Cancer Letters, (1992) 43 .- 49

Elsevier Scientific Publishers Ireland Ltd.

43

Decreased interleukin-2 P-chain receptor expression by interleukin-4 ton LGL: influence on the IL-2 induced cytotoxicity and proliferation

Christer Lindqvist aTb, Ann-Len &trnan b, Christian Okerblorn b and Karl A kerrnan b

‘Centre for Biotechnology, SF-20521, Turku and bDepartment of Biochemistry and Pharmacy, ,&o Akademi

University, SF-20500, Abo (Finland)

(Received 18 February 1992) (Accepted 12 March 1992)

Summary

Addition of incr’easing amounts of inter- leukin-4 (IL-4) to large granular lymphocytes (LGL) had a selective downregulative effect on the interleukin-2 P-chain (~70) receptor ex- pression. A 40% inhibition of the p70 expres- sion compared to untreated cells was already observed after a 24-h incubation with IL-4. This decrease in ~7’0 receptor expression had a marked suppressive effect on their pro- liferative response to IL-Z. In addition, LGL cultured in the pre.sence of both IL-2 and IL-4 substantially decreased the cytotoxic activity against the erythroleukemia cell line K562. Our data therefore indicate an important regulatiue role for IL-4 on the LAK-generation.

Key words: interleukin-4; large granular lymphocytes; interleukin-2 receptor.

Introduction

Large granular lymphocytes (LGL) are a population of morphologically homogeneous

Correspondence to: Christer Lindqvist, Department of

Biochemistry and Pharmacy, Abe Akademi University,

SF-20500, Turku, Finland.

cells with cytoplasmic azurophilic granules which contain cytochemically detectable acid hydrolases [1,22]. In addition to the cytotoxic functions described for LGL, they can also release a variety of lymphokines, such as IL-l, IL-2, CSFs and interferons [23,6]. The LGL mainly responsible for the cytotoxic activity are characterized by expression of the CD56 and CD16 markers, but lacking the CD3 TCR com- plex [8,19]. In addition, they also predomi- nantly express the p70 subunit and low levels of the p55 subunit of the IL-2 receptor [3,12,18]. Due to this IL-2R expression, they are also predominantly responsible for the in- creased ability of peripheral blood mono- nuclear cells (PBL) to lyse NK-resistant targets after treatment with IL-2, defined as LAK ac- tivity [16,17].

IL-4, like IL-2, is a multifunctional lym- phokine mainly produced by T-cells [4]. It was originally characterized for its ability to stimu- late the proliferation of B-cells, but properties such as enhanced expression of class II MHC molecules, CD 23 and its own receptor have later been described [15,20,28]. We have previously demonstrated that IL-4 has a selec- tive downregulative effect on the IL-2 P-chain receptor expression on PBL in the presence or absence of phytohemagglutinin (PHA) and OKT 3 [lo]. These studies have now been

0304-3835/92/$05.00 (3 1992 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland

44

extended to LGL and the effects of IL-4 on LGL proliferation/LAK cell generation and cytotoxic capacity have been analyzed. The role of IL-4 in LAK-ceil generation and therapy is discussed.

Materials and Methods

Antibodies and reagents The anti-Tat (~55) and the Mik-l/3 (~70)

anti-IL-2R monoclonal antibodies were a kind gift from Dr. T.A. Waldmann and Dr. M. Tsudo, respectively. Recombinant human IL-4 were kindly provided by Dr. S. Gillis (Im- munex Corporation). Recombinant IL-2 were purchased from Genzyme (Boston, MA).

Cells Peripheral blood mononuclear cells were

obtained from normal healthy donors by spin- ning down the anti-coagulant treated blood cells at 600 x g for 10 min. The plasma was collected and the cells were separated over a Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden) [ 11. The lymphocytes separated in a distinct layer, were first depleted of macro- phages by 30-min plastic adherence (37OC) and thereafter passed over nylon wool col- umns [5]. These cells were then fractionated by a seven-step discontinuous Percoll gradient (Percoll, Pharmacia, Sweden) and centrifuged for 30 min at 550 x g (18OC) [25]. The col- lected cell fractions were finally washed twice before the depletion of CD3 positive cells.

Depletion of CD3 positive cells The isolated nylon wool passed and Percoll

fractionated (fraction 3 - 5) LGL, were de- pleted of CD3 positive cells by Dynabeads M-450 (Dynal A.S, Oslo, Norway) (4 x lo* beads/ml) coated with covalently bound goat- anti mouse Ig. In brief, the isolated LGL were adjusted to 1 x lO’/ml in serum-free RPMI 1640 and incubated with anti-CD3 mono- clonal antibodies (10 pg/ml) [27] for 2 x 10 min (centrifuged for 5 min at 500 x g). Cells were then washed twice in PBS, before the im- munomagnetic beads were added. The mix-

ture was incubated for 2 x 60 min at 4OC, with gentle shaking every 10 min. Cells form- ing rosettes with the beads were removed with a magnet and the remaining cells (CD3-) were used and treated as described in the legends to the figures and Tables.

Proliferation assay Cells (1 x 106/ml) purified as described

were stimulated for 72 h in 200 ~1 cultures in flat-bottomed microtitre plates (Nunc, Roskilde, Denmark), with the indicated amounts of lymphokines. [3H]Thymidine in- corporation was determined after a 4-h pulse with 1 pCi/well of [3H]thymidine (Amersham International 25 Ci/mmol) at the end of the culture.

Target cells The target cell used in these experiments

was the erythroleukemia cell line K562 [ 111.

Fluorescence staining Cells treated as described in the legends to

the figures were distributed to v-bottomed 96-well microtiter plates (Costar, Badhoeve- dorp, The Netherlands) (1 x 106/well). Cells were then incubated with either anti-Tat (~55) or Mik-l/3 (~70) monoclonal antibodies for 45 min at +4OC. After two washes in RPM1 1640 + 5% FCS, an incubation step with FITC labelled rabbit anti-mouse Ig (Dakopatts Ab, Hggersten, Sweden) was performed for an additional 45 min (+4OC). Cells were then washed twice in RPM1 1640 + 5% FCS and finally analyzed on a FACSCAN flow cyto- meter (Becton Dickinson, Mountain View, CA).

LAK-generation and cytotoxicity assay LAK activity was generated by culturing cells

at 1 x 106/ml with lymphokines in either 50 ml culture flasks (Costar) or 24-well plates (Costar) for l-5 days. A 4-h 51Cr release assay was used to determine cytotoxicity. In brief, target cells were incubated with 50 &i of NaZ51Cr04 in 100 ~1 for 1.5 h in FCS (37OC) and washed twice in PBS. A graded number of

45

effector cells were incubated with a constant number (5 x 103) of 51Cr-labelled target cells. Following a 4-h incubation at 37OC, cells were centrifuged and supernatants collected and counted in a gamma counter for release of 51Cr from lysed cells. Percent specific lysis was calculated accordirq to the formula:

cpm,,,. dease - wmspont. deaSe

Lysis (%) =- x 100

cpmmax. re~ease - cpmSpont. re~eaSe

Results

IL-4 inhibits the IL-2 induced LGL pro- liferation

Table I shows that adding increasing con- centrations of IL-4, always decreased the LGL proliferation in the: presence of a constant amount of IL-Z. The same pattern was observ- ed no matter if a high (200 units/ml) or a low (down to 0.020 unil:s/ml) IL-2 dose was used. An evident inhibition was observed in both 48- and 72-h cultures, although it was necessary to add a higher IL-4 dose to the 72-h cultures in order to achieve thle same growth inhibition.

Table 1. Inhibition of IL-2 induced LGL proliferation by IL-4a.

IL-2 induced LAK-activity is affected by IL-4 We next analyzed if the IL-4 mediated

decrease in proliferation, as demonstrated in Table I for the IL-2 induced LGL-cells, and also to determine if it had any influence on LAK-activity. The results-as shown in Fig. 1, a 3-day incubation of LGL-cells with lympho- kines-clearly show the efficiency with which 200 units/ml IL-2 can induce LAK-activity. This was demonstrated by the capacity to kill the erythroleukemia cell line K562. Addition of IL-4 had a similar impact on the IL-2 induced LAK-activity as on the proliferation, namely a marked decrease of the IL-2 mediated effect. Freshly prepared LGL-cells, that were mixed with target cells in the presence of the lym- phokines (no preculture/generation of LAK- activity) gave a similar cytotoxic pattern as for the 72-h cultured effector cells, although the difference between the treated groups were much smaller (data not shown).

Reduced p70 expression on IL-4 treated LGL The FACS diagram illustrating the p7O/p55

chain expression of the IL-2R is shown in Fig. 2. A presentation of the data in a numerical

Dose of rIL-4 (units/ml) Dose of rIL-2 (units/ml)

200 2 0.02 0

48-h dim. time

0

0.5 5

50

17583 zt 1990 10681 + 892 6829 zt 840 6404 + 2666 12400 f 2447 8839 + 1517 6748 +z 472 7461 f 1187 12769 zt 1316 5636 + 1332 3847 zt 1660 1505 f 449 9789 zt 1059 6475 f 389 3785 zt 387 1783 zt 1059

72-h dim. time

0 44 957 f 3505 39799 f 1114 24933 f 7790 22609 A 7019 0.5 43 939 zt 8041 37539 f 1475 30877 f 3440 25 985 zt 878 5 37885 + 9819 28814 zt 6476 18532 f 962 18717 zt 3382

50 7983 f 7152 371 f 59 908 zt 596 131 + 72

“The indicated values are representative of four experiments. For further information, see Materials and Methods.

46

100

80

a

3

1 g 60

1:25

T/E ratio

Fig. 1. IL-2 induced LAK activity is affected by rIL-4. LGL were cultured for 72 h in the presence of medium (O-O) ; 200 units/ml IL-2 (O-O); 5 units/ml IL-4 (O-Cl); and 200 units/ml IL-2 + 5 units/ml IL-4

(m-m). The generated cells were then tested for cyto- toxic activity against K562. Similar results were obtained in three independent experiments. For further informa- tion, see Materials and Methods.

form, demonstrate that incubating LGL with 500 units/ml r-IL-4 for 24 h reduces the p70 expression by almost 40% compared with cells grown in normal culture medium (Table II). The p55 expression, which is normally absent or very low on LGL cells [3,12], were in these experiments only 3.9% for cells cultured in medium and 2.7% for 500 units/ml IL-4 treated LGL (Fig. 2, Table II). As a lym- phokine control we used both IL-la or IL-lo, but these lymphokines did not affect the p70

expression to any greater extent, whereas a slight increase (10 - 15%) was observed for the p55 expression (data not shown).

Discussion

It has previously been shown in the human system [24,7] and now confirmed by us in Fig. 1, that IL-4 does not induce LAK activity. In addition, IL-4 clearly suppressed IL-2 induced LAK cell activity (Refs. 14 and 24; Fig. 1). The mechanism behind the observed effect by IL-4 on LGL/LAK-cells has so far been unclear, although there have been speculations of (1) inhibitory factors induced by IL-4 and released from LAK precursor cells as well as (2) overgrowth of non-lytic cells, that have been reported to exist in the effector cell population [9]. However, the latter statement can at least be ruled out, since an IL-4 dose dependent in- hibition of the IL-2 dependent growth could be observed in the cultures (Table I).

Earlier studies by Nagler et al., [14] have demonstrated that IL-4 has no effect on LGL activation with o-IFN. Therefore, based on their results, it is likely that the IL-4 mediated effect is directly acting on either the IL-2 molecule itself, its receptor or on the signal transduction mechanism for IL-2. Trials to displace ‘251-radiolabelled IL-2 with IL-4 always failed, indicating that the actual binding of IL-2 to its receptor is not affected (data not shown). We recently demonstrated that IL-4 can selectively downregulate the p70 chain ex- pression of the IL-2R, whereas the p55 chain is unaffected on peripheral blood mononuclear cells [lo]. Since this was also likely to be the explanation for the results observed with LGL and the fact that the p70 chain of the IL-2 receptor is mediating the signal transduction [18,26], we measured the p70 and p55 ex- pression on highly purified and IL-4 treated LGL. Here, a close to 40% inhibition of the p70 expression, compared to untreated cells was already observed after a 24-h incubation with IL-4 (Fig. 2, Table II), whereas IL-lo and IL-lb did not have any effect in this system (data not shown). Longer incubation times

47

I U-0

loo IO’ ld Id loo IO’ 102 ld loo 101 102 Id lo’

FLUORESCENCE INTENSITY

Fig. 2. Influence of rIL-4 on the expression of the p55/p70 IL-2 binding proteins on LGL. p55/p70 expression on

cells grown for 24 h in i he presence of medium (A), (B) , (C) ; 50 units/ml rIL-4 (D), (E) , (F) ; and 500 units/ml rlL-4 (G), (H), (I). The control fluorescence (FITC-labelled sec. ab.) is presented in figures (A), (D) and (G). Figures (B), (E) and (H) present the p55 expression, whereas (C), (F) and (I) show the p70 expression. The relative fluorescence intensity

is indicated on the x-axis and the relative cell number on the y-axis; the result is based on 5000 counted cells.

Table 11. Expression of IL-2 binding proteins in the presence of rIL-4 on purified LGL-Fig. 2 in a numerical form”.

Treatment of cells

0 units/ml rIL-4 50 units/ml rIL-4

500 units/ml rlL-4

% p55 positive

3.9 3.5

2.7

% p70 positive

47.5 42.9

29.4

‘For further information, see legend to Fig. 1 and Materials and Methods.

with IL-4, such as 72 h, did not increase the difference in p70 expression between treated and untreated LGL any further, instead a decrease in the difference could often be observed (data not shown). We were never able to obtain a complete inhibition in the p70 expression. This may indicate that IL-4 also interferes with the signal transduction via the p70 protein, provided that there is no limit in the number of receptors that need to be stimulated in order to achieve a signal.

Mule and coworkers found that IL-4 alone,

48

in the murine system, in contrast to the human system, can induce LAK activity from murine splenocytes and is able to augment LAK activ- ity induced by IL-2 against fresh tumors [13]. Whether this species difference in the ability to induce LAK activity by IL-4 is also linked to the possibility that murine IL-4 does not down- regulate the murine equivalent to p70 of the IL-ZR, remains to be seen.

Altogether, IL-4 is clearly a potent inhibitor of IL-2 induced LAK-cell activation. The sup- pression can at least partly be explained by a selective downregulation of the p70 chain of the IL-PR, which is known to be responsible for the IL-2 induced activation of LGL [26]. If IL-4 and its influence on LAK induction can also explain the failure in tumor regression observ- ed with in vivo administered IL-2 is open to 121,291. Iiowever, IL-4 in combination with serological reagents may be a useful tool for further dissecting the activation pathways in- volved in the induction of LAK cells.

AcknowIedsements

This work was supported by the Academy of Finland,. the Sigrid Juselius Foundation, the Oskar Oflunds Foundation, the Kathe and Olof Rundbergs Foundation, the Wallenius Foundation, the Borgs Foundation, the Magnus Ehrnrooth Foundation and the Swedish Medical Research Council.

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