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Cyclosporin A inhibition of macrophage colony-stimulating factor (M-CSF) production by activated human T lymphocytes Ste´phanie Fre´tier,* Arnaud Besse,† Adriana Delwail,* Martine Garcia,* Franck Morel,* Vale´rie Leprivey-Lorgeot,‡ John Wijdenes,§ Vincent Praloran,† and Jean-Claude Lecron* *Laboratoire Cytokines, FRE 2224, IFR FR 59, IBMIG, Universite´ de Poitiers, Cedex, France; †Laboratoire Universitaire d’He´matologie, EA 482, Universite´ de Bordeaux 2, France; ‡Laboratoire de Physiologie, Faculte´ de Me´decine, Universite´ de Limoges, Cedex, France; and §Laboratoire Diaclone, BP 1985, Besançon, France

Abstract: M-CSF is a pleiotropic cytokine involved in the survival, proliferation, and differentiation of cells of the monocyte/macrophage lineage. M-CSF is produced by numerous cells including CD3-activated T cells. M-CSF serum levels are increased during acute graft rejection. We tested the in vitro production of M-CSF, GM-CSF, IL-2, and IL-4 by T-cell clones costimulated by CD3 and accessory activation pathways and the effects of cyclosporin A and methylprednisolone. The nine clones studied and CD4ⴙ cells purified from peripheral blood mononuclear cells (PBMC) spontaneously produced low levels of M-CSF, which PMA and CD3 mAb strongly enhanced. In contrast to IL-2, CD28 mAb did not further enhance this production. CsA inhibited M-CSF production by clones and purified CD4 T cells. Addition of IL-2, anti IL-2, or anti CD25 mAb to the cultures demonstrated that CsA down-regulated M-CSF synthesis by activated T cells through its inhibition of IL-2 synthesis. These results could help to better understand the complex mechanisms of acute graft rejection and immunosuppression. J. Leukoc. Biol. 71: 289 –294; 2002. Key Words: immunosuppression 䡠 graft-versus-host 䡠 TNF-␣ 䡠 PMA

INTRODUCTION The glycoprotein macrophage colony-stimulating factor (MCSF or CSF-1) is a hematopoietic growth factor required for the survival, proliferation, and differentiation of cells of the monocyte/macrophage lineage [1, 2]. M-CSF was shown later to participate in the immunological defenses, bone metabolism, lipoprotein clearance, fertility, and pregnancy, demonstrating that it is a pleiotropic cytokine (reviewed in refs. [3, 4]). M-CSF is produced spontaneously, or after stimulation, by numerous cell types such as endothelial cells, fibroblasts, monocytesmacrophages, or bone marrow-derived stromal cells [3]. Activated normal B cells and spontaneously outgrown Epstein-Barr virus (EBV)-B cell lines [5], as well as activated T cells [6], also produce M-CSF. Previously, we demonstrated that activa-

tion of normal T cells by phorbol 12-myristate 13-acetate (PMA) and the A23187 calcium ionophore, cytokines [tumor necrosis factor ␣ (TNF-␣), interleukin (IL)-1␣] or anti-CD3 monoclonal antibodies (mAb), and IL-2 induced the production of M-CSF [6, 7]. A unique gene located on the short arm of chromosome 1 [8] encodes M-CSF. Multiple alternative mRNA splicing and complex co- and/or posttranslational glycosylations together with proteolysis of M-CSF from the cell surface generate soluble and membrane-associated mature isoforms of M-CSF (reviewed in refs. [9, 10]). M-CSF binds to a specific cell-surface tyrosine kinase receptor (CSF-1-R or M-CSF-R), which is the product of the c-fms protooncogene [11]. Because T cells do not express this receptor, the M-CSF that they produce acts locally or by humoral route on other cells, such as monocytes, participating in the regulation of the inflammatory and immune response [11]. The binding of the T-cell receptor leads to signal transduction when it is associated with costimulatory signals provided by cytokines and/or cell-cell interactions [12–14]. It induces T-cell activation, proliferation, and various functions, such as cytokine synthesis. CD28, a constitutive T-cell surface glycoprotein [15–18], constitutes an important accessory pathway for T-cell activation and survival. Its natural ligands, B7-1 (CD80) and B7-2 (CD86), are two monomeric transmembrane glycoproteins expressed by the antigen-presenting cells [16, 19]. In addition to the signals provided by the activation of the CD2 or CD3 pathways, anti-CD28 mAb induces a long-lasting and monocyte-independent T-cell proliferation [12]. This proliferation is associated with the induction of a prolonged secretion of high levels of cytokines, such as IL-1␣, IL-2, TNF-␣, and M-CSF [17, 19, 20]. The accessory molecules modulate the pattern of cytokines produced in response to these different signals. Conversely, cytokine production can be down-regulated by immunosuppressors. Cyclosporin A (CsA), an immunosuppressive drug used in treating rejection of allogeneic transplants, inhibits cytokine synthesis, in particular IL-2 [21].

Correspondence: J. C. Lecron, Laboratoire Cytokines, FRE 2224, IFR FR 59, IBMIG, Universite´ de Poitiers, 40, avenue du Recteur Pineau, 86022 Poitiers, Cedex, France. E-mail: [email protected] Ste´phanie Fre´tier and Arnaud Besse contributed equally to this work. Received April 1, 2001; revised September 5, 2001; accepted September 6, 2001.

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Methylprednisolone (MP), another immunosuppressor, inhibits IL-6 synthesis [22]. We demonstrated several years ago that serum and tissue levels of M-CSF were increased during an acute graft-versus-host (GVH) reaction model in mice [23] and recently that high serum levels of M-CSF accompanied the onset of kidney graft-rejection episodes in humans [24]. This work was focused on the production of M-CSF by activated CD4 and CD8 T-cell clones and CD4⫹ lymphocytes purified from peripheral blood mononuclear cells (PBMC) and on the mechanisms of inhibition of this secretion by the immunosuppressors CsA and MP.

MATERIALS AND METHODS T-cell clones and culture conditions The cloned CD4⫹ T-cell lines SP-B21, TA 20.6, TA 23.6 [25], and the EBV-lymphoblastoid cell line JY used in this study were kindly provided by Dr. H. Yssel (INSERM U454, Montpellier, France). The cloned CD4⫹ T-cell lines AB14 and AO22 and the cloned CD8⫹ T-cell lines AO15, AO23, NN82, and NN84 were obtained in the laboratory as described by Spits et al. [26]. T-cell clones (2⫻105 cells/ml) were stimulated every 2 weeks by 0.1 ␮g/ml purified phytohemagglutinin (PHA; Murex, Chaˆ tillon, France) on a feeder cell mixture consisting of 106 irradiated (50 GY), allogenic PBMC and 105 irradiated (50 GY) JY cells per ml in Yssel’s medium [27] supplemented with 1%

fetal calf serum (FCS; Sigma Chemical Co., Saint Quentin Fallavier, France) and were seeded in 24-well plates (Nunc, Paisley, Scotland). Three to 4 days after PHA stimulation and every 2 days until days 10 –12, the T-cell clones were split and further expanded in Yssel’s medium containing 10 ng/ml rIL-2 (Eurocetus, Amsterdam, The Netherlands). The JY cell line was cultured in RPMI-1640 medium (Gibco BRL, Cergy Pontoise, France) supplemented with 10% FCS. All cells were cultured at 37°C in a humidified atmosphere of 6% CO2. T-cell clones were collected for cytokine assays 10 –12 days after stimulation with PHA and feeder cells.

Purification of CD4⫹ T cells from PBMC PBMC from normal donors were prepared by centrifugation of heparinized blood on Ficoll-Hypaque (Nycomed, Oslo, Norway). CD4⫹ T lymphocytes were isolated with magnetic anti-CD4 microbeads (Miltenyi Biotec, Paris, France) using a magnetic cell sorter (VarioMacs, Miltenyi Biotec), according to the manufacturer’s instructions. The lymphocyte fraction was enriched up to 98% of CD4⫹ T cells as assessed by flow cytometry.

T-cell stimulations T cells were washed and incubated (106 T cells) during 24 h in 24-well plates containing 1 ml Yssel’s medium with different combinations of antibodies and/or PMA (1 ng/ml). The antibodies used were anti-CD3 (10 ␮g/ml; OKT3, purified from ascite), anti-CD25 mAb (15 ␮g/ml; gift of Dr. Y. Jacques, INSERM U463, Nantes, France), anti-IL-2 mAb (0.2, 2, 20 ␮g/ml B-G5; Diaclone, Besanç on, France), anti-CD28 L293 (1 ␮g/ml; a gift of Dr. L. Lanier, DNAX Research Institute, Palo Alto, CA), and the combination of anti-CD2 D66 and X11 mAb (2 ␮g/ml; gift of Pr. A. Bernard, INSERM U243, Nice,

Fig. 1. Production of M-CSF by CD4⫹ and CD8⫹ T-cell clones. The CD4⫹ (䡲) (SP-B21, 20.6, 23.6, AO22, and AB14) and CD8⫹ (䡩) T-cell clones (NN82, NN84, AO23, and AO15) were stimulated for 24 h with a combination of PHA, PMA with anti-CD2 or anti-CD3 mAb, and/or anti-CD28 mAb. M-CSF levels were measured by ELISA.

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TABLE 1.

Effects of CsA and MP on the Production of Cytokines by SP-B21 and Purified CD4⫹ T Cells

Unstimulated SP-B21 PMA/␣CD3 activated SP-B21 PMA/␣CD3/␣CD28 activated SP-B21 Unstimulated CD4⫹ PMA/␣CD3 activated CD4⫹

IL-2 (ng/mL)

IL-4 (ng/mL)

GM-CSF (ng/mL)

M-CSF (Ul/mL)

0.02 ⫾ 0.01 0.03 ⫾ 0.02 0.02 ⫾ 0.01 77 ⫾ 44 0.9 ⫾ 0.5 53 ⫾ 30 278 ⫾ 161 1.5 ⫾ 0.9 228 ⫾ 132 0.05 ⫾ 0.03 0.05 ⫾ 0.03 0.04 ⫾ 0.02 45 ⫾ 4.9 0.2 ⫾ 0.06 29 ⫾ 2.3

ND ND ND 1.6 ⫾ 0.9 ND 1.3 ⫾ 0.8 1.6 ⫾ 0.9 ND 1.3 ⫾ 0.8 ND ND ND 0.17 ⫾ 0.01 0.01 ⫾ 0.01 0.05 ⫾ 0.01

ND ND ND 7⫾4 0.02 ⫾ 0.01 3.2 ⫾ 1.9 12 ⫾ 7 0.5 ⫾ 0.3 6.5 ⫾ 4 1 ⫾ 0.1 1 ⫾ 0.1 0.1 ⫾ 0.01 8.6 ⫾ 0.8 1.3 ⫾ 0.2 3.7 ⫾ 0.7

9⫾5 10 ⫾ 6 8⫾5 156 ⫾ 90 25 ⫾ 15 96 ⫾ 56 164 ⫾ 95 51 ⫾ 30 94 ⫾ 54 14 ⫾ 1 10 ⫾ 1.2 10 ⫾ 1 42 ⫾ 4 15 ⫾ 0.6 19 ⫾ 1.7

Control CsA MP Control CsA MP Control CsA MP Control CsA MP Control CsA MP

ND ⫽ Not determined. France). Cultures were realized with or without MP (10⫺5 M; Sigma Chemical Co.) or CsA (5 ␮g/ml or concentrations indicated in legends; Sandoz, Basel, Switzerland). Culture supernatants were centrifuged (400 g, 5 min, 20°C), aliquoted, and stored at ⫺20°C until cytokine measurements.

Cytokines measurements Concentrations of IL-2 (sensitivity: 44 pg/ml) and M-CSF (sensitivity: 5 IU/ml) were measured by specific enzyme-linked immunosorbent assay (ELISA), as described previously [28, 29]. IL-4 (sensitivity: 31 pg/ml; Biosource, Fleurus, Belgium) and granulocyte-macrophage colony-stimulating factor (GM-CSF; sensitivity: 4 pg/ml; Endogen, Montluç on, France) measurements were performed as indicated by the manufacturers’ instructions.

RESULTS Production of M-CSF by T-cell clones The low spontaneous production of M-CSF (16 –70 IU/ml) by the five CD4⫹ and four CD8⫹ T-cell clones was not modified by anti-CD28 mAb stimulation alone (Fig. 1). PMA, anti-CD3 mAb, anti-CD2 mAb, and PHA significantly increased this basal synthesis. The combination of PMA with anti-CD3 or anti-CD2 mAb further increased the M-CSF production in comparison with either of these agents alone. Although antiCD28 mAb was ineffective on the anti-CD3 mAb- or PMA/ anti-CD3 mAb-induced M-CSF secretion, its effect was greater than additive when added to PMA (Fig. 1 and Table 1). In contrast, the addition of anti-CD28 mAb to PMA/anti-CD3 mAb strongly enhanced IL-2 production, as evidenced for the SP-B21 clone in Table 1. Production of M-CSF by CD4⫹ T-cell clones stimulated with PMA/anti-CD3 mAb or PMA/anti-CD3/ anti-CD28 mAb stimulations appears more important than in CD8⫹ clones. In addition (Fig. 2), the kinetic analysis of induced production of several cytokines showed that it peaked earlier for IL-2 (6 h) than for IL-4, GM-CSF, and M-CSF (72–96 h).

Effect of CsA and MP on the production of M-CSF, IL-2, IL-4, and GM-CSF by SP-B21 T-cell clones and CD4⫹-purified T cells CsA strongly inhibits (up to sevenfold) the production of IL-2, IL-4, and GM-CSF by SP-B21 T cells (activated by

PMA/anti-CD3 mAb and PMA/anti-CD3/anti-CD28 mAb) and by purified CD4⫹ cells (activated by PMA/anti-CD3 mAb), whereas its inhibition of M-CSF production is lower (three- to sixfold; Table 1). This CsA inhibition of the production of IL-2 and M-CSF by SP-B21 cells or CD4⫹purified T cells (Fig. 3, a and b) is dose-dependent. By contrast, in the same conditions of stimulation, MP was a weak inhibitor of the production of IL-2, IL-4, GM-CSF, and M-CSF (Table 1).

Mechanism of the CsA-induced M-CSF inhibition We hypothesized that the inhibition of the M-CSF production could be indirect and linked to the decrease of IL-2 production by T-cell clones. Addition of exogenous IL-2 to PMA/anti-CD3 mAb-stimulated T-cell clones in the presence of CsA partially restored the production of M-CSF, whereas it did not restore the GM-CSF and IL-4 production, also inhibited by CsA. The

Fig. 2. Kinetics of cytokine production by the CD4⫹ T-cell clone SP-B21. Cells were stimulated with a combination of PMA and anti-CD3 mAb. Cytokine levels were measured in the culture supernatants by ELISA. Mean ⫾ SE of three experiments.

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T cells instead of T-cell clones. More probably, there were discrepancies because the production and release of M-CSF are regulated mainly by posttranscriptional processes [30 –32]. In contrast, the production of IL-2 and GM-CSF was increased by the addition of anti-CD28 mAb to PMA/anti-CD3 mAb stimulation. It suggests that induction of M-CSF production by T cells is associated with a limited number of regulatory mechanisms. As shown previously by Price et al. [10] with mouse L929 cells, Western blots of T-cell extracts and supernatants (resting or induced) evidenced several types of M-CSF of similar molecular weight (unpublished results). We suggest that in vivo, the activation of resting T cells strongly enhances their ability to produce soluble and membrane-anchored MCSF glycoproteins as well as to release the large proteoglycan forms bound to heparansulfates of the extracellular matrix. These three different types of M-CSF display, respectively, endocrine, cell-to-cell, and stroma-to-cells effects. We hypothesize that this enhanced production of the different forms of M-CSF induces the recruitment and activation of monocytes at inflammatory sites. In agreement with this hypothesis, increased M-CSF serum and organ levels have been detected

Fig. 3. Inhibition by CsA of M-CSF (●) and IL-2 (䡲) production by SP-B21 (a) or purified CD4⫹ T cells (b). T cells were stimulated with PMA and anti-CD3 mAb, with increasing concentrations of CsA. M-CSF and IL-2 levels were measured by specific ELISA. Mean ⫾ SE of three experiments.

addition of IL-2 was also ineffective to reverse the MP-induced inhibition of M-CSF, IL-4, or GM-CSF production or to enhance the spontaneous or induced M-CSF production (Fig. 4). Finally, addition of anti-CD25 (Fig. 4)- as well as anti-IL-2 (Fig. 5)-blocking mAb decreased the PMA/anti-CD3 mAbinduced M-CSF production. This is in accordance with our hypothesis that the inhibition of CsA on M-CSF production is mediated indirectly via a decrease of IL-2 production.

DISCUSSION Chemical inducers (PMA, PHA, calcium ionophore) or cytokines (IL-1␣, TNF-␣) induced M-CSF production by T cells [6, 7]. Its induction by costimulatory signals, which more closely mimic in vivo interactions, has not been tested. The nine T-cell clones studied constitutively produce low amounts of M-CSF, which PHA, PMA, anti-CD2, or anti-CD3 mAb but not antiCD28 mAb significantly increased. These results apparently contradicted those of Cerdan et al. [12], showing that purified T cells activated by the CD28 pathway display a transient rise of the M-CSF transcripts but remain insensitive to CD2 activation. These discrepancies may be related to the use of blood 292


Fig. 4. Effect of anti-CD25 mAb and IL-2 on the production of M-CSF, IL-4, and GM-CSF by SP-B21. Cells were stimulated or not with PMA and anti-CD3 mAb, with or without IL-2 (50 ng/ml) or anti-CD25 mAb (15 ␮g/ml). Cytokine levels were measured by ELISA. Mean ⫾ SE of three experiments.


Fig. 5. Effect of anti-IL2 mAb on the production of M-CSF by SP-B21. Cells were stimulated or not with PMA and anti-CD3 mAb, with or without CsA (5 ␮g/ml) or anti-IL-2 mAb (0.2, 2, 20 ␮g/ml). M-CSF levels were measured by ELISA. Mean ⫾ SE of three experiments.

already in a mouse-acute GVH reaction model and during kidney graft-rejection episodes [24]. These results could partly explain the rapid and massive macrophage infiltration of the grafted organ that accompanies acute graft rejection [33, 34]. The various combinations of costimulatory molecules that we used induced similar productions of M-CSF by CD4 and CD8 T-cell clones, even if PMA/anti-CD3 mAb or PMA/anti-CD3 mAb/anti-CD28 mAb induced a higher production of M-CSF by CD4⫹ rather than CD8⫹ T-cell clones. The low number of clones tested herein does not allow a statistical analysis of these differences. CD4⫹ T cells have been classified in different subpopulations characterized by their patterns of cytokine production and functions. The T helper cell type (Th)1 clones produce IL-2, interferon-␥ (IFN-␥), and TNF-␤, whereas Th2 clones produce IL-4, IL-5, and IL-10. Both types produce similar amounts of GM-CSF and IL-3. Because we found no correlation between the M-CSF production and those of IL-4 or IL-2 by T-cell clones tested (unpublished results), its production, as for IL-3 or GM-CSF, is probably not linked to a polarized T-cell response. To investigate a potential down-regulation of the increased M-CSF production during graft-rejection processes, we tested the effect of the two classical immunosuppressive drugs CsA and MP on activated, purified, peripheral CD4⫹ T cells and T-cell clones. MP inhibits the IL-6 production directly by a large number of cell types [22], whereas it weakly inhibits the production of IL-2, IL-4, GM-CSF, and M-CSF by activated T cells. By contrast, CsA strongly inhibits the T-cell production of IL-2, IL-3, and IFN-␥, mainly accounting for its immunosuppressive action [21, 35, 36]. We showed that CsA strongly inhibits the production of IL-2, IL-4, and GM-CSF by T-cell clones as well as freshly isolated CD4 T cells. We partially contradict the results of Bickel et al. [21], showing that CsA inhibited the production of IL-2 but not of GM-CSF by murine T cells, whereas to a lesser extent, CsA also reduced the production of M-CSF, a phenomenon that similarly affects the various isoforms of M-CSF (unpublished results). Because M-CSF is produced later than IL-2, and its inhibition by CsA is lower than for the other cytokines, we hypoth-

esized that its down-regulation by CsA could be indirectly related to the inhibition of IL-2 production. The partial reversion of CsA inhibition by IL-2 addition and the blocking effect of anti-CD25 (anti-IL-2 receptor) and anti IL-2 mAb on the PMA/anti-CD3-induced M-CSF synthesis confirmed this hypothesis. These data also suggest that the induction of M-CSF synthesis by T cells is independent of the calcineurin/NF-AT pathway, which CsA inhibits strongly and directly [37]. It is interesting that the range of concentrations of CsA that inhibits the M-CSF production in vitro in this work was in the same range as those in the serum of patients treated with CsA after renal transplantation [38]. Previously, and in this study, we showed [7] that activated T lymphocytes produced M-CSF and that acute GVH reaction strongly increased their serum and tissue concentrations [23]. Then, the in vivo increase of serum and tissue M-CSF concentrations could be mainly a result of a dramatic T-lymphocyte activation that accompanies the acute GVH reaction. Several studies showed that the extent of infiltration and activation of macrophages in the grafted organs is linked to the rejection processes [3, 34, 39 – 41]. They could, themselves, be related to the high local concentrations of the proteoglycan forms of M-CSF bound to the stroma. The efficiency of CsA to prevent rejection in transplantation is a result of various well-known effects on T cells, such as the inhibition of IL-2 synthesis [38]. It could also be related to the inhibition of M-CSF production by T cells that reduces the graft infiltration by monocytes and the deleterious effects of locally activated macrophages.

ACKNOWLEDGMENTS S. F. and A. B. were, respectively, supported by grants from “la Ligue contre le cancer des Deux-Se`vres” and the “Conseil re´ gional du Limousin”.

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