ALAN L. SCHOCKET and PETER F. KOHLER Sera from patients with systemic lupus erythematosus (SLE) and clinically related diseases were examined for cold-reactive lymphocytotoxic antibodies (LCA). The incidence of LCA was significantly increased in SLE (93%), discoid lupus (SO%), and “lupuslike” syndromes associated with congenital complement deficiencies (63%) as compared to normal controls (3%) and patients with drug-induced lupus (11%), mixed connective tissue disease (MCTD) (17%), and necrotizing vasculitis (1%). The diagnostic and pathogenetic implications of these Merences are discussed.

An increased incidence of cold-reactive lymphocytotoxic antibodies (LCA) has been noted in sera from patients with a variety of diseases. Of these diseases, the highest incidence has been noted in patients with systemic lupus erythematosus (SLE) (14). In some studies, the incidence of lymphocytotoxic antibodies in lupus patients has exceeded 8W0 (3). Several other diseases have clinical features in common with those seen in SLE. These diseases include discoid lupus, drug-induced lupus, “lupus-like” syndromes associated with congenital complement deficiencies, mixed connective From the Division of Clinical Immunology, University of Colorado Medical Center, Denver, Colorado. Publication No. 5 of the Lupus Study Group, University of

Colorado Medical Center. Supported by NIH Grant RR51, General Clinical Research Center Program Grants AI-TI0031 and NS-12394, and donations from the Colorado Lupus Foundation. Alan L.Schocket, MD; Peter F. Kohler, MD. Address reprint requests to Alan L. Schocket, MD, University of Colorado Medical Center, Clinical Immunology, Box B-164, 4200 East Ninth Avenue, Denver CO 80262. Submitted for publication April 19, 1979; accepted June 7, 1979. Arthritis and Rheumatism, Vol. 22, No. 10 (October lW9)

tissue disease, and leukocytoclastic v a d t i s . In some of these SLE variants, the incidence of LCA has not previously been studied. Furthermore, no study has examined the incidence of LCA in all of these diseases at one time. The present study compares the incidence of LCA in patients with SLE and clinical variants of this disease to determine if LCA can be diagnostically useful to differentiate these syndromes and possibly to provide some insight into pathogenesis.

MATERIALS AND METHODS Patient population. Serum was obtained from 59 patients with systemic lupus erythematosus (SLE), 8 patients with discoid lupus erythematosus (DLE), 8 patients with “lupus-like” syndromes in association with congenital complement deficiencies including C1, C2, and C4 INH deficiencies, 19 patients with “lupus-like” syndromes and positive antinuclear antibodies (ANA) associated with drug ingestions (procainamide, apresoline, practalol) (some sera obtained from Drs. Marvin Fritzler and Eng Tan), 12 patients with mixed connective tissue disease (MCTD), 80 patients with leukocytoclastic vasculitis, and 3 1 volunteers and normal laboratory personnel. Serum samples were stored at -70°C. Assay for lymphocytotoxic antibodies. Lymphocytotoxic antibodies (LCA) were measured in serum at 15°C by the modified microtiter assay of Terasaki and McClelland (5). Mononuclear cells separated on a Ficoll-Hypaque gradient from 12 normal donors were used as target cells in the assay. Target cells, 2 X lo3, were incubated with 1 p1 of test serum for 1/2 hour at 15°C in a well of a microtiter plate. Five microliters of normal rabbit serum (NRS)were then added to the well, and the plate was incubated for an additional 3 hours at 15°C. Three microliters of Eosin Y and 5 pl of formalin were added to the wells. Each well was then examined under an inverted phase microscope. Cell death was determined by vital dye uptake, and the percentage of dead cells was recorded for each well. A negative control using 5% human serum albumin and a positive control using a standard lupus



sera which consistently killed between 8O-lWo of cells were used on each microtiter plate. Assays run with greater than 20% killing by the negative control were repeated. Results were expressed as a cytotoxic index (CI) (6):

CI =

Average % cytotoxicity of tested serum minus negative control (albumin) Average % cytotoxicity of known positive control minus negative control

x 100

This method of quantitation was used so that any small changes in test conditions from run to run could be adjusted for by comparing the results back to the same positive control standard. We were then able to directly compare results obtained at different times and in different diseases. In addition, this technique of reporting results provides a rough quantitation of the amount of lymphocytotoxic antibodies present in the serum. Terasaki reported (9,and we have also observed, that the level of killing by undiluted serum correlates with titers determined by serial dilution. In our laboratory, a serum with a cytotoxic index greater than IS is considered positive for lymphocytotoxic antibodies (6). Statistical evaluation. The incidence of lymphocytotoxic antibodies in different groups was compared using a modified Chi-square method (Fisher’sexact text).

RESULTS The incidence of lymphocytotoxic antibodies and mean cytotoxic index for each study group are noted in Table 1. The incidence and mean CI are significantly greater in the systemic lupus erythematosus group than in any other of the groups (P< 0.01). The incidence of LCA is greater in both the discoid lupus erythematosus and the complement deficiency groups than in the normal control group (P c 0.01). On the other hand, there is no significant increase in the incidence of serum LCA in the drug-induced lupus,

mixed connective tissue disease, or vasculitis groups. Although there were 2 patients in the drug-induced lupus group who had serum positive for lymphocytotoxic antibodies, the mean cytotoxic index in this group is comparable to mean cytotoxic index in the normal group. The incidence of elevated lymphocytotoxic antibodies in the mixed connective tissue disease and vasculitis groups is not significantly greater than the incidence in the normal group. The few patients in these two groups who were positive, however, had high levels of LCA averaging 78 (MCTD) and 53 (vasculitis), similar to the levels seen in SLE.

DISCUSSION The results of this study are consistent with previous reports of a high incidence of lymphocytotoxic antibodies in sera from patients with systemic lupus erythematosus (1-3). Furthermore, although the groups are small, the increased incidence of lymphocytotoxic antibodies found in patients with discoid lupus has not previously been noted. The increased lymphocytotoxic antibodies in complement deficiencies have previously been reported by our laboratory (7). The low incidence and levels of LCA in drug-induced lupus are also new observations. On the other hand, the absence of a significantly increased incidence of LCA in sera from patients with mixed connective tissue disease and necrotizing venulitis is contrary to the findings of 2 previous studies (8,9) in which LCA was found to be increased in frequency in both diseases. It is interesting, however, that although our study fails to show a significant increase in the incidence of LCA in these two groups, those patients with sera positive for LCA had levels comparable to those seen in SLE. Whether these patients are ANA negative SLE patients (10) is not clear.

Table 1. lncidence of lymphocytotoxic antibodies (LCA) in patients with systemic lupus erythematosus, its clinical variants and in controls Disease* SLE Discoid lupus Complement deficiency Drug-induced SLE MCTD Vasculitis Normal controls

Total patients

Positive LCA


55 4 5

8 8 19 12

2 2









93 50 63 11 17 19 3

76 21 39 6 13 13 4

* SLE = systemic lupus erythematosus; MCTD = mixed connective tissue disease.

t C1= cytotoxic index; see Materials and Methods.



A finding of a low incidence and low level of lymphocytotoxic antibodies in the drug-induced patients as compared to the idiopathic SLE patients is very interesting. Although the factor responsible for the induction of LCA production in the host has not yet been determined, there is increasing evidence that LCA production is related to exposure or infection with an environmental agent or virus. This hypothesis is supported by studies demonstrating an increased incidence of LCA in serum from family members of patients with SLE (1 1,12), multiple sclerosis (6), inflammatory bowel disease (13), and Hodgkin’s disease (14). In addition, LCA has been found in patients with acute and chronic viral infections (15-17) and in normal controls after vaccination with viral vaccines (18). Patients with diseases known to be related to chronic viral infections, such as subacute sclerosing panencephalitis (19), also have a significantly increased incidence of serum LCA. If indeed the production of lymphocytotoxic antibodies is related to exposure to (or infection with) a viral agent, one would predict a low incidence and level of LCA in patients with drug-induced lupus, a pharmacologically induced disease. The findings of the present study are compatible with this prediction. Whether the finding of an increased incidence of LCA in patients with discoid lupus and “lupus-like” syndromes associated with complement deficiencies and a decreased incidence of LCA in mixed connective tissue disease and necrotuing venulitis indicates that the first 2 diseases are virus related and the latter 2 are not is purely hypothetical. The small subgroup of the vasculitis patients with high LCA levels suggests that this is a heterogenous disease, and some of these patients indeed have virus related vasculitis. This syndrome has been previously described in association with hepatitis B infection (20). Finally, since there is a striking difference in the incidence of LCA between patients with drug-induced lupus and those with idiopathic lupus, LCA determination might be useful in the clinical delineation of these syndromes. Also, the low incidence of LCA in patients with MCTD and necrotizing venulitis may also be helpful in distinguishing these diseases from systemic lupus erythematosus, but this is controversial. Unfortunately, at the present time, the assay for LCA is tedious and not practical for routine laboratory use. If in the future, however, the techniques for determining LCA become greatly simplified, this may become a useful test in the differentiation of systemic lupus erythematosus from other clinically related syndromes.

REFERENCES 1. Mittal KK, Rossen RD, Sharp JT, Lidsky MD, Butler WT: Lymphocyte cytotoxic antibodies in systemic lupus erythematosus. Nature 225:1255-1256, 1970 2. Terasaki PI, Mottironi VD, Barnett EV: Cytotoxins in disease: autocytotoxins in lupus. N Engl J Med 283:724-728, 1970 3. Ooi BS, Orlina AR, Pesce AJ, Mendoza N, Masaitis L, Pollak VE: Lymphocytotoxic antibodies in patients with systemic lupus erythematosus. Clin Exp Immunol 17:237243, 1974 4. Winfield JB, Winchester RJ, Wernet P, Fu SM,Kunkel HG: Nature of cold-reactive antibodies to lymphocyte surface determinants in systemic lupus erythematosus. Arthritis Rheum 18:l-8, 1975 5. Terasaki PI, McCleUand JD: Microdroplet assay of human serum cytotoxins. Nature 204:998-1000, 1964 6. Schocket AL, Weiner HL: Lymphocytotoxic antibodies in family members of patients with multiple sclerosis. Lancet 1~571-573,1978 7. Kohler PF: Lymphocytotoxic antibodies and genetic complement deficiencies. Proceedings of the International Symposium on Clinical Aspects of the Complement System. Edited by W Opferkuch, K Rother, DR Schultz. Stuttgart, Georg Thieme Publishers, 1978, pp 226-231 8. Diaz-Jouanen E, Llorente L, Ramos-Niembro F, Alar-

con-Segovia D: Cold-reactive lymphocytotoxic antibodies in mixed connective tissue disease. J Rheumatol 44-10, 1977 9. Raum D, Glass D, Soter NA, Stillman JS, Carpenter CB, Schur PH: Lymphocytotoxic antibodies: HLA antigen associations, disease associations, and family studies. Arthritis Rheum 20933-936,1977 10. Fessel, WJ: ANA-negative systemic lupus erythematosus. Am J Med M80-86, 1978 11. DeHoratius RJ, Messner RP: Lymphocytotoxic antibodies in family members of patients with systemic lupus erythematosus. J Clin Invest 55: 1254-1258, 1975 12. Malave I, Papa R, Layrisse Z: Lymphocytotoxic antibodies in SLE patients and their relatives. Arthritis Rheum 19:70&704, 1976 13. Korsmeyer SJ, Williams RC Jr, Wilson ID, Strickland RG: Lymphocytotoxic antibody in inflammatory bowel disease: a family study. N Engl J Med 293:1117-1120, 1975 14. Mendius JR, DeHoratius RJ, Messner RP, Williams RC Jr: Family distribution of lymphocytotoxins in Hodgkin’s disease. Ann Intern Med 84:151-156, 1976 15. Mottironi VD, Terasaki PI: Lymphocyiotoxins in disease. I. Infectious mononucleosis, rubella, and measles, Histocornpatability Testing. Edited by PI Terasaki. Copenhagen, Munksgaard, 1970, pp 301-308



16. Huang S-W, Lattos DB, Nelson DB, Reeb K, Hong R: Antibody-associated lymphotoxin in acute infection. J Clin Invest 52:1033-1040, 1973 17. DeHoratius RJ, Henderson C, Strickland RG: Lymphocytotoxins in acute and chronic hepatitis: characterization and relationship to changes in circulating T lymphocytes. Clin Exp Immunol26:21-27,1976 18. Kreisler MJ, Hirata AA, Terasaki PI: Cytotoxins in dis-

ease. 111. Antibodies against lymphocytes produced by vaccination. Transplantation 10411 4 1 5 , 1970 19. Weiner HL, Schocket AL, Lehrich JR: Lymphocytotoxic antibodies in subacute sclerosing panencephalitis and amyotrophic lateral sclerosis. Lancet I:1013-1014, 1977 20. Gower RG, Sausker WF, Kohler PF, Thorne GE, McIntosh RM: Small vessel vasculitis caused by hepatitis B virus immune complexes. J All Clin Immunol62:222-228, 1978

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Lymphocytotoxic antibodies in systemic lupus erythematosus and clinically related diseases.

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