IL-21 Enhances Phagocytosis in Mononuclear Phagocyte Cells: Identification of Spleen Tyrosine Kinase as a Novel Molecular Target of IL-21

CD132 (γc)-dependent cytokines (CD132-DCs), including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, play crucial roles in the development, proliferation, survival, and differentiation of multiple cell lineages of both the innate and adaptive immune systems (1–6). IL-21, the most recently discovered of the CD132-DC, mediates biological activities via its receptor, which is composed of CD132 and IL-21Rα subunits (7–9). Binding of IL-21 to its receptor leads to activation of the Jak–STAT pathway, predominantly Jak-1, Jak3, STAT1, and STAT3 (1, 7, 8, 10). In addition to the Jak–STAT pathway, IL-21 also activates MAPKs and PI3K pathways (10, 11). Although its roles in lymphoid cells have been (and are still) intensively studied (12–14), the effects of IL-21 in myeloid cells have attracted much less attention (15). In fact, although several studies have demonstrated that IL-21 can inhibit dendritic cell activation and maturation (16–19), its role in other myeloid cells is poorly documented. Moreover, there is presently no evidence that monocytes and/or macrophages express IL-21Rα on their cell surface. Although human neutrophils express CD132, they do not express IL-21Rα, consistent with the inability of IL-21 to directly activate these cells (11). In contrast, IL-21Rα was detected in human preomyelocyte HL-60 cells differentiated toward the monocyte or macrophage phenotype and IL-21Rα was also detected by Western blot analysis in both human monocytes and monocyte-derived macrophages (11). A recent study indicated that IL-21 modulated its effect on monocytes by maintaining CD16 expression via the production of IL-10 by human naive CD4⁺ T cells (20). Despite this, the role of IL-21 on phagocytosis, a major function of myeloid cells, is presently unknown. In this study, we report for the first time, to our knowledge, that mononuclear cells express IL-21Rα on their cell surface and that IL-21 enhances FcR-mediated phagocytosis in the human monocytic cell line THP-1, as well as in isolated monocytes and GM-CSF–monocyte-derived macrophages. In addition, using a pharmacological approach as well as an antisense strategy, we have demonstrated that spleen tyrosine kinase (Syk) is a novel and important target of IL-21 for enhancing phagocytosis.

RPMI 1640 medium, HEPES, penicillin/streptomycin, heat-inactivated FBS, Opti-MEM, and HBSS were purchased from Invitrogen (Grand Island, NY). The anti–phospho-specific Erk1/2 Ab was purchased from BioSource International (Camarillo, CA), and the rabbit polyclonal Erk1/2 Ab was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-sheep RBCs, FITC-dextran, piceatannol, and trypan blue were from Sigma-Aldrich (St. Louis, MO). Ficoll-Paque was obtained from GE Healthcare BioScience (Uppsala, Sweden). Cytokines (GM-CSF, IL-15, and IL-21) were purchased from PeproTech (Rocky Hill, NJ). Anti–IL-21Rα Ab was from R&D Systems (Minneapolis, MN), and anti–IL-2Rγ (CD132) and anti-Syk Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–phosphorylated Syk and Akt were from Cell Signaling Technology (Danvers, MA). Sheep RBCs (SRBCs) were purchased from Lampire Biological Laboratories (Pipersville, PA). All secondary Abs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

THP-1 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 2.05 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FBS and incubated in a 5% CO₂ atmosphere at 37°C. Cells were subcultured before reaching a concentration of 8 × 10⁵ cells/ml, as recommended by the American Type Culture Collection. Human PBMC were isolated from venous blood of healthy volunteers by centrifugation over Ficoll-Paque. Blood donations were obtained from informed and consenting individuals as per institutionally approved procedures.

For monocyte isolation, 25 × 10⁶ PBMC were incubated at 37°C in a 5% CO₂ atmosphere for 2 h in RPMI 1640 medium and 10% autologous heat-inactivated serum in petri dishes. Monocytes obtained by removing the nonadherent PMBC were further incubated in RPMI 1640 medium–10% heat-inactivated FCS HEPES and penicillin/streptomycin for another 12 h. The monocytes were washed twice with HBSS without Ca²⁺ and Mg²⁺ with 2 mM EDTA and harvested with a cell scraper. Human monocyte–derived macrophages (HMDM) were generated by incubating 2 × 10⁶ PBMC at 37°C in a 5% CO₂ atmosphere for 2 h in RPMI 1640 medium supplemented with 10% heat-inactivated autologous serum in 48-well plates. Monocytes obtained by removing the nonadherent PMBC were further incubated in RPMI 1640 medium–10% heat-inactivated FCS HEPES, penicillin/streptomycin supplemented with 2 ng/ml GM-CSF to obtain macrophages. In all cases, cell viability was assessed by trypan blue exclusion prior to experiments.

Cells (1 × 10⁶) were incubated with 20 μg/ml anti–IL-2Rγ, anti–IL-21Rα or with the appropriate isotypic control Abs for 30 min on ice, and then washed twice with ice-cold PBS. Cells were then incubated for another 30 min on ice in the dark with the appropriate secondary FITC-conjugated Ab. Cells were washed twice with ice-cold PBS before being resuspended at 1 × 10⁶ cells/ml. Cell surface expression was immediately detected using a FACscan (BD Biosciences, San Jose, CA). In some experiments, cells were treated with 50 ng/ml IL-21 for 30 min prior to investigating cell surface expression of IL-21R components.

SRBCs were washed three time in ice-cold PBS, resuspended at 50 × 10⁶ cells/ml, and then opsonized with anti-SRBC Ab (1:200) at 37°C for 45 min, as documented previously (21). THP-1 cells (10 × 10⁶/ml) or monocytes (5 × 10⁶ cells/ml) were treated with the agonists indicated in the corresponding figure legends in RPMI 1640 medium for 30 min. The medium was removed following centrifugation and opsonised SRBCs were added to the cell pellet at a 5:1 ratio and incubated at 37°C in a 5% CO₂ atmosphere for 45 min. After incubation with SRBCs, the samples were centrifuged at 200 × g for 10 min at 4°C. Supernatants were discarded, and pellets were subjected to osmotic shock by resuspending the cells with 400 μl H₂O for 15 s. Osmolarity was then restored by the addition of 4.5 ml ice-cold PBS. The samples were then washed twice, and the final pellets were suspended in 400 μl PBS. A minimum of 250 cells/condition were counted, and the percentage of cells having ingested at least one opsonized SRBC was calculated for each condition, and the results are presented as the fold of increase, which was obtained by dividing the percentage value of the tested condition by the percentage value of the control or basal level. In some experiments, cells were preincubated for 30 min at 37°C with piceatannol (30 mM) or Syki II (1 mM) before the phagocytosis assay.

Before the assay, HMDM were washed twice with warm HBSS and then stimulated with IL-21 at 50 ng/ml RPMI 1640 medium in a total volume of 100 μl for 30 min at 37°C. These macrophages, and 10 × 10⁶ SRBCs were cocultured for 15 min at 37°C. The plates were then removed and placed on ice for 5 min. Cells were washed once with ice-cold HBSS to remove excess of noningested SRBCs. An osmotic shock was performed by adding 100 μl ice-cold H₂O for 15 s. Osmolarity was restored by adding 1 ml ice-cold HBSS. Wells were then stained with Hema-3 stain set (Biochemical Sciences, Swedesboro, NJ) and observed under a light microscope. A minimum of 250 cells/condition was counted, and the results are presented as the fold increase, obtained by dividing the percentage value of the tested condition by the percentage value of the control. In some experiments, cells were preincubated for 30 min at 37°C with piceatannol (30 mM) or Syki II (1mM) before the phagocytosis assay.

THP-1 cells were incubated with 1 mg/ml FITC-dextran for 30 min at 37°C. Cellular uptake of FITC-dextran was then monitored by flow cytometry at 525 nm. A negative control was performed in parallel by incubating cells with FITC-dextran at 4°C instead of 37°C. Uptake of FITC-dextran was expressed as the Δ mean fluorescence intensity (MFI); MFI (uptake at 37°C) – MFI (uptake at 4°C) as described previously (22).

THP-1 (5 × 10⁵/ml) were incubated with a mixture of two Syk antisense oligonucleotides previously used (23, 24): (5′-CTCGGATCAGGAACTTTCCAT-3′ and 5′-CATGGAAACCTGATGAACCAG-3′) or scrambled (Scr) antisense: (5′-TCGACA-AGTCGACTTTCATCG and 5′-GATGGAAACCTGCAGATACCA) with a phosphorothioate backbone at a final concentration of 5 μM each at 37°C in Opti-MEM without addition of serum for 6 h to increase uptake of the oligonucleotides. Subsequently, cells were cultured for 22 h in Opti-MEM supplemented with 5% FBS. Transfection efficiency was >90% as determined by using the same oligonucleotide sequences with a Cy3 fluorophore coupled to the 5′ end and then visualized by fluorescence microscopy. After transfection, phagocytosis was determined as described above. The level of Syk protein expression was monitored by Western blot analysis.

Cells in RPMI 1640 medium were stimulated with IL-21 (50 ng/ml), GM-CSF (65 ng/ml), or the diluent (HBSS) for indicated periods of time at 37°C, followed by lysis in Laemmli’s sample buffer (0.25 M Tris-HCl [pH 6.8], 8% SDS, 40% glycerol, and 20% 2-ME). Aliquots of extracts corresponding to 3.5 × 10⁵ cells were loaded onto 7.5% SDS-PAGE gels, followed by electrophoresis, and then transferred to nitrocellulose membranes for detection of p-Syk, p-Akt, p-ERK, Syk, and ERK. Membranes were blocked for 1 h at room temperature in TBS-Tween containing 3% BSA. After blocking, the primary Abs were added at a final dilution of 1:1000 in TBS-Tween (0.10%). The anti-Syk Ab was added at a final concentration of 1:4000. The membranes were incubated overnight at 4°C, then washed with TBS-Tween and incubated for 1 h at room temperature with the appropriate HRP-conjugated secondary Abs diluted 1:20,000 in TBS-Tween, followed by several washes. Protein expression was revealed using a Luminata Forte HRP substrate (Millipore, Temecula, CA). Membranes were stripped with Reblot Plus Strong solution (Millipore) and reprobed to verify protein loading. Densitometric analyses were performed using Quantity One, version 4.6.6 (Bio-Rad, Hercules, CA).

THP-1 (10 × 10⁶ cells/condition) were treated with the indicated agonists for the indicated period of time at 37°C, centrifuged, and lysed in nondenaturing cold lysis buffer (50 mM Tris-HCl [pH 8], 50 mM NaCl, 1% Triton X-100, 125 mM PMSF, 10 g/ml trypsin inhibitor, aprotinin, leupeptin, and pepstatin, and 1 mM orthovanadate) for 1 h on ice and sonicated three times for 20s. The lysates were preincubated with protein G–Sepharose (GE Healthcare BioScience). After 1 h, samples were centrifuged to remove Sepharose beads and then incubated with 2 μg/ml mouse anti-human Syk or IL-21Rα at 4°C with gentle agitation overnight. Protein G–Sepharose was then added for 4 h with gentle agitation at 4°C. The solid matrix was collected and washed three times with lysis buffer before adding an equivalent volume of sample buffer. The samples were then boiled at 100°C for 10 min. Immunoprecipitates were electrophoresed on an SDS-polyacrylamide gel, followed by Western blot analysis.

Statistical analyses were performed using Student t test or ANOVA, and if significance was identified, individual comparisons were subsequently made by Bonferroni test using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Statistical significance was set at p < 0.05.

We first determined whether monocyte-like THP-1 cells expressed IL-21R components. Fig. 1A illustrates that resting THP-1 cells expressed IL-21Rα (MFI = 11.3 ± 1.5 for IL-21Rα versus 6.1 ± 0.3 [n = 3] for the corresponding isotypic control given a ratio of 1.9) as assessed by flow cytometry. The other component of the IL-21R, CD132, was also expressed on the cell surface of these cells (MFI = 34.8 ± 9.9 for CD132 versus 5.9 ± 1.2 for the corresponding isotypic control (n = 3) (Fig. 1B). The presence of IL-21Rα was also confirmed at the protein level by Western blot experiments in three different THP-1 cell lysates (Fig. 1C). These results demonstrated that THP-1 cells expressed the full IL-21R, and this prompted us to verify whether IL-21 could activate these cells. Because IL-21 is not mitogenic by itself and knowing that it activates Erk-1/2 in human promelocytic HL-60 cells and monocytes (11, 25), we next determined whether IL-21 could activate ERK-1/2 in THP-1 cells. As illustrated in Fig. 1D, IL-21 induced a rapid phosphorylation of ERK-1/2 in THP-1 cells where the peak of activation occurred after ∼1 min and start to decline after 5 min of treatment. These results support the fact that THP-1 cells possess a functional IL-21R.

Because phagocytosis is one of the most important functions exerted by phagocytes, we next determined whether IL-21 could enhance FcR-mediated phagocytosis using opsonized SRBCs in THP-1 monocyte-like cells. By testing different concentrations of IL-21 (0–500 ng/ml), we found that this cytokine enhanced phagocytosis at maximum biological effect at a ratio of 1.5 ± 0.12 (mean ± SEM, n = 3) at the concentration of 50 ng/ml (Fig. 2A). Therefore, all subsequent experiments with IL-21 were performed at this concentration. In another set of experiments, we compared the potency of IL-21 as compared with that of GM-CSF, a non-CD132-DC known to increase phagocytosis in THP-1 cells (26). As illustrated in Fig. 2B, IL-21 is as potent as the cytokine GM-CSF for enhancing phagocytosis, with a slightly diminished ratio of 1.9 ± 0.4 versus 2.2 ± 0.4 (mean ± SEM, n = 4). Because IL-21 is known to act in synergy with IL-15 (27, 28), we then determined whether a similar response could be elicited in monocyte during phagocytosis. As illustrated in Fig. 2C, IL-15 also enhanced phagocytosis in monocytes when used alone, although to a lower extent than IL-21. However, IL-15 did not further increase the ability of IL-21 to enhance phagocytosis, indicating an absence of synergy. Then, we investigated the possibility that IL-21 modulate endocytosis in THP-1 cells. As illustrated in Fig. 2D, IL-21 did not influence endocytosis, as assessed by flow cytometry using the FITC-dextran assay, because the fluorescent signal obtained after IL-21 treatment gave similar results to control cells with a G mean of ∼25 in both conditions.

The ability of a cytokine to modulate the expression of its own receptor components is a common phenomenon. We determined the cell surface expression of both CD132 and IL-21Rα after 30 min, following cell activation with IL-21, because this is the period of time required to observe that IL-21 enhances phagocytosis. As illustrated in Fig. 3, IL-21 did not significantly modulate cell surface expression of IL-21Rα (Fig. 3A) and CD132 (Fig. 3B). However, a slight (nonstatistically significant) decrease of both receptor components (more observable with CD132) was observed. This may correspond to endocytosis of the receptor complex following ligand binding. Taken together, the results indicate that the ability of IL-21 to enhance phagocytosis could not be attributed to its ability to increase cell surface expression of one or the other IL-21R components.

Syk is an enzyme well-known for its involvement in phagocytosis, and we investigated the possibility that IL-21 activates this kinase. As illustrated in Fig. 4A, upper panel, kinetic experiments revealed that IL-21 activated Syk as quickly as after 1 min of treatment. Such activation was sustained for 15 min and started to decline after ∼30 min. IL-21 also activated the Syk substrate Akt. The peak of activation was obtained after 5 min of activation (Fig 4A, bottom panel). We next performed immunoprecipitation experiments and demonstrated that the IL-21–induced Syk phosphorylation decreased to the basal level in the presence of piceatannol (Fig 4B) and that Syk is physically associated with IL-21Rα (Fig. 4C).

Inhibition of Syk activity and its protein expression markedly decreases the effect of IL-21 on phagocytosis.

We next used two approaches to establish the role of Syk in IL-21–induced cell phagocytosis. First, using two different Syk pharmacological inhibitors (SykII and piceatannol), we found that the biological activity of IL-21 was markedly inhibited by both inhibitors, because the ratio decreased from ∼1.8 for IL-21 alone to a ratio close to 1 when cells were pretreated with one or the other inhibitor (Fig. 5A). Second, we used an antisense strategy to downregulate Syk expression. As illustrated in Fig. 5D, the expression of Syk remained similar to that of control cells when Scr-antisense was added to the culture but markedly decreased when Syk-antisense was added, confirming that Syk-specific oligonucleotides downregulated Syk expression. This correlates with a loss of IL-21 biological activity on phagocytosis only in the presence of Syk-antisense, even though both Scr- and Syk-antisenses were able to enter the cells at the same efficiency, as assessed by immunofluorescence (Fig 5B, 5C).

Peripheral blood–isolated human monocytes are known to express CD132 on their cell surface (1, 29, 30), but the expression of IL-21Rα in these cells is not clear. Therefore, we next decided to clarify this situation, and as assessed by flow cytometry, primary monocytes were found to express a modest level (but significantly) of IL-21Rα on their surface (p = 0.018; n = 3) (Fig. 6A). Interestingly, the basal level of phagocytosis was increased by IL-21 treatment by a factor 1.24 ± 0.04 (n = 3) (Fig. 6B), indicating that monocytes express a functional IL-21R. Interestingly, unlike THP-1 cells, we observed that, in general, more than one SRBC was observed in each cell. Therefore, we decided to calculate a phagocytic index (total number of SRBCs/counted monocytes) and found that the index was 1.5 ± 0.2 in IL-21–treated monocytes versus control cells (Fig. 6B, inset).

We next investigated cell surface expression of CD132 and IL-21Rα in GM-CSF–HMDM. Although CD132 was found to be highly expressed (Gmean of 112.3 ± 9.8 versus 8.2 ± 0.5 for the corresponding isotypic control [n = 5]) (Fig. 7B), IL-21Rα was modestly, but significantly, expressed (Gmean of 11.6 ± 0.7 versus 9.4 ± 0.4 for the isotypic control [n = 6]) (Fig. 7A). We then demonstrated that Syk and Akt were activated by IL-21 treatment (Fig. 7C). As for monocytes, IL-21 enhanced phagocytosis in these macrophages by a factor 1.7 ± 0.3 (n = 5) (Fig. 7D). Because several SRBCs/macrophages were observed, resulting in a poor resolution for counting the number of SRBC/cells with certainty, we did not calculate the phagocytic index, but this is certainly greater than that observed for monocytes. As for monocytic THP-1 cells (Fig. 5A), inhibition of Syk by the two different pharmacological inhibitors decreased the ability of IL-21 to enhance phagocytosis in macrophages (Fig. 7E).

The role of IL-21 is well documented in lymphoid cells (2). This cytokine is known for its ability to augment proliferation of T cells, to drive B cell differentiation into both memory cells and terminally differentiated plasma cells, as well as to enhance NK cell activity (3, 14, 31–34). Except for its ability to negatively regulate dendritic cell functions (16, 19), the role of IL-21 in cells of myeloid origin, such as monocytes and macrophages, is not well documented. Furthermore, data regarding the expression of IL-21R in monocytes and/or macrophages as well as the role of IL-21 in these cells is not clear and is even contradictory. Although Fuqua et al. (25) reported that IL-21 increased the production of IL-8 in human monocytes, we previously demonstrated that IL-21 did not exert such an effect in monocytes but did in monocyte-derived macrophages from the same blood donors (11). Although monocytes were similarly isolated in the two studies, Fuqua et al. (25) pretreated the cells with 5 mM orthovanadate (an inhibitor of protein tyrosine phosphatases) for 30 min, and then, 50 ng/ml IL-21 was added to a cell suspension of 30 × 10⁶ cells/ml for different periods of time (2–24 h), whereas in our study, monocytes were directly treated with IL-21 (0–500 ng/ml) for 24 h but at a cell density of 1 × 10⁶ cells/ml. Whether these different experimental conditions are the basis of this discrepancy is not clear. Despite this, Fuqua et al. (25) confirmed our previous observation that ERK-1/2 is activated by IL-21 (11). This is agreement with our present study indicating that IL-21 activates ERK-1/2 in THP-1 cells, supporting the fact that these cells express a functional IL-21R. In addition, they demonstrated that inhibition of ERK with the pharmacological inhibitor U0126 reversed the ability of IL-21 to increase the production of GM-CSF, IL-1, IL-2, IL-7, IL-15, IFN-γ, and TGF-β. Few studies have indicated/suggested that monocytes and/or macrophages express the specific IL-21Rα component. IL-21Rα was found to be expressed in rat mononuclear leukocytes, as assessed by RT-PCR and Western blot experiments (35). Previously, we reported that human monocytes and monocyte-derived macrophages, but not neutrophils, expressed IL-21Rα, as determined by Western blotting experiments (11). Using RT-PCR, in situ hybridization, and Western blotting experiments, IL-21Rα was detected in human synovial macrophages from rheumatoid arthritis patients (36). Intriguingly, it has been reported that 1-d monocytes were virtually devoid of IL-21Rα mRNA expression, while 3-d– and 7-d–differentiated dendritic cells expressed IL-21R mRNA in high levels, as assessed by Northern blotting (19); curiously, there are no data regarding naive monocytes. In addition, IL-21Rα mRNA expression was detected at low levels in cells committed to macrophage differentiation or in mature macrophages. Although we have shown in this study that THP-1 cells expressed IL-21Rα on their cell surface, the expression of this component has been tested by others in 19 different cell lines, including THP-1 and U937 cell lines, but the authors reported that these two cell lines did not possess IL-21Rα, because they considered a ratio of relative fluorescence intensity < 1.2 as a negative result (37). Also, it was not specified whether their results were from several experiments or simply one, and unlike our results (ratio of 1.9), there was no illustration of the FACS data. In addition, although they also reported that Raji cells do not express IL-21Rα, this cell line has been (and is still) used as a positive control in the data sheet of some companies (e.g., Santa Cruz Biotechnology) for the detection of IL-21Rα by Western blot experiments, using an Ab recognizing an immunogen directed against the extracellular domain of human IL-21Rα. Moreover, U-937 cells have been used in the study of Fuqua et al. (25), as discussed above, to demonstrate that IL-21 activated ERK-1/2 and by Jüngel et al. (36) to demonstrate the presence of IL-21Rα component by Western blot, in which a clear band of ∼52 kDa was detected.

We also show in this study that Syk is an important target of IL-21 and that this enzyme is physically associated with IL-21Rα, similarly to IL-4Rα (23), and to the best of our knowledge, this is the first study demonstrating that IL-21 activates Syk. Therefore, Syk should be added to a growing list of enzymes known to be part of the IL-21–induced cell signaling events, including Jak1, Jak-3, STAT1, STAT3, suppressor of cytokine signaling 1, suppressor of cytokine signaling 3, Shc, Akt, and ERK-1/2 (10, 11, 19, 25, 38). We show not only that IL-21 activated Syk but also that inhibition of Syk by pharmacological inhibitors and downregulation of Syk protein expression by an antisense technique reversed markedly the ability of IL-21 to enhance phagocytosis in THP-1 cells. This strategy has been previously used by us (23) to demonstrate the role of Syk in IL-4–induced neutrophil phagocytosis and adhesion, as well as its ability to delay neutrophil apoptosis, and by others (24) to demonstrate the role of Syk in lemellipodium formation and chemotaxis in leukocytes. Interestingly, phosphorylation and activation of Syk has previously been reported in THP-1 cells after clustering of FcγRII in undifferentiated cells (39). These observations open the possibility that IL-21, in mononuclear cells, exerts functions other than phagocytosis, which involve the participation of Syk, an enzyme that is becoming recognized to play crucial roles in several biological functions (40). Moreover, knowing that targeting Syk is relevant to the treatment of inflammatory diseases (41–43), and because IL-21 is associated with a variety of inflammatory disorders, including psoriasis, systemic lupus erythematosus, and arthritis (44–46), our results support the targeting of the IL-21/IL-21R system for the development of therapeutic strategies.

In this study, we have demonstrated that IL-21 did not enhance phagocytosis in THP-1 monocyte-like cells by a mechanism involving upregulation of IL-21R components on the cell surface and that no synergy was noted between IL-21 and IL-15. Therefore, the previously reported synergistic effect of IL-21 and IL-15 in lymphocytes is not a generalized phenomenon and appears to occur, for example, for cell proliferation (27) and cytokine production (28) but not for monocyte phagocytosis (this article). Interestingly, IL-21 was found to be more potent than IL-15 for enhancing phagocytosis in these cells, indicating that cytokines from a same subclass, the CD132-DCs, modulate phagocytosis at different intensities. Also, we observed that IL-21 enhanced phagocytosis as strongly as GM-CSF (not a CD132-DC) in THP-1 cells known to express functional GM-CSFR (47). Interestingly, using radiolabeled recombinant human GM-CSF, THP-1 cells were reported to express fewer than 74 receptors on their surface (48). This observation is consistent with the fact that a weak expression of IL-21Rα is sufficient to induce important biological functions in these cells and argue against the previous findings of Akamatsu et al. (37), which reported that THP-1 cells did not express IL-21R. Although IL-21 enhances FcR-mediated phagocytosis of opsonized SRBCs in monocyte-like THP-1 cells, we also report in parallel that this cytokine does not increase endocytosis of FITC-dextran, a sugar known to bind to mannose receptors (49). Therefore, IL-21 is not a modulator of mannose receptors in THP-1 cells known to possess such receptors that, for example, were previously found to promote phagocytosis of Mycobacterium tuberculosis (50).

In addition to THP-1 monocyte-like cells, we have demonstrated in this study that primary human monocytes as well as GM-CSF–HMDM not only express IL-21R on their cell surface, but also, to our knowledge, this is the first study demonstrating that IL-21 enhances phagocytosis in these cells. Also, we demonstrated that IL-21 activates Syk in GM-CSF–HMDM and that inhibition of Syk by pharmacological inhibitors diminished the ability of IL-21 to enhance phagocytosis. The expression of IL-21Rα was not investigated previously by flow cytometry but rather by RT-PCR, Northern blot, in situ hybridization, or Western blot experiments (11, 35, 36). Curiously, except for the ability to increase the production of cytokines in mononuclear cells, no other functions of IL-21 have been reported in these cells before our present investigation. Recently, however, Liu et al. (20) reported that IL-21 maintained the expression of CD16 on monocytes via the production of IL-10 by human naive CD4⁺ T cells, which is not in itself a direct function, but one can imagine that this observation could be associated with other biological processes.

We present in this study new data demonstrating not only that THP-1, primary monocytes, and GM-CSF–HMDM express IL-21R at their cell surfaces but also that the receptor is fully functional, based on the fact that IL-21 enhances one of the most important biological responses exert by monocytes, macrophages, and phagocytosis. Therefore, monocytes and macrophages functionally respond to IL-21 and future development of therapeutic strategies targeting the IL-21/IL-21R system should consider that monocyte and macrophage cell physiology may be affected by this system.

We thank Mary Gregory for reading the manuscript.

The authors have no financial conflicts of interest.