Natural killing in systemic lupus erythematosus: Inhibitory effects of serum

CLINICAL

IMMUNOLOGY

Natural

AND

17, 219-226

IMMUNOPATHOLOGY

(1980)

Killing in Systemic Lupus Erythematosus: Inhibitory Effects of Serum S.L.

AND E.S.

SILVERMAN,'

CATHCART

The Arthritis and Connective Tissue Disease Section, Department of Medicine and Thorndike Memorial Laboratory, Boston City Hospital, and the Evans Memorial Department of Clinical Research. and Department of Medicine, University Hospital Boston University Medical Center, Boston, Massachusetts 02118 Received

October

30, 1979

The natural killing (NK) of a tumor cell line, K-562, was studied using lymphocytes of patients with systemic lupus erythematosus (SLE) and normal subjects. NK varied greatly in both populations but was significantly decreased in those patients with untreated SLE 17 ? 5%, versus those patients with treated SLE, 44 ? 15% and controls, 44 + 13%. The NK of SLE cells increased following overnight incubation, suggesting the role of membrane-bound factors. Serum from patients with untreated SLE markedly inhibited NK (45 2 17%) in contrast to control sera (2 2 3%). A significant correlation was found between the NK of a given patient and the inhibition by autologous sera of the NK of mononuclear cells from normal subjects.

In spontaneous cell-mediated cytotoxicity or natural killing (NK), target cells are lysed by unsensitized effector cells in the absence of antibody (1). Studies in mice suggest that NK may be of importance in tumor surveillance (2), early host response to bacterial and viral infection (due to interferon activation) (3), and in bone marrow graft rejection (4). The effector cells responsible for NK in man have not been definitely characterized, but it is suggested they belong to a T-cell subpopulation bearing low-avidity receptors for sheep erythrocytes (5). Patients with active systemic lupus erythematosus show significant reduction in antibody-dependent cellular cytotoxicity (ADCC) and mitogen-induced cytotoxicity (6,7). In this study we demonstrate that patients with untreated SLE who are clinically active also show significantly decreased NK. In addition, serum from patients with untreated SLE was found to inhibit the NK of normal controls. MATERIALS

AND METHODS

Patients

Fourteen patients with SLE were studied. All patients met four or more criteria for SLE of the American Rheumatism Association and were fully evaluated for disease activity by standard clinical and laboratory parameters. Six of the 14 patients had untreated disease and were clinically active as defined by the presence of acute signs and symptoms-e.g., pleuritis, pericarditis, nephritis, arthritis, and cerebritis, plus two or more of the following: Westergren sedimentation rate I Current Pennsylvania

address: 19104

Arthritis

Section,

Hospital

of the University

of Pennsylvania,

Philadelphia,

219 0090-1229/80/100219-08$01.00/O Copyright AU rights

@ 1980 by Academic Fess, Inc. of reproduction in any form reserved.

220

SILVERMAN

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CATHCART

greater than 20 mm, serum C3 less than 55 mg/lOO ml (normal range 55- llO), antinuclear antibody titer more than l/32 or DNA binding more than 25% using the Farr technique (8). Eight of the 14 had treated SLE which was inactive by the above criteria. The treatment consisted of salicylates or prednisone in a daily dose of 20 mg or less. Age- and sex-matched healthy volunteers served as control patients. Twenty-four SLE sera were also studied. The sera were frozen at -70°C until used. All sera were heat inactivated at 56°C for 30 min prior to testing. All blood was drawn at a specified morning hour. Cytotoxicity

Assays

Heparinized peripheral blood mononuclear cells were separated on a Ficoll-Hypaque gradient (9). The mononuclear cells were then resuspended in RPM1 with 25 mm Hepes (Microbiological Associates, Bethesda, Md.) supplemented with L-glutamine and antibiotics. The human myeloid cell line, K-562, was used in the NK assay. Cells were obtained from Dr. Grace Cannon of Litton Bionetics and Dr. Michael Williams of the Sydney Farber Cancer Institute. The cell line was maintained in suspension culture and subcultured three times weekly. The NK assay was modified from that used by West (1). K-562 cells were decanted and washed once in HBSS: Cells, 2 x 106, were resuspended in HBSS and labeled with 100 &i chromium-51 (New England Nuclear, Boston, Mass.) by incubation for 60 min. The cells were washed three times and resuspended in media at lo5 cells/ml. Effector cells were resuspended at 1.2 x 10Vml. Effector cells, 0.1 ml, and target cells, 0.1 ml, were incubated in microtiter plates (Falcon 304) for 4 hr at 37°C in 5% COZ and harvested using a Titertek harvester (Flow Lab). Assays were run in triplicate or quadruplicate. Data were calculated by the following formula: (E-S/MAX-S) x 100 = 51Cr specific release, where E = 51Cr released from K-562 target cells plus effector cells; S = 51Cr released spontaneously from target cells; and MAX = 51Cr released after addition of 5 vol of distilled water. Serum Inhibition

Studies

The method used is a modification of that of Fye (10). Two tenths percent of bovine serum albumin (Miles Laboratories, Elkhart, Ind.) was used in the tissue culture media. Mononuclear cells were adjusted to 5 x lOVm1 in media, incubated for 1 hr at 37°C with an equal volume of heat-decomplemented serum, and washed twice. After dilution to the appropriate concentrations, the NK assay was performed as above. Mononuclear cells were incubated with autologous normal serum or media as controls. Percentage inhibition was determined by the following formula: Percentage where E = 51Cr specific 51Cr specific release of In some experiments from Dr. K. Atul Bhan

specific inhibition

= E-F/E

x

100,

release of mononuclear cells incubated in media and F = mononuclear cells incubated in serum. varying concentrations of heat-aggregated IgG obtained of the Massachusetts General Hospital were also added.

NATURAL

KILLING

IN

221

SLE

Fluorescent-Binding Studies K-562 cells, 0.1 ml, resuspended at 2 x 107/ml in phosphate-buffered saline, pH 7.2, with 0.2% bovine serum albumin (PBS-BSA) were incubated with 0.2 ml of undiluted test serum at room temperature for 30 min, washed, and resuspended to 0.1 ml in PBS-BSA. The cells are then incubated for 30 min with 0.1 ml of fluorescent-labeled polyvalent anti-human immunoglobulin (Cappel Labs.), washed, and the pellet resuspended in PBS-glycerin. The pellet was placed on a slide and read on a Leitz fluorescent microscope. Statistical Analysis All values were expressed as the mean -+ standard deviation of triplicate or quadruplicate values. (Student’s t test for nonpaired values was used as the test for statistical significance.) RESULTS

Direct NK of Patients

with SLE and Controls

Preliminary experiments established that an effector to target cell ratio of 12: 1 was near the maximal plateau for a 4-hr NK assay. The mean NK of 15 controls was variable 44 + 13% (Table l), although results for a single subject were reproducible on successive days (e.g., control No. 5, 48 + 6%). Six patients with TABLE 1 NK IN SLE Untreated Direct Patient

No.

NKn

SLE

Treated

Serum

(%I

inhibition* (%)

U-l u-2 u-3 u-4 u-5 U-6 u-7 U-8 u-9 u-10 u-11

10 14 25 16 18 22 ND’ ND ND ND ND

54 68 30 37 59 40 25 75 30 47 34

Mean

17.5 + 5.4

45.4 ? 16.6

Patient

43.9 k 13.3% (n = 15)

Direct NK” (%)

T-l T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10 T-11 T-12 T-13 Mean

Controls Mean

No.

38 54 21 48 51 33 70 35 ND ND ND ND ND 43.8

L 15.2

SLE Serum

inhibition* (%) 12 29 29 5 3 19 0 21 3 10 19 0 14

12.6 k 10.2

2.1 k 2.6% (n = 25)

’ 0.1 of mononuclear cells at 1.2 K-562 cells. The release of Wr into b Control mononuclear cells at 5 serum, washed, and the NK assay c ND, not determined.

x 10’Vml are incubated for 4 hr with 0.1 ml of 1OVml jlCr-labeled medium was measured as described under Materials and Methods. x 10Vml in 0.2% BSA are incubated with an equal volume of test is performed as above.

222

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untreated SLE had significantly decreased NK as compared to the controls with a mean NK of 18 + 5%, P < 0.01 (Table 1). The mean NK of eight patients with treated disease, 44 r 15%, did not differ significantly from the mean of the 15 controls, 44 f 13%. To determine whether the decreased NK seen in patients with untreated SLE was due to a decreased number of cells, dose-response curves were performed. Patients with untreated SLE showed reduced NK activity at all ratios studied in contrast to controls (Table 2). Overnight Incubation The NK of peripheral blood mononuclear cells from two patients with active untreated SLE and two controls was studied: (a) as soon as possible after venupuncture and (b) 24 hr later after overnight incubation at 37°C. The NK of the SLE patients showed a marked increase, while the NK of the control patients decreased or remained the same after overnight incubation (Table 3). The NK of SLE mononuclear cells incubated overnight decreased to levels seen with fresh cells after addition of autologous serum (Table 3). Serum Inhibition Studies To confirm the presence of inhibitory factors in the serum or patients with active SLE, lymphocytes obtained from a panel of normal subjects were incubated tiith sera of 11 patients with active SLE, 13 patients with inactive disease, and 25 controls from normal healthy volunteers or media (Table 1). Incubation of normal lymphocytes with control sera always resulted in less than 10% inhibition of NK activity with a mean of 2 + 3%. The percentage inhibition of normal NK by 11 active SLE sera ranged from 25 to 75% with a mean of 45 2 17%. This was significantly greater (P < 0.01) than the mean for 13 clinically inactive patients (13 ? 10%). In those SLE patients in whom direct NK and serum inhibition was studied, direct NK was inversely proportional to the serum inhibition (r = 0.83). This inhibition by serum was not due to binding to target cells. No binding of SLE TABLE

2

EFFECT OF EFFECTOR TO TARGET CELL RATIOS ON NK OF CONTROLS j’Cr

release

AND SLE

PATIENTS”

(%)

2.5:1

5:l

10: 1

2O:l

40: 1

Control 1 2

44 26

52 35

55 38

56 46

44 46

SLE 1 2 3

13 ND 6

20 9 8

26 15 10

28 22 11

24 20 ND

LI 0.1 ml of FicollHypaque-purified mononuclear cells and 0.1 ml of lO~/ml chromium-5 1 from controls or patients with SLE labeled K-562 cells were incubated for 4 hr at the above effector-target cell ratios. The percentage Chromium-51 release into the medium was measured as described under Materials and Methods. ND, not determined.

NATURAL

KILLING TABLE

EFFECT

OF OVERNIGHT

IN

223

SLE

3

INCUBATION SLE

No.

ON NK 1

IN SLE” SLE

(%o) A.

NK

B. NK C. NK

of Fresh

Cells”

after overnight incubation” after overnight incubation and subsequent addition of autologous serumd

No.

2

(%I

Control (%I

27 5 3 40 k 4

10 k 2 33 k 6

38 k 5 33 * 3

25 r 3

15 * 3

30 f 2

a All assays were performed at the same time. Numbers represent the mean of triplicate determinations and are corrected for mean spontaneous release. b The NK of fresh cells was determined as described under Materials and Methods. P 2 x 10Vml mononuclear cells were incubated for 18 hr at 37”C, resuspended at 1.2 x lOVml, and the NK was determined. d Cells were incubated as above, resuspended at 5 x lOVml, and incubated with an equal volume of heat-inactivated autologous sera. After washing the NK was determined.

sera to K-562 cells was seen by indirect immunofluorescence using a fluorescinated goat polyvaient antihuman immunoglobulin (Cappel Labs.). NK Inhibition

by Heat-Aggregated

ZgG

Various amounts of heat-aggregated IgG (30-500 &ml) or media were added to effector cells as above. There was no inhibition until 125 pg/ml. The inhibition then increased linearly until 500 pg/ml (see Fig. 1). DISCUSSION

The effector cells capable of spontaneously lysing K-562 in the NK assay appear to be Fc receptor-bearing cells of the T-cell lymphocyte subpopulation. The

JIM HEAT-AGGREGATED

IgG/ml

FIG. 1. Inhibition of NK by heat-aggregated IgG. 5 x 10Yml control mononuclear bated in the presence of varying amounts of heat-aggregated IgG for 1 hr and washed assay. Numbers represent the mean of triplicate determinations.

cells were incutwice prior to NK

224

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presence of Fc receptors on NK effector cells has been shown by the removal of NK cell activity by adsorption of effector cells onto IgG-sensitized monolayers (11, 12). West ef al. (1978) have found that NK cell activity is associated with a subpopulation of T cells bearing low-avidity E-RFC receptors, which could only be isolated under optimal experimental conditions (5). The non-E-RFC were also capable of NK activity. This activity may represent dissociated low-avidity E-RFC since T cells are present in non-E-RFC fractions as judged by anti-T-cell antisera (11). T cells with Fc receptors for IgG, i.e., T, cells, are capable of NK against K-562 (13). Adherent monocytes and B lymphocytes appear not to be directly involved in NK cell lysis (1, 11). We have studied the NK of peripheral blood mononuclear cells of patients with SLE against K-562 cells. Patients with untreated SLE had significantly reduced NK as compared to treated SLE patients and controls. Treatment with predinosine cannot explain the higher values seen. In rheumatoid arthritis Burmester has shown no effect of steroids in low dose on NK (16). The increase in NK following overnight incubation suggests the presence of membrane-bound serum factors, such as immune complexes and/or lymphocytoxic antibodies, which are found in SLE. The subsequent decrease in NK following addition of autologous sera (Fig. 2), as well as the finding that sera from patients with active SLE markedly inhibited the NK of normal mononuclear cells, support the conclusion that serum factors in SLE patients reduce NK. Heat-aggregated IgG also inhibited NK in a linear fashion, mimicking possible effects of immune complexes. Using soluble rabbit anti-ovalbumin immune complexes, Bolhuir (11) has recently shown a partial (40%) reduction in human NK. Finally, we cannot exclude an absolute reduction in NK in patients with active SLE. The number of T cells and in particular To cells is decreased in patients with

50”60 . 1 . 50 i z zI

40

.

.

30

0.

0

0

20

0 0

10 1

Oo 0

I 10

I 20

I 30

I 40 DIRECT

I 50

I 60

7b

I 80

NK

FIG. 2. Correlation of Direct NK as measured by percentage specitic Wr release and percentage inhibition by autologous serum of the NK of control mononuclear cells. (0) Six patients with untreated active SLE. (0) Eight patients with treated SLE. 0.1 ml of 1.2 x 10Vml Ficoll-Hypaque-purified mononuclear cells were incubated for 4 hr with 0.1 ml of lOVm1 Wr-labelled K-562 cells and the percentage Wr release into the medium was measured as described under Materials and Methods.

NATURAL

KILLING

225

IN SLE

active SLE. This abnormality is most prominent in patients with severe hypocomplementemia (14) and increased levels of immune complexes (15). A reduction in the number of TG cells in SLE is probably relative rather than an absolute, since IgG Fc receptor sites necessary for enumeration appear to be blocked by immune complexes (5). It is also possible that IgG Fc receptors have been lost after interaction with immune complexes in vivo. This has been demonstrated under in vitro conditions with the TG cells of healthy subjects (17) and needs to be tested in patients with SLE. APPENDIX: Patient No.

Direct NK” (%I

C-l c-2 c-3 c-4 c-5 C-6 c-7 C-8 c-9 c-10 c-11 c-12 c-13

37 38 23 46 48 57 50 33 40 31 80 51 42

NK IN CONTROLS Direct NK” (%o)

Serum inhibitor? (%) 1 8 9 0 0 0 3 0 0 5 0 1 0

Serum inhibition* (%)

c-14 c-15 C-16 c-17 C-18 c-19 c-20 c-21 c-22 C-23 C-24 C-25

46 37 ND’ ND ND ND ND ND ND ND ND ND

1 0 2 4 6 3 0 0 2 1 4 0

Mean

43.9 2 13.3

2.1 + 2.6

n 0.1 ml of Ficoll-Hypaque mononuclear cells at 1.2 x 106/ml were incubated for 4 hr with 0.1 ml of 105/mI chromium-51-labeled K-562 and the release of chromium-51 into the medium measured as described under Materials and Methods. * Control mononuclear cells at 5 x 106/ml in 0.2% BSA are incubated with an equal volume of test serum, washed, and the NK assay is performed as above. I’ ND, not determined.

ACKNOWLEDGMENTS This investigation was Metabolism and Digestive Resources General Clinical Foundation and the Lupus

supported by grants from the USPHS, National Institute of Arthritis, Diseases (AM 26451, AM 07014, and AM 04599); Division of Research Research Branch, NIH (RR 533); the Massachusetts Chapter of Arthritis Erythematosus Foundation.

REFERENCES 1. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. C., and Herberman, R. B., J. Immunol. 118, 355, 1977. 2. Haller, O., Hansson, M., Kiessling, R., and Wigzell, H. Nature (London) 270, 609, 1977. 3. Gidlund, M., Orn, A., Wigzell, H., Senik, A., and Gresser, I., Nature (London) 273,759, 1978. 4. Kiessling, R., Klein, E., Pross, H., and Wigzell, H., Eur. .I. Immunol. 5, 117, 1975. 5. West, W., Boozer, R. D., and Herberman, R. B., J. Immunol. 120, 90, 1978. 6. Scheinberg, M A., and Cathcart, E. S., Clin. Exp. Immunol. 24, 317, 1976. 7. Namba, H. and Sarurami, T., “Proceedings of XIV International Congress of Rheumatology,” San Francisco, Calif. (Abst. 315), 1977. 8. Pincus, T., Schur, P. H., Ror, J. A., Decker, J. L., and Talal, N., N. Engl. J. Med. 281,701, 1969.

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9. Boyum, A., &and. J. Clin. Lab. Invest. 21 (suppl. 97), 77, 1%8. 10. Fye, K. H., Becker, M. J., Theofilopoulos, A. N., Moutsopolos, M., Feldman, J. C., and Talal, N., Amer. J. Med. 62, 783, 1977. 11. Bolhuis, R. L. H., Schuit, H. R. E., Nooyen, A. M., and Ronteltap, C. P. M., Eur. 1. Zmmunol. 8, 731, 1978. 12. Kay, H. D., Bonnard, G. D., West, W. H., and Heberman, R. B., J. Zmmunol. 118,2058, 1977. 13. Gupta, S., Femandes, G., Nair, M., and Good, R., Proc. Nat. Acad. Sci. USA 75, 5137, 1978. 14. Hamilton, M. E., and Winfield, J. B., Arth. Rheum. 22, 1, 1979. 15. DeHoratius, R. J., Santoli, D., and Pincus, T., Arth. Rheum. 21, 553, 1978. 16. Burmester, G. R., Kalden, J. R., Peter, H. H., Schedel, I., Beck, P., and Wittenborg, A., &and. J. Zmmunol.

7, 405,

17. Moretta, L., Mingori,

1978.

M. C., Romanzi, C. A., Nature

(London)

272,

618, 1978.