The influence of culture conditions on the suppression of lymphocyte transformation by salicylate

Journal o f Immunological Methods, 49 (1982) 65--73

65

Elsevier Biomedical Press

THE INFLUENCE OF CULTURE CONDITIONS ON THE SUPPRESSION OF LYMPHOCYTE TRANSFORMATION BY SALICYLATE

W.R. WILLIAMS and L.A.G. DAVIDSON

Asthma Research Unit, Sully Hospital, Penarth, South Glamorgan CF6 2 Y A , U.K. and Commonwealth Institute o f Health, University o f Sydney, Sydney, Australia (Received 5 June 1981, accepted 3 September 1981)

Therapeutic concentrations of salicylate enhanced or inhibited human lymphocyte PHA transformation, depending on the conditions of culture. Responses to salicylate were influenced by serum and cell concentration, PHA mitogenic activity and concentration. In particular, pre-incubation of the mononuclear cell population, in salicylate-free medium, made lymphocyte transformation more susceptible to inhibition. The results suggest that the inhibitory effect of salicylate on lymphocyte transformation may be mitigated by a factor released from adherent mononuclear cells. The extent to which culture conditions influence the results of this test emphasise its limitations for assessing the effects of drugs on l y m p h o c y t e function in vivo. Key words: l y m p h o c y t e transformation -- salicylate -- drug inhibition o f l y m p h o c y t e

proliferation

INTRODUCTION

Extensive studies into the effect of aspirin on lymphocyte transformation to assess the in vitro anti-inflammatory action of this drug have produced variable results. Pachman 11971) reported that aspirin and sodium salicylate were potent inhibitors of phytohaemagglutinin (PHA) lymphocyte transformation (94% suppresssion by 300 pg/ml salicylate). In contrast, Viken (1976) found salicylate had little effect (20% suppression by 800 pg/ml). Dissimilar results were also obtained following oral administration of aspirin. Ganter and Zuckner (1965) claimed that a single therapeutic dose of aspirin completely inhibited in vitro lymphocyte transformation, for up to 36 h. Crout et al. (1975), however, observed a moderate suppression (49%) peaking at 12 h, with no correlation between plasma salicylate and the degree of suppression. The inconsistency of these reports is disturbing. Little confidence should be placed in results of lymphocyte transformation tests with other less well studied drugs until the reasons for such variation are established. This paper 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press

66 investigates some technical aspects of the test that might account for these anomalies. MATERIALS AND METHODS

Subjects Blood was taken from normal healthy volunteers, who were n o t taking aspirin or any other therapy. Culture medium Eagle's (MEM) 10 × conc. (Wellcome) was reconstituted with sterile pyrogen-free water, NaHCO3 (20 mM) and supplemented with complement inactivated, pooled normal human serum. No antibiotics were used. Sodium salicylate was from B.D.H. Freshly made up medium had a pH of 7.2--7.4 and was filtered (pore size <0.2 pm) before use. Samples of purified phytohaemagglutinin (PHA) of different mitogenic activity (WeUcome) were reconstituted and diluted with sterile 0.9% saline. [Methyl-3H]thymidine (specific activity 15--20 Ci/mmol) was purchased from the Radiochemical Centre, Amersham and diluted with sterile 0.9% saline (10 Ci/mmol). Lymphocyte separation and culture Mononuclear cens were separated from heparinised blood (20 units/ml, preservative-free heparin) using 1.078 Sg Ficoll-Hypaque (BSyum, 1968) and washed 3 × in phosphate-buffered saline (PBS). They consisted primarily of lymphocytes, with up to 20% monocytes. These were cultured in medium at 1 × 106/ml in flat-bottomed microtitre plates. Replicate cultures (6) were set up in a randomised plate design, to minimise inconsistencies due to evaporation. Transformation tests c o m m e n c e d with the addition of 10 pl. PHA or saline, to 200 pl cell suspension. After 48 or 68 h culture at 37°C, in a humidified atmosphere containing CO2, 50 gl o f tritiated thymidine were added to each well. Cultures were terminated after a further 4 h incubation. In some experiments, mononuclear cells were pre-incubated in medium with 5% serum for 18 h, in plastic Petri dishes. Non-adherent l y m p h o c y t e s were removed with a Pasteur pipette and washed before use in transformation cultures. Culture harvesting and scintillation counting Each well was emptied into a test tube containing 2 ml cold 0.9% saline, to which was added 3 saline washes o f the well. Tube contents were vacuum filtered through separate 0.9 cm glass-fibre paper discs, followed by t w o 2 ml washes of 5% TCA, 2 ml 95% ethanol and 2 ml absolute ethanol. Dry filter discs were treated with 40 pl 1 M hyamine hydroxide and the radioactivity was counted using a double vial assembly (Robbins et al., 1972).

67 RESULTS

The effects of salicylate on lymphocyte transformation in 10% serum cultures proved to be va~riable (Fig. 1). In the presence of 2 0 0 / l g / m l salicylate, only 2 cultures from 9 different individuals were inhibited (mean 24%) and thymidine incorporation was enhanced in 6 of the other cultures (mean 35%). Spontaneous thymidine incorporation (saline control) was inhibited in 4 of 5 cultures (mean 47%). For substantial inhibition o f lymphocyte transformation in 10% serum cultures, 800/zg/ml salicylate was necessary. However, in cultures with a higher serum concentration (Fig. 2) inhibition was achieved with 200 pg/ml (mean inhibition 59%, 40% serum). The influence of serum supplementation of culture medium on spontaneous thymidine incorporation was more variable than in cultures stimulated with PHA. Short term or overnight incubation o f cells with salicylate, before addition of PHA, did not increase the sensitivity of transforming lymphocytes to the drug. Small changes in PHA concentration (Table 1) or a reduction in cell num-

• cultures

with

o cultures

with

BO

60

4C • • u

20

u

oE

o

•

• o

o

•

•

•

o

8 -2c o

o

c_ ~ E

@o gill 0

- 4C .o

U ~ 612

oo o

o

2OO

40O

-812 o

Salicylate (Hg/ml )

Fig. 1.

800

PI-IA sahne serum

t0%

68 100

8C

8

h

6c

40

2 20

o

g ~ c

-20

g g 5

-4o

-60

-80 10

20

30 40 10 % serum concentrcltiorl

sahne 20

30

40

Fig. 2.

bers per culture could increase the sensitivity of the test to salicylate. There was often a considerable difference in the salicylate sensitivity of cultures stimulated with different batches of PHA. A period of pre-incubation in drug-free medium, before transformation, significantly increased the sensitivity of lymphocytes to salicylate (Table 2). This difference between pre-incubated and non pre-incubated cultures was particularly evident with a PHA concentration of 0.5 pg/ml. Comparing transformation responses of pre-incubated cells, the increased sensitivity obtained by using 1/~g/ml PHA instead of 0.5 ~ug/ml was not significant (P > 0.1). This was also true for non-pre-incubated cultures. A longer incubation period with thymidine (20 h) decreased the sensitivity of tests to salicylate, in pre-incubated and non-pre-incubated cultures, though again this was not significant (P > 0.1 ). The addition of lymphocytes to wells containing adherent cells (Table 3, D) increased thymidine incorporation in lymphocyte cultures (C) by more than 20%. Thymidine incorporation was increased to a greater extent in salicylate inhibited cultures, by adherent cens (A and D). This reduced the differences in thymidine incorporation between control and salicylate inhibited

Serum 20%. Salicylate 300 ~g/ml, 52 h culture. * Significant inhibition (P < 0.05).

% change

cpm

0.I

0.2

cpm

% change

0.3

0.4

0.5

(31mitogenic units) 1

1

cpm

% change

cpm

% change

cpm

% change

cpm

% change

cpm(± S.D.)

(19mitogenic units)

PHA (/~g/ml)

24 645 (±2914) --25*

23621 (±2376) 0

26 064 (±3364) +9

25 567 (±4 879) --30*

30121 (±6276) --22*

38 546 (±2 666) --31"

27 317 (±4 670) --56*

33600 (±6700) --70*

31339 (±4064) --62*

37328 (±1 777) --66*

24 181 (-+4 075) --80*

7 119 (±4085) --34

30190 (±6130) --34*

38 226 (-+3999) --46*

40 016 (±2 284) --74*

33 892 (-+2852) --38*

39 232 (-+7 475) --43*

29030 (-+3 547) --74*

5

4

3

1

2

Lymphocytes 0.5 x 106/ml

Lymphocytes I x 106/ml

Subjects

Inhibition of lymphocyte transformation by salicylate. Effects of changes in PHA activity and concentration.

TABLE 1

16 807 (-+4 003) --22

31 015 (+6 467) --56*

24 785 (-+8 792) --49*

21447 (-+5 505) --82*

6

O~

54.3 (-+19.9)

% inhibition by salicylate (-+S.D.)

51.3 (-+26.6)

82 248 (218 375) 73.3 (-+20.0)

31 137 (-+1 974) 61.3 (-+24.2)

74 600 (-+19 684) 80.5 (-+10.8)

29 609 (-+7 611)

4

78.5 (-+10.6)

75 972 (-+7 806)

20

PHA (0.5 /.tg/ml)

Pre-incubation

Results are average values for 4 subjects. Serum 20%. Salicylate 300 #g/ml. PHA batch activity°19 mitogenic units. Lymphocytes 0.5 x 106/ml, 52 h culture. Significance of changes in salicylate inhibition pre-incubated vs. non pre-incubated cultures F o r all cultures --P < 0.01. With PHA 0.5 pg/ml --P < 0.02. With PHA 1 pg/ml --P 0.1--0.05.

35 278 (+1 560)

Control response (cpm ~ . D . )

20

4

4

20

PHA (1 /.tg/ml)

PHA (0.5 gg/ml)

No pre-incubation

Incubation period with thymidine (h)

87.5 (-+4.7)

24 873 (-+8 782)

4

86.3 (-+4.4)

58 341 (-+9 784)

20

PHA (1 /./g/ml)

TABLE 2 Inhibition of lymphocyte transformation by salicylate. Effects of cell pre-incubation, PHA concentration and different incubation periods with thymidine.

Q

71 TABLE 3 Inhibition of lymphocyte transformation by salicylate. The effect of an adherent cell population. Protocol

A B

C D A B

C D

Subject 1

Subject 2

Control cultures (cpm -+S . D . )

% change with salicylatea

Control cultures (cpm -+S.D.)

% change with salicylate a

34 37 28 36

--45 --68 --52 --43

35 216 33 546 26 343 31753

--39 --82 --68 --49

103 977 282 026

(+6 538( (+8 054) (+5 138) (+4 528)

(+11 980) (+4 718) (-+4 342) (-+6 385)

transformation test set up immediately PHA and salicylate added to cultures after 24 h transformation test set up after removal of adherent cells by pre-incubation (24 h) as C lymphocytes added back to wells containing adherent cells

Serum 20%. Salicylate 300 /2g/ml. Lymphocytes 0.5 × 106/ml, 52 h culture. a Significant inhibition (P < 0.05).

cultures. The increased salicylate sensitivity of pre-incubated cells was n o t entirely related to the removal o f adherent cells. Cultures left for 24 h before adding PHA and salicylate (B) were also more sensitive. DISCUSSION

The inhibitory effect of therapeutic concentrations of salicylate (200-300 pg/ml) proved to be d e p e n d e n t on culture conditions. The test was made more sensitive to salicylate b y pre-incubation of cells before culture and the discriminate use o f serum and different PHA concentrations. Sensitivity was also affected by cell concentration and the mitogenic activity of PHA used for l y m p h o c y t e stimulation. The culture conditions regulating salicylate p o t e n c y , at first, appear unrelated b u t may all act on an adherent cell with the ability to mitigate the inhibitory effect of the drug. M o n o c y t e s are known to augment PHA lymp h o c y t e responses, through the release o f a soluble factor (Hansen et al., 1977). This factor m a y prevent salicylate binding to l y m p h o c y t e s (Anthony and Panusch, 1978). Removal of adherent cells b y pre-incubation would allow salicylate to bind to lymphocytes. An increase in cell culture concentration (without pre-incubation) would increase the effect of adherent cells in relation to the salicylate concentration. Lipsky and Ziff (1977) similarly found that l y m p h o c y t e transformation inhibition b y gold salts was increased in m o n o c y t e depleted cultures and reversed by supplementation o f cultures

72 with monocytes. Sohnle and Collins-Lech (1979), however, considered that changes in the responding cells, rather than destruction of a suppressor cell population, were responsible for augmentation of lymphocyte transformation after a 24 h pre-incubation period. Inhibition of thymidine incorporation in cultures without PHA, by low salicylate levels (200 ~zg/ml), indicates that release of the adherent cell factor is PHA dependent. High serum concentrations may increase salicylate sensitivity by limiting the effect of PHA on monocytes, or the activity of the adherent cell factor. The stimulatory effect of indomethacin on lymphocyte transformation has also been noted to be dependent on mitogen concentration (Rao et al., 1979). The requirement for suitable culture conditions to demonstrate inhibition by salicylate explains the large variation in sensitivity reported by different authors. Use of aspirin, instead of salicylate, is unlikely to account for any large discrepancy, as aspirin rapidly hydrolyses to salicylate in vitro (Pachman et al., 1971) and in vivo (Rowland and Riegelman, 1968). The greatest and most consistent suppression has been obtained with highly purified lymphocyte cultures, achieved by passage of cells through cotton or nylon (Pachman et al., 1971). Others have used as few cells as 1 × l 0 s per culture (Opeltz et al., 1973; Crout et al., 1975) or have used a large volume of culture medium in relation to cell concentration, e.g., 8 × 10 s lymphocytes in 4 ml medium (Dam et al., 1975). The use of foetal calf serum (FCS) is also associated with high inhibitory values (Pachman et al., 1971; Dam et al., 1975). Values for 800 pg/ml salicylate in transformation cultures were 57% in medium with FCS and 20% with human serum (Viken, 1976). Lymphocyte transformation has been w i d e l y u s e d to test properties of therapeutic drugs on lymphocyte function. Technical aspects of the test for which there are in vivo values (drug:albumin molar ratios and cell populations) should reflect these as closely as possible. The in vitro response of transforming lymphocytes to drugs is, however, complex and very dependent on culture conditions. This emphasises the artificial nature of the test and its limitation for assessing the effects of therapeutic drugs on lymphocyte function in patients. ACKNOWLEDGEMENT

The authors thank the Welsh Medical Research Fund for financial support. REFERENCES

Anthony, C.R. and R.S. Panusch, 1978, Clin. Exp. Immunol. 31,482. BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 31. Crout, J.E., B.H. Hepburn and R.E. Ritts, 1975, New Engl. J. Med. 292, 221. Dam, W.C., F.D. Malkinson and H. Gewurz, 1975, Experientia 31,375. Ganter, Jr., G.E. and J. Zuckner, 1965, Arthr. Rheum. 8,443. Hansen, G.S., B. Rubin and S.F. Sorensen, 1977, Clin. Exp. Immunol. 29, 295.

73 Lipsky, P.E. and M. Ziff, 1977, J. Clin. Invest. 59,455. Opeltz, G., P.I. Teraski and A.A. Hkata, 1973, Lancet i, 478. Pachman, L.M., N.B. Esterly and R.D.A. Peterson, 1971, J. Clin. Invest. 50, 226. Rao, K.M.K., S.A. Schwartz and R.A. Good, 1979, Cell. Immunol. 48, 155. Robbins, J.H., J.J. Gart, W.R. Levis and P.G. Burk, 1972, Clin. Exp. Immunol. 11,629. Rowland, M. and S. Riegelmann, 1968, J. Pharm. Sci. 57, 1313. Sohnle, P.G. and C. Collins-Lech, 1979, Clin. Immunol. Immunopathol. 13, 47. Viken, K.E., 1976, Acta Pathol. Microbiol. Scand. Sect. C 84,465.