Prothymosin α enhances interleukin 2 receptor expression in normal human T-lymphocytes

lnt. J. lmmunopharmac., Vol. 13, No. 8, pp. 1059- 1065, 1991. Printed in Great Britain.

0192-0561/91 $3.00 + .00 Pergamon Press pie. International Society for lmmunopharmaeology.

PROTHYMOSIN a ENHANCES INTERLEUKIN 2 RECEPTOR E X P R E S S I O N IN N O R M A L H U M A N T - L Y M P H O C Y T E S * O. J. CORDERO,t C. S. SARANDESES,t J. L. LOPEZ, t E. CANCIO,:~ B. J. REGUEIRO:~ and M. NOGUEIRAt§ tDepartamento de Bioquimica y Biologia Molecular, Facultad de Biologia y *Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad de Santiago, Santiago de Compostela, Spain (Received 29 April 1991 and in final form 23 June 1991)

-We investigated whether the enhancement, by prothymosin alpha (Proa), of the phytohaemagglutinin-stimulated proliferation of human peripheral blood mononuclear cells (PBMC) is due to its affect on the number of cells expressing the interleukin 2 receptor (IL-2R) or the surface density of IL-2R on PBMC. Peripheral blood mononuclear cells were obtained from 21 donors. For both an optimal phytohaemagglutinin (PHA) concentration (H) and a 10-fold dilution (L), their responses fell in two classes, high (h) and low (1), making four dose-response situations. Proa significantly increased the number and IL-2R density of cells expressing IL-2R only when the response in its absence was about half maximal, i.e. for PBMC responding well to the low PHA stimulus (group Lh) or PBMC responding poorly to the optimal stimulus (group HI). The enhancement of IL-2R expression in group Lh by Proa was dose-dependent and paralleled by increased proliferative response. It appears not to be mediated by IL-2, since it was unaffected when IL-2 production was suppressed by cyclosporin A. The early interaction of Proa with lymphocytes did not require the presence of macrophages, but macrophages were necessary during lymphocyte activation for modulation of PHA-stimulated IL-2R expression to be affected. The immunoregulatory activity of Proa may prove useful for improving the decreased T-cell function associated with immunodeficiency, or for restoration of normal IL-2R expression by the lymphocytes of aged individuals. Abstract

The lymphokines, a group of mediators produced by activated lymphocytes, have important effects in the cascade of events following lymphocyte stimulation. Interleukin 2 (IL-2) is an inducible lymphokine produced by T-lymphocytes upon antigenic or mitogenic stimulation (Morgan, Ruscetti & Gallo, 1976; Giilis & Smith, 1977). It has been shown that the strength of the immune response is regulated largely by the amount of IL-2 available for T-lymphocyte growth (Gillis, Baker, Ruscetti & Smith, 1978; Zarling & Bach, 1979; Henney, Baker, Ruscetti & Smith, 1978); interaction of IL-2 with its specific, high-affinity cell surface receptors induces cellular proliferation and other physiological responses (Robb, Greene & Rusk, 1984). Study of the control of IL-2 production, and of the regulation of the expression of its receptors on lymphoid cells, is therefore important for understanding the molecular basis of T-cell functions. The expression

of the IL-2 receptor as defined by the CD25 antigen (IL-2R) and the expression of IL-2 are both thought to require the accessory function of macrophages (Wakasugi, Bertoglio, Tursz & Fradelizi, 1985); there is in fact evidence that interleukin 1 (IL-1) plays a major role in the direct activation of T-cells by inducing IL-2 secretion and IL-2R expression (Lowenthal, Cerottini & MacDonald 1986; Truneh, Simon & Schmitt-Verhulst, 1986). The thymosins are a family of thymic humoral factors that may have important immunoregulatory effects (Franco, Diaz, Barcia, Arias, GomezMarquez, Soriano, Mendez & Freire, 1989; Sztein & Goldstein, 1986; Sztein & Serrate, 1989; Zatz, Oliver, Samuels, Stotnicki, Sztein & Goldstein, 1984; Zatz, Oliver, Sztein, Skotnicki & Goldstein, 1985). Thymosin alpha 1 (al), thymosin fraction 5 (TF5) (Goldstein et al., 1977) and other thymosin molecules have been shown to modulate T-cell

*This work was supported in part by a grant from the Xunta de Galicia (XUGA 80212/89). §Author to whom correspondence should be addressed. 1059

O. J. CORDEROet al.

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maturation and the induction of differentiation markers. TF5 enhances the ability of human peripheral blood mononuclear cells (PBMC) and purified T- and natural killer (NK) cell populations to proliferate, secrete IL-2 and interferon y (IFN-),) and express IL-2R in response to mitogenic stimulation (Serrate, Schulof, Leondaridis, Goldstein & Sztein, 1987; Sztein, Serrate & Golstein, 1986). Additionally, there are reports that TF5 and al modulate the expression of high-affinity IL-2R, which is essential for biological responsiveness to IL-2 (Leichtling, Serrate & Sztein, 1989). In previous work (Cordero, Sarandeses & Nogueira, 1990), we found that Proa, a polypeptide of the a thymosin family (Panmeerselvam, Haritos, Caldarella & Horecker, 1987), can increase the PHAinduced proliferation of fresh human peripheral blood mononuclear cells (PBMC), possibly by affecting the IL-2-dependent proliferation of T-cells. In this paper we report that Proa enhances the expression of IL-2R on peripheral blood lymphocytes from normal human volunteers in a dose-dependent fashion, and that this is not due to IL-2R up-regulation by IL-2.

EXPERIMENTAL PROCEDURES

Reagents Ficoll - Hypaque was supplied by Pharmacia Fine Chemicals (Uppsala, Sweden); PHA by Sigma Chemical Company (St. Louis, MO, U.S.A.) and anti-CD25 monoclonal antibody fluorescein conjugate, which specifically reacts with human IL-2R by Becton Dickinson (CA, U.S.A.); and human r l L - l a was provided from Boehringer Mannheim (F.R.G.). Cyclosporin A (CsA) was a gift from Sandoz Pharmaceutical. Prothymosin alpha, isolated from natural sources and tested for purity by amino acid analysis and high voltage electrophoresis, was supplied by Peninsula Laboratories (U.K.). Non-retained peptide fractions (NRF) were obtained in our laboratory from calf thymocytes by the method of Haritos, Goodall & Horecker (1984). Both NRF and Proa were endotoxin-free according to the Limulus assay. Preparation o f PBMC, PBL and monocytes Peripheral blood mononuclear cells obtained from heparinized blood of normal adults volunteers were collected by F i c o l l - H y p a q u e gradient centrifugation. The washed PBMC were resuspended at a density of 1 × 10 6 cell/ml in RPMI-1640

medium (Gibco, Grand Island, NY, U.S.A.) containing 1007o heat-inactivated foetal calf serum (Gibco), 100 ~g/ml streptomycin and 100 IU/ml penicillin (Gibco). Peripheral blood lymphocytes and monocytes were obtained by sorting 5 × 10 6 cells/ ml PBMC in a Coulter EPICS-V cell-sorter, which distinguishes lymphocytes from monocytes and macrophages on the basis of forward and 90 ° light scattering. The sorted lymphocytes were always more than 9607o pure, and the monocytes always more than 9907o pure.

Culture conditions Peripheral blood mononuclear cells and PBL were resuspended in RPMI-1640 without serum and preincubated for 3 0 m i n at 37°C under 5°7o CO2 (Cordero et al., 1990) in the presence or absence of PHA (0.5 or 5 tag/ml), alone or with various concentrations of Proa ( 0 . 1 - 10/ag/ml), BSA ( 1 - 10 ~g/ml), NRF ( 0 . 1 - 10tag/ml) and CsA (1 /~g/ml). After this period, heat-inactivated foetal calf serum was added to a final concentration of 10070 and the cells were cultured for a further 24 or 72 h ( 1 0 6 per ml, 37°C, 507o CO2). In the experiments in which the involvement of macrophages in modulation by Proa was investigated, PBL were preincubated alone or over a monolayer of 1007o autologous macrophages in the presence or absence of Proa. After 30 min at 37°C in 5070 CO2, T-cells were collected, washed twice and incubated alone or over a monolayer of 1007o autologous macrophages in the presence of PHA (0.5 ~g/ml) with or without IL-1 (20 U/ml). Under all the experimental conditions employed, more than 95°70 of cells were trypan-blue positive within 72 h of culture.

Proliferative assays Proliferative responses were determined by measuring 3[H]-thymidine incorporation. Cells seeded in triplicate were cultured in 96-well flatbottomed plates (Costar, Cambridge, MA, U.S.A.). After 60 h they were dosed for 12 h with 0.5 ~Ci of 3[H]-thymidine/well (New England Nuclear, spec. act. 15 Ci/mmol), and were then harvested onto glass-fibre filters with a multiple sample harvester (Cambridge Technology Inc. USA, M-200A). The filter discs were placed in plastic vials containing 4 ml of scintillation liquid, and radiactivity was measured in a Kontron/3-matic scintillation counter. Percentage activation by Proa was calculated as follows:

1061

Proa Enhances IL-2R Expression Control

Pro

57.77% (732)

25.49% (311)

PHA 0.5 pg/ml

H

t~

E

t'+li'i+k..t'-+'tI,,+..~.,,... j..,.,. ,

c O

PHA 5 pg/ml

35.35% (1094)

55.36% (1186)

Fluorescence intensity

Fig. 1. Enhancement by Proa of IL-2R expression by PHA-stimulated PBMC. Cells were cultured in medium alone (unstimulated) or with PHA (0.5 or 5/ag/ml) in the presence or absence of Proa (1/ag/ml). After 72 h of incubation cells were harvested and analysed by flow cytometry for expression of IL-2R. Percentages show the proportion of cells expressing IL-2R, and values in parentheses the mean fluorescence intensity of the positively stained cells. In the absence of PHA, IL-2R expression was very slight (<7% CD25+). Data shown are those of one experiment representative of several with similar results (see text).

~(counts/min of cultpres contining Proa) °7°A = ~_ (counts/rainof control cultures)

1J × 100.

Cyclosporin A did not affect DNA synthesis by CTLL-2 cells.

IL-2 bioassay

IL-2R expression

Supernatants were collected 24 h after initiation of the cultures and their IL-2 content was estimated from their ability to support the growth of CTLL-2 IL-2-dependent cell line (European Collection of Animal Cultures, PHLS, U.K.). Samples were diluted serially in 96-well plates (Costar), with complete RPMI-1640 medium. Before assays the CTLL-2 cells were thoroughly washed in RPMI-1640 and then incubated in the same medium in order to remove any IL-2. Then 104 cells were added to each well. A standard IL-2 preparation was included in every assay. The activity of each dilution was assayed in triplicate. Three wells containing cells plus medium were included to estimate background proliferation. After 24 h of incubation at 37°C in 5070 COz, 0.5 ~Ci of 3[H]-thymidine was added to each well and the plates incubated for 6 h. The cells were then harvested onto filter pads, which were transferred to scintillation vials for radiactivity measurement. Since the same standard IL-2 preparation was used in every assay, results obtained in separate experiments were compared directly. Results are expressed as Units/ml referred to BRMP human IL-2 (Jurkat).

Cells were cultured at 10 6 per ml in 24-well plates (Costar) as indicated above. The cells were then washed, stained with saturating concentrations of anti-CD25 fluorescein conjugated monoclonal antibody for 30 min in ice, washed again, suspended in Isoton 10°70 paraformaldehyde and sorted by flow cytometry using an EPICS-V cell-sorter with laser excitation at 488 nm. Five thousand viable cells were sorted for each histogram.

Statistical analysis Statistical analysis was performed using a twotailed Student's t-test for paired and unpaired observations, as appropriate.

RESULTS

Enhancement of PHA-induced IL-2R expression on PBMC by Proa The induction of IL-2R on PBMC from each of 21 donors by optimal and low concentrations of P H A (5 and 0.5/ag/ml, respectively) was estimated by measuring the percentage of CD25+ cells (in the

O. J. CORDERO et al.

1062

accompanied by a proliferation (Table 1).

lOG

8G + Lq CN

significant

increase

in

Role of 1L-2

6(:

/J

5

//

,g

4£

20 , L , , o \ .0 5 1 0.1

, 2

, 3

, 4

, 5

Proa (pg/ml) Fig. 2. Dose-dependence of enhancement of IL-2R expression by Proa. PBMC from six donors were incubated with PHA (0.5/ag/ml) and several concentrations of Proa (O) or NRF (O). After 72 h, cells were harvested, stained with anti-CD25 and analysed by flow cytometry. Results are expressed as o7o CD25" cells. Each point represents the mean of six independent experiments, with bars showing S.E.M. absence of P H A , less than 7% of cells were CD25 + after incubation). For each P H A concentration, the IL-2R induction response unequivocally defined two groups of donors (though the groups were not identical for the two P H A concentrations): the percentage of cells made CD25 + by the optimal P H A concentration was 62.43 _+ 2.75 for high responders (n = 14; group Hh) and 35.13 _+ 2.27 for low responders (n = 7; group HI); while the corresponding percentages for the low P H A concentration were 25.15 _+ 2.68 (n = 17; group Lh) and 15.65 +_ 3.16 (n = 4; group LI). Proa did not significantly alter the response of groups Hh or LI, but did significantly increase the responses of groups HI and Lh as regards both the percentage of cells made CD25 + (P<0.02 for HI, P<0.005 for Lh) and the density of IL-2R on cell surfaces (Fig. 1). In all subsequent work we used PBMC and stimulus conditions ensuring the maximum potential effect of Proa, i.e. PBMC from group Lh and stimulation by 0.5/ag/ml PHA. Under these conditions, the effects of Proa were dose-dependent (Fig. 2), the maximum effect being achieved with 1 /ag/ml Proa (NRF was used as the control in these experiments).

Correlation between proliferation

IL-2R

expression

Purified IL-2 can directly up-regulate synthesis and expression of its own receptors on T-cells during the early phase of their PHA-induced expression, and on cells whose IL-2R expression has declined (Depper, Leonard, Drogula, Krfnke, Waldmann & Greene, 1985). To determine whether IL-2 is involved in the effects of Proa on PHA-stimulated IL-2R expression, we incubated PBMC with P H A (0.5 ~g/ml) and Proa in the presence of CsA, which inhibits IL-2 production but not IL-2 expression (Kr6nke, Leonard, Depper, Ayra, Wong-Staal, Gallo, Waldmann & Greene, 1984). In five experiments, a significant increase in IL-2R expression occurred in cells activated by P H A in the presence of Proa and CsA, while IL-2 production was practically abrogated (Fig. 3). Thus the increase of IL-2R expression by Proa is not due to upregulation of IL-2R by IL-2. In the absence of CsA, IL-2 production was increased by Proa (Fig. 3).

Role of macrophages In our previous work (Cordero et al., 1990) we found that the enhancing effect of Proa on the PHAstimulated proliferation of fresh human PBMC was greatly reduced if monocytes were previously removed from the PBMC. To relate this to our findings on IL-2R expression on PBMC we separated lymphocytes from monocytes and macrophages with a cell-sorter, preincubated the lymphocytes with or without macrophages in the presence or absence of Proa, and then washed them and incubated them with P H A in the presence or absence of autologous macrophages or IL-I. Proa increased IL-2R expression (Fig. 4) and IL-2 production (results not shown) under all conditions except when neither IL-1 nor macrophages were present during activation by PHA. Thus macrophages seem not to be required during the initial stages of interaction between lymphocytes and Proa, but macrophages or IL-I are required subsequently for the initial l y m p h o c y t e - P r o a interaction to increase the effect of activation by PHA. IL-I was slightly less efficient than whole macrophages in permitting the effect of Proa on stimulation by PHA.

and

In experiments in which the effects of Proa on PHA-stimulated IL-2R expression and on PHAstimulated proliferation were measured in parallel, we found that increased IL-2R expression was always

DISCUSSION

The proliferation of human T-lymphocytes is regulated, in part, by the co-ordinated expression of genes encoding the T-cell growth factor (IL-2) and

1063

Proa Enhances 1L-2R Expression Table 1. Effect of Proa on PBMC IL-2R expression and proliferation induced by PHA Proliferation Donor

IL-2R expression

Proa

Counts/min

%A

%CD25 +

%A

+

43,652 ± 2251 72,893 ± 3198

67

23.13 71.29

208

+

35,402 ± 2244 81,763 ± 3970

131

25.67 49.26

92

+

15,942 ± 2950 41,509 ± 1321

160

25.83 68.95

167

+

37,102 ± 2125 76,338 ± 4023

106

25.65 67.17

162

1

2

3

4

PBMC were cultured in replicates with PHA (0.5/ag/ml) in the presence or absence of Proa (1 t~g/ml) as described in Experimental Procedures. Parallel cultures were set up in 96-well plates to determine proliferation. IL-2R expression and proliferation were determined 72 h after initiation of cultures. Results are expressed as 07o CD25 ÷ cells and as incorporation of 3[H]-thymidine (mean counts/min of triplicate cultures _+ S.E.M.).

70

-

~

CsA

60

T

50

5

+

40

c~ o

O4

30

s

-

20 10

PHA

P H A + Proa

PHA

PHA + Proo=

Fig. 3. Enhancement of IL-2R expression by Proa in the presence of CsA. PBMC were cultured in duplicate in medium alone or in the presence of PHA (0.5 tag/ml) with or without Proa (I /ag/ml). Replicate plates were cultured in the presence of CsA (1 ~g/ml). Supernatants for determination of IL-2 levels were collected after 24 h of culture. For the determination of 07oCD25 +, cells were harvested after 72 h and then stained with anti-CD25. Columns show the mean of five independent experiments, with S.E.M. represented by bars. IL-2 receptors (Depper et al., 1985). In previous experiments ( C o r d e r o et al., 1990) we f o u n d that P r o a can increase the response o f P H A - s t i m u l a t e d fresh h u m a n P B M C m u c h m o r e effectively t h a n t h y m o s i n a l . A l t h o u g h its activity was greatly reduced if m o n o c y t e s were f r o m the P B M C , we argued that it did not act solely by inducing IL-1 p r o d u c t i o n by m o n o c y t e s (which would then activate IL-2 production), since IL-1 alone or with P r o a

c a n n o t p e r f o r m all accessory cell functions in T-cell activation by mitogens (Cordero et al., 1990; ShuKuamg Hu, Badamchian, Mitcho & Goldstein, 1989). The alternative possibility that P r o a modulates the expression o f IL-2R on activated P B M C was s u p p o r t e d by the finding ( C o r d e r o et al., 1990) that an IL-2R-blocking polyclonal a n t i b o d y was able to suppress the induction o f proliferation by P r o a + IL-I. In the present study we c o n f i r m e d that P r o a

1064

O . J . CORDEROet al.

[ ] BSA

9o

[ ] Proa 70

lO I1 Preincubation

-M~

-M~

+M~

+M~

Incubation (PHA)

+M~)

+11.-1

+M(1)

-M(]D

Fig. 4. Influence of macrophages or IL-1 on enhancement of IL-2R expression by Proa. Lymphocytes separated from PBMC in a cell-sorter were preincubated at room temperature with or without 10% of autologous macrophages in the presence or absence of Proa. After 30 min, the cells were collected, washed and incubated at 37°C with PHA (0.5 /ag/ml) in the presence or absence of 10% autologous macrophages or in the presence of IL-1 (20 U/ml). After 72 h, cells were collected and stained with anti-CD25 for measurement of IL-2R expression. Columns show the mean of four independent experiments, with S.E.M. represented by bars. can enhance the PHA-induced expression of IL-2R, though only when the state of the PBMC is such that, in the absence of Proa, PHA-induced IL-2R expression is significant but submaximal. The expression of IL-2R on the surface of activated cells appears to be modulated by a number of signals, including IL-2 itself. Purified IL-2 can upregulate the synthesis and expression of its own receptors on activated T-cells during the early phase of their expression, but only when the concentration of mitogen or antigens is low (Depper et al., 1985; Welte, Andreef, Platzer, Holloway, Rubin, Moore & Mertelsmann, 1984). Enhancement of IL-2R expression by Proa, however, is not mediated by IL-2, since it occurred even when IL-2 production was suppressed by CsA. In keeping with previous results (Sztein & Serrate, 1989; Cordero et al., 1990), PHA-induced IL-2 and IL-2R production by lymphocytes separated from PBMC was enhanced by preincubation with Proa followed by activation in the presence of macrophages or IL-1, but not when neither

macrophages nor IL-1 were present during incubation with P H A (even when macrophages had been present during preincubation with Proa). These results suggest that although the early interaction of Proa with lymphocytes does not require the presence of macrophages, the latter are necessary at the later stage if Proa is to enhance lymphocyte activation. Thymosins have been successfully administered to improve certain parameters of patients with cancer or other diseases associated with alterations of the immune function (Gravenstein, Duthie, Miller, Roecker, Drinka, Prathipati & Ershler, 1989; Reddy & Grieco, 1987; Sztein & Goldstein, 1986). The results presented here seem to go some way towards explaining the mechanism of such activity, since it seems likely that the activation of lymphocytes and/or macrophages may be modulated not only by Proa but also by other thymic factors. However, it should be borne in mind that although we have shown modulation by Pro¢r to involve enhancement of IL-2R and IL-2 production, other thymosins may affect other points of proliferation process.

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Proa Enhances IL-2R Expression

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