Monoclonal anti-double-stranded DNA antibodies cross-react with phosphoglycerate kinase 1 and inhibit the expression and production of IL-2 in activated Jurkat T cell line

doi:10.1016/j.clim.2006.06.002

Clinical Immunology

Volume 120, Issue 3, September 2006, Pages 326-334

https://doi.org/10.1016/j.clim.2006.06.002 Get rights and content

Abstract

Anti-double strand DNA antibodies (anti-dsDNA) involve in lupus nephritis. However, their role in tissue damage mechanism remains unclear. In this study, a 45-kDa cognate antigen of anti-dsDNA monoclonal antibodies 9D7 was identified by two-dimensional gel electrophoresis and determined to be human phosphoglycerate kinase 1 (PGK-1) by MALDI-TOF analysis. The binding of 9D7 to PGK-1 was not affected by DNase I but was inhibited by thymus dsDNA. Human SLE sera with high anti-dsDNA titers had a high affinity with PGK. In activated Jurkat T cells, 9D7 decreased the PGK-1 mRNA production and IL-2 promoter activity. Reduction in IL-2 gene expression and protein production were observed in the 9D7-treated cells. Because PGK-1 deficiency may cause mental tardy and hemolytic anemia, interaction between anti-dsDNA and PGK-1 may be important in lupus pathogenesis. Moreover, reduction in IL-2 production by anti-dsDNA suggests their role in increasing infection rate and decreasing proper generation of activation-induced cell death.

Introduction

Autoantibodies to double-stranded DNA are highly specific for SLE. The titers of these antibodies fluctuate with the disease activity and they appear to participate in the development of organ damage, especially in the kidney [1]. However, their crucial role in lupus nephritis and tissue damage remains unclear. In addition to non-specifically deposit in the kidney by binding to DNA, anti-double strand DNA antibodies (anti-dsDNA) may also directly cross-react with protein antigens to trigger cell dysfunction and tissue damage [2], [3], [4], [5], [6], [7]. Moreover, a 30-amino acid peptide corresponding to the combined sequences of CDR 2 and 3 of heavy chain variable region of anti-DNA has been demonstrated to penetrate into the cells [8]. These findings suggest the role of protein antigens in lupus pathogenesis.

The modulation of cytokines has been considered to be important in the pathogenesis of SLE, especially the involvement of Th1 cytokine IL-12, Th2 cytokine IL-10, proinflammatory cytokine TNF-α, and cytokine BLyS [9]. Proinflammatory cytokines produced by activated macrophages and monocytes may have potent stimulatory effects on T cells, B cells, and neutrophils in increasing the biosynthesis of prostaglandins and acute-phase proteins [10]. IL-12 may induce the differentiation of Th1 to produce IL-2 and IFN-γ and modulate cellular immune responses. IL-4 may induce the differentiation of Th2 to produce IL-4 and IL-10 and modulate humoral immune reactions [11]. Anti-dsDNA may stimulate the release of IL-1 and IL-6 from endothelial cells and cause inflammatory injury of vascular endothelium in SLE [12]. Moreover, anti-dsDNA monoclonal antibodies (anti-dsDNA mAb) has been demonstrated to penetrate Jurkat T cells and stimulate IL-1β, IL-8, TNF-α, and IL-10 production in human mononuclear cells [7].

To identify new cross-reactive antigens bound by anti-dsDNA, we established the anti-dsDNA mAb 9D7 and found two possible cognate protein antigens with molecular weights of 35 kDa and 45 kDa. The 35-kDa antigen was identified to be heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) [13]. In this study, the 45-kDa antigen was determined to be phosphoglycerate kinase-1 (PGK-1). In Jurkat T cells, the 9D7 mAb was also found to affect PGK-1 mRNA expression and decrease IL-2 expression and production. These findings indicate that anti-dsDNA may involve in the pathogenesis of SLE by interacting with PGK-1 and decreasing IL-2 production.

Section snippets

Cell lines

Human acute T cell leukemia (Jurkat) and RAW 264.7 mouse macrophage cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). RAW 264.7 were cultured in DMEM and Jurkat T cells were cultured in RPMI-1640. These culture media were supplemented with 10% FBS and the cell cultures were maintained in 5% CO₂ at 37°C.

Sera

Serum samples were collected from SLE patients and normal subjects according to the IRB protocol in the Section of Arthritis, Immunology and Rheumatology,

The 45-kDa cognate protein of anti-dsDNA mAb 9D7 is human PGK-1

By immunoblotting, we identified a 45-kDa protein to be a cognate antigen of anti-dsDNA. However, this protein was not detectable in a small amount (1 × 10⁶) of cell lysates by 2D gel electrophoresis and Coomassie brilliant blue staining [13]. In this study, 2D gel electrophoresis (Fig. 1A) was performed on a larger amount (9 × 10⁶) of Jurkat lysates and then immunoblotted with the 9D7 mAb. Two cognate protein antigens of anti-dsDNA mAb with molecular weights of 35 kDa and 45 kDa were visualized (

Discussion

SLE is a systemic autoimmune disease that affects many organs and tissues of the body, causing glomerulonephritis, arthritis, anemia, and cerebritis. This disease is characterized by B cell hyperactivity and the presence of autoantibodies. Among the autoantibodies, anti-dsDNA are most commonly found in SLE specimens [16]. Because polyreactivity is a common property of natural and disease-associated human autoantibodies [17], anti-DNA may cross-react with DNase I [2], myosin 1 [3], calreticulin

Acknowledgments

This work was supported in part by grants from the National Science Council (NSC 93-2314-B-010-016), Mackay Memorial Hospital-Yang-Ming University (MMHYM 93-P-010-006), Veteran General Hospital-University System of Taiwan (VGHUST93-P7-34), and a grand from Ministry of Education, Aim for the Top University Plan, ROC.

References (39)

F.M. Brennan et al.
Cytokines in autoimmunity
Curr Opin. Immunol.
(1996)
L.B. Nicholson et al.
Manipulation of the Th1/Th2 balance in autoimmune disease
Curr. Opin. Immunol.
(1996)
M. Reichlin
Cellular dysfunction induced by penetration of autoantibodies into living cells: cellular damage and dysfunction mediated by antibodies to dsDNA and ribosomal P proteins
J. Autoimmun.
(1998)
O. Popanda et al.
Modulation of DNA polymerases α, δ and ε by lactate dehydrogenase and 3-phosphoglycerate kinase
Biochim. Biophys. Acta
(1998)
T. Ogino et al.
Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription
J. Biol. Chem.
(1999)
A.P. Kraus et al.
Red cell phosphoglycerate kinase deficiency. A new cause of non-spherocytic hemolytic anemia
Biochem. Biophys. Res. Commun.
(1968)
R. Rosa et al.
A new case of phosphoglycerate kinase deficiency: PGK Creteil associated with rhabdomyolysis and lacking hemolytic anemia
Blood
(1982)
G.C. Tsokos et al.
Rewiring the T cell: signaling defects and novel prospects for the treatment of SLE
Trends Immunol.
(2003)
B.J. Hamilton et al.
Association of heterogeneous nuclear ribonucleoprotein A1 and C proteins with reiterated AUUUA sequences
J. Biol. Chem.
(1993)
T. Henics et al.
Enhanced stability of interleukin-2 mRNA in MLA 144 cells. Possible role of cytoplasmic AU-rich sequence-binding proteins
J. Biol. Chem.
(1994)

B.H. Hahn

Antibodies to DNA

N. Engl. J. Med.

(1998)

A. Puccetti et al.

Anti-DNA antibodies bind to DNase I

J. Exp. Med.

(1995)

K. Yanase et al.

Receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies via myosin

J. Clin. Invest.

(1997)

N. Seddiki et al.

Calreticulin, a potential cell surface receptor involved in cell penetration of anti-DNA antibodies

J. Immunol.

(2001)

L.A. Giorgio et al.

A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus

Nat. Med.

(2001)

B. Deocharan et al.

α-Actinin is a cross-reactive renal target for pathogenic anti-DNA antibodies

J. Immunol.

(2002)

K.H. Sun et al.

Monoclonal anti-double-stranded DNA autoantibody stimulates the expression and release of IL-1β, IL-6, IL-8, IL-10 and TNF-α from normal human mononuclear cells involving in the lupus pathogenesis

Immunology

(2000)

A. Avrameas et al.

Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules

Proc. Natl. Acad. Sci. U. S. A.

(1998)

A. Rahman

Cytokines in systemic lupus erythematosus

Arthritis Res. Ther.

(2003)

Cited by (14)

Specific HDAC6 inhibition by ACY-738 reduces SLE pathogenesis in NZB/W mice
2016, Clinical Immunology
Citation Excerpt :
Following treatment with ACY-738 (20 mg/kg), IL-6 was undetectable in the kidneys of NZB/W mice. The proinflammatory cytokine IL-6 is upregulated in SLE and contributes to overproduction of IgG [84,85]. The reduction in IL-6 correlated with a decrease in total IgG and the IgG2a isotype levels in the sera.
We sought to determine if a selective HDAC6 inhibitor (ACY-738) decreases disease in NZB/W mice. From 22 to 38 weeks-of-age, mice were injected intraperitoneally with 5 or 20 mg/kg of ACY-738, or vehicle control. Body weight and proteinuria were measured every 2 weeks, while sera anti-dsDNA, Ig isotypes, and cytokine levels were measured every 4 weeks. Kidney disease was determined by evaluation of sera, urine, immune complex deposition, and renal pathology. Flow cytometric analysis assessed thymic, splenic, bone marrow, and peripheral lymphocyte differentiation patterns. Our results showed HDAC6 inhibition decreased SLE disease by inhibiting immune complex-mediated glomerulonephritis, sera anti-dsDNA levels, and inflammatory cytokine production and increasing splenic T_reg cells. Inhibition of HDAC6 increased the percentage of cells in the early-stage developmental fractions of both pro- and pre-B cells. These results suggest that specific HDAC6 inhibition may be able to decrease SLE disease by altering aberrant T and B cell differentiation.
Der p 2 can induce bystander activation of B cells derived from patients with systemic lupus erythematosus
2014, Immunobiology
Citation Excerpt :
When the expression of autoantigens increases, the chance of immune cells recognizing these autoantigens concomitantly increased, resulting in production of autoantibodies which then attack the autoantigens. Other research has shown that anti-double strand DNA antibody has a cross-reaction with PGK-1 (Luan et al. 2006). In addition, a recent study showed that TIM and enolase-1 can be recognized by autoantibodies (Lee et al. 2009; Llanos et al. 2009; Pratesi et al. 2000; Sasajima et al. 2006; Uchibori et al. 2005; Watanabe et al. 2004).
Although many patients with SLE also have allergies, the immunological events triggering the onset and progression of the clinical manifestations of SLE by allergens have yet to be clarified. A total of three autoantigens, phosphoglycerate kinase 1 (PGK-1), triosephosphate isomerase (TIM) and enolase were identified by autologous serum in B cell lysate derived from HDM allergic SLE patients after Der p 2 stimulation. Autoantigen, TRIM-21 expression were also significantly increased in B cells derived from HDM allergic SLE patients. In PBMCs derived from SLE patients, the concentration of anti-PGK-1 was significantly upregulated after Der p 2 stimulation compared to HDM allergic without SLE patients and healthy subjects. Inflammatory related cytokines and chemokines include IL-1β, IL-6, IL-8, CXCL5 could be upregulated after Der p 2 stimulation in PBMCs derived from HDM allergic SLE patients. In conclusion, our data demonstrated that long-term allergen exposure could be a contributing factor in the development of SLE.
Transgenic overexpression of anti-double-stranded DNA autoantibody and activation of Toll-like receptor 4 in mice induce severe systemic lupus erythematosus syndromes
2010, Journal of Autoimmunity
Citation Excerpt :
The 9D7 anti-dsDNA monoclonal antibody (9D7 mAb) was an IgG2b isotype and derived from an MRL-lpr/lpr mouse. This antibody has been shown to have 9 positively charged amino acids within the heavy chain CDR, to penetrate cells via electrostatic interactions [13], and to cross-react with the intracellular mRNA binding proteins hnRNP A2 [14] and PGK-1 [15]. The 9D7 mAb also has been known to stimulate secretions of IL-1β, IL-6, IL-8, IL-10, and TNF-α from human mononuclear cells [16], while inhibiting IL-2 production in T cells [15].
Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characteristized by the presence of autoantibodies against double-stranded DNA (anti-dsDNA) in sera at high levels. Bacterial infections in SLE are associated with higher morbidity and mortality. Our goal was to observe the interaction between these 2 factors in the pathogenesis of lupus. We generated transgenic mice with monoclonal anti-dsDNA to investigate the development of lupus. By challenging the mice in vitro and in vivo with Toll-like receptor 4 (TLR4) ligand lipopolysaccharides (LPS), we were able to examine the role of bacterial infection in SLE. In our study, the transgenic mice with a secreted form of anti-dsDNA were found to have higher levels of anti-nuclear antibodies, anti-dsDNA, blood urea nitrogen, and proteinuria. The splenocytes of the mice stimulated with LPS secreted more anti-dsDNA, IFN-γ, and IL-10. After injecting them with LPS in vivo, we further found higher immune complex depositions and IL-10 in the kidneys of the transgenic mice. Moreover, the LPS-injected transgenic mice had higher mortality rate. This is the first transgenic model to demonstrate that only 2 risk factors, pathogenic anti-dsDNA and TLR4 activation, induce severe SLE syndromes in normal mice through the overproduction of IL-10 and IFN-γ. These findings imply that anti-dsDNA and bacterial infections have pivotal roles in the pathogenesis of SLE; the inhibition of TLR4 may be regarded as a therapeutic target.
Cytosolic internalization of anti-DNA antibodies by human monocytes induces production of pro-inflammatory cytokines independently of the tripartite motif-containing 21 (TRIM21)-mediated pathway
2018, Frontiers in Immunology
Featured Article: Interactions of surface-expressed TLR-4 and endosomal TLR-9 accelerate lupus progression in anti-dsDNA antibody transgenic mice
2014, Experimental Biology and Medicine
The lupus-derived anti-double-stranded DNA IgG contributes to myofibroblast-like phenotype in mesangial cells
2012, Journal of Clinical Immunology

View all citing articles on Scopus

View full text

Monoclonal anti-double-stranded DNA antibodies cross-react with phosphoglycerate kinase 1 and inhibit the expression and production of IL-2 in activated Jurkat T cell line

Abstract

Introduction

Section snippets

Cell lines

Sera

The 45-kDa cognate protein of anti-dsDNA mAb 9D7 is human PGK-1

Discussion

Acknowledgments

Curr Opin. Immunol.

Curr. Opin. Immunol.

J. Autoimmun.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Blood

Trends Immunol.

J. Biol. Chem.

J. Biol. Chem.

Antibodies to DNA

N. Engl. J. Med.

Anti-DNA antibodies bind to DNase I

J. Exp. Med.

Receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies via myosin

J. Clin. Invest.

Calreticulin, a potential cell surface receptor involved in cell penetration of anti-DNA antibodies

J. Immunol.

A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus

Nat. Med.

α-Actinin is a cross-reactive renal target for pathogenic anti-DNA antibodies

J. Immunol.

Monoclonal anti-double-stranded DNA autoantibody stimulates the expression and release of IL-1β, IL-6, IL-8, IL-10 and TNF-α from normal human mononuclear cells involving in the lupus pathogenesis

Immunology

Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules

Proc. Natl. Acad. Sci. U. S. A.

Cytokines in systemic lupus erythematosus

Arthritis Res. Ther.