Mechanism involved in interleukin‐21‐induced phagocytosis in human monocytes and macrophages

Summary

The interleukin (IL)‐21/IL‐21 receptor (R) is a promising system to be exploited for the development of therapeutic strategies. Although the biological activities of IL‐21 and its cell signalling events have been largely studied in immunocytes, its interaction with human monocytes and macrophages have been neglected. Previously, we reported that IL‐21 enhances Fc gamma receptor (FcRγ)‐mediated phagocytosis in human monocytes and in human monocyte‐derived macrophages (HMDM) and identified Syk as a novel molecular target of IL‐21. Here, we elucidate further how IL‐21 promotes phagocytosis in these cells. Unlike its ability to enhance phagocytosis of opsonized sheep red blood cells (SRBCs), IL‐21 did not promote phagocytosis of Escherichia coli and zymosan by monocytes and did not alter the cell surface expression of CD16, CD32 and CD64. In HMDM, IL‐21 was found to enhance phagocytosis of zymosan. In addition, we found that IL‐21 activates p38, protein kinase B (Akt), signal transducer and activator of transcription (STAT)‐1 and STAT‐3 in monocytes and HMDM. Using a pharmacological approach, we demonstrate that IL‐21 enhances phagocytosis by activating some mitogen‐activated protein kinases (MAPKs) and phosphoinositide 3‐kinase (PI3K)–Akt and Janus kinase (JAK)–STAT pathways. These results obtained in human monocytes and macrophages have to be considered for a better exploitation of the IL‐21/IL‐21R system for therapeutic purposes.

Introduction

Interleukin (IL) 21 is the latest member of the CD132 (γc)‐dependent cytokines that have been discovered 1, 2. This family of cytokines is also composed of IL‐2, ‐4, ‐7, ‐9 and ‐15, all of which are known to play important roles in regulating immune system development and responses 3. Upon ligation to its heterodimeric receptor, which is composed of a specific IL‐21Rα (CD360) chain and the shared CD132 component (or γc), IL‐21 is known to mediate its biological effects through activation of the Janus kinase (JAK) and signal transducer and activator of transcription pathway (STAT), mainly JAK1, JAK3, STAT‐1, STAT‐3 and, to a lesser extent, STAT‐5 2, 4, 5, 6. Some studies have reported that IL‐21 could also activate mitogen‐activated protein kinase (MAPK) and phosphatidylinositide 3‐kinase (PI3K) pathways 7, 8. This cytokine plays an important role in the physiopathology of various inflammatory and autoimmune diseases. Indeed, different studies have reported that blockade of IL‐21 has a therapeutic effect, as observed in systemic lupus erythematosus and rheumatoid arthritis murine models, where this approach reduced the disease severity/symptoms and increase animal survival 9, 10, 11, 12.

The effects of IL‐21 on lymphoid cell functions are well characterized. IL‐21 is known to regulate T cell survival, differentiation and proliferation, B cell survival, differentiation into plasma cells and immunoglobulin (Ig) production as well as natural killer (NK) cells proliferation and cytoxicity 13. However, the effects of IL‐21 on myeloid cells have attracted much less attention, in particular in humans. Initially, myeloid cells were not recognized as cells expressing CD360. Over time, increasing evidence indicates that monocytes, macrophages and dendritic cells express this IL‐21 receptor component 8, 14, and it is only recently that primary human monocytes and human monocyte‐derived macrophages (HMDM) were found to express CD360 (initially IL‐21Rα) at their cell surface 15. Some functions of myeloid‐origin cells were found to be modulated by IL‐21. For example, stimulation of dendritic cells with IL‐21 inhibits their activation and maturation 16. IL‐21 was also found to inhibit lipopolysaccharide (LPS)‐induced cytokine secretion 17. Recently, it has been shown that IL‐21 stimulation on conventional dendritic cells induced IL‐1β secretion by a non‐canonical mechanism involving STAT‐3 activation 18. Using THP‐1 cells, primary human monocytes and HMDM, we reported that IL‐21 enhances Fc gamma receptor (FcγR)‐mediated phagocytosis of opsonized sheep red blood cells (SRBCs) and that spleen tyrosine kinase (Syk) is a molecular target of IL‐21 15. Also in macrophages, IL‐21 was found to promote antigen uptake, stimulate protease activity and attenuate the cytokine secretion induced by LPS 19, 20.

There is an increasing interest to develop IL‐21 blockers and to exploit the IL‐21/IL‐21R system therapeutically in the establishment of new clinical trials 21, 22, 23. Clearly, based on the above observations, it becomes important to understand more clearly how IL‐21 can alter the biology of human monocytes and macrophages in order to achieve this goal. In this study, we demonstrate that IL‐21 enhances the ability of human monocytes and HMDM to exert phagocytosis by a mechanism involving MAPKs [p38, extracellular signal regulated kinase (Erk)‐1/2], PI3K/Akt and JAK/STAT pathways.

Materials and methods

Chemicals and reagents

>RPMI‐1640, HEPES, penicillin/streptomycin, heat‐inactivated fetal bovine serum (FBS), phosphate‐buffered Anti‐sheep red blood cell (SRBC) antibodies and trypan blue were purchased from Sigma‐Aldrich (St Louis, MO, USA). Ficoll‐paque was obtained from GE Healthcare Bioscience AB (Uppsala, Sweden). Cytokines [granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and IL‐21] were purchased from Peprotech (Rocky Hill, NJ, USA) and SRBCs from Lampire Biological Laboratories (Pipersville, PA, USA). Antibodies directed against p‐p38, p‐Erk1/2, Erk1/2, p‐Akt, p‐STAT‐1, STAT‐1, p‐STAT‐3 and STAT‐3 were purchased from Cell Signaling Technologies (Danvers, MA, USA). The anti‐p38 and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All secondary antibodies were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA). The JAK‐2/JAK‐3 STAT‐1, ‐3, ‐5a and ‐5b inhibitor tyrphostin B42 (or AG490), MAPK/ERK inhibitor PD98059 and p38 MAPK inhibitor SB203580 were obtained from Sigma Aldrich and the Akt inhibitor wortmannin was purchased from EMD Bioscience (San Diego, CA, USA). Mouse IgG1 isotypic control‐fluorescein isothiocyanate (FITC), mouse anti‐human CD16‐FITC, mouse anti‐human CD32‐FITC and mouse anti‐human CD64‐FITC antibodies were all obtained from BD Biosciences (San Diego, CA, USA).

Cell culture and preparation of HMDM

THP‐1 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and were cultured in RPMI‐1640 supplemented with 2·05 mM L‐glutamine, 100 mM HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin (referred hereafter as RPMI‐1640) and 10% FBS and incubated in a 5% CO₂ atmosphere at 37°C, as described previously 15. Cells were subcultured before reaching a concentration of 8 × 10⁵ cells/ml, as recommended by the ATCC.

Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood of healthy volunteers by centrifugation over Ficoll‐paque. Blood donations were obtained from informed and consenting individuals by an institutionally approved procedure. HMDM (also designated here as macrophages) were generated by incubating 4 × 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 non‐adherent PMBCs were incubated further for 7 days in RPMI‐1640 medium supplemented with 10% heat‐inactivated FBS in the presence of 2 ng/ml GM‐CSF to obtain macrophages, as described previously 15. Cell viability was determined systematically by trypan blue exclusion assay prior all experiments and was always ≥ 95%.

Cell surface expression of CD16, CD32 and CD64

THP‐1 monocytes (1 × 10⁶ cells/ml) were incubated with buffer or 50 ng/ml IL‐21 for 30 min at 37°C, 5% CO₂. After several washes, cells (1 × 10⁶ cells/ml) were stained with 10 µl of FITC‐conjugated anti‐CD16, ‐CD32, ‐CD64 or the appropriate isotypic control antibodies for 30 min on ice. Cells were washed twice with ice‐cold PBS before being resuspended at 1 × 10⁶ cells/ml. Cell surface expression was determined immediately using a fluorescence activated cell sorter (FACS)Calibur (BD Biosciences, San Jose, CA, USA). In some experiments, after the 30‐min treatment with buffer or IL‐21, cells were washed and then kept for an additional 60 min in the presence of culture medium only. Then, cell surface expression of CD16, CD32 and CD64 was determined as above.

THP‐1 phagocytosis of Escherichia coli, Zymosan and opsonized SRBCs

Phagocytosis was assessed by flow cytometry using Alexa Fluor^© 488‐conjugated E. coli or Alexa Fluor^© 488‐conjugated zymosan bioparticles (Life Technologies, Eugene, OR, USA). To this end, cells were stimulated with buffer or 50 ng/ml IL‐21 at 37°C or 4°C (as a negative technical control) for 30 min before incubation with E. coli or zymosan opsonized or not with serum. Cells were then centrifuged and supernatants were discarded. Bacteria or zymosan particles were diluted in HBSS and were added to THP‐1 cells (10 : 1 THP‐1). After 30 min, bacteria or zymosan particles that were not ingested by the cells were washed twice with PBS and removed by centrifugation onto a 4·5‐ml gradient of RPMI‐1640 medium containing 5% bovine serum albumin (BSA). Bacteria remaining at the surface were then removed and cells located in the pellet were quenched with trypan blue. After three washes, cells were suspended in 500 μl PBS for analysis. Cellular phagocytosis was monitored by flow cytometry at 525 nm. Phagocytosis of Alexa Fluor^© 488‐conjugated E. coli and Alexa Fluor^© 488‐conjugated zymosan was expressed as the percentage of FL‐1‐positive cells compared with cell autofluorescence.

Phagocytosis of opsonized SRBCs by THP‐1 cells or macrophages

SRBCs were washed three times in ice‐cold PBS and resuspended at 50 × 10⁶ cells/ml and then opsonized with anti‐SRBCs (1 : 200) (subagglutination titre) at 37°C for 45 min, as published previously 15, 24. THP‐1 cells were treated with buffer or IL‐21 for 30 min, as above. RPMI‐1640 was removed after centrifugation and opsonized SRBC were added onto the pellet in a 5 : 1 ratio and incubated at 37°C in a 5% CO₂ atmosphere for 1 h. After incubation with SRBCs, the samples were centrifuged at 200 g for 10 min at 4°C. Supernatants were discarded and an osmotic shock was performed on the pellets by resuspending the cells with 400 μl H₂O for 15 s, and osmolarity was then recovered by the addition of 4·5 ml of ice‐cold PBS. The samples were then washed twice and the final pellets were suspended in 400 μl PBS, cytocentrifuged and stained with the Hema‐3 stain set (Biochemical Sciences, Swedesboro, NJ, USA). A minimum of 250 cells per condition were counted and phagocytosis was expressed as the percentage of THP‐1 cells ingesting at least one opsonized SRBC. In some experiments, cells were preincubated for 30 min at 37°C with p38 inhibitor SB203580 (5 µM), mitogen‐activated protein kinase kinase (MEK)1/2/Erk‐1/2 inhibitor PD98059 (10 µM), JAK/STAT inhibitor AG490 (30 µM) or PI3K/Akt inhibitor wortmannin (50 nM) before IL‐21 stimulation. For phagocytosis by HMDM, cells were washed twice with warm HBSS and then stimulated with or without IL‐21 in a final volume of 100 µl for 30 min at 37°C. These macrophages were incubated with 10 × 10⁶ SRBCs for 15 min at 37°C in a 5% CO₂ atmosphere. The plates were then removed and placed on ice for 5 min. Cells were washed once with ice‐cold PBS to remove excess non‐ingested 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 PBS. Cells were then stained with the Hema‐3 stain kit and ∼250 cells/condition were used to determine phagocytosis. As above, cells were preincubated for 30 min at 37°C with the indicated inhibitors prior to the IL‐21 treatment.

Phagocytosis of zymosan by macrophages

Before the assay, HMDM were washed twice with warm HBSS and then stimulated with or without IL‐21, as above. Macrophages were then incubated with 200 µg/ml of pHrodo green zymosan (Life Technologies) resuspended in live cell imaging solution (Life Technologies, Grand Rapid, NY, USA) for 1 h at 37˚C, 5% CO₂. Cells were then washed three times with live cell imaging solution and 200 µl of ice‐cold live cell imaging solution was added. Nuclei were stained with NucBlue live cell stain (Life Technologies). Phagocytosis was determined by fluorescence microscopy. In some experiments, cells were pretreated for 30 min at 37°C with different inhibitors prior to the IL‐21 stimulation, as above.

Western blot experiments

Cells (1 × 10⁶ cells/ml) were incubated with buffer or IL‐21 (50 ng/ml) for 0·25–60 min at 37°C. At the end of the incubation periods, the cells were lysed in Laemmli's sample buffer and aliquot corresponding to 3·5 × 10⁵ cells were loaded onto 7·5% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to nitrocellulose membranes for the detection of specific protein. Membranes were blocked for 1 h at room temperature in Tris‐buffered saline (TBS)‐Tween (0·15%) containing 3% BSA. After washing, primary antibodies were added at a final dilution of 1 : 1000 in TBS‐Tween 0·15%. The membranes were kept overnight at 4°C with gentle agitation, then washed with TBS‐Tween and incubated for 1 h at room temperature with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies (1 : 20 000 in TBS‐Tween) followed by several washes. Protein expression was revealed using Clarity ECL substrate with ChemiDoc MP imaging system (Bio‐Rad, Hercules, CA, USA). Membranes were stripped with re‐blot plus strong antibody stripping solution (Millipore, Billerica, MA, USA) and reprobed to confirm equal loading of proteins. In some experiments, cells were preincubated for 30 min at 37°C with the indicated inhibitors before the IL‐21 stimulation, as above.

Statistical analysis

Experimental data are expressed as means ± standard error of the mean (s.e.m.). Repeated‐measures analysis of variance (anova) (Tukey's multiple‐comparison test) or paired Student's t‐test were performed using Graph‐Pad Prism (version 5.01) as specified in the figure legend. Differences were considered statistically significant as follows: *P < 0·05, **P < 0·01 and ***P < 0·005 versus buffer or the appropriate diluent.

Results

IL‐21 does not enhance the capacity of human monocytic THP‐1 cells to phagocytose bacteria or zymosan

As we have documented previously that IL‐21 enhances the FcγR‐mediated phagocytosis of opsonized SRBSa in human THP‐1 cells 15, we decided to determine first if IL‐21 could increase the phagocytosis of other agents that could also involve complement components. As illustrated in Fig. 1, IL‐21 did not increase the basal level of phagocytosis of serum‐opsonized or non‐opsonized E. coli (Fig. 1a,b, respectively) as well as serum‐opsonized or non‐opsonized zymosan (Fig. 1c,d), respectively. However, cells were responsive to IL‐21 because, as expected 15, the cytokine treatment enhances the basal level of phagocytosis of opsonized SRBCs from 23 ± 2·6% (mean ± s.e.m., n = 8) to 30·7 ± 2·9% (Fig. 1e).

Figure 1.

Interleukin (IL)‐21 does not promote phagocytosis of bacteria or zymosan opsonized or not with human serum in human monocytic THP‐1 cells. THP‐1 cells were treated with buffer (Ctrl) or IL‐21 (50 ng/ml) for 30 min and then phagocytosis of human serum opsonized (a) or non‐opsonized Escherichia coli, (b) serum opsonized (c) or non‐opsonized zymosan (d) and opsonized sheep red blood cells (SRBCs) (e) was determined as described in Materials and methods. The assay was performed at 37°C (black bar) or 4°C (open bar). Data are means ± standard error of the mean (n = 4).

IL‐21 does not modulate the cell surface expression of FcγR in human monocytes

Although we have reported previously that IL‐21 enhances the phagocytosis of opsonized SRBCs in human monocytes and macrophages by a Syk‐dependent mechanism 15, the mode of action of IL‐21 is still unclear. Therefore, we determined next whether or not IL‐21 could modulate the cell surface expression of FcγR in THP‐1 monocytes and, as illustrated in Fig. 2, IL‐21 did not alter the expression of CD16, CD32 or CD64.

Figure 2.

Interleukin (IL)‐21 does not modulate the cell surface expression of Fc gamma receptor (FcRγ) in human monocyte‐like THP‐1 cells. Separate batches of THP‐1 cells were cultured and used to determine the cell surface expression of Fc receptors by flow cytometry using specific antibodies against CD16, CD32, CD64 and the corresponding isotypic controls (Iso), as described in Materials and methods. Cells were untreated (a), treated with buffer or IL‐21 (50 ng/ml) for 30 min (b) or treated with IL‐21 (50 ng/ml) for 30 min, washed and incubated for 60 min (c). Results are expressed as mean fluorescence intensity (MFI). Data are means ± standard error of the mean (n = 4).

Cell signalling events induced by IL‐21

The cell signalling events induced by IL‐21 in human monocytes is documented poorly. As we have demonstrated previously that IL‐21 can activate Erk‐1/2 in THP‐1 cells 15, we then decided to investigate further the cell signalling events induced by IL‐21. As illustrated in Fig. 3a, in addition to Erk/1‐2 (used here as a positive control), IL‐21 activates p38 and Akt rapidly within 5 min of stimulation. Also, we found that IL‐21 can activate STAT‐1 and STAT‐3 (Fig. 4b).

Figure 3.

Activation of signal transducer and activator of transcriptions (STATs), mitogen‐activated protein kinases (MAPKs) and protein kinase B (Akt) in human monocyte‐like THP‐1 cells by IL‐21. THP‐1 cells were treated with buffer (Ctrl) or IL‐21 (50 ng/ml) for the indicated periods of time, and activation of extracellular signal regulated kinase (Erk)‐1/2, p38, Akt (a) and STAT‐1 and STAT‐3 (b) was determined by Western blot experiments as described in Materials and methods. Equal loading was verified after stripping and reprobing the membranes with antibodies directed against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) or the corresponding non‐phosphorylated form of Erk‐1/2 and p38. Results are from one representative experiment of three.

Figure 4.

Role of some mitogen‐activated protein kinases (MAPKs) and phosphoinositide 3‐kinase–protein kinase B (PI3K/Akt) and Janus kinase–signal transducer and activator of transcription (JAK/STAT) pathways in the ability of interleukin (IL)‐21 to enhance Fc gamma receptor (FcRγ)‐mediated phagocytosis in THP‐1 cells. (a) THP‐1 cells were treated with the indicated inhibitors or corresponding diluent prior activation with IL‐21 (50 ng/ml) for the indicated periods of time and activation of extracellular signal regulated kinase (Erk)‐1/2, p38, Akt and STAT‐3 (a) or phagocytosis (b) was performed as described in Materials and methods. (a) Results are from one representative experiment of three; (b) data are means ± standard error of the mean (n = 6). #P < 0·05 versus diluent; **P < 0·01 or ***P < 0·001 versus Ctrl‐IL‐21. PD = PD98059; SB = SB203580; Wort = wortmannin; AG = AG490.

IL‐21 enhances phagocytosis by a mechanism involving activation of Erk‐1/2, Akt and STATs but not p38 in human monocytes

Next, we performed experiments in order to determine the role of several intracellular proteins activated by IL‐21 in FcγR‐mediated phagocytosis. First, we tested the ability of different pharmacological inhibitors to reverse the ability of IL‐21 to induce tyrosine phosphorylation of a given protein. As illustrated in Fig. 4a, pretreatment of cells with the corresponding diluent, PD98059 (MEK1/MEK2 and Erk‐1/2 sensitive inhibitor), SB203580 (p38 inhibitor), wortmannin (PI3K/Akt inhibitor) and AG490 (JAK/STAT inhibitor) indicates that all inhibitors are able to reverse the activation/phosphorylation of the tested proteins; namely, Erk‐1/2, p38, Akt and STAT‐3. Next, we used these inhibitors to determine if they can reverse the ability of IL‐21 to enhance FcRγ‐mediated phagocytosis. As illustrated in Fig. 4b, all inhibitors but SB203580 were able to decrease significantly the percentage of phagocytosis at values close to the basal level.

IL‐21 activates STATs in human monocyte‐derived macrophages

We have reported previously that IL‐21 activates Erk‐1/2 in HMDM 15. Because of the present results indicating that IL‐21 can activate other MAPKs and STAT‐1 and STAT‐3 in human THP‐1 monocytes, we then performed experiments in order to determine if this is also observed in GM‐CSF‐derived HMDM. As illustrated in Fig. 5, IL‐21 induces a very rapid tyrosine phosphorylation of p38 (within 15 s) in macrophages. Also, this cytokine activates both STAT‐1 and STAT‐3 after ∼5 min of stimulation in these cells, which disappears after ∼60 min. As expected 15, macrophages were fully responsive to IL‐21, as evidenced by its ability to enhance phagocytosis of opsonized SRBCs (inset).

Figure 5.

Interleukin (IL)‐21 activates p38, signal transducer and activator of transcription (STAT)‐1 and STAT‐3 in human macrophages. Macrophages were treated with buffer (Ctrl) or IL‐21 (50 ng/ml) for the indicated periods of time and activation of p38, STAT‐1 and STAT‐3 was determined by Western blot experiments as described in Materials and methods. Inset, the ability of IL‐21 to enhance Fc gamma receptor (FcRγ)‐mediated phagocytosis was performed in parallel as described in Materials and methods. Results are from one representative experiment of at least three. Inset = results are means ± standard error of the mean (n = 6).

IL‐21 enhances phagocytosis in HMDM by a MAPK‐ and STAT‐dependent mechanism

Fig. 6a illustrates that, as for THP‐1 monocytes, the tested inhibitors also reversed the phosphorylation of MAPKs, STAT‐1 and STAT‐3 in HMDM. In addition, all these inhibitors, with the exception of PD98059, were able to decrease IL‐21‐induced phagocytosis, indicating that the weak to moderate phosphorylation of Erk‐1/2 is not involved in this process (Fig. 6b).

Figure 6.

Cell signalling events involved in interleukin (IL)‐21‐induced enhancement of Fc gamma receptor (FcRγ)‐mediated phagocytosis in human macrophages. Macrophages were treated with the indicated inhibitors or corresponding diluent prior to activation with IL‐21 for the indicated periods of time and activation of protein kinase 3 (Akt), extracellular signal regulated kinase (Erk)‐1/2, signal transducer and activator of transcription (STAT)‐1 and STAT‐3 (a) or phagocytosis (b) was performed as described in Materials and methods. (a) Results are from one representative experiment of three; (b) data are means ± standard error of the mean (n = 4). #P < 0·05 versus diluent; **P < 0·01 or ***P < 0·001 versus corresponding diluent. PD = PD98059; AG = AG490; Wort = wortmannin.

The capacity of human macrophages to phagocytose zymosan is enhanced by IL‐21 via a MAPK‐, PI3K/Akt‐ and JAK/STAT‐dependent mechanism

Unlike THP‐1 cells, HMDM are extremely adherent during culture conditions, and instead of harvesting them by a drastic procedure in order to determine phagocytosis by FACS, we adopted another strategy based on the use of the pHrodo green zymosan bioparticles conjugate for phagocytosis that are pH‐sensitive and non‐fluorescent extracellularly but fluoresce brightly inside cells, in the phagosomes (green spots in Fig. 7b). As illustrated in Fig. 7a, IL‐21 enhances significantly the basal level of zymosan phagocytosis from 18·1 ± 3·4% to 31·7 ± 2·4% (mean ± s.e.m., n = 3). In one other cohort of experiments, we next used the pharmacological inhibitors to determine if IL‐21 also acts by different intracellular pathways under these experimental conditions. As illustrated in Fig. 7c, all the tested inhibitors decreased the capacity of IL‐21 to enhance zymosan phagocytosis, indicating the role of MAPKs, PI‐3K/Akt and STATs in human macrophages.

Figure 7.

Involvement of mitogen‐activated protein kinases (MAPKs) and phosphoinositide 3‐kinase–protein kinase B (PI3K/Akt) and Janus kinase–signal transducer and activator of transcription (JAK/STAT) pathways in the ability of interleukin (IL)‐21 to enhance phagocytosis of zymosan in human macrophages. (a) Macrophages were treated with buffer (Ctrl) or IL‐21 (50 ng/ml) for 30 min before being incubated with 200 µg/ml of pHrodo green zymosan bioparticles for 1 h and phagocytose was determined as described in Materials and methods; (b) representative microphotographs of the pHrodo green zymosan bioparticle phagocytosis assay; (c) macrophages were pretreated for 30 min with the indicated inhibitors prior to activation with buffer or IL‐21 for 30 min and phagocytosis was then determined. (a) Results are means ± standard error of the mean (n = 3); (c) results are means ± standard error of the mean (n = 4). [Colour figure can be viewed at wileyonlinelibrary.com]

Discussion

In most of the review papers published on IL‐21 22, 23, 25, 26, 27, only two major observations regarding its biological activity in monocytes–macrophages are reported: IL‐21 enhances phagocytosis and IL‐8 production. These observations originate from a few papers: one where treatment with IL‐21 in murine bone marrow‐derived macrophages was found to increase FITC‐dextran uptake 19; one other where we found that IL‐21 enhances the phagocytosis of opsonized SRBCs in THP‐1 cells, primary human monocytes and HMDM 15; one where the production of IL‐8 by human monocytes was increased by IL‐21 28; and finally, one where we reported that IL‐21 induces IL‐8 production in human macrophages 8. Of note, even recently, other excellent reviews simply ignore the fact that monocytes and macrophages are targets of IL‐21. Therefore, even if the IL‐21/IL21R system is attractive to be exploited for the development of therapeutic strategies 9, 21, 22, 29, 30, 31, there is currently a serious lack of information regarding how this system operates in human monocytes and macrophages. Herein, we demonstrate that IL‐21 does not enhance phagocytosis of E. coli and zymosan by human monocytes, even in the presence of human serum, leading to the observation that, as yet, this cytokine increases FcRγ‐mediated phagocytosis of SRBCs only in human monocytes, as ovine erythrocytes were opsonized specifically here with anti‐SRBC antibodies. This suggests that even if Syk is activated in these cells 15, the downstream events involved in these types of phagocytosis are not activated efficiently. However, we reported that IL‐21 does not alter the cell surface expression of the FcRγ CD16, CD32 and CD64 in monocytes directly after 30 min of stimulation with IL‐21 or when cells were kept 1 h after their activation. To the best of our knowledge, this is the first time that the expression of these receptors has been studied in IL‐21‐treated human monocytes. However, in one study, IL‐21 was found to maintain the expression of CD16 on monocytes, but this was observed in an experimental condition requiring the presence of IL‐10 in the supernatants from CD3/CD28/IL‐21‐activated T cells, and this was determined after 24 h of culture 32.

Even if the cell signalling events and the roles of IL‐21 are well established in most of the immune cell types, in particular in lymphoid B, T and NK cells 21, 33, 34, 35, 36, this is not the case for human monocytes and macrophages. Therefore, the results of this present study, demonstrating that IL‐21 enhance phagocytosis in human monocytes and HMDM by a mechanism requiring activation of several cell signalling molecules, are novel observations, increasing our general knowledge on the mode of action of IL‐21. Indeed, in addition to Syk 15 and Erk‐1/2 15, 28, we determined here that p38, Akt, STAT‐1 and STAT‐3 are new molecular targets of IL‐21 in these cells.

In our previous study, we reported that IL‐21 can enhance phagocytosis of opsonized SRBCs in THP‐1, primary monocytes and HMDM 15. In this study we found that IL‐21 can enhance phagocytosis of zymosan by HMDM, but not by THP‐1 cells. It is interesting to note that, in one other study, we reported previously that IL‐21 can increase the production of IL‐8 by HMDM, but not by primary monocytes 8, suggesting that mature differentiated cells are more responsive to IL‐21. However, and in contrast to our results, only one study reported that IL‐21 was able to increase the IL‐8 production by primary human monocytes 28. Monocytes were plated at very high cell density (30 × 10⁶ cells/ml versus 1 × 10⁶ cells/ml in our experimental conditions), and the basal level of secreted IL‐8 was not monitored over time in corresponding untreated cells.

Also in this study we report that IL‐21, in contrast to monocytes, enhance the ability of HMDM to phagocytose zymosan, again suggesting that mature differentiated cells are more responsive to IL‐21. In general, the yeast cell wall component zymosan is known to bind to Toll‐like receptor (TLR)‐2 during inflammatory responses whereas phagocytosis of zymosan, as performed here, is known to involve different receptors that could recognize the same or different of its components. For example, receptors to sugars (in particular mannose receptors) are known to be involved and to lead to zymosan uptake/internalization/phagocytosis by phagocytes, including macrophages 37, 38. However, as human monocytes such as THP‐1 cells do not express the mannose receptor 39, it is tempting to speculate that IL‐21 enhances the phagocytosis of zymosan in HMDM by altering the expression of the mannose receptors and/or the subsequent intracellular signalling events. Interestingly, pharmacological inhibition of PI3K/Akt was found previously to block non‐opsonized zymosan by macrophages 39. Thus, our present results support this further, as in addition to activation of Akt by IL‐21, we also demonstrate that wortmannin (known to inhibit PI3K/Akt) blocks IL‐21‐induced phagocytosis of not only opsonized SRBCs but also that of non‐opsonized zymosan in HMDM. Nevertheless, we are aware that several other receptors/components may be involved and that several experiments need to be performed to determine fully the mode of action of IL‐21 during human macrophage phagocytosis of opsonized (SRBCs) and non‐opsonised (zymosan) particles.

In addition to the very few previous studies reporting that IL‐21 enhance phagocytosis in rodents 19 or human monocytes and macrophages 15, our present data demonstrating that IL‐21 activates different cell signalling events in human monocytes and macrophages, including MAPKs, PI3K/Akt and JAK‐STAT, provide new mechanistic insights into IL‐21. In addition, our data indicate that the modulatory activity of IL‐21 in human monocytes and macrophages is complex and may have relevance in the development of new or ongoing therapies, in particular for the use of IL‐21 blockers. As we have used GM‐CSF‐induced monocyte differentiation into macrophages in this study, it will be interesting in future to determine if IL‐21 will use these same cell signalling pathways when macrophages will be derived from M‐CSF‐induced monocytes having certain phenotypical differences.

Disclosure

The authors declare no commercial or financial conflicts of interest.

Author contributions

The conception and design of the study was by D. G.; F. V. performed all the experiments. The analysis and interpretation of data was performed by F. V. and D.G.; D. G. and F. V. wrote the paper.