Identification of DNA-Binding Lymphocytes

in Patients with Systemic Lupus Erythematosus

ARTHURD. BANKHURSTand RALPHC. WILLIAMS, JR.

From the Department of Medicine, Bernalillo County Medical Center,University of New Mexico School of Medicine, Albuquerque, New MIexico 87131

A B S T R A C T Antigen-binding lymphocytes capable ofbinding native DNA (DNA-ABC) were identified inthe peripheral blood of normal controls and patientswith systemic lupus erythematosus (SLE) by auto-radiography with 'I-nDNA. 12 patients with activeSLE had 404±273 (mean±SD) DNA-ABC/105 lym-phocytes, while 7 inactive SLE patients and 13 normalshad 120+48 and 48±36, respectively. All three groupswere significantly different from one another (P < 0.01).No significant correlation was detected between thequantity of anti-native DNA (nDNA) antibody andnumber of DNA-ABC; however, most patients withlarge amounts of anti-nDNA antibody had both activedisease and large numbers of DNA-ABC. Numbers ofDNA-ABCand lymphocytes with surface immunoglobu-lin (Ig) did not change significantly after anl 18-h incu-bation at 37°C. After depletion of B-lymphocytes bypassage over bead columns coated with a complex ofIgG and anti-IgG, the great majority of DNA-ABCwere removed in both normal subjects and SLE pa-tients. Labeling lymphocytes sequentially with 'JI-nDNA, followed by an indirect fluorescence techniquefor identification of surface Ig, indicated that the greatmajority of radiolabeled cells had surface Ig by fluores-cence microscopy in four normals (average 93%) andfive patients with active SLE (average 82%).

The predominance of nDNA-sensitive B-lymphocytesin the peripheral blood of both normals and SLE pa-tients is consistent with the concept that the induction ofthe anti-nDNA antibody response is due to the stimula-tion of preexisting nDNA-specific B lymphocytes bymechanisms other than those necessarily involving par-ticipation of nDNA-specific T lymphocytes.

Dr. Bankhurst is a Senior Investigator of the ArthritisFoundation.

Received for publicationt 4 Decemiber 1974 antd int revisedform 17 July 1975.

INTRODUCTIONSystemic lupus erythematosus (SLE)1 was first recog-nized as a disease accompanied by widespread connectivetissue damage to segments of the vascular system andvarious serous surfaces (1). The finding that hema-toxvlin bodies were altered DNA (2) and later that LEcells contained ingested phagocytized nuclear material(3) indicated possible participation of nuclear sub-stances in the genesis of the disease. Subsequently, theapplication of a variety of immunologic methods estab-lished that sera from patients with SLE contain a widespectrum of antibodies to nuclear components, includ-ing nucleoprotein, extractable nuclear antigens, histones,and DNA itself (4-10). Antibodies to DNAappear toplay a central role in some of the immune lesions ofSLE, since several lines of evidence clearly link themto renal injury. Analyses of immunoglobulin eluted fromisolated SLE glomeruli have indicated high relativeconcentrations of anti-DNA antibody (11, 12). A closetemporal relationship between the appearance of DNAin serum and subsequent anti-DNA antibody associatedwith exacerbations of lupus nephritis and lowering ofserum complement components (13-17) emphasizes thepathogenetic significance of such antibodies.

Other antibodies to nuclear components, as well asantibodies to various types of RNA (18), may also beof importance in the pathogenesis of SLE, yet nativeDNA (nDNA) and anti-nDNA antibodies have clearlybeen defined as an important potential source of immunecomplexes in this disorder.

'Abbreviations utsed in this paper: ABC, antigen-bindingcell; DNA-ABC, antigen-binding lymphocyte for nativeDNA; DNP10-BSA, dinitrophenylated bovine serum albu-min; FCS, fetal calf serum; MEM, minimal essential me-dium; nDNA, native or double-stranded DNA; PBS, phos-phate-buffered saline, pH 7.2; SLE, systemic lupus erythe-matosus; Tg, thyroglobulin.

The Journal of Clinical Investigation Volume 56 December 1975 1378-13851378

The present study was designed to examine the typesof nDNA-antigen-binding cells (DNA-ABC) presentin the peripheral blood of normal subjects and lupuspatients. Current data support the concept that the inter-action of an antigen with a specific, predetermined lym-phocyte surface receptor initiates a chain of events lead-ing to detectable antibody formation. Studies in themouse with heterologous isotopically labeled antigenshave shown that the antigen-sensitive lymphocyte is in-cluded among the ABC identified with autoradiographictechniques (19). Previous studies identified the presenceof ABC for human thyroglobulin in normal subjects(20), as well as in chronic thyroiditis (21). Since theformer study suggested that the ABC for thyroglobulin(Tg) were primarily B lymphocytes, it is possible thatself-tolerance to Tg may be due to the absence of helperT lymphocytes (22). It was unclear from these Tgstudies whether the anti-Tg response was related to theappearance of specific T helper cells in patients withchronic thyroiditis. The current study was directed tothe question whether ABC capable of binding the po-tential autoantigen nDNA in normal subjects and lupuspatients were B and/or T lymphocytes. The evidencepresented here seems to indicate that loss of toleranceto nDNA in patients with SLE may be accompanied byproliferation principally of antigen-sensitive B lympho-cytes.

METHODSSubjects. Blood samples were obtained from 12 patients

with active SLE, 7 patients with inactive SLE, and 13normal subjects. The diagnosis of SLE was based onAmerican Rheumatism Association criteria (23). Patientswith active SLE generally showed decreased serum com-plement (C3) and signs of activity such as synovitis, leu-kopenia, rash, nephritis, or serositis. Patients with inactiveSLE showed normal complement and no evidence of clinicaldisease activity. The SLE patients generally were on atherapeutic regime that included corticosteroids (10-30 mgprednisone) and azathioprine (50-150 mg). Several un-treated patients were studied, including the patients L. P.and V. C., referred to in Tables I and IV.

Lymphocyte preparation. Peripheral venous blood wascollected in heparinized glass tubes. Purified lymphocytesuspensions were then prepared with Ficoll-Hypaque gradi-ent (24) followed by three washings in PBS. The lympho-cytes were incubated on 100 X 15-mm plastic Petri dishesfor 45 min at 37°C in minimal essential medium (MEM)containing 10% fetal calf serum (FCS) to deplete phago-cytic mononuclear cells. The nonadlherent cells were aspi-rated from the Petri dishes and again washed three timesin PBS before use in the procedures described below.

Preparation of antigens. Native calf thymus DNA(Sigma Chemical Co., St. Louis, Mo.; Type I, sodium salt,high-polymerized, D 1501) was deproteinized by three suc-cessive phenol extractions by the method of Harbeck et al.(25).The DNA was then sonicated with a Sorvall sonifer(Branson Sonic Power Co., Danbury, Conn., Model W185D, position 5 for 1 min). The sonicated, deproteinized DNAwas finally passed through a Sepharose 4B column in 0.05

M Tris buffer, pH 8.0. Finally, the sonicated, deproteinizedDNA was passed through a cellulose nitrate filter (typeHAWP, Millipore Corp., Bedford, Mass.) to remove anycontaminating, single-stranded DNA bound to the filter(26), dialyzed against PBS, and stored at concentrations of0.5 mg/ml at - 200C. The resulting DNAproduced a single,narrow peak on analytical ultracentrifugation with a sedi-mentation coefficient of approximately 22S. There was nodetectable protein in this DNA preparation by the Folinmethod (27).

Staphylococcal protein A and human kappa light chainwere used as controls in the blocking experiments. Pro-tein A was prepared according to the method of Sjoquist etal. (28); human kappa light chains were prepared fromlyophilized urine by zone electrophoresis on starch block.Both protein A and the purified light chains were dialyzedagainst PBS before use.

Labeling of DNA with "I. The deproteinized, sonicatednDNA was labeled with 1'I as previously described (29).The incubation volume was 0.15 ml and contained 20 ,tgnDNA, 0.1 M acetate buffer, pH 5.0, 2.5 X 104 M KI,3 mCi radioactive iodine (carrier-free "I, sodium salt in0.1 M NaOH, Amersham/Searle Corp., Arlington Heights,Ill.), and 1.5 X 10' M thallium trichloride (K and K Labor-atories, Inc., Plainview, N. Y.). After the incubation for1 h at 60°C, DNAwas dialyzed against phosphate-bufferedsaline (PBS) (500: 1, dialysate volume: dialysis tube vol-ume) for 48 h with four changes of dialysis fluid. The "JI-DNA was passed through a Millipore filter again, as de-scribed above, to remove any DNAdenatured by the iodina-tion procedures. The specific activity of the 'I-nDNA was4-7 IACilAg.

Labeling of lymphocytes with ..51-nDNA. Conditions forlymphocyte labeling with radioiodinated antigens have beendescribed (30). 3 X 10' lymphocytes prepared as above wereincubated in 0.5 ml of PBS, 0.1% sodium azide, with 800-1,200 ng of 'I-nDNA for 30 min at 0°C. The cells werethen washed alternately with PBS or FCS gradients fourtimes. In the case of FCS gradients, the cells were intro-duced on the surface of a 3.0 ml gradient of FCS and PBS.The lower third of the gradient consisted of 100%o FCS,the middle third 75%o FCS, and the top third 50%o FCS and50%o PBS. In blocking experiments with unlabeled antigens,the lymphocytes were incubated for 1 h at 0°C with either340 Ag nDNA, 100 ,ug protein A, or 1,700 ,ug human kappalight chain in a volume of 0.5 ml and washed through oneFCS gradient before incubation under standard conditionswith 'I-nDNA. The specificity of binding of native DNAwas also studied by preincubating the lymphocytes as de-scribed above with 500 ,ug dinitophenylated bovine serumalbumin (DNP1o-BSA), 340 gtg denatured DNA, as wellas unlabeled nDNA itself. DNP1o-BSA was prepared bythe method of Davie and Paul (31), as previously described.Denatured DNAwas prepared by heating purified, sonicatednDNA to 100°C for 15 min in boiling water and then im-mediately cooling it in ice water. Finally, binding specificitywas additionally checked with a DNase-treated preparationof 'I-nDNA. The DNasle (Type I deoxyribonuclease) wasobtained from Sigma Chemical Co., and used as describedby Harbeck et al. (25).

Autoradiographic detection of antigen-binding lympho-cytes. Cell suspensions for autoradiography were spreadupon gelatin-coated slides. The slides were dipped in NBT-2 liquid emulsion (Eastman Kodak Co., Rochester, N. Y.).The techniques for the fixation of cells, exposure of slides,and the development of slides have been described (30).Slides were exposed for 12-18 days. The cells were stained

DNA-Binding Lymphocytes in Systemic Lupus Erythematosus 1379

with Giemiisa andl at least 5-10 X 10 cells were counted()ii eaclh sli(le. A positive cell was i(lenltifle(l ly thle fol-lowing characteristics: (a) it had to be morphologicallya small lynmphocyte (less than 8 ,um in diameter) ; (b1) ithad to have an intact cytoplasmic membrane with no otherobvious damage; anid (-) it could not be in contact wvithdlebris or aniother cell. All cells thit lhadl en ugh grainls to

)bscure the illlderlyinig miiorphology xwere treate(l with asolution of 1% I)otassium ferricyalld(le to dissolve the grainis(2.35 mg potassium ferricyani(le and 30 Imlg sodiuIml thiosul-fate/1.0 ml). This technique was iml)ortanit, since debrisand monocytes were occasionally labeled that would other-wise have been indistinguishable from positive lymphocytes.The background grain count was generally in the range of0-6 grains. A positive cell was identified by a minimum of20 grains on its surface or within one-half cell diameterfrom its surface.

Antti-h1um7tant Ig columt antd produictiont of T lynplhocytesuspensionis. Double-layer (anti-human Ig-Ig) columnswere prepared by a minor modification of the method ofWigzell et al. (32). Degalan V-26 plastic beads (DegussaWolfgang AG, Hanau am Mfain, Germany) were coatedwith human IgG (Cohn II fraction) and then equilibratedin Pasteur pipettes with a large excess of rabbit anti-humanIg. Generally only 0-3% of cells in the effluent from sucha columniii were lymphocytes with detectable amounts ofsurface Ig.

Idcnttificationt of lymiiphocyte sutrface Ig. Lymphocyte sur-face Ig was identified by an indirect fluorescent technique.Generally 2-3 X 10' lymphocytes were incubated at 0'C for30 min with a 1: 4 dilution in PBS of a high titer rabbitanti-polyvalent human Ig antiserum. This was followed bythree washes with PBS and another 30-min incubation at0°C with 0.1 ml of fluoresceinated goat anti-rabbit IgG(IgG fraction, 2-3 mg/ml, Meloy Laboratories Inc., Spring-field, Va.). The cells were again washed three times afterthe second incubationi, ancd at least 200 cells were countedunder the fluorescent microscope.

AnialYsis of ABC by, autoradiograplhy anid/or immun71it11o-fluiorescenzce. Lymphocyte suspeinsions were labeled suc-cessively with '"I-nD.NA and fluoresceinated goat anti-rabbit IgG by the indirect sandwich technique. The firststep, involving the incubation with '25I-nD -A, was doneas described above. The cells were waslhed three times witlFCS gradients before incubation with rabbit anti-polyvalenthuman Ig. The indirect sandwich technique was performedas above, except that the third wash after the incubationwith fluoresceinated goat anti-rabbit IgG was done througha FCS gradient before fixation on gel slides with ethanolfor 5 min. The slides were then dipped in nuclear emulsionas described above and developed 4-10 days later.

Microscopy. The preparations were examined with aZeiss Photomicroscope equipped with a vertical IlluminatorIII RS and a mercury light source (Carl Zeiss, Inc., NewYork). The following filter combination was used for fluoro-chrome visualization: BG12 exciter filter, Fl 500 chromaticbeam-splitter, and barrier filter 50. Experiments evaluatingthe percentage of labeled lymphocytes in cell suspensionswere performed by counting fluorescent cells in the ultra-violet field, then switching to light-field illumination to countthe total cell numbers. When fixed cells were examinedunder bright-field for autoradiographic labeling as well as

under ultraviolet for fluorochrome labeling, the slide was

covered with several drops of a mixture of glycerine and10% Tris buffer, pH 9.5 (9: 1 volume ratio), and a coverslip. When only bright-field microscopy was performed, thefixed preparations were examined without a cover slip

witlh a high-power objective (40X), so that the grains couldbe dissolved w\-hen necessar.y. \Wlen com?blined bright-field-ultraviolet microscopy was done on fixed preparations, theonly radiolabeled cells selecte(d for fluorochromne examinlatiolnwere those with grains that did not obscure the morphologyof the cell, since dissolution of grains was impossible whentlle cover slil) as present.

J1lf dSur'Co,.,t Of tmti-L).iDAA anitiblodv. This was donewith ["4CnDNl)NA in a primary binding tecili(lue by theammiloniumll sulfate (Farr) method (33). ["C]nD)NA wasobtaine(d froml Amershlamii/Searle Corp. (CFB 170). TheD-NA-binding capacity was expressed as percentage of total[l4CnDNA bound by a 1: 10 dilution of test serum (nor-mal serum bound, 5.1±1.5c, mean±1 SD).

RESULTSJN'um;ibers of DNA-ABC in SLE patients and niio0rm-al

people. A comparison was made between the numbersof DNA-ABC in normal people and patients with inac-tive or active SLE (Fig. 1). 12 patients writh activeSLE had 416±270 (mean+SD) DNA-ABC/105 lym-phocytes, whlile 7 inactive SLE patients and 12 normalshad 120±48 and 49±40, respectively. No clear correla-tion between DNA-ABCand any distinct clinical profilewsas detected; however, most patients with active SLEalso showed lupus nephritis. The three groups were allsignificanitly different from one another (P < 0.01).

The relationslhip between the numtber of DNA-ABCanid the quantity of anti-niD-A anitibody. No significantcorrelationi 'was detected between the quantity of anti-niDNA antibody and the total number of lymplhocytesthat bounid '251-nDNA (Fig. 2). However, the great mia-

Cf)0

LJ

z

z

C')

900

800

7001-

600F-

500e

400+-

300F-

200+

loop-

:

100~~~~~~

I I

ACTIVE SLE INACTIVE SLE

soNORMAL

FIGURE 1 Numbers of DNA-ABC in SLE patients andnormal people. The mean±SD are indicated.

1380 A. D. Bankhurst and R. C. WVilliams, Jr.

ol

1001 * Active SLE0I/nJctive SLE

80k-

60K-

40

200

0

N04Lqo 100 200 300 400 500 600

DNA-Binding Cells/ 105 Lymphocytes

FIGURE 2 The relationship between the nunABC and the quantity of anti-nDNA antibnDNA titer is expressed as the percentaglabeled nDNAprecipitated by a 1: 10 dilutior(Normal serum binding is 5.1±1.5%, mean±

jority of patients with DNA-binding actifhad both active disease and large numbABC. Several interesting discrepanciesamount of anti-nDNA antibody and tbDNA-ABC were noted such as in an ac

tient with 560 DNA-ABCand no anti-nDNA antibodyand in one inactive SLE patient with a normal numberof DNA-ABCbut a high titer of anti-nDNA antibody(34%).

Tests for specificity of "I5-nDNA binding. The spe-cificity of the surface labeling of lymphocytes with ra-diolabeled nDNAwas investigated by preincubating thelymphocytes with excess unlabeled nDNA, human kappalight chain, staphylococcal protein A, DNP1o-BSA, anddenatured DNA (Table I). 100% inhibition of 'I-

j |ww nDNAbinding was observed in active SLE patients and700 80 9a) in normals only with nDNA, and not with controls such

as protein A or human kappa light chain. Of interestnber of DNA- was that only minor inhibition of binding (9 and 12%)ody. The anti- was recorded with DNP1o-BSA. A small degree of in-e of the total hibition (25-28%) was recorded with denatured DNA.~of test serum.:1 SD). (Table I). Finally, prior DNase treatment of 'I-nDNA

completely abolished DNAbinding in all instances.vity over 30% The effect of prolonged incubation upon the numbersers of DNA- of DNA-ABC. To investigate the question of whether

between the anti-lymphocyte antibody, anti-nDNA antibody, orie number of nDNA-anti-nDNA complexes were present on the sur--tive SLE pa- face of lymphocytes, the numbers of radiolabeled lym-

TABLE IBlocking of the Formation of DNA-ABCby Pretreatment with Unlabeled Materials

Radio-labeled

Human subject Pretreatment Labeled compound cells Inhibition

cells/105 %lymphocytes

D. F. (active SLE) PBS 1251-nDNA 400DNA (340u) 1251-nDNA 0 100Protein A (100,ug) '251-nDNA 320 20Humankappa light chain (1,700,ug) 251-nDNA 380 5

A. R. (normal) PBS 125I-nDNA 100DNA (340,ug) 1251-nDNA 0 100

L. P. (active SLE) PBS 1251-nDNA 800Protein A (100 jg) 1"5I-nDNA 780 2.5Humankappa light chain (1,700,4g) 251 -nDNA 900 0

V. C. (active SLE) PBS 1251-nDNA 550nDNA (340 ,g) 1251-nDNA 0 100Denatured DNA (340,ug) 1251-nDNA 400 28DNP-BSA (500,ug) 125I-nDNA 500 9PBS DNase-treated 1251-nDNA 0 100

J. D. (normal) PBS 12-IInDNA 80nDNA (340 ,ug) "251-nDNA 0 100I)enatured DNA (340 Ag) 121I-niDNA 60 25DNP-BSA (500 Mg) I'5I-nDNA 70 12PBS DNase-treated 251-nDNA 0 100

3 X 106 cells were preincubated for 30 min at 2°C with the indicated quantities of materials in a volume of 0.1 cm3. The cellswere subsequently washed and incubated with 251-nDNA (1.000 ,ug) as described under Methods.

DNA-Binding Lymphocytes in Systemic Lupus Erythematosus

E

CP

z

Q-

1381

TABLE I IThe Effect of Prolonged Incubation upon the Number

of DNA-ABCand Lymphocytes withDetectable Surface Ig

Number of radiolabeledlymphocytesil

Before AfterExp. Subject incubatior.* incubationt

1 D. R. (normal) 40 60S. Z. (inactive SLE) 220 220

2 R. R. (normal) 80 100S. Z. (inactive SLE) 120 180

4 V. P. (active SLE) 240 260

Percentage of cells witlsur-face Ig§

1 S. A. (active SLE) 21 232 S. Z. (inactive SLE) 16 193 S. 0. (active SLE) 19 26

C. 0. (active SLE) 23 18P. C. (normal) 30 27

* Lymphocytes were prepared on Ficoll gradients and incubated in plasticPetri dishes for 45 min at 37°C. The nonadherent, washed cells were thenfixed on slides after labeling with fluoresceinated antiserum or 125I-nDNA.I These cells were further incubated for 18-20 h at 37°C in MEMcontaining10% FCS before surface labeling with either l25I-nDNA or fluoresceinatedantiserum. Essentially no loss of viability wvas observed during thisincubation.§ 200 cells on a fixed preparation were counted. No significant difference wasobserved between the percentage of cells before and after prolonged incu-bation (P > 0.8).

[l At least 5,000 cells were counted. No significant difference wvas observedbetween the number of labeled cells before and after incubation (P > 0.10).

phocytes and lymphocytes with surface immunoglobulindetectable by fluorescent antibody were measured be-fore and after an 18-h incubation at 37°C. There wasno significant difference between the numbers of DNA-ABC (Table II, top) (P > 0.8) and lymphocytes withsurface Ig (Table II, bottom) (P > 0.10) before andafter incubation in active or inactive SLE patients andnormals.

The identification of DNA-ABC as eitlier B or Tlymtphocytes. The first approach to the question ofwhether DNA-ABCwere B- or T-cells was to compare

the number of DNA-ABCbefore and after depletion ofB lymphocytes by passage over Degalan bead columnscoated with human IgG and anti-Ig (Table III). Thegreat majority of cells capable of binding labeled nDNAwere removed by this procedure in two normals (100%and 75% depletion) and one active SLE patient (90%).

The second approach was to label lymphocytes se-quentially with 'I-nDNA and an indirect fluorescenttechnique for surface immunoglobulin (Table IV).Using such a technique, Nve could tell by visible, bright-field microscopy whether a cell bound 'I-nDNA andwhether the same cell had surface immunoglobulin. Anaverage of 92% of DNA-ABC in four normals and 82%of DNA-ABC in five patients with active SLE showedconcomitant surface Ig. One active SLE patient hadonly 50%I, of radiolabeled cells with surface immuno-globuliin.

DISCUSSION

One of the most convincing associations between auto-antibody and tissue lesions is exemplified by the relation-ship of anti-nDNA antibody and the glomerulonephritisof SLE (13-17). To cast light on the events initiatingsuch an aberrant B-cell response, we presumably haveto focus on the nDNA-sensitive lymphocyte. Identifica-tion of this functional subpopulation by morphologiccriteria (nDNA-binding lymphocyte) is based on thework done by Ada and Byrt in the mouse (19). Thepresence of DNA-ABC in normal subjects, demonstratedin the current study, is contrary to any simplistic theoryof self-tolerance that presupposes the absence of auto-antigen-sensitive lymphocytes in normal individuals. Inother words, the activation of the anti-nDNA responseinvolves the triggering of preexisting specific B lympho-cytes and not merely the emergence de novo of nDNA-sensitive B lymphocytes. The present data also showedincreased numbers of DNA-ABC in patients with activeor inactive SLE. Previous studies with human thyro-globulin have shoN-il parallel changes in ABC for human

TABLE IIIThe Decrease in the Nuniber of DNA-ABCafter Column Depletion of Lymphocytes with

a Large Amount of Surface Ig (B Lymphocytes)

Number of DNA-ABC B Lyn-.phocytes*

Subject Before column After column Decrease Before column After column

labeled lymphocytes, 105 lymphocytes C% %

M. K. (normal) 80 0 100 28 1

D. R. (normal) 80 20 75 25 0S. A. (active SLE) 200 20 90 32 1

* B lymphocytes were identified by their large amount of surface Ig by ultraviolet microscopy. The

tlepletion of B lymphocytes was effected by their passage through a double-layer head column thatpossessed free anti-Ig combining sites.

1382 A, D Bankhurst and R. C. Williams, Jr.

TABLE IVA Study of the Percentage of Cells that Simultaneously

Have Surface Ig and Bind 'l25-nDNA

125I-nDNA-binding cells

Cells with also havingSubject DNA-ABC* surface Ig$ surface Ig§

J. D. (normal) 60 18 100F. P. (normal) 80 31 80M. P. (normal) 80 36 90M. F. (normal) 100 26 100D. F. (active SLE) 550 36 80L. P. (active SLE) 680 17 50S. A. (active SLE) 560 20 100V. C. (active SLE) 440 18 90L. B. (active SLE) 300 15 90

* At least 5,000 cells were counted. Results are expressed as number ofradiolabeled cells/105 lymphocytes.t Fixed cells were examined for fluorescence by the sandwich techniquedescribed in Methods.§ At least 10 radiolabeled cells were examined for the presence of fluores-cence by the double-label technique described under Methods.

thyroglobulin in normals (20) and patients with chronicthyroiditis (21). The increase in DNA-ABC with dis-ease activity was not unexpected since studies in micehave clearly shown that immunization causes an in-crease in the number of ABCfor that antigen (34).

The DNA-ABCwere primarily B lymphocytes in bothnormal individuals and SLE patients, whether the Blymphocytes were identified by column depletion or thedouble-labeling technique. This would suggest that theSLE patients did not differ from normals simply becauseof the presence of a larger number of antigen-sensitive Tlymphocytes that could enable a cooperative B-T lympho-cyte anti-nDNA response to occur. One might arguethat the antigen-binding techniques preferentially iden-tify B lymphocytes, and any T lymphocytes that areDNA-ABC would evade detection. However, mouse Tlymphocytes that are ABC (35) and ABC in the humanthymus (36) have been identified by the autoradio-graphic method used in this paper. Our results providea possible explanation for some forms of tolerance tonDNA and the induction of the anti-nDNA responsein SLE. The presence of DNA-ABC in normal indi-viduals presumably excludes the simple explanation thattolerance to nDNA is due to a lack of immunocompetentcells able to react with nDNA. If we assume that theformation of antibodies against nDNA requires theparticipation of two types of lymphocytes, it is possiblethat the absence of either B or T lymphocytes couldresult in self-tolerance. Since the DNA-ABC in normalpeople were primarily B lymphocytes, the observationssuggest that T lymphocytes able to react with suchB-cells are relatively scarce or absent. However, thevery presence of DNA-ABC in normal subjects is of

considerable interest and should temper a differentiationbetween normals and patients with SLE only on thebasis of presence or absence of such cells. ABC de-tection is a subject of some controversy and appears tovary according to antigen used and methodology (37).Despite these technical problems, the inhibition data asshown in Table I support apparent primary specificity ofABC for nDNA in both normal subjects and patientswith SLE. The initiation of the anti-nDNA responsecould presumably have arisen by one of three mecha-nisms: (a) the emergence of a population of helper Tlymphocytes, (b) the disappearance of a population ofsuppressor T lymphocytes, or (c) another mechanismthat bypasses the requirement for a helper T lymphocyte.If one assumes that specific helper or suppressor T cellscould be identified as ABC by autoradiography, thefirst two possibilities seem less likely, since there wasno apparent alteration in the percentage of lymphocytesthat were both B lymphocytes and DNA-ABC in nor-mal people versus patients with active SLE. This leavesthe T cell "bypass" mechanism as another possibilityfor the initiation of the anti-nDNA response. The needfor a nDNA-sensitive T lymphocyte could be overcomeif helper function could be supplied by stimulation ofother T lymphocytes reactive with a cross-reacting anti-gen, or if the nDNAwas functioning as a hapten com-plexed to a carrier.

Several problems are involved in the interpretation ofDNA-ABC. First, the specificity of the reaction be-tween lymphocytes and nDNAmust be examined. Datapresented in Table I support the primary nDNA speci-ficity of the reactions measured here. Very little in-hibition was observed with unrelated proteins such asprotein A, kappa light chain, or DNP1o-BSA. Completeinhibition was repeatedly observed with excess unla-beled nDNA, whereas only 25-28% inhibition was re-corded with heat-denatured DNA. It was recognizedthat the sonicated preparations of nDNA used in ourexperiments may have contained fragments of DNAwith exposed terminal ends partially denatured by thesonication process. However, the technical requirementsnecessary for the antigen-binding assay required suchprior treatment.

Second, one has to be certain that the DNA-ABCarelymphocytes and not monocytes that bind and/or pino-cytose antigens and antigen-antibody complexes (38,39). Morphologic separation of large monocytes versuslarge lymphocytes is extremely difficult without specialstains, such as nonspecific esterase in the case of mono-cytes. However, it is well established that cells identifiedmorphologically as small lymphocytes are never stainedby the sensitive, nonspecific esterase method (40). Nolymphocytes greater than approximately 8 /Am werecounted in the present study, so that monocyte labeling

DNA-Binding Lymphocytes in Systemic Lupus Erythematosus 1383

Was essentiatly excluded. Grains overlying cells were(lissolved wllhen necessar) to facilitate positive id(enti-fication of cells as simiall 1lmphlocytes. 1Pinocytosis \V,;sprevented by the cold incubation conditions and thepresence of sodium azide.

Third, it is conceival)le tllat cvtoplhilic alnti-uDN.Aanltilb()odv all(l or nlDNA-anlti-lnDNA comlplexes miiay bep)resellt on the lymiphlocyte suirface. This consideratiolnwas of special importance, since our data showed atendency for increased numbers of DNA-ABC to be as-sociated with a larger amount of anti-nDNA antibody.B lymphocytes are capable of binding certain types ofIg. This Ig binding is not as secure as wrhen the Ig iscomplexed with antigen, and generally this cytophilicantibody can be removed by washing (41). There areseveral reasons wlhy cytophilic anti-nDNA antibody isprobably not important in the present study. Thesereasons may be listed as follows: (a) Patients werefound writh high numbers of DNA-ABC without de-tectable serum anti-nDNA. Obviously one wtould notexpect this if cytophilic antibody were responsible forthe elevated numbers of DNA-ABC found in SLE pa-tients. (b) Incubation in cell culture for 18-20 h didnot affect the number of DNA-ABC. Because of thewell-documented dynamic turnover of the lymphocytemembrane (42), one would expect cytophilic antibodyto be shed along with membrane components. (c) Lym-phocyte suspensions were washed at least tlhree timesafter preparation, wNhich ordinarily removes cytophilicantibody (41). For an adherent nDNA-anti-nDNAcomplex to interact witlh radiolabeled nDNA, it is neces-sary that the complex be formed in antibody excess sothat the complex would retain free anti-nDNA com-bining sites. However, it is clear from the present studythat large numbers of DNA-ABC can exist in the ab-sence of anti-nDNA antibody. Also dynamiic membranechanges would presumably involve the receptors forantigen-antibody complexes and result in the sheddingof the complex in a relatively short time. Our long-term incubation studies showed no significant changein the number of DNA-ABC after overnight incubationat 37°C, thus making the possibility of 1"I-nDNA bind-ing by complexes unlikely.

B lymphocytes were identified in the present study bymethods that detected relatively large amounts of sur-face Ig. Since patients with SLE have antibodies di-rected against both B and T cells (43, 44), the presenceof surface Ig could theoretically include some T lym-phocytes as w,vell as B lymphocytes. This potential prob-lem was overcome in the present study by an initial in-cubation at 37° C, which allowed any anti-lymphocyteantibody to be shed from the cell surface (45). The effi-cacy of such a preliminary short incubation in inducingcomplete antibody shedding was confirmed by the lackof a significant change in the number of cells with sur-

face Ig after a furtlher, prolonged, 18-h incubationi at370C.

Thrle i(leltificatioll of DNA\-ANBC in normal peopleanid patients wvith SLE was an attempt to define the cel-lular mechanisms involved in the induction of the anti-nDN:A antibody response. The resuilts reported here are(NCoMiSistenlt with 1-'iriiet's Concept (46) thlat the emier-

gence of a, 'forbidden clonie" of B lymniphlocytes is theprimary event in the production of auitoantibodies. Atleast in the case of the anti-nDNA antibody response illSLE, it appears that the response may be initiated bythe stimulation of preexisting nDNA-specific B lym-phocvtes by several mechanisms other than the partici-pation of nDNA-specific helper T lymphocytes.

ACKNOWLEDGMENTSThe authors thank 'Ms. Julia Buckelew for her skillful tech-nical assistance and to Ms. Bernadette NIarquez for her helpwith the manuscript.

This work was supported in part by grants AMIAI 13824-05 and T01AI000353-4 from the U. S. Public HealthService and in part by a grant from the Arthritis Foun-dation.

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