Granulocyte-macrophage colony-stimulating factor (GM-CSF) has opposing effects on the capacity of monocytes versus monocyte-derived dendritic cells to stimulate the antigen-specific proliferation of a human T cell clone

Abstract

GM-CSF is widely used in combination with IL-4 to differentiate monocytes into potent T cell stimulatory cells, referred to as monocyte-derived dendritic cells (MoDC). These cytokines further increased the stimulatory function of MoDC when present during their incubation with antigen, as determined by the proliferative response of an allergen-specific T cell clone. Conversely, the incubation of freshly isolated monocytes with antigen in the presence of GM-CSF or GM-CSF and IL-4 strongly inhibited the specific stimulation of the T cells, compared with monocytes pulsed in the absence of cytokines. This suppression was partly due to the secretion of prostaglandin E₂ (PGE₂) and IL-10 by GM-CSF-treated monocytes, since the combined use of indomethacin and anti-IL-10 antibodies during GM-CSF incubation and antigen pulsing restored T cell growth to about 65% of control levels. As confirmed by culture supernatant transfer experiments, maximal inhibition of T cell stimulation was also dependent on the direct contact between the T cells and GM-CSF-treated monocytes during antigen presentation. Collectively, these results imply that GM-CSF can either inhibit or enhance the re-stimulation of primed T cells by antigen-presenting monocytes or MoDC, respectively.

Introduction

GM-CSF stimulates growth and differentiation of granulocyte and monocyte/macrophage precursor cells (reviewed in [1]). It is also known to affect the function of mature myeloid cells by priming monocytes and neutrophils for enhanced adhesion [2], tumour cytotoxicity [3], or leukotriene production [4]. However, in addition to stimulatory effects on myeloid cells, previous reports showed that GM-CSF may also have suppressive effects via the induction of prostaglandin E₂ (PGE₂) synthesis. Lipopolysaccharide (LPS)- and interferon-gamma (IFN-γ)-induced tumour necrosis factor-alpha (TNF-α) release by GM-CSF-primed macrophages was inhibited due to GM-CSF-induced PGE₂ production [5], and PGE₂ has been demonstrated to inhibit the proinflammatory activity of monocytes/macrophages by decreasing the production of IL-12 [6] while augmenting IL-10 release [7]. Pronounced inhibitory effects by IL-10 on monocytes/macrophages have been reported (reviewed in [8]). In the presence of monocyte/macrophage antigen-presenting cells (APC), hIL-10 inhibited not only cytokine synthesis [9], but also proliferation of human T cells and T cell clones [10,11]. Moreover, PGE₂ directly inhibits T cell proliferation by decreasing the expression of both IL-2 and IL-2Rα chain [12,13]. This may partly explain the observation that prostaglandins released by monocytes may mediate the suppression of T cell function in cancer patients [14–16] and that CD14⁺ cells present in GM-CSF-mobilized peripheral blood stem cell products can inhibit T cell function.

GM-CSF together with IL-4 promotes the differentiation of monocytes into dendritic cells in vitro[17,18]. Monocyte-derived dendritic cells (MoDC) are amongst the most potent stimulators of naive T cells but they are also superior compared with other APC at inducing antigen-specific recall responses of memory T cells. We wanted to investigate whether GM-CSF alone or in combination with IL-4 would also support a stronger APC function of monocytes as it does for MoDC. As a model system, we used a human T cell clone (TCC) specific for the major protein of house dust mites, Der p1, in association with the HLA-DPw4 class II molecule [19,20]. In this study we show that GM-CSF alone or in combination with IL-4 drastically inhibits the capacity of allogeneic, HLA-DP-matched monocytes to induce antigen-specific proliferation of the TCC if the cytokines are present during the period of antigen pulsing. The relevance of this finding for regulation of memory T cells by different types of APC in chronic inflammation will be discussed.

Materials and methods

Reagents, antibodies and cytokines

The basal culture medium (CM) was RPMI 1640 that was further supplemented with NaHCO₃ 2 mg/ml, penicillin 50 μg/ml, streptomycin 50 μg/ml, l-glutamine 2 mm, and 10% fetal calf serum (FCS; Gibco, Grand Island, NY). This culture was endotoxin-free as determined by the Limulus amebocyte lysis assay (< 10 pg/ml of endotoxin). Recombinant human (rh) GM-CSF (specific activity 6 × 10⁶ U/mg), rhIL-4 (specific activity 6 × 10⁶ U/mg), and rhIL-2 (specific activity 10⁷ U/mg) were produced by and obtained from Novartis Pharma (Basel, Switzerland). In some experiments, GM-CSF obtained from R&D Systems (Minneapolis, MN) was used to confirm the specific biological activity of the material produced by Novartis Pharma. The neutralizing anti-IL-10 antibody (JES3-9D7) was purchased from PharMingen (San Diego, CA). Lyophilised protein extract of Dermatophagoides pteronyssinus (Dpt) was purchased from ARTU Biologicals (Lelystad, The Netherlands). The content of the major allergen Der p1 in the lot used was 18·5 μg/mg of Dpt protein extract. Indomethacin was obtained from Sigma (St Louis, MO).

Isolation and culture of cells

The human T helper cell clone CFTS 4:3.1 (TCC) was isolated from a skin punch biopsy of a patient with atopic dermatitis as described previously [19,20]. It specifically recognizes the major allergen of house dust mite, Der p1, in association with the MHC class II restriction molecule HLA-DPw4 (P. Baselmans et al., Hum Immunol, in press). The TCC was propagated by stimulating the T cells every 14–16 days via immobilized anti-CD3 MoAb (Leu-4; Becton Dickinson, San Jose, CA) or by presentation of Dpt antigen on irradiated autologous Epstein–Barr virus (EBV)-transformed B cells (EBV-B) in the presence of rhIL-2 and rhIL-4 (50 U/ml each) as described [19].

Primary human monocytes were obtained by countercurrent elutriation [21] of leukapheresis samples donated by healthy individuals. To obtain a higher purity of monocytes, residual T cells and B cells were negatively depleted using magnetic beads covalently coupled to anti-CD2 and anti-CD19 MoAb (Dynal Inc., Oslo, Norway) using a magnetic bead-to-target cell ratio of 5:1. The resulting monocyte preparation contained > 95% CD14⁺ cells, as judged by flow cytometry, and was further cultured in CM in the absence or presence of different supplements as indicated in the text.

MoDC were obtained by culturing purified human monocytes at 8 × 10⁵ cells/ml in CM supplemented with rhGM-CSF (300 U/ml) and rhIL-4 (120 U/ml). Cultures were fed every other day (days 2, 4 and 6) by exchanging half of the medium for fresh CM that had been supplemented with cytokines. MoDC were harvested on day 7 of culture and frozen in liquid nitrogen for subsequent use as APC in T cell proliferation assays. Cell viability was > 90% after thawing, as determined by trypan blue exclusion.

Antigen-specific lymphocyte proliferation assay

For antigen-specific stimulation of the T cells, each type of APC was incubated in CM for 16–20 h with Dpt (250 μg/ml for EBV-B and 50 μg/ml for monocytes and MoDC) or without antigen as negative control. Where indicated, rhGM-CSF (300 U/ml), rhIL-4 (120 U/ml), indomethacin (100 ng/ml), or anti-IL-10 antibody (10 μg/ml) were added during the preincubation of APC with antigen. For the determination of soluble mediators secreted by treated or untreated monocytes, cell-free culture supernatants were harvested after 16–20 h. Subsequently, APC were irradiated (40 Gy), washed and added to the T cells that were used not earlier than 14–16 days after stimulation with Dpt or plate-bound anti-CD3 to ascertain a resting state of the cells. Cloned T cells (4 × 10⁴ cells/well) were mixed with autologous EBV-B (4 × 10⁴ cells/well), HLA-DPw4-matched human monocytes (4 × 10⁴ cells/well), or HLA-DPw4-matched human MoDC (4 × 10³ cells/well) and further cultured in a volume of 200 μl CM per well of 96-well round-bottomed plates (Corning Costar, Badhoevedorp, The Netherlands). Indomethacin (100 ng/ml), rhGM-CSF (300 U/ml) or rhIL-2 (50 U/ml) were also present during the lymphocyte proliferation assay, where indicated. At day 3, 1 μCi of ³H-thymidine was added per well during the last 16 h and incorporated radioactivity was quantified by liquid scintillation counting. Results are expressed as ct/min ± s.d. and represent the mean of triplicate cultures.

Polyclonal stimulation of CD4⁺ T cells

Ninety-six-well round-bottomed plates were seeded with 2 × 10⁵ monocytes for incubation in CM with increasing amounts of GM-CSF in the presence or absence of indomethacin (100 ng/ml). After 16 h, monocytes were washed twice with CM in the microtitre plates. The monocytes were not irradiated. T cells were collected from high density fractions of elutriations of the same peripheral blood mononuclear cell (PBMC) donors as used for the preparation of monocytes. T cells (1 × 10⁸/ml) were incubated with paramagnectic beads covalently coupled to anti-CD4 MoAbs (CD4⁺ T cell isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany) as described by the manufacturer. Subsequently, 1 × 10⁵ purified CD4⁺ T cells were seeded into the wells that contained cytokine-treated or untreated monocytes or no APC as a control. T cell activation was induced by adding soluble anti-CD3 MoAb (200 ng/ml; PharMingen). After 48 h of co-culture, the proliferative response of the T cells was assessed by adding 1 μCi/well of ³H-thymidine for another 16 h. Results are expressed as ct/min ± s.d. and represent the mean of quadruplicate cultures.

Immunoassays

Cell-free supernatants were harvested at 20 h from cytokine-treated or untreated monocyte cultures or at 24 h from the co-culture of the T cells and monocytes for the determination of prostaglandins and cytokines. PGE₂ was determined with the Immunoassay kit from R&D (Abingdon, UK). The sensitivity of this assay was 36·2 pg/ml. Production of IL-10 was measured by cytokine-specific ELISA from Predicta (Genzyme, Cambridge, MA). The lower detection limit of this ELISA was 5 pg/ml. IL-1β, TNF-α, IL-6, transforming growth factor-beta 1 (TGF-β1), TGF-β2, and heterodimer IL-12-specific ELISA were obtained from R&D Systems (Minneapolis, MN). The sensitivity of these assay kits was 1 pg/ml, 4·4 pg/ml, 0·7 pg/ml, 7 pg/ml, 7 pg/ml, 5 pg/ml, respectively.

Results

GM-CSF profoundly decreases the capacity of monocytes to induce antigen-specific growth of TCC

The human house dust mite-specific T cell clone CFTS4:3.1 was used to assess the stimulatory capacity of different APC. Autologous, EBV-transformed B cells, HLA-DP-matched, allogeneic MoDC or monocytes were pulsed with specific antigen and subsequently added in varying numbers to cultures of resting 4:3.1 TCC. Consistent with published data, MoDC were superior to monocytes or EBV-B cells in their capacity to stimulate T cell proliferation over a wide range of APC/T cell ratios. As shown in Fig. 1 and also indicated by results obtained in a series of similar experiments (n = 8), the proliferative response induced by monocytes when used at a 1:1 ratio (4 × 10⁴ APC/well) was comparable to the degree of T cell proliferation stimulated by MoDC at a 1:10 ratio (4 × 10³ APC/well). Therefore, these numbers of APC/well were used in subsequent experiments to allow for a comparison between the relative stimulatory capacities of MoDC and monocytes.

Fig. 1.

Monocyte-derived dendritic cells (MoDC) are more potent stimulators of T cell clones (TCC) than monocytes or autologous Epstein–Barr virus (EBV)-B cells. Different numbers of antigen-pulsed antigen-presenting cells (APC), autologous EBV-transformed B cells (▪) or allogeneic MoDC (▴) and monocytes (•) obtained from HLA-DPw4 matched donors, were co-cultured with TCC (4 × 10⁴ cells/well). The proliferative response was determined at day 3. The results represent the mean ct/min (± s.e.m.) of three different experiments. Proliferation values of non-Dermatophagoides pteronyssinus-stimulated TCC have been subtracted.

MoDC were generated in accordance with published protocols by culture of monocytes in media containing GM-CSF and IL-4 for 7 days, after which they are referred to as immature dendritic cells based on their phenotype [17]. MoDC were subsequently washed and pulsed with antigen in the presence or absence of freshly added GM-CSF or GM-CSF and IL-4 to address the influence of both cytokines on antigen presentation and stimulatory capacity. As shown in Fig. 2, the proliferative response of the specific T cell clone was slightly increased by adding fresh cytokines, particularly GM-CSF, to MoDC when pulsing with antigen. This result led us to investigate the influence of these cytokines on the stimulatory capacity of monocytes or autologous EBV-B cells. Unexpectedly, the proliferative response of the TCC was greatly diminished when antigen was presented by monocytes pulsed in the presence of GM-CSF and IL-4 compared with monocytes pulsed in the absence of either cytokine (Fig. 2). T cell growth stimulation was further decreased (90%) if GM-CSF alone was present during the incubation of monocytes with antigen. As determined in GM-CSF titration experiments, the inhibition of TCC proliferation reached a plateau when using 300 U/ml of GM-CSF, which therefore was the concentration used in all subsequent experiments. This was also confirmed by using a commercial sample of GM-CSF (R&D Systems) in some of the titration experiments (data not shown). In contrast, the APC function of autologous EBV-B cells was unaffected by GM-CSF ± IL-4.

Fig. 2.

Treatment of monocytes, but not Epstein–Barr virus (EBV)-B cells or monocyte-derived dendritic cells (MoDC) with GM-CSF during antigen pulsing strongly inhibits antigen-specific stimulation of T cells. The T cell clones (TCC) was stimulated by autologous EBV-B cells (B), allogeneic monocytes (Mo) or MoDC that were matched for the expression of HLA-DPw4 as restriction element. Antigen-presenting cells (APC) were incubated with antigen (Dermatophagoides pteronyssinus (Dpt)) for 20 h in presence or absence of GM-CSF (300 U/ml) or with GM-CSF and IL-4 (300 U/ml and 120 U/ml, respectively). Data represent the mean ct/min (± s.e.m.) of at least four independent experiments, except for GM-CSF-treated MoDC, where two experiments were performed. Proliferation values of non-Dpt-stimulated TCC have been subtracted.

GM-CSF-treated monocytes secrete PGE₂ and a variety of immunomodulatory cytokines

GM-CSF has been reported to induce the secretion of prostaglandins by monocytes [5,22,23]. Therefore, supernatants harvested after 20 h from monocyte cultures containing GM-CSF or the combination of GM-CSF and IL-4 were assayed for the presence of PGE₂ by specific ELISA. As shown in Table 1, high levels of PGE₂ were found in the supernatants of GM-CSF-treated monocytes. The production of PGE₂ was almost completely suppressed when indomethacin was present during the incubation of monocytes with GM-CSF. IL-4 also inhibited GM-CSF-induced PGE₂ production to about 90%.

Table 1.

Effect of GM-CSF, IL-4, and indomethacin on cytokine production by monocytes^*

	TGF-β1†pg/ml	PGE2	IL-10	IL-1β ng/ml	TNF-α	IL-6
Medium	25·4 ± 17·5	0·3 ± 0·1	< 0·05	< 0·05	< 0·05	0·3 ± 0·1
GM-CSF	40·5 ± 18·2	41·0 ± 7·9	4·7 ± 0·7	14·1 ± 2·5	7·5 ± 3·8	218·6 ± 22·3
GM-CSF/indo	NT	0·3 ± 0·1	5·0 ± 1·1	15·6 ± 2·0	5·7 ± 3·2	277·3 ± 10·9
GM-CSF/IL-4	NT	3·5 ± 0·6	2·9 ± 0·3	3·8 ± 0·5	0·6 ± 0·2	80·1 ± 12·2

^*Freshly isolated monocytes (1 × 10⁶/ml) were stimulated with GM-CSF in the absence or presence of indomethacin (indo) or with GM-CSF and IL-4. Data represent mean cytokine production (± s.e.m.) of at least four independent experiments.

^†Values for transforming growth factor-beta 1 (TGF-β1) found in serum containing medium were subtracted.

NT, Not tested.

Since GM-CSF may also modulate or directly induce the secretion of other mediators, the same supernatants were assayed for the presence of various cytokines. As can be seen in Table 1, IL-10, IL-1β, TNF-α and IL-6 were produced by monocytes treated with GM-CSF regardless of whether indomethacin was present or not. The addition of IL-4 reduced the production of IL-10 by about 40%, but it also inhibited the secretion of the potentially T cell stimulatory cytokines IL-1β, TNF-α and IL-6. After subtraction of values found in FCS-containing medium (approx. 0·75 ng/ml), the levels detected for TGF-β1 were always < 0·1 ng/ml and not significantly affected by GM-CSF. TGF-β2 or IL-12 could not be detected in supernatants of GM-CSF-treated monocytes.

PGE₂ but not IL-10 is the major monocyte-derived soluble inhibitor of antigen-specific T cell proliferation

The issue of whether PGE₂ and/or IL-10 were directly responsible for T cell inhibition was addressed in culture supernatant transfer experiments. Supernatants were harvested from cytokine-treated or untreated monocytes and added to the co-culture of the TCC with either antigen-pulsed monocytes, autologous B cells or MoDC. As shown in Fig. 3, supernatants harvested from GM-CSF-treated monocytes were found to inhibit T cell clone proliferation regardless of the type of APC used for antigen presentation. In contrast, supernatants harvested from untreated or GM-CSF- and indomethacin-treated monocytes did not significantly affect T cell growth. This result suggests that PGE₂ is the major soluble factor secreted by GM-CSF-treated monocytes that negatively and directly affects allergen-specific T cell proliferation.

Fig. 3.

Indomethacin relieves inhibition of T cell growth mediated by supernatant of GM-CSF-treated monocytes. Human T cell clones (TCC) (4 × 10⁴/well) were stimulated by antigen-pulsed Epstein–Barr virus (EBV)-B cells (4 × 10⁴/well), monocytes (4 × 10⁴/well), or monocyte-derived dendritic cells (MoDC; 4 × 10³/well) in a 1:1 mixture of culture medium and SN of monocytes that were incubated for 20 h in the absence (□) or presence of either GM-CSF (▪) or GM-CSF and indomethacin (hatched bars). The degree of T cell proliferation induced by antigen-presenting cells (APC) in the presence of SN of monocytes not treated with GM-CSF (□) was set at 100% response and compared with the antigen-specific T cell proliferation induced by the relevant type of APC in the presence of SN(GM-CSF) (▪) or SN(GM-CSF/indo) (hatched). Data represent the mean (± s.e.m.) of three independent experiments. Proliferation values of non-Dermatophagoides pteronyssinus-stimulated TCC have been subtracted.

In the next series of experiments we asked which of the mediators produced during the co-culture of antigen-pulsed monocytes and TCC were actually responsible for the inhibition of T cell proliferation. Therefore, culture supernatants were harvested 24 h after addition of APC to the T cells and the content of IL-10 and PGE₂ was determined by specific ELISA. As shown in Table 2, high PGE₂ levels correlated with strong TCC suppression (13% growth response). On the other hand, the highest level of IL-10 was found in the supernatant of the medium control, e.g. T cells stimulated by monocytes pulsed with antigen in the absence of cytokines. It is of note that an inhibition of T cell growth was still observed compared with the medium control, although PGE₂ synthesis was decreased by 90% or even 99% through inclusion of indomethacin or IL-4, respectively, in combination with GM-CSF during antigen pulsing of monocytes (Table 2). These data indicate that (i) IL-10 did not mediate T cell growth inhibition directly, and (ii) GM-CSF plus IL-4-treated monocytes were able to inhibit T cell proliferation during co-culture although PGE₂ synthesis was reduced to background levels.

Table 2.

Cytokine production during antigen-specific T cell clone (TCC) stimulation by monocytes

Monocyte treatment	TCC response (%)	PGE2 (ng/ml)	IL-10 (ng/ml)
Medium	100	0·5 ± 0·2	2·3 ± 0·8
GM-CSF	13·0 ± 1·9	24·2 ± 8·1	0·6 ± 0·1
GM-CSF/indo	36·1 ± 3·8	2·3 ± 1·4	1·0 ± 0·3
GM-CSF/IL-4	32·6 ± 2·4	0·2 ± 0·1	1·0 ± 0·3

Prior to co-culture with TCC (5 × 10⁵ cells), monocytes (5 × 10⁵ cells) were incubated with Dermatophagoides pteronyssinus in the absence or presence of either GM-CSF or GM-CSF and indomethacin (indo) or IL-4. Cell-free supernatant was collected 24 h after specific T cell stimulation. The percent of TCC response (± s.e.m.) is shown relative to that induced by antigen-pulsed non-treated monocytes. The levels of IL-10 and PGE₂ are expressed as the mean (± s.e.m.) of at least three independent experiments.

TCC proliferation is partially restored by indomethacin, anti-IL-10 antibody or exogenous IL-2

Since previous reports demonstrated IL-10 to inhibit antigen presentation by mononuclear cells, we tested whether the addition of neutralizing anti-IL-10 antibody alone or in combination with indomethacin would restore the ability of monocytes to fully activate the TCC. As shown in Fig. 4, antigen pulsing of monocytes in the presence of GM-CSF and anti-IL-10 antibody slightly relieved inhibition of TCC proliferation. This effect was comparable to that achieved by blocking PGE₂ through the addition of indomethacin. However, by combining anti-IL-10 antibody with indomethacin at the time of monocyte incubation with antigen and GM-CSF, T cell proliferation was restored to about two-thirds of the level that was stimulated by monocytes pulsed in the absence of GM-CSF. Moreover, if indomethacin was supplemented to the co-culture of monocytes and T cells in order to block ongoing PGE₂ synthesis, T cell growth was still inhibited by about 35% compared with control levels of TCC proliferation induced by non-treated antigen-pulsed monocytes in the presence of exogenous indomethacin.

Fig. 4.

T cell clone (TCC) proliferation is partially restored by indomethacin, anti-IL-10 or exogenous IL-2. Prior to co-culture with TCC, monocytes were incubated with Dermatophagoides pteronyssinus (Dpt) in culture medium that was further supplemented with GM-CSF, indomethacin (indo), or anti-IL-10 antibody as indicated. Indomethacin or IL-2 was also added to the co-culture of T cells and cytokine-treated or non-treated monocytes. Shown is the percent of growth response of TCC relative to that induced by antigen-pulsed non-treated monocytes. Data represent mean (± s.e.m.) of at least four experiments. Proliferation values of non-Dpt-stimulated TCC have been subtracted.

One of the most obvious reasons for reduced growth response would be the failure to synthesize sufficient IL-2, perhaps due to small quantities of PGE₂ that were not detected in the ELISA. To exclude this possibility, excess amounts of IL-2 (50 U/ml) were added at the start of the T cell stimulation assay. As shown in Fig. 4, GM-CSF- or GM-CSF and indomethacin-treated, antigen-pulsed monocytes induced not more than about half-maximal proliferation of the TCC when compared with the control level of TCC proliferation induced by non-treated antigen-pulsed monocytes in the presence of exogenous IL-2.

GM-CSF-mediated inhibition of monocyte APC function is transient

Generally, GM-CSF is believed to convert monocytes into APC with enhanced T cell stimulatory function. Therefore, we reasoned that the suppression of T cell growth by monocytes presenting antigen after their culture in the presence of GM-CSF should be transient. To address this question, monocytes were treated for 1, 2 or 5 days with GM-CSF and were subsequently co-cultured with the TCC. The TCC response induced by monocytes treated for 1 day with GM-CSF was inhibited, as shown before (Fig. 5). However, monocytes treated for 2 days with GM-CSF induced a TCC reponse comparable to the control level of TCC proliferation induced by monocytes pulsed 1 day with antigen in the absence of cytokines. Monocytes treated for 5 days with GM-CSF were superior to non-treated monocytes in their capacity to stimulate T cell proliferation.

Fig. 5.

Inhibitory effect by GM-CSF-treated monocytes on T cell clone (TCC) proliferation is transient. Monocytes were either pulsed 1 day with antigen in the absence of cytokines, washed, and used as antigen-presenting cells (APC) (•) or were incubated with GM-CSF for 1 day (▪), 2 days (▾), or 5 days (▴). During the last day of cytokine treatment the medium was supplemented with Dermatophagoides pteronyssinus. Different numbers of antigen-pulsed monocytes were co-cultured with TCC (4 × 10⁴ cells/well). The proliferative response was determined at day 3. Data represent the mean ct/min (± s.e.m.) of four independent experiments.

GM-CSF dose-dependently decreases the accessory function of monocytes for anti-CD3 activated autologous polyclonal CD4⁺ T cells

Having demonstrated the reduced capacity of GM-CSF-treated monocytes to stimulate the antigen-specific proliferation of an HLA-DP-matched TCC, we asked whether the same phenomenon could be seen in a non-cognate model of T cell activation. To address this question we used highly purified CD4⁺ T cells that were activated by soluble anti-CD3 MoAb in the presence of autologous monocytes treated before with increasing doses of GM-CSF. Since PGE₂ induced by GM-CSF is likely to be one of the mediators responsible for inhibition of T cell proliferation, we also included indomethacin during the incubation of the monocytes and their co-culture with the T cells. The results of a representative experiment are shown in Fig. 6. It is obvious that GM-CSF incubation of the monocytes prior to the addition of T cells and anti-CD3 antibody dose-dependently decreased their accessory function. The use of indomethacin both during the incubation of monocytes with GM-CSF and during the co-culture period partially restored the growth response of T cells cultured in the presence of monocytes treated with higher doses of GM-CSF.

Fig. 6.

GM-CSF dose-dependently decreases monocyte accessory function for polyclonally stimulated autologous T cells. Two × 10⁵ monocytes were seeded per 96-well and treated with increasing concentrations of GM-CSF in the absence (▪) or in the presence of indomethacin (▾). After 16 h they were washed in culture medium and 1 × 10⁵ autologous CD4⁺ T cells were added to each well followed by the addition of soluble anti-CD3 MoAb (200 ng/ml final concentration). The proliferation was determined at day 3. No proliferation was detected in wells that contained only monocytes or T cells (ct/min < 250). Results obtained in one of three similar experiments are shown (means of ct/min ± s.d.).

Discussion

In this study, we have shown that GM-CSF mediated a strong inhibition (approx. 90%) of specific memory T cell proliferation when present during the incubation of monocytes with antigen. The strong suppressive effect of GM-CSF was slightly reversed by IL-4 since the combination of both cytokines with antigen during APC pulsing still resulted in about 70% inhibition of TCC proliferation. In contrast, a small stimulatory effect on T cell growth was obtained if MoDC were pulsed with antigen in the presence of GM-CSF or GM-CSF and IL-4. Because both cytokines are widely used to promote the differentiation of monocytes into potent APC in vitro[17,18], a profound suppression of specific recall T cell responses as a result of treating monocytes with GM-CSF alone or in combination with IL-4 was not anticipated. Therefore, we also investigated the stimulatory capacity of monocytes that were incubated with GM-CSF for 2 or 5 days. This experiment revealed that the inhibition of T cell growth by GM-CSF-treated monocytes was transient.

As demonstrated by others and in this study, GM-CSF can induce monocytes to secrete mediators that negatively affect T cell functions, particularly PGE₂ and IL-10. In fact, PGE₂ appeared to be the major soluble inhibitor, as shown by the transfer of supernatants obtained from GM-CSF- or GM-CSF and indomethacin-treated monocytes to co-cultures of T cells and antigen-pulsed APC (EBV-B cells, MoDC or untreated monocytes). Conversely, IL-10 did not seem to inhibit T cell growth directly, because high IL-10 levels were found in the supernatant of T cells co-cultured with untreated antigen-pulsed monocytes (Table 2). The results shown here would be more consistent with an indirect inhibition of antigen presentation via the release of IL-10 from GM-CSF-treated monocytes. This is in line with a previous report showing IL-10 mediated inhibition of antigen-specific T cell proliferation only if monocytes but not if EBV-transformed B cells were used as APC [10]. Later it was shown that IL-10 decreased the level of antigen/MHC class II complexes by reducing ability of monocytes to efficiently transport newly synthesized, peptide-loaded MHC class II molecules to the plasma membrane [24]. Although the level of expression of HLA class II antigens or, more specifically, HLA-DP molecules on monocytes was not decreased after 20 h of incubation with GM-CSF (FACS data not shown), it still remains possible that IL-10 secreted during antigen pulsing caused a decrease of the fraction of HLA-DP molecules that became successfully loaded with specific peptides. Consistent with this assumption is the partial restoration of T cell growth induced by monocytes treated with GM-CSF in the presence of anti-IL-10 antibody.

It is noteworthy that IL-2 added to the co-culture of the T cell clone and antigen-pulsed GM-CSF-treated monocytes failed to relieve growth inhibition to more than about 50%. Even the combination of indomethacin and anti-IL-10 did not completely restore the T cell response. Taken together, these data indicate a cell contact-dependent inhibition by GM-CSF-treated monocytes as opposed to insufficient costimulation. The latter was addressed by FACS analysis of the relative expression level of CD40, CD80, and CD86 on monocytes treated with GM-CSF compared with untreated monocytes. We could not detect a significant modulation of the expression of these surface receptors (data not shown).

Several reports describe GM-CSF-induced immune suppressor cells that resemble immature cells of the monocyte lineage [14,15,25]. Recently, CD14⁺ monocytes present in mobilized stem cell products have been reported to inhibit T cell function only via cell–cell contact [16]. At least in part, this is consistent with the results obtained in this study, since the inhibition seen in co-culture experiments was always far more pronounced than the suppression obtained in supernatant transfer experiments. Soluble factors like PGE₂ and IL-10 are known as general inhibitors of naive T cell proliferation. This led us to investigate the GM-CSF-mediated reduction of monocyte accessory function in a non-cognate stimulation system. As shown in Fig. 6, GM-CSF dose-dependently decreased the capacity of purified monocytes to support the polyclonal proliferation of autologous T cells activated by soluble anti-CD3 MoAb. As seen in the cognate situation, the accessory function was partially restored by addition of indomethacin during monocyte incubation and T cell co-culture.

Recently Kalinski et al. [26] showed that PGE₂ facilitates the cytokine-induced final maturation of MoDC and that these PGE₂-matured MoDC could bias the differentiation of naive Th cells toward Th2 due to their greatly diminished capacity to secrete bioactive IL-12. These data together with the results shown in the present study would imply that monocytes infiltrating an inflammatory environment, for example the synovial joints in rheumatoid arthritis, where GM-CSF is abundantly expressed [27,28], are activated to secrete high levels of PGE₂ and thereby influence not only the final maturation process of MoDC but also contribute to the direct inhibition of Th1 cell proliferation and an anti-inflammatory effect via immune deviation from a Th1 into a Th2 dominated response.