S215
SIGNIFICANCE OF ANTI-LYMPHOCYTE ANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS JOHN B. WINFIELD, PETER I. LOBO, and ALFRED SINGER
Sera from patients with SLE frequently contain IgM and IgG antibodies with multiple specificities for lymphocyte surface determinants, including autologous antigens. The IgM antibodies are of relatively low binding avidity and exhibit broad reactivity with B and T lymphocytes from most individuals. IgG antibodies are reactive selectively with PBL from different individuals and appear to be more specific for B cell and a minor proportion of T cells. The molecular nature of the surface determinants involved and their relationship with known antigens and receptors remain largely undefined. Interest in anti-lymphocyte antibodies in SLE relates in part to data suggesting a causal role in the abnormal immune system function in this disorder. In this regard, possible mechanisms that are supported by indirect data include: a ) antibody-mediated lymphocyte depletion in vivo, perhaps involving functional subsets specifically; b) antibody blockade of surface receptors operant in cellFrom the University of Virginia School of Medicine, Charlottesville, Virginia 22901, and The Rockefeller University, New York, New York 10021. Supported in part by NIH grants RR-102, AM 19096, AM 11766, and grants from the Whitehall Foundation, the John A. Hartford Foundation, Inc., the Virginia Affiliate of the American Heart Association, and the New York Chapter of the Lupus Foundation. John B. Winfield, M.D.: Associate Professor of Internal Medicine, University of Virginia, Senior Investigator of the Arthritis Foundation: Peter I. Lobo, M.D.: Assistant Professor of Internal Medicine, University of Virginia; Alfred Singer, M.D.: Clinical Associate, Immunology Branch, National Cancer Institute, Bethesda, Maryland. Address reprint requests to John B. Winfield, M.D., Department of Internal Medicine, Univerisity of Virginia School of Medicine, Charlottesville. Virginia 22901. Arthritis and Rheumatism, Vol. 21, No. 5 Supplement (June 1978)
cell and in cell-soluble antigen interactions. Certain data have raised the possibility that anti-lymphocyte antibodies represent serum markers for infection with virus as etiologic in SLE, but this question is controversial. Nevertheless, further investigation may yet reveal viral or genetically determined “SLE-specific” lymphocyte surface antigens. Clinically, anti-lymphocyte antibodies may have potential for mediating tissue injury in SLE, either directly or indirectly as circulating complexes in association with “shed” lymphocyte surface antigen. Direct evidence in support of such a role in the natural history of this disorder has not been forthcoming. Among the antibodies reactive with self- constituents in SLE, anti-lymphocyte antibodies recently have been of particular interest. This interest derives primarily from evidence that such antibodies may be related causally to immune system abnormalities in this disorder. In addition, considerable attention is being directed to the possibility that anti-lymphocyte antibodies might provide a means for defining genetic and environmental factors contributing to the development of SLE. Anti-lymphocyte antibodies in SLE may be separated operationally into those of the IgM and of the IgG classes. Although of uncertain substance with regard to basic differences in the types of reactive surface determinants, this dichotomy nevertheless is convenient for describing the special properties of these antibodies. Cold-reactive IgM antibodies predominate and are easily quantitated by complement-dependent micro-
S216
cytotoxicity assays (l,2). The IgM antibodies are broadly reactive with both B- and T-cell subpopulations of most individuals (2,3). These general properties are shared by similar antibodies that have been described in a large number of other immunologic or infectious diseases. IgG antibodies are less frequently detected in the sera of patients with SLE, perhaps because of their more limited cytotoxicity for peripheral blood lymphocytes (PBL). IgG antibodies are often warm-reactive, binding to lymphocytes equally well at 4°C and at 37"C, and appear t o exhibit relatively more restricted .antigenic specificities than IgM antibodies (43).
WINFIELD ET AL
a -.
74r
20
...
25°C.
B
B
ROLE OF ANTI-LYMPHOCYTE ANTIBODIES IN ABNORMAL IMMUNE SYSTEM FUNCTION Abnormal cellular immunity (CMI) and an exaggerated humoral response with production of autoantibodies are characteristic immunologic phenomena in SLE. Demonstrable abnormalities in CMI are more prominent during periods of active disease, with partial or complete resolution during remission (6,7). Considerable indirect evidence suggests that autoantibodies directed toward lymphocyte surface determinants may be of fundamental importance in this regard (4-14). The present consensus is that there is no intrinsic defect in the lymphocytes per se, although this issue is controversial and by no means resolved. The role of anti-lymphocyte antibodies could involve lymphocyte depletion, functional blockade of surface receptors, or both. Figure 1 illustrates the relationship between serum lymphocytotoxic activity and absolute peripheral lymphocyte counts in 26 patients with SLE studied serially during a 12-month period (9). In this prospective study, regression analysis indicated a highly significant linear inverse correlation between anti-lymphocyte antibody activitiy and absolute peripheral lymphocyte counts in these patients. Significant associations between anti-lymphocyte antibody activity and other clinical indices-e.g., DNA binding activity or serum complement levels-were not found. Although a causal relationship between anti-lymphocyte antibody and lymphopenia cannot be established by a study of this type, the data strongly support the probability of such an association. If anti-lymphocyte antibodies do function in vivo to remove circulating lymphocytes, a critical and as yet unresolved question concerns whether or not there is specific depletion of functionally important lymphocyte subpopulations. Winchester and colleagues carefully ex-
0
0 0
OEgO-9
0 0 0 0 0
04-
0 0
I
I
I
I
0 0 0 0 I
Figure 1. Inverse linear correlation between serum cytotoxicity and peripheral lymphocyte counts in SLE (10).( a ) Data obtained with wdiluted sera assayed at 2S°C against PBL ( r = 0.6667). ( b )Data obtained with serum diluted 1:4 in lS°C assays ( r = 0.4664).
amined this question using a variety of cell surface markers to define peripheral B and T cells in patients with lymphopenia and found no change in the proportion of these major subpopulations (15). Alteration in minor subpopulations was not excluded, however. More recently, several groups have been able to demonstrate depletion of a specific subset of peripheral T cells in SLE (7,14,16). Using a discontinuous Ficoll gradient technique for fractionating PBL, Glinski ef uf. (7,14) found that a distinct T-cell subpopulation was reduced in patients with active disease. Furthermore, evidence was
SIGNIFICANCE OF ANTI-LYMPHOCYTE ANTIBODIES
obtained suggesting that IgG antibodies mediated lysis of these T cells. Data obtained in functional studies by Bresnihan and Jasin (16) raise the possibility that patients with active SLE lack circulating short-lived suppressor cells. Early work clearly demonstrated that IgG antilymphocyte antibodies suppress the mixed leukocyte culture response (43). The data of Wernet and Kunkel indicated that this suppression involved specific blockage of surface receptors functional in this type of cellcell interaction (4). Stobo and colleagues (see page S210 of this symposium) have observed that autologous Tcell reactivity to a Fc receptor-bearing, surface Ig negative lymphocyte-like population was inhibited by coldreactive antibody, possibly through receptor blockade on both target and responding cells. Other recent data suggest that 7s IgG from SLE sera has the capacity to inhibit antibody-dependent cell-mediated cytotoxicity, although neither the specificity of this blockade nor the antibody nature of the inhibiting IgG was defined completely (1 1-13). Singer and colleagues have examined the suppressive effects of SLE sera on certain specific antigen-induced blastogenic responses (17). The data in Table 1 demonstrate that SLE serum markedly inhibited the tetanus toxoid-induced DNA synthetic reTable 1. Inhibition of Tetanus Toxoid-Induced Blastogenic Response by SLE Serum (17) Tetanus Toxoid Inhibicpm tion,% AB pool (control)
PHA cpm
84,458
I 14,000
93,046 89,863 97,248 85.247
142,853 152,206 125.4 12 126.91 2
Inhibition,%
Normal serum: 1
2 3 4
SLE serum: Es Ha Sn
sc
12.125 52,189 17,791 10,709
86 38 79 19
114,518 100,979 138,362 100,860
S217
Table 2. Inhibition of Tetanus Toxoid Blaslogenic Response by Immunoglobulin from SLE Patients ( I 7); Ig (pg/ml) Required for 50% Inhibition? SLE Patients
IgM
IgG
Ha Es Sa Sm
11 40 28 130
270 260 52 160
* Culture conditions
are described in Table 1. IgM and IgG were isolated from SLE serum by gel filtration or sucrose density gradient centrifugation using pH 3.5 buffers. Concentrations of Ig represent pg/ml of culture medium.
sponse of PBL but had little effect on the PHA response. IgM and IgG fractions were obtained from a number of these sera by gel filtration or by sucrose density gradient centrifugation using low pH buffers. The IgM fractions were relatively more inhibitory of the tetanus toxoid blastogenic response than the IgG fractions (Table 2). Certain evidence was obtained in lymphocyte absorption and elution experiments using purified SLE serum IgM suggesting that cold-reactive anti-lymphocyte antibody was the major element inhibiting the tetanus toxoid response. Data from a representative experiment of this type are shown in Table 3. Because cell viability was not decreased in the experimental cultures, the most probable mechanism for inhibition was receptor blockade. It remains to be determined, however, whether this inhibitory effect involved lymphocytes alone, macrophages through surface antigens cross-reactive with those on lymphocytes, or both. Table 3. Inhibition of Tetanus Toxoid-Induced Blastogenic Response by Cold-Reactive IgM Anti-Lymphocyte Antibody ( I 7); Blastogenic Response (cpm)
15°C Cyto-
toxicity
0 11 0 12
Optimal concentrations of tetanus toxoid (100 &well) or PHA were added to triplicate cultures of isolated peripheral mononuclear cells at a density of 1.5 X lV/ml. The medium contained 10% heatinactivated AB serum in control cultures or 5% AB serum and 5% normal or SLE serum in experimental cultures. The DNA synthetic response was quantitated by incorporation of "-thymidine added 16 hours before harvest. Maximal response occurred on day 5 for tetanus toxoid and day 3 for PHA.
SLE IgM
Tetanus Toxoid
PHA
Bkqd
Cells)t
Unabsorbed Tonsil absorbed Tonsil eluate$
8,208 50,397 8,199
188,000 301,000 150,000
1,055 2,585 650
85
SIGNIFICANCE OF ANTI-LYMPHOCYTE ANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS JOHN B. WINFIELD, PETER I. LOBO, and ALFRED SINGER
Sera from patients with SLE frequently contain IgM and IgG antibodies with multiple specificities for lymphocyte surface determinants, including autologous antigens. The IgM antibodies are of relatively low binding avidity and exhibit broad reactivity with B and T lymphocytes from most individuals. IgG antibodies are reactive selectively with PBL from different individuals and appear to be more specific for B cell and a minor proportion of T cells. The molecular nature of the surface determinants involved and their relationship with known antigens and receptors remain largely undefined. Interest in anti-lymphocyte antibodies in SLE relates in part to data suggesting a causal role in the abnormal immune system function in this disorder. In this regard, possible mechanisms that are supported by indirect data include: a ) antibody-mediated lymphocyte depletion in vivo, perhaps involving functional subsets specifically; b) antibody blockade of surface receptors operant in cellFrom the University of Virginia School of Medicine, Charlottesville, Virginia 22901, and The Rockefeller University, New York, New York 10021. Supported in part by NIH grants RR-102, AM 19096, AM 11766, and grants from the Whitehall Foundation, the John A. Hartford Foundation, Inc., the Virginia Affiliate of the American Heart Association, and the New York Chapter of the Lupus Foundation. John B. Winfield, M.D.: Associate Professor of Internal Medicine, University of Virginia, Senior Investigator of the Arthritis Foundation: Peter I. Lobo, M.D.: Assistant Professor of Internal Medicine, University of Virginia; Alfred Singer, M.D.: Clinical Associate, Immunology Branch, National Cancer Institute, Bethesda, Maryland. Address reprint requests to John B. Winfield, M.D., Department of Internal Medicine, Univerisity of Virginia School of Medicine, Charlottesville. Virginia 22901. Arthritis and Rheumatism, Vol. 21, No. 5 Supplement (June 1978)
cell and in cell-soluble antigen interactions. Certain data have raised the possibility that anti-lymphocyte antibodies represent serum markers for infection with virus as etiologic in SLE, but this question is controversial. Nevertheless, further investigation may yet reveal viral or genetically determined “SLE-specific” lymphocyte surface antigens. Clinically, anti-lymphocyte antibodies may have potential for mediating tissue injury in SLE, either directly or indirectly as circulating complexes in association with “shed” lymphocyte surface antigen. Direct evidence in support of such a role in the natural history of this disorder has not been forthcoming. Among the antibodies reactive with self- constituents in SLE, anti-lymphocyte antibodies recently have been of particular interest. This interest derives primarily from evidence that such antibodies may be related causally to immune system abnormalities in this disorder. In addition, considerable attention is being directed to the possibility that anti-lymphocyte antibodies might provide a means for defining genetic and environmental factors contributing to the development of SLE. Anti-lymphocyte antibodies in SLE may be separated operationally into those of the IgM and of the IgG classes. Although of uncertain substance with regard to basic differences in the types of reactive surface determinants, this dichotomy nevertheless is convenient for describing the special properties of these antibodies. Cold-reactive IgM antibodies predominate and are easily quantitated by complement-dependent micro-
S216
cytotoxicity assays (l,2). The IgM antibodies are broadly reactive with both B- and T-cell subpopulations of most individuals (2,3). These general properties are shared by similar antibodies that have been described in a large number of other immunologic or infectious diseases. IgG antibodies are less frequently detected in the sera of patients with SLE, perhaps because of their more limited cytotoxicity for peripheral blood lymphocytes (PBL). IgG antibodies are often warm-reactive, binding to lymphocytes equally well at 4°C and at 37"C, and appear t o exhibit relatively more restricted .antigenic specificities than IgM antibodies (43).
WINFIELD ET AL
a -.
74r
20
...
25°C.
B
B
ROLE OF ANTI-LYMPHOCYTE ANTIBODIES IN ABNORMAL IMMUNE SYSTEM FUNCTION Abnormal cellular immunity (CMI) and an exaggerated humoral response with production of autoantibodies are characteristic immunologic phenomena in SLE. Demonstrable abnormalities in CMI are more prominent during periods of active disease, with partial or complete resolution during remission (6,7). Considerable indirect evidence suggests that autoantibodies directed toward lymphocyte surface determinants may be of fundamental importance in this regard (4-14). The present consensus is that there is no intrinsic defect in the lymphocytes per se, although this issue is controversial and by no means resolved. The role of anti-lymphocyte antibodies could involve lymphocyte depletion, functional blockade of surface receptors, or both. Figure 1 illustrates the relationship between serum lymphocytotoxic activity and absolute peripheral lymphocyte counts in 26 patients with SLE studied serially during a 12-month period (9). In this prospective study, regression analysis indicated a highly significant linear inverse correlation between anti-lymphocyte antibody activitiy and absolute peripheral lymphocyte counts in these patients. Significant associations between anti-lymphocyte antibody activity and other clinical indices-e.g., DNA binding activity or serum complement levels-were not found. Although a causal relationship between anti-lymphocyte antibody and lymphopenia cannot be established by a study of this type, the data strongly support the probability of such an association. If anti-lymphocyte antibodies do function in vivo to remove circulating lymphocytes, a critical and as yet unresolved question concerns whether or not there is specific depletion of functionally important lymphocyte subpopulations. Winchester and colleagues carefully ex-
0
0 0
OEgO-9
0 0 0 0 0
04-
0 0
I
I
I
I
0 0 0 0 I
Figure 1. Inverse linear correlation between serum cytotoxicity and peripheral lymphocyte counts in SLE (10).( a ) Data obtained with wdiluted sera assayed at 2S°C against PBL ( r = 0.6667). ( b )Data obtained with serum diluted 1:4 in lS°C assays ( r = 0.4664).
amined this question using a variety of cell surface markers to define peripheral B and T cells in patients with lymphopenia and found no change in the proportion of these major subpopulations (15). Alteration in minor subpopulations was not excluded, however. More recently, several groups have been able to demonstrate depletion of a specific subset of peripheral T cells in SLE (7,14,16). Using a discontinuous Ficoll gradient technique for fractionating PBL, Glinski ef uf. (7,14) found that a distinct T-cell subpopulation was reduced in patients with active disease. Furthermore, evidence was
SIGNIFICANCE OF ANTI-LYMPHOCYTE ANTIBODIES
obtained suggesting that IgG antibodies mediated lysis of these T cells. Data obtained in functional studies by Bresnihan and Jasin (16) raise the possibility that patients with active SLE lack circulating short-lived suppressor cells. Early work clearly demonstrated that IgG antilymphocyte antibodies suppress the mixed leukocyte culture response (43). The data of Wernet and Kunkel indicated that this suppression involved specific blockage of surface receptors functional in this type of cellcell interaction (4). Stobo and colleagues (see page S210 of this symposium) have observed that autologous Tcell reactivity to a Fc receptor-bearing, surface Ig negative lymphocyte-like population was inhibited by coldreactive antibody, possibly through receptor blockade on both target and responding cells. Other recent data suggest that 7s IgG from SLE sera has the capacity to inhibit antibody-dependent cell-mediated cytotoxicity, although neither the specificity of this blockade nor the antibody nature of the inhibiting IgG was defined completely (1 1-13). Singer and colleagues have examined the suppressive effects of SLE sera on certain specific antigen-induced blastogenic responses (17). The data in Table 1 demonstrate that SLE serum markedly inhibited the tetanus toxoid-induced DNA synthetic reTable 1. Inhibition of Tetanus Toxoid-Induced Blastogenic Response by SLE Serum (17) Tetanus Toxoid Inhibicpm tion,% AB pool (control)
PHA cpm
84,458
I 14,000
93,046 89,863 97,248 85.247
142,853 152,206 125.4 12 126.91 2
Inhibition,%
Normal serum: 1
2 3 4
SLE serum: Es Ha Sn
sc
12.125 52,189 17,791 10,709
86 38 79 19
114,518 100,979 138,362 100,860
S217
Table 2. Inhibition of Tetanus Toxoid Blaslogenic Response by Immunoglobulin from SLE Patients ( I 7); Ig (pg/ml) Required for 50% Inhibition? SLE Patients
IgM
IgG
Ha Es Sa Sm
11 40 28 130
270 260 52 160
* Culture conditions
are described in Table 1. IgM and IgG were isolated from SLE serum by gel filtration or sucrose density gradient centrifugation using pH 3.5 buffers. Concentrations of Ig represent pg/ml of culture medium.
sponse of PBL but had little effect on the PHA response. IgM and IgG fractions were obtained from a number of these sera by gel filtration or by sucrose density gradient centrifugation using low pH buffers. The IgM fractions were relatively more inhibitory of the tetanus toxoid blastogenic response than the IgG fractions (Table 2). Certain evidence was obtained in lymphocyte absorption and elution experiments using purified SLE serum IgM suggesting that cold-reactive anti-lymphocyte antibody was the major element inhibiting the tetanus toxoid response. Data from a representative experiment of this type are shown in Table 3. Because cell viability was not decreased in the experimental cultures, the most probable mechanism for inhibition was receptor blockade. It remains to be determined, however, whether this inhibitory effect involved lymphocytes alone, macrophages through surface antigens cross-reactive with those on lymphocytes, or both. Table 3. Inhibition of Tetanus Toxoid-Induced Blastogenic Response by Cold-Reactive IgM Anti-Lymphocyte Antibody ( I 7); Blastogenic Response (cpm)
15°C Cyto-
toxicity
0 11 0 12
Optimal concentrations of tetanus toxoid (100 &well) or PHA were added to triplicate cultures of isolated peripheral mononuclear cells at a density of 1.5 X lV/ml. The medium contained 10% heatinactivated AB serum in control cultures or 5% AB serum and 5% normal or SLE serum in experimental cultures. The DNA synthetic response was quantitated by incorporation of "-thymidine added 16 hours before harvest. Maximal response occurred on day 5 for tetanus toxoid and day 3 for PHA.
SLE IgM
Tetanus Toxoid
PHA
Bkqd
Cells)t
Unabsorbed Tonsil absorbed Tonsil eluate$
8,208 50,397 8,199
188,000 301,000 150,000
1,055 2,585 650
85
