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(Journal of Leukocyte Biology. 2001;70:46-51.)
© 2001 by Society for Leukocyte Biology

Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils

Hiroaki Asaga*, Katsuhiko Nakashima{dagger}, Tatsuo Senshu*, Akihito Ishigami* and Michiyuki Yamada{dagger}

* Department of Bioactivity Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo, and
{dagger} Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan

Correspondence: Michiyuki Yamada, Ph.D., Graduate School of Integrated Science, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama 236 0027, Japan. E-mail: myamada{at}yokohama-cu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptidylarginine deiminase, registered as PAD V in the DDBJ/GenBank/EMBL data banks, is expressed in HL-60 cells differentiated into granulocytes or monocytes. We analyzed PAD activities in density-fractionated human peripheral blood cell fractions. PAD activity with similar substrate specificity to that of PAD V was found in the eosinophil and neutrophil fractions, which showed single bands comigrating with authentic PAD V on immunoblotting with an anti-PAD V antibody. Both the biochemical and immunoblotting analyses showed marked enrichment of PAD V in the eosinophil fraction. Its immunoreactivity appeared to localize in eosinophilic granules at high density and in myeloperoxidase-negative cytoplasmic granules of neutrophils at low density, as determined by confocal laser-scanning microscopy. Possible roles of PAD V in myeloid differentiation and granulocyte function are discussed. In addition, we present evidence for the presence of PAD(s) that are antigenically different from PAD V in monocytes and lymphocytes.

Key Words: citrullinated proteins • HL-60 cells • peripheral blood cells • granulocytes • monocytes • posttranslational modification enzyme • protein deimination


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptidylarginine deiminases (PADs) are a group of enzymes that convert protein arginine residues to citrulline residues in the presence of calcium ions [1 , 2 ]. Earlier studies based on protein biochemistry and enzymology suggested the presence of at least three types of PADs (types I, II, and III) in mammalian tissues [3 , 4 ]. With recent advances in molecular biological techniques, not only has the presence of these three PAD types been confirmed but that of an additional type IV and another possible type has been revealed (5–13). PADs might be involved in hair follicle biology, epidermal keratinization, and myelination or demyelination of nerve axons. The possibility of PAD involvement has been postulated from the presence of citrulline residues in trichohyalin [1 , 14 , 15 ], filaggrin, and keratins [15 16 17 ], and myelin basic proteins [18 ]. Only limited information is available about PADs in hemopoietic cells. An earlier report from our laboratory describes PAD activities in rat granulocytes and mouse peritoneal macrophages [19 ]. A brief note from another laboratory implicates the presence of PAD in mouse yolk sac erythroid cells immunocytochemically [20 ]. We have also presented evidence suggesting selective deimination of vimentin by endogenous PAD in mouse peritoneal macrophages undergoing calcium ionophore-induced apoptosis [21 ]. However, the types of PAD involved in these observations were rather obscure. We recently reported that a novel type of PAD, tentatively called PAD V, appears in human myeloid leukemia HL-60 cells that have been induced to differentiate into granulocytes with retinoic acid or dimethyl sulfoxide and into monocytes with 1{alpha},25-dihydroxyvitamin D3, and we also observed that isolated relevant cDNA clones could be expressed as a catalytically active recombinant enzyme [11 ]. These findings prompted us to investigate whether PAD V is expressed in human peripheral blood cells. Here, we present evidence showing that eosinophils and neutrophils express PAD that is indistinguishable from PAD V with respect to its substrate specificity with synthetic substrates and its immunoblotting profile. Both immunoblotting and immunocytochemical studies showed marked enrichment of PAD V in eosinophils. In addition, we present evidence for the presence of one or more PADs antigenically different from PAD V in monocytes and lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and fractionation of blood cells
HL-60 cells were cultured in the presence of 1 µM all trans retinoic acid (Sigma Chemical Co., St. Louis, MO) for three days to induce granulocyte differentiation [11 ]. Human peripheral-blood-cell fractions were obtained from heparinized blood of healthy donors, and the cell compositions of the fractions were estimated by Wright’s staining, double esterase cytochemical staining of {alpha}-naphthyl butyrate esterase and naphthol AS-D chloroacetate esterase (Sigma) [22 ], and cytochemical staining of peroxidase.

To obtain eosinophils and neutrophils, red blood cells were removed by sedimentation with dextran 200,000. The cells remaining in the supernatant were sedimented through LymphoprepTM (NYCOMED AS, Norway) to remove the mononuclear cells from granulocytes. The resulting pellet enriched with granulocytes was exposed briefly to water to rupture residual red blood cells and then subjected to Percoll density-gradient centrifugation as described by Sedgwick et al. [23 ]. Briefly, the hypotonically treated granulocytes (values are means plus or minus standard deviations), which consisted of 83.3 ± 4.1% neutrophils and 14.5 ± 3.6% eosinophils, were resuspended in Percoll in calcium- and magnesium-free Hank’s balanced salt solution containing 5% fetal bovine serum (density, 1.070 g/mL) and layered onto multiple discontinuous Percoll gradients consisting of densities of 1.085, 1.090, 1.095, and 1.100 g/mL. After centrifugation for 20 min at 700 g, the cells banded at the 1.095/1.100 g/mL interface and were collected as an eosinophil fraction. The cells remaining in the top layer were collected as a neutrophil fraction. Mean purities obtained from the neutrophil and eosinophil fractions were 96.6 ± 1.6% SD and 88.7 ± 4.4% SD, respectively.

To obtain monocytes and lymphocytes, diluted blood was directly layered onto LymphoprepTM and centrifuged at 800 g for 20 min. The cells retained at the interface were collected as mononuclear cell fractions, which consisted of 11.6 ± 3.6% monocytes, 86.2 ± 3.8% lymphocytes, and 2.2 ± 0.2% neutrophils. These cell fractions were incubated in plastic flasks for 1 h to isolate adherent cells as a monocyte fraction. The cells remaining nonadherent were used as a lymphocyte fraction. Purities obtained from the monocyte and lymphocyte fractions were 96.9 ± 1.0% and 95.5 ± 1.2%, respectively. Red blood cells were obtained by sedimentation through PolymorphprepTM (NYCOMED AS, Norway) according to the manufacturer’s instruction.

Assay of PAD activity
For determination of PAD activity, sample cells were ruptured by sonication, and the entire lysates were incubated with benzoyl-L-arginine ethyl ester (BAEE) or benzoyl-L-arginine (BzArg) as a substrate as described previously [3 ]. One unit was defined as the amount of enzyme catalyzing the formation of 1 µmol of citrulline derivative in 1 h at 50°C. Protein concentrations were determined by the method of Lowry et al. [24 ] using bovine serum albumin as a standard.

Preparation of antibody against recombinant PAD V
A glutathione S-transferase-PAD V fusion protein was expressed in BL-21 cells transformed with pGEX-hPAD containing a 3C protease site and affinity purified using a glutathione-Sepharose 4B column as previously described [11 ]. The PAD V moiety was isolated by PreScission 3C protease digestion followed by glutathione-Sepharose chromatography. A rabbit was immunized with purified recombinant PAD V (about 300 µg) in complete Freund’s adjuvant and boosted by the same antigen twice at 3week intervals. The antiserum collected 3 weeks after the final injection was affinity purified using an immobilized PAD V column, which was prepared by conjugating purified recombinant PAD V to activated cellulofine (2-fluoro-1-methylpyridinium toluene-4 sulfonate cellulofine) (Seikagaku Corp., Tokyo, Japan).

Western blotting
Sample cells prewashed with ice-cold phosphate-buffered saline (PBS) were quickly resuspended in ice-cold 10% trichloroacetic acid and kept on ice for 10–15 min. The acid-fixed cells were collected by centrifugation and washed twice with ice-cold acetone by resuspension and centrifugation. These preparatory steps were necessary to minimize secondary changes due to proteolytic degradation. The resulting pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the method of Laemmli [25 ]. Recombinant rat PAD types I and IV [9 ], purified rat muscle PAD type II [3 ], recombinant human PAD type III (a gift from H. Takahara, Ibaraki University, Ibaraki, Japan) [13 ], and recombinant PAD V were used as references. The resolved bands were transferred to nitrocellulose membranes for staining total proteins with Amido Black 10B or immunoblotting of PAD V using the affinity-purified anti-PAD V antibody (0.5 µg/mL) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (Bio-Rad Laboratories, Hercules, CA) as previously described [11 ]. For preabsorption of the primary antibody, the antibody (0.5 µg/mL) was preincubated with or without a 40-fold molar excess of recombinant PAD V or rat PAD IV for 30 min at room temperature and centrifuged at 13,000 g for 20 min at 4° C. The supernatant was used for probing blots.

Immunofluorescence staining
Sample cell suspensions were fixed for 30 min with ice-cold 4% paraformaldehyde in PBS prior to cytospin preparation on gelatin-coated slides. This process hindered spreading of cells on the slides, thereby impairing microscopic resolution. However, prefixation was necessary because we found secondary relocalization of PAD V immunoreactivity when cytospins were prepared from unfixed cells. The cytospins were postfixed for 30 min with ice-cold 4% paraformaldehyde in PBS, made permeable with 0.1% Triton X-100 in PBS and preincubated with 1% normal goat serum containing 2% bovine serum albumin. They were then incubated successively with affinity-purified anti-PAD V antibody (0.5 µg/mL) and fluorescein isothiocyanate -labeled goat anti-rabbit IgG (Cappel, Organon Teknika Co., Durham, NC). Nuclei were stained with 0.005% propidium iodide (Calbiochem Novabiochem Intl., La Jolla, CA). For double immunofluorescence staining of PAD V and myeloperoxidase, the cytospins were first incubated with a mixture of anti-PAD V (0.5 µg/mL) and anti-myeloperoxidase monoclonal IgG 3-2H3 [26 ] (0.5 µg/mL), next with a mixture of fluorescein isothiocyanate -labeled goat anti-mouse IgG and biotinylated goat anti-rabbit IgG (Vecter Laboratories, Inc., Burlingame, CA), and finally with streptavidin-labeled Cy3 conjugate (Sigma). Stained cells were mounted using a fluorescence antifader, SlowFade antifade kit (Molecular Probes, Leiden, The Netherlands) for fluorescence microscopy. For improved resolution of cytoplasmic granules, cytospins were horizontally scanned as 1-µm-thin sections at 5 µm above the slide surface using a confocal laser scanning microscope (MRC 1000; Nippon Bio-Rad Laboratories, Tokyo, Japan). To identify cell types of the immunofluorescence-positive cells, we subjected cytospins to cytochemical poststaining for peroxidase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PAD activities in peripheral blood cell fractions
Table 1 shows specific PAD activities of various blood cell fractions. Those of control HL-60 cells and HL-60 granulocytes are included as references. The eosinophil fraction showed the highest activity towards BAEE of all the blood cell samples—about 2.6-fold that of the neutrophil fraction and 6.2-fold that of HL-60 granulocytes. It should be noted that both eosinophil and neutrophil fractions showed about 1.5-fold-higher activities towards BzArg than that towards BAEE as HL-60 granulocytes. This is a characteristic of PAD V [11 ]. The monocyte fraction showed activity towards BAEE comparable with that of HL-60 granulocytes. However, the monocyte fraction differed from HL-60 granulocytes in the relative activity towards BAEE and BzArg. The lymphocyte fraction showed low but significant activities towards BAEE and BzArg, which could not be accounted for by contaminating neutrophils and monocytes. The enzyme activity was negligible in the red blood cell fraction as in control HL-60 cells.


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  		Table 1. PAD Activities of Various Peripheral Blood Cell Fractions

 
Western blot analyses of authentic PADs and peripheral blood cell lysates
To examine whether the PAD activities detected as described above represented PAD V, we performed Western blot analyses of the peripheral blood cell fractions. We first tested cross-reactivity of the affinity-purified antibody using authentic PAD preparations. HL-60 cells and HL-60 granulocytes were included as controls. Each preparation gave a single band on protein staining (Fig. 1A , lanes 1–5). Recombinant PAD V appeared to comigrate with recombinant rat PAD type I. Recombinant rat PAD type IV migrated slightly behind, and purified rat muscle PAD type II migrated further behind. Recombinant human PAD type III migrated slightly ahead of recombinant PAD V. Neither recombinant rat PAD type I nor purified rat muscle PAD type II showed a measurable reaction with the affinity-purified antibody (Fig. 1A , lanes 1 and 2). The antibody reacted very weakly with recombinant human PAD type III and strongly with recombinant rat PAD type IV (lanes 3 and 4, respectively). It reacted most strongly with recombinant PAD V, which comigrated with the band detected in HL-60 granulocytes but not in control HL-60 cells (Fig. 1B , lanes 5–7).



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  		Figure 1. Western blot analyses of authentic PADs in comparison with PAD induced in HL-60 granulocytes. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blot analysis as described in the text. (A) Total protein profiles; (B) immunoblot profiles. Lanes 1, recombinant rat PAD type I; lanes 2, purified rat muscle PAD type II; lanes 3, recombinant human PAD type III; lanes 4, recombinant rat PAD type IV; lanes 5, recombinant PAD V; lanes 6, control HL-60 cell lysate; lanes 7, HL-60 granulocyte lysate. HL-60 cell lysate and HL-60 granulocyte lysate were run as negative and positive controls, respectively. Amounts of proteins loaded on lanes 1–5 were about 0.5 µg in panel A and 25 ng in panel B. Those on lanes 6 and 7 were about 30 µg in both panels.

 
We then performed immunoblot analyses of the peripheral blood cell fractions. The neutrophil fraction showed a single strong band (about 67 kDa) comigrating with authentic recombinant PAD V (Fig. 2B , lanes 1 and 2). The eosinophil fraction gave a much stronger band of the identical mobility (lane 3), indicating marked enrichment of PAD V in accordance with results of enzyme assay. The band intensity in these samples was obviously diminished by preabsorption of the antibody with recombinant rat PAD IV (Fig. 2C) . Preabsorption with recombinant PAD V abolished the band completely (Fig. 2D) . No immunopositive bands were detected in the monocyte and lymphocyte (Fig. 2B , lanes 4 and 5). Rabbit anti-rat muscle PAD serum [3 ], which was confirmed to react with human muscle PAD type II [27 ], showed no signals in any blood cell samples examined (data not shown). These data suggested that most of the PAD activity found in the eosinophils and neutrophils could be accounted for by PAD V, while those activities found in monocytes and lymphocytes appeared to be antigenically distinguishable from not only PAD V but also PAD type II.



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  		Figure 2. Western blot analyses of PAD V in neutrophils and eosinophils. The eosinophil and neutrophil fractions were immunoblotted with anti-PAD V in parallel with the authentic PAD. (A) Total protein profiles; (B) immunoblot profiles using anti-PAD V without preabsorption; (C) immunoblot profile using the antibody preabsorbed with recombinant rat PAD IV; (D) immunoblot profiles using the antibody preabsorbed with recombinant human PAD V. Lanes 1, recombinant human PAD V; lanes 2, neutrophil lysate; lanes 3, eosinophil lysate; lanes 4, monocyte lysate; lanes 5, lymphocyte lysate. The amounts of proteins loaded on lanes 1 were 0.5 µg in panel A and 10 ng in panels B–D. Those on lanes 2 and 3 were 30 µg in panel A and 10 µg in panels B–D. Those on lanes 4 and 5 were 30 µg in panels A and B.

 
Immunocytochemical localization of PAD V
To confirm cell types expressing PAD V morphologically and to study its intracellular localization, we performed immunofluorescence microscopy using the affinity-purified antibody. Blood cell fractions at intermediate stages of purification were chosen, so that multiple cell types could be observed under the same microscopic view. First, we examined the granulocyte fraction. More than 95% of cells were PAD V positive, all of which were characterized as granulocytes based on their nuclear morphology, although the intensity varied in individual granulocytes (Figs. 3A and B ). No immunofluorescence signals were observed when normal rabbit IgG was used as a control (data not shown). The PAD V immunofluorescence was localized in the cytoplasm, showing a granular appearance, and was distinguishable from the propidium iodide-positive nucleus. It is interesting that about 15% of PAD V-positive granulocytes exhibited stronger immunofluorescence than that of the rest of the positive granulocytes (Fig. 3A , large arrow). The strongly positive cells were identified as eosinophils based on their enrichment of relatively large cytoplasmic granules by phase-contrast microscopy (Fig. 3C , small arrows) and strong cytochemical peroxidase poststain (Fig. 3F , large arrow). These were confirmed by staining purified eosinophils (data not shown). The rest of the positive granulocytes were regarded as neutrophils based on their moderate peroxidase staining. Preabsorption of the antibody with PAD V virtually abolished the immunofluorescence staining, confirming specificity of the antibody (data not shown). These data were consistent with those obtained by biochemical and Western blot analyses. We next examined the mononuclear cell fraction. Neither monocytes nor lymphocytes were PAD V positive (Fig. 3D and 3E) as could be anticipated from the Western blot analyses. The results also supported specificity of the immunocytochemical staining of PAD V.



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  		Figure 3. Immunocytochemical localization of PAD V in granulocytes. Cytospins of the granulocyte fraction (A–C, F) and the mononuclear cell fraction (D, E) were subjected to immunofluorescence staining of PAD V combined with other appropriate examinations as described in the text. (A, D) Immunofluorescence stain of PAD V; (B) propidium iodide stain of nuclei; (C, E) phase views corresponding to panels A and D, respectively; (F) peroxidase poststain corresponding to panels A–C. Most positive cells were neutrophils. The large arrow shows an eosinophil exhibiting strongly positive signals. Note its marked enrichment with relatively large cytoplasmic granules (small arrows) and strong peroxidase staining. Monocytes (large arrowhead) and lymphocytes (small arrowheads) are negative. Scale bar, 10 µm.

 
To study the localization of PAD V in granulocytes more precisely, we performed confocal laser scanning microscopy after double immunofluorescence staining of PAD V and myeloperoxidase. Strong immunofluorescence of PAD V was detected as relatively large granular signals in the cytoplasm of myeloperoxidase-negative eosinophils (Fig. 4A and B , *). Moderate immunofluorescence of PAD V was detected as relatively small granular signals in the cytoplasm of myeloperoxidase-positive neutrophils (Fig. 4A and 4B , arrowheads). It should be noted that the PAD V-positive signals coincided poorly with the myeloperoxidase-positive signals. This suggests the possible localization of PAD V in neutrophilic granules other than azurophilic granules.



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  		Figure 4. Double immunofluorescence staining of PAD V and myeloperoxidase in neutrophils. Cytospins of the granulocyte fraction were stained with anti-PAD V and antimyeloperoxidase and examined by confocal laser microscopy as described in the text. (A) PAD V; (B) myeloperoxidase. The asterisk shows an eosinophil exhibiting strongly positive granular signals of PAD V without detectable myeloperoxidase signals. Note less intense granular signals of PAD V showing poor coincidence with those of myeloperoxidase. The arrowheads indicate reference points. Scale bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous reports suggest the presence of multiple types of PADs in mammalian tissues that show a specific distribution; i.e., type I in the epidermis and uterus [2 3 4 , 12 ], type II in a wide variety of tissues [3 4 5 6 ], and type III in hair follicles and epidermis [1 2 3 4 , 7 , 8 , 13 ]. The activity of PAD type IV has not been detected in natural tissues, although its transcripts analyzed by reverse transcriptase-polymerase chain reaction suggest its wide occurrence in rat tissues [10 ]. PAD V showed higher amino acid sequence similarity to rat PAD type IV (73%) than to the other types of rat PADs (50–55%). However, we tentatively named it "PAD V," because the kinetic property of recombinant PAD V towards synthetic substrates differed significantly from that of recombinant rat PAD type IV [11 ]. Whether there is human type IV PAD is an open question. The affinity-purified anti-PAD V used in the present study did not show measurable reactions with either recombinant rat PAD type I or purified rat muscle PAD type II. Human recombinant PAD type III showing weak cross-reactivity could be distinguished from PAD V by its electrophoretic mobility. This proved the usefulness of the antibody for probing PAD V in human tissues. The antibody cross-reacted strongly with recombinant rat PAD type IV. This and the relatively high amino acid sequence similarity between PAD V and rat type IV suggest that the two might be orthologous PADs. Future investigation of the localization of PAD type IV in rodent tissues may provide an answer to this problem. In addition, the present study revealed significant activities of PAD(s) in purified monocyte and lymphocyte fractions that could not be detected with either anti-PAD V or anti-rat muscle PAD. These findings and the limited distribution of PAD types I and III indicated the possible presence of still another type or types of PAD in monocytes and lymphocytes that await future investigation.

The present study provided evidence for the presence of PAD V in eosinophils and neutrophils in human peripheral blood. We were unable to study PAD in basophils because of their small number. Although human PAD type III showed weak cross-reactivity with the antibody, it seemed unlikely to account for a significant part of the activity observed in the blood cells. This notion is supported by the comigration of the immunoreactive band with authentic PAD V as well as the positive band in HL-60 granulocytes and widely different relative activities towards synthetic substrates [11 , 13 ].

We recently postulated that expression of the PAD V gene is tightly linked with myeloid differentiation [11 ]. Our recent finding that it was induced in HL 60 granulocytes is consistent with our report here of its localization in eosinophils and neutrophils. However, it is puzzling that PAD V found in HL-60 cells induced to differentiate into monocytes with 1{alpha},25-dihydroxyvitamin D3 [11 ] was not detectable in the isolated monocytes that appeared to express yet unidentified type(s) of PAD as described above. Prolonged cultivation of the isolated monocytes resulted in differentiation into macrophages at least morphologically with loss of PAD activity (data not shown). This agreed with our recent observation that HL-60 cells induced to differentiate into macrophages with 12-O-tetradecanoylphorbol 13-acetate did not show measurable PAD activities [11 ]. However, these findings are not readily reconciled with our earlier reports showing PAD activities in rodent peritoneal macrophages [19 , 21 ]. It is probable that PAD V is expressed at a specific stage of monocyte differentiation in vivo. It should be mentioned that HL-60 cells are not always induced to express all the phenotypes expressed during normal myeloid differentiation in vivo and may be induced to express even phenotypes that are not normally expressed in blood cells [28 ]. Further studies using human macrophages and/or human monocytic leukemia cell lines such as THP-1 [29 ] may yield useful information on the regulation of PAD V during differentiation of cells of human monocyte/macrophage lineage.

The presence of PAD V in eosinophils and neutrophils and its possible localization in the cytoplasmic granules indicate its biological significance. Multiple types of granules present in leukocytes play roles in their bactericidal action and are released into the extracellular space by external signals during bacterial infection, inflammation, and immune responses [30 , 31 ]. Various posttranslational protein modifications during apoptosis or cellular injury have been postulated to be involved in the development of autoantibodies in autoimmune diseases [32 ]. The PAD V-positive granules released from leukocytes infiltrating diseased tissues are likely to cause deimination of various proteins in the extracellular, high-calcium environment. The deiminated proteins formed may be recognized as foreign to the immunological surveillance system leading to formation of antibodies. It is interesting that we reported preferential deimination of vimentin in mouse macrophages undergoing calcium ionophore-induced apoptosis [21 ]. Conceivably the proteins deiminated in apoptotic cells might also be recognized as foreign. Actually, citrulline residues have been shown to constitute epitopes recognized by autoantibodies in patients with rheumatoid arthritis [33 , 34 ]. The exact localization of PAD remains to be clarified by immunoelectron microscopy. Further studies on PAD V and its reaction products are likely to open novel aspects of inflammation, apoptosis, and autoantibody formation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Sasakawa Scientific Research Grant from The Japan Science Society (to K.N.).

We thank Dr. Hidenari Takahara, School of Agriculture, Ibaraki University, for a generous supply of recombinant human PAD type III. We also thank the medical staff of Tokyo Metropolitan Geriatric Hospital for cooperation in collecting blood samples.

Received August 28, 2000; revised January 2, 2001; accepted January 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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