Differential inhibition of lymphocyte function by 2-chloroadenosine

Int. J. lmmunopharmac., Vol. 6, No. 4, pp. 335-338, 1984. Printed in Great Britain.

0192-0561/84 $3.00+ .00 © 1984 International Society for Imrnunopharmacology

DIFFERENTIAL INHIBITION OF LYMPHOCYTE FUNCTION BY 2-CHLOROADENOSINE Y. NISHIDA, N. KAMATANI, T. MORITO and T. MIYAMOTO Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo Bunkyo-ku, Hongo, 7-3-1 Japan, 113

(Received 29 June 1983 and in final form 5 January 1984)

Abstract -- The effects of 2-chloroadenosine, a poorly metabolized adenosine analogue, on some human

lymphocyte functions were studied. Mixed lymphocyte responses were strongly inhibited by very low concentrations of 2-chloroadenosine. The mitogen-induced proliferation of human lymphocytes was also generally suppressed by 2-chloroadenosine in a dose dependent manner. Blastogenesisinduced by Con A and PWM was severelyinhibited by low doses of 2-chloroadenosine while its inhibition of that induced by PHA was less marked. Natural killer cell activity was inhibited only about 55°70 by high concentrations of 2-chloroadenosine. These results suggested that many subsets of human lymphocytes are controlled by adenosine receptor.

A number of lines of evidence indicated that in the human subject, many cells have a specific receptor for adenosine, (Daly, Bruns & Synder, 1981; Londos, Cooper & Wolff, 1980). This receptor is known to modulate adenylate cyclase activity and hence cyclicAMP concentrations and cellular functions (Marone, Plaut & Lichtenstein, 1978). However, the adenosine receptor has been shown to have different effects according to the species and tissue source. The existence of a specific receptor for adenosine on the surface of h u m a n lymphocytes was demonstrated by Marone et al. (1978) and Schwarz, Stern & Polmar (1978). However, Moroz, Bessler, Djaldetti & Stevens (1981) and Saxon, Thiele, Moroz & Stevens (1981) showed that the percentage of adenosine receptor positive cells is very small. As a subset of the lymphocyte population, adenosine receptor positive cells are recognized in E-rosetting cells and null cell fractions (Moroz et aL, 1981; Saxon et aL, 1981). On the other hand it has been shown that 2-chloroadenosine is neither taken up nor metabolized by cells (Schnebli, Hill & Bennett, 1967). It has been used as an adenosine receptor agonist (Churchill, 1982; Wu & Phillis, 1982) and therefore in this experiment, we studied the effects of 2-chloroadenosine on the various lymphocyte functions.

MATERIALS A N D METHODS

Lymphocyte suspensions Venous blood was collected from healthy subjects in a heparinized syringe. The lymphocytes were separated by discontinuous centrifugation in FicollHypaque solution as previously described (BOyum, 1968) and washed three times in large volumes of RPMI 1640 medium. Incorporation of JH-thymidine into mixed lymphocyte cultures The lymphocytes were suspended at 1 x 106 cells ml -~ in the RPMI 1640 medium containing 10°70foetal calf serum, 200 U ml-' penicillin G and 100/~g ml-' streptomycin. The mixed lymphocyte culture reactions (MLC) were carried out in sterile microplates with round-bottom wells and done in triplicate. 1 x l0 s lymphocytes and 1 x l0 s another human lymphocytes in a total volume of 200 ~1 of culture medium which contained the various concentrations of 2-chloroadenosine were put into same well. They were incubated for 3 days at 37°C in a humidified atmosphere with 5o/0 CO2 and 95°7o air. 1 /aCi of 3H-thymidine (2 Ci mmol-~: Radiochemical Centre) was added to each well for a further 24 h incubation. Cells were harvested on glass-fiber filter paper using a Millipore

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Y. NISHIDA, N. KAMATANI,T. MORITO and T. MIYAMOTO

semiautomated harvester. Radio-activity of thymidine incorporated into lymphocyte DNA was measured in a liquid-scintillation counter.

Lymphocyte blastogenesis with mitogens The lymphocytes were suspended at 1 × l06 cells ml-~ in the RPMI-1640 medium containing 10% foetal calf serum, 200 U m1-1 penicillin G, 100 ~g ml-~ streptomycin and the various concentrations of sterilized 2-chloroadenosine. Cultures were carried out in sterile microplates with round-bottom wells and were done in triplicate. 1 × 10~ of lymphocytes in 100 /~l of culture medium and 100/~l of mitogens, which were dissolved at appropriate concentrations in culture medium as follows (4/~g mP 1 of phytohemagglutinin (PHA): Difco Laboratories, 5/~g ml-~ of concanavalin A (Con A): Pharmacia Fine Chemicals, and l:100 diluted pokeweed mitogen (PWM): Grand Island Biological Company) were put into the same well. They were incubated for 96 h at 37°C in a humidified atmosphere with 5°7oCO2 and 95% air. Following 72 h of culture, I/~Ci of 3H-thymidine was added to each well. Cells were harvested on glass fiber filter paper using a Millipore s e m i a u t o m a t e d harvester. Radioactivities of thymidine incorporated into lymphocyte DNA were measured in a liquid scintillation counter.

Natural killer cell activity The activity of natural killer cells was measured by in vitro s'Cr release assay with K-562 target cells as described in detail elsewhere (Koren, Anderson, Fischer, Copeland & Jensen, 1981). K-562 cells were labeled with s~Cr by incubating 5 × 106 cells m1-1with 100 gCi of the sodium ~xCrO4 (New England Nuclear) for 1 h at 37°C. After washing three times with cold RPMI 1640 medium, the cells were adjusted to 2 × l0 s cells ml -~ in RPMI 1640 medium and used as target. Effector lymphocytes were suspended at a density of 8 × 106 m1-1 in RPMI 1640 medium. 50 ~l of effector lyrnphocytes and 50 ~l of target K-562 cells were incubated at a 40:1 ratio in U bottom microtiter trays. 2-chloroadenosine was added to the wells at the final concentrations stated in the figure. After centrifugation at 500 G for 5 min., plates were incubated at 37°C for 4 h. At the end of incubation the microtiter trays were centrifuged at 500 G for 5 rain. Then 100 /~l of supernatant were carefully removed from each well and counted. The results were calculated as: percent specific lysis = (clamtest samplecpm spontaneous release) + (total cpm-spontaneous release) × 100. Spontaneous release represents the release of 5'Cr from K-562 cell incubated alone, total

cpm is the ~'Cr activity in 1 × 104 labeled cells treated with IN-HCL. Effects of various concentrations of 2-chloroadenosine without effector lymphocytes on the release of ~'Cr from K-562 cell is also examined. These experiments were made in triplicate.

Cell viability Effect of 2-chloroadenosine on the cell viability was studied. Lymphocyte suspensions at a density of I × 106 cells ml -' in culture medium containing various concentration of 2-chloroadenosine were incubated for 4 days at 37°C in a humidified atmosphere with 5o7o CO2 and 95°7o air. After one to 4 days, cell viability was assayed by the trypan blue dye exclusion test.

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50 o

01 0.2 0.4

0.8

concentration

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31

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125

25.0 ,.M

of 2-chloroadenosine

Fig. 1. The effects of 2-chloroadenosine on mixed lymphocyte reactions (11-II L blastogenesis stimulated with mitogens (Con A: ©----O, PHA: O - - O , PWM: /x..... A) and natural killer cell activity ( e e). Results are the mean values from triplicate measurements.

RESULT Figure 1 shows the results of the effects of 2-chloroadenosine on the various lymphocyte functions. 3H-thymidine incorporation in allogenic mixed lymphocyte culture was most severely suppressed by 2-chloroadenosine with a detectable effect at 0.1 eM and a maximal effect at approximately 6.2 ~M. This inhibitory effect was dose-dependent. Adding 1.6 glVland 3.1 ~ 2-chloroadenosine resulted 30% and 60% i n h i b i t i o n of 3H-thymidine incorporation respectively. Similarly, the mitogen-induced proliferation of human lymphocytes was also generally suppressed by 2-chloroadenosine with a detectable effect at 1.6 ~M

Differential Inhibition of Lymphocyte Function by 2-Chloroadenosine and a maximal effect at approximately 25/~M. The inhibitory effect was dose dependent. Blastogenesis induced by Con A and P W M was most inhibited by low doses of 2-chloroadenosine while its inhibition of that induced by P H A was less marked. Inhibition rates by 3.1 /~M 2-chloroadenosine of lymphocyte blastogenesis induced by Con A, PWM and PHA were 430/o, 37% and 290/0 respectively. In contrast to those results, natural killer cell activity was not inhibited by 2-chloroadenosine to the same degree. 2-chloroadenosine at 1.6 /~M to 25.0 /JM concentrations resulted in a 4 1 - 55% inhibition of natural killer cell activity, slCr release from K-562 cells without human lymphocytes was not influenced by up to 25/~M 2-chloroadenosine. Within one day there were no significant differences in cell viability between the cells treated with 2-chloroadenosine and untreated control cells. After 4 days, the viability of lymphocytes treated with 2-chloroadenosine at 3.1 gM, 6.2 paM and 12.5/~M was 88.5%, 78.6% and 60.4% respectively.

DISCUSSION Adenosine has been shown to alter T lymphocyte functions such as the mitogenic response to PHA, Con A or lymphocyte mediated cytolysis (Carson & Seegmiller, 1976; Wolberg, Zimmerman, Hiemstra, Winston & Chu, 1975). It is confirmed that adenosine treatment results in elevation of cellular cyclic-AMP levels (Hershfield, Synder & Seegmiller, 1977). Adenosine is taken up by the cell and easily phosphorylated to adenine nucleotides and converted to cyclic-AMP. Another possibility is that adenosine stimulates a special receptor. Indeed, Birch & Polmer (1982); Birch, Rosenthal & Polmar (1982); Bessler, Djaldetti & Moroz (1982); and Moroz & Stevens (1980) showed that adenosine regulates the expression of T lymphocyte for receptors due to stimulation of adenosine receptors. Previous studies concerning adenosine receptors in lymphocytes have shown that about 10°7o of lymphocytes have this receptor (Moroz

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et ak, 1981; Saxon et al., 1981).

It is well recognized that 2-chloroadenosine is a poorly metabolized adenosine analogue and it binds to cell surface receptors in many tissues (Churchill, 1982; Clanachan & Muller, 1980; Crawley, Patel & Marangos, 1981; Schnebli et al., 1967; Wu & Phillis, 1982). Therefore, the effects of 2-chloroadenosine on lymphocyte function may be explained by stimulation of the adenosine receptor. In this experiment, 2-chloroadenosine caused a dramatic suppression in mixed lymphocyte reactions. In addition, the mitogen-induced blastogenesis of lymphocytes was generally suppressed by 2-chloroadenosine. Blastogenesis induced by Con A was most suppressed by low concentration of 2-chloroadenosine. These results are compatible with those of Moroz et al. (1981) who reported that the adenosine receptor positive cells are found in E-rosetting cells and null cell fractions. However, this experiment also showed that PWM stimulated blastogenesis was also inhibited by 2-chloroadenosine. Natural killer ceils are a subset of lymphocytes that appear to have a specific capacity for spontaneous cytolysis of tumor and virus-infected cells. Natural killer cell activity does not imply cell proliferation. When the effect of 2-chloroadenosine on human natural killer cell activity was tested, this activity was suppressed about 55% by high concentrations of 2-chloroadenosine. 2-chloroadenosine itself caused no direct cytotoxic effect assessed by the inability to alter ~lCr release from labeled K-562 cells. The mechanisms by which 2-chloroadenosine inhibits natural killer cell activity are a matter of speculation but is possible that 2-chloroadenosine functions by partly inhibiting all natural killer cells or alternatively by activating suppressor ceils. In summary, many subsets of human lymphocytes have been suppressed by 2-chloroadenosine and these results suggest that many subsets of human lymphocytes have an adenosine receptor. We are attempting to separate different cell types and to study the receptor density and affinity on these lymphocyte subsets.

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