impaired cell-mediated immunity in systemic lupus erythematosus (sle): a controlled study of 23...

Impaired Cell-Mediated Immunity in Systemic Lupus

Erythematosus (SLE)

A Controlled Study of 23 Untreated Patients

JOHN G. PATY, Jr., M.D.’

CHARLES W. SIENKNECHT, M.D.’

ALEXANDER S. TOWNES, M.D. +

ARAM S. HANISSIAN, M.D.

JOHN B. MILLER, B.S.

ALFONSE T. MASI, M.D., DR. P.H.

Memphis, Tennessee

From the Section of Rheumatology, Division of Connective Tissue Diseases, Department of Medicine, University of Tennessee Center for the Health Sciences, Memphis, Tenn. This study was supported in part by Grants AM-12049 and AM-05055 of the National Institute of Arthritis, Metabolism, and Digestive Diseases, General Research Support @ant USPHS RR05423, a grant from the West Tennessee Chapter of the Arthritis Foundation, and by Grant RR021 1 from the General Research Centers Program of the Division of Research Resources National Insti- tutes of Health. Requests for reprints should be addressed to Dr. A. S. Townes. Manuscript ac- cepted June 25, 1975.

l Present address: Section of Rheumatology, University of Tennessee Clinical Education Cen- ter, Baroness Erlanger Hospital, Chattanooga, Tennessee 37403.

+ Present address: Veterans Administration Hospital, 1030 Jefferson Avenue, Memphis, Tennessee 38 104.

Cell-mediated immunity was evaluated in 23 patients with systemic lupus erythematosus (SLE) prior to therapy and in 23 control subjects. The patients with SLE who had moderate to severe disease activity had significantly fewer positive delayed skin tests to streptokinase-streptodornase (SK-SD) and Candkta than the control subjects, and a higher frequency of anergy than either the control subjects or the patients with mild SLE. Significant impairment of lymphocyte transformation to all common antigens tested was found in patients with SLE as compared to both normal subjects and control subjects with disease. Phytohemagglu- tinin response was reduced in patients with SLE as compared to normal subjects but not to the control subjects with disease. Lymphocyte transformation responses to SK-SD and Candida were also significantly lower in patients with moderate to severe SLE as compared to patients with mildly active SLE. Primary Im- mune response to keyhole limpet hemocyanin (KLH) was impaired in patients with SLE as measured by lymphocyte transformation and total KLH antibody, but not 2-mercaptoethanol resistant antibody. The data indicate defective T-cell function in SLE, and suggest that the Impairment relates in part to disease activi-

ty*

Impairment of cell-mediated immunity has been postulated as an important mechanism in the pathogenesis of systemic lupus erythematosus (SLE) [ 1,2]. However, previous studies of cell-mediated immune response in patients with SLE have often shown con- flicting results [2-i I]. In these reports, concomitant skin test and in vitro responses have often been lacking, and the influence of disease activity and therapy has not always been considered. We re- port here results of skin tests and measures of in vitro lymphocyte transformation response in 23 consecutive initially untreated patients with SLE in whom a broad spectrum of clinical disease activity and severity was demonstrated. When compared to results in 23 control patients, significant impairment of cell-mediated immune response to specific antigens was found in the patients with SLE

December 1975 The American Journal of Medlclne Volume 59 769

TABLE I Clinical Findings in 23 Patients with SLE Initially Untreated with Corticosteroids

Patient

Age iv), II isease Race and DNA

Sex Act. Our. SLE Criteria Positive Prot.* Cr.* C3 CH50 ANF %B PBL

2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23

Mean total: 27.0 2.6 29.4 5.2 1.40 . . . 105 14 mild: 30.0 1.9 36.9 4.8 0.80 . . . 129

9 others: 22.0 3.4 17.9 5.9 2.20 . . . 73

14,B,F 4 1 1,6-8,10-14 17,B,F 4 4 4,7,8,11,12 52,B,F 4 99+ 2,4,7,8,13,14 22,B,F 4 4 1,3.4,6,7,8,11,12,14 18,B,F 3 4 1,2,4,7,8.11,14 26,W,F 3 2 1,4,7,8,11,12,14 14,W,F 3 11 1,2,7,8 21,B,F 3 12 7,10,11,12,14 16,B,F 3 24 1,7,8,10,14 13,B,F 2 1 7.8,11,13,14 29,W,M 2 99+ 1,5,7,8,13 23,B,F 2 5 4.7,11,12 46,B,F 2 14 7,8,12,14 40,W,M 2 18 1,2,5,7,12,14 45,B,F 2 99+ 7,8,10,11,12,14 20,W.F 2 1 7,8,12,14 28,B,F 2 4 2,4,7,8,12.14 40,B,F 2 99+ 2.3,6,7,14 lO,W,F 2 6 1,3.7,14 32,B,F 2 13 1,3,4,7,8 25,B,F 2 1 4,7.8,14 28,B,F 1 96 7,8,11,12,14 42,B.F 1 60 5,7,9,12

5.80 0.20 0.50 0.40 2.50 0.10 0.10 6.20 4.20 0.20

d.bb 0.20 0.00 5.30 0.30 0.40 0.10 0.10 1.10 0.10 . . . . . .

0.6 120 1.2 60 1.1 40 0.9 50 0.9 60 1.7 50 0.8 180 0.6 60 0.8 36 1.4 220 1.1 100 1.0 40 0.9 80 1.4 70 1.1 150 1.0 150 0.7 110 0.8 0.7 ii0 0.8 170 0.9 1.2 ‘id . . . 190

. . . 7

21 19 17 23 28 . . .

. .

ii . 40 25 81 56 23 31

ii’ . . .

44 .

31.7 35.3 19.2

1,024+

256

1,024+ 64 64

256 1,024+

256 256

1,024+ 64

4 256

16 256

64 16 64 64 64 16

256 64

96

65 44 66 94 70 24 21 19

ii 45 28 25 12 38 12

7 13

8 12

8

128 35 64 19

256 60

0.4 0.7 0.5 0.8 2.4 0.7 3.1 0.6 1.7 2.5 1.8 1.1 1.4 1.5 1.3 0.5 2.0 0.4 1.6 1.6 1.1 2.3 . . .

1.3 1.5 1.2

NOTE: Disease Act. is over-all clinical evaluation of activity coded: 1 = remission or no activity, 2 = mild, 3 = moderate and 4 = severe activity. Patients are ordered according to clinically determined categories of disease activity. Disease Dur. is duration from onset to study in months. Criteria Positive are preliminary ARA criteria positive by number as listed in reference [ 121. *Prot. is 24 hour urinary profein excretion in grams; Cr. is serum creatinine in mg/lOO ml, C3 is B,C complement (normal range 80 to 120 mg/lOO ml); CH50 is hemolytic complement (normal range 29 to 45 CH50/ml). ANF = reciprocal antinuclear factor titer; DNA %B is per cent serum binding of native DNA; PBL is absolute numbers of peripheral blood lymphocytes x 10e3.

which correlated significantly with the level of disease activity. Consideration of SLE disease activity and methods of measuring cell-mediated response help rationalize the differences found in the results of previous studies.

MATERIALS AND METHODS

Patlents. Twenty-three consecutive patients with SLE re- ceiving neither corticosteroid nor immunosuppressive therapy who were available for study and fulfilled preliminary ARA criteria for the classification of SLE [ 121 were com-

pared with 23 control subjects, 10 hospitalized patients

and 13 normal subjects. The group with SLE consisted of

17 patients with newly diagnosed SLE, four with exacerba- tion of known SLE and two with long-standing but currently asymptornatic SLE. There were two males and 21 females of ages 10 to 52 years, with a mean age of 27. Seventeen

patients with SLE were black and six were white. All were untreated with corticosteroids or immunosuppressive agents during the initial in vivo and in vitro studies. Only two patients had ever received corticosteroids in the past, and they had had none within three months prior to study. Six patients, all with mild disease activity, had been taking small doses of aspirin. Patients were classified as having mild, moderate or severe clinical disease activity based on the concensus of at least two independent evaluations of the patients by us without reference to the immune response data. Of 14 patients with SLE considered to have mild clinical activity, 12 exhibited active arthritis, serositis or skin lesions without high fever or marked constitutional symptoms, and with evidence of minimal renal involvement in only four patients. Two additional patients were clinically asymptomatic, although they had laboratory findings of chronic anemia, leukopenia or reduced serum complement which suggested mild disease activity. The remaining nine patients had moderate to severe clinical

779 December 1975 The Amerkan Journal ot Medkh Volume 59

CELL-MEDIATED IMMUNITY IN SLE-PATY ET AL.

TABLE II Measures of Cell-Mediatec! Immunity in 23 Patients with SLE Initially Untreated with Corticosteroids

Skin Tests Transformation

Patient &SD PPD Cand Con PHA SK-SD PPD Cand KLH-1 Pred KLH-2 _.__

n 13 31 7 nc

0 0 0 5

8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23

. . . 2 . . . 7 0

10 6

;; . 22 41

0 0 . . . . . . . . .

0 0 0.20 62.9 0.7 0 . . . 0.10 38.2 0.1 0 9 0.05 17.4 0.1 0 0 0.78 34.0 0.1 0 0 0.10 50.0 0.3 3 20 0.62 22.3 11.4 0 . . . 0.1 54.2 0.6 0 0 0.30 69.5 3.8

. . . . . . 0.60, 21.9 0.7 0 17 0.97 40.5 13.8 0 19 0.30 63.5 12.9 7 0 0.80 30.5 0.5 0 11 0.42 63.7 19.8

. . 0

G . 0.56 75.9 12.0 0.19 76.6 30.3

0 11 0.20 58.3 3.5 6 0 0.29 25.9 6.4 0 15 1.50 38.6 8.1 0 5 0.13 50.2 6.9

. . . . . . 0.08 26.4 0.5

. . . . . . 0.15 32.8 5.4

. . . . . 1.09 65.1 32.2

0.1 0.3 0.1 0.1 0.1 0.2 2.1 0.1 0.2 0.8 3.5 5.6 0.3

27.6 2.2 0.7 0.8 0.5 0.2 0.3 0.1 0.2 6.4

0.7 4.9 0.1 0.1 0.2 0.1 5.4 0.9 0.2 1.6 2.6 9.7 0.7 9.9 3.7 8.3 5.4 2.0 6.2 1.3 0.1 1.8 2.5

. . 1.70 & 0.10 60

0 d.ib 60 0.10 60 1.30 10 0.30 0 0.20 60 0.40 0 0.30 15 9.90 15 0.40 0 0.60 0 1.30 0 2.20 0 1.70 0 0.10 0 1.30 5 0.10 15 0.10 0 0.30 . . 1.20 0

Y.2

‘d.; 2.0 0.4 3.2 0.1 .

‘ii 12.2

2.3 2.7 3.4

21.2 12.7

G.9 0.7 6.3

‘d.4

Mean total: 9.0 0.9 8.1 0.40 45.7 7.4 2.3 3.0 1.10 18 5.7 14 mild: 13.4 1.4 10.3 0.52 47.8 10.9 3.5 4.0 1.40 3.8 8.1

9 others: 2.3 0.4 4.8 0.26 42.4 2.0 0.4 1.4 0.50 44.2 1.2

NOTE: Skin tests are millimeter maximum induration diameter at 48 hours using antigens specified. Transformation is cpm x 10e3 per lo6 lymphocytes with Con as control culture without antigen for each patient. All mitogen-antigens, except KLH-2, were used to stimulate lymphocytes at entry to study before Pred (milligramsprednisone daily) therapy was initiated, and KLH-2 is the result three weeks after KLH-1 therapy was started and onset of prednisone therapy, if

disease activity which was multisystem in character, was Lymphocyte Transformation. Lymphocyte rich mononu- often associated with fever or severe constitutional symp- clear cell suspensions were obtained from heparinized toms and for which they required hospitalization. These whole blood by Ficoll-Hypaque separation at room temper- nine patients had significantly lower mean total hemolytic ature [ 131. Following the final wash and after trypan blue complement (CH50) levels (19.2 versus 35.3), greater exclusion, cell counts were corrected to the number of DNA binding (60 versus 19), higher antinuclear antibody viable cells per cubic millimeter. The average viability was (ANF) titers (256 versus 64), and more renal disease (7/9 approximately 99 per cent. In most experiments, a versus 4114) than the 14 patients with mild disease activi- Wright’s stain of the separated mononuclear cells re- ty. None of the patients with renal involvement was uremic vealed approximately 95 per cent cells having the mor- at the time of study, and only one patient had a serum cre- phology of lymphocytes. One million viable lymphocytes atinine level over 2 mg/lOO ml. The control group consist- were then cultured in 2 cc of minimal essential medium- ed of 19 females and four males, with a mean age of 25.6 Spinner (MEM-S) containing 20 per cent heat inactivated years, 15 of whom were black and eight white. The 10 pooled normal human serum, 1 per cent glutamine, 100 hospitalized control subjects included two with gastrointes- units of penicillin, 100 @g of streptomycin and 0.075 per tinal disease, five with infectious disease, and one each cent sodium bicarbonate. The cells were cultured in 5 per with cardiomyopathy, diabetes and chronic renal disease. cent carbon dioxide at 37’C in triplicate, either without an- The latter patient was the only subject who had uremia tigen (control cultures) or with optimal amounts of anti- (blood urea nitrogen 43 mg/lOO ml, creatinine 4.5 mg/lOO gens, 100 units streptokinase-streptodornase (SK-SD), 0. I ml) and she did not exhibit impairment of skin test or in cc of dermatophytin “0” undiluted, 10 pg of tuberculin pu- vitro response. All study subjects gave their informed con- rified protein derivative (PPD), 200 pg of keyhole limpet sent prior to entering the study. Tabular presentation of hemocyanin (KLH) and 0.1 cc phytohemagglutinin as clinical and laboratory data on individual patients with SLE PHA-M or 1:lO dilution of PHA-P (Difco). Results of time is provided in Table I. and dose response curves in patients with SLE indicated

December 1975 The American Journal of Medklne Volume 59 771


TABLE III Measures of Ceil-Mediated Immunity in 10 Subjects with Non-Rheumatic Disease and 13 Normal Control Subjects

Patient Age (yr),

Skin Tests Transformation

Race and Sex SK-SD PPD Cand Con PHA SK-SD PPD Cand KLH-1 Pred KLH-2

1 20, B, F 2 42, B, M 3 34, B, F 4 28, B, F 5 21, B, F 6 35, B, F 7 14, B, F 8 18, B, F 9 16, B, F

10 18, W, M 11 23, W, F 12 27, W, F 13 30, W, F 14 25, W, F 15 25, B, F 16 24, W, F 17 24, B, F 18 27, B, F 19 21, B, F 20 25, B. F 21 31, W, M 22 42, W, M 23 18, B, F

Mean total: 25.6 Disease: 24.6 Normal: 26.3

-

0 0 8 14 0 0 9 0 0 0

26 8 . . . . . . 8 0 0 20

lY *-. 0 10 0

5 0 27 . . .

0 0 3 0

25 0 3 0

13 0 20 0 19 22 ;; .

7 6

0 17 35 11 18 36 . . . 0

17

id . 5

12 9

12 2 7

11 0

19 15 18

9

0.2 0.3 0.3 0.7 0.2 0.3 0.1 0.2 0.2 0.4 0.5 1.4 1.3 0.5 0.2 0.5 0.5 0.5 0.5 2.2 0.8 0.2 0.4

61.1 71.9 75.9 35.4 19.0 51.3 47.8 62.4 45.7

id.; 99.9 95.7 42.3 75.2 59.8 67.8 65.9 93.9 52.2 69.0 66.7 62.6

13.6 1.1 4.7 23.1 37.7 3.5 19.3 18.0 35.0 25.8 10.7 1.5 12.2 1.0 32.1 48.9 39.8 14.5

7.1 0.9 1.7 17.2 0.9 7.8

5.1 48.1 13.2 7.7 4.2 4.1

65.9 18.5 44.0 19.1 11.6 8.5 48.5 24.4 30.9 33.2 1.6 4.9 11.3 13.6 6.1

8.3 16.2 1.2 30.9 6.1 19.3 23.1 22.5 8.7 12.3 4.7 5.4 27.5 12.4 41.1 59.3 16.0 25.7 31.6 22.1 17.7 29.0 4.4 8.6

0.7 2.2 1.5 0.7 0.8 8.5 2.2 1.2 3.1 2.7

ii . . .

ii 0.2 1.2 2.8 0.7 0.1

iid 3.2

0 0

30 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

11.3 . . .

18.5 18.7 17.3 13.9 16.0 31.5 . . .

2&J 29.5 . . .

ii-l 39.0 17.2 36.5 29.7 31.5 30.7 32.3 29.2

10.5 3.7 13.0 0.5 63.4 25.2 14.6 14.8 2.2 . . . 25.6 6.4 5.3 16.8 0.3 52.3 18.0 16.2 11.8 2.4 3 18.2

13.0 2.5 10.7 0.7 71.2 30.8 13.4 17.1 2.1 0 30.3

NOTE: Subjects 1 through 10 are control subjects with disease diagnosis as follows: 1 cardiomyopathy, 2 bacterial endocarditis, 3 granulomatous colitis, 4 leptospirosis, 5 sickle cell disease with infection, 6 nephrotic syndrome, 7 diabetes, 8 aseptic meningitis, 9 gonococcal arthritis, and 10 upper gastrointestinal bleeding. Subjects 11 through 23 are normal. Abbreviations are the same as indicated for Tables I and II.

that the optimal antigen doses and time for pulsing with ra- diolabeled thymidine did not differ from this. Thus, after three days of incubation for the PHA and five days for the other antigen-stimulated and control cultures, 0.1 Ci of 14C-thymidine (New England-Nuclear, specific activity 55 mCi.‘mmol) was added to each tube. After an additional 24 hours incubation, the cultures were prepared for scintillation counting according to the method of Dutton and Page [ 141 and counted in a unilux liquid scintillation counter (Nu- clear Chicago) corrected for quenching. Results were ra- corded as the mean of the absolute value of triplicate cultures in counts per minute (cpm) X 10-3/106 lymphocytes. Preparation of Antigens. SK-SD (Lot No. 5-2201-66 obtained from the Lederle Laboratories Division, American Cyanamide Co., Pearl River, N.Y.), and Candida (dermatophytin “0” undiluted, Lot No. E467614 obtained from Hol- lister-Stier) were dialyzed in a 5 cc volume against 200 volumes of normal saline solution with three ex’changes over a 36 hour period. Dialysis tubing was prepared by leaving the tubing in 5 per cent acetic acid for 10 minutes, boiling in 1 per cent sodium bicarbonate and then rinsing thoroughly in distilled water. PPD was obtained freeze- dried without preservative (ministry of Agriculture, Fish-

eries and Food, Surrey, England). All three antigens were subsequently passed through a 0.22 p millipore filter and kept frozen at -20% until used. KLH was obtained from California Biochemicals or Pacific Biomarine in ammonium sulfate and further prepared according to the method of Weigle [ 151. It was then dialyzed for 36 hours in a 10 cc volume against 200 volumes of phosphate buffered saline solution, pH 7.0, with a total of three exchanges. Because of the size of the antigen, it couM not be sterilized by pas- sage through a millipore filter. Also, freezing of the KLH produced denaturation of the protein. Consequently, 100 units of penicillin and 100 pug/cc of streptomycin were added to the KLH after dialysis and it was stored at 4’C. Following dilution of an aliquot of the dialyzed KLH in bo- rate buffer pH 6.9, protein nitrogen content of the antigen was determined using a standard ultraviolet absorption curve at 277 rnp based on a KLH standard with the nitrogen content determined by the Kjeldahl method (courtesy of Dr. M. W. Brandriss). For immunization to KLH. patients received 100 rg subcutaneously and were tested three weeks later, at the time of the usual peak response in normal subjects [ 161. All antigens were prepared every three weeks, and the same antigens were used for both in vivo and in vitro studies [ 171. The PHA was reconstituted in 5

772 December 1975 The Amerkan Journal of Modklne Volume 59


cc sterile water every three weeks and kept at 4’C until USed.

Skin Testing. After blood for lymphocyte transformation was obtained, 10 units of SK-SD, 0.1 cc of dermatophytin “0” undiluted and 0.1 pg of PPD were administered intra- dermally. All antigens were administered in volume 0.1 cc, and skin tests were recorded as the diameter of the milli- meters of maximum induration at 48 hours. KLH Antlbody. Antibody to KLH was measured by hemagglutination using antigen prepared as described coupled to sheep erythrocytes with 0.01 per cent chromic chloride [ 181. Test serums were heated at 56’C for 30 minutes, absorbed with washed packed sheep erythrocytes, and mixed in doubling dilutions in microtiter plates with an equal volume of 0.5 per cent KLH-coupled erythrocytes. An aliquot of each serum was also treated overnight with an equal volume of 0.2 M P-mercaptoethanol followed by 0.04 M iodoacetate and dialysis as described by Deutsch and Morton [ 191. Anti-KLH hemagglutination titers of whole serum are designated total anti-KLH antibody, and M-R refers to mercaptoethanol resistant (presumably 7s) antibody. Other Studies. Erythrocyte sedimentation rates were determined using the Westergren method. Tests for lupus erythematosus cells used the method of Zinkham and Con- ley [20]. Complement was measured as @I&/3IA globulin using radial immunodiffusion plates from Hyland Laborato- ries, and as total hemolytic complement as previously described [21]. Antibody to DNA was determined by the DNA-binding method [22] using 14C-labeled DNA from thy- mine-requiring strains of Escherichia coli (Amersham/ Searle). Any binding above 20 per cent was considered abnormal. Antinuclear antibody titers were determined by a previously described method using a standard indirect immunofluorescent technic with fresh frozen rat kidney sections [ 231. Analysis. A detailed IBM preceded form was used to rec- ord data pertaining to personal, clinical, laboratory, special immunologic and therapeutic aspects of patients with SLE and control subjects. Data were processed with the aid of a computer. Statistical analyses of dichotomous variables were made with the Chi-square test with Yates’ correc- tion, when appropriate, and continuous variables were analyzed with the Student’s t-test. Absolute values of stimulated lymphocyte transformations were analyzed and presented in the tables. Two-tailed probability levels were used throughout in testing the null hypothesis.

RESULTS

Lymphocyte Transformation. Results of in vitro lymphocyte transformation to PHA and the common antigens for the patients with SLE and the control subjects are presented in Tables II and Ill. The mean and ranges for each antigen are recorded in absolute values of cpm X 1 0m3/ 1 O6 lymphocytes for antigen- or mitogen-stimulated and unstimulated (control) cultures [24]. Table IV shows that the mean lymphocyte transformation responses to all the common antigens tested (SK-SD, PPD, Candida) were significant-

December 1975 The Amsrkan Journal of Msdklne Volume 59 773

CELL-MEDIATED IMMUNITY *I SLE-PATY ET AL.

TABLE V Mean (and Range) Lymphocyte Transformation (cpm x 10-3/106 Lymphocytes) in Patients with Different SLE Activity

Antigen Moderate-Severe

Activity Mild Activity p Values*

None 0.3 (0.1-0.8) 0.5 (0.1-1.1) N.S. PHA 42.4 (17.4-69.5) 47.8 (21.9-76.6) N.S. SK-SD 2.0 (0.1-11.4) 10.9 (0.5-32.2) p < 0.05 PPD 0.4 (0.1-2.1) 3.5 (0.1-27.6) N.S. Candida 1.4 (0.1-5.4) 4.0 (0.1-9.9) p < 0.05 KLH 1.2 (0.1-3.2) 8.1 (0.3-26.9) p < 0.05

*Student's t test.

ly lower in the patients with SLE when compared with those in normal subjects (p <O.OOl) and in control subjects with disease (SK-SD p <0.02, others p <O.OOl). Although the mean response of patients with SLE to PHA was also significantly lower than that of normal subjects (p <O.OOl), the difference from the control group with disease was not statistically significant. The latter undoubtedly relates to a decreased mean response to PHA in control subjects with disease as compared with normal subjects (p <0.05). Mean values of unstimulated control cultures were also lower in the patients with SLE (p <0.02) and control subjects with disease (p <0.05) than in

normal subjects. The relationship of activity of SLE to lymphocyte

transformation response was also evaluated (Table

TITERS TOTAL ANTIBODY M-R ANTIBODY

. .

. .

32 -c- . . . . 1 ,,I,,

SLE CONTROL SLE CONTROL

P ( ,001 N.S.

Figure 1. Indivklual and mean (-4 KLH total and mercaptoethanol resistant (M-R) antibody titers (reciprocal) in patients with SLE and control subjects. P value is significance of difference in geometric mean values by Stu- dent’s t test. N.S. = not significant.

V). The mean lymphocyte transformation responses to SK-SD, Candida and KLH were significantly lower (p KO.05) in patients with SLE classified as having moderate to severe disease activity than in those with mild activity. Response to PPD was also less in the patients with moderate-severe activity (mean 0.4; range 0.1 to 2.1) versus the patients with mildly active SLE (mean 3.5; range 0.1 to 27.6). but this difference is not significant (p <O.l). Considering patients with very low lymphocyte transformation responses to each of the common antigens (less than l.O), there were significantly more such patients with moderate to severe disease activity as compared to those with mild disease activity (SK-SD p <O.Ol; PPD and Candida p <0.05). Interestingly, response to PHA did not differ appreciably in relation to disease activity in the patients with SLE. Response to Immunization with KLH. A pro- nounced impairment of the immune response of patients with SLE was observed three weeks after primary immunization with KLH. This was evident both in the lymphocyte transformation response to KLH (Table II) and in the level of serum antibody (Figure 1). It should be noted that 10 of the 18 patients immunized with KLH were started on corticosteroid therapy (5 to 60 mg/day) in the interim between immunization and evaluation of response. However, with respect to lymphocyte transformation, there was no significant difference between the treated and untreated groups with SLE (mean 4.8 and 6.1, respectively). Further- more, the lymphocyte transformation response in both the treated and untreated patients with SLE was significantly lower than in the control subjects (p <O.OOl).

There was also a decreased response to KLH in control subjects with disease as compared to normal subjects, but this was a different order of magnitude than in SLE. Only four patients with SLE had a transformation response as high as the lowest in the group with disease; all these patients had mild SLE.

The mean total antibody response to KLH as measured by hemagglutination was also significantly decreased in patients with SLE (p <O.OOl) compared

with control subjects as shown in Figure 1. In contrast, titers of mercaptoethanol resistant (M-R) antibodies, although less, were not statistically different from those in control subjects, and considerable overlap occurred between the two groups. KLH antibody responses were not significantly differert in those who received prednisone and those who did not, and there was no correlation of titer with prednisone dose. Patients with SLE and moderate to severe disease activity had no significant difference in the mean total anti-KLH titers when compared to those with mild disease activity (l/64 versus l/32, respec-

774 December 1975 The Amarkan Journal of Medklm Volume 59


TABLE VI Proportion Positive Skin Tests at 5+ and IO+ mm of Maximum Induration (and Mean) in Patients with SLE and Control Subjects

_~

Antigen mm

SK-SD SK-SD Candida Candida PPD PPD

fW fW -.-

5+ 10+

5+ to+

5+ 10+

5+ 10+

(1) All Patients

with SLE

(2) Moderate-

Severe (3)

Mild

(4) Control Subjects

Significance”

1 vs. 4 2 vs. 4 2 vs. 3 3 vs. 4

12/15 9115

i;E (2.3)

;;; (4.8)

“0;; (0.4)

316 l/6

;;; (10.5) 1;;;; (10.5)

$ (10.3) ;;;;; (13.0)

;;; (1.4) :;;; (3.7)

g/g 19121

819 17/21

N.S. N.S. N.S. p < 0.05 N.S. p < 0.025 N.S. p < 0.05 N.S. N.S. N.S. N.S. N.S. p < 0.025 N.S. p < 0.005

* Chi square test.

tively). However, mean M-R anti-KLH titers were higher in the group with moderate-severe disease than in those with mild SLE (l/32 versus l/8, p <0.02). Skin Tests. The delayed skin test responses to PPD, Candida and SK-SD are shown for individual patients and control subjects in Tables II and Ill and summarized in Table VI. Patients with SLE had less mean induration and a smaller proportion reacting to each antigen as compared with control subjects, although the differences are not statistically significant when all patients with SLE are considered as a group. How- ever, if the patients with SLE are separated into those with moderate-severe and mild disease activity, significant differences are observed with decreased reactivity in the groups with moderate-severe activity compared with control subjects using SK-SD and Candida antigens. None of the eight patients with moderate-severe activity tested reacted to PPD as compared with five of 19 control subjects. Regardless of whether a positive skin test is considered to be 5 mm or 10 mm of induration, the proportion of patients with SLE and moderate to severe disease activity who demonstrated anergy (i.e., failed to react positively to any of the antigens tested) is significantly greater than that of the control subjects or patients with mildly active SLE (p <0.025). The prev- alence of anergy in the patients with mild SLE was low and similar to that of the control subjects. Signifi- cant correlation of skin test responses (mm induration) with corresponding lymphocyte transformation (in cpm/106 lymphocytes) was found in control subjects for each of the antigens tested (p <O.Ol). However, in patients with SLE correlation of skin test results and lymphocyte transformation was incom- plete, being significant only for Candida antigen (r = 0.681, p <O.Ol). There was no correlation of skin test and in vitro transformation response to PPD (r = 0.13) in confirmation of previous results [ 51.

N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.

p < 0.025 N.S. p < 0.01 N.S.

COMMENTS

This study demonstrates significant impairment of the mean in vitro lymphocyte transformation response to common antigens in a group of patients with untreated SLE as compared to control subjects. Results of delayed skin tests to these same antigens also showed a trend toward decreased size of reaction and a smaller proportion with positive reactions in the patients with SLE. Further, the degree of impairment of these responses was found to correlate significantly with the clinical assessment of disease activity, being greatest in the patients with highly active disease.

Previous reports of cell-mediated immune responses in patients with SLE as summarized in Table VII have indicated both normal and decreased reactions [2-l 11. Four prior publications have described results of lymphocyte transformation to specific antigens in SLE. Two are in agreement with our findings [ 7,111, and in the other two [5, lo], no significant difference was reported between the patients with SLE and control subjects. The discrepancy between the latter results and our study may be explained in part by differences in methods. For example, in these reports [5, lo] ratios of thymidine uptake in antigen stimulated to unstimulated cultures (stimulation index) were reported rather than absolute counts per culture. Either high base line counts which may occur [24] with fetal calf serum in the medium [5] or low counts in unstimulated cultures as found in patients with SLE [lo] may alter the sensitivity of the assay, so that the use of absolute counts seems preferable for comparisons over the wide range of observed response [ 251.

The relationship of the level of disease activity and therapy to cell-mediated immune responses in patients with SLE has not been uniformly considered in previous reports. In this study of untreated patients

December 1975 The American Journal ol Medkine Vohme 59 775

TABL

E VI

I S

um

mar

y o

f 11

Stu

die

s o

f C

ell-

Med

iate

d

Imm

un

ity

in P

atie

nts

wit

h

SL

E V

ersu

s C

on

tro

l S

ub

ject

s G

Patie

nts

with

SL

E

Per

cent

N

o.

SLE

Vers

us C

ontr

ol

Gro

up

Diffe

renc

es

with

Sk

in T

ests

-Con

trol

(in

Par

enth

eses

) an

d Ly

mph

ocyt

e Tr

ansf

orm

atio

n b

Refe

renc

e To

tal

No.

Ac

tive

Trea

ted

Subj

ects

PH

A PP

D SK

-SD

Cand

Tr

ich

Hist

o O

ther

%

Ane

rgy

Com

men

ts

5 iz

Blo

ck

et a

l. [3

] 20

55

60

11

2*

(0.0

05)

(N-S

.)

(N.S

.)

(N.S

.)

(N.S

.)

(0)

On

ly

sig

nif

ican

t d

iffe

r-

ence

fo

un

d

in s

kin

Z

test

s w

as w

ith

P

PD

2 t

Ab

e,

Ho

mm

a [8

] 20

35

50

20

(0

.01)

(0

.01)

(3

5)

Bo

th

anti

gen

s sh

ow

ed

r

(DN

CB

) si

gn

ific

antl

y d

ecre

ased

re

spo

nse

g

2 H

orw

itz

[2]

14

(?)

21

18*

N.S

. (0

.001

) (0

.025

) (0

.01)

(8

6)

Su

gg

este

d

that

sk

in

test

re

acti

vity

in

S

LE

co

r-

1

rela

ted

w

ith

ac

tivi

ty

?

_ __

_~

Bit

ter

et a

l. 10

10

0 0

10*

No

p

atie

nt

wit

h

SL

E

reac

ted

(M

um

ps)

(1

00)

O/1

0 S

LE

vs

. 9/

10

con

- (a

bst

ract

) [4

] (C

occ

id.)

tr

ol

sub

ject

s re

acte

d

0.00

1

0.00

1 to

an

y te

st

ML

C

Go

ldm

an

et a

l. [6

] 24

Hah

n

et a

l. [5

]

Su

ciu

-Fo

ca

et a

l. [9

]

Ro

sen

thal

; F

ran

klin

[7

] 12

39

40

Sen

yk

et a

l. [l

o]

16

(?)

50

24

(N.S

.)

(N.S

.)

(N.S

.)

(N-S

.)

(13)

A

ner

gic

p

atie

nts

w

ere

acu

tely

ill

100

10

30

(0.0

05)

N.S

.

(0.0

05)

Co

rrel

atio

n

of

PP

D

skin

te

sts

wit

h

tran

sfo

r-

mat

ion

in

no

rmal

s b

ut

no

t in

SL

E

(?)

52.5

74

N

o

dif

fere

nce

in

PH

A

resp

on

se

wh

en

SL

E

lym

ph

ocy

tes

wer

e 0.

001

0.00

1 cu

ltu

red

in

ho

mo

- M

LC

lo

go

us

or

auto

log

ou

s se

rum

50

33

50

50

17t

N.S

. (N

o

pat

ien

t w

ith

ac

tive

S

LE

re

acte

d)

(Mu

mp

s)

(50)

A

ner

gy

in 6

act

ive

un

- A

ll an

tig

ens

gro

up

ed

p <

0.

001

(Als

o)

trea

ted

p

atie

nts

; im

- p

rove

men

t in

ski

n

and

in

vit

ro

test

s w

ith

A

ll an

tig

ens

gro

up

ed

p <

0.

001

trea

tmen

t

12

N.S

. N

.S.

N.S

. N

.S.

<0.0

5 O

nly

h

alf

of

SL

E

pa-

N

ucl

ear

tien

ts

con

sid

ered

ac

- ti

ve

and

w

ere

also

o

n

ster

oid

th

erap

y

co d

co 4

CELL-MEDIATED IMMUNITY IN SLE---PATY F-T AL

with SLE and variable levels of disease activity, im-

pairment of skin test reactivity was not found to be

statistically significant when our entire group of pa-

tients with SLE was compared with control subjects.

However, those patients with moderate or severe ac-

tivity had significantly less reactivity to Candida and

SK-SD antigens than control subjects, and had a sig-

nificantly higher frequency of anergy than either con-

trol subjects or patients with SLE and mild disease

activity (Table VI).

Careful analysis of previous reports (Table VII)

tends to confirm these results since delayed skin test

and lymphocyte transformation hyporeactivity was

found predominately in series of patients with SLE

and active disease [4,7,1 l] in contrast to findings in

groups with less active disease on therapy

[3,6,8,10]. We [26] and others [7] have also ob-

served an increase in the level of transformation re-

sponse in patients with SLE after corticosteroid ther-

apy. In contrast to findings in this study with lymphocyte

transformation response to specific antigens (Candi-

da and SK-SD) in SLE, response to the nonspecific

mitogen PHA showed no correlation with the degree

of SLE activity, and no significant difference from

that in control subjects with disease. It is possible

that the use here of a single optimal stimulatory dose

of PHA may have obscured potential differences in

the transformation response of SLE lymphocytes to

PHA as has been recently reported using smaller

doses of PHA [ 271. However, no correlation of PHA

response to SLE disease activity was observed using

smaller amounts of PHA when SLE lymphocytes

were cultured in normal plasma [ 111.

Impairment of cell-mediated immune response to

specific antigens in patients with SLE is also corrobo-

rated by the marked decrease in lymphocyte trans-

formation response observed after primary immuni-

zation with KLH in patients with SLE as compared to

that in control subjects. Horwitz [2] has previously

reported impairment of transformation response to

KLH in a patient with SLE in whom serum antibody

response was normal. In this study, total hemaggluti-

nation titers to KLH were decreased in patients with

SLE as compared to that in control subjects, al-

though mercaptoethanol resistant antibodies were

similar. These results are like those of Baum and Ziff

[28] who showed impaired 19s but normal 7s anti-

body response in SLE following immunization with

Brucella antigen. It is also important to note that pa-

tients with SLE and moderate to severe disease activity demonstrated a significantly higher 7s response

than those with mild activity. This increase in 7s anti-

body response observed only in patients with more active SLE suggests that there is an imbalance in

December 1975 The American Journal ol Medicine Volume 59 777

CELL-MEDIATED I~~~MUNITY IN SLE-PATY ET AL.

regulation of this antibody response which also cor- of anergic patients with SLE and active disease by relates with disease activity. Horwitz and Cousar [ 111.

Alteration of cell-mediated immune responses has been reported in a variety of seemingly unrelated disease states [29-331. In SLE, lymphocytotoxins have been described [34] with relative T-cell specificity [35]. Reduction in total circulating lymphocytes and a decrease in the per cent of cells with T-cell markers have also been reported in SLE [36,37]. Thus, selective elimination of T cells or certain T-cell sub- sets from the peripheral blood of patients with SLE as a result of lymphocytotoxins could contribute to the findings of impaired measures of cell-mediated immunity and altered immune response.

The methods employed in this study minimize or exclude an in vitro effect of the cold-reactive IgM lymphocyte antibody [38] which has been reported to interfere with response of SLE lymphocytes in mixed lymphocyte cultures [39,40]. Lymphocytes were routinely isolated and washed at room tempera- ture and cultured in normal serum, not in serum from patients with SLE. In addition, in two experiments, there was no change in depressed lymphocyte response after incubation of lymphocytes in media containing 20 per cent normal serum for 24 hours at 37OC, followed by washing, in which shedding of the inhibitory factors was previously reported [39]. How- ever, we have found in preliminary experiments, as did Horwitz [ 111, Suciu-Foca et al. [9] and Malave et al. [27], that some serum specimens from patients with SLE used in the culture media suppressed transformation values of SLE and normal lymphocytes without cytotoxicity. Thus, a serum factor in SLE re- sulting in irreversible functional alteration of peripheral blood lymphocytes could explain the diminished responses observed. An IgG serum factor with such an effect has been recently demonstrated in the serum

Talal [l] and Talal and Steinberg [41] have pro- posed that immunologic imbalance with depressed cellular immunity and decreased activity of T-sup- pressor cells which modulate B-cell response and antibody production are important in the pathogenesis of autoimmune disease in New Zealand mice, and possibly also in human SLE. The data reported here are consistent with this hypothesis in that impairment of cell-mediated responses and an increased production of 7s antibody in response to primary immunization were found in patients with SLE. Since both of these abnormalities were demonstrated to correlate significantly with disease activity, our findings suggest that the abnormal immune responses observed are more likely to be a secondary effect of the disease rather than a primary event in pathogenesis of SLE. This problem however, is extremely complex [41] and deserves further study. In either event, this impairment of T-cell function may be a factor in the perpetuation of the disease as well as contributory to the increased susceptibility of some patients with SLE to intercurrent infection [42] in which cell-mediated immunity is important in host defense.

ACKNOWLEDGMENT

We thank Mrs. Astghik K. Martin for her valuable laboratory assistance; Mr. Seth L. Feigenbaum, Biomet- ric Computer Center, University of Tennessee Center for the Health Sciences, for valuable assistance in the computer processing of the data; Dr. Carolyn F.

Blackwell, Director of Clinical Chemistry, Baptist Me- morial Hospital, Memphis, for DNA binding studies: and Dr. Robert W. Chandler, Associate Professor of Immunology in Medicine, University of Tennessee, for fluorescent antinuclear antibody studies.

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December 1975 The Am&can Journal of Medkine Vohme 59 779

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