Studies of rheumatoid synovial fluid lymphocytes

CLINICALIMMUNOLOGY

Studies

39, 159-167 (19x6)

ANDIMMUNOPATHOLOGY

of Rheumatoid

III. Phenotypic

Synovial

Fluid Lymphocytes

and Functional Analysis of Natural Killer-like Cells’ RICHARD M. SILVER

Synovial fluid and peripheral blood lymphocytes from patients with rheumatoid arthritis have been compared in terms of phenotypic and functional expression of natural killer (NK) activity. Following pretreatment with the monoclonal antibodies anti-HNK-I (anti-Leu-7) or anti-Leu-I lb and complement. NK activity against K563 cells was assessed in 4-hr chromium release assays. Rheumatoid peripheral blood lymphocytes resembled those of normal individuals. in that complement-mediated lysis of Leu-I lb. cells virtually eliminated all NK activity. Complement-mediated lysis of Leu-7’ cells resulted in only a modest reduction in NK activity. Rheumatoid synovial fluid lymphocytes differed from autologous peripheral blood lymphocytes: the overall cytotoxicity was generally less and only a fraction of the NK-like activity appeared to be mediated by Leu- I lb cells. This apparent lack of Leu-I lb expression by rheumatoid synovial fluid NK-like cells could not be accounted for by blockage or alteration of the Leu-I lb antigen by factors present in synovial fluid. These observations support and extend previous data showing a discordance of phenotype and function of lymphocytes in rheumatoid 3ynovial fluid possibly representing activation of such cells within the rheumatoid joint. G 1486 Ac.tdemlc Prec,. Inc

INTRODUCTION

Cytotoxic cells potentially present in a given host include antigen-specific cytotoxic T lymphocytes (CTL)? (l), nonspecific cytotoxic cells or natural killer (NK) cells (2). and activated nonspecific cytotoxic cells. The latter have been variously designated as anomalous killers (AK) (3). activated lymphocyte killers (4-.5), and cytotoxic cells generated by the autologous mixed lymphocyte reaction (AMLR killers) (6- 10). We have reported previously that patients with rheumatoid arthritis (RA) have normal NK cells in their peripheral blood but that the phenotype and functional activity of their synovial fluid cytotoxic lymphocytes differ from peripheral blood NK cells and resemble that of AMLR killers (9). It has recently been shown that NK cells have the ability to regulate B-cell differentiation and antibody synthesis (1 1- 13). Since the rheumatoid synovium is the site of an ongoing immune response characterized by intense lymphoid proliferation and synthesis of large amounts of immunoglobulin (14). one might specu1 Supported in part by the Medical University of South Carolina State Appropriations for Research l98l-1982. 2 Abbreviations used: NK, natural killer: CTL. cytotoxic T lymphocyte; AK, anomalous killer: ALK, activated lymphocyte killer: AMLR. autologous mixed lymphocyte reaction; RA. rheumatoid arthritis; SF, synovial fluid: PB, peripheral blood; FCS. fetal calf serum: C’. complement; PBS, phosphate-buffered saline; HAGG. heat-aggregated IgG: E:T. effector to target. 1.59 0090- 1229186 $ I .50 Copynpht I’ 1986 hy Academic Pre*\. Inc All rIghI\ of reproduction in any form rewwcd

I60

KIC’HAKD

M.

SII.\Jt.K

late that NK cells are absent or abnormal, Indeed. immunohistologic studies ol rheumatoid synovium have shown natural killer cells to be reduced in comparison to peripheral blood ( IS- 17). This study was undertaken to compare the phcnotype and function of rheumatoid \ynovial fluid cytotoxic lymphocytes to that ol‘ paired peripheral blood lymphocytes, employing monoclonal antibodies directed against NK antigens. MATERIALS

AND METHODS

S(rbjrc.t.s. Heparinized venous blood was obtained from patients with definite or classical RA (IX) with active disease and from normal controls. Synovial lluid (SF) was obtained from seven patients with RA by arthrocentesis of the knee at the same time that blood was obtained. Ccl1 .srprr~rrtic,n. Heparinired SF was treated with hyaluronidase (Wydase, Wyeth. Philadelphia. Pa.). I .5 U/ml for 30 min at 37°C to reduce viscosity. Paired heparinized venous blood was treated identically. After three washes in RPM1 1640 with 10% FCS. PB and SF mononuclear cells were obtained by Ficoll-Hypaque density gradient centrifugation. Cells at the interface were washed three times and resuspended at I x IOf3/ml in RPM1 1640 containing 105%FCS. IO mM Hepes (Research Organica. Inc.. Cleveland. Ohio). 2 mM L-glutamine (GIBCO, Grand Island. N.Y.). penicillin ( 100 U/ml). and streptomycin 100 kg/ml (GIBCO). SF lymphocytes (SFL) and PB lymphocytes (PBL) were obtained by removing adherent cells after overnight incubation on plastic dishes (Corning No. 25020. Corning, N.Y.) at 37°C in a humidified 5qf CO, and 9Tc/ air atmosphere. C~,/@r,~~r/lt (C’)-/~lc~dicrtc~d (.(,/I /x.vi.s. Following overnight incubation. PBL and SFL were washed three times and IO x IO” viable cells were incubated in 0.5 ml media alone or media containing monoclonal antibody HNK-I. 50 pg/l * IOh cells (19) (kindly provided by T. Abe and C. M. Balch, University of Alabama. Birmingham). or Leu-7 (HNK-I 1 or Leu-I I b. 0. IO &I s 1Of1cells (10) (BectonDickinson. Mountain View. Calif.). After 45 min incubation at 77°C. the cells were centrifuged at 1000 I-pm for IO min and the supcrnatant was discarded. The cells were resuspended in 0.5 ml baby rabbit complement (I:?) (Pcl-Freez. Rogers, Ark.) and incubated for 60 min at 23°C. The cells were then centrifuged at 1000 rpm for IO min. resuspended in PBS. and rocked for IS min at 21°C. Finally. the cells were centrifuged and resuspended in RPM1 1640 t- 10% FCS for functional cytotoxicity assays. In control experiments. normal PBM were incubated for various periods of time with heat-aggregated IgG (HAGG) prior to use in cytotoxicity assays. HAGG was prepared by heating human Cohn fraction 11 (Sigma. St. Louis. Mo.) for 30 min at 63°C. followed by centrifugation at 1800 I-pm to remove particulate material. In some experiments. rheumatoid PBM were incubated overnight in RPM1 1640 containing 10% autologous synovial tluid plus 10% FCS. C’ytoto.l-icit>’ tr,s.srr~~. K.562 target cells (31) (obtained from C. Lozzio. University of Tennessee, Knoxville) wjere labeled with Na, 5iCr0, (New England Nuclear. Boston, Mass.). 200 yCi per 3 to 5 x 106 cells at 37°C for 2 hr. The target cells were washed three times in large volumes of cold RPMI 1640 + 10% FCS and resuspended at I x IO~iml in RPM1 1640 + 10% FCS. Chromium-labeled target

NK-LIKE

CELLS

IN

RA

SYNOVIAL

cells (I x 104) were incubated with graded numbers microtiter plates (Cooke Labs, Alexandria, Va.) in 37°C in humidified 5% CO? and 95%~ air. After 4 hr. 0.1 ml supernatant was removed, and radioactivity gamma counter (Beckman No. 4000, Irvine. Calif.). as percentage 2lCr release by the formula: T-S ~ x 100. :%I - s

FLUID

161

of effector cells in V-bottom a total volume of 0.2 ml at the plates were centrifuged, released was measured in a Cytotoxicity was expressed

in which T = cpm released by effector cells, S = spontaneous cpm released in medium alone, and M = maximal cpm released by the addition of detergent (0.1% sodium dodecyl sulfate). All assays were done in triplicate and individual SlCr-release values varied less than 5%. Percentage reduction in cytotoxicity was defined as the cytotoxicity of the whole cell population minus the cytotoxicity of the monoclonal antibody-treated population, divided by that of the whole cell population, multiplied by 100. In some cases. cytotoxicity was expressed in terms of lytic units/l06 cells, with a lytic unit defined as the number of effector cells required to produce 50% lysis. All data presented in terms of lytic units were derived from points on the linear portion of the cytotoxicity curve, and comparisons of lytic units between two populations were made only when the lines were parallel. RESULTS Expression of NK-associated antigens 011 rheumatoid PBM: analysis by compfement-mediated cell fysis. In preliminary experiments, the NK activity of PBM from normal donors was compared to that of PBM preincubated with either antiLeu-1 lb or anti-Leu-7 (HNK-1) and complement. As depicted in Fig. 1, complement-mediated depletion of PBM bearing Leu-I 1b resulted in virtual elimination of all NK activity against K562. This was true in all donors tested and at all effector:target ratios. As also shown in Fig. I, the elimination of Leu-7 bearing PBM had a modest effect on NK activity at an effector target ratio of 50: 1, but no effect at 100: I. A comparison of cytotoxicity curves at points parallel to one another reveals that pretreatment with anti-Leu-7 and C’ resulted in a reduction of lytic units from 8.3 to 2.5. In contrast, elimination of Leu-1 lb bearing PBM resulted in marked reduction of NK activity, even at effector:target ratio of 100: I (99% reduction). The virtual elimination of all NK activity against K562 precludes the meaningful calculation of lytic units, but it is clear that depletion of Leu-11 bearing PBM eliminates NK activity more efficiently than the depletion of Leu-7 bearing PBM. In subsequent experiments, therefore, anti-Leu-1 lb was used to deplete NK activity against K562. PBM from 16 patients with rheumatoid arthritis showed a similar reduction in NK activity as normal PBM when Leu-1 lb+ cells were eliminated by complement-mediated lysis. As shown in Table 1, the mean reduction in NK activity was approximately 90% at all effector:target ratios tested. A representative experiment is depicted in Fig. 2. Expression of the NK-associated antigen Leu-ilb on rheumatoid SFM. In

162

RICHARD M. SI1.VF.K

II;

I 6

3

I 12

I 25

I 50

h I 100

E:T Ratio

FIG. I. NK activity of normal PBL pretreated with complement (C’) alone (O), anti-Leu-7 and C’ (0). and anti-Leu-I lb and C’ (A). The results are expressed as the percentage of specific release of Yr-labeled K562 cells in a 4-hr assay vs the effector:target ratio.

contrast to rheumatoid PBM, there was only a modest reduction in SFM NK activity after elimination of Leu-I 1b+ cells (see Table 2). In seven-experiments the mean reduction in NK activity was approximately 30% at an effector:target ratio of 50: 1 and 20% at 100: I, compared to a 90% reduction in NK activity of autologous PBM at the same effector:target ratios. A representative experiment involving rheumatoid SFM is depicted in Fig. 3. 50-I

E:T Ratio

FIG. 2. NK activity of representative rheumatoid PBL (patient number I I) pretreated with complement (C’) alone (0) or with anti-Leu-1 lb and C’ (0). With the latter. 90% of NK activity is eliminated at each effector:target ratio. The results are expressed as the percentage of specific release of 51Cr-labeled K562 cells in a 4-hr assay vs the effector:target ratio.

NK-LIKE

CELLS IN RA SYNOVIAL

1:.5-

FLUID

163

100

Ratio FIG. 3. NK activity of representative rheumatoid SFL (patient number 1) pretreated with complement (C’) alone (0) or with anti-Leu-I lb and C’ (0). With the latter, there is mild reduction of NK activity at effector:target ratios of 12.5 and 25 but no significant effect on NK activity at effector:target ratios of 50 and 100. The results are expressed as the percentage of specific release of 51Cr-labeled K.562 cells in a 4-hr assay vs the effector:target ratio. E:T

Effect of synovialfluid or immune complexes on expression of Leu-1 I. Experiments were performed to determine whether the different behavior of rheumatoid SFM compared to PBM was due to blockage or alteration of the Leu-1 1 antigen on SFM by immune complexes or other factors present in synovial fluid. Rheumatoid PBM were cultured overnight in media containing 10% autologous synovial fluid and then tested for cytotoxicity against K562. Rheumatoid PBM incubated in autologous synovial fluid exhibited a reduction of NK activity by approximately 75% at all effector:target ratios tested when pretreated with anti-Leu-llb and complement (data not shown). Thus. the behavior of rheumatoid SFM cannot be explained by blockage or alteration of the Leu-1 1 antigen by factors present in rheumatoid synovial fluid. Similarly, normal PBM exposed to HAGG (100-500 pg/ml) for 12-72 hr at 37°C were also significantly depleted of NK activity by complement-mediated lysis of Leu- 11b+ cells (see Fig. 4). Similar results were obtained when normal PBM were exposed to HAGG for 1 hr at 4°C (data not shown). DISCUSSION

Natural killer (NK) cells may be operationally defined by their ability to lyse certain tumor cell lines (2). It has become clear that considerable heterogeneity may exist within this population, which may be indicative of differences in cell lineage or differences in state of activation. This study was performed to compare the phenotype and function of rheumatoid synovial fluid NK cells to that of peripheral blood NK cells, using the monoclonal antibodies anti-Leu-7 (HNK-1) and anti-Leu-1 lb, both of which are said to identify NK cells (19, 20). From the data presented, it may be concluded that (1) the monoclonal antibody anti-leuI lb identifies virtually all NK cells present in the peripheral blood of normal

164

KICHAKD

bl.

SIl.Vl~.K

60

E:T Ratlo

FIG. 4. NK activity of normal PBL, cultured for 71 hr in 500 kg/ml (0). Despite prolonged exposure to HAGG. NK with monoclonal antibody anti-Leu-I lb plus complement centage of specific release of r’Cr-labeled K562 cells in a

RPM1 1640 + IOG FCS containing HAGG. activity can still he eliminated by treatment (0). The results are expressed a\ the per4-hr assay vs the effector:target ratio.

individuals and patients with RA, whereas anti-Leu-7 reacts with only a fraction of such cells: (2) unlike peripheral blood NK cells, the majority of rheumatoid synovial fluid NK cells do not express Leu-1 lb: (3) this difference in Leu-I lb expression by rheumatoid synovial fluid NK cells does not appear to be due to blockage or alteration of the Leu- 1I b antigen by factors present in synovial fluid: (4) and finally, it confirms previous observations that in most cases the rheumatoid synovial fluid NK activity is less than that of peripheral blood (9). The observation that anti-Leu-I 1b identifies essentially all peripheral blood lymphocytes expressing NK activity and that Leu-7p cells may express NK activity is consistent with the data of Lanier et ul. (20). The Leu-1 1 antigen is believed to be an Fc receptor: the present observation that rheumatoid synovial fluid NK cells are Leu-11 is consistent then with our earlier observation that these cells lack Fc receptors for IgG (9). The blocking experiments with synovial fluid or HAGG indicate that the Leu-I 1 antigen on peripheral blood lymphocytes is re-expressed after exposure to synovial fluid or immune complexes (experiments performed at 37°C). or that the monoclonal antibody anti-Leu-1 lb is capable of successfully competing with such factors (experiments performed at 4°C). In either case. the inability of anti-Leu-1 lb and complement to eliminate rheumatoid synovial fluid NK activity does not appear to be due to blockage OI alteration of the Leu-I 1 antigen, but rather appears to be due to a relative lack of expression of the Leu-I I antigen.

NK-LIKE

CELLS

IN RA SYNOVIAL TABLE

162

FLUID

I

RHEUMATOID PBL NK ACTIVITY AGAINST KS61 Whole

PBL

Leu- I I depleted

PBL

Patient numher

29 77 66 66 6Y hi XI 64, 33 ‘5 Ii 11

5’) 63 63 1-l Mean 2 SD
30 2 I7

39 f

20

37 I

IY

54 2 20

_T+!._ 93’;

3r! 911 i

5 t 3 89C’r

624 89’7

ratio

The precise origin of the rheumatoid synovial fluid NK cell remains unknown, but it resembles in many respects the cytotoxic cell generated by the AMLR (9, 10). Goto and Zvaifler have shown that the AMLR killer cell lacks the Leu-7 marker (IO). The same investigators have reported that the rheumatoid synovial fluid NK-like cell does not express Leu-7, OKM-I, or OKT-8; it does express OKTll and 4F2 (22). Although these authors employed monoclonal antibodies which identify subsets of NK cells (OKMl and Leu-7). it has been shown that the most potent NK effector cell in peripheral blood expresses the Leu-11 antigen, even in the absence of OKM-1 or Leu-7. Thus, we elected to use anti-Leu-I 1b since it recognizes virtually all NK cells in peripheral blood. Our data suggest that the rheumatoid synovial fluid cytotoxic cell is unique, in that it is functionally cytotoxic to K562 cells, yet lacks the NK surface marker, Leu-lib. Studies are underway to determine if AMLR cytotoxic cells express Leu- I I. In most cases, the rheumatoid synoviai fluid cytotoxic activity was less than that of peripheral blood (see Tables 1 and 2, patients l-7). This is consistent with earlier observations by us and others (9, 23, 24). Combe et ~1. have recently shown that adherent cells enhance synovial fluid cytotoxic activity but suppress peripheral blood cytotoxic activity (24). One potential explanation for the decreased synovial fluid cytotoxic activity in these experiments might be a differential effect of adherent cell removal. It would not explain our previous observation that overnight culture on tissue culture plates resulted in some increase in cytotoxicity (9). Other potential explanations for the decreased synovial fluid cyto-

166

Patient nun1het

.--. 12.5,

1 2 3 4 5 h 7

17 ‘0 I3 Ii I-1 47

KIC‘HARD

M.

SIL.Vt-.R

37 T IS

-15 2 IJ

?i

Mean ? SD 9 Reduction

‘I

9’ Effector:target

Y- 21

2x 7 I4

13 -t IO 3x<;

IX i I7 36’ ;

26 T I‘l 30”;

35 1 I7 zz’,;

ratio.

toxic activity include the presence of suppressor cells (25, 26) in synovial fluid or the presence of inhibitory substance (27) in synovial fluid. In favor of the latter is our prior observation that the addition of cell-free autologous synovial fluid resulted in a decrease in NK activity of PBM (9). Preliminary experiments using a single-cell cytotoxicity assay have shown that the decrease in synovial fluid cytotoxic activity is not due to decreased target cell binding (to be published). but inhibitory substance may act at a postbinding lytic stage (27). It is clear that rheumatoid synovial fluid NK cells differ from peripheral blood NK cells in several respects, both phenotypically and functionally (28. 29). It remains to be seen whether these differences are important to the pathogenesis of rheumatoid synovitis or merely a reflection of the ongoing immune response within the rheumatoid joint. With the recent observation that peripheral blood NK cells are capable of regulating B-cell differentiation and immunoglobulin synthesis (12. 13). it will be important to assess the FcRm, Leu-I I - synovial fluid NK cells for such regulation. NK cells have also been shown to suppress the AMLR in \ritw. A recent report suggests that this suppression occurs through an interaction of NK cells with antigen-presenting dendritic cells (30). It has been shown that rheumatoid synovial fluid contains cells which are very potent stimulators of the AMLR (28), and that such effusions contain 5- 10 times more dendritic cells than blood (3 I ). Thus, the rheumatoid joint is the site of an ongoing AMLR. With the recent observation that NK cells are capable of regulating Tlymphocyte proliferation by interacting with dendritic cells. it will be important to assess the FcR-- , Leu-I 1~ synovial fluid NK cell for such regulatory function. ACKNOWLEDGMENTS The author for secretarial

gratefully acknowledges assistance.

Ms. K. Prioleau

for technical

assistance

and Ms. J. Anderson

NK-LIKE

CELLS

IN RA SYNOVIAI.

FLUID

167

REFERENCES I. Gillis, S.. Baker, I’. E., Ruscetti, E W.. and Smith. K. A.. ./. Erp. Med. 148, 1093. 1978. 2. Herberman. R. B.. Djeu, J. Y., Kay. D. H.. Ortaldo. J. R.. Riccardi. C.. Bonnard. G. D.. Holden. H. T.. Santoni. A.. Santoni. P.. and Pucetti. P.. I~n,?~lr&. Ret,. 44. 43. lY7Y. 3. Seeley. J. K.. and Golub, S. H.. J. I~t~nzrrnol. 120, 1415. 1978. 4. Masucci, M. J.. Klein. E.. and Argov. S.. J. /rn!?~rc/~ol. 124. 245X. IY80. 5. Grimm. E. A.. Mazuder. A.. Zhang, H. Z., and Rosenberg. S. A.. J. E.t-p. n!e(i. 155, 1823. 1982. 6. Van de Stouwe. R. A.. Kunkel. H. G.. Halper. J. P.. and Wrksler. M. E.. J. Lip. Med. 146, 1809. 1977. 7. Miller. R. A.. and Kaplan, H. S.. J. Ir)r/t~lrr~o/. 121, 2165. IY7X. 8. Tomonari. K.. J. ltrrmrtnol. 124, I I I I. 1980. 0. Silver. R. M.. Redelman. D.. Zvaifler. N. J.. and Naides. S.. J. I~r~rnrrr&. 128, 1958. 1982. IO. Goto. M.. and Zvaifler, N. J.. J. E-1-p. Med. 157, 1309. 1983. I I. Abruzro. L. V.. and Rowley. D. A.. Sc,ic>rlc,c (Woshin~~o~r. D.C.) 222, 5X1. 1983. 12. Brieva. J. A., and Stevens. R. H.. J. I~nr77w7ol. 133, 13-88. 1984. 13. Arai. S.. Yamamoto. H.. Itah. K.. and Kumagai. K.. J. f/nl,~lr/~c~/. 131. 651. 1983. 14. Zvaifler. N. .I.. Ad1 I777n77rnol. 16, 165, 1973. 15. Lindblad. S.. Klal-eskog. L., Hedfor\. E.. Forsum. U.. and Sundstrom. C.. Ar111riti.s Rhrro~. 26, 1321. 1983. 16. FOX. K. I.. Fang. S.. Tsoukds. C.. and Vaughan. J. H., J. I,tiftllinol. 132, 1883. 1984. 17. Malone. D. G., Wahl. S. M.. Tsoko\. M.. Cattell. H.. Decker. J. L.. and Wilder. R. L.. .I. c‘litl. itrt~,.\r. 74, 1173. IY84. IX. Ropes. M. W.. Bennett. E. A., Cobbs. S.. Jacox. R.. and Jessar. R.. B//l/. Rht,rou. Dis. 9, 175. 1958. 19. Abo. I’.. and Balch. C. M.. J. Io7n7rrt7d. 127, 1024. 1981. 20. Lanier. L. L.. Le. A. M., Phillips. J. H.. Warner. N. L.. and Babcock. C. F.. J. Iu7n77rnd. 131, l78Y. 1983. 21. LozLio. C. B.. and Lozzio. B. B.. B/o& 45, 321. IY75. 22. Goto. M.. and Zvaifler. N. J.. J. I~~7munol. 134, 1483. 19X5. 93. Armstrong. R. D.. and Panayi, G. S.. C/in. Rhrrrr~r. 2, 243, 1983. 74. Combe. B.. Pope. R.. Darnell. B.. Kincaid. W.. and Talal. N.. J. Im~771rnol. 133, 709, 1984. 25. %rkkanen. J.. Saksela. E., and Paavolainen, M.. Cli~z. Ir~rn7wtd. fr,7,,7rr,7optrt/rc,1. 28, 29. 1983. 26. D’Amore. P. J.. and Golub, S. H.. J. /mrnrrnof. 134. 272. 1985. 27. Lichtenstein, A.. Mickel. R.. and Zighelboim. J.. C(,//. Ir~7~77unol. 80, 66. 1983. 28. Silver, R. M.. Redelman, D.. and Zvaifler, N. J.. C/in. I7r7t~7rrnd. lmr77rrr7oprrrl7. 27, IS. 1983. 29. Silver. R. M., and Zvaifler. N. J.. !,I “Rheumatoid Arthritis: Eliology. Diagnosis, Management” (P. D. Utsinger. N. J. Zvaifler. and G. E. Ehrlich. Eds.). pp. 71-89. J. B. Lippincott Co.. Philadelphia. 1985. 30. Shah. P. D.. Gilbertson. S. M.. and Rowley, D. A.. J. Exp. Me
September

20. 198.5: accepted

November

4. 1985