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Cross-reactivity of antilymphocyte and antinuclear antibodies in systemic lupus erythematosus
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Cross-reactivity Antibodies
14, 292-299 (1979)
of Antilymphocyte and Antinuclear in Systemic Lupus Erythematosus’
ROBERTP. SEARLES,~RONALD P. MESSNER, AND ARTHUR D. BANKHURST," Department
of Medicine, Lovelace Medical
University Center,
of Net+ Mexico, School Albuquerque. New Mexico
of Medicine
and
87131
Received February 2 1, 1979 Both antinuclear antibody (ANA) and antilymphocyte antibody (ALA) are prevalent m systemic lupus erythematosus (SLE). This report presents data supporting crossreactivity between these two antibodies. Differential absorption of SLE sera showed that chicken erythrocyte nuclei absorbed IgM ALA while intact human and chicken erythrocytes had no effect. RNase digestion of chicken nuclei fragments partially removed the ALA absorbing effect. Both IgM and IgG ANA were also absorbed by the chicken nuclei. Absorptions with intact human lymphocytes removed ALA activity and absorption with human mononuclear cells removed ANA activity. ALA and ANA were both found in PBS eluates from absorbed chicken nuclei, demonstrating specificity of nuclear absorption.
INTRODUCTION
Systemic lupus erythematosus (SLE) is a connective tissue disease characterized by multiple autoantibodies including antinuclear (ANA) and antilymphocyte antibodies (ALA) (l-4). Both antibodies fluctuate with SLE disease activity and each has been postulated to play a major role in the pathogenesis of this disease (5-8). ANA are a heterogenous group of antibodies directed against multiple nuclear antigens, including deoxyribonucleic acid (DNA) (9), saline extractable proteins (lo), soluble nucleoprotein (1 l), and ribonucleic acid (RNA) (12, 13). ALA are also heterogenous and are directed at lymphocyte surface antigens (14, 15). Lymphocytotoxic ALA are predominantly cold reactive IgM and react with both T and B lymphocytes (15, 16). In addition, ALA react with brain extract and are increased in lupus patients with CNS disease (17, 18). They also react with trophoblastic antigens and may play a role in spontaneous abortion in SLE patients (19). Recently the possibility of cross-reactions between ANA and ALA has been suggested (20). This report presents further data demonstrating the existence of cross-reactivity between ANA and ALA. MATERIALS Subjects.
American
Fourteen Rheumatism
AND METHODS
patients with SLE, as determined by the criteria of the Association (21), were screened for ANA and ALA. From
’ Supported by Career Development Award, Grant 5K04 AM 70301, Grant 2 ROl AM 13789-08 from the National Institute of Health, and grants from the National and New Mexico Arthritis Foundation. ’ Postdoctoral Research Fellow, Arthritis Foundation. 3 Senior Investigator. Arthritis Foundation. 292 OWO-1229179/110292-08$01.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
ALA
AND
ANA
IN
SLE
293
this group, six patients with high titers of both ANA and ALA were selected for further investigation. Those tested included three acute untreated patients and three patients who became active while on maintenance prednisone. All six patients had low C3 complement; five of six patients had skin rash and arthritis; four of six had nephritis and direct Coombs positivity; and two of six had Raynaud’s phenomenon, pleuritis, and thrombocytopenia. Lymphocytes. Lymphocytes were separated from heparinized whole blood of normal human donors by Ficoll- Hypaque gradient centrifugation (22). Monocyte depletion was effected through glass adherence on petri dishes at 37°C for 30 min. T cell-enriched populations of human lymphocytes were prepared using rabbit immunoglobulin-coated bead columns (23). T-Cell preparations had 95% or greater E rosettes with sheep erythrocytes (24). Peroxidase positive cells using a benzidine dihydrochloride stain (25) were generally 2% or less in T cell-enriched lymphocytes and monocyte-depleted preparations. Antilymphocyte antibody. ALA were determined by both indirect immunofluorescence of suspensions of lymphocytes and complement-mediated cytotoxicity (3, 14). In the suspension procedure, 2 x lo6 monocyte-depleted or T cell-enriched lymphocytes were incubated serially with test serum and with fluorescein-labeled (FITC) goat antihuman IgM or IgG (Meloy, Springfield, Va.) for 1 hr at 4°C. Specificity for IgM and IgG was measured by immunoelectrophoresis. In each test, 300 cells were counted and results were recorded as the total number of fluorescent cells per 100 counted. Cell viability tested by trypan blue exclusion was 95% or greater before and after ALA assay. Heat inactivation of SLE sera did not effect ALA suspension determination. Antilymphocyte antibodies were also determined using the two-step microcytotoxicity assay of Terasaki (3) with a complement incubation of 3 hr at 15°C. Dilutions of SLE sera were made with minimum essential medium (MEM) and 10% human AB positive serum. Normal human serum controls had fewer than 5% dead cells in each experiment. Antinuclear antibody. Antinuclear antibodies were measured in an indirect immunofluorescent technique with mouse liver as substrate (26). Fluoresceinlabeled goat anti-human IgM and IgG (Meloy) were employed as the second antibody. Abiorptions. A 50% suspension of chicken nuclei in phosphate-buffered saline (PBS) was prepared from fresh chicken erythrocytes using a saponin lysis method (27). Unlysed chicken and human erythrocytes were employed as controls and absorptions were carried for 1 hr at 4°C. Chicken nuclei and fresh erythrocytes were greater than 99% intact before and after absorption by phase contrast microscopy. In addition, preparations of chicken erythrocyte nuclear fragments prepared by sonication (28) of intact chicken nuclei were used as absorbents. Absorptions were also carried out using Ficoll-separated human mononuclear cells and monocyte-depleted lymphocytes. To remove ANA 50% mononuclear cell solutions each containing 3 x lOa cells in PBS were used to absorb one-half the volume of serum. Fewer cells were used for ALA absorptions and contained only lymphocytes as prepared by monocyte depletion in glass petri dishes. Twenty percent lymphocyte suspensions in PBS were incubated with one-half serum volumes. Percentage absorption was calculated by dividing number of positive cells
294
SEARLES,
MESSNER,
AND
BANKHURST
per 100 counted after absorption by number of positive cells before absorption. Lymphocyte cell viability by trypan blue exclusion was greater than 95% before and after absorption. Efution. Antibody was eluted from chicken erythrocyte nuclei used in the absorption of SLE sera by incubation in PBS (pH 7.2) at 4°C for 6 hr. PBS was changed after each 2-hr period and the three eluates were combined and concentrated before ANA and ALA determination. Enzyme digestion. Chicken nuclei fragments prepared by sonication of intact nuclei were used for enzyme digestion experiments. Digestion was carried out at 37°C for 45 min with 0.2 mg/ml ribonuclease (RNase) (Worthington, Freehold, N.J.) and 0.2 mg/ml deoxyribonuclease (DNase) (Sigma, St. Louis, MO.) and was terminated by washing the cells in PBS at room temperature (29). Absorption of SLE sera was carried out with both undigested and enzyme-digested fragments (50% in PBS). RESULTS
ALA activity in sera of patients with active SLE was approximately equivalent using monocyte-depleted and T cell-enriched preparations of lymphocytes as targets. T lymphocytes were employed in most experiments because of the greater homogenicity of these preparations and to eliminate the nonspecific cytophilic attachment of ALA to Fc or complement receptors present on monocytes or B lymphocytes (15, 30, 31). Fc receptors are also present on T lymphocytes but the present assay method does not satisfy the necessary experimental conditions required for their detection (32. 33). Positive cells with normal human serum (NHS) controls using FITC anti-IgM were always 2% or less in the T-cell suspension system. Therefore, pepsin digestion of the second antibody was not routinely employed in these experiments. Only SLE sera positive for ALA by cytotoxicity method were found to have ALA using suspension immunofluorescence. ALA identified in suspension was primarily of the IgM class. Titers of ALA in SLE were found to vary from 1: 16 to 1: 128 depending on the combination of serum, lymphocyte target, and assay employed. Absorption of Antilymphocyte Antibody To evaluate the cross-reactivity of ALA with nuclear antigens, SLE serums were absorbed with freshly prepared chicken erythrocytes, chicken erythrocyte nuclei, and human lymphocytes. ALA was determined using the T-lymphocyte suspension technique. Representative data from SLE Patient 124 demonstrate the results of two sequential absorptions (Fig. 1). Absorption with chicken nuclei resulted in a 94% reduction in the percent of cells labeled by the SLE serum. A decrease of 71% of preabsorption values was found after absorption with human lymphocytes. A 94% reduction of ALA was found after a third adsorption with human lymphocytes. Absorption with intact chicken erythrocytes essentially had no effect on the preabsorption titers. The range of absorption of ALA by the chicken nuclei preparations was 55 to 94% in four SLE patients tested. The range for human lymphocytes was 71 to 100% absorption. The dilution factor intrinsic to this absorption system was considered in final interpretation of the postabsorption titers.
ALA AND ANA IN SLE
ABSORPTION
295
NUMBER
FIG. 1. Sequential absorption of SLE Patient 124 with chicken erythrocytes, chicken erythrocyte nuclei and human lymphocytes. Antilymphocyte antibodies (ALA) were measured by indirect immunofluorescence of suspension of T lymphocytes.
Specificity of the reaction of ALA with nuclear antigens was further investigated by eluting the ALA from postabsorption chicken erythrocyte nuclei in PBS for 6 hr at 4°C. In this experiment the three sequential absorptions of SLE Patient 126 with chicken nuclei reduced the percentage of positive cells from 20 to 9 and completely eliminated ANA activity at the lowest dilution tested (Table 1). ALA and ANA were both found in PBS eluates from the absorbed nuclei, but the ALA activity recovered was greater than that of ANA. These data demonstrate differential elution of ALA and ANA and suggest ALA has a lower avidity for the nuclei. To further identify which nuclear antigens have ALA reactivity, chicken erythrocyte nuclei were sonicated into nuclear fragments and digested with RNase and DNase. The undigested nuclear fragments were as effective as intact nuclei in the absorption of ALA (67- 100% absorption). DNase digestion of nuclear fragments had no effect on ALA absorption. RNase-Digested preparations were less effective and yielded 57% absorption compared to 91% absorption with the untreated nuclear fragments. These data suggest RNase-sensitive nuclear antigens participate in ALA cross-reactivity. To confirm these absorption experiments, ALA were tested by the cytotoxic procedure. Similar results were found in four SLE patients after absorption with TABLE 1 ALA AND ANA BEFORE AND AFTER ABSORPTION WITHCHICKENNUCLEI IN SLE PATIENT 126 ANDELUATES OF ABSORBED NUCLEI ALA Dilution Before After Eluate Control
1:6 1:6 1:3 PBS
ANA Percent positive” 20 9 15 1
” ALA, Percentage positive cells using indirect immunofluorescence cytes as targets. ’ ANA, Negative at lowest titer tested 1: 16.
I&f 1:256 Negb 1:12 Neg
I& 1: 1024 Neg 1:32 Neg
of suspensions of T lympho-
SEARLES,
296
MESSNER,
AND BANKHURST
TABLE CHICKEN
NUCLEI
ABSORPTIONS
2 ALA IX SLE P.41IEN I s
OF CYTOTOXI~
ALA SLE
~__
Patient 120 Patient 121 Patient 122 Patient 125 NHS” NHS MEMlAB
titers
Pre
PO9
256” 32 256 8 Neg Neg
c Nep 4 Neg NQ! Neg
-
” Positive 2 30% dead cells. b NHS and MEMIlOYF AB+ were <5% dead cells.
chicken erythrocyte nuclei (Table 2). In addition, absorptions with intact chicken and human erythrocyte preparations showed no significant change in cytotoxic ALA effect. Possible interference by chicken nuclei absorption with the two-step cytotoxic method was also evaluated. Lymphocytes were preincubated with nonabsorbed and absorbed cytotoxic serum. This was followed by addition of guinea pig complement and cells were evaluated for numbers killed. Reestablishment of lymphocyte cytotoxicity by the nonabsorbed cytotoxic serum demonstrated that the integrity of the cytotoxic method was not altered by anticomplementary activity in chicken nuclei-absorbed SLE sera. Absorption of Antinuclear Antibody The effect of absorption on ANA in SLE sera was also evaluated. Chicken erythrocytes, erythrocyte nuclei, human erythrocytes, and human mononuclear cells (50% in PBS) were used as absorbents of SLE sera over three consecutive 1-hr periods at 4”C, and IgM and IgG ANA were determined by indirect immunofluorescence. A representative experiment is shown in Table 3. Absorption of both IgM and IgG ANA in Serum 124 by the chicken nuclei was 98% or greater at the lowest dilution tested (1: 16). Absorption with human mononuclear cells (9 x IO8 cells) also removed greater than 80% of IgM and IgG ANA. In all human mononuclear cell absorptions, residual ANA activity was present, Investigation of the dilution effect during antibody absorption was tested by absorption of IgG ANA with intact human and chicken erythrocytes. The titer changes after absorption with chicken erythrocytes were one tube dilution or less.
ABSORPTION
TABLE 3 OF SLE SERUM 126ANA
EFFWT
ANA titer Absorbent
kM
kG
Chicken nuclei Human mononuclear cells
640 Neg” 32
640 16 128
Preabsorption
n No immunofluorescent
at lowest titer tested (1: 16).
--
ALA
AND
ANA
IN
SLE
297
DISCUSSION The data presented here demonstrate that antibodies reacting with intact lymphocytes can be absorbed with nuclei. The corollary observation that antinuclear antibodies can be absorbed with intact human mononuclear cells was also confirmed (20). Using ANA positive, ALA positive SLE sera, significant ALA absorption occurred with fresh chichen erythrocyte nuclei (55-94%) and paralleled ALA absorption by human lymphocytes (71- 100%). No significant ALA reactivity was found with intact chicken or human erythrocytes. Similar results occurred with ANA absorption where chicken nuclei absorbed 98% or greater of IgM and IgG ANA. Extensive absorption with human mononuclear cells removed greater than 80% of IgM and IgG ANA. These latter results are similar to the observations that whole leukocyte preparations will absorb IgG ANA (20). They further demonstrate that human mononuclear cells absorb IgM ANA which might be expected considering the IgM nature of cold reactive ALA. Elution of antibodies from the chicken nuclei yielded significant recovery of absorbed ALA, demonstrating specificity of nuclear absorption. Experiments with ALA by the cytotoxic method produced similar results with nuclear and lymphocyte absorptions. The data presented are not meant to be representative of all SLE patients. They represent selected sera with high titers of both ALA and ANA. Further studies of SLE sera with other combinations of ALA and ANA positivity are needed for the total profile. The concept that the autoantibodies of SLE have shared specificities will warrant further investigation. As previously mentioned, ANA are heterogenous with specificities for multiple nuclear antigens. ALA are predominantly IgM antibodies and are also heterogenous as indicated by differences in reactivity with brain (17) and with lymphocytes from different donors (34). Available data suggest that all ALA and ANA do not share identical specificities. Sera containing one of these activities but not the other do exist, demonstrating that antibodies without overlapping specificities occur in viva. The variable reactivity of ALA with lymphocytes from different donors contrasts with lack of substrate specificity found with ANA. Finally, our data on ANA/ALA cross-reactivity showed residual ALA activity despite exhaustive nuclear absorption and elution experiments with PBS suggested differential ALA and ANA affinity for nuclear antigens. The differences between sera in the effect of absorption with nuclei and lymphocytes noted in this study are thus not unexpected and probably reflect differences in the repertoire of antibodies contained in each serum. Besides the cross-reactivity between ALA and ANA, rheumatoid factor also reacts with ANA (35, 36). Cross-reactions of rheumatoid factor with ANA and peripheral blood leukocytes with ANA were found to be mutually exclusive which further supports specificity of these reactions. Most theories on antibody cross-reactivity suggest these reactions occur only when structural similarity exists between the offending antigens (37). On the other hand, Richards ef al. (38) proposed the existence of polyfunctional antibody combining sites to explain reactions between diverse haptens with the same monovalent myeloma proteins. These myeloma proteins should be unifunctional with definite antigen specificity and yet they react with diverse ligands like menadione and dinitrophenol.
298
SEARLES,
MESSNER,
AND
BANKHURST
Several types of nuclear antigens have been described (9-13), and some of these may be structurally similar to lymphocyte surface antigens. Our data showed DNase had no effect on ALA absorption but RNase digestion of nuclei fragments removed 37% of ALA cross-reactivity. Thus, RNase-sensitive nuclear antigens may share specificities with the lymphocyte surface antigens. Recent observations have shown both DNA (39) and DNA receptors (40) are present on lymphocyte membranes suggesting this antigen may also participate in crossreactions between ALA and ANA. Further investigations are needed to identify the specific nuclear antigens involved in ALA cross-reactivity before structural comparisons are made. Resolution of ANA and ALA cross-reactivity may eventually give insight into whether, and if so, how the autoantibodies are involved in the pathogenesis of SLE. REFERENCES 1. Hargraves, M. M., Richmond, H., and Morton, R., Presentation of two bone marrow elements, the Tart cell and the LE cell. Proc. Stuff Meeting, Mayo Clinic. 23, 25-28. 1948. 2. Friou, G. J., In “The LE Cell Phenomenon and Antinuclear Antibodies in Arthritis and Rheumatism” (J. L. Hollander and D. J. McCarty, Eds.). p. 172. Lea & Febiger. Philadelphia. 1972. 3. Terasaki, P. I., Mottironi, V. D., and Barnett. E. V., Cytotoxins in disease: Autocytotoxins in lupus. N. Engl. .I. Med. 283, 724-728, 1970. 4. Mittal. K. K., Rossen, R. D., Sharp, U. T. et al., Lymphocytotoxic antibodies in systemic lupus erythematosus. Nature (London) 225, 1255- 1256, 1970. 5. Norman, D. D., Kurata, N., and Tan, E. M., Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann. Inc. Med. 83, 464-469, 1975. 6. Alarcon-Segovia. D., Ruiz-Arguelles. A., and Fishbein, E., Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature Ilondon) 271,67-69. 1978. 7. Messner, R. P., In Lymphocytes and Their Interactions (R. C. Williams, Jr.. Ed.), p. 169. Raven Press, New York, 1975. 8. Winfield, J. B., Winchester, R. J., and Kunkel. H. G.. Association of cold-reactive antilymphocyte antibodies with lymphopenia in systemic lupus erythematosus. Arthritis Rherrm. 18.587-594, 1975. 9. Tan, E. M., Schur, P. H.. Carr, R. I., and Kunkel. H. G.. Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J. C/in. Invest. 45, 1732, 1966. IO. Sharp, G. C., Irvin, W. S.. Tan, E. M., Gould, R. G., and Holman, H. R., Mixed connective tissue disease-an apparently distinct rheumatic disease associated with a specific antibody to an extractable nuclear antigen (ENA). Amer. J. Med. 52, 148, 1972. 11. Tan, E. M., An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus erythematosus. J. Clin. Invest. 46, 735-745. 1967. 12. Ritchie, R. F.. Antinucleolar antibodies. N. Engl. J. Med. 282, 1174-I 178, 1970. 13. Pinas, J. L.. Northway. J. D., and Tan, E. M.. Antinucleolar antibodies in human sera. ./. Inrmunol. 111,9%-1004, 1973. 14. Messner. R. P.. Kennedy. M. S., and Jelinek, J. G.. Antilymphocyte antibodies in syatemrc lupus erythematosus: effect on lymphocyte surface characteristics. Arthritis Rheum. 18,201-206, 1975. 15. Winfield. J. B., Winchester. R. J.. and Wemet, P.. et al., Nature of cold-reactive antibodies to lymphocyte surface determinants in systemic lupus erythematosus. Arthritis Rheum. 18. I-8. 1975. 16. Michlmayr, G., Pathonci, C., Huber. C.. and Huber, H., Antibodies for T lymphocytes in 3ystemic lupus erythematosus. Ckn. Exp. Immunol. 24, 18-25. 1976. 17. Bluestein, H. G., and Zvaifler, N. J.. Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. J. C/in. Zn~~rst. 57, 509, 1976. 18. Bresnihan. B., Oliver, M.. Gerigor. R., and Hughes, G. R.. Brain reactivity of lymphocytotoxtc
ALA
AND
antibodies in systemic lupus erythematosus Zmmunol.
30,
333-337,
ANA
IN
SLE
with or without cerebral involvement.
299 Clin. Exp.
1977.
19. Bresnihan, B., Grigor, R. R., Oliver, M., Lewkonia, R. M., Hughes, G. R., Livings, R. E., and Fauck, W. P., Immunologic mechanism for spontaneous abortion in systemic lupus erythematosus. Lancet 2, 1205-1297, 1977. 20. Rekvig, 0. P., and Hannestad, K., Certain polyclonal antinuclear antibodies cross react with the surface membrane of human lymphocytes and granulocytes. Stand. .Z. Zmmunol. 6, 1041- 1054, 1977. 21. Cohen, A., and Canoso, J. J., Criteria for the classification of systemic lupus erythematosus states. Arthriris Rheum. 15, 540-543, 1972. 22. Boyum, A., Isolation of mononuclear cells and granulates from human blood. Stand. J. Lab. C&t. Invest. 21 (Suppl. 97), 77, 1968. 23. Goldstein, P., Wigzell, H., Blomgren, H., and Swedmyr, E. A. J., Cells mediating specific in vitro cytotoxicity. II. Probable autonomy of thymus-processed lymphocytes (T cells) for the killing of allogeneic target cells. J. Exp. Med. 135, 890-906, 1972. 24. Sasaki, M., Sekizawa, T., and Takahaski, H., Heterogeneity of human T lymphocytes to bind sheep erythrocytes and mitogenic responses of their subpopulations. J. Zmmunol. 115,1509, 1975. 25. Kaplow, L. S., Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood 26, 215-219, 1965. 26. Rothfield, N., Detection of Antibodies to Nuclear Antigens by Immunofluorescence, In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, Eds.), p. 647. Am. Sot. Microbial., Washington D.C. 1976. 27. Tenveen, J. H., and Keptkamp, T. E. W., Formalinized chicken red cell nuclei as a simple antigen for standardized antinuclear factor determination. C/in. Exp. Zmmunol. 5,673-678, 1968. 28. Zeutgraf, H., Deumling, B., Jarasch, E. D., and Franke, W., Nuclear membranes and plasma membranes from hen erythrocytes. J. Biol. Chem. 246, 2986-2995, 1972. 29. Holman, H., and Deicher, H. R. , The reaction of the lupus erythematosus (LE) cell factor with deoxynucleoprotein of the cell nucleus. J. C/in. Invest. 38, 2059-2072, 1959. 30. Bankhurst, D. D., Hastain, E., Husby, G., Diaz-Jouanen, E., and Williams, R. C., Human lymphocyte subpopulations defined by double surface markers. J. Lab. C/in. Med. 91, 15-23, 1978. 31. Huber, C., and Wigzell, H., A simple rosette assay for demonstration of complement receptor sites using complement-coated zymosan beads. Eur. J. Zmmunol. 5, 432, 1975. 32. Ferrarini. M., Moretta, L., Abrile. R., and Durante, M. L., Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells. Eur. J. Zmmunol. 5, 70, 1975. 33. Moretta, L.. Ferrarini, M., Durante, M. L., and Mingari, M. C.. Expression of a receptor for IgM by human T cells in vitro. Eur. J. Zmmunol. 5, 565, 1975. 34. DeHoratius. R. J., and Messner, R. P.. Lymphocytotoxic antibodies in family members of patients with systemic lupus erythematosus. J. C/in. Invest. 55, 1254, 1975. 35. Hannestad, K., Certain rheumatoid factors react with both IgG and an antigen associated with cell nuclei. Stand. J. Zmmunol. 7, 127. 1978. 36. Agnello, V., Kasep, G., and Arbetter, A., A redefinition of rheumatoid factors (RFs) of clinical and investigative significance. C/in. Res. 26, 51 lA, 1978. 37. Kabat. E. A., “Structural concepts in immunology and immunochemistry.” Holt, Rinehart & Winston, New York, 1968. 38. Richards, F. F., Konigsberg, W. H., Rosenstein, R. W., and Varga, J. M., On the specificity of antibodies. Biochemical and biophysical evidence indicates the existence of polyfunctional antibody combining regions. Science 187, 130, 1975. 39. Golub, E. S., Personal communication. 40. Bankhurst, A. D., and Williams, R. C., Jr., Identification of DNA-binding lymphocytes in patients with systemic lupus erythematosus. J. Clin. Invest. 56, 1378, 1975.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Cross-reactivity Antibodies
14, 292-299 (1979)
of Antilymphocyte and Antinuclear in Systemic Lupus Erythematosus’
ROBERTP. SEARLES,~RONALD P. MESSNER, AND ARTHUR D. BANKHURST," Department
of Medicine, Lovelace Medical
University Center,
of Net+ Mexico, School Albuquerque. New Mexico
of Medicine
and
87131
Received February 2 1, 1979 Both antinuclear antibody (ANA) and antilymphocyte antibody (ALA) are prevalent m systemic lupus erythematosus (SLE). This report presents data supporting crossreactivity between these two antibodies. Differential absorption of SLE sera showed that chicken erythrocyte nuclei absorbed IgM ALA while intact human and chicken erythrocytes had no effect. RNase digestion of chicken nuclei fragments partially removed the ALA absorbing effect. Both IgM and IgG ANA were also absorbed by the chicken nuclei. Absorptions with intact human lymphocytes removed ALA activity and absorption with human mononuclear cells removed ANA activity. ALA and ANA were both found in PBS eluates from absorbed chicken nuclei, demonstrating specificity of nuclear absorption.
INTRODUCTION
Systemic lupus erythematosus (SLE) is a connective tissue disease characterized by multiple autoantibodies including antinuclear (ANA) and antilymphocyte antibodies (ALA) (l-4). Both antibodies fluctuate with SLE disease activity and each has been postulated to play a major role in the pathogenesis of this disease (5-8). ANA are a heterogenous group of antibodies directed against multiple nuclear antigens, including deoxyribonucleic acid (DNA) (9), saline extractable proteins (lo), soluble nucleoprotein (1 l), and ribonucleic acid (RNA) (12, 13). ALA are also heterogenous and are directed at lymphocyte surface antigens (14, 15). Lymphocytotoxic ALA are predominantly cold reactive IgM and react with both T and B lymphocytes (15, 16). In addition, ALA react with brain extract and are increased in lupus patients with CNS disease (17, 18). They also react with trophoblastic antigens and may play a role in spontaneous abortion in SLE patients (19). Recently the possibility of cross-reactions between ANA and ALA has been suggested (20). This report presents further data demonstrating the existence of cross-reactivity between ANA and ALA. MATERIALS Subjects.
American
Fourteen Rheumatism
AND METHODS
patients with SLE, as determined by the criteria of the Association (21), were screened for ANA and ALA. From
’ Supported by Career Development Award, Grant 5K04 AM 70301, Grant 2 ROl AM 13789-08 from the National Institute of Health, and grants from the National and New Mexico Arthritis Foundation. ’ Postdoctoral Research Fellow, Arthritis Foundation. 3 Senior Investigator. Arthritis Foundation. 292 OWO-1229179/110292-08$01.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
ALA
AND
ANA
IN
SLE
293
this group, six patients with high titers of both ANA and ALA were selected for further investigation. Those tested included three acute untreated patients and three patients who became active while on maintenance prednisone. All six patients had low C3 complement; five of six patients had skin rash and arthritis; four of six had nephritis and direct Coombs positivity; and two of six had Raynaud’s phenomenon, pleuritis, and thrombocytopenia. Lymphocytes. Lymphocytes were separated from heparinized whole blood of normal human donors by Ficoll- Hypaque gradient centrifugation (22). Monocyte depletion was effected through glass adherence on petri dishes at 37°C for 30 min. T cell-enriched populations of human lymphocytes were prepared using rabbit immunoglobulin-coated bead columns (23). T-Cell preparations had 95% or greater E rosettes with sheep erythrocytes (24). Peroxidase positive cells using a benzidine dihydrochloride stain (25) were generally 2% or less in T cell-enriched lymphocytes and monocyte-depleted preparations. Antilymphocyte antibody. ALA were determined by both indirect immunofluorescence of suspensions of lymphocytes and complement-mediated cytotoxicity (3, 14). In the suspension procedure, 2 x lo6 monocyte-depleted or T cell-enriched lymphocytes were incubated serially with test serum and with fluorescein-labeled (FITC) goat antihuman IgM or IgG (Meloy, Springfield, Va.) for 1 hr at 4°C. Specificity for IgM and IgG was measured by immunoelectrophoresis. In each test, 300 cells were counted and results were recorded as the total number of fluorescent cells per 100 counted. Cell viability tested by trypan blue exclusion was 95% or greater before and after ALA assay. Heat inactivation of SLE sera did not effect ALA suspension determination. Antilymphocyte antibodies were also determined using the two-step microcytotoxicity assay of Terasaki (3) with a complement incubation of 3 hr at 15°C. Dilutions of SLE sera were made with minimum essential medium (MEM) and 10% human AB positive serum. Normal human serum controls had fewer than 5% dead cells in each experiment. Antinuclear antibody. Antinuclear antibodies were measured in an indirect immunofluorescent technique with mouse liver as substrate (26). Fluoresceinlabeled goat anti-human IgM and IgG (Meloy) were employed as the second antibody. Abiorptions. A 50% suspension of chicken nuclei in phosphate-buffered saline (PBS) was prepared from fresh chicken erythrocytes using a saponin lysis method (27). Unlysed chicken and human erythrocytes were employed as controls and absorptions were carried for 1 hr at 4°C. Chicken nuclei and fresh erythrocytes were greater than 99% intact before and after absorption by phase contrast microscopy. In addition, preparations of chicken erythrocyte nuclear fragments prepared by sonication (28) of intact chicken nuclei were used as absorbents. Absorptions were also carried out using Ficoll-separated human mononuclear cells and monocyte-depleted lymphocytes. To remove ANA 50% mononuclear cell solutions each containing 3 x lOa cells in PBS were used to absorb one-half the volume of serum. Fewer cells were used for ALA absorptions and contained only lymphocytes as prepared by monocyte depletion in glass petri dishes. Twenty percent lymphocyte suspensions in PBS were incubated with one-half serum volumes. Percentage absorption was calculated by dividing number of positive cells
294
SEARLES,
MESSNER,
AND
BANKHURST
per 100 counted after absorption by number of positive cells before absorption. Lymphocyte cell viability by trypan blue exclusion was greater than 95% before and after absorption. Efution. Antibody was eluted from chicken erythrocyte nuclei used in the absorption of SLE sera by incubation in PBS (pH 7.2) at 4°C for 6 hr. PBS was changed after each 2-hr period and the three eluates were combined and concentrated before ANA and ALA determination. Enzyme digestion. Chicken nuclei fragments prepared by sonication of intact nuclei were used for enzyme digestion experiments. Digestion was carried out at 37°C for 45 min with 0.2 mg/ml ribonuclease (RNase) (Worthington, Freehold, N.J.) and 0.2 mg/ml deoxyribonuclease (DNase) (Sigma, St. Louis, MO.) and was terminated by washing the cells in PBS at room temperature (29). Absorption of SLE sera was carried out with both undigested and enzyme-digested fragments (50% in PBS). RESULTS
ALA activity in sera of patients with active SLE was approximately equivalent using monocyte-depleted and T cell-enriched preparations of lymphocytes as targets. T lymphocytes were employed in most experiments because of the greater homogenicity of these preparations and to eliminate the nonspecific cytophilic attachment of ALA to Fc or complement receptors present on monocytes or B lymphocytes (15, 30, 31). Fc receptors are also present on T lymphocytes but the present assay method does not satisfy the necessary experimental conditions required for their detection (32. 33). Positive cells with normal human serum (NHS) controls using FITC anti-IgM were always 2% or less in the T-cell suspension system. Therefore, pepsin digestion of the second antibody was not routinely employed in these experiments. Only SLE sera positive for ALA by cytotoxicity method were found to have ALA using suspension immunofluorescence. ALA identified in suspension was primarily of the IgM class. Titers of ALA in SLE were found to vary from 1: 16 to 1: 128 depending on the combination of serum, lymphocyte target, and assay employed. Absorption of Antilymphocyte Antibody To evaluate the cross-reactivity of ALA with nuclear antigens, SLE serums were absorbed with freshly prepared chicken erythrocytes, chicken erythrocyte nuclei, and human lymphocytes. ALA was determined using the T-lymphocyte suspension technique. Representative data from SLE Patient 124 demonstrate the results of two sequential absorptions (Fig. 1). Absorption with chicken nuclei resulted in a 94% reduction in the percent of cells labeled by the SLE serum. A decrease of 71% of preabsorption values was found after absorption with human lymphocytes. A 94% reduction of ALA was found after a third adsorption with human lymphocytes. Absorption with intact chicken erythrocytes essentially had no effect on the preabsorption titers. The range of absorption of ALA by the chicken nuclei preparations was 55 to 94% in four SLE patients tested. The range for human lymphocytes was 71 to 100% absorption. The dilution factor intrinsic to this absorption system was considered in final interpretation of the postabsorption titers.
ALA AND ANA IN SLE
ABSORPTION
295
NUMBER
FIG. 1. Sequential absorption of SLE Patient 124 with chicken erythrocytes, chicken erythrocyte nuclei and human lymphocytes. Antilymphocyte antibodies (ALA) were measured by indirect immunofluorescence of suspension of T lymphocytes.
Specificity of the reaction of ALA with nuclear antigens was further investigated by eluting the ALA from postabsorption chicken erythrocyte nuclei in PBS for 6 hr at 4°C. In this experiment the three sequential absorptions of SLE Patient 126 with chicken nuclei reduced the percentage of positive cells from 20 to 9 and completely eliminated ANA activity at the lowest dilution tested (Table 1). ALA and ANA were both found in PBS eluates from the absorbed nuclei, but the ALA activity recovered was greater than that of ANA. These data demonstrate differential elution of ALA and ANA and suggest ALA has a lower avidity for the nuclei. To further identify which nuclear antigens have ALA reactivity, chicken erythrocyte nuclei were sonicated into nuclear fragments and digested with RNase and DNase. The undigested nuclear fragments were as effective as intact nuclei in the absorption of ALA (67- 100% absorption). DNase digestion of nuclear fragments had no effect on ALA absorption. RNase-Digested preparations were less effective and yielded 57% absorption compared to 91% absorption with the untreated nuclear fragments. These data suggest RNase-sensitive nuclear antigens participate in ALA cross-reactivity. To confirm these absorption experiments, ALA were tested by the cytotoxic procedure. Similar results were found in four SLE patients after absorption with TABLE 1 ALA AND ANA BEFORE AND AFTER ABSORPTION WITHCHICKENNUCLEI IN SLE PATIENT 126 ANDELUATES OF ABSORBED NUCLEI ALA Dilution Before After Eluate Control
1:6 1:6 1:3 PBS
ANA Percent positive” 20 9 15 1
” ALA, Percentage positive cells using indirect immunofluorescence cytes as targets. ’ ANA, Negative at lowest titer tested 1: 16.
I&f 1:256 Negb 1:12 Neg
I& 1: 1024 Neg 1:32 Neg
of suspensions of T lympho-
SEARLES,
296
MESSNER,
AND BANKHURST
TABLE CHICKEN
NUCLEI
ABSORPTIONS
2 ALA IX SLE P.41IEN I s
OF CYTOTOXI~
ALA SLE
~__
Patient 120 Patient 121 Patient 122 Patient 125 NHS” NHS MEMlAB
titers
Pre
PO9
256” 32 256 8 Neg Neg
c Nep 4 Neg NQ! Neg
-
” Positive 2 30% dead cells. b NHS and MEMIlOYF AB+ were <5% dead cells.
chicken erythrocyte nuclei (Table 2). In addition, absorptions with intact chicken and human erythrocyte preparations showed no significant change in cytotoxic ALA effect. Possible interference by chicken nuclei absorption with the two-step cytotoxic method was also evaluated. Lymphocytes were preincubated with nonabsorbed and absorbed cytotoxic serum. This was followed by addition of guinea pig complement and cells were evaluated for numbers killed. Reestablishment of lymphocyte cytotoxicity by the nonabsorbed cytotoxic serum demonstrated that the integrity of the cytotoxic method was not altered by anticomplementary activity in chicken nuclei-absorbed SLE sera. Absorption of Antinuclear Antibody The effect of absorption on ANA in SLE sera was also evaluated. Chicken erythrocytes, erythrocyte nuclei, human erythrocytes, and human mononuclear cells (50% in PBS) were used as absorbents of SLE sera over three consecutive 1-hr periods at 4”C, and IgM and IgG ANA were determined by indirect immunofluorescence. A representative experiment is shown in Table 3. Absorption of both IgM and IgG ANA in Serum 124 by the chicken nuclei was 98% or greater at the lowest dilution tested (1: 16). Absorption with human mononuclear cells (9 x IO8 cells) also removed greater than 80% of IgM and IgG ANA. In all human mononuclear cell absorptions, residual ANA activity was present, Investigation of the dilution effect during antibody absorption was tested by absorption of IgG ANA with intact human and chicken erythrocytes. The titer changes after absorption with chicken erythrocytes were one tube dilution or less.
ABSORPTION
TABLE 3 OF SLE SERUM 126ANA
EFFWT
ANA titer Absorbent
kM
kG
Chicken nuclei Human mononuclear cells
640 Neg” 32
640 16 128
Preabsorption
n No immunofluorescent
at lowest titer tested (1: 16).
--
ALA
AND
ANA
IN
SLE
297
DISCUSSION The data presented here demonstrate that antibodies reacting with intact lymphocytes can be absorbed with nuclei. The corollary observation that antinuclear antibodies can be absorbed with intact human mononuclear cells was also confirmed (20). Using ANA positive, ALA positive SLE sera, significant ALA absorption occurred with fresh chichen erythrocyte nuclei (55-94%) and paralleled ALA absorption by human lymphocytes (71- 100%). No significant ALA reactivity was found with intact chicken or human erythrocytes. Similar results occurred with ANA absorption where chicken nuclei absorbed 98% or greater of IgM and IgG ANA. Extensive absorption with human mononuclear cells removed greater than 80% of IgM and IgG ANA. These latter results are similar to the observations that whole leukocyte preparations will absorb IgG ANA (20). They further demonstrate that human mononuclear cells absorb IgM ANA which might be expected considering the IgM nature of cold reactive ALA. Elution of antibodies from the chicken nuclei yielded significant recovery of absorbed ALA, demonstrating specificity of nuclear absorption. Experiments with ALA by the cytotoxic method produced similar results with nuclear and lymphocyte absorptions. The data presented are not meant to be representative of all SLE patients. They represent selected sera with high titers of both ALA and ANA. Further studies of SLE sera with other combinations of ALA and ANA positivity are needed for the total profile. The concept that the autoantibodies of SLE have shared specificities will warrant further investigation. As previously mentioned, ANA are heterogenous with specificities for multiple nuclear antigens. ALA are predominantly IgM antibodies and are also heterogenous as indicated by differences in reactivity with brain (17) and with lymphocytes from different donors (34). Available data suggest that all ALA and ANA do not share identical specificities. Sera containing one of these activities but not the other do exist, demonstrating that antibodies without overlapping specificities occur in viva. The variable reactivity of ALA with lymphocytes from different donors contrasts with lack of substrate specificity found with ANA. Finally, our data on ANA/ALA cross-reactivity showed residual ALA activity despite exhaustive nuclear absorption and elution experiments with PBS suggested differential ALA and ANA affinity for nuclear antigens. The differences between sera in the effect of absorption with nuclei and lymphocytes noted in this study are thus not unexpected and probably reflect differences in the repertoire of antibodies contained in each serum. Besides the cross-reactivity between ALA and ANA, rheumatoid factor also reacts with ANA (35, 36). Cross-reactions of rheumatoid factor with ANA and peripheral blood leukocytes with ANA were found to be mutually exclusive which further supports specificity of these reactions. Most theories on antibody cross-reactivity suggest these reactions occur only when structural similarity exists between the offending antigens (37). On the other hand, Richards ef al. (38) proposed the existence of polyfunctional antibody combining sites to explain reactions between diverse haptens with the same monovalent myeloma proteins. These myeloma proteins should be unifunctional with definite antigen specificity and yet they react with diverse ligands like menadione and dinitrophenol.
298
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MESSNER,
AND
BANKHURST
Several types of nuclear antigens have been described (9-13), and some of these may be structurally similar to lymphocyte surface antigens. Our data showed DNase had no effect on ALA absorption but RNase digestion of nuclei fragments removed 37% of ALA cross-reactivity. Thus, RNase-sensitive nuclear antigens may share specificities with the lymphocyte surface antigens. Recent observations have shown both DNA (39) and DNA receptors (40) are present on lymphocyte membranes suggesting this antigen may also participate in crossreactions between ALA and ANA. Further investigations are needed to identify the specific nuclear antigens involved in ALA cross-reactivity before structural comparisons are made. Resolution of ANA and ALA cross-reactivity may eventually give insight into whether, and if so, how the autoantibodies are involved in the pathogenesis of SLE. REFERENCES 1. Hargraves, M. M., Richmond, H., and Morton, R., Presentation of two bone marrow elements, the Tart cell and the LE cell. Proc. Stuff Meeting, Mayo Clinic. 23, 25-28. 1948. 2. Friou, G. J., In “The LE Cell Phenomenon and Antinuclear Antibodies in Arthritis and Rheumatism” (J. L. Hollander and D. J. McCarty, Eds.). p. 172. Lea & Febiger. Philadelphia. 1972. 3. Terasaki, P. I., Mottironi, V. D., and Barnett. E. V., Cytotoxins in disease: Autocytotoxins in lupus. N. Engl. .I. Med. 283, 724-728, 1970. 4. Mittal. K. K., Rossen, R. D., Sharp, U. T. et al., Lymphocytotoxic antibodies in systemic lupus erythematosus. Nature (London) 225, 1255- 1256, 1970. 5. Norman, D. D., Kurata, N., and Tan, E. M., Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann. Inc. Med. 83, 464-469, 1975. 6. Alarcon-Segovia. D., Ruiz-Arguelles. A., and Fishbein, E., Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature Ilondon) 271,67-69. 1978. 7. Messner, R. P., In Lymphocytes and Their Interactions (R. C. Williams, Jr.. Ed.), p. 169. Raven Press, New York, 1975. 8. Winfield, J. B., Winchester, R. J., and Kunkel. H. G.. Association of cold-reactive antilymphocyte antibodies with lymphopenia in systemic lupus erythematosus. Arthritis Rherrm. 18.587-594, 1975. 9. Tan, E. M., Schur, P. H.. Carr, R. I., and Kunkel. H. G.. Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J. C/in. Invest. 45, 1732, 1966. IO. Sharp, G. C., Irvin, W. S.. Tan, E. M., Gould, R. G., and Holman, H. R., Mixed connective tissue disease-an apparently distinct rheumatic disease associated with a specific antibody to an extractable nuclear antigen (ENA). Amer. J. Med. 52, 148, 1972. 11. Tan, E. M., An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus erythematosus. J. Clin. Invest. 46, 735-745. 1967. 12. Ritchie, R. F.. Antinucleolar antibodies. N. Engl. J. Med. 282, 1174-I 178, 1970. 13. Pinas, J. L.. Northway. J. D., and Tan, E. M.. Antinucleolar antibodies in human sera. ./. Inrmunol. 111,9%-1004, 1973. 14. Messner. R. P.. Kennedy. M. S., and Jelinek, J. G.. Antilymphocyte antibodies in syatemrc lupus erythematosus: effect on lymphocyte surface characteristics. Arthritis Rheum. 18,201-206, 1975. 15. Winfield. J. B., Winchester. R. J.. and Wemet, P.. et al., Nature of cold-reactive antibodies to lymphocyte surface determinants in systemic lupus erythematosus. Arthritis Rheum. 18. I-8. 1975. 16. Michlmayr, G., Pathonci, C., Huber. C.. and Huber, H., Antibodies for T lymphocytes in 3ystemic lupus erythematosus. Ckn. Exp. Immunol. 24, 18-25. 1976. 17. Bluestein, H. G., and Zvaifler, N. J.. Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. J. C/in. Zn~~rst. 57, 509, 1976. 18. Bresnihan. B., Oliver, M.. Gerigor. R., and Hughes, G. R.. Brain reactivity of lymphocytotoxtc
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19. Bresnihan, B., Grigor, R. R., Oliver, M., Lewkonia, R. M., Hughes, G. R., Livings, R. E., and Fauck, W. P., Immunologic mechanism for spontaneous abortion in systemic lupus erythematosus. Lancet 2, 1205-1297, 1977. 20. Rekvig, 0. P., and Hannestad, K., Certain polyclonal antinuclear antibodies cross react with the surface membrane of human lymphocytes and granulocytes. Stand. .Z. Zmmunol. 6, 1041- 1054, 1977. 21. Cohen, A., and Canoso, J. J., Criteria for the classification of systemic lupus erythematosus states. Arthriris Rheum. 15, 540-543, 1972. 22. Boyum, A., Isolation of mononuclear cells and granulates from human blood. Stand. J. Lab. C&t. Invest. 21 (Suppl. 97), 77, 1968. 23. Goldstein, P., Wigzell, H., Blomgren, H., and Swedmyr, E. A. J., Cells mediating specific in vitro cytotoxicity. II. Probable autonomy of thymus-processed lymphocytes (T cells) for the killing of allogeneic target cells. J. Exp. Med. 135, 890-906, 1972. 24. Sasaki, M., Sekizawa, T., and Takahaski, H., Heterogeneity of human T lymphocytes to bind sheep erythrocytes and mitogenic responses of their subpopulations. J. Zmmunol. 115,1509, 1975. 25. Kaplow, L. S., Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood 26, 215-219, 1965. 26. Rothfield, N., Detection of Antibodies to Nuclear Antigens by Immunofluorescence, In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, Eds.), p. 647. Am. Sot. Microbial., Washington D.C. 1976. 27. Tenveen, J. H., and Keptkamp, T. E. W., Formalinized chicken red cell nuclei as a simple antigen for standardized antinuclear factor determination. C/in. Exp. Zmmunol. 5,673-678, 1968. 28. Zeutgraf, H., Deumling, B., Jarasch, E. D., and Franke, W., Nuclear membranes and plasma membranes from hen erythrocytes. J. Biol. Chem. 246, 2986-2995, 1972. 29. Holman, H., and Deicher, H. R. , The reaction of the lupus erythematosus (LE) cell factor with deoxynucleoprotein of the cell nucleus. J. C/in. Invest. 38, 2059-2072, 1959. 30. Bankhurst, D. D., Hastain, E., Husby, G., Diaz-Jouanen, E., and Williams, R. C., Human lymphocyte subpopulations defined by double surface markers. J. Lab. C/in. Med. 91, 15-23, 1978. 31. Huber, C., and Wigzell, H., A simple rosette assay for demonstration of complement receptor sites using complement-coated zymosan beads. Eur. J. Zmmunol. 5, 432, 1975. 32. Ferrarini. M., Moretta, L., Abrile. R., and Durante, M. L., Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells. Eur. J. Zmmunol. 5, 70, 1975. 33. Moretta, L.. Ferrarini, M., Durante, M. L., and Mingari, M. C.. Expression of a receptor for IgM by human T cells in vitro. Eur. J. Zmmunol. 5, 565, 1975. 34. DeHoratius. R. J., and Messner, R. P.. Lymphocytotoxic antibodies in family members of patients with systemic lupus erythematosus. J. C/in. Invest. 55, 1254, 1975. 35. Hannestad, K., Certain rheumatoid factors react with both IgG and an antigen associated with cell nuclei. Stand. J. Zmmunol. 7, 127. 1978. 36. Agnello, V., Kasep, G., and Arbetter, A., A redefinition of rheumatoid factors (RFs) of clinical and investigative significance. C/in. Res. 26, 51 lA, 1978. 37. Kabat. E. A., “Structural concepts in immunology and immunochemistry.” Holt, Rinehart & Winston, New York, 1968. 38. Richards, F. F., Konigsberg, W. H., Rosenstein, R. W., and Varga, J. M., On the specificity of antibodies. Biochemical and biophysical evidence indicates the existence of polyfunctional antibody combining regions. Science 187, 130, 1975. 39. Golub, E. S., Personal communication. 40. Bankhurst, A. D., and Williams, R. C., Jr., Identification of DNA-binding lymphocytes in patients with systemic lupus erythematosus. J. Clin. Invest. 56, 1378, 1975.