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Decreased interleukin-2 β-chain receptor expression by interleukin-4 on LGL: influence on the IL-2 induced cytotoxicity and proliferation
43
Cancer Letters, (1992) 43 .- 49 Elsevier Scientific Publishers Ireland Ltd.
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
‘Centre for Biotechnology, SF-20521, University, SF-20500, Abo (Finland) (Received (Accepted
&trnan
b, Christian
Turku and bDepartment
of Biochemistry
b and Karl A kerrnan b and Pharmacy,
,&o Akademi
18 February 1992) 12 March 1992)
Summary Addition of incr’easing amounts of interleukin-4 (IL-4) to large granular lymphocytes (LGL) had a selective downregulative effect on the interleukin-2 P-chain (~70) receptor expression. A 40% inhibition of the p70 expression 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 proliferative 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 lymphocytes; interleukin-2 receptor.
granular
Introduction Large granular lymphocytes population of morphologically Correspondence to: Christer Biochemistry and Pharmacy, SF-20500,
Okerblorn
Turku,
(LGL) are a homogeneous
Lindqvist, Department of Abe Akademi University,
Finland.
0304-3835/92/$05.00 Printed and Published
(3 1992 Elsevier Scientific Publishers in Ireland
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 complex [8,19]. In addition, they also predominantly 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 increased ability of peripheral blood mononuclear cells (PBL) to lyse NK-resistant targets after treatment with IL-2, defined as LAK activity [16,17]. IL-4, like IL-2, is a multifunctional lymphokine mainly produced by T-cells [4]. It was originally characterized for its ability to stimulate 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 selective 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 Ireland Ltd
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
ture was incubated for 2 x 60 min at 4OC, with gentle shaking every 10 min. Cells forming 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.
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 (Immunex Corporation). Recombinant IL-2 were purchased from Genzyme (Boston, MA). Cells Peripheral blood mononuclear cells were obtained from normal healthy donors by spinning 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 macrophages by 30-min plastic adherence (37OC) and thereafter passed over nylon wool columns [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 collected 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 depleted of CD3 positive cells by Dynabeads M-450 (Dynal A.S, Oslo, Norway) (4 x lo* beads/ml) coated with covalently bound goatanti mouse Ig. In brief, the isolated LGL were adjusted to 1 x lO’/ml in serum-free RPMI 1640 and incubated with anti-CD3 monoclonal 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 immunomagnetic beads were added. The mix-
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 incorporation 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, Badhoevedorp, 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 cytometer (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
x 100 Lysis (%) =cpmmax. re~ease - cpmSpont.re~eaSe Results IL-4 inhibits the IL-2 induced LGL proliferation Table I shows that adding increasing concentrations of IL-4, always decreased the LGL proliferation in the: presence of a constant amount of IL-Z. The same pattern was observed 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 48and 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.
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 lymphokines-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 lymphokines (no preculture/generation of LAKactivity) 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
Inhibition of IL-2 induced LGL proliferation by IL-4a.
Dose of rIL-4 (units/ml)
Dose of rIL-2 (units/ml) 200
2
0.02
17583 zt 1990 12400 f 2447 12769 zt 1316 9789 zt 1059
10681 8839 5636 6475
+ 892 + 1517 + 1332 f 389
6829 6748 3847 3785
44 957 43 939 37885 7983
39799 37539 28814 371
f 1114 f 1475 zt 6476 f 59
24933 30877 18532 908
0
48-h dim. time 0
0.5 5 50
zt +z zt zt
840 472 1660 387
6404 7461 1505 1783
+ 2666 f 1187 f 449 zt 1059
22609 25 985 18717 131
A 7019 zt 878 zt 3382 + 72
72-h dim. time 0
0.5 5 50
f zt + f
3505 8041 9819 7152
f 7790 f 3440 f 962 zt 596
“The indicated values are representative of four experiments. For further information, see Materials and Methods.
46
expression to any greater extent, whereas a slight increase (10 - 15%) was observed for the p55 expression (data not shown).
100
Discussion
80 a 3 1 g
60
1:25
T/E ratio Fig.1. IL-2induced 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 cytotoxic activity against K562. Similar results were obtained in three independent experiments. For further information, 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 lymphokine control we used both IL-la or IL-lo, but these lymphokines did not affect the p70
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 inhibition 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 expression 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 expression 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
U-0
I
IO’
loo
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 50 units/ml 500 units/ml
rIL-4 rIL-4 rlL-4
‘For further information, Materials and Methods.
% p55 positive
% p70 positive
3.9 3.5 2.7
47.5 42.9 29.4
see
legend
to Fig.
1 and
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 activity 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 downregulate 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 suppression 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 observed 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 involved 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 the Borgs Foundation, the Foundation, Magnus Ehrnrooth Foundation and the Swedish Medical Research Council.
of a T cell derived B cell growth factor distinct from interleukin 2. J. Exp. Med., 155, 914-923. 5
6
7
Kawakami, Y., Custer, M.C., Rosenberg, S.A. and L&e, M.T. (1989) IL-4 regulates IL-2 induction of iymphokineactivated killer activity from human lymphocytes. J. Immunol., 142, 3452-3461.
8
Lanier, L.L. and Phillips, J.H. (1986) Evidence for three types of human cytotoxic lymphocytes. lmmunoi. Today, 7, 132- 135. Lebow, L.A. and Bonavida, B. (1990) Purification and
9
10
11
12
13
14
References 15 Bijyum, A. (1968) Isolation of mononuclear cells and granuiocyte from human blood. Stand. J. Ciin. Lab. Invest., 21, (suppl.97) 77-91. Grossi, C.E., Cadoni, A., Zicca. A., Leprini, A. and Ferrarini, M. (1982) Large granular lymphocytes in human peripheral blood: ultrastructural and cytochemical characterization of the granules. Blood, 59, 277-283. Hori, T., Uchiyama, T., Onishi, R., Kamio, M., Umadome, H., Tamori, S., Motoi, T., Kodaka, T.-I. and Uchino, H. (1988) Characteristics of the IL-2 receptor expressed on large granular lymphocytes from patients with abnormally expanded large granular lymphocytes. Impiication of a non-Tat binding peptide. J. Immunoi., 140, 4199-4203. Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, T., Hamaoka, T. and Paul, W.E. (1982) Identification
Julius, M.H., Simpson, E. and Herzenberg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol., 3, 645-649. Kasahara, T., Djeu, J.Y., Dougherty, SF. and Oppenheim, J.J. (1983) Capacity of human large granular iymphoc~es (LGL) to produce multiple lymphokjnes: interleukin-2, interferon and coiony stimulating factor. J. lmmunol., 131, 2379 - 2383.
16
17
18
19
20
characterization of cytoiytic and noncytolytic human natural killer subsets. Proc. Natl. Acad. Sci., 87, 6063 - 6067 Lindqvist, C., Nihlmark, E.L., Nordstrom, T. and Andersson, L.C. (1991) Interleukin-4 downregulates the ~70 chain of the IL-2R on peripheral blood mononuclear ceils. Ceil. Immunoi., 136, 62-68. Louio, C.B. and Lozzio, B.B. (1975) Human chronic myelogenous ieukemia ceiline with positive Philadelphia chromosome. Blood, 4.5, 321- 334. Mukaida, N., Kasahara, T., Hosoi, J., Shioi~-Nakano, K. and Kawai, T. (1986) Effects of anti-Tat antibody on the response of large granular lymphocytes to interleukin-2. Immunol., 57, 137 - 143. Mule, J.J., Smith, C.A. and Rosenberg, S.A. (1987) Interleukin 4 (B cell stimuiatory factor I) can mediate the induction of lymphokine activated killer cell activity directed against fresh iumor cells. J. Exp. Med., 166, 792 - 797. Nagler, A., Lanier, L.L. and Phillips, J.H. (1988) The effects of IL-4 on natural killer cells: a potent regulator of IL-2 activation and proliferation. J. immunol., 141, 2349-2351. Ohara, J. and Paul, W.E. (1988) Up-regulation of interleukin 4/B-cell stimuiatory factor 1 receptor expression. Proc. Nati. Acad. Sci., 85, 8221-8225. Ortaldo, J.R., Mason, A. and Overton, R. (1986) Lymphokine-activated killer cells: analysis of progenitors and effecters. J. Exp. Med., 164. 1193- 1205. Phillips, J.H. and Lanier, L.L. (1986) Dissection of the lymphokine activated killer phenomenon: relative contribution of peripheral blood natural killer cells and T Iymphocytes to cytoiysis. J. Exp. Med., 164, 814-818. Phillips, J.H., Takeshita, T., Sugamura, K. and Lanier, L.L. (1989) Activation of natural killer cells via the p75 interleukin receptor. J. Exp. Med., 170, 291- 296. Reynolds, C.W. and Ortaido, J.R. (1987) Natural killer activity: the definition of a function rather than a cell type. Immunol. Today, 8, 172 - 173. Roehm, N.W., Leibson, H.J., Ziotnik, A., Kappier. J.,
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21
22
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24
Marrck, P. and Cambier, J.C. (1984) Interleukin-induced increase in la expression by normal mouse B cells. J. Exp. Med., 160, 679 - 694. Rosenberg, S.A., Lotze, M.T., Muul, L.M., Chang, A.E., Avis, F.P., Leitman, S., Linehan, W.M., Robertson, C.N., Lee, R.E., Rubin, J.T., Seipp, C.A., Simpson, C.G. and White, D.E. (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokineactivated killer cells and interleukin-2 or high-dose interleukin-2 alone. hew. Engl. J. Med., 316, 889-897. Saksela, E., Timonen, T., Ranki, A. and H%yry, P. (1979) Morphological and functional characterization of isolated effector cells responsible for human natural killer cell activity to fetal fibroblasts and to cultured cell line targets. Immunol. Rev., 44, 71- 123. Scala, G., Allavena, P., Djeu, J.Y., Kasahara, T., Ortaldo, J.R., Herberman, R.B. and Oppenheim, J.J. (1984) Human large granular lymphocytes are potent producers of interleukin-I. Nature, 309, 56 - 59. Spits, H., Yssel, H., Paliard, X., Kastelein, R., Figdor, C. and De Vries, J.E. (1.988) IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of anti’gen-specific cytotoxic T lymphocytes in mixed leukocyte cultures. J. Immunol., 141, 29 - 36.
25
26
27
28
29
Timonen, T., Reynolds, C.W., Ortaldo, J.R. and Herberman, R.B. (1982) Isolation of human and rat natural killer cells. J. Immunol. Methods, 51, 269- 277. Tsudo, M.. Goldman, C.K., Bongiovanni, K.F., Chan, W.C., Winton, E.F., Yagita, M., Grimm, E.A. and Waldmann, T.A. (1987) The p75 peptide is the receptor for interleukin 2 expressed on large granular lymphocytes and is responsible for the interleukin 2 activation of these cells. Proc. Natl. Acad. Sci., 84, 5394- 5398. Van Wauwe, J.P., DeMey, J.R. and Goosens, J.G. (1980) OKT3: A monoclonal anti-human T-lymphocyte antibody with potent mitogenic properties. J. Immunol., 124, 2708 - 2713. Vercelli, D., Leung, D.Y.M., Jabara. H.H. and Geha, R.S. (1989) lnterleukin 4 dependent induction of IgE synthesis and CD23 expression by the supernatants of a human helper T cell clone. Int. Arch. Allergy Appl. Immunol., 88, 119- 121. West, W.H., Tauer, K.W., Yannelli, J.R.. Marshall, G.D., Orr, D.W., Thurman, G.B. and Oldman, R.K. (1987) Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. New. Engl. J. Med., 316, 898 - 905.
Cancer Letters, (1992) 43 .- 49 Elsevier Scientific Publishers Ireland Ltd.
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
‘Centre for Biotechnology, SF-20521, University, SF-20500, Abo (Finland) (Received (Accepted
&trnan
b, Christian
Turku and bDepartment
of Biochemistry
b and Karl A kerrnan b and Pharmacy,
,&o Akademi
18 February 1992) 12 March 1992)
Summary Addition of incr’easing amounts of interleukin-4 (IL-4) to large granular lymphocytes (LGL) had a selective downregulative effect on the interleukin-2 P-chain (~70) receptor expression. A 40% inhibition of the p70 expression 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 proliferative 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 lymphocytes; interleukin-2 receptor.
granular
Introduction Large granular lymphocytes population of morphologically Correspondence to: Christer Biochemistry and Pharmacy, SF-20500,
Okerblorn
Turku,
(LGL) are a homogeneous
Lindqvist, Department of Abe Akademi University,
Finland.
0304-3835/92/$05.00 Printed and Published
(3 1992 Elsevier Scientific Publishers in Ireland
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 complex [8,19]. In addition, they also predominantly 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 increased ability of peripheral blood mononuclear cells (PBL) to lyse NK-resistant targets after treatment with IL-2, defined as LAK activity [16,17]. IL-4, like IL-2, is a multifunctional lymphokine mainly produced by T-cells [4]. It was originally characterized for its ability to stimulate 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 selective 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 Ireland Ltd
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
ture was incubated for 2 x 60 min at 4OC, with gentle shaking every 10 min. Cells forming 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.
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 (Immunex Corporation). Recombinant IL-2 were purchased from Genzyme (Boston, MA). Cells Peripheral blood mononuclear cells were obtained from normal healthy donors by spinning 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 macrophages by 30-min plastic adherence (37OC) and thereafter passed over nylon wool columns [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 collected 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 depleted of CD3 positive cells by Dynabeads M-450 (Dynal A.S, Oslo, Norway) (4 x lo* beads/ml) coated with covalently bound goatanti mouse Ig. In brief, the isolated LGL were adjusted to 1 x lO’/ml in serum-free RPMI 1640 and incubated with anti-CD3 monoclonal 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 immunomagnetic beads were added. The mix-
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 incorporation 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, Badhoevedorp, 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 cytometer (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
x 100 Lysis (%) =cpmmax. re~ease - cpmSpont.re~eaSe Results IL-4 inhibits the IL-2 induced LGL proliferation Table I shows that adding increasing concentrations of IL-4, always decreased the LGL proliferation in the: presence of a constant amount of IL-Z. The same pattern was observed 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 48and 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.
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 lymphokines-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 lymphokines (no preculture/generation of LAKactivity) 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
Inhibition of IL-2 induced LGL proliferation by IL-4a.
Dose of rIL-4 (units/ml)
Dose of rIL-2 (units/ml) 200
2
0.02
17583 zt 1990 12400 f 2447 12769 zt 1316 9789 zt 1059
10681 8839 5636 6475
+ 892 + 1517 + 1332 f 389
6829 6748 3847 3785
44 957 43 939 37885 7983
39799 37539 28814 371
f 1114 f 1475 zt 6476 f 59
24933 30877 18532 908
0
48-h dim. time 0
0.5 5 50
zt +z zt zt
840 472 1660 387
6404 7461 1505 1783
+ 2666 f 1187 f 449 zt 1059
22609 25 985 18717 131
A 7019 zt 878 zt 3382 + 72
72-h dim. time 0
0.5 5 50
f zt + f
3505 8041 9819 7152
f 7790 f 3440 f 962 zt 596
“The indicated values are representative of four experiments. For further information, see Materials and Methods.
46
expression to any greater extent, whereas a slight increase (10 - 15%) was observed for the p55 expression (data not shown).
100
Discussion
80 a 3 1 g
60
1:25
T/E ratio Fig.1. IL-2induced 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 cytotoxic activity against K562. Similar results were obtained in three independent experiments. For further information, 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 lymphokine control we used both IL-la or IL-lo, but these lymphokines did not affect the p70
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 inhibition 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 expression 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 expression 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
U-0
I
IO’
loo
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 50 units/ml 500 units/ml
rIL-4 rIL-4 rlL-4
‘For further information, Materials and Methods.
% p55 positive
% p70 positive
3.9 3.5 2.7
47.5 42.9 29.4
see
legend
to Fig.
1 and
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 activity 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 downregulate 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 suppression 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 observed 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 involved 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 the Borgs Foundation, the Foundation, Magnus Ehrnrooth Foundation and the Swedish Medical Research Council.
of a T cell derived B cell growth factor distinct from interleukin 2. J. Exp. Med., 155, 914-923. 5
6
7
Kawakami, Y., Custer, M.C., Rosenberg, S.A. and L&e, M.T. (1989) IL-4 regulates IL-2 induction of iymphokineactivated killer activity from human lymphocytes. J. Immunol., 142, 3452-3461.
8
Lanier, L.L. and Phillips, J.H. (1986) Evidence for three types of human cytotoxic lymphocytes. lmmunoi. Today, 7, 132- 135. Lebow, L.A. and Bonavida, B. (1990) Purification and
9
10
11
12
13
14
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