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Rheumatology 2001; 40: 851-858
© 2001 British Society for Rheumatology

Original Papers

Monoclonal anti-double stranded DNA antibody is a leucocyte-binding protein to up-regulate interleukin-8 gene expression and elicit apoptosis of normal human polymorphonuclear neutrophils

S.-C. Hsieh, K.-H. Sun1, C.-Y. Tsai2, Y.-Y. Tsai2, S.-T. Tsai2, D.-F. Huang2, S.-H. Han2, H.-S. Yu3 and C.-L. Yu4,

Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei,
1 Faculty of Medical Technology, National Yang-Ming University, Taipei,
2 Department of Medicine, Veterans General Hospital-Taipei, Taipei,
3 Department of Dermatology, Kaohsiung Medical University, Kaohsiung and
4 Department of Medicine and Institute of Molecular Medicine, National Taiwan University College of Medicine, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To determine whether anti-double stranded DNA (anti-dsDNA) autoantibody could bind and affect the functions of normal human polymorphonuclear neutrophils (PMN).

Methods. Normal human PMN were incubated with different concentrations of a monoclonal mouse anti-dsDNA antibody (12B3) or mouse isotype-matched IgG2a. The binding of anti-dsDNA and PMN was measured by flow cytometry and interleukin-8 (IL-8) gene expression in PMN was detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription–polymerase chain reaction (RT–PCR). PMN apoptosis was justified by morphological changes. The cognate antigen(s) of anti-dsDNA on the PMN surface was identified by membrane biotinylation, immunoprecipitation and Western blot.

Results. The binding of PMN with anti-dsDNA was much higher than with non-specific mouse IgG2a (70.8 vs 2.0%). Anti-dsDNA at concentrations higher than 12.5 ng/ml significantly enhanced the production and mRNA expression of IL-8 by PMN. However, anti-dsDNA facilitated PMN apoptosis after 3 h incubation. Western blot analysis of biotinylated PMN cell lysates demonstrated that a 50–52 kDa membrane molecule is the cognate antigen of anti-dsDNA.

Conclusions. Anti-dsDNA autoantibody up-regulates IL-8 gene expression and elicits activation-induced cell death (AICD) of human PMN via binding to a 50–52 kDa membrane-expressed molecule.

KEY WORDS: Monoclonal anti-dsDNA antibody, Polymorphonuclear neutrophils, Interleukin-8, Apoptosis, Activation-induced cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Anti-double stranded DNA antibodies (anti-dsDNA) are not exclusively found in patients with systemic lupus erythematosus (SLE). IgG class anti-dsDNA has a high specificity for SLE and the serum titre can reflect the disease activity of this disorder [1, 2]. Increased susceptibility to infections is clinically a major cause of morbidity and mortality in SLE patients [3, 4]. Although treatment with glucocorticoids or immunosuppressants is an important contributory factor, decreased number [5, 6] as well as impaired functions [7–9] of polymorphonuclear neutrophils (PMN) per se are crucial to the susceptibility to bacterial infections in these patients. Many authors have demonstrated that the presence of antineutrophil antibodies (anti-PMN) correlated well with neutropenia in patients with SLE [10–15]. However, the real pathological role and the identity of cognate antigen(s) of anti-PMN autoantibodies have not been reported. In our previous study, we demonstrated that polyclonal and monoclonal anti-dsDNA bound to human acidic ribosomal phosphoproteins (P0, P1 and P2) expressed on the surface of many cells and exerted cytostatic/cytotoxic effects on these cells [16]. The target cells of anti-dsDNA included glomerular mesangial cells, fibroblasts, astrocytes, hepatocytes, splenocytes and mononuclear cells (MNC) [16–18]. These results suggest that anti-dsDNA is probably a neutrophil-binding antibody capable of deranging the neutrophil functions. To test this hypothesis, a monoclonal anti-dsDNA antibody, 12B3, derived from autoimmune MRL-lpr/lpr mice was used. Several novel biological/immunological activities of this antibody on human PMN were found.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Generation and characterization of mouse monoclonal anti-dsDNA antibody
Monoclonal anti-dsDNA antibody, 12B3, was generated by fusing NS-1 myeloma cells and spleen cells from a 5-month-old autoimmune MRL-lpr/lpr mouse (Jackson Laboratories, Bar Harbor, ME, USA). The antibody was determined to be IgG2a subclass by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Boehringer Ingelheim Biochemicals, GmbH, Mannheim, Germany). The calf thymus dsDNA binding capacity of 12B3, NS-1 cultured supernatant, and mouse isotype- and subclass-matched non-specific IgG2a purchased from Sigma Chemical Company (St. Louis, MO, USA) was measured by ELISA as described elsewhere [19]. Briefly, polystyrene microtitre plates (Corning Co., New York, NY, USA) were pre-coated with 150 µl of 0.5 mg/ml protamine chloride (Sigma) per well at 37°C for 2 h and then washed with phosphate-buffered saline (PBS), pH7.2. Commercially obtained calf thymus dsDNA (Sigma) was further purified and added to the microwells following the method of Rauch et al. [20]. The dsDNA binding capacity of 50 ng/ml 12B3 was 1.85 compared with 0.103 of 50 ng/ml mouse non-specific IgG2a at OD410 absorbance.

Isolation and culture of normal human PMN
Heparinized venous blood obtained from normal individuals was mixed with a quarter volume of 2% dextran solution (molecular weight 500 000) and incubated at 37°C for 20 min. The supernatant enriched in leucocytes was collected and diluted with the same volume of Hanks’ balanced salt solution. After centrifugation at 150 g for 30 min over a Ficoll-Hypaque density gradient cushion (specific gravity 1.077) (Pharmacia Biotech Far East Ltd., Hong Kong) PMN were obtained from the bottom. The purity and viability of the PMN suspension were greater than 95%, confirmed by Wright's stain and Trypan blue dye exclusion. The concentration of PMN was adjusted to 2 x 106/ml. PMN (0.5 ml) were mixed with 0.1 ml of different concentrations (6.25–100 ng/ml) of 12B3 or mouse non-specific IgG2a and 0.4 ml of RPMI-1640 containing 10% fetal bovine serum (10% FBS-RPMI). The mixture was incubated at 37°C in 5% CO2–95% air for 24 h. The cell-free cultured supernatants were stored at - 20°C until used.

Detection of the binding of anti-dsDNA and PMN by flow cytometry
The binding of anti-dsDNA or mouse non-specific IgG2a and PMN was detected by FACSort flow cytometry (Becton-Dickinson, San Jose, CA, USA). Briefly, 0.5 ml of PMN (2 x 106/ml) was incubated with 0.1 ml of 12B3 (500 ng/ml) or mouse non-specific IgG2a (500 ng/ml) in an ice bath for 30 min. After three washes with PBS, pH 7.2, the cell pellets were then incubated with 1 : 2000 diluted FITC-labelled goat anti-mouse IgGs in an ice bath for another 30 min. After three washes, the percentage binding and mean fluorescence intensity (MFI, represented by the mean channel number) of PMN and anti-dsDNA were measured by flow cytometry at 488 nm excitation.

Measurement of interleukin-8 (IL-8) concentration in PMN cultured supernatants by ELISA
The IL-8 concentration of PMN cultured supernatants was measured by a commercially available ELISA kit (Quantikine, R&D System, Minneapolis, MN, USA). The minimal detectable concentration of IL-8 was 18.l pg/ml by the kit.

Detection of IL-8 mRNA expression in PMN by reverse transcription–polymerase chain reaction (RT–PCR)
Total cellular RNA extraction and cDNA synthesis
Total cellular RNA was extracted from PMN (1 x 107/ml) after 2 h incubation with anti-dsDNA (50 ng/ml) or mouse non-specific IgG2a (50 ng/ml) at 37°C in 5% CO2–95% air according to the method reported by Chomczynski and Sacchi [21]. cDNA was synthesized by priming l µg/ml of total RNA at 42°C for 1 h in a final volume of 20 µl containing l µg of oligo-dT primer (Pharmacia Fine Chemicals, Piscataway, NJ, USA), 200 nmol of each dNTP (Pharmacia), and mMLV reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD, USA) at 200 U/µg RNA.

Amplification of cDNA by PCR
An aliquot of cDNA was amplified by PCR using paired oligonucleotide primers specific for human IL-8 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH, as an internal control) (Clontech Laboratories, Inc., Palo Alto, CA, USA).

Human IL-8:

5'ATGACTTCCAAGCTGGCCGTGGCT3' (sense)
5'TCTCAGCCCTCTTCAAAAACTTCTC3' (anti-sense)

Human G3PDH:

5'ACCACAGTCCATGCCATCAC3' (sense)
5'TCCACCACCCTGTTGCTGTA3' (anti-sense)

A Hybaid OmniGene DNA thermal cycler (Teddington, Middlesex, UK) was used to run 35 cycles of denaturation at 95°C for 1 min and annealing/extension at 60°C for 2 min for IL-8 and 26 cycles of denaturation at 94°C for 1 min and annealing/extension at 65°C for 2 min for G3PDH. The PCR products were then electrophoresed in 1.8% agarose gels using {phi}X174 digested with HaeIII enzyme as the calibration markers. The cDNA fragments obtained by these sets of primers were 289 bp for IL-8 and 452 bp for G3PDH.

Identification of PMN apoptosis by morphology after haematoxylin–eosin stain
PMN (2 x 106/ml) were incubated with anti-dsDNA (50 ng/ml) or mouse non-specific IgG2a (50 ng/ml) for 1, 3 and 24 h at 37°C in 5% CO2–95% air. After the incubation, the cell suspension was cytospun at 2000 r.p.m. for 20 min. The cells attached to a glass slide were then dried, fixed and stained using a standard haematoxylin–eosin method. The cells with karyopyknosis/karyorrhexis in the nuclei were considered to be undergoing apoptosis.

Detection of the cognate antigen(s) of anti-dsDNA on the surface of human PMN
Membrane biotinylation of human PMN
Freshly isolated PMN (5 x 106/ml) were washed with borate buffer (50 mM sodium borate in 150 mM NaCl, pH 8.0). D-biotinoyl-{varepsilon}-aminocaproic acid N-hydroxy-succinimide ester (biotin-7-NHS; Boehringer Mannheim) was diluted with borate buffer pH 8.0 to a concentration of 100 µg/ml. Equal volumes of cell suspension and biotin-7-NHS solution were incubated at 4°C for 30 min with gentle agitation. The cells were then washed twice with Tris-buffer, pH 8.3. The cell lysates were prepared by repeated sonication in extraction buffer (50 mM borate, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 2.5 mM phenylmethylsulphonylfluoride, pH 8.0) and incubated for 30 min in an ice bath. The homogenates were further cleared by centrifugation at 12 000 r.p.m. for 20 min.

Detection of the cognate antigen(s) of anti-dsDNA in biotinylated PMN cell lysates after immunoprecipitation by Western blot
The biotinylated PMN lysates were subjected to two rounds of immunoprecipitation, starting with human non-specific {gamma}-globulin-conjugated agarose beads (Sigma) and then reacted with streptavidin-conjugated beads (Pierce, Rockford, IL, USA) at 4°C overnight. After PBS washes, the beads were suspended in 2x sodium dodecyl sulphate (SDS) sample buffer and heated at 100°C for 5 min to separate the membrane proteins and beads. After centrifugation, the clear supernatants were electrophoresed using 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and probed by anti-dsDNA in a Western blot. The signal was detected by an enhanced chemiluminescence protein detection kit (Amersham International plc, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions.

In addition, a number of cells including human MNC and red blood cells (RBC), K-562 (human erythroleukaemia cell line), HL-60 (human promonocytic leukaemia cell line), Jurkat cells (human T-lymphoma cell line), RBA-1 (rat brain astrocyte cell line), and RKO cells (human colon cancer cell line) were also used in the same experiment.

Statistical analysis
The results are presented as mean ± standard deviation (S.D.). Statistical significance was assessed by Kruskal–Wallis one-way analysis of variance on ranks (Dunnett's method) for multiple comparisons in the Sigma Stat software program.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Binding of anti-dsDNA with PMN
A representative case showing the binding of PMN with anti-dsDNA is demonstrated in Fig. 1Go. The percentage binding of PMN with anti-dsDNA [70.8% in Fig. 1Go(4)] is much higher than that of mouse non-specific IgG2a [2.0% in Fig. 1Go(3)]. The same experiment was repeated four times with a similar tendency.



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  		FIG. 1. A representative case demonstrating the binding of anti-dsDNA (50 ng/ml) or mouse non-specific IgG2a (50 ng/ml) with normal human neutrophils (PMN) detected by flow cytometry. (1) PMN + medium (no antibody added); (2) PMN + medium + secondary antibody (2000x diluted FITC-labelled goat anti-mouse IgGs); (3) PMN + mouse non-specific IgG2a + secondary antibody; (4) PMN + anti-dsDNA + secondary antibody. %, percentage binding; MFI, mean fluorescence intensity (mean channel number) of the cells. The same experiment was repeated four times with a similar tendency.

 

Effect of anti-dsDNA on IL-8 gene expression of normal human PMN
To determine whether anti-dsDNA can modulate IL-8 gene expression of PMN, different concentrations of anti-dsDNA from 6.25 to 100 ng/ml were tested. We found that anti-dsDNA at concentrations higher than 12.5 ng/ml significantly enhanced IL-8 protein production (Fig. 2Go) and mRNA expression (Fig. 3Go) of PMN with a maximal effect at 25 ng/ml compared with the mouse non-specific IgG2a (100 ng/ml).



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  		FIG. 2. Dose–response effect of monoclonal anti-dsDNA antibody (6.25–100 ng/ml) on IL-8 production by normal human PMN after 24 h incubation. Mouse non-specific IgG2a (100 ng/ml) and bacterial lipopolysaccharide (LPS, 100 ng/ml) were used as negative and positive controls, respectively. *P < 0.05.

 


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  		FIG. 3. Effect of anti-dsDNA (50 ng/ml) and mouse non-specific IgG2a (50 ng/ml) on IL-8 mRNA expression in normal human PMN after 3 h incubation detected by RT–PCR. Lane l, G3PDH (internal control; 452 bp) in PMN + medium; lane 2, IL-8 (289 bp) in PMN + medium; lane 3, G3PDH in PMN + mouse non-specific IgG2a; lane 4, IL-8 in PMN +mouse non-specific IgG2a; lane 5, G3PDH in PMN + anti-dsDNA; lane 6, IL-8 in PMN + anti-dsDNA. The same experiment was repeated twice with a similar tendency.

 

Kinetic effect of anti-dsDNA on normal human PMN apoptosis
In an attempt to determine whether anti-dsDNA affects PMN apoptosis, normal human PMN were incubated with 50 ng/ml of anti-dsDNA or mouse non-specific IgG2a at 37°C for 1, 3 and 24 h. The cells were then cytospun, fixed and stained with haematoxylin–eosin solution for morphological observation. As shown in Fig. 4Go, karyopyknotic/karyorrhexic nuclei began to appear in anti-dsDNA-treated, but not in mouse non-specific IgG2a-treated PMN after 3 h incubation. In addition, the number of apoptotic cells in anti-dsDNA-treated PMN was much greater than in mouse non-specific IgG2a-treated PMN after 24 h incubation. The same experiment was repeated five times with a similar tendency.



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  		FIG. 4. Kinetic effects of anti-dsDNA (50 ng/ml) and mouse non-specific IgG2a (50 ng/ml) on the apoptosis of normal human PMN after incubation for 1, 3 and 24 h. A-(1) PMN + mouse non-specific IgG2a for 1 h; A-(2) PMN + mouse non-specific IgG2a for 3 h; A-(3) PMN + mouse non-specific IgG2a for 24 h; B-(1) PMN + anti-dsDNA for 1 h; B-(2) PMN + anti-dsDNA for 3 h; B-(3) PMN + anti-dsDNA for 24 h. The arrows indicate PMN with karyopyknotic/karyorrhexic nuclei, a typical change in apoptosis (original magnification 1000x in all pictures). The same experiment was repeated five times with a similar tendency.

 

Detection of the cognate antigen(s) of anti-dsDNA on the surface of PMN and other cells
A number of cells, including human PMN, MNC and RBC, K-562, HL-60, Jurkat, RBA-1 and RKO, were membrane biotinylated, lysed, immunoprecipitated and probed by anti-dsDNA in Western blot to detect the cognate antigen(s) of anti-dsDNA on the cell surface. As shown in Fig. 5Go, a 50–52 kDa molecule expressed on the surface of human PMN and MNC, K-562, HL-60, and Jurkat T cells, but not on RKO (human colon cancer cell line) or human RBC, bound with anti-dsDNA. Instead, high (115 kDa) and low (30 kDa) molecular weight molecules expressed on the surface of the rat brain astrocyte cell line (lane 4) reacted with anti-dsDNA.



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  		FIG. 5. Expression of the cognate antigen(s) on the cell surface of human PMN and other cells probed by monoclonal anti-dsDNA. Lane 1, K-562; lane 2, HL-60; lane 3, Jurkat T cells; lane 4, RBA-1 (rat brain astrocytes); lane 5, RKO (human colon cancer cell line); lane 6, human erythrocytes; lane 7, human MNC; lane 8, human PMN. A 50–52 kDa membrane-expressed protein is the major cognate antigen for monoclonal anti-dsDNA antibody. A 36–38 kDa molecule reactive with anti-dsDNA was found in Jurkat cells (lane 3) in addition to the 50–52 kDa protein. High (115 kDa) and low (30 kDa) molecular weight molecules reactive with anti-dsDNA appeared on the surface of RBA-1 instead of 50–52 kDa (lane 4).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Anti-dsDNA antibodies are the classical autoantibodies that characterize SLE and play a major role in lupus pathogenesis. The DNA–anti-DNA or DNA–histone–anti-DNA immune complexes formed in the circulation or in situ deposit in different tissues eliciting tissue damage [22]. However, many authors have demonstrated that antinuclear or anticytoplasmic autoantibodies can bind directly to different kinds of cells and cause cellular cytotoxicity through an immunological cross-reactivity [16–19, 23, 24]. In our previous reports, we found that polyclonal anti-dsDNA purified from active SLE exerted diverse effects on human MNC [18, 25], cultured rat glomerular mesangial cells and different cell lines [17, 26–28] by binding to the cell surface-expressed acidic ribosomal phosphoproteins P0, P1 and P2 [16, 17, 27]. Increased susceptibility to infections is clinically a major cause of morbidity and mortality in patients with SLE. It is conceivable that elevated lupus disease activity, administration of glucocorticoids/immunosuppressants, hypoalbuminaemia, neutropenia and functional impairment of PMN are associated with increased infections in patients with SLE [3, 4, 14, 15].

Mature neutrophils have long been regarded as terminally differentiated cells incapable of protein synthesis and committed to death within 72 h via an apoptotic mechanism [29, 30]. Recently, many authors reported that PMN could express and produce a number of important cytokines and chemokines mRNA, including IL-1, IL-6, IL-8, IL-12, tumour necrosis factor {alpha} (TNF-{alpha}), granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor, macrophage-chemoattractant protein 1, macrophage inflammatory protein 1{alpha} and macrophage inflammatory protein 1ß [31, 32]. Among these, IL-8 is of particular importance because: (a) IL-8 is produced in abundance by normal PMN either spontaneously or after stimulation; (b) IL-8 is a potent specific chemotactic factor and activator of PMN for stimulating chemotaxis, release of lysosomal enzymes and generation of reactive oxygen species [33, 34]; (c) IL-8 acts as an autocrine regulator of PMN to increase the recruitment and activation of PMN in the inflammatory sites [9, 31, 32]. Accordingly, an abnormal regulation of the IL-8–PMN–IL-8 amplification loop would remarkably influence the altitude of the inflammatory responses leading to overwhelming or defective tissue inflammation [9]. It is conceivable that the presence of neutrophil-binding autoantibodies in the circulation correlated with neutropenia in the patients with active SLE [10–13]. However, the pathological roles of antineutrophil autoantibodies in abnormal neutrophil functions and the identification of the cognate antigens of the antibodies have not been reported in the literature. Because of the polyreactive and cross-reactive properties of anti-dsDNA [16–18, 23, 24, 27, 28], it is possible that anti-dsDNA is a potential antineutrophil antibody capable of deranging the functions of PMN. In the present study, three original findings were noted: (a) anti-dsDNA can bind to a 50–52 kDa membrane-expressed molecule on normal human PMN [Figs 1Go and 5Go (lane 8)]; (b) anti-dsDNA up-regulates the IL-8 gene expression of normal human PMN (Figs 2Go and 3Go); (c) anti-dsDNA accelerates PMN apoptosis via an activation-induced cell death (AICD) mechanism (Fig. 4Go). These original observations suggest that anti-dsDNA is a kind of antineutrophil autoantibody, but not a non-specific neutrophil binder by adhering to the surface IgG Fc receptors, as reported by Starkebaum and Arend [10]. The binding of anti-dsDNA to PMN simultaneously turns on IL-8 and apoptosis-related genes. However, these in vitro effects of a monoclonal anti-dsDNA antibody derived from an autoimmune MRL-lpr/lpr mouse in this study cannot directly reflect the in vivo or in vitro pathological effects of polyclonal anti-dsDNA autoantibodies obtained from patients with SLE. Immunologically, IL-8 and other {alpha}-chemokines are implicated as major participants in the initiation and amplification of the acute inflammatory reaction [35]. IL-8 can stimulate the vascular endothelial cells and facilitate the adhesion and emigration of PMN from blood vessels into tissues such as lungs or kidneys to cause tissue damage [36–39]. Our findings are quite similar to the report of Rekvig and Hannestad [40] who demonstrated that certain polyclonal antinuclear antibodies cross-reacted with the molecules expressed on the surface membrane of human lymphocytes and granulocytes. In the present study, we further identified that a 50–52 kDa membrane protein is the cognate antigen of anti-dsDNA. In our preliminary study, we found that the amino acid sequence of this 50–52 kDa membrane-expressed molecule showed a high homology with hnRNP-A2 molecule. But the real nature of this molecule needs further investigation.

Biologically, apoptosis is intimately linked to cell stimulation and proliferation [41, 42]. Cell activation and cell death are inseparable and become a continuous spectrum of the fate of the cells after activation. This AICD phenomenon is commonly found in antigen- or mitogen-activated immune cells [43, 44] to maintain the immune homeostasis. Carvalho et al. [45] reported that IgG class anti-endothelial cell autoantibodies derived from SLE or systemic vasculitis stimulated endothelial cells to release IL-1 and an as-yet-unidentified mediator which enhance intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin expression and leucocyte adhesion in an autocrine manner. However, the outcome of these activated endothelial cells was not mentioned in that study. Lai et al. [46] demonstrated that polyclonal anti-dsDNA autoantibodies enhanced the gene expression of IL-8, transforming growth factor ß, and nitric oxide synthase in cultured umbilical vein endothelial cells. But the number of endothelial cells in apoptosis increased in parallel with gene expression. Our study suggests that the presence of anti-dsDNA in the serum of patients with active SLE not only stimulates circulating PMN to produce more IL-8 but also transduces signals for AICD to maintain immunological homeostasis and prevent overwhelming tissue inflammation in vivo.

Undoubtedly, there are at least four drawbacks in this study: (a) we did not characterize the nature of the 50–52 kDa cognate antigen of anti-dsDNA expressed on human PMN; (b) the signal transduction pathway involving IL-8 and AICD gene expression in PMN after binding with anti-dsDNA was not studied, although protein kinase C{theta} is a potential candidate for it [47]; (c) the effect of anti-dsDNA in apoptosis-related gene expression of PMN, such as Fas, FasL, p53, c-myc, and bcl-2 family was not investigated [28, 30]; (d) the correlation of anti-dsDNA, IL-8, leucopenia, and bacterial infection rate in the patients with active SLE was not elucidated. However, our results are quite important in understanding the pathological role of anti-dsDNA in accelerating apoptosis and abnormal functions of neutrophils in patients with SLE.

In conclusion, anti-dsDNA not only enhances neutrophil IL-8 gene expression but also accelerates the apoptosis of normal human PMN via a mechanism of AICD which is frequently found in patients with active SLE.


   Acknowledgments
 
This study was supported by grants from National Science Council (NSC87-2314-B010-022) and National Health Research Institute (DOH87-HR-508), Taipei, Taiwan.


   Notes
 
Correspondence to: C.-L. Yu, Department of Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan 100. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 20 August 1999; Accepted 7 March 2001


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