NATURAL KILLER CELL ACTIVITY OF MONONUCLEAR CELLS FROM RHEUMATOID PATIENTS MEASURED BY A CONJUGATE-BINDING CYTOTOXICITY ASSAY

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1 1440 NATURAL KILLER CELL ACTIVITY OF MONONUCLEAR CELLS FROM RHEUMATOID PATIENTS MEASURED BY A CONJUGATE-BINDING CYTOTOXICITY ASSAY ELIZABETH REINITZ, P. ANDREW NEIGHBOUR, and ARTHUR I. GRAYZEL The natural killer cell activity of synovial fluid mononuclear cells and synovial membrane mononuclear cells from patients with rheumatoid arthritis was compared with that of paired peripheral blood mononuclear cells from many of these patients. Natural killer cell activity was measured by the use of a conjugate-binding cytotoxicity assay with an erythroleukemic cell line, K562, as target. There was no significant difference when 15-minute target binding by peripheral blood mononuclear cells was compared with synovial fluid mononuclear cells. Target binding of synovial membrane mononuclear cells was significantly greater (P < 0.05) than that of peripheral blood cells. Three-hour target killing, however, was significantly greater when synovial fluid mononuclear cells were compared with the peripheral blood cells, P < 0.01, and when synovial membrane cells were compared with the peripheral blood cells (P < 0.05). The products of binding and 3- hour killing, a reflection of the total number of mononuclear cells participating in cytotoxicity, were significantly greater when either synovial fluid or synovial membrane mononuclear cells were compared with pe- From the Departments of Medicine, Pathology, and Microbiology & Immunology, Montefiore Hospital & Medical Center, and the Albert Einstein College of Medicine, Bronx, NY. Supported in part by a Clinical Research Center Grant from the Arthritis Foundaton and research grants NS-15541, NS-I 1920, AI and AM from the National Institutes of Health. Elizabeth Reinitz, MD: Assistant Professor of Medicine, formerly Fellow in Rheumatology; P. Andrew Neighbour, PhD: Assistant Professor of Pathology and Microbiology & Immunology; Arthur I. Grayzel, MD: Professor of Medicine, the Albert Einstein College of Medicine. Address reprint requests to Arthur I. Grayzel, MD, Montefiore Hospital & Medical Center, 111 E. 210 Street, Bronx, NY Submitted for publication March IS, 1982; accepted in revised form July 19, Arthritis and Rheumatism, Vol. 25, No. 12 (December 1982) ripheral blood mononuclear cells (both P < 0.01). Electron microscopy confirmed the presence of large granular lymphocytes, representing 20% of the peripheral blood cells and 37% of the synovial fluid mononuclear cells. Interferons were detected in 10/12 rheumatoid and 3/12 nonrheumatoid synovial fluid samples studied. These findings indicate that functional natural killer cells are selectively increased in the rheumatoid joint and may contribute to the overall increase in immunologic activity found in the joints of these patients. Some mononuclear cells bearing Fc receptors with no surface immunoglobulins have been recognized to have spontaneous cytotoxicity for a variety of target cells in the absence of prior sensitization. These target cells have included tumor, virus-infected, and hematopoietic cells. This cytotoxicity is antibody independent and does not require major histocompatibility complex identity. The effector mononuclear cells have become known as natural killer (NK) cells. In the human, they are thought to play an important role in tumor surveillance and in resistance to acute and chronic viral infections (1). They have been morphologically described as large granular lymphocytes. These are mononuclear cells that are larger than most lymphocytes and characterized by crescent-shaped nuclei and abundant cytoplasm with azurophilic granules (2). Many patients with certain chronic diseases, such as systemic lupus erythematosus, multiple sclerosis, and Crohn s disease, have low or absent NK cell activity in their peripheral blood mononuclear cells (PBMC) as compared with normal donors, and many of these cells fail to respond in vitro to interferon with the increase of NK cell activity seen with normal

2 NK CELL ACTIVITY IN RA 1441 donors (3-5). Patients with rheumatoid arthritis, however, have normal NK cell activity in their PBMC, and this activity can be boosted with interferon as in normal donors (4). Because the site of disease activity in patients with rheumatoid arthritis is in the synovium, we used a conjugate-binding cytotoxicity assay to study the NK cell activity of synovial fluid mononuclear cells (SFMC) and synovial membrane mononuclear cells (SMMC). We also measured interferon levels in synovial fluids and quantitated the large granular lymphocytes in several mononuclear cell samples. MATERIALS AND METHODS Patients. Effector cells were isolated from the synovial fluid and simultaneous peripheral blood samples of 10 patients with definite or classic rheumatoid arthritis. SFMC without paired blood samples were obtained from another 3 patients. SFMC were isolated from 6 patients; from 3 of these patients, PBMC were also obtained. Three patients were receiving gold injections, 3 were receiving penicillamine, 2 were on low doses of corticosteroids, and all were on nonsteroidal antiinflammatory drugs. SFMC were obtained from 1 patient with rheumatoid arthritis before, and 2 and 4 weeks after, intraarticular injection with cortocosteroids. Synovial fluid samples were obtained from patients with rheumatoid arthritis, osteoarthritis, traumatic arthritis, and crystal-induced arthritis for measurement of interferon levels. Efector cells. PBMC were isolated by Ficoll-Hypaque (Pharmacia Fine Chemicals) density gradient centrifugation. These cells were then put through a nylon wool column to remove adherent monocytes and B cells (6), a procedure known not to deplete NK cells. The nonadherent cells were then used as effector cells. Synovial fluids were incubated with DNase (50 pg/ml) (Sigma #D-4763) and bovine testicular hyaluronidase (20 pg/ml) (Sigma #H-2376) for 30 minutes on a rocking tray at 37 C. They were filtered through gauze and then treated in the same way as blood samples. Synovial membranes were dissected free of fat tissue and minced. In addition to DNase and testicular hyaluronidase (added in the same concentrations used in synovial fluids), collagenase (50 pghl) (Sigma #C-0130) was added. After incubation for 5 hours at 37"C, the cells were filtered through sterile gauze. The effector cells were then isolated by the above method for peripheral blood. To control for the effect of these enzymes on NK cell function, 2 blood samples were divided in half. One-half was treated with the enzymes used for synovial fluids and membranes at the same concentrations; the other half was not. The medium used throughout was RPMI (Gibco) with 10% fetal calf serum. Target cells. K562 cells, a human erythroleukemic cell line, were maintained in continuous suspension culture. These cells are known to be susceptible to NK cell-mediated lysis. Conjugate formation and cytotoxicity assay. We determined the ability of single effector cells to bind and lyse K 562 target cells by using a modification of the method of Grimm and Bonavida (7) described in detail elsewhere (8). Equal volumes of effector and target cells were mixed at a concentration of 4 x lo5 cells and centrifuged at 1,000 revolutions per minute (rpm) for 5 minutes. After the pellets were incubated for 10 minutes at 37T, we gently resuspended them by pipetting up and down 6 times with a Pasteur pipette. The percent of viable mononuclear cells binding to target cells (percent TBC) was determined. The effector-target mixtures were resuspended in 1% agarose in medium, spread on glass slides, and allowed to cool. Slides were then incubated at 37 C in medium. At 3 hours, the slides were stained with 1% trypan blue at room temperature for 5 minutes and then washed thoroughly in phosphate buffered saline. The percent of conjugates containing dead cells was determined microscopically. The minimum number of conjugates scored was 200. The percent NK cells was the product of percent TBC and percent conjugates with dead targets. Each sample was prepared and scored in duplicate. Measurement of interferon levels in synovial fluid samples. Twelve synovial fluid samples from rheumatoid patients and 12 synovial fluid specimens from patients with other diseases were assayed for interferon levels. In 6 patients the same synovial fluid was analyzed for both interferon and NK cell activity. Interferon was titrated by the inhibition of vesicular stomatitis virus plaques on human amniotic cells (WISH-2; American type culture collection) (9). Titers were expressed as the reciprocal of the dilution that produced 50% reduction of plaques after standardization to the NIH human reference interferon (G ). Statistical methods. The group mean values of percent TBC, dead conjugates, and percent NK cells for synovial fluid and membrane samples were compared with PBMC effectors by the Mann-Whitney test. Mean interferon titers were compared by Student's t-test on log,,, data transference. Significance was accepted throughout at the P < 0.05 level. Table 1. Target binding and killing activity of synovial fluid, synovial membrane, and peripheral blood mononuclear cells from patients with rheumatoid arthritis Source* Binding Killing % natural killer (n) (15 minutes) Pt (3 hours) Pi (binding x killing) Pi PBMC (13) 20.8 t_ f SFMC (13) 21.2? t SMMC (6) 25.7? _t * PBMC = peripheral blood mononuclear cells; SFMC = synovial fluid mononuclear cells; SMMC = synovial membrane mononuclear cells. 1- P value as compared with results for PBMC (Mann-Whitney test).

3 1442 REINITZ ET AL I2- RESULTS The recovery of cells after nylon wool passage was the Same for blood (63 * 7.6%), SYnOvial fluid ( %), or synovial membrane (65 * 9.3%). 5l 3 PBMC SFMC SMMC for each blood, synovial fluid, and synovial membrane examined; the mean values are summarized in Table 1. The percentage of mononuclear cells bound at 15 minutes was not significantly different when PBMC were compared with SMFC but was significantly greater when the SMMC were compared with PBMC The percentage Of mononuclear bound at 15 minutes, the percentage Of bound target results. Three-hour target cell killing was significantly and the percentage of NK cells are shown in Figure I greater when SFMC were with pbmc and when SMMC were compared with PBMC. The products of binding and 3-hour cytotoxicg1 ity, a measure of the isolated PBMC with NK cell activity, showed significantly greater results when the SFMC values or the SMMC values were compared 7 with PBMC values. To test whether enzyme treatment of synovial fluids and membranes affected NK cell activity, PBMC from 2 patients were examined with and without enzyme treatment at the same concentrations as used in synovial samples. These experiments showed that the enzymes had no significant effect on NK cell activity, (3.42% and 4.32% without enzymes; 3.60% and 4.00% with enzymes). SFMC from 1 patient were studied before and 2 and 4 weeks after intraarticular steroid injection. The initial percentage of NK cells (5.7%) showed a decline 2 weeks after injection (3.5%) and partial recovery at 4 weeks (5.5%). Since some large granular lymphocytes are thought to mediate NK cell activity, we used electron microscopy to look for such lymphocytes. Characteristic large granular lymphocytes accounted for 20% of the peripheral blood nonadherent cells and 37% of the synovial fluid nonadherent cells (Figure 2). 12 I Figure 3 shows the interferon levels detected in the synovial fluid samples. Twelve rheumatoid samples had a mean of log,, units of interferon; samples from patients with osteoarthritis or crystal- 2 induced arthritis had a mean of 0.80 *.55 log,, units of 28-7 & interferon. These values were found to be significantly different (P = ). In the 6 synovial fluids analyzed a for both interferon and NK cell activity, there was no 20 significant correlation between the titer of interferon am - and cytotoxicity. Figure 1. The percent target cell binding (bottom panel), 3-hour target cell killing (middle panel), and percent natural killer (NK) (upper panel) of mononuclear cells isolated from the peripheral blood (PBMC), synovial fluid (SFMC), and synovial membrane (SMMC) of patients with rheumatoid arthritis. The shaded area indicates the mean? I SD. DISCUSSION The synovium in rheumatoid arthritis is the site of an intense immune response (10) that is reflected in the cellular contents of rheumatoid synovial fluid. In addition to the local synthesis of immunoglobu~ins and rheumatoid factor (11, 12), activated dendritic macrophages and lymphocytes are present in synovial membranes and fluids (13, 14). Our studies show that

4 NK CELL ACTIVITY IN RA 1443 Figure 2. Electron micrographs of lymphocytes showing typical large granular lymphocyte-like characteristics from a synovial infiltrate of a patient with rheumatoid arthritis. Characteristic features include a high cytoplasmic-to-nuclear ratio, crescentic nucleus, intracytoplasmic granules, and abundant short microvilli. Cells were fixed in suspension with 2.5% gluteraldehyde in Millonings phosphate buffer and postfixed with 1% osmium tetroxide. Thin sections were stained with uranyl acetate followed by lead citrate and examined in a Siemens 101 electron microscope at 80 kv. rheumatoid synovial membranes and synovial fluid contained more functional NK cells than did blood samples from patients with rheumatoid arthritis. Recently Fox et al (15), using monoclonal antibodies to T lymphocyte surface antigens, showed a difference in the proportion of T cell subsets between synovial fluid and peripheral blood lymphocytes in patients with rheumatoid arthritis. These antibodies, however, do not permit one to enumerate NK cells. We have also demonstrated the presence of large granular lympho-.- c 3 0 a A crystal induced arthritis m osteoarthritis 0 rheumatoid arthritis 0 Rheumatoid Non Rheumatoid Figure 3. The interferon levels in log,, transformed units of synovia1 fluid samples from rheumatoid and nonrheumatoid patients. The shaded areas indicate the geometric mean interferon level 2 I SD. Values less than 1 unit are considered negative for interferon. cytes, some of which are responsible for NK cell activity (2). Finally, interferon, known to increase NK cell activity, was found in rheumatoid synovial fluids. The inflamed rheumatoid joint also contains prostaglandin E (PGE). PGE and interferon may have antagonistic actions on NK cell activity (16), although the interaction, even in vitro, is complex (17). One cannot be certain of the net effect in vivo, but the evidence from the in vitro assays indicates an increase in functional NK cells in the synovium as compared with peripheral blood in these patients. All patients in our study were treated with nonsteroidal antiinflammatory drugs known to decrease the production of PGE-a process that might also tip the balance in favor of increasing NK cell activity. It is of interest that in 1 patient, SFMC studied before and after intraarticular steroid injection showed an initial decline and then recovery of NK cell activity. This is consistent with our previous report in which corticosteroids were found to depress NK cell activity (4). Burmester et a1 (18) investigated the NK cell activity of PBMC and SFMC from patients with rheumatoid arthritis and found significantly higher activity using SFMC. In their study, SFMC NK cell activity was often 3 times greater than the PBMC NK cell activity, but they used a chromium release cytotoxicity assay with a 12-hour incubation in contrast to the conjugate binding assay with a 3-hour incubation used in the present study. The former allows for effector cell recycling. Each NK cell can bind and lyse more

5 1444 REINITZ ET AL than 1 target cell (19). In addition, there may be an increase in interferon levels during a longer period of incubation; this may serve to recruit or activate additional NK cells. Because of the immobilization in agar, recycling does not take place, and only bound effectors can be recruited with the conjugate binding assay. This assay measures a short-term event involving lymphocytes, presumably activated in vivo, whereas the chromium release assay may measure recycling and more extensive recruitment as well. Taken together, however, the finding of increased NK cell activity in the "Cr release assay and increased numbers of NK cells by the conjugate binding assay suggest an absolute increase in the number and function of NK cells in the rheumatoid joint. We found a larger percentage of large granular lymphocytes in SFMC than in PBMC. Since at least some of these cells are thought to mediate NK activity, it is possible that we are seeing a selective accumulation of these cells in the joint, along with interferon. Interferon titers are known to rise in response to viral infections and to be involved with immunologic processes. It is intriguing to speculate about the reason NK cell activity is high in the synovium of patients with rheumatoid arthritis, yet diminished in the blood of patients with some other diseases, such as systemic lupus erythematosus. Perhaps in rheumatoid arthritis we are seeing a reaction to an offending agent or antigen localized in the joint, whereas in systemic lupus we are seeing the reflection of a primary immunoregulatory abnormality. At present we can characterize and describe these phenomena but we cannot define their role in the disease process. ACKNOWLEDGMENT We wish to thank Yvonne Kress for the electron microscopy. REFERENCES Herberman RB, editor: Natural Cell-Mediated Immunity against Tumors. New York, Academic Press, 1980, pp Timonen T, Ortaldo JR, Herberman RB: Characteristics of human large granular lymphocytes and relationship to natural killer and K cells. J Exp Med 153: , 1981 Hoffman T: Natural killer function in systemic lupus erythematosus. Arthritis Rheum 23:30-35, 1980 Neighbour PA, Grayzel AI, Miller AE: Endogenous and interferon augmented natural killer cell activity of human peripheral blood mononuclear cells in vitro. Clin Exp Immunol 49: , Auer 10, Ziemer E, Sommer H: Immune status in Crohn's disease V. Decreased in vitro natural killer activity in peripheral blood. Clin Exp Immunol 42:41-49, 1980 Julius MH, Simpson E, Herzenberg LA: A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur J Immunol 3: , 1973 Grimm E, Bonavida B: Mechanism of cell-mediated cytotoxicity at the single cell level. I. Estimation of cytotoxic T lymphocyte frequency and relative lytic efficacy. J Immunol 123: , 1979 Neighbour PA, Huberman HS: Sr++-induced inhibition of human natural killer (NK) cell mediated cytotoxicity. J Immunol 128: , 1982 Neighbour PA, Grayzel AI: Interferon production in vitro by leukocytes from patients with systemic lupus erythematosus and rheumatoid arthritis. Clin Exp Im- munol 45: , Ziff M: Relation of cellular infiltration of rheumatoid synovial membrane to its immune response. Arthritis Rheum 17: , Smiley JD, Sachs C, Ziff M: In vitro synthesis of immunoglobulin by rheumatoid synovial membrane. J Clin Invest 47: , Munthe E, Natvig JB: Immunoglobulin classes, subclasses and complexes of IgG rheumatoid factor in rheumatoid plasma cells. Clin Exp Immunol , Burmester GR, Yu DTC, Irani A, Kunkel HG, Winchester RJ: Ia' T cells in synovial fluid and tissues of patients with rheumatoid arthritis. Arthritis Rheum 24: , Janossy G, Duke 0, Poulter LW, Panayi G, Bofill M, Goldstein G: Rheumatoid arthritis: a disease of T- lymphocytehacrophage immunoregulation. Lancet , Fox RI, Fong S, Sabharwal N, Cartens SA, Kung PC, Vaughan JH: Synovial fluid lymphocytes differ from peripheral blood lymphocytes in patients with rheumatoid arthritis. J Immunol 128: , Koren HS, Anderson SJ, Fischer DG, Copeland CS, Jensen PJ: Regulation of human natural killing. I. The role of monocytes, interferon and prostaglandin. J Immunol 127: , Targan SR: The dual interaction of prostaglandin E2 (PGE)2 and interferon (IFN) on NK lytic activation: enhanced capacity of effector-target lytic interactions (recycling) and blockage of prenk cell recruitment. J Immunol 127: , Burmester GR, Kalden JR, Peter HH, Schedel I, Beck P, Wittenborg A: Immunological and functional characteristics of peripheral blood and synovial fluid lymphocytes from patients with rheumatoid arthritis. Scand J Immunol 7: , Ullberg M, Jandal M: Recycling and target binding capacity of human natural killer cells. J Exp Med 153: I981