodies against neutrophils and monocytes: Tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1985, i:425-429 2. Gross WL, LOdemann G, Kiefer G, Lehmann H: Anti-cytoplasmic antibodies in Wegener's granulomatosis. Lancet

1986, i:806 3. Ludemann G, Gross WL: Autoantibodies against cytoplasmic structures of neutrophil granulocytes in Wegener's granulomatosis. Clin Exp Immunol 1987, 69:350-357 4. Parlevliet KJ, Henzen-Logmans SC, Oe PL, Bronsveld W, Balm AJM, Donker A: Antibodies to components of neutrophils cytoplasm: A new diagnostic tool in patients with Wegener's granulomatosis and systemic vasculitis. Q J Med 1988, 66:55-63 5. Goldschmeding R, Van der Schoot CE, Ten Bokkel Huinink D, Hack CE, Van den Ende ME, Kallenberg CGM, Von dem Borne AEGK: Wegener's granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding proteine in the lysosomes of normal human neutrophils. J Clin Invest 1989, 84:1577-1587 6. Niles JL, McCluskey RT, Ahmad MF, Arnaout MA: Wegener's granulomatosis autoantigen is a novel neutrophil serine proteinase. Blood 1989, 74:1888-1893 7. Ludemann J, Utecht B, Gross WL: Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme. J Exp Med 1990, 171:357-362 8. Baggiolini M, Bretz U, Dewald B, Feigenson ME: The polymorphonuclear leukocyte. Agents Actions 1987, 8:3-10 9. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal GR: Proteinase 3: A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988, 82:1963-1973 10. Nolle B, Specks U, Ludemann J, Rohrbach MS, DeRemee RA, Gross WL: Anticytoplasmic autoantibodies: Their immunodiagnostic value in Wegener granulomatosis. Ann Intern Med 1989, 111:28-40 11. Gross WL: Wegener's granulomatosis: New aspects of the disease course, immunodiagnostic procedures and stageadapted treatment. Sarcoidosis 1989, 6:15-29 12. Specks U, DeRemee RA: Significance of antibodies to cytoplasmic components of neutrophils. Thorax 1989, 44:369370

13. Jennette JC, Falk RJ: Anti-neutrophil cytoplasmic autoantibodies: New insight into crescentic glomerulonephritis, pulmonary-renal syndrome, and systemic vasculitis. AKF Nephrology Letter 1989, 6:11-18 14. Rasmussen N, Borregaard N, Wiik A: Anti-neutrophil-cytoplasm antibodies in Wegener's granulomatosis are not directed against alkaline phosphatase. Lancet 1989, i:1488 15. Goldschmeding R, Ten Bokkel Huinink D, Faber N, Tetteroo PAT, Vroom TM, Hack CE, von dem Borne AEG Kr: Identification of the ANCA-antigen as a novel myeloid lysosomal serine protease. Acta Pathol Microbiol Immunol Scand 1989, 97(Suppl 6):46 16. Borregaard N, Rasmussen N: Constituents of the human neutrophil cytoplasm: Methods for examination and methodological problems with special reference to the binding side of the anti-neutrophil cytoplasmic antibodies. Acta Pathol Microbiol Immunol Scand 1989, 97(suppl. 6):10-11 17. Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988, 318:1651-1657 18. LUdemann J, Utecht B, Gross WL: Detection and quantification of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by ELISA using affinity-purified antigen. J Immunol Methods 1988,114:167-174 19. Friguet B, Djavadi-Ohaniance L, Pages J, Bussard A, Goldberg M: A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic sites. Application to hybridomas specific for the ,B2-subunit of Escherichia coli tryptophan synthase. J Immunol Methods 1983, 60:351-358 20. Bainton DF, Miller LJ, Kishimoto TK, Springer TA: Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils. J Exp Med 1987,166:1641-1653 21. Cramer E, Pryzwansky KB, Villeval JL, Testa U, Breton-Gorius J: Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold. Blood 1985, 65: 423-432 22. Ganz T, Selsted ME, Szklarek D, Harwig SSL, Daher DF, Bainton DF, Lehrer RI: Defensins, natural peptide antibiotics of human neutrophils. J Clin Invest 1985, 76:1427-1435 23. Calafat J, Goldschmeding R, Ringeling PL, Janssen H, Van der Schoot CE: In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophil and monocytes. Blood 1990, 75: 242-250 24. Gabay JE, Scott RW, Campanelli D, Griffith J, Wilde C, Marra MN, Seeger M, Nathan CF: Antibiotic proteins of human polymorphonuclear leukocytes. Proc NatI Acad Sci USA 1989, 86:5610-5614

Acknowledgments The authors thank Yvonne Jaques and Bert Utecht for their professional assistance.

Ultrastructural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener's granulomatosis.

To investigate the distribution of proteinase 3, the target antigen of anti-cytoplasmic antibodies (ACPA or C-ANCA), within the organelles of resting ...
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