Abstract
Interleukin (IL)-21 is a T cell-derived cytokine which uses a heterodimeric receptor, composed of the common γ-chain (CD132) and an IL-21Rα-chain. IL-21 activates lymphoid T and B cells, modulates antibody production but also suppresses maturation of myeloid dendritic cells; however, its role in the differentiation and function of other myeloid cells remains less clear. In this study we analysed IL-21/IL-21Rα effects on macrophage (MΦ) differentiation and function. MΦ could be generated readily from bone marrow with MΦ–colony-stimulating factor in the presence of IL-21 (designated IL-21MΦ) or from IL-21Rα–/– mice. IL-21Rα–/– mice had normal MΦ numbers, suggesting a non-essential role of both IL-21 and the IL-21Rα for MΦ generation. We could demonstrate that mature MΦ express the IL-21Rα and the common γ-chain. However, short-term IL-21 stimulation did not enhance MΦ proliferation but induced anti-apoptotic cell-cycle regulators p21waf1/p27Kip1 and expression of suppressors of cytokine signalling (SOCS)2/SOCS3. Moreover, IL-21 enhanced phagocytosis by MΦ via IL-21Rα signalling and supports protease activity and matrix metalloproteinase 12 expression. Stimulating MΦ with IL-21 enhanced their capacity to induce antigen-specific CD4+ T cell proliferation in dependence from the IL-21Rα, which was not the case for CD8+ T cells. Taken together, IL-21 plays a previously unrecognized role in modulating innate and acquired effector mechanisms of murine MΦ by linking these different functions to support CD4+ T cell-mediated immune responses.
Keywords: cytokines, inflammation, macrophages, T cells
Introduction
Interleukin (IL)-21 is a cytokine with close structural similarities to IL-15, IL-2 and IL-4 [1]. IL-21 binds to a heterodimeric receptor composed of a private high-affinity α-chain, and in addition shares the common γ-chain (CD132) as functional subunit of its IL-21 receptor complex [2] (reviewed in Brandt et al.) [3]. We can show expression of the complete receptor in dendritic cells (DCs), and IL-21R expression has been found in synovial macrophages (MΦ) of patients with rheumatoid arthritis [4]. IL-21 polymorphism has been associated with other autoimmune diseases as lupus erythematosus [5], suggesting that this receptor/ligand pair could play an important role both in innate and acquired immunity and in the biology of myeloid cells. In fact, it has been demonstrated recently that IL-21 stimulation of myeloid MΦ resulted in up-regulation of IL-4Rα and IL-13Rα1 [6]. Interestingly, IL-21Rα expression was also found in bone marrow (BM) cells, matching the fact that IL-21 promotes the differentiation of lymphoid cells [7] and, as we have shown recently, also modulates the differentiation of murine myeloid DCs. The suppressive effects of IL-21 on DCs had also been confirmed recently in human cells [8]. Unlike the expression of its receptor, IL-21 itself appeared more restricted; production was found mainly in activated but not resting peripheral T cells [1]. These observations open up the possibility that IL-21 might be involved in the interaction of antigen-presenting cells (APCs) such as MΦ with T cells at inflammation sites. It has been speculated recently that IL-21 is an important stimulus for the development of highly functional alternatively activated MΦ, which are efficient stimulators of CD4+ (T helper type 2) T cells [6]; however, direct evidence for the potency of IL-21-activated MΦ to induce antigen-specific CD4+ T cell activation is still lacking. In antigen-unspecific, cytokine-driven T cell activation, IL-21 enhanced significantly the cytokine-driven proliferation of CD4+ helper T cells synergistically with IL-7 and IL-15, suggesting that IL-21 produced from CD4+ memory T cells may have a supportive role in the maintenance of CD4+ T cell subsets [9]. In contrast, it has been demonstrated that IL-21 induces apoptosis of antigen-specific CD8+ T cells [10].
In this study we have analysed how IL-21 and IL-21Rα influence the differentiation function and activation of BM-derived MΦ as well as the MΦ–T cell interaction. To this end, we generated MΦ with MΦ–colony-stimulating factor (M–CSF) alone or in the presence of IL-21 from BM of wild-type and IL-21Rα-deficient mice and evaluated the effects of IL-21 on MΦ proliferation and phagocytosis as well as the modulatory role of IL-21 on the capacity of MΦ to activate antigen-specific CD4+ and CD8+ T cells. We used primary BM-derived MΦ culture to ensure a homogeneous MΦ population that responds to physiological proliferative or activating stimuli.
Taken together, we could show here that IL-21 is not essential for the in vivo and in vitro differentiation of MΦ, but our data imply that IL-21 enhances innate functions such as phagocytosis and protease activity of MΦ. After a short stimulation, IL-21 does not support the proliferation of MΦ but significantly induced cell-cycle regulators and suppressors of cytokine signalling (SOCS) molecules. During acquired immune functions with specific antigens in the context of MΦ/T–cell interaction, IL-21 enhances antigen-specific CD4+ T cell activation by MΦ via the IL-21Rα, but had no effect on antigen-specific CD8+ T cell responses.
This report expands knowledge on IL-21 significantly and demonstrates a functional role of this cytokine on murine MΦ. Our data suggest that IL-21 has complex effects on MΦ function and biology and regulates innate as well as acquired immune functions.
Methods
Mice and culture medium
Bone marrow was taken from C57BL/6 wild-type and IL-21Rα-deficient mice on a BL/6 background. In addition, we used (also on a BL/6 background) OTIItg mice transgenic for a CD4+ T cell-restricted T cell receptor (TCR), recognizing ovalbumin (OVA(323−2339)) peptide, and OTItg mice transgenic for a CD8+-restricted TCR recognizing OVA(257−5264) peptide. All mice were bred at the Research Center Borstel, maintained in specific pathogen-free conditions and used between 8 and 10 weeks of age.
Cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (Biochrom, Berlin, Germany), 2 mM l-glutamine (Invitrogen, Karlsruhe, Germany), 50 μM β-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Pasching, Germany) at 37°C in 5% CO2.
Macrophage preparation
Bone marrow cells from femurs were freshly isolated and red blood cells were lysed by osmotic shock. Washed cells were seeded (1 × 106/ml) in bacterial-grade Petri dishes (BD Falcon, Heidelberg, Germany) and induced to differentiate for 8 days in 10 ml medium plus 20 ng/ml M–CSF (Tebu, Offenbach, Germany) alone or M–CSF plus 20 ng/ml IL-21 (R&D Systems, Wiesbaden, Germany). After 3 days, 10 ml fresh medium with M–CSF or M–CSF + IL-21 was added. At day 8, cells were harvested using Accutase (PAA).
Reverse transcription–polymerase chain reaction
Total RNA was extracted after an additional 24-h stimulation with medium or IL-21 (100 ng/ml) using Trizol (Gibco, Karlsruhe, Germany), following the manufacturer's instructions. cDNA was synthesized using random hexanucleotide primers and the Superscript pre-amplification system II (Invitrogen). cDNA was amplified using 1 U AmpliTaq DNA polymerase (Roche, Grenzach, Germany). The final concentration of each primer was 0·5 μM. Cycling conditions for 30 cycles were for 5 min at 94°C, for 30 s at 60°C, for 30 s at 72°C and finally for 10 min at 72°C. To exclude contamination, all experiments were run with a mock polymerase chain reaction (PCR). β-actin was used to normalize cDNA amount. Primer sequences (Metabion, Martinsried, Germany) are shown in Table 1.
Table 1.
Primer sequences.
| IL-21Rα sense 5′-CTCAGCCAGGCACTTCATTCAGG-3′ |
| IL-21Rα anti-sense 5′-ATCACAGGAAGGGCATTTAGC-3′ |
| γc sense 5′-GTCGACAGAGCAAGCACCATGTTGAAACTA-3′ |
| γc anti-sense 5′-GGATCCTGGGATCACAAGATTCTGTAGGTT-3′ |
| IL-12p40 sense 5′-ATCGTTTTGCTGGTGTCTCC-3′ |
| IL-12p40 anti-sense 5′-CTTTGTGGCAGGTGTACTGG-3′ |
| TNF-α sense 5′-AACTAGTGGTGCCAGCCGAT-3′ |
| TNF-α anti-sense 5′-CTTCACAGAGCAATGACTCC-3′ |
| p21waf1 sense 5′-GGGATGGCAGTTAGGACTCA-3′ |
| p21waf1 anti-sense 5′-ACCCTAGACCCACAATGCAG-3′ |
| p27kip1 sense 5′-AATCTCATCACCCCACTTGC-3′ |
| p27kip1 anti-sense 5′-GGCCATTTTCCATCTCTGAA-3′ |
| MMP12 sense 5′-TGAAAGTGACCGGGCAACTGGACA-3′ |
| MMP12 anti-sense 5′-TGTGCATCCCCTCCAATGCCAGA-3′ |
| SOCS2 sense 5′-GGGACTGCCTTTACCAACAA-3′ |
| SOCS2 anti-sense 5′-AAAGGGCCATTTGATCTTGA-3′ |
| SOCS3 sense 5′-TGCAAGGGGAATCTTCAAAC-3′ |
| SOCS3 anti-sense 5′-AGCTCACCAGCCTCATCTGT-3′ |
| β-actin sense 5′-GTGGGGCGCCCCAGGCACCA-3′ |
| β-actin anti-sense 5′-CTCCTTAATGTCACGCACGATTTC-3′ |
IL, interleukin; MMP, matrix metalloproteinase; SOCS, suppressors of cytokine signalling; TNF, tumour necrosis factor.
Fluorescence activated cell sorter analysis
Macrophage were characterized using a panel of antibodies including anti-F4/80 [fluorescein isothiocyanate (FITC)-labelled, (Serotec) Düsseldorf, Germany] and anti-CD80, CD86 and major histocompatibility complex (MHC) class II (IA/IE, phycoerythrin-labelled; Pharmingen Heidelberg, Germany). Propidium iodide (Sigma, Seelze, Germany) was added to exclude dead cells from analysis. Flourescence activated cell sorter (FACS) analysis was performed on F4/80+ gated MΦ on a FACSCalibur with CellQuest software (Becton Dickinson, Heidelberg, Germany).
Analysis of endocytosis by FACS
To quantify the endocytic activity, FITC–dextran (Molecular Probes, Karlsruhe, Germany) uptake was monitored as described by Stumbles et al. [11]. In brief, 5 × 105 MΦ were resuspended in 100 μl RPMI-1640/FCS containing 0·5 mg/ml FITC–dextran (molecular weight: 70·000) for 30 min at 37°C or on ice. Cells were washed twice with ice-cold phosphate buffered saline/bovine serum albumin and fluorescence intensity was determined by FACS. The mean fluorescence intensity is given.
Analysis of protease activity
This assay was performed using an EnzCheck Protease assay kit (Molecular Probes). Results are presented as relative fluorescence units.
Proliferation assays
T cells were isolated from lymph nodes (LN) of OTI and OTII TCR transgenic mice; 1 × 105 MΦ were aliquoted into 96-well flat-bottomed culture plates (Costar Corning, Schiphol, Netherlands) and allowed to adhere for 2 h. CD4+ cells were added at 2 × 105 cells per well. OVA(323−2339) peptide, 0·1 μM (optimal concentration defined by titration experiments), was supplemented to a final volume of 200 μl. The cultures were incubated for 72 h and labelled for an additional 18 h with 0·2 μCi/well [3H]-thymidine (Amersham Biosciences Buckinghamshire, UK).
Antigen-specific CD8+ T cell proliferation assays were performed as follows: MΦ were labelled with 2 μg OVA(257−5264) peptide for 2 h at 37°C in 500 μl medium, washed twice and plated-out into 96-well round-bottomed culture plates (Greiner, Flacht, Germany) at a density of 1 × 104 cells per well along with 1 × 105 LN cells. After 48 h, plates were pulsed for 18 h with 0·2 μCi/well [3H]-thymidine. [3H]-thymidine uptake was analysed by liquid scintillation counting (Wallac/PerkinElmer, Waltham, MA, USA). The experiments were repeated three times with six replicates each.
Statistical analysis
Results are presented as mean ± standard deviation from the pooled data of two to three identical experiments. FACS and PCR data from one representative experiment are shown. Student's t-test for unpaired samples was used for the determination of statistical differences (*P ≤ 0·05; **P ≤ 0·01).
Results
IL-21 and IL-21Rα do not modulate murine MΦ differentiation
The expression of the IL-21 receptor has already been demonstrated in BM cells and we could show further that the presence of IL-21 during myeloid DC's differentiation modulates DC's maturation and function [12, 13]. However, little is known as yet about the role of IL-21 on differentiation and function of MΦ. Here we analysed whether IL-21 and signalling via the IL-21Rα could modulate the M–CSF-induced generation of MΦ. Therefore, freshly isolated BM cells were cultured following well-established protocols with M–CSF alone to induce differentiation of MΦ or M–CSF combined during the entire culture period of 8 days with 20 ng/ml IL-21 (subsequently designated IL-21MΦ). In parallel, BM from IL-21Rα−/− mice was cultured with M–CSF alone (IL-21Rα−/− MΦ). Using FACS analysis, after 8 days of culture all three different MΦ showed comparable size and granularity (Fig. 1a) and the percentage of F4/80+ cells (a specific MΦ marker in mice) was similarly high in all conditions (Fig. 1a). Moreover, using FACS analysis we assessed that adding IL-21 to BM cells in combination with M–CSF did not lead to increased differentiation of other cell types such as B, natural killer, T cells, DCs or granulocytes (not shown). In accordance, the cell shape and morphology of the differentiated MΦ was comparable (Fig. 1). The total differentiation efficiency was also equal; seeding 1 × 107 BM cells per 9-cm tissue culture plate resulted in the differentiation of similar numbers of MΦ, irrespective of the addition of IL-21 or the presence of the IL-21Rα (Fig. 1b). Moreover, ex vivo isolation of peritoneal MΦ revealed no differences in number in wild-type and IL-21Rα−/− mice (data not shown). Message-expression for the private IL-21Rα subunit was found previously in lymphoid tissues [1] but has not been analysed in MΦ. In order to elucidate whether MΦ express IL-21 receptor components, the IL-21Rα subunit and the common γ-chain, we performed reverse transcription–polymerase chain reaction analysis and found both receptor components expressed constitutively in MΦ (Fig. 1c). Different stimuli, such as the structurally related IL-15 or IL-21 itself, but also danger signals such as lipopolysaccharide, did not affect the message expression for both receptor chains. In addition, we detected expression of the chemokine receptors CCR1 and CCR5 mRNA, which were also not regulated by IL-21 (data not shown). Taken together, these findings reveal that IL-21 is not essential for MΦ differentiation in vitro and in vivo. However, mature MΦ express the complete dimeric IL-21 receptor, suggesting a role of IL-21 in the biology of differentiated MΦ.
Fig. 1.
Interleukin (IL)-21 did not alter macrophage (MΦ) differentiation. (a) MΦ were differentiated from murine bone marrow (BM) cells with MΦ–colony-stimulating factor (M–CSF) or M–CSF plus IL-21 (IL-21MΦ) from wild-type mice or from BM of IL-21Rα−/− mice and analysed by fluorescence activated cell sorter at day 8. They show comparable size (forward scatter) and granularity (side scatter), as well as F4/80 expression (given in percentage) and comparable cell morphology. (b) Equally efficient differentiation of MΦ with M–CSF in the presence of IL-21 or absence of the IL-21Rαin vitro. Shown is the number of recovered MΦ per culture plate, differentiated out of 1 × 107 BM cells. (c) MΦ express constitutively the complete heterodimeric IL-21 receptor. Polymerase chain reaction was performed on unstimulated or 24-h stimulated MΦ as indicated for both components: the IL-21Rα and the common γ-chain.
IL-21MΦ shows a high proliferation rate
Expansion of MΦ in response to growth factors is important to increase the number of phagocytosing MΦ, i.e. at infection sites. In this study we tested the proliferation of 8 days' differentiated MΦ, IL-21MΦ and IL-21Rα−/− MΦ in response to an additional 48-h stimulation with IL-21. As shown in Fig. 2a, IL-21MΦ showed significantly higher proliferation compared with MΦ and IL-21Rα−/− MΦ. However, the addition of IL-21 to the normal MΦ and IL-21Rα−/− did not enhance their proliferative response, clarifying that IL-21 shifts MΦ during differentiation states towards highly dividing cells but has less effect on the proliferation of readily differentiated MΦ.
Fig. 2.
Interleukin (IL)-21 enhances macrophage (MΦ) survival and stimulates expression of cell-cycle regulators and inhibitory molecules. (a) Proliferation is enhanced significantly in IL-21MΦ and 48-h IL-21 stimulation has no effect on MΦ and IL-21Rα−/− proliferation; cpm: counts per minute. (b) Stimulation of MΦ with IL-21 for 24-h induced message expression of the cell-cycle regulator p21waf1 and p27Kip1 as well as of suppressors of the cytokine signalling (SOCS)2 and SOCS3 message.
IL-21 induces expression of cell-cycle inhibitors
Cell survival, development, proliferation and differentiation are controlled tightly by cell-cycle promoting and inhibiting regulators, of which cyclin-dependent kinases and their inhibitors are the best-studied. We reasoned that the enhanced MΦ survival after IL-21 stimulation (not shown) could be due to altered expression of anti-apoptotic cell-cycle regulators. Therefore, we analysed the message expression (which correlates with protein expression [14]) of two important cyclin-dependent kinase (CDK) inhibitors and found that p21Waf1, which protects MΦ from apoptosis [15], is indeed induced in MΦ by IL-21 (Fig. 2b).
Another very important regulator of the MΦ life-cycle is p27Kip1, which has been shown to be regulated tightly during terminal maturation and MΦ proliferation [15]. Our data demonstrate that p27Kip1 is induced clearly by IL-21 stimulation (Fig. 2b). In summary, expression of these CDK inhibitors correlates with the effects of IL-21 on normal differentiated MΦ, which failed to induce proliferation, but protects MΦ from apoptosis. In addition, expression of SOCS-2 and SOCS3 is up-regulated (Fig. 2b), which also might contribute to enhanced survival as they are associated with counteracting MΦ cell death [16].
IL-21 promotes phagocytosis and protease activity in MΦ
To analyse functional consequences of the stimulation of MΦ with IL-21, we used several assays for unspecific, innate MΦ functions. MΦ are important in the context of immunological surveillance. One of the major tasks of MΦ is the phagocytosis of bacteria or dead cells, but also of soluble antigens. It is accepted widely that soluble antigens taken up by the mannose receptor are presented by not only MHC class II molecules to CD4+ T cells but also cross-presented by MHC class I to CD8+ T cells. We analysed first the mannose receptor-mediated uptake of FITC–dextran by the different MΦ, as described previously [17], and the influence of IL-21 during this process. Stimulation of MΦ with IL-21 during the 30-min incubation enhanced phagocytosis significantly (Fig. 3a). This was also true for the long-term IL-21-exposed IL-21MΦ, which displayed a significantly enhanced baseline phagocytosis capacity compared with normal MΦ, which could be triggered further by additional short-term IL-21 stimulation. Interestingly, the IL-21Rα−/− MΦ took up antigen to the same extent as wild-type MΦ, which could not be elevated by the addition of IL-21, pointing to an essential role of the IL-21Rα chain for stimulation of this innate MΦ function.
Fig. 3.
Interleukin (IL)-21 enhances macrophage (MΦ) phagocytosis, protease activity and MMP12 expression. (a) IL-21 significantly stimulates MΦ phagocytosis of fluorescein isothiocyanate (FITC)–dextran, which was more pronounced in IL-21MΦ. MFI: mean fluorescence index. In addition, original fluorescence activated cell sorter data from one representative experiment are shown; the upper row shows FITC–dextran in medium, the lower row shows FITC–dextran with IL-21 added for the 30-min incubation period. Numbers in the blots give the MFI. (b) In accordance with high antigen-uptake, IL-21MΦ display significantly higher unspecific protease activity after lipopolysaccharide activation. RFU: relative fluorescence units. (c) Reverse transcription–polymerase chain reaction showed message induction for MMP12 by short-term IL-21 incubation and higher baseline expression in IL-21MΦ.
After phagocytosis, the material which has been taken up has to be degraded subsequently, while the MΦ migrate through the periphery. Both processes are mediated by several classes of enzymes. To gain information on the role of IL-21 on MΦ enzyme activity, we checked general, unspecific protease activity and found it to be enhanced significantly only when MΦ were cultured long-term with IL-21 into IL-21MΦ (Fig. 3b) but not, however, in IL-21Rα−/−MΦ or after short-term stimulation with IL-21 (not shown). It has been published recently that IL-21 induces matrix metalloproteinase (MMP) expression in fibroblasts [18]. We could demonstrate that MMP12 was induced at message level in MΦ after 24-h stimulation with IL-21 and also in IL-21MΦ, which had been exposed to IL-21 during differentiation (Fig. 3c). Our data revealed that IL-21 modulates protease activity, and especially MMP12, known to be important for MΦ migration. In future studies we will analyse the underlying processes in more detail.
IL-21Rα−/− MΦ display reduced MHC class II expression
After uptake and degradation of antigen and migration of MΦ to lymphoid organs, MΦ present soluble antigen to naive T cells. Antigen presentation to CD4+ T cells and induction of T cell activation by MΦ is mediated by MHC class II and a variety of co-stimulatory molecules on the surface of MΦ. To assess the repertoire of surface molecules, we performed FACS phenotyping after 8 days of MΦ culture. Interestingly, generation in the presence of IL-21 did not change the expression of MHC class II but enhanced CD80 and CD86 on IL-21MΦ. In the absence of IL-21Rα, MΦ showed a reduction of all three molecules tested (Fig. 4a), suggesting a role of IL-21Rα for the expression of antigen-presenting and co-stimulatory molecules.
Fig. 4.
Interleukin (IL)-21Rα is important for expression of co-stimulatory molecules on macrophage (MΦ) and promotes the capacity of MΦ to induce antigen-specific CD4+ T cell proliferation. (a) The different MΦ were generated for 8 days in vitro by cultivating bone marrow cells with MΦ–colony-stimulating factor (M–CSF) and expression of co-stimulatory molecules, and major histocompatibiltity complex (MHC) class II was analysed by fluorescence activated cell sorter. Positive cells are given in percentage. (b) MΦ were stimulated for 2 h with IL-21, washed and co-cultured with 0·1 μM ovalbumin (OVA) peptide and OVA T cell receptor transgenic CD4+ T cells for 48 h. Proliferation was analysed by [3H]-thymidine incorporation; cpm: counts per minute.
IL-21 enhances MΦ capacity to induce antigen-specific CD4+ T cell proliferation via the IL-21Rα
To analyse the effect of IL-21 on MΦ/T cell interactions, and in order to dissect specifically the immunostimulatory capacity of IL-21MΦ and IL-21Rα−/− MΦ, we used a co-culture assay with TCR-transgenic, syngeneic CD4+ T cells. The MΦ were labelled with peptides from OVA and cultured with OVA TCR transgenic T cells in vitro. As shown in Fig. 4b, peptide-labelled MΦ induced profound CD4+ T cell proliferation, which is increased significantly when the MΦ were pre-incubated for 2 h with IL-21. IL-21MΦ induced a significantly higher T cell proliferation as normal MΦ (correlating with the elevated CD80 and CD86 expression) which, again, could be enhanced further if they were stimulated for 2 h with IL-21 during antigen uptake. Interestingly, despite lower MHC class II expression, IL-21Rα−/− MΦ induce baseline CD4+ T cell proliferation comparable with wild-type MΦ, but incubation with IL-21 did not affect their capacity for activating CD4+ T cells. Our results suggest that the IL-21 effect on MΦ is mediated by IL-21Rα and not the second IL-21 receptor, the common γ-chain.
IL-21Rα−/− MΦ induce higher antigen-specific CD8+ T cell proliferation
As shown above, IL-21 supports MΦ to induce CD4+ T cell expansion. However, it remains to be clarified how IL-21 affects the capacity of MΦ to induce antigen-specific CD8+ T cell expansion. To test this, we used OVA peptide-specific, transgenic CD8+ T cells. First, MΦ were pulsed for 2 h with OVA peptide alone or additionally IL-21 was added in parallel with OVA peptide. After washing away peptide and IL-21, T cells from LN of OTI mice, expressing a CD8+ T cell-restricted, transgenic TCR which recognizes the OVA peptide specifically, were given to the peptide/cytokine-labelled MΦ. As depicted in Fig. 5a, OVA peptide-labelled MΦ and IL-21MΦ induced a profound, antigen-specific T cell proliferation. However, 2 h of MΦ stimulation with IL-21 did not affect the MΦ-mediated CD8+ T cell proliferation (Fig. 5a). Interestingly, using IL-21Rα−/− MΦ to present the antigen and stimulate the CD8+ T cells resulted in a significantly higher T cell proliferation which, as in normal MΦ, could not be modulated further by previous IL-21 incubation of the MΦ (Fig. 5a).
Fig. 5.
Interleukin (IL)-21 does not enhance antigen-specific CD8+ T cell proliferation, but the IL-21Rα is a suppressor of CD8+ T cell response and macrophage (MΦ) IL-12p40 production. (a) MΦ were labelled for 2 h with ovalbumin (OVA) peptides alone or peptide plus IL-21, washed and co-cultured with OVA T cell receptor transgenic CD8+ T cells for 48 h. Proliferation was analysed by [3H]-thymidine incorporation; cpm: counts per minute. (b) MΦ, IL-21Rα−/−MΦ and IL-21 MΦ were stimulated for 24 h with medium or IL-21 and expression of IL-12p40 and tumour necrosis factor-α was analysed by reverse transcription–polymerase chain reaction.
IL-21Rα−/− MΦ express more IL-12p40 message
Finally, we analysed the possible mechanisms whereby MΦ, without IL-21Rα, induces significantly higher CD8+ T cell proliferation. To this end, we searched for cytokine expression in the MΦ. Using PCR, we found an enhanced IL-12p40 message in the IL-21Rα−/− MΦ compared with wild-type MΦ and IL-21MΦ, which was specific for this cytokine as tumour necrosis factor-α expression was comparable in IL-21Rα−/− MΦ and MΦ (Fig. 5b).
Discussion
Macrophages are derived from undifferentiated stem cells in the BM and reach different tissues through the blood where, in most cases, they undergo apoptosis. In the presence of specific growth factors or cytokines, MΦ receive survival signals and are subsequently able to proliferate, become activated or differentiate. To carry out their functional activities as phagocytosis or antigen presentation, MΦ must become activated. Thus, after interaction with T cell-derived cytokines, released by activated T cells, MΦ undergo biochemical, morphological and functional modifications that allow them to perform their functional activities. IL-21 is a newly described and, in the context of MΦ biology to date, not well-studied T cell-derived cytokine which might affect MΦ functions. Here we present the first evidence that IL-21 indeed modulates several innate and acquired immune functions of murine MΦ. Our results show that IL-21 might be important for the transition from unspecific to specific CD4+ T cell-mediated immune responses induced by MΦ.
Recently, it could be demonstrated that IL-21 is expressed by stromal cells of lymphoid organs [19]; therefore, it might also be secreted by BM stromal cells and might influence MΦ differentiation. However, our data show that IL-21Rα signalling is redundant during the differentiation of murine MΦ from haematopoietic stem cells (HSC), as phenotypically normal MΦ could be differentiated in vitro with comparable efficiency from IL-21Rα-deficient as from wild-type HSC. Also, addition of IL-21 during the entire MΦ differentiation did not shift the MΦ morphology, purity and efficiency of the M–CSF-triggered MΦ generation. Recently we could also demonstrate similar findings for the differentiation of murine DCs from HSC [13]. Because IL-21R is expressed in BM, where activated T cells (the main source of IL-21) are also found, it is likely that in vivo HSC are also exposed to this cytokine. We demonstrate further that upon maturation, MΦ expresses both chains of the heterodimeric IL-21 receptor, similar to DCs. This opens up a new possibility of how MΦ and activated T cells might communicate, e.g. at infection or inflammation sites in the body, but also in lymphoid organs. During infection, MΦ phagocytose, invading pathogens as well as apoptotic and necrotic cells. Interestingly, IL-21 stimulation enhances this process significantly by binding to IL-21Rα. In DCs, IL-21 also enhanced the capacity for phagocytosis by keeping murine DCs in an immature state [12, 13]. Long-term exposure to IL-21 contributes to the capacity of MΦ to degrade the phagocytosed material and to migrate to or from the inflammation site by enhancing total protease activity and expression of MMP12 as one exemplary protease. This is in line with recent findings that in other clinically relevant settings IL-21 also increases MMP expression [18]. This has been demonstrated for inflammatory bowel disease, where MMP expression by fibroblasts is enhanced after IL-21 stimulation [18]. Taken together, our data support a proinflammatory effect of IL-21 on innate MΦ functions.
Expansion and proliferation of monocytes is controlled tightly and normally takes place in BM, from where they are released into the circulation and migrate to peripheral tissues, where they differentiate into tissue MΦ. In this study we have analysed how IL-21 affects the proliferation of readily differentiated MΦ (after 8 days of culture with M–CSF). Interestingly, the addition of IL-21 for another 3 days did not increase proliferation, but long-term IL-21-exposed MΦ showed a significantly higher cell division rate. Proliferation and apoptosis are regulated tightly by CDK and several CDK inhibitors; the expression of two of these was analysed by us. In particular, p27Kip1 has been shown to be responsible in inhibiting the proliferation of MΦ, whereas p21Waf1 expression is necessary to protect MΦ from apoptosis [15]. Both molecules are induced in MΦ by short-term IL-21 stimulation, suggesting that in the context of immune responses IL-21 delivers a survival signal to MΦ.
In addition, IL-21 induces expression of the feedback inhibitors SOCS2 and SOCS3, which in turn down-regulate signal transducer and transcription activation, and therefore important signalling pathways of several cytokines, including IL-21 itself [7].
The induction of SOCS3 in myeloid cells affects their innate and acquired immune function significantly. We could demonstrate that murine DCs pre-incubated with IL-21 are inhibited in their maturation and are therefore kept in an immature state, where they are unable to induce potent CD8+ T cell expansion. This is in accordance with reports using human DC's and CD8+ T cells, where pre-incubation of DC's with IL-21 also did not trigger CD8+ expansion; however, giving IL-21 directly to CD8+ T cells led to their expansion [20]. Here, we show that murine MΦ are not affected in their capacity to induce antigen-specific CD8+ T cell proliferation by short-term stimulation or long-term generation with IL-21. Interestingly, however, IL-21Rα−/− MΦ were more potent in inducing CD8+ T cell responses, correlating with enhanced IL-12p40 expression in these IL-21Rα−/− MΦ. These data show that the IL-21Rα on MΦ mediates a suppressive signal for (IL-12-induced) CD8+ T cell responses by controlling MΦ IL-12 expression and CD8+ expansion. In contrast with our results, where we analysed the consequence of IL-21 stimulation of MΦ and subsequent capacity to stimulate CD8+ T cells, Casey et al. demonstrated with artificial APCs that the direct stimulation of CD8+ T cells with IL-21 acts directly on the T cells [21] and supports their expansion. Clearly, IL-21 can provide the direct signal required by naive CD8+ T cells to differentiate in response to antigen and co-stimulation, and the resulting effector T cells represent a unique phenotype with highly effective cytolytic activity, but a deficient capacity to secrete interferon-γ. Interestingly, in another well-performed experimental setting published recently, the authors provided evidence that, rather, IL-21 induces apoptosis of antigen-specific CD8+ T cells [10]. This contradicts partially the first report, or might suggest a dichotomic role of IL-21 in initiating and ending a CD8+ T cell response, showing an urgent need for further research on this cytokine.
Taken together, our data demonstrate a functional role of IL-21 on MΦ functions. The IL-21 released by activated T cells in the context of inflammation during infection but also during allergic sensitization could stimulate MΦ to induce a transition from innate to acquired T cell-mediated immune responses. Therefore, IL-21 should be investigated in further studies and additional in vitro and in vivo models for its pathophysiological relevance, investigating the option for therapeutic modulation of the IL-21 function.
Acknowledgments
We thank Katrin Seeger and André Jenckel for excellent technical assistance.
References
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