Clin. exp. Immunol. (1979) 35, 210-217.

Heterogeneous neurocytotoxic antibodies in systemic lupus erythematosus I-I. G. BLUESTEIN Department of Medicine, University

ofCalij;irnia Medical Center, San Diego, Califorrnia 92103 US- 1

(Received 1 August 1978)

SUMMARY

Sera from eighteen patients With systemic lupus erythematosus (SLE) were tested for cytotoxic antibody to three neuronal and two glial continuous cell lines of human origin. Eighty per cent (fifteen out of eighteen) of the sera were cytotoxic to at least one of the cell lines, but only seven sera were active against all five lines. Three sera had anti-neuronal but not anti-glial reactivity. No sera were gliocytotoxic without neurocytotoxicity. Three SLE sera with relatively strong cytotoxicity to all five cell lines were absorbed with each of the cell lines separately and the absorbed sera were then tested for residual cytotoxicity to each of the cell lines. The absorptions uncovered at least six different antibody specificities directed at antigens expressed on some but not all of the neuronal and glial cell lines. Each patient's serum had its own profile of antibody specificities reactive with membrane antigens on nervous tissue-derived cells.

INTRODUCTION Central nervous system (CNS) involvement is a major cause of morbidity in systemic lupus erythematosus (SLE) and yet its pathogenesis remains obscure. The observations that lymphocyte-reactive antibodies in SLE sera bind to antigens on human brain tissue, and that there is more lymphocytotoxic antibody in the serum of patients with CNS manifestations than in others has led to the speculation that autoantibodies reactive with CNS cell membranes may be responsible (Bluestein & Zvaifler, 1976; Bresnihan et al., 1977). Subsequent studies have shown that most SLE sera do, in fact, contain antibodies reactive with surface antigens on human neuronal cells (Bluestein, 1978), thus increasing the plausibility of an autoantibody-mediated pathogenesis for CNS disease. The specificities of the neural cell-reactive antibodies have not been fully defined. The neurocytotoxic antibodies do not bind to adult human erythrocytes and only a small part of them react with human lymphoid cells. However, most of the SLE sera with anti-neuronal activity are also cytotoxic to human glial cells (Bluestein, 1978). The shared reactivity with different cell types from nervous tissue could reflect common non-differentiated surface antigens on those cells. Alternatively, SLE sera may contain multiple antibody specificities each reactive with unique antigenic determinants expressed on the neuronal or glial cells. To distinguish between those alternatives, three neuronal and two glial cell lines, all of human origin, were used as absorbents of SLE sera cytotoxic to all five lines. The absorbed sera were then tested for residual cytotoxicity to each of the cell lines. The results reveal that SLE sera contain heterogeneous populations of antibodies directed at several different antigenic determinants found on nervous tissue-deriv ed cells. Correspondence: Dr H. G. Bluestein, Department of Medicine, University of California Medical Center, San Diego, California 92103, USA. 0099-9104/79/0020-0210S02.00 (Ct 1979 Blackwell Scientific Publications

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MATERIALS AND METHODS Clinical material. Sera were obtained from eighteen SLE patients under the care of the Rheumatology Service at University Hospital, San Diego. All fulfilled the preliminary American Rheumatism Association diagnostic criteria for SLE (Cohen & Canoso, 1972), and they all had antinuclear antibody (titre greater than 1:40). Sera were stored - 20'C without preservatives. All sera were heated at 56 C for 45 min prior to use. Tissue culture cell lines. The origin and source of the human neuronal and glial cell lines used in these studies is presented in Table 1. In our laboratory, all of the cell lines were grown as monolayer cultures adherent to plastic in Eagle's minimal essential medium (MEM) supplemented with 15% foetal calf serum (FCS), 2 0 mm glutamine and non-essential amino acids (Microbiological Associates, Rocks ille, Maryland). The cultures were maintained at 37 C in a 5% CO2 in air atmosphere. Serum absorptions. The SLE sera, at a 1:2 dilution in MEM, were absorbed with each of the cell lines at a density of 4 x 106 cells per ml, at 4VC for 1 hr. The cells were then remov ed by centrifugation at 600 g for 10 min at 4VC. An aliquot of the serum supernate was removed for testing, fresh cells were added to the same density, and the process repeated for the number of absorptions indicated. Absorptions of sera with homogenized human brain and liver tissue were performed as described previously (Bluestein & Zvaifler, 1976). TABLE 1. Origins of tissue culture cell lines used in this study

Type

Origin*

Sourcet

Neuronal Neuronal Neuronal Glial Glial

Neuroblastoma Neuroblastoma Neuroblastoma Glioblastoma Glioblastoma

J. Biedler (S) R. Seeger (L) R. Seeger (L) J. Fogh (S) J. Fogh (S)

Name SK-N-SH LA-N-I IMR-32 A-172 U-118MG

* The tissue culture cell lines were established from fragments of the human tumours indicated. The characteristics of their growth were described in the following references: SK-N-SH (Biedler et al., 1973), LA-N-1 (Seeger et al., 1977), IMR-32 (Tumilowicz et al., 1970). A-172 (Giard et al., 1973) and U-1 18MG (Ponten & Macintyre, 1968). t The india iduals who supplied our laboratory with each cell line are listed with their institutions. (S) = Sloan-Kettering Cancer Research Institute, Rye, New York. (L) = Department of Pediatrics, University of California, Los Angeles School of Medicine.

Cytotoxic assay. Each of the cell lines, as indicated, were cultured in flat bottom microtitre trays (Linbro Plastics, New Haven, Connecticut). Cultures were initiated at a density of 105 cells per well in MEM supplemented with 15% FCS (MEMFCS) and were incubated at 37 C in a humidified 5% CO2 in air atmosphere until confluent (24-48 hr). The medium was removed by aspiration, and the cells incubated with 2 0 pCi5'Cr in the form of sodium chromate (Amersham-Searle, Chicago, Illinois) in 0 1 ml MEM at 37 C for 1 hr. The 51Cr-containing medium was then removed by aspiration and the cultures washed three times with 0 1 ml of MEM-FCS. A 1:2 dilution of test serum and fresh undiluted rabbit serum as a source of complement were each added in 25 l volumes. The rabbit serum had been pre-absorbed with SK-N-SH cells. After a 1 hr incubation at 370C, 50 ul of MEM-FCS were added to each culture, followed by the removal of 50 p1 aliquot of the supernatant for counting in a gamma spectrometer. The total 51Cr in the cells available for release was determined by solubilizing a 5 Cr-labelled monolayer culture in 0 2 ml of 0 4 N NaOH and counting the contents of the well in a gamma spectrometer. The 51Cr-released from cultures treated with a standard normal human serum (NHS) was taken as the background spontaneous chromium release. Cytotoxicity is expressed as the percentage 51Cr released according to the formula: percentage

cytotoxicitv# #

=

100 x test serum ct/min- NHS ct/min. ~~~total ct/min -NHS ct/min

The upper limit of the percentage 5"Cr release for each cell line was calculated as the mean --2 s.d. obtained with a panel of fifteen normal human sera. Those values were SK-N-SH, 14%; LA-N-1; 9%, IMR-32; 12%, U-118MG, 8%.

D

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RESULTS Most of the SLE sera were cytotoxic in the complement-dependent 51Cr release assay to the three neuronal and two glial continuous human cell lines. Out of eighteen SLE sera tested, fifteen were cytotoxic to at least one of the cell lines. Neuronal line SK-N-SH was the most susceptible with fourteen sera causing greater than 14% 51"Cr release. Eleven sera were cytotoxic to each of the other cell lines, but only seven were active against all five cell lines. The cytotoxicity to all of the neuronal and glial lines was completely removed by absorptions with homogenized human brain tissue but not by absorptions with human liver or by cultured human fibroblasts. Although the number of sera cytotoxic to each of the lines was similar, the percentage 5 Cr released from each cell type by the SLE sera differed (Table 2). SK-N-SH was the most sensitive with a mean 5"Cr release of 4000. The neuronal line IMR-32 and the glial line A-172 were intermediate, while the neuronal line LA-N-1 and glial line U-118MG released the least 51Cr. Individual SLE sera showed similar relative cytotoxic potency against each of the cell lines. The correlation coefficients, calculated by linear regression analysis (r) obtained when comparing cytotoxic activities against any two cell lines, were 0'74 or greater (P< 0.002), except for comparisons involving LA-N-1 cells. There was no significant correlation between anti-LA-N-1 and anti-IMR-32 activities (P>0 1) and there was only a weakly positive correlation between cytotoxicity to LA-N-1 and U-118MG or SK-N-SH (0.05>P>001). Cytotoxicity to LA-N-1 correlated best with activity against A-172 (Pn

73 en

a)

4 0 Number of absorptions

FIG. 1. Removal of neurocytoxicity from SLE sera by absorptions with SK-N-SH cells. Sera A (0), B (9), ) or IMR-32 cells (- - - ) as described in the Materials and C (A) were absorbed with SK-N-SH cells ( and Methods section. (a) Residual cytotoxicity to IMR-32 cells. (b) Residual cytotoxicity to LA-N-1 cells.

100

(a)

(b)

00 0X -03

400 30

2

3

4 Number of absorptions

2

3

4

FIG. 2. Removal of gliocytotoxicity from SLE sera by absorptions with IMR-32 cells. Sera A (0), B (e) and C (A) were absorbed with IMR-32 cells as described in the Materials and Methods section for the number of times indicated. (a) Residual cytotoxicity to A-172 cells. (b) Residual cytotoxicity to U-1 18MG cells.

Absorptions with the glial lines A-172 and U-i 18MG did not highlight any differences in the specificities of cytotoxic antibodies among the three SLE sera. However, the absorptions did reveal that those two glial lines each express one or more membrane antigens not shared by the other. Anti-A-172 activity persisted in all three SLE sera after absorption with U-i 18MG, and anti-U-i 18MG cytotoxic activity remained after absorption with A-172. Those non-shared antigens are also present on some but not all of the neuronal cells. In Fig. 3, the residual cytotoxic activity against A-172 and U-1 18MG in serum B after absorption with both neuronal and glial cell lines is shown. The anti-A-172 activity was readily removed by neuronal cells IMR-32. However, absorption with LA-N-1, as well as U-118MG, left behind considerable residual activity (Fig. 3a). A reciprocal relationship is apparent when testing for

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Neurocytotoxic antibodies in SLE (b)

50-

4030

20-

10.~ ~~ 10

2

3

2

3

Number of absorplions

FIG. 3. Identification of distinct antibody specificities in an SLE serum that react with two different membrane antigens shared by some glial and neuronal cells. SLE serum B was absorbed with A-172 (0), IMR-32 (D), U-1 18MG (n) or LA-N-1 (A) cells and tested for residual cytotoxicity to A-172 (a) or U-1 18MG (b).

cytotoxic activity to U-1 18MG following absorption with those cell lines (Fig. 3b). Both A-172 and IMR32 left considerable residual activity, while LA-N-I and U- 1i8MG effectively eliminated the cytotoxic antibody to U-i 18MG. Thus, SLE serum B defines one or more antigens present on A-172 and IMR-32 not found on LA-N-1 or U-118MG, and reciprocally there is an antigen(s) present on LA-N-1 and U-118MG that is not present on IMR-32 or A-172. DISCUSSION Sera from the majority of patients with SLE contain antibodies to cells of tissue culture lines derived from human neuronal or glial tumours. The neuronal cell lines SK-N-SH, LA-N-1 and IMR-32 were derived from human neuroblastoma. Following their establishment as permanent cell lines, these cells retained some of the differentiated characteristics of neurons. SK-N-SH has dopamine fl-hydroxylase (Biedler, Helson & Spengler, 1973), and the action potential Na+ ionophore characteristic of electrically excitable membranes (Bluestein, 1977; West et al., 1977). LA-N-I also has the Na + ionophore and the enzyme tyrosine hydroxylase, which is also found in IMR-32 (West et al., 1977). The glial cell lines, A-172 and U- 1i8MG, derived from human glioblastoma lack the action potential Na+ ionophore (unpublished personal observation). Antibodies generated in animals immunized with SK-N-SH, LA-N-1, or IMR-32 react with human brain tissue (Akeson & Seeger, 1977), indicating that the cultured neuronal cells have also retained some normal brain antigens. The neuro- and gliocytotoxic antibodies in the SLE sera are also directed at normal antigens found in human brain. Absorption with brain homogenates removes all of the cytotoxicity. Fibroblasts from human skin and human liver failed to absorb the cytotoxic activity against the neuronal and glial cell lines. Thus the target antigens are not widely represented on tissue outside the nervous system. Individual SLE sera generally were similarly cytotoxic to each of the neuronal and glial cells. However, a few of the sera reacted with some but not all of the cell lines, suggesting that SLE sera may contain several different anti-neural specificities. Assessment of the residual cytotoxicity in SLE sera following absorption with each of the cell lines confirmed that suggestion. At least six different antibody specificities were detected in the three SLE sera subjected to the absorptions: (1) sera A and B but not C had at least

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one antibody specificity directed at an antigenic determinant on LA-N-1 that was not present on SK-N-SH cells. That membrane antigen is present on IMR-32 and A-172. (2) Anti-U-1 18MG activity in serum A had at least one specificity directed at an antigen that was not expressed on A- 172, but was present on all of the other cell lines. (3) Serum B had different antibody specificities reactive with U-i 18MG which were directed at determinants also present on LA-N-I cells but not on the other cell lines. (4) Serum B also had antibody to an antigen on A-172 and IMR-32 but not on SK-N-SH, LA-N-1, or U-118MG. (5) Serum C had a different anti-A-172 specificity directed at an antigen that was not found on any of the other cell lines. (6) Sera A, B and perhaps C had an anti-SK-N-SH specificity that reacted with all of the other cell lines except U-1 18MG. In addition to those six restricted specificities, there may well have been antibody specificities reactive with all of the cells, since partial depletion of cytotoxic activity occurred after absorption with any of the cell lines. In addition to the antibodies that react with both neuronal and glial cells, SLE sera may contain specificities which distinguish between those cell types. Serum C in this study had an antibody that reacted only with the glial line A-172. Furthermore, although none of the specificities identified in the three absorbed SLE sera were restricted in their activity to neuronal cells, several other SLE sera have been identified which are cytotoxic to the neuronal line SK-N-SH, but not to the glial lines A-172 or U-i 18MG (Bluestein, 1978). The multiple antigenic determinants on human neuronal and glial cells defined by the heterogeneous antibody populations in SLE sera are similar to those identified on rodent cell lines using heterologous antisera induced by immunization with rat neuronal and glial cells (Stallcup & Cohn, 1976). At least six different determinants shared in various combinations by nerve, muscle, glial and fibroblastic cell lines were identified, in addition to three antigens restricted to neuronal cells and two to glial lines. Rodent and human neuronal cells share at least two common membrane antigens. One of these, called INMA (interspecies neuronal membrane antigen), is expressed on the human lines used in this study, SK-N-SH, IMR-32 and LA-N-1, but not on SK-N-MC, another human neuroblastoma line. It is also found on the mouse neuroblastoma NK-119 and N18. Antibody to INMA is readily absorbed with human and mouse brain, but not with kidney or liver (Akeson & Seeger, 1977). A second humanrodent antigen, MBA-2, is detected by naturally occurring antibody in normal mouse serum. It is present on SK-N-MC as well as the three human neuroblastomas used in this study, but not on the glial lines A-172 or U-118MG (Martin & Martin, 1975b). MBA-2 is expressed on mouse neuronal line NB1, but not on NK-l 19 or N18. Anti-MBA-2 activity is reactive with human and mouse kidney as well as brain tissue from both species (Martin & Martin, 1975a). The relationship of the membrane antigens detected by antibodies in the SLE sera to the antigens detected in the analysis of interspecies neuronal antigens has yet to be defined. Anti-MBA-2 is IgM antibody, as are the neurocytotoxic antibodies in SLE sera (Bluestein, 1978). However, all of the antineuronal activity in the three SLE sera was removed by absorption with A-172 cells. MBA-2 is not expressed on those cells. A different, naturally occuring antibody reactive with cerebellar cells has been identified in the serum of New Zealand mice which spontaneously develop lupus-like autoimmune disease (Harbeck et al., 1978). This antibody was not detected in normal mouse serum. It, too, isIgM and may be the analogue of the neurocytotoxic antibodies in SLE sera. The antigenic stimulus responsible for the induction of anti-neural antibodies in SLE has not been identified,but there are a number of possibilities. The first nervous tissue reactive antibodies identified in lupus sera were also lymphocytotoxic (Bluestein & Zvaifler, 1976), suggesting that the antibodies were lymphocyte induced. Their brain reactivity was attributed to antigenic determinants shared by lymphocytes and brain (Reif & Allen, 1974; Golub, 1971). However, the subsequent demonstration that lymphocyte-reactive antibodies are only a small part of the pool of neurocytotoxic antibodies in SLE sera (Bluestein, 1978), indicated that lymphocyte membrane antigens did not induce most of the antineuronal antibodies. Nervous tissue itself may have induced the antibodies against its cellular constituents. The immune system does not normally have ready access to the central nervous system, but pathogenetic mechanisms in SLE may disrupt the usual barriers. The heterogeneity of antibody specificities reactive with both neuronal and glial cells favours nervous tissue as the antigenic stimulus. A third

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alternative is developmental antigens expressed on immature cell types. Studies in mice have demonstrated that brain shares some antigenic determinants in common with haematopoietic stem cells (Golub, 1972; Meyer-Hamme & Bluestein, 1978). In fact, Duckham et al. (1975) have shown that some lupus sera can suppress the development of mouse bone marrow colonies in vitro. Each of the three SLE sera examined in this study contained different combinations of anti-neural antibodies. This may reflect individual differences in the antigenic stimulus inducing the antibodies. Nervous tissue, stem cells, lymphocytes, or other sources may be functioning alone or in various combinations in each patient. Alternatively, individual differences in immune responsivity to common antigens may be responsible for the different patterns of anti-neuronal specificities. In either case, cataloguing the individual antibody specificity profiles will permit analysis of their relationship to the various clinical manifestations of SLE. The generosity of Drs J. Biedler and J. Fogh of the Sloan-Kettering Cancer Research Institute and Dr R. Seeger of the Department of Pediatrics, UCLA in providing the cell lines used in this study is gratefully acknowledged. Cheryl OgdenBell provided valuable technical assistance, and Deborah Ann Frank excellent secretarial support. This study supported in part by State of California Department of Health Contract 76-57087, research grant AI-10931 and Training grant AM-07062 from the National Institutes of Health.

REFERENCES AKESON, R. & SEEGER, R.C. (1977) Interspecies neural membrane antigens on cultured human and murine neuroblastoma cells. 7. Immunol. 118, 1995. BIEDLER, J.L., HELSON, L. & SPENGLER, B.A. (1973) Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 33, 2643. BLUESTEIN, H.G. (1977) Antineuronal activity in systemic lupus erythematosus serum. Arthr. and Rheum. 19, 805A. BLUESTEIN, H.G. (1978) Neurocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. Proc. Nat. Acad. Sci. (Wash.), 75, 3965. BLUESTEIN, H.G. & ZVAIFLER, N.J. (1976) Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. J7. c/in. Invest. 57, 509. BRESNIHAN, B., OLIVER, M., GRIGOR, G. & HUGHES, G.R.V. (1977) Brain-reactivity of lymphocytotoxic antibodies in systemic lupus erythematosus with and without cerebral involvement. C/in. exp. Immunol. 30, 333. COHEN, A. & CANOSO, J.J. (1972) Criteria for the classification of systemic lupus erythematosus status. Arthr. and Rheum. 15, 540. DUCKHAM, D.J., RHYNE, R.L., JR., SMITH, F.E. & WILLIAMS, R.C., JR. (1975) Retardation of colony growth of in vitro bone marrow culture using sera from patients with Felty's syndrome, disseminated lupus erythematosus (SLE), and other disease states. Arthr. and Rheum. 18, 323. GIARD, D.J., AARONSON, S.A., RODARO, G.J., ARNSTEIN, P., KERSEY, J.H., DOSIK, H. & PARKS, W.P. (1973) In vitro cultivation of human tumors; establishment of cell lines derived from a series of solid tumors. 7. Nat. Cancer Inst. 51, 1417. GOLUB, E.S. (1971) Brain-associated 0-antigen: reactivity of rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 2, 353. GOLUB, E.S. (1972) Brain-associated stem cell antigen: an

antigen shared by brain and hemopoietic stem cells. 5. exp. Med. 136, 369. HARBECK, R.J., HOFFMAN, A.A., HOFFMAN, S.A., SHUCARD, D.W. & CARR, R.I. (1978) A naturally occurring antibody in New Zealand mice cytotoxic to dissociated cerebellar cells. C/in. exp. Immunol. 31, 313. MARTIN, S.E. & MARTIN, W.J. (1975a) Interspecies brain antigen detected by naturally occurring mouse antibrain autoantibody. Proc. Nat. Acad. Sci. (Wash.), 72, 1036. MARTIN, S.E. & MARTIN, W.J. (1975b) Expression by human neuroblastoma cells of an antigen recognized by naturally occurring mouse anti-brain autoantibody. Cancer Res. 35, 2609. MEYER-HAMME, S. & BLUESTEIN, H.G. (1978) Antibodymediated suppression of bone marrow colony formation i1n Vitro. _7. Cell. Physiol. 94, 47. PONTEN, J. & MACINTYRE, E.H. (1968) Long term culture of normal and neoplastic human glia. Acta Path. microbiol. Scanid. 74, 465. REIF, A.E. & ALLEN, J.M.V. (1964) The AKR thymic antigen and its distribution in leukemias and nervous tissues. 7. exp. Med. 120, 413. SEEGER, R.C., RAYNER, S.A., BANERJEE, A., CHUNG, H., LAUG, W.E., NEUSTEIN, H.B. & BENEDICT, W.F. (1977) Morphology, growth, chromosomal pattern, and fibronolytic activity of two new human neuroblastomi cell lines. Cancer Res. 37, 1364. STALLCUP, W.B. & COHN, M. (1976) Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system. Exp. Cell. Res. 98, 285. TUMILOWICZ, J.J., N ICHOLS, W.W., CHOLON, J.J. & GREENE, A.E. (1970) Definition of a continuous human cell line derived from neuroblastoma. Canzcer Res. 30, 2110. WEST, G.J., UKI, J., HERSCHMAN, H.R. & SEEGER, R.C. (1977) Adrenergic, cholinergic, and inactive human neuroblastoma cell lines with the action potential NA' ionophore. Cancer Res. 37, 1372.