Expression and functionality of type I interferon receptor in the megakaryocytic lineage


Ricardo M. Gomez, Instituto de Biotecnologia y Biologia Molecular, CCT-La Plata, 49 y 115, La Plata (1900), Buenos Aires, Argentina.
Tel.: +54 221 422 6977; fax: +54 221 422 4967.

Correspondence: Mirta Schattner, Laboratorio de Trombosis I, Academia Nacional de Medicina, Pacheco de Melo 3081, Buenos Aires (1425), Argentina.
Tel.: +54 11 4805 5759; fax: +54 11 4805 0712.


Summary. Background: Type I interferons (IFN-I) negatively regulate megakaryo/thrombopoiesis. However, expression of the IFN-I receptor (IFNAR) in the megakaryocytic lineage is poorly characterized. Objectives: To study the expression and functionality of IFNAR in the megakaryocytic lineage. Methods and results: Although IFNAR mRNA was found in every cell type studied, its protein expression showed differences between them. According to flow cytometry and immunofluorescence, IFNAR1 was observed in Meg-01, Dami, CD34+ cells and megakaryocytes, but not in proplatelets or platelets. Immunoblotting assays showed that IFNAR1 and IFNAR2 were highly expressed in all cell types, except in platelets where it was barely detectable. Regarding IFNAR1, 130- and 90-kDa bands were detected in Meg-01 and Dami, whereas 130- and 60-kDa bands were found in CD34+ cells and megakaryocytes. Activation of megakaryocytic IFNAR by IFN-β induced pSTAT1/2 and upregulated the antiviral genes IRF7 and MXA. The latter response was completely suppressed by IFNAR blockade. In contrast, the low levels of IFNAR in platelets were not functional as pSTAT1/2, aggregation and P-selectin expression were not induced by IFN-I. In addition, megakaryocytes increased IFN-I transcript levels and produced IFN-β upon stimulation with PolyI:C, a synthetic dsRNA that mimics viral infection. Conclusions: Early progenitors and mature megakaryocytes, but not platelets, express functional IFNAR and synthetize/release IFN-β, revealing not only that megakaryo/thrombopoiesis regulation by IFN-I is associated with a specific interaction with its receptor, but also that megakaryocytes may play a role in the antiviral defense by being both IFN producers and responders.


The type I interferons (IFN-I), IFN-β and the numerous IFN-α subtypes, are cytokines which act as antiviral effectors by eliciting a broad range of responses in a variety of cell types [1]. IFN-I can be produced by almost any cell type in the body. These inducible cytokines mediate their activity by binding to a common IFN-I receptor (IFNAR), which through downstream signaling activates a multitude of IFN-I-stimulated genes with antiviral, antiproliferative and immunomodulatory activities [1,2]. This wide range of physiological responses has made IFN-I therapeutically useful in the treatment of human diseases, including viral hepatitis [3], AIDS-associated Kaposi’s sarcoma [4], leukemias [5], essential thrombocythemia [6], systemic autoimmunity [7] or multiple sclerosis [8]. However, IFN-I treatment may have some negative side effects. For example, in patients treated with IFN-α for viral hepatitis, the development of thrombocytopenia often leads to a dose reduction or discontinuation of IFN-α therapy [9,10].

Although initial studies suggested that thrombocytopenia was linked to an autoimmune reaction and capillary sequestration [9], several experimental in vitro studies in animal and human cells have shown that IFN-α inhibits megakaryocyte colony formation [10–12]. In clinical studies, however, the administration of human IFN-α to patients with chronic hepatitis, solid tumors and myeloproliferative disorders does not affect the number of megakaryocytes in the bone marrow [9,10]. These controversial findings were recently clarified by Yamane et al., [13] who demonstrated that IFN-α failed to reduce endomitosis of megakaryocytes from human CD34+ hematopoietic stem cells, but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. In addition, more recent results suggested that the fate of megakaryopoiesis seems to be linked with the concentration of IFN-I as high levels of IFN-I markedly decrease megakaryocyte proliferation, whereas reduced levels only impair platelet production [14]. Moreover, the selective effect of IFN-I on platelet biogenesis was also observed in CD34+ cells infected with the arenavirus Junín virus (JUNV) (a causal agent of Argentine hemorrhagic fever) or after treatment with polyriboinosinic polyribocytidylic acid (PolyI:C), a synthetic double-stranded RNA that triggers IFN-I production, suggesting that the IFN-I produced in virus-infected bone marrow cells could be a major pathogenic mechanism involved in the thrombocytopenia associated with viral infection [14].

All IFN-I subtypes utilize a ubiquitously expressed heterodimeric IFN-α/-β receptor (IFNAR) and transduce similar, although not necessarily identical, intracellular signals [15]. It is notable that, in spite of the increasing evidence for the role of IFN-I in controlling megakaryopoiesis, except for some megakaryocytic cell lines [16,17] and at the mRNA level in primary megakaryocytes [14], the presence of the IFNAR in the megakaryocytic lineage has not yet been reported. Herein, we characterize the expression and functionality of this receptor and the production of IFN-I in the early megakaryocytic progenitor cell lines Meg-01 and Dami, as well as in primary cells including early precursors and mature megakaryocytes, proplatelets and platelets.

Materials and methods

Ethics statement

The present study was approved by the Institutional Review Board of the National Academy of Medicine, Argentina. All individuals provided written informed consent for the collection of samples and subsequent analysis.


Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) against CD34, glycoprotein (GP) IIb (CD41), PE-conjugated anti-GP IIIa (CD61), a fixation/permeabilization kit and anti-actin Ab were purchased from BD Biosciences (FranklinLakes, NJ, USA). FITC-conjugated anti-rabbit immunoglobulins (Igs) Ab were obtained from Dako A/S (Glostrup, Denmark). Alexa 488- and 546-conjugated goat anti-mouse and anti-rabbit IgG Abs, respectively, were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). Rabbit anti-human IFNAR1 Abs were purchased from Abcam (Cambridge, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human Stat1 p84/p91 (E-23) and STAT2 (phospho Y690) were from Santa Cruz and Abcam, respectively. Rabbit anti-human IFNAR2 and mAb to human interferon-a/b R2 neutralizing (clone MMHAR-2) were from PBL InterferonSource (Piscataway, NJ, USA). The synthetic dsRNA PolyI:C and recombinant human IFNβ1a were obtained from InvivoGen (San Diego, CA, USA).

Cell line cultures

Meg-01 and Dami cells (ATCC, Manassas, VA, USA) were grown in suspension in RPMI (10% fetal calf serum and penicillin/streptomycin) at 37 °C in a humidified atmosphere of 5% CO2 and were split every 3–4 days. Viral infection was performed with the attenuated vaccine Candid no 1 strain of JUNV at a multiplicity of infection (MOI) of 1 as previously described [14]. Cells were further cultured for 72 h and then collected for RNA isolation.

Isolation of CD34+ cells, progenitors and mature megakaryocytes

Isolation of CD34+ cells from human umbilical cord blood was performed using a magnetic cell-sorting system (Miltenyi Biotec, Gladbach, Germany) as previously described [18]. The purity of the cell suspension was determined by flow cytometry and typically ranged from 95% to 99%. Cell viability was > 90%. CD34+ cells were cultured in the presence of stem cell factor (SCF, 50 ng mL−1) and thrombopoietin (TPO, 100 ng mL−1) (Peprotech, Veracruz, Mexico). Fresh TPO was added at day 7. For PCR and immunoblotting experiments, progenitors and mature megakaryocytes were purified by immunomagnetic positive selection from cultures on days 6 and 12, respectively, using anti-CD61 magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of the final cell suspension was > 97% and cell viability was above 80%.

Preparation of human platelets

Blood samples from healthy donors were drawn directly into plastic tubes containing 3.8% sodium citrate (9:1) and platelet-rich plasma (PRP) was obtained by centrifugation (180 × g for 10 min). PRP was then centrifuged in the presence of prostacyclin I2 (PGI2) (75 nm) and after washing, platelets were resuspended in Tyrode’s buffer. Highly purified platelets were obtained using a high-efficiency leukoreduction filter (Purecell PL; PALL Biomedical Products, East Hills, NY, USA) as previously described [19], which decreased contamination to < 1 leukocyte every 104 platelets.


Western blots were performed as previously described [19] with minor differences. Briefly, cells were resuspended in loading buffer and incubated for 5 min at 95 °C. Samples were separated by 12% SDS-PAGE and electro-transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). After blocking, the membranes were incubated overnight at 4 °C with primary Abs followed by an HRP-conjugated secondary Ab. Each membrane was reprobed with an antibody against actin to ensure equal loading. Protein bands were visualized using an Amersham ECL kit (GE Healthcare).


Cells fixed with 1% paraformaldehyde were centrifuged and permeabilized with 0.1% Triton X-100. After blocking with 6% BSA, cells were incubated overnight with the primary Ab followed by Alexa-conjugated secondary Abs. To visualize nuclei, the cells were stained with a DAPI solution (0.75 μg mL−1) for 5 min. Slides were then mounted with PolyMount and analyzed under an epifluorescent microscopy (Olympus BX60, Tokyo, Japan).

Proplatelet formation assay

Coverslips were coated with 100 μg mL−1 fibrinogen (Sigma) for 2 h and subsequently blocked with 1% bovine serum albumin (BSA) for 1 h. Cells at day 12 were plated on the precoated coverslips and further cultured for 16 h. Cells were double-stained with tetramethyl rhodamine isothiocyanate-conjugated phalloidin (Sigma) and IFNAR1 Ab or isotype followed by a FITC-conjugated secondary Ab. Proplatelet structures were then evaluated by confocal microscopy. The fluorescence intensity within megakaryocytes and proplatelets was analyzed in more than 50 proplatelet-bearing cells with the Olympus FV100-ASW Version 2.1c software [18].

Flow cytometry studies

Cells were incubated with a PE-conjugated anti-CD61 mAb or an isotype control for 20 min and then fixed with 1% paraformaldehyde (PFA). After washing, cells were permeabilized with 0.1% saponin, and then incubated with a IFNAR1 mAb at 4 °C followed by a FITC-conjugated swine anti-rabbit Igs. Forward and side scatter were used to distinguish megakaryocytes from culture-derived platelets. The percentages of positive cells and the median of fluorescence intensity (MFI) in the green channel of the CD61+ events were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

Semi-quantitative and quantitative RT-PCR

Total RNA was isolated from cell pellets using TriReagent (Genbiotech, BsAs, Argentina). RNA was quantified with a spectrophotometer (Nanodrop 1000). Before cDNA synthesis, DNase treatment was performed with an RNase-free DNase kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from 1.5 ug of total RNA using 15 mm of random hexamers (Byodinamics, BsAs, Argentina) and MMLV reverse transcriptase (Promega, BsAs, Argentina). Bands were analyzed with GelPro software (Bethesda, MD, USA) and the actin density was used to normalize the values.

Quantitative-PCR (q-PCR) was performed as previously described [20]. Briefly, PCR amplification and analysis were performed with a Line-Gene instrument (Bioer Technology, Hangzhou, China) and LineGene K Fluorescence Quantitative Detection System Version 4.0 software. The 5 × HOT FIREPol® EvaGreen® q-PCR Mix Plus (Solis Biodyne, Tartu, Estonia) was used for all reactions. Normalized expression values were calculated from the absolute quantities of the gene of interest and the housekeeping gene (actin). Standard curves for absolute quantification were generated from purified amplicons, upon confirmation of their sequences. The primers sequences and sizes of the amplified fragments are shown in (Table S1).

Determination of IFN-β levels by ELISA

The concentrations of IFN-β in the culture’s supernatants were determined using a commercially ELISA kit (PBL InterferonSource) according to the manufacturer’s instructions.

Statistical analysis

Results are expressed as mean ± standard error of the mean (SEM). Student’s paired t-test was employed to determine the significance of differences between the groups. A P-value < 0.05 was considered to be statistically significant.


Meg-01 express IFNAR

The expression of IFNAR in the megakaryocytic lineage was first evaluated in the megakaryocytic cell line Meg-01. RT-PCR studies revealed transcripts of the expected size for IFNAR subunits 1 and 2 in these cells (Fig. 1A).

Figure 1.

 Type I interferon receptor (IFNAR) is expressed in the Meg-01 cell line. (A) mRNAs encoding IFNAR subunits 1 (R1) and 2 (R2) were detected by semi-quantitative RT-PCR. (n = 3). (B) IFNAR1 protein was determined by flow cytometry (n = 4). (C) IFNAR1 expression (red) was confirmed in Meg-01 by immunofluorescence microscopy. Nuclei were visualized with a (DAPI) counterstain (blue) (n = 4). Original magnification, 60 × (scale bar: 10 μm). (D) IFNAR1 and IFNAR2 bands were detected in cell lysates by western blot (n = 3).

To verify that the mRNA was ultimately translated into protein, we studied the protein expression levels of IFNAR using two different primary Abs. The data shown from all the experiments in the present study correspond to those obtained using the Abcam anti-IFNAR1 Ab (No ab45172). Similar results were observed when the Cell Signaling Ab (No sc-845) was used (data not shown). Flow cytometric and immunofluorescence analysis showed that IFNAR1 was expressed in Meg-01 (Fig. 1B,C). Immunoblotting studies showed that the typical bands of 130 and 90 kDa, corresponding to the heavily glycosylated isoforms of IFNAR1 [21], were present in Meg-01 (Fig. 1D). Moreover, IFNAR2 was also expressed in this cell line (Fig. 1D).

The presence of a high-affinity binding site for IFN-α in the megakaryoblastic cell line Dami has been previously demonstrated [16]. We further analyzed the expression of IFNAR and found that, like Meg-01, Dami cells express high levels of the 130 and 90 kDa bands of IFNAR1 and IFNAR2 (Fig. S1).

Expression of IFNAR in CD34+ cells, megakaryocyte progenitors, mature megakaryocytes and platelets

To confirm these results in primary cells, we further explored IFNAR expression and functionality throughout the different stages of megakaryopoiesis. The results showed that the mRNAs for IFNAR subunits 1 and 2 were expressed in CD34+ cells, progenitors and mature megakaryocytes as well as in peripheral blood platelets (Fig. 2A). The mRNA transcripts observed in the platelet and megakaryocyte samples were not because of leukocyte contamination as CD2 and CD20, both very highly expressed by mononuclear cells (MNC), were undetectable in platelet (Fig. 2A) and megakaryocyte samples (data not shown).

Figure 2.

 Type I interferon receptor (IFNAR) expression in hematopoietic progenitors, megakaryocytes and platelets. CD34+ cells, megakaryocytes (Mks), culture- and peripheral blood-derived platelets (Plat) were assessed for IFNR expression. (A) mRNAs encoding IFNAR subunits 1 (R1) and 2 (R2) were detected by RT-PCR. Mononuclear cell (MNC) antigens CD2 and CD20 were assessed in platelet samples to disregard contamination (n = 4). (B) The expression of IFNAR1 was determined in CD34+ cells, megakaryocytes and platelets by flow cytometry. For megakaryocytes, the IFNAR1-associated fluorescence within the CD61+ cell population is shown (n = 5). (C) IFNAR1 and IFNAR2 bands were detected in cell lysates by western blot. The number of cells per lane is shown in parentheses (n = 3).

Flow cytometry analysis indicated that IFNAR was expressed at the protein level in CD34+ cells and throughout all stages of megakaryocyte development, from early hematopoietic progenitors (megakaryocytes at day 6 of culture) to mature megakaryocytes (day 12 and 18 of culture) (Fig. 2B). On the other hand, IFNAR1 expression levels on either culture- or peripheral blood-derived platelets seemed to be very low compared with megakaryocytes, as only a small shift on the MFI (from 17 ± 2 to 21 ± 1, n = 3) was observed (Fig. 2B).

Interestingly, immunoblotting studies revealed a different pattern of bands in each cell type studied. In CD34+ cells and megakaryocyte precursors (day 6), not only the typical 130 kDa band of IFNAR1 (a heavily glycosylated isoform) but also an additional weak band of 60 kDa (a non-glycosylated isoform) [21] was observed, showing a similar pattern to that seen in peripheral MNC (Fig. 2C). Regarding mature megakaryocytes (day 12), these cells expressed both 130- and 60-kDa bands, although the intensity of the latter was always higher. In agreement with flow cytometry data, the expression levels of IFNAR1 in platelets detected by immunoblotting were extremely low as only a weak 60-kDa band was displayed in lysates from 107 platelets (20 μg of protein per lane), which is usually enough to detect most of the phosphoproteins involved in platelet signaling (data not shown). Detection of both 130 and 60 kDa bands was only possible when lysates were prepared from 108 platelets (200 μg of protein per lane) (Fig. 2C). Leukocyte contamination was also disregarded as no band was obtained in lysates derived from the maximal amount of mononuclear cells (104 MNC) present in 108 platelet samples (Fig. 2C). On the other hand, IFNAR2 expression, like subunit 1, was strong in megakaryocytes but weak in platelets (Fig. 2C).

Immunofluorescence assays were consistent with flow cytometry analysis. IFNAR expression was detected in CD34+ cells and in every stage of megakaryocyte development (day 6, 12 and 18), but was absent or undetectable in platelets (Fig. S2A). Immunostaining for the receptor (red) displayed a diffused pattern and did not co-localize with von Willebrand factor (VWF) (green), which was observed as a granular staining pattern as a result of its location inside megakaryocytic α-granules (Fig. 3A,B). Interestingly, in proplatelet-bearing megakaryocytes, while VWF is present in the proplatelet arm, IFNAR staining was observed on a side of the cell different to the one where the proplatelet structure was elongated (Fig. 3B). To further clarify this point, we performed a double staining with phalloidin/IFNAR1 Ab. A differential interference contrast microscopy (DIC) image revealed cellular elongations and swelling characteristics of proplatelet structures and phalloidin staining showed more clearly the thin shams along them. We confirmed that IFNAR is expressed in the megakaryocyte body but not in proplatelets (Fig. 3C and Fig. S2B). Moreover, the fluorescence intensity analysis along the two lines drawn on two different proplatelets indicated that while the IFNAR-associated signal was high in megakaryocytes, a value close to the baseline was observed in proplatelets (Fig. S2B). However, considering that IFNAR was detected by immunoblotting when lysates were prepared with 108 platelets and proplatelets are very thin structures, we do not rule out that IFNAR might be present in proplatelets but not detectable by immunofluorescence.

Figure 3.

 Type I interferon receptor (IFNAR) expression in megakaryo/thrombopoiesis by immunofluorescence. (A–B) IFNAR1 expression (red) and/or von Willebrand factor (VWF) (green) were analyzed in megakaryocytes and proplatelets on day 12 of culture. Nuclei were visualized with a DAPI counterstain (blue). Images were taken on an epifluorescence microscope (n = 3). Original magnification, 60 × (scale bar: 10 μm). (C) Megakaryocytes on day 12 of culture were co-stained with phalloidin (red) and IFNAR1 Ab (green) or the irrelevant isotype. Differential interference contrast (DIC) and fluorescence images were analyzed by confocal microscopy (n = 4). Original magnification, 60 × (scale bar: 10 μm).

IFNAR functionality in megakaryocytes and platelets

A type I IFN–IFNAR interaction triggers the activation of intracellular signaling pathways that mediate the cellular anti-viral response. Among these signals, Myxovirus A (MXA) and Interferon Regulatory Factor-7 (IRF-7) are well-known downstream proteins that, once upregulated, create a positive feedback mechanism that augments the host defense against infection [22,23]. In order to test receptor functionality during megakaryocyte development, transcription levels of the downstream target genes MXA and IFR7 were measured after stimulation with IFN-β in purified CD34+ and CD61+ cells at 6, 12 and 18 days of culture. Both MXA and IRF7, but not GATA1, were significantly upregulated by IFN-β at every stage of megakaryopoiesis (Fig. 4A) as well as in Meg-01 cells (Fig. 4B) compared with unstimulated controls. In order to prove that the IFN-β response was specific, we repeated these experiments in the presence of an IFNAR neutralizing Ab (MMHAR2). As shown in Fig. 4C, preincubation of megakaryocytes with the MMHAR2 Ab, but not the isotype, completely blocked the MXA and IRF7 upregulation induced by IFN-β.

Figure 4.

 The expression of type I interferon receptor (IFNAR) downstream targets is upregulated upon IFN-β stimulation. (A) CD34+ cells and megakaryocytes at 6, 12 and 18 days of culture were stimulated with 500 U mL−1 IFN-β for 24 h and MXA, IFR7 and GATA1 levels were quantified by quantitative (q)-PCR. (B) Meg-01 cells were stimulated with 500 U mL−1 IFN-β for 24 h and MXA and IFR7 levels were quantified by qPCR. (C) Megakaryocytes at day 12 of culture were incubated with 10 μg mL−1 IFNAR neutralizing Ab (MMHAR2) or an equal amount of the isotype for 15 min and then stimulated with 500 U mL−1 IFN-β for 24 h. MXA and IFR7 levels were quantified by q-PCR. Data in A–C are shown as expression values relative to untreated controls (n = 3–7, mean ± standard error of the mean [SEM], *P < 0.05 vs. control). (D) Megakaryocytes at day 12 of culture were stimulated with 500 U mL−1 IFN-β for 24 h and pSTAT1/2 were detected in cell lysates by western blot (n = 3).

Phosphorylation of STAT1 and 2 is also a typical response triggered by the interaction of IFN-I with its receptor in many cell types [24]. We confirmed that in megakaryocytes, both STAT1 and 2 are phosphorylated upon IFN-β stimulation (Fig. 4D).

To clarify whether the low amount of IFNAR detected in platelets was functional, we analyzed different platelet responses after the stimulation of IFNAR with IFN-I. In contrast to megakaryocytes, we failed to detect pSTAT1 and pSTAT2 in platelets stimulated with neither IFN-α nor -β (data not shown). Similarly, both IFN-α and -β failed to induce any changes in basal or thrombin-induced P-selectin expression, fibrinogen binding (Fig. S3) or platelet aggregation (data not shown).

Megakaryocytes can produce IFN-I

It has been shown that IFN-I are upregulated as a result of viral infection in TPO-stimulated CD34+ cells [14]. However, the cell responsible for the initial synthesis of IFN-I was not determined. We analyzed the expression of mRNA for IFN-I in Meg-01, Dami and purified megakaryocytes. The expression of IFN-I transcripts was strongly upregulated in a time-dependent manner by infection or stimulation of Meg-01 cells with either JUNV or PolyI:C, respectively (Fig. 5A,B). Similar results were obtained with Dami cells (Fig. S1E). Although primary megakaryocytes were restrictively infected by JUNV [14], they efficiently upregulated the IFN-I transcripts and IFN-β production at protein levels when stimulated with PolyI:C (Fig. 5C,D).

Figure 5.

 Type I interferons (IFN-I) are upregulated in megakaryocytes after polyriboinosinic polyribocytidylic acid (PolyI:C) stimulation or Junín virus (JUNV) infection. (A) Meg-01 cells were stimulated or incubated with 100 μg mL−1 PolyI:C or JUNV for 24 or 1 h, respectively. IFN-α and -β expression were determined by RT-PCR (n = 5). (B) Meg-01 cells were stimulated with 100 μg mL−1 PolyI:C for 6, 24 or 48 h. IFN-α and -β expression levels were measured by quantitative (q)-PCR and the results are shown as expression values relative to untreated controls (n = 4, mean ± standard error of the mean [SEM], *P < 0.05 vs. 0 h). (C) Megakaryocytes at day 12 of culture were incubated with 100 μg mL−1 polyriboinosinic polyribocytidylic acid (PolyI:C) for 24 h and IFN-α and -β expression were determined by RT-PCR (n = 3). (D) Megakaryocytes at day 12 of culture were incubated with 100 μg mL−1 PolyI:C for 24 or 48 h and IFN-β concentration was quantified by ELISA (n = 5, mean ± SEM, *P < 0.05 vs. 0 h).


In the present study, we demonstrated that IFNAR1 and 2 were found at mRNA levels not only in the megakaryocytic cell lines Meg-01 and Dami, but also in CD34+ cells, precursors and mature megakaryocytes as well as in platelets. However, IFNAR protein was detected in the cell lines, CD34+ cells and megakaryocytes but was absent or weak in platelets. The expression of IFNAR1 at the protein level was previously reported in Dami and UT-7 cells [16,17]. We now extended these findings and show that although Meg-01 and Dami cells express different IFNAR1 isoforms than primary cells, they not only express a functional IFNAR but also are able to produce IFN-I, suggesting that these cell lines could be useful models to study the IFN pathway. The observation of IFNAR1 on hematopoietic progenitors (CD34+ cells) is in agreement with previous studies [24]. However, to the best of our knowledge, this is the first characterization of the expression and functionality of this receptor at different stages of megakaryo/thrombopoiesis.

Expression of the receptor in circulating platelets was barely detected by either flow cytometry or immunofluorescence. Moreover, in mature megakaryocytes forming proplatelets, the expression of IFNAR1 was only observed in the cell body. Of note, only a weak 60-kDa band of IFNAR1, corresponding to the core protein [21], was observed in western blot assays with standard platelet concentrations. However, a 10-fold increase in the total amount of protein revealed that the highly glycosylated band at 130 kDa was also present in platelet samples. Together, these data suggest that although platelets contain mRNA encoding for IFNAR and express the core protein, they lack the highly glycosylated, surface-expressed functional receptor. In the same line of evidence, an early study showed the presence of the IFN-γ but not the IFN-α receptor in platelets [17]. Interestingly, this feature appears to be distinctive of megakaryocytes as IFN type I and II receptors are found in all immune cells.

IFN-I signaling exerts anti-proliferative and anti-viral effects through the IFNAR1 and IFNAR2 subunits (also designated as IFNαR or IFNβR, respectively) that are associated with the cytoplasmic protein tyrosine kinase Tyk2, which controls IFNAR1 cell surface expression [21], and Jak1, respectively. Dimerization of these receptor subunits in response to the binding of IFN-I causes the activation of Tyk2 and Jak1, which eventually activate the signal transducers and activators of transcription Stat1 and Stat2 [25]. The Stat proteins mediate transcriptional activation of IFN-regulated genes. Among them, MXA and IRF-7 are anti-viral proteins that are specifically induced by treatment with IFN-I and are proven to be reliable biomarkers of IFN-β bioactivity [26]. The observation that the phosphorylation of STAT1 and 2 as well as the expression of MXA and IRF7 were increased at the transcriptional level after megakaryocyte stimulation with IFN-β strongly suggests that IFNAR is functional in these cells. Moreover, upregulation of MXA and IRF7 transcripts after IFN-β stimulation of CD34+ cells, immature and mature megakaryocytes confirmed the IFNAR functionality through all the stages of megakaryocyte development. The IFNβ-mediated response was specific as neutralization of IFNAR with a blocking Ab completely suppressed the increased transcription of MXA and IRF7, and off-target genes such as GATA-1 were not modified upon IFN-β stimulation.

To analyze whether the presence of the 60- and 130-kDa isoforms were relevant in platelet function, we studied different parameters of platelet activation. The failure of IFN-α or -β to modulate pSTAT1/2, fibrinogen binding, P-selectin externalization or platelet aggregation argues against a putative role of IFNAR in platelets. These data are in contrast to those reported by Bhattacharyya et al., [21] who demonstrated that IFN-α inhibits human platelet aggregation. Nevertheless, it is important to point out that this inhibition was only observed after 40-min incubation of platelets with IFN-α. Interestingly, it has recently been showed that IFN-I are required and sufficient to cause the platelet dysfunction seen in the course of infection by the lymphocytic choriomeningitis virus (LCMV) and other selected viruses. Since in vitro incubation of platelets with IFN-β had no effect on the aggregation of mouse platelets, the authors suggested that IFN-I might act on parent megakaryocytes, leading to the production of altered platelets [27]. Although further experiments are required to confirm this hypothesis, our present results strongly support the notion that functional responses of IFN-I in the megakaryocytic lineage are linked to a selective interaction of IFN-I with megakaryocytes but not platelets.

Although the main role of platelets is to regulate hemostasis and thrombosis, increasing evidence shows that they are also key elements in the innate immune response [28–30]. In fact, platelets and megakaryocytes express several members of the toll-like receptor (TLR) family [28,31]. Although several experimental studies have addressed the role of platelets in the immune response, much less information is available regarding the immunomodulatory properties of megakaryocytes in the bone marrow microenvironment. In this context, some recent elegant studies have shown that Meg-01 cells and murine megakaryocytes express TLR2 and its stimulation by a synthetic ligand promotes megakaryocyte maturation [31]. The observation that megakaryocytes are capable of synthesizing IFN-I upon PolyI:C stimulation together with the presence of TLR3 transcripts in these cells (Fig. S4) further support the novel role of TLR in the megakaryocytic lineage.

Our present data show that megakaryocytes not only express functional IFNAR but are also capable of producing IFN-I led us to hypothesize that, as a consequence of a viral infection, the interaction between IFN-I and its receptor in megakaryocytes elicits downstream signals that not only regulate megakaryopoiesis but also allows these cells to take an active role in the anti-viral defense by being both IFN-I producers and responders. Whether both responses are regulated by a common signaling pathway as well as the in vivo relevance of these findings need further investigation.


S. Negrotto performed most of the experiments, analyzed the data and wrote the paper. C. J. de Giusti, L. Rivadeneyra and R. G. Pozner performed and analyzed the data of the RT-PCR experiments. R. M. Gomez and M. Schattner designed, directed the study and wrote the paper.


This work was supported by grants from Universidad Nacional de La Plata (Project X592), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT 2008-0230 (M.S.), 2010-1393 (S.N.), 2010-0411 (R.G.P.), 2007-00642 and 2007-00028 (R.M.G.), and CONICET PIP 1142009010016301 (S.N.) and 11420080100603 (R.G.P.). S.N., R.G.P., R.M.G. and M.S. are scientific researchers from CONICET; C.J.G., J.E. and L.R. hold fellowships from CONICET and M.J.L. holds a fellowship from ANPCyT.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.