1404
QUANTITATION OF IgM- AND IgG-SECRETING B CELLS IN THE PERIPHERAL BLOOD OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS DENNIS M. KLINMAN, AKIRA SHIRAI, YOSHIAKI ISHIGATSUBO, JACQUELINE CONOVER, and ALFRED D. STEINBERG An enzyme-linked immunospot assay was used to quantitate the number of autoantibody-secreting B cells in the peripheral blood of 67 patients with systemic lupus erythematosus. These patients had 1.5-4-fold more lymphocytes secreting IgG and I@ per million peripheral blood lymphocytesthan did normal controls. There was a concomitant increase in the number of B cells secreting antibodies reactive with a diverse panel of foreign and self antigens (including actin, myosin, trinitrophenylated keyhole limpet hemocyanin, ovalbumin, and retroviral gp160). By comparison, the number of B cells producing anti-DNA antibodies was increased disproportionately. The magnitude of this anti-DNA response correlated significantly with disease activity. Thus, B cell activation in human systemic lupus erythematosus had characteristics of both generalized (polyclonal) B cell activation and (autolantigen-specific immune stimulation. From the Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration, Bethesda, Maryland, the Cellular Immunology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, and the Yokohama City University Medical School, Yokohama, Japan. Dennis M. Klinman, MD,PhD: Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration; Akira Shirai, M D Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration; Yoshiaki Ishigatsubo. MD: Yokohama City University Medical School; Jacqueline Conover, BS: Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration; Alfred D. Steinberg, MD: Cellular Immunology Section, Arthritis and Rheumatism Branch, NIAMS. National Institutes of Health. Address reprint requests to Dennis M. Klinrnan, MD. PhD, Building 29A, Room 3 D 02, CBEWFDA, Bethesda, MD 20892. Submitted for publication March 21, 1991; accepted in revised form June 25, 1991. Arthritis and Rheumatism, Vol. 34,No. 11 (November 1991)
Systemic lupus erythematosus (SLE) is a n autoimmune disease characterized by B cell hyperproliferation (1) and the spontaneous production of antibodies reactive with a variety of self antigens (for review, see ref. 2). While there is a strong correlation between the degree of lupus activity and the number of Ig-secreting cells in the peripheral blood, the extent to which particular autoantibodies contribute to the disease process is unclear (3,4). No single autoantibody is invariably found in all patients with lupus. Whereas an excess of IgG antiDNA is classically found in SLE serum, anti-DNA antibodies are absent in up to 30% of individuals with active disease (5-7). In addition, antibodies reactive with one of a large number of other autoantigens (such as Ro, La, actin, myosin, and cardiolipin) are present at elevated concentrations in some patients (8-12), while other patients exhibit increased reactivity against multiple autoantigens simultaneously. These observations have raised questions concerning the contribution of specific autoantibodies to the disease process and suggest that a more generalized process of B cell activation might also be involved. Several studies of human and murine lupus have emphasized the apparent contribution of idiotypic or antigenic stimulation to the production of specific autoantibodies, such as anti-DNA (13-18). Others have found evidence of generalized B cell activation (associated with excess T cell help) (1,3,9,19,20).These processes are not mutually exclusive, and recent work suggests that distinct subsets of lupus B cells may be responding to polyclonal versus (auto)antigen-specific stimuli (21). Researchers at our laboratory and others have been using a sensitive and specific enzyme-linked
B CELLS IN SLE
1405
Table 1. Laboratory findings (mean rt SEM)in patients with systemic lupus erythematosus grouped according to degree of disease activity
Clinical disease status
normal
normal
IgM (mg/dl; normal
normal
Anti-DNA (I/titer; normal
(n)*
6504,600)
65415)
50-320)
16-40)
2 standard deviations from the mean value in the control group. In contrast, 30% of the patients expressed repertoires in which IgG anti-DNA production was preferentially increased. As expected, patients with active disease more frequently expressed repertoires skewed toward anti-DNA reactivity, i.e., 69% of those with severe disease, versus only 12% of those with inactive disease (Table 2).
DISCUSSION An ELISPOT assay was used to monitor the number and antigenic specificityof Ig-secreting B cells in the peripheral blood of patients with SLE. The absolute number of IgM- and IgG-secreting cells was increased 1.54-foldl depending on disease activity, over normal levels. Patients with lupus had disproportionately increased numbers of lymphocytes secreting IgG anti-DNA antibodies, with the frequency of antiDNA-producing lymphocytes correlating significantly with disease activity (r2 = 0.47). By comparison, the number of B cells producing antibodies against myosin, actin, ovalbumin, TNP, and retroviral gp160 increased in proportion to the total increase in the number of Ig-secreting cells. When the repertoires expressed by individual patients were analyzed, skewing of the repertoire toward increased anti-DNA production was found in 69% of the patients with active lupus, whereas skewing against the other antigens was generally present in 4%of the subjects. These findings indicate that I) lupus patients have increased numbers of B cells secreting antibodies reactive with a number of different antigens (consistent with the influence of polyclonal activation), but 2) anti-DNAproducing cells are preferentially activated, reflecting (auto)antigen-specificimmune stimulation. ELISPOT assays have been used to determine
B CELLS IN SLE
1407
DNA
TNP
0
A
A
A
A
A
0
-t
0
I
ACT
OVA A
400
A
A
A
A
A
a
0
200
Irnt
A
1W
m
0
0
OD
MY0
0
:
GP160 mT
I a
0
40
0
8 0
20
0
'f 0
A 0 0
m a
SLE
NL
SLE
NL
0
SLE
SLE
NL
Figure 2. Absolute number ofIgM- and IgG-secreting B cells, from 67 patients with systemic lupus erythematosus (SLE) and I5 normal controls ( N U that were reactive with the autoantigens DNA, actin (ACT), and myosin (MYO) or with the conventional antigens tnnitrophenylated keyhole limpet hemocyanin (TNP) and ovalbumin (OVA). An analysis of reactivity to GP160, the envelope glycoprotein of human immunodeficiency virus-I, was also performed in a subgroup of the subjects.
KLINMAN ET AL
1408
DISEASE ACTIVITY
=
25
IgM
EM EXl
HIGH MODERATE
INACTIVE 0 CONTROL
Figure 3. Percent of B cells producing antibodies reactive with each member of the antigen panel. The proportion of IgG anti-DNA secreting cells correlated closely with disease activity (r = 0.62,
Spearman-Pearson rank correlation). None of the other antigens examined showed statistically significant correlations with disease activity (r < 0.4). Values are the mean and SD of results from 16 patients with highly active systemic lupus erythematosus (SLE), 25 patients with moderately active SLE, 26 patients with inactive SLE, and 15 controls. See Figure 2 for additional definitions.
the number and specificity of antibody-secreting B cells in murine lupus (22,35,36) and to quantitate antibody-secreting B cells in cultured normal human PBL (31) and, most recently, in CD5+ and CD5- PBL (as analyzed by fluorescence-activated cell sorter) from patients with SLE (21). The accuracy of these assays has been documented by antigen-inhibition tests and through use of hybridomas of known antigenic specificity (22,3 1,36,38). Prior to the development of the ELISPOT assay, Ig secretion by individual human B cells was quantitated primarily by the hemolytic plaque assay. Studies using this assay demonstrated that patients with active SLE had 10,000-20,000 cells secreting Ig
per lo6 PBL (4,39), a finding confirmed by the present work. Cells that contained cytoplasmic Ig (and were therefore capable of immediate Ig secretion) were detected in 2-10% of SLE PBL (4). Few studies have examined the specificity of the antibodies secreted by these B cells in vivo. Budman et al demonstrated that 1 ,000/106PBL from SLE patients produced antibodies that reacted with sheep red blood cells, and that approximately onetenth this number reacted with a variety of chemical haptens (9). Earlier studies of bulk-cultured PBL demonstrated that lupus patients had increased numbers of anti-DNA-producing lymphocytes (40), but the assays used were not sufficiently sensitive to quantitate individual Ig-secreting cells. Moreover, results from studies of in vitro cultured B cells might not reflect the B cell repertoire actually expressed in vivo (41). A recent study used the ELISPOT assay to study CD5+ and CD5- B cells isolated from SLE patients, and the investigators concluded that the CD5- pool was polyclonally activated to produce anti-DNA, whereas the CD5+ pool seemed to reflect (auto)antigen-specific stimulation (21). In the present experiments, the ELISPOT assay was used to quantitate autoantibody-secreting B cells from a large number of patients with differing degrees of disease activity. We found that the frequency of lupus B cells producing anti-DNA antibodies increased as a function of disease activity, suggesting persistent (auto)antigenicstimulation. In contrast, the number of lymphocytes producing antibodies reactive with other antigens increased as a function of
-
Percentage of systemic lupus erythematosus (SLE) patients and normal controls expressing antigenically skewed repertoires*
Table 2.
Antigen DNA
TNPKLH
Actin
4 12 6
0 8 6
4
4
ovalMyosin bumin
~
Inactive SLE Moderately active SLE Very active SLE Controls
12 24 69 6
a 4 0 3
0 4 13 4
* The proportion of B cells producing antibodies reactive with DNA, trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), actin. myosin, and ovalbumin was calculated as a percentage of the total number of IgG-secreting cells (22). The percentage of individuals expressing repertoires preferentially biased toward reactivity with each antigen (i.e.. exceeding the control mean by >2 standard deviations) is shown. Expressed repertoires of patients with active disease were significantly skewed toward the overproduction of anti-DNA (P< 0.001, by Yates’ mean score).
B CELLS IN SLE the total increase in Ig-secreting cells, reflecting polyclonal activation. The possibility that anti-DNAsecreting B cells were responsible for the polyclonal stimulation (through nonspecific antigenic crossreactivity or bystander stimulation) seems unlikely, since the number of B cells producing anti-DNA did not correlate significantly with the number of B cells of other specificities (r2 = 0.08-0.19). Earlier studies from our laboratory have provided evidence that autoantibody production in murine lupus is a 2-stage process which is initiated by polyclonal B cell activation and subsequently magnified and perpetuated by specific (auto)antigenic stimulation (27). Similarly, polyclonal B cell expansion has been detected in humans with early untreated lupus (9), while patients with established disease have been shown to generally express repertoires skewed toward reactivity against 1 or more different autoantigens (30). This pattern of B cell reactivity suggests that autoantibody production in human SLE, like its murine counterpart, may be influenced by both antigenspecific and antigen-independent factors. Unfortunately, discrimination between these processes is difficult in humans, since it requires identifying at-risk individuals before their disease becomes clinically evident. One approach would be to analyze family members (especially identical twins) of lupus patients. It is our hope that continued analysis of the B cell repertoire expressed by patients with autoimmune diseases will aid in the early diagnosis and treatment of such diseases before the development of serious immunopathologic sequelae.
1409
systemic lupus erythematosus. N Engl J Med 281:701705, 1970
6. Feldman MD, Huston DP, Karsh J, Balow JE, Steinberg
AD: Correlation of serum IgG, IgM and anti-native DNA antibodies with renal and clinical indices of activity in SLE. J Rheumatol 952-58, 1982 7. Koffler D, Carr R, Agnello V, Thoburn R, Kunkel HG: Antibodies to human polynucleotides in human sera: antigenic specificityand relation to disease. J Exp Med 134:29&3 12, I971 8. McCarty GA, Rice JR, Bembe ML, Pisetsky DS: Independent expression of autoantibodies in SLE. J Rheumatol 9:691495, 1982 9. Budman DR, Merchant EB, Steinberg AD, Doft B, Gershwin ME, Lizzio E, Reeves JP: Increased spontaneous activity of antibody-formingcells in the peripheral blood of patients with active SLE. Arthritis Rheum 20:829-833, 1977
10. Morimoto C, Abe T, Hara M, Homma M: In vitro
TNP-specific antibody formation by peripheral lymphocytes from patients with SLE. Scand J Immunol6575579, 1977 11. Guilbert B, Dighiero G,Avrameas S: Naturally occur-
ring antibodies against nine common antigens in human sera. J Immunol 128:277%2787, 1982 12. Stollar BD, Schwartz RS: Monoclonal anti-DNA antibodies: the targets and origins of SLE autoantibodies. Ann N Y Acad Sci 465:192-199, 1986 13. Kalunian KC,Panosian-Sahakian N, Ebling FM, Cohen AH, Louie JS, Kaine J, Hahn BH: Idiotypic characteristics of immunoglobulins associated with systemic lupus erythematosus: studies of antibodies deposited in glomeruli of humans. Arthritis Rheum 32513-522, 1989 14. Horsfall AC, Isenberg DA: Idiotypes and autoimmunity: a review of their role in human disease. J Autoimmun 1:7-30, 1988
REFERENCES 1. Becker TM, Lizzio EF, Merchant LP, Reeves JP, Stein-
2.
3.
4.
5.
berg AD: Increased multiclonal antibody-forming cell activation in the peripheral blood of patients with SLE. Int Arch Allergy Appl Immunol66:293-298, 1981 Steinberg AD, Klinman DM: Pathogenesis of systemic lupus erythematosus. Rheum Dis Clin North Am 14:2541, 1988 Blaese RM, Grayson J, Steinberg AD: Elevated immunoglobulin secreting cells in the blood of patients with active systemic lupus erythematosus: correlation of laboratory and clinical assessment of disease activity. Am J Med 69:345-350, 1980 Tan PLJ, Pang GTM, Wilson JD, Cullinane G: Immunoglobulin secreting cells in SLE: correlation with disease activity. J Rheumatol 7:807-813, 1980 Pincus T, Schur PH, Rose JA, Decker JL, Tala1 N: Measurement of serum anti-DNA binding activity in
IS. Ebling FM, Ando DG, Sahakian NP. Kalunian KC, Hahn BH: Idiotypic spreading promotes the production of pathogenic autoantibodies. J Autoimmun 1 :47-61, 1988 16. Shlomchik
M, Marshak-Rothstein A, Wolfowicz CB, Rothstein TL, Weigert MG: The role of clonal selection and somatic mutation in autoimmunity. Nature 328:80581 I , 1987 17. Laskin CA, Haddad G. Soloninda CA: The regulatory role of NZB T lymphocytes in the production of antiDNA antibodies in vitro. J Immunol 137:1867-1872, 1986
18. Datta SK,Patel H,Berry D: Induction of a cationic shin
in IgG anti-DNA autoantibody. J Exp Med 165:12521268, 1987 19. Izui SP, McConahey PJ, Dixon FJ: Increased spontaneous polyclonal activation of B lymphocytes in mice with spontaneous autoimmune disease. J Immunol 121 :22132219, 1978
1410
20. Theofilopoulos AN, Shawler DC, Eisenberg RA, Dixon FJ: Splenic Ig-secreting cells and their regulation in autoimmune mice. J Exp Med 151:446466,1980 21. Suzuki N, Sakane T,Engleman EG:Anti-DNA antibody production by CDS+ and CD5- B cells of patients with systemic lupus erythematosus. J Clin Invest 85:238-247, 1990 22. Klinman DM, Steinberg AD: Systemic autoimmune disease arises from polyclonal B cell activation. J Exp Med 1651755-1760, 1987 23. Klinman DM: Polyclonal B cell activation in lupusprone mice precedes and predicts the development of autoimmune disease. J Clin Invest 86: 1249-1254, 1990 24. Ebling F, Hahn BH: Restricted subpopulations of DNA antibodies in kidneys of mice with systemic lupus: comparison of antibodies in serum and renal eluates. Arthritis Rheum 23:392403, 1980 25. Eisenberg RA, Craven SY,Cohen PL: The stochastic control of anti-Sm autoantibodies in MRL/Mp-IprApr mice. J Clin Invest 80:691497, 1987 26. Portanova JP, Arndt RE, Kotzin BL: Selective production of autoantibodies in graft-versus-host-induced and spontaneous murine lupus: predominant reactivity with histone regions accessible in chromatin. J Immunol 140755-760, 1988 27. Klinman DM, Eisenberg RA, Steinberg AD: Development of the autoimmune B cell repertoire in MRL-lpr/lpr mice. J Immunol 1440:506512, 1990 28. Cohen AS, Reynolds WE, Franklin EC, Kulka JP. Ropes MW, Shulman LE, Wallace SL: Preliminary criteria for the classification of systemic lupus erythematosus. Bull Rheum Dis 21:643-648, 1971 29. Klinman DM, Mushinski JF, Honda M, Ishigatsubo Y, Mountz JD, Raveche ES, Steinberg AD: Oncogene expression in autoimmune and normal peripheral blood mononuclear cells. J Exp Med 163:1292-1307, 1986 30. Ishigatsubo Y, Sakamoto H, Hagiwara E, Aoki A, Shirai A, Tani K, Okubo T, Klinman DM: Quantitation of autoantibody-secreting B cells in systemic lupus erythematosus. Autoimmunity 551-78, 1989 31. Longtenberg T , Kroon A, Gmelig-Meyling FH, Ballieux
KLINMAN ET AL RE: Antigen-specific activation of autoreactive B cells in normal human individuals. Eur J Immunol 16:14971501, 1986 32. Monestier M, donin B, Migliorini P, Dang H, Datta S, Kuppers R, Rose N, Maurer N, Tala1 N, Bona C: Autoantibodies of various specificities encoded by genes from the Vh J558 family bind to foreign antigens a n d share idiotypes of antibodies specific for self and foreign antigens. J Exp Med 166:1109-1 124, 1987 33. Klinman DM, Steinberg AD: Proliferation of anti-DNA producing NZB B cells in a non-autoimmune environment. J Immunol 137:69-75, 1986 34. Boyum A: Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 21:77-90, 1968 35. Sedgwick JD, Holt PG: A solid phase immunoenzymatic technique for the enumeration of specific antibody secreting cells. J lmmunol Methods 57:301-309, 1983 36. Ando DG, Ebling FM, Hahn BH: Detection of native and denatured DNA antibody forming cells by the enzyme-linked immunospot assay: a clinical study of (New Zealand black x New Zealand white)F, mice. Arthritis Rheum 29: 1139-1 146, 1986 37. Klinman DM, Steinberg AD: Similar in vivo expansion of B cells from normal D B M and autoimmune N Z B mice in xid recipients. J Immunol 139:2284-2289, 1987 38. Klinman DM, Steinberg AD: Novel ELISA spot assays to quantitate B cells specific for T cell and bromelated red blood cell autoantigens. J Immunol Methods 102: 157-164, 1987 39. Ginsburg WW, Finkelman FD,Lipsky PE: Circulating and pokeweed mitogen-induced immunoglobulinsecreting cells in systemic lupus erythematosus. Clin Exp Immunol 35:76-88, 1979 40. Hoch S, Schur PH, Schwaber J: Frequency of anti-DNA antibody producing cells from normals and patients with SLE. Clin Immunol Immunopathol 27:28-37, 1983 41. Freitas AA, Guilbert B, Holmberg D, Wennerstron G, Coutinho A, Avrameas S: Analysis of autoantibody reactivities in hybridoma collections derived from normal adult BALBlC mice. Ann Inst Pasteur Immunol 137D:3345, 1986
QUANTITATION OF IgM- AND IgG-SECRETING B CELLS IN THE PERIPHERAL BLOOD OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS DENNIS M. KLINMAN, AKIRA SHIRAI, YOSHIAKI ISHIGATSUBO, JACQUELINE CONOVER, and ALFRED D. STEINBERG An enzyme-linked immunospot assay was used to quantitate the number of autoantibody-secreting B cells in the peripheral blood of 67 patients with systemic lupus erythematosus. These patients had 1.5-4-fold more lymphocytes secreting IgG and I@ per million peripheral blood lymphocytesthan did normal controls. There was a concomitant increase in the number of B cells secreting antibodies reactive with a diverse panel of foreign and self antigens (including actin, myosin, trinitrophenylated keyhole limpet hemocyanin, ovalbumin, and retroviral gp160). By comparison, the number of B cells producing anti-DNA antibodies was increased disproportionately. The magnitude of this anti-DNA response correlated significantly with disease activity. Thus, B cell activation in human systemic lupus erythematosus had characteristics of both generalized (polyclonal) B cell activation and (autolantigen-specific immune stimulation. From the Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration, Bethesda, Maryland, the Cellular Immunology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, and the Yokohama City University Medical School, Yokohama, Japan. Dennis M. Klinman, MD,PhD: Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration; Akira Shirai, M D Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration; Yoshiaki Ishigatsubo. MD: Yokohama City University Medical School; Jacqueline Conover, BS: Laboratory of Retrovirus Research, Division of Virology, Food and Drug Administration; Alfred D. Steinberg, MD: Cellular Immunology Section, Arthritis and Rheumatism Branch, NIAMS. National Institutes of Health. Address reprint requests to Dennis M. Klinrnan, MD. PhD, Building 29A, Room 3 D 02, CBEWFDA, Bethesda, MD 20892. Submitted for publication March 21, 1991; accepted in revised form June 25, 1991. Arthritis and Rheumatism, Vol. 34,No. 11 (November 1991)
Systemic lupus erythematosus (SLE) is a n autoimmune disease characterized by B cell hyperproliferation (1) and the spontaneous production of antibodies reactive with a variety of self antigens (for review, see ref. 2). While there is a strong correlation between the degree of lupus activity and the number of Ig-secreting cells in the peripheral blood, the extent to which particular autoantibodies contribute to the disease process is unclear (3,4). No single autoantibody is invariably found in all patients with lupus. Whereas an excess of IgG antiDNA is classically found in SLE serum, anti-DNA antibodies are absent in up to 30% of individuals with active disease (5-7). In addition, antibodies reactive with one of a large number of other autoantigens (such as Ro, La, actin, myosin, and cardiolipin) are present at elevated concentrations in some patients (8-12), while other patients exhibit increased reactivity against multiple autoantigens simultaneously. These observations have raised questions concerning the contribution of specific autoantibodies to the disease process and suggest that a more generalized process of B cell activation might also be involved. Several studies of human and murine lupus have emphasized the apparent contribution of idiotypic or antigenic stimulation to the production of specific autoantibodies, such as anti-DNA (13-18). Others have found evidence of generalized B cell activation (associated with excess T cell help) (1,3,9,19,20).These processes are not mutually exclusive, and recent work suggests that distinct subsets of lupus B cells may be responding to polyclonal versus (auto)antigen-specific stimuli (21). Researchers at our laboratory and others have been using a sensitive and specific enzyme-linked
B CELLS IN SLE
1405
Table 1. Laboratory findings (mean rt SEM)in patients with systemic lupus erythematosus grouped according to degree of disease activity
Clinical disease status
normal
normal
IgM (mg/dl; normal
normal
Anti-DNA (I/titer; normal
(n)*
6504,600)
65415)
50-320)
16-40)
2 standard deviations from the mean value in the control group. In contrast, 30% of the patients expressed repertoires in which IgG anti-DNA production was preferentially increased. As expected, patients with active disease more frequently expressed repertoires skewed toward anti-DNA reactivity, i.e., 69% of those with severe disease, versus only 12% of those with inactive disease (Table 2).
DISCUSSION An ELISPOT assay was used to monitor the number and antigenic specificityof Ig-secreting B cells in the peripheral blood of patients with SLE. The absolute number of IgM- and IgG-secreting cells was increased 1.54-foldl depending on disease activity, over normal levels. Patients with lupus had disproportionately increased numbers of lymphocytes secreting IgG anti-DNA antibodies, with the frequency of antiDNA-producing lymphocytes correlating significantly with disease activity (r2 = 0.47). By comparison, the number of B cells producing antibodies against myosin, actin, ovalbumin, TNP, and retroviral gp160 increased in proportion to the total increase in the number of Ig-secreting cells. When the repertoires expressed by individual patients were analyzed, skewing of the repertoire toward increased anti-DNA production was found in 69% of the patients with active lupus, whereas skewing against the other antigens was generally present in 4%of the subjects. These findings indicate that I) lupus patients have increased numbers of B cells secreting antibodies reactive with a number of different antigens (consistent with the influence of polyclonal activation), but 2) anti-DNAproducing cells are preferentially activated, reflecting (auto)antigen-specificimmune stimulation. ELISPOT assays have been used to determine
B CELLS IN SLE
1407
DNA
TNP
0
A
A
A
A
A
0
-t
0
I
ACT
OVA A
400
A
A
A
A
A
a
0
200
Irnt
A
1W
m
0
0
OD
MY0
0
:
GP160 mT
I a
0
40
0
8 0
20
0
'f 0
A 0 0
m a
SLE
NL
SLE
NL
0
SLE
SLE
NL
Figure 2. Absolute number ofIgM- and IgG-secreting B cells, from 67 patients with systemic lupus erythematosus (SLE) and I5 normal controls ( N U that were reactive with the autoantigens DNA, actin (ACT), and myosin (MYO) or with the conventional antigens tnnitrophenylated keyhole limpet hemocyanin (TNP) and ovalbumin (OVA). An analysis of reactivity to GP160, the envelope glycoprotein of human immunodeficiency virus-I, was also performed in a subgroup of the subjects.
KLINMAN ET AL
1408
DISEASE ACTIVITY
=
25
IgM
EM EXl
HIGH MODERATE
INACTIVE 0 CONTROL
Figure 3. Percent of B cells producing antibodies reactive with each member of the antigen panel. The proportion of IgG anti-DNA secreting cells correlated closely with disease activity (r = 0.62,
Spearman-Pearson rank correlation). None of the other antigens examined showed statistically significant correlations with disease activity (r < 0.4). Values are the mean and SD of results from 16 patients with highly active systemic lupus erythematosus (SLE), 25 patients with moderately active SLE, 26 patients with inactive SLE, and 15 controls. See Figure 2 for additional definitions.
the number and specificity of antibody-secreting B cells in murine lupus (22,35,36) and to quantitate antibody-secreting B cells in cultured normal human PBL (31) and, most recently, in CD5+ and CD5- PBL (as analyzed by fluorescence-activated cell sorter) from patients with SLE (21). The accuracy of these assays has been documented by antigen-inhibition tests and through use of hybridomas of known antigenic specificity (22,3 1,36,38). Prior to the development of the ELISPOT assay, Ig secretion by individual human B cells was quantitated primarily by the hemolytic plaque assay. Studies using this assay demonstrated that patients with active SLE had 10,000-20,000 cells secreting Ig
per lo6 PBL (4,39), a finding confirmed by the present work. Cells that contained cytoplasmic Ig (and were therefore capable of immediate Ig secretion) were detected in 2-10% of SLE PBL (4). Few studies have examined the specificity of the antibodies secreted by these B cells in vivo. Budman et al demonstrated that 1 ,000/106PBL from SLE patients produced antibodies that reacted with sheep red blood cells, and that approximately onetenth this number reacted with a variety of chemical haptens (9). Earlier studies of bulk-cultured PBL demonstrated that lupus patients had increased numbers of anti-DNA-producing lymphocytes (40), but the assays used were not sufficiently sensitive to quantitate individual Ig-secreting cells. Moreover, results from studies of in vitro cultured B cells might not reflect the B cell repertoire actually expressed in vivo (41). A recent study used the ELISPOT assay to study CD5+ and CD5- B cells isolated from SLE patients, and the investigators concluded that the CD5- pool was polyclonally activated to produce anti-DNA, whereas the CD5+ pool seemed to reflect (auto)antigen-specific stimulation (21). In the present experiments, the ELISPOT assay was used to quantitate autoantibody-secreting B cells from a large number of patients with differing degrees of disease activity. We found that the frequency of lupus B cells producing anti-DNA antibodies increased as a function of disease activity, suggesting persistent (auto)antigenicstimulation. In contrast, the number of lymphocytes producing antibodies reactive with other antigens increased as a function of
-
Percentage of systemic lupus erythematosus (SLE) patients and normal controls expressing antigenically skewed repertoires*
Table 2.
Antigen DNA
TNPKLH
Actin
4 12 6
0 8 6
4
4
ovalMyosin bumin
~
Inactive SLE Moderately active SLE Very active SLE Controls
12 24 69 6
a 4 0 3
0 4 13 4
* The proportion of B cells producing antibodies reactive with DNA, trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), actin. myosin, and ovalbumin was calculated as a percentage of the total number of IgG-secreting cells (22). The percentage of individuals expressing repertoires preferentially biased toward reactivity with each antigen (i.e.. exceeding the control mean by >2 standard deviations) is shown. Expressed repertoires of patients with active disease were significantly skewed toward the overproduction of anti-DNA (P< 0.001, by Yates’ mean score).
B CELLS IN SLE the total increase in Ig-secreting cells, reflecting polyclonal activation. The possibility that anti-DNAsecreting B cells were responsible for the polyclonal stimulation (through nonspecific antigenic crossreactivity or bystander stimulation) seems unlikely, since the number of B cells producing anti-DNA did not correlate significantly with the number of B cells of other specificities (r2 = 0.08-0.19). Earlier studies from our laboratory have provided evidence that autoantibody production in murine lupus is a 2-stage process which is initiated by polyclonal B cell activation and subsequently magnified and perpetuated by specific (auto)antigenic stimulation (27). Similarly, polyclonal B cell expansion has been detected in humans with early untreated lupus (9), while patients with established disease have been shown to generally express repertoires skewed toward reactivity against 1 or more different autoantigens (30). This pattern of B cell reactivity suggests that autoantibody production in human SLE, like its murine counterpart, may be influenced by both antigenspecific and antigen-independent factors. Unfortunately, discrimination between these processes is difficult in humans, since it requires identifying at-risk individuals before their disease becomes clinically evident. One approach would be to analyze family members (especially identical twins) of lupus patients. It is our hope that continued analysis of the B cell repertoire expressed by patients with autoimmune diseases will aid in the early diagnosis and treatment of such diseases before the development of serious immunopathologic sequelae.
1409
systemic lupus erythematosus. N Engl J Med 281:701705, 1970
6. Feldman MD, Huston DP, Karsh J, Balow JE, Steinberg
AD: Correlation of serum IgG, IgM and anti-native DNA antibodies with renal and clinical indices of activity in SLE. J Rheumatol 952-58, 1982 7. Koffler D, Carr R, Agnello V, Thoburn R, Kunkel HG: Antibodies to human polynucleotides in human sera: antigenic specificityand relation to disease. J Exp Med 134:29&3 12, I971 8. McCarty GA, Rice JR, Bembe ML, Pisetsky DS: Independent expression of autoantibodies in SLE. J Rheumatol 9:691495, 1982 9. Budman DR, Merchant EB, Steinberg AD, Doft B, Gershwin ME, Lizzio E, Reeves JP: Increased spontaneous activity of antibody-formingcells in the peripheral blood of patients with active SLE. Arthritis Rheum 20:829-833, 1977
10. Morimoto C, Abe T, Hara M, Homma M: In vitro
TNP-specific antibody formation by peripheral lymphocytes from patients with SLE. Scand J Immunol6575579, 1977 11. Guilbert B, Dighiero G,Avrameas S: Naturally occur-
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