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Lupus. Author manuscript; available in PMC 2016 December 01. Published in final edited form as: Lupus. 2015 December ; 24(14): 1567–1569. doi:10.1177/0961203315603144.
Polyreactive antibodies in the circulation of patients with systemic lupus erythematosus Sreenivasulu Guntia, Eleni I. Kampylafkab, Athanasios G. Tzioufasb,*, and Abner L. Notkinsa,* aExperimental
Medicine Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892 USA.
Author Manuscript
bDepartment
of Pathophysiology, School of Medicine, National Kapodistrian University of Athens,
Greece. Sir,
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Polyreactive antibodies are now recognized as a major component of the natural antibody repertoire. These antibodies can bind to a variety of structurally unrelated host and foreign antigens and in the presence of complement are capable of lysing bacteria and enhancing the phagocytosis of apoptotic cells 1–3. Normal serum contains millions of polyreactive antibodies with different binding patterns and affinities. In order to quantify these antibodies, we used a 2,4-Dinitrophenol (DNP) surrogate assay and showed that in mice stimulation of Toll-Like Receptors (TLRs) by their respective exogenous or endogenous ligands led to a substantial increase in the secretion of polyreactive antibodies4. It has been estimated that as much as 50% of serum IgM in mice and IgG in humans is polyreactive 5. Although there have been a number of studies on natural antibodies in humans6, 7, relatively little is known about the quantity of polyreactive antibody in the serum of normal subjects or patients with various diseases. The present investigation was initiated to quantify the titer of polyreactive antibodies in sera of systemic lupus erythematosus (SLE) patients. Sixty-five sera from 53 patients with SLE (37+/- years, 91% females), who fulfilled the 1997 ACR classification criteria for SLE, were studied. Sera from 30 age- and sex-matched healthy individuals (34+/−12years, 77% females) served as normal controls.
Author Manuscript
A surrogate assay was used to measure polyreactive antibodies. After comparing a number of different molecules, dinitrophenol (DNP) was chosen as the surrogate because it is a synthetic molecule, not present in the environment, and individuals are not normally exposed to it. Therefore, if antibodies in sera bind to DNP, these antibodies would almost certainly have to be polyreactive antibodies3. In brief, ELISA plates were coated with DNP and serially two-fold dilutions of serum was used to determine the polyreactive antibody titer and the titer is reported as the reciprocal of highest serum dilution that produced an
Correspondence to: Abner L. Notkins or Athanasios Tzioufas. [email protected], [email protected]. Conflict of interest Statement The authors have no conflicts of interest to declare.
Gunti et al.
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absorbance above the plate background activity. Serum immunoglobulin concentrations were determined by sandwich ELISA using a standard curve.
Author Manuscript
As seen in Figure 1A, the titer of polyreactive IgG antibody in SLE patients was significantly higher than in normal controls but, in contrast, the concentration of serum IgG in SLE patients did not differ from that of normal controls (Figure 1B). Of particular interest, the ratio of the polyreactive IgG antibody titer to the IgG concentration in the SLE patients was significantly higher than in the normal controls (Figure 1C). This argues that the serum IgG of SLE patients contains more polyreactive IgG antibody than the serum of controls. In contrast to polyreactive IgG, the titer of polyreactive IgM antibody in the serum of SLE patients was higher, but not significantly higher, than in normal controls (Figure 1E). Moreover, the concentration of serum IgM was significantly lower in SLE patients than in normal controls (Figure 1F). However, the ratio of the polyreactive IgM antibody titer to the IgM serum concentration (Figure 1G) was significantly higher in the SLE patients. This argues that the serum IgM of SLE patients contains more polyreactive IgM antibody than the serum of normal controls. Although there was a positive trend when comparing the titer of polyreactive antibodies and the SLEDAI score, it did not reach statistical significance (Figure 1D). A positive trend was not found with polyreactive IgM (Figure 1H), nor was there a statistically significant positive correlation between the titer of polyreactive IgG or polyreactive IgM and the titer of anti-dsDNA antibody or the level of serum complement (data not shown).
Author Manuscript
A possible explanation for the increase in the ratio of the serum polyreactive antibody titer to the serum IgG or IgM concentration in SLE patients may be related to chronic inflammation, excessive tissue destruction or the inability to clear apoptotic cells. Tissue damage by any of these means could result in the release of a number of different endogenous TLR ligands 8 which could stimulate the secretion of polyreactive antibodies. To what extent the increase in polyreactive antibodies in SLE serum contributes to the protective effects attributed to natural antibodies9 remains to be determined.
Acknowledgments Funding: This research was funded by the Research Council of the University of Athens-Greece (AGT and EK) and, in part, by the Intramural Research Program of the US National Institutes of Health, NIDCR (ALN). The clinical study was approved by institutional review board of National University of Athens, Greece (IRB approval number 5107) and signed consent was obtained.
References Author Manuscript
1. Zhou ZH, Tzioufas AG, Notkins AL. Properties and function of polyreactive antibodies and polyreactive antigen-binding B cells. J Autoimmun. 2007; 29:219–228. [PubMed: 17888628] 2. Zhou ZH, Wild T, Xiong Y, et al. Polyreactive antibodies plus complement enhance the phagocytosis of cells made apoptotic by UV-light or HIV. Sci Rep. 2013; 3:2271. [PubMed: 23881356] 3. Notkins AL. Polyreactivity of antibody molecules. Trends Immunol. 2004; 25:174–179. [PubMed: 15039043] 4. Gunti S, Notkins AL. Polyreactive Antibodies: Function and Quantification. J Infect Dis. 2015; 212(S1):S42–S46. [PubMed: 26116731]
Lupus. Author manuscript; available in PMC 2016 December 01.
Gunti et al.
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5. Berneman A, Guilbert B, Eschrich S, Avrameas S. IgG auto- and polyreactivities of normal human sera. Mol Immunol. 1993; 30:1499–1510. [PubMed: 8232336] 6. Matsiota P, Druet P, Dosquet P, Guilbert B, Avrameas S. Natural autoantibodies in systemic lupus erythematosus. Clin Exp Immunol. 1987; 69:79–88. [PubMed: 3498588] 7. Chou MY, Fogelstrand L, Hartvigsen K, et al. Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans. J Clin Invest. 2009; 119:1335–1349. [PubMed: 19363291] 8. Marshak-Rothstein A. Toll-like receptors in systemic autoimmune disease. Nat Rev Immunol. 2006; 6:823–835. [PubMed: 17063184] 9. Gronwall C, Akhter E, Oh C, Burlingame RW, Petri M, Silverman GJ. IgM autoantibodies to distinct apoptosis-associated antigens correlate with protection from cardiovascular events and renal disease in patients with SLE. Clin Immunol. 2012; 142:390–398. [PubMed: 22297166]
Author Manuscript Author Manuscript Author Manuscript Lupus. Author manuscript; available in PMC 2016 December 01.
Gunti et al.
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Figure 1. Level of polyreactive antibodies in SLE patients
Author Manuscript
Upper panel: (A) Polyreactive IgG antibody titer, (B) serum IgG concentration and (C) ratio of polyreactive IgG antibody titer to IgG concentration in SLE patients compared to normal donors. Data represents mean ±SEM (Mann-Whitney U Test, n.s.; not significant). (D) Pearson rank correlation analysis of polyreactive IgG antibodies with patient’s SLEDAI. Lower panel: (E) Polyreactive IgM antibody titer, ( F) serum IgM concentration and (G) ratio of polyreactive IgM antibody titer to IgM concentration in SLE patients compared to normal donors. Data represents mean ±SEM (Mann-Whitney U Test, n.s.; not significant). (H) Pearson rank correlation analysis of polyreactive IgM antibody titer with patient’s SLEDAI.
Author Manuscript Lupus. Author manuscript; available in PMC 2016 December 01.
Lupus. Author manuscript; available in PMC 2016 December 01. Published in final edited form as: Lupus. 2015 December ; 24(14): 1567–1569. doi:10.1177/0961203315603144.
Polyreactive antibodies in the circulation of patients with systemic lupus erythematosus Sreenivasulu Guntia, Eleni I. Kampylafkab, Athanasios G. Tzioufasb,*, and Abner L. Notkinsa,* aExperimental
Medicine Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892 USA.
Author Manuscript
bDepartment
of Pathophysiology, School of Medicine, National Kapodistrian University of Athens,
Greece. Sir,
Author Manuscript
Polyreactive antibodies are now recognized as a major component of the natural antibody repertoire. These antibodies can bind to a variety of structurally unrelated host and foreign antigens and in the presence of complement are capable of lysing bacteria and enhancing the phagocytosis of apoptotic cells 1–3. Normal serum contains millions of polyreactive antibodies with different binding patterns and affinities. In order to quantify these antibodies, we used a 2,4-Dinitrophenol (DNP) surrogate assay and showed that in mice stimulation of Toll-Like Receptors (TLRs) by their respective exogenous or endogenous ligands led to a substantial increase in the secretion of polyreactive antibodies4. It has been estimated that as much as 50% of serum IgM in mice and IgG in humans is polyreactive 5. Although there have been a number of studies on natural antibodies in humans6, 7, relatively little is known about the quantity of polyreactive antibody in the serum of normal subjects or patients with various diseases. The present investigation was initiated to quantify the titer of polyreactive antibodies in sera of systemic lupus erythematosus (SLE) patients. Sixty-five sera from 53 patients with SLE (37+/- years, 91% females), who fulfilled the 1997 ACR classification criteria for SLE, were studied. Sera from 30 age- and sex-matched healthy individuals (34+/−12years, 77% females) served as normal controls.
Author Manuscript
A surrogate assay was used to measure polyreactive antibodies. After comparing a number of different molecules, dinitrophenol (DNP) was chosen as the surrogate because it is a synthetic molecule, not present in the environment, and individuals are not normally exposed to it. Therefore, if antibodies in sera bind to DNP, these antibodies would almost certainly have to be polyreactive antibodies3. In brief, ELISA plates were coated with DNP and serially two-fold dilutions of serum was used to determine the polyreactive antibody titer and the titer is reported as the reciprocal of highest serum dilution that produced an
Correspondence to: Abner L. Notkins or Athanasios Tzioufas. [email protected], [email protected]. Conflict of interest Statement The authors have no conflicts of interest to declare.
Gunti et al.
Page 2
Author Manuscript
absorbance above the plate background activity. Serum immunoglobulin concentrations were determined by sandwich ELISA using a standard curve.
Author Manuscript
As seen in Figure 1A, the titer of polyreactive IgG antibody in SLE patients was significantly higher than in normal controls but, in contrast, the concentration of serum IgG in SLE patients did not differ from that of normal controls (Figure 1B). Of particular interest, the ratio of the polyreactive IgG antibody titer to the IgG concentration in the SLE patients was significantly higher than in the normal controls (Figure 1C). This argues that the serum IgG of SLE patients contains more polyreactive IgG antibody than the serum of controls. In contrast to polyreactive IgG, the titer of polyreactive IgM antibody in the serum of SLE patients was higher, but not significantly higher, than in normal controls (Figure 1E). Moreover, the concentration of serum IgM was significantly lower in SLE patients than in normal controls (Figure 1F). However, the ratio of the polyreactive IgM antibody titer to the IgM serum concentration (Figure 1G) was significantly higher in the SLE patients. This argues that the serum IgM of SLE patients contains more polyreactive IgM antibody than the serum of normal controls. Although there was a positive trend when comparing the titer of polyreactive antibodies and the SLEDAI score, it did not reach statistical significance (Figure 1D). A positive trend was not found with polyreactive IgM (Figure 1H), nor was there a statistically significant positive correlation between the titer of polyreactive IgG or polyreactive IgM and the titer of anti-dsDNA antibody or the level of serum complement (data not shown).
Author Manuscript
A possible explanation for the increase in the ratio of the serum polyreactive antibody titer to the serum IgG or IgM concentration in SLE patients may be related to chronic inflammation, excessive tissue destruction or the inability to clear apoptotic cells. Tissue damage by any of these means could result in the release of a number of different endogenous TLR ligands 8 which could stimulate the secretion of polyreactive antibodies. To what extent the increase in polyreactive antibodies in SLE serum contributes to the protective effects attributed to natural antibodies9 remains to be determined.
Acknowledgments Funding: This research was funded by the Research Council of the University of Athens-Greece (AGT and EK) and, in part, by the Intramural Research Program of the US National Institutes of Health, NIDCR (ALN). The clinical study was approved by institutional review board of National University of Athens, Greece (IRB approval number 5107) and signed consent was obtained.
References Author Manuscript
1. Zhou ZH, Tzioufas AG, Notkins AL. Properties and function of polyreactive antibodies and polyreactive antigen-binding B cells. J Autoimmun. 2007; 29:219–228. [PubMed: 17888628] 2. Zhou ZH, Wild T, Xiong Y, et al. Polyreactive antibodies plus complement enhance the phagocytosis of cells made apoptotic by UV-light or HIV. Sci Rep. 2013; 3:2271. [PubMed: 23881356] 3. Notkins AL. Polyreactivity of antibody molecules. Trends Immunol. 2004; 25:174–179. [PubMed: 15039043] 4. Gunti S, Notkins AL. Polyreactive Antibodies: Function and Quantification. J Infect Dis. 2015; 212(S1):S42–S46. [PubMed: 26116731]
Lupus. Author manuscript; available in PMC 2016 December 01.
Gunti et al.
Page 3
Author Manuscript
5. Berneman A, Guilbert B, Eschrich S, Avrameas S. IgG auto- and polyreactivities of normal human sera. Mol Immunol. 1993; 30:1499–1510. [PubMed: 8232336] 6. Matsiota P, Druet P, Dosquet P, Guilbert B, Avrameas S. Natural autoantibodies in systemic lupus erythematosus. Clin Exp Immunol. 1987; 69:79–88. [PubMed: 3498588] 7. Chou MY, Fogelstrand L, Hartvigsen K, et al. Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans. J Clin Invest. 2009; 119:1335–1349. [PubMed: 19363291] 8. Marshak-Rothstein A. Toll-like receptors in systemic autoimmune disease. Nat Rev Immunol. 2006; 6:823–835. [PubMed: 17063184] 9. Gronwall C, Akhter E, Oh C, Burlingame RW, Petri M, Silverman GJ. IgM autoantibodies to distinct apoptosis-associated antigens correlate with protection from cardiovascular events and renal disease in patients with SLE. Clin Immunol. 2012; 142:390–398. [PubMed: 22297166]
Author Manuscript Author Manuscript Author Manuscript Lupus. Author manuscript; available in PMC 2016 December 01.
Gunti et al.
Page 4
Author Manuscript Author Manuscript
Figure 1. Level of polyreactive antibodies in SLE patients
Author Manuscript
Upper panel: (A) Polyreactive IgG antibody titer, (B) serum IgG concentration and (C) ratio of polyreactive IgG antibody titer to IgG concentration in SLE patients compared to normal donors. Data represents mean ±SEM (Mann-Whitney U Test, n.s.; not significant). (D) Pearson rank correlation analysis of polyreactive IgG antibodies with patient’s SLEDAI. Lower panel: (E) Polyreactive IgM antibody titer, ( F) serum IgM concentration and (G) ratio of polyreactive IgM antibody titer to IgM concentration in SLE patients compared to normal donors. Data represents mean ±SEM (Mann-Whitney U Test, n.s.; not significant). (H) Pearson rank correlation analysis of polyreactive IgM antibody titer with patient’s SLEDAI.
Author Manuscript Lupus. Author manuscript; available in PMC 2016 December 01.
