The response of cultured lymphocytes from patients with systemic lupus erythematosus to DNA.код для вставкиСкачать
The Response of Cultured Lymphocytes from Patients with Systemic Lupus Erythematosus to DNA By AIUA PATHUCCO, NAOMI F. KOTHFIELD AND KURT HIRSCHHORN that the immunologic responses of an animal are effected by he activity of fixed and circulating cells, which, in aggregate, make up the lymihoid tissue. The small lymphocytes, which ire the predominant cells in this tissue, lave been shown to have many potential 'unctions: They recirculate in the organism, hey have a long life span, and, when ippropriately stimulated, they develop into arge pyroninophilic cells and begin to clivide.'g2 During the past few years it has been shown that human peripheral blood lymphocytes can respond to a variety of stimuli in cell culture by enlarging and dividing. These cells respond to specific antigens to which the donor is sensitized, as well as to certain nonspecific stimulants such as phytohemagglutinin (PHA). It has been suggested that this in vitro system may be representative of the donor's immune status. The significance of immunologic reactions in the pathogenesis of systemic lupus erythematosus (SLE) is obscure. Different antibodies to nuclei, deoxyribonucleic acid (DNA), histones, and nucleoproteins found in this disease have been investigated, but T IS KNOWN their role in the clinical manifestations of the disease has not been established."-" The purpose of the present study was to examine the in vitro response of peripheral lymphocytes from patients with SLE when these cells were cultured in the presence of DNA. The results suggest that these lymphocytes, unlike those of normal subjects, do respond to stimulation with DNA. MATERIALS AND METHODS The cells were cultured by the method described in detail elsewhere.7 Forty ml. of heparinized blood was allowed to sediment at 37 C. The supernatant plasma was removed and left on glass at 37 C. for from one to two hours to allow attachment of polymorphonuclear leukocytes and monocytes to the glass. The supernatant was carefully removed. This contained between 60 and 95 per cent small mononuclear cells. The cells were washed three times and then suspended in minimal essential Eagle's medium modified for suspension culture* with 20 per cent fetal calf serum, penicillin, streptomycin, and 1-glutamine. The final cell concentration was 750,000 mononuclear cells per ml. Four ml. cultures were set up in replicate tubes at 37 C., and 0.1 ml. of PHA-Mf was added to one tube from each blood obtained. Two control tubes were set up for each blood which contained no additive. Duplicate tubes containing 1.0 gamma/ml. of highly polymerized calf thymus DNA$ were set up for each blood Professor of Medicine, New York Uniuersit3 School of Medicine, New York, N . Y. KURI HIRSCHHORN, M.D.: Associate Professor of Medi. cine, New York University School of Medicine Career Scientist, New York City Health Researd Council, 1-41 6, present address: Mt. Sinai Schoo of Medicine, New York, N. Y. OMEM-Spinner, Grand Island Biological Corn pany, Grand Island. N. Y. Difco Laboratories, Detroit, Mich. 4Mann Research Laboratories, New York, N. Y From the Department of Medicine and the Rheumatic Diseases Study Group, New York University School of Medicine, New York, N. Y. This study w a s supported by grants from the USPHS (HD-00542 and AI-068531, T h e New York City Health Research Council Grant U-1030, The American Heart Association, and The Lupus Foundation, N e w York. AIDA PATRUCCO, M.D.: Fellow, Michael I . Schaffer Foundation. Present address: Hospital Privado, Cordoba, Argentina. NAOMIF. ROTHFIELD,M.D.: Clinical Scholar, Arthritis Foundation; Assistant + 32 A N D RHEUMATISM, VOL. 10, No. 1 (FEBRUARY, 1967: ARTHRITIS 33 RESPONSE OF CULTURED LYMPHOCYTES obtained. DNA was denatured by heating a t 100 C. for ten minutes and then rapidly cooling. The denatured DNA was added to culture tubes with the final concentration of 1.0 gamma/ml. Cultures containing PHA were harvested at three days, and control and DNA tubes a t five days. After harvesting, the cells were washed in 1 per cent sodium citrate, fixed in 3:l methanol: glacial acetic acid, and stained with 0.5 per cent acetic orcein. Slides were examined using phase contrast illumination. One thousand cells from each culture tube were classified as large, small, or in mitosis. ‘The degree of response was defined as the percentage of large cells plus cells in mitosis in the experimental cultures less the percentage found in the cultures without additive. A positive response was defined as an increase of 5 per cent or more in the experimental tubes.8 In each instance, the blood was drawn, numbered, and clinical data obtained by one investigator. Cultures were set up and analyzed by a second investigator without knowledge of the diagnosis. The fluorescent antinuclear antibody test was carried ont as previously described using mouse liver as a source of nuclei and rabbit antihuman gamnia globulin conjugated to fluroescein isothiocyanate.9 Twelve patients with SLE were studied. All patients had multiple system disease compatible with SLE and positive LE cell tests at some time during the course of the disease. Ten healthy normal individuals and eleven patients with other diseases were also studied. All patients were studied either while they were patients at Bellevue Hospital or as outpatients in the New York University Arthritis C h i c of Bellevue Hospital. Table 1.-Response to Phytohemagglutinin % Increase of Large Cells and Mitoses NO. SLE Rheumatoid Arthritis Other Diseases Normal Studied Range Mean 14 60.1-80.2 74.6 4 7 10 64.5-86.6 74.0-82.0 67.1-84.4 73.1 77.7 76.5 Table 2.-Response of Lymphocytes to Stimulation with Native D N A NO. Studied No&al Individuals G-I Disease Seizure Disorder Cardiovascular Disease Rheumatoid Arthritis IIepatitis Hepatitis l’:orias s SLE % Increase Large Cells & Mitoses Range 10 1 1 2 4 0-2 0 3 0 0-3 1 0 1 1 10 11 12 4-34 - 11 patients with other diseases also showed no response to DNA. One of the two patients with hepatitis and one patient with psoriasis and arthritis had 10 per cent and 11 per cent responses to DNA respectively. Eleven of the 12 patients with SLE had a positive response to DNA (Fig. 1). The patient whose lymphocytes did not respond RESULTS significantly to DNA was being treated Response in control tubes (tubes with with 80 mg. of prednisone daily. Other no additive): The pattern of response-i.e., patients with SLE were taking 25 mg. or numbers of large cells and cells in mito- less of prednisone. Lymphocytes were obsis-in tubes with no additive was similar tained from another patient with SLE who in patients with SLE, patients with other was being treated with 60 mg. of preddiseases, and healthy normal individuals. nisone daily, but the lymphocytes failed Response to PHA: The per cent of re- to grow in the control culture tubes so sponse in tubes with PHA was similar that the response to DNA could not be in patients with SLE, patients with other evaluated. Four patients had taken chlorodiseases, and healthy normal individuals quine for from three months to four years, but this did not prevent the response of (Table 1 ) . Response to DNA: There was essentially their cells to DNA. The mean degree of no response to DNA in the ten healthy response to DNA of lymphocytes from panormal individuals (Table 2). Nine of the tients with SLE was 12.6 per cent com- 34 PATRUCCO, ROTHFIELD AND HInSCHHOnN 30 X increase Large cells + 25 mitoses 20 15 10 5 ... .., < b ......... ......t n " - I 2 3 4 5 6 7 8 9 10 II 12 L mean ,,f21 controls Fig. 1.-The response of lymphocytes from patients with SLE to DNA. For comthe average of the response in the 21 other individuals tested is also shown. The asterisk indicates the average of three experiments at monthly intervals using lymphocytes from the same patient. parison DISCUSSION pared to the mean of 1.6 per cent for the 21 others studied. The difference is statisIt is now well established that morphotically highly significant ( t test with Coch- logic changes and mitoses occur in cultured rane's correction: p < .OOl). peripheral blood lymphocytes as a response Response to Denatured DNA: Cells of to specific antigens to which the donor is control patients which did not respond to native DNA did not respond to stimulaTable 3.-Response of Lymphocytes from tion with denatured DNA (Table 3). The Controls and Patients with SLE to hepatitis patient who had a response to Native and Denatured DNA native DNA also responded to denatured 70Increase Large Cells & Mitoses DNA. Of the four patients with SLE tested, N Tt 've Denatured the cells of three responded to both native DNA DNA and denatured DNA, while the other reNormal 0 0 sponded only to native DNA. Normal 0 0 Relation of response of lymphocytes to Rheumatoid Arthritis 2 0 Rheumatoid Arthritis 1 2 DNA and titer of antinuclear antibody: Rheumatoid Arthritis 0 0 There was no correlation between the titer Hepatitis 10 10 of antinuclear antibody and the degree of SLE 34 24 response to DNA in patients with SLE. SLE 9 8 Antinuclear antibodies were not present SLE" F 11 SLE 13 3 in sera from the patients with other diseases or the healthy individuals (Fig. 2). *Average result of one patient studied 3 times. 35 RESPONSE OF CULTURED LYMPHOCYTES 35 30 X increase of large cells + 25 mitoses of tar native DNA 15 0 IC 5 C i/ 8 16 32 64 128 256 512 1024 I / A N F titer Fig. 2.-The relation of titer of antinuclear antibody and degree of lymphocyte response to stimulation with DNA. sensitized.8J0 This response has been dem- immunologically competent cells are capaonstrated for such varied substances as ble of producing antibodies to more than bacterial and viral extracts,1° antibiot- one nuclear antigen.17 The finding that ics,11J2 and other allergens.1° Sensitivity lymphocytes from patients with SLE are to allogeneic cells based on histocompati- sensitized to DNA is, therefore, not unexbility differences as well as to the patients’ pected, even when a low titer of antinucown skin cells in infantile excema have lear antibodies is present in the serum. also been described.13J4 The latter find- The lymphocyte stimulation experiments ing indicates that autoimmunity can be reflect the capacity of the cells to recogdemonstrated by this culture system, and nize the antigen, rather than the amount has prompted us to study the response of of antibody that is currently being produced or circulating. cells from patients with SLE to DNA. The clear-cut response of the lymphoAntibodies to various nuclear antigens have been reported by many investigators cytes from SLE patients to DNA and the to occur in serum from patients with SLE. lack of the response in most other individThese nuclear antigens include the whole uals suggests a specific capacity of the nucleus, nucleoprotein, DNA, histone, a cells to respond to DNA. Recent prelimiphosphate buffer nuclear extract, and a nary experiments using lymphocytes from recently reported additional nuclear anti- other SLE patients have confirmed this gen.3-6J5J6Antibodies to more than one finding and have, in addition, revealed that of these nuclear antigens are commonly the lymphocytes from SLE patients negapresent in SLE sera, and it is likely that tive to PPD will not respond to another 36 PATRUCCO, polyanion, purified lipopolysaccharide, or to PPD, but do respond to DNA. SLE lymphocytes responded not only to native, but also to denatured DNA. Stollar and Levine found that most SLE sera reacted more effectively with denatured DNA than with native DNA.1s The positive reaction of lymphocytes from the patient with psoriasis and arthritis remains to be explained. The patient did not have antinuclear antibodies nor did 16 of 17 other patients with psoriasis and arthritis tested in our l a b o r a t ~ r y .The ~ ~ positive test of one of our two patients with actue hepatitis may relate to the finding that LE cells have been reported in this disorder.”J1 There was no spontaneous increase in cellular enlargement or mitosis in cells from L,E patients, beyond that found in normals, at the time the cultures were harvested. The findings by Cooper and Firkin that there is an increased spontaneous rate of mitosis and DNA incorporation during the first hour of cluture reflect in vivo activity of these cells.22 These active cells are no longer found by the second day of culture and are certainly no longer active by the fifth day, when our assays were performed. ROTHFIELD AND HIRSCHIIORN The similarity of results in cultures without stimulant in SLE patients and normals also eliminates the possibility that the minute amount of DNA in the culture medium. released from dying cells, plays any role in these results. I n a recent study, it was shown that there was perfect correlation between the in vitro lymphocyte response and immediate wheal and flare reaction to penicillin or penicilloyl antigens in patients with clinical penicillin allergies.12 In that study, there was no correlation between the in vitro response and the titer of circulating antipenicillin antibody of either IgG or IgM type. There was no clear-cut correlation with delayed hypersensitivity. We suggest that a positive response to DNA by cells from patients with SLE may, as with penicillin, be a sensitive indicator of the presence of circulating cells previously sensitized to this antigen. ACKNOWLEDGhlENT We wish to thank Dr. Beatriz Pogo of Rockefeller University for performing the DNA assays, and Mrs. Maureen Toulon and h4iss llianca Pollo for technical assistance. SUMMARY Lymphocytes from normal individuals and from most patients with other diseases did not respond to stimulation with DNA in vitro by enlarging and dividing. Lymphocvtes from 11 of 12 patients with SLE showed a response to stimulation with DNA. The response to heat-denatured DNA in controls and SLE patients was similar to the response to native DNA. No relationship between degree of response of the SLE lymphocytes to DNA and the titer of circulating antinuclear antibody could be demonstrated. IN INTERLINGUA SUMMARIO Lymphocytos ab subjectos normal e ab le majoritate de patieiites con morbos altere clue disseminate lupus erythematose non respondeva per allargamento e division a1 stimulation in vitro con acido deoxyribonucleic. Lymphocytos ab 11 de 12 patientes con disseminate lupus erythematose respondeva positivemente a stimulation con acido deoxyribonucleic. Le responsa a thermo-denaturate acido deoxyribonucleic in subjectos de control0 e in patientes con disseminate lupus erythematose esseva simile a1 responsa a native acido deoxyribonucleic. Nulle relation inter le grado del responsa del lymphocytos in disseminate lupus erythematose a acido deoxyribonucleic e le titro del circulante anticorpore antinucleari poteva esser demonstrate. 37 RESPONSE OF CULTURED LYMPHOCYTES REFERENCES 1. Gowans, J. L., and McGregor, D. D.: The immunological activities of lymphocytes. Progr. Allerg. 9:1, 1965. 2. Fitzgerald, P. H. The immunological role and long fife-span of small lymphocytes. 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