Indomethacin augments inhibitory effects of interferons on lymphoproliferative response

lmmunok>gv Letters. 7 (1984) 321 324 Elsevier lmlet 460

INDOMETHACIN

AUGMENTS INHIBITORY LYMPHOPROLIFERATIVE

EFFECTS OF INTERFERONS RESPONSE

ON

N. AOKI, Y. M A R U Y A M A , Y. O H N O and Y. A Z U M A I Department ~f Medicine, Kinki University School ~/ Medicine. Savama. Osaka. and tNational Kyoto Hospital, Kvoto, Japan (Received 19 October 1983) (Modified version received 27 December 1983) (Accepted 10 January 1984)

I. Summary

Lectin-induced lymphoproliferative response was enhanced by the addition of indomethacin to the culture and in contrast, suppressed by the addition of interferon. However, when indomethacin and interferon were concomitantly present in the lectindriven cultures, the suppressive effect of interferon on the lymphocyte blastogenesis surpassed the enhancing effect of indomethacin on the response. Furthermore, and unexpectedly, the inhibitory effect on the response due to interferon was definitely augmented in the presence of indomethacin as compared with the response in the absence of indomethacin.

are known to possess counteracting effects on the expression of mitogen-induced lymphoproliferative response in culture conditions where they are added separately. It is not known, however, if there are interactive effects of interferon and indomethacin on the blastogenic response when the two agents are concomitantly present in mitogen-driven lymphocyte cultures. This kind of investigation would be important, since indomethacin has been used frequently in association with the administration of interferon preparations in order to suppress fever occurring often as a side effect caused by interferon preparations. The present study was undertaken to examine in vitro interactive effects of interferon and indomethacin on the expression of lectin-induced lymphocyte blastogenesis.

2. Introduction 3. Materials and methods

It is now well established that interferon exerts a variety of biological effects [1] distinct from its 'classical' antiviral activity [2]. For instance, interferon could regulate immune responses as shown by the inhibition of DNA synthesis in lymphoproliferative response, suppression of antibody synthesis, and enhancement of natural killer activity. On the other hand, indomethacin has been reported to enhance mitogen-induced lymphoproliferative response through the inhibition of prostaglandin synthesis [3]. Thus, interferon and indomethacin K
interferon

PHA

lymphocyte

0165 2478 / 84 / $3.00 © Elsevier Science Publishers B.V.

Peripheral mononuclear cells (PMC) were obtained from heparinized blood utilizing Ficoll Hypaque (Pharmacia) separation [4] and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (Gibco) at 37 °C in 5% CO 2 in moist air. The PMC were cultured in quadruplicates in fiat-bottom wells of microculture plates (Falcon), each well containing 105 cells in 0.25 ml of the culture medium with or without additives. The additives include phytohemagglutinin P (PHA, Gibco), indomethacin (Sigma) and interferon preparations. Interferon-a and interferon-C/were gifts from the Green Cross Corporation, Osaka, Japan. Specific 321

line 1 vs. left of line 3). Furthermore, it is clear that a combination of indomethacin (1 # g / m l ) and interferon-c~ at various concentrations never affected the lymphocyte response which was not stimulated with P H A (line 3). With respect to PHA-driven lymphocyte blastogenesis, it is notable that the inhibitory effects of interferon-~ on the PHA-driven cultures were dose-dependent in the culture conditions where interferon and P H A were concomitantly present, despite the fact that the decline of PHA-driven blastogenic response between 0 and 12.5 of interferon concentration was more markedly observed in the presence of indomethacin than in the absence of it (line 2 vs. line 4). When no interferon was included in the cultures, reported indomethacin effects [3] were apparently confirmed in the present study, i.e., an enhancement of P H A induced lymphocyte blastogenesis due to the addition of indomethacin was significantly observed (19,557 _+ 480 vs. 21,125 _+ 510, p = 0.02). However, this indomethacin-induced enhancing effect on P H A - d r i v e n lymphocyte blastogenesis was no longer noted in the culture conditions where a further 12.5 or 25 U / m l of interferon was added, or more precisely it should be noted that the addition of interferon at these concentrations caused a converse reduction of PHA-driven lymphocyte blastogenesis in the cultures which included indomethacin

activity w a s 107 U / m g protein for both interferon preparations. The additives were dissolved in the culture medium and adjusted in a final culture volume of 0.25 ml to concentrations of a 1:100 dilution for P H A , 1 p.g/ml for indomethacin and several doses ranging 12.5 50 U / m l for interferons. The P M C were cultured for 72 h at 37 ° C and the cells were pulsed with [1251]iododeoxyuridine ([125I-1UdR], NEN, 0.5/~Ci/well) for the last 4 h of culture to monitor D N A synthesis due to the lymphoproliferative response. Subsequently, the cells were harvested on glass wool filters using an automated cell harvester (Titertek). Incorporation of [1251-1UdR] into D N A was measured in a g a m m a counter and expressed in cpm. Statistical evaluation for significant difference between the two groups was performed using Student's t-test.

4. Results and Discussion As shown in Table l, interferon-~ alone, at each concentration tested, never altered significantly the baseline response of the lymphocyte cultures which included neither indomethacin nor P H A (line 1 of the table). Also, indomethacin at a concentration of 1 # g / m l never changed significantly the lymphocyte response when it was not driven with P H A (left of

Table I Interaction of interferon-c~ and indomethacin in PHA-induced lymphoproliferative response Cultures

Concentration of interferon-a (U / ml) 0

I.IMC() PHA ( ) 2. IMC ( ) PHA, 1:100

244+ 72 19557+_480

12.5

25

50

283_+ 56 (NS)

291+ 60 (NS)

252+_ 33 (NS)

13317_+973 (p - 0.001)

12539_+700 (p - 0.001)

9259_+870 (p - 0.0001)

3.1MC, l # g / m l PHA ( )

257+115

296_+105 (NS)

301+ 98 (NS)

272+ 44 (NS)

4. IMC, I #g/ml PHA, I:100

21 125+510

11 543+573 (p 0.0001)

10925+935 (p=0.0001)

10241 _+490 (p=0.0001)

Values are the mean _+ SD (n = 4) being expressed in cpm. Statistical values show comparison of cpm results between the cultures without interferon and the cultures with interferon at each concentration between 12.5 and 50 U/ml included in the same line. NS, not significant; IMC, indomethacin; PHA, phytohemagglutinin P.

322

CPM

b)

a)

L

2xlO"

..** O ............. C k

"" * .*......

lXl0"

I

0

IFN-a

I

12.5

I

25

I

50

concentration(u/mt)

~)

IF'N-,6'

I

12.5

** .... Q .

I

25

I

50

concentration(u/mt)

Fig. 1. Effects of various concentrations of interferon on PHAinduced lymphoproliferativcresponse. (a) Interferon-a, and (b) interferon-B, ...O..., PHA-driven culture without addition of indomethacin and • , PHA-driven culture with addition of indomethacin ( 1 #g/ml). Each point represents the mean of quadruplicate cultures. Statistical evaluation (Student's t-test) for the difference of DNA synthesis(cpm) between with- and withoutindomethacin groups at each interferon concentration is shown by NS (not significant), * (p - 0.05) and ** (p - 0.02). IFN, interferon.

(13,317 + 973 vs. 11,543 + 573, p = 0.02; 12,539 + 700 vs. 10,925 + 935, p = 0.02). This crossrelationship between PHA-cultures with and without indomethacin along with the increase in the concentration of interferon is shown in Fig. 1 (left panel). At an interferon-c~ concentration of 50 U/ml, there was no statistically significant difference between P H A - d r i v e n lymphocyte cultures with or without indomethacin. A n o t h e r run of experiment using interferon-/3 instead of interferon-a was performed in order to compare the effects between the interferons (Fig. 1). It is obvious that basically similar results were obtained for interferon-/3 (Fig. I b) to the data for interferon-a described in the table or Fig. l a, indicating that interaction of indomethacin and interferon in P H A - d r i v e n lymphocyte cultures resulted in an unexpected further suppression of the blastogenic response as compared to the suppression due to interferon alone. Basically similar results to the above were obtained in studies repeated three times dealing with interactive effects of indomethacin and interferon. Previously, it has been shown that suppressor cells could be generated

generally in conventional lymphocyte cultures including the conditions employed in the present study [5,6]. Recent studies have yielded conflicting results on the effect of indomethacin on T-cell response [7-9]. Precise mechanism for the observed phenomenon described in the present study is not known at this moment, although interplays between modulation of suppressor cell functions due to indomethacin and immune regulation attributable to interferon activity would be complexly involved. Concerning the relation of inflammation to interferon, it has been reported by Yaron et al. [10] that interferon could stimulate production of prostaglandin E which has emerged as one of the principal mediators of inflammation, although Koltai and Mecs have suggested that interferon may have antiinflammatory properties [11]. Except for the report by Yaron et al., the present culture condition which included both interferon and indomethacin would lead to an environment with two counteracting activities for prostaglandin E synthesis, interferoninduced stimulation and indomethacin-induced suppression by inhibiting prostaglandin synthetase activity. Naturally, lymphoproliferative response in the present culture condition should have been enhanced if interferon-induced biological effects on lymphocytes were limited to the mechanism through prostaglandin increase, since the interferon effect was reportedly prevented by suppression of prostaglandin production [10]. As was the case, PHA-induced lymphoproliferative response was more severely suppressed in the presence of both indomethacin and interferon than in the presence of interferon alone, suggesting that interferon would affect the lymphocyte culture in other mechanisms than prostaglandin production. The present report suggested a possibility that interferon effects on lymphocyte responses might be augmented in the presence of indomethacin. Thus, since clinical use of interferon as an anti-cancer agent depends largely on interferon effects on immunological responses, clinical benefits due to an additive use of indomethacin to interferon treatment might be expected. The interaction of both agents in each immunological response is not yet clear and is under further study.

323

Acknowledgements T h e a u t h o r s a r e g r a t e f u l t o D r . H. A r i m u r a a n d D r . H. M o r i s e , t h e G r e e n C r o s s C o r p o r a t i o n , J a p a n , f o r t h e gift o f i n t e r f e r o n p r e p a r a t i o n s . H e l p f u l disc u s s i o n w i t h P r o f e s s o r T. Y a m a m o t o w a s g r e a t l y a p p r e c i a t e d . T h e a u t h o r s a l s o t h a n k M s . T. A o k i f o r her secretarial assistance. This work was supported in p a r t b y a g r a n t f r o m t h e E d u c a t i o n M i n i s t r y o f Japan.

References [1] Stewart 11, W. E. (1981) in: The Interferon System (W. E. Stewart 11, Ed.) pp. 223, Springer-Verlag, New York. [2] Isaacs, A. and Lindenmann, J. (1957) Proc. R. Soc. Ser. B 147, 258 267.

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[3] Goodwin, J. S., Messner, R. P. and Peak, G. I. (1978),1. Clin. Invest. 62, 753 760. [4] Aoki, N., Pinnamaneni, K. and DeGroot, L. ,1. (1979)J. Clin. Endocrinol. Metab. 48, 803 810. [5] Aoki, N. and DeGroot. L. ,1. (1979) Experientia 35, 1515 1517. [6] Aoki, N. and DeGroot, L. J. (1981) Experientia 37, 901 902. [7] Rao, K. M. K., Schwartz, S. A. and Good, R. A. (1979) (.'ell lmmunoL 48, 155 165. [8] Soppi, E., Eskola, J. and Ruuskancn, O. (1982) Immunopharmacology 4, 235 242. [9] Badger, A. M., Griswold, D. E. and Walz, D. T. (1982) lmmunopharmacology 4. 149 162. [10] Yaron, M., Yaron, I., Gurari-Rotman, D., Revel, M., Lindner, H. R. and Zor, U. (1977) Nature (London) 267, 457 459. [11] Koltai, M. and Mecs. E. (1973) Nature(London) 242, 525 526.