Background
A major function of macrophages is phagocytosis of cellular and acellular debris during inflammation and wound healing, and the activation response of macrophages following phagocytosis can be varied depending on the local extracellular environment [
1‐
6]. The importance of phagocytosis in the resolution of inflammation is emphasized by pathological conditions involving impaired phagocytosis, which may manifest as persistent infections or chronic inflammatory lesions such as diabetic ulcers and atherosclerotic plaques [
7‐
11].
During their differentiation from primary monocytes, macrophages acquire specialized receptors and machinery for recognizing and clearing both apoptotic and infected cells [
12]. In clearing apoptotic cells, macrophages use receptors such as scavenger receptors and integrins that function independently or in cooperation with each other depending on the type of cell targeted for phagocytosis [
13‐
19].
Platelets are anucleate cells which play an integral role in maintaining vascular integrity. Within their 8-10 day lifespan, platelets can become activated either in the circulation or during adherence at a site of injury, and during this process they become targeted for destruction by macrophages [
20]. The process of platelet activation involves several changes to the cell surface, including expression of P-selectin and loss of membrane asymmetry [
21‐
23]. These changes in the platelet membrane may provide molecular signals to macrophages that trigger phagocytosis, although the precise mechanism by which macrophages recognize and phagocytose activated platelets remains to be identified.
Cells that have become apoptotic as part of their normal life cycle are recognized and cleared by phagocytosis in a manner that usually inhibits pro-inflammatory responses [
24‐
27]. Although circulating platelets can exert a pro-inflammatory effect on circulating monocytes [
28,
29], their effect on differentiated macrophages, particularly at sites of inflammation, is not clear. We show here that phagocytosis of autologous platelets results in an pro-inflammatory profile that is opposite to the macrophage response following phagocytosis of apoptotic cells. Importantly, the platelet-enhanced, pro-inflammatory response of macrophages can be inhibited when the platelets are loaded with the glucocorticoid dexamethasone. In addition to novel insight into the macrophage inflammatory profile that exists in several diseases, these results also provide evidence that platelet-macrophage interactions are an important therapeutic target for reducing inflammation.
Methods
Monocyte-derived Macrophages
Human monocytes were isolated and cultured using techniques similar to those previously described [
30,
31]. Briefly, blood from healthy human donors was collected into citrate and peripheral blood mononuclear cells (PBMCs) were isolated by using Lymphoprep (Accurate Chemical) according to the manufacturer's instructions. Monocytes were further isolated by plating the PBMCs on gelatin-coated tissue culture flasks for 45 min at 37°C followed by 10 washes with phosphate buffered saline (PBS) to remove non-adherent lymphocytes. Monocytes were then detached from the flasks by incubation in 10 mM EDTA for 2 min at 37°C. Monocytes (250,000 in 500 μl volume) were then plated in 24-well plates overnight in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml recombinant human GM-CSF (R&D Systems). Monocytes were plated on glass coverslips for scanning electron microscopy (SEM) analysis and plastic tissue-culture plates for TEM analysis and phagocytosis experiments. Media was changed on day 2 and day 5, and after 7 days of culture the MDMs were used for phagocytosis and cytokine assays.
Platelets
Platelets were isolated from whole blood collected into acid-citrate-dextrose (ACD) from healthy human donors and centrifuged for 15 min at 500 ×
g to generate platelet-rich plasma (PRP). PRP was pelleted by centrifugation for 10 min at 800 ×
g and the platelet pellet subsequently washed 2 times in citrated saline (pH 6.8). A portion of the platelet samples were degranulated by incubating 1 ml of platelets (250,000/μl in citrated saline) with 10 μl of 10 μM calcium ionophore A21387 (Sigma) for 15 min with rocking at room temperature, followed by three centrifugational washes with citrated saline. For phagocytosis experiments, platelets were fluorescently labeled with Cell Tracker Green CMFDA (Invitrogen) as previously described [
32]. After the final wash, and prior to use in phagocytosis assays, platelets were resuspended in warm serum-free RPMI for 15 min at 37°C.
For analysis of surface P-selectin and phosphatidylserine, platelets (250,000/μl) were first incubated in either citrated saline or serum-free RMPI media for 1 hr at 37°C. A portion of the platelets were activated by including thrombin (King Pharmaceuticals, 1 U/ml final concentration) in the incubation reaction. To detect surface expression of activation markers in each platelet treatment group, a 10 μl aliquot of platelets was stained with either FITC-anti-CD62P (Biolegend) or FITC-Annexin-V (Biolegend) for 30 min at room temperature, after which the cells were fixed and analyzed immediately by flow cytometry. Flow cytometry analysis was performed using a CyAn flow cytometer (Beckman Coulter) and the Summit analysis software.
Dexamethasone-loaded platelets were prepared by incubating 1 ml of platelets (250,000/ul) in citrated saline with 5 ul of dexamethasone (Sigma, 10 mM in DMSO) for 15 min on a rocker at room temperature. Platelets were then washed three times with citrated saline to remove unbound dexamethasone. For subsequent experiments, 5 × 106 dex-platelets were added to each well of macrophages in a 24-well plate.
To prepare apoptotic cells, PBMCs were isolated as above, and following monocyte-adherence to gelatin-coated flasks the non-adherent lymphocytes were collected. Cells were rendered apoptotic (Annexin-V positive) by UV-irradiation for 10 min followed by overnight incubation in RPMI + 10% FBS at 37°C + 5% CO2.
Phagocytosis Experiments
Thirty minutes prior to the start of each experiment, 7-day old MDMs were washed 3 times with PBS and incubated with 500 μl fresh RPMI media. In some experiments the media was supplemented with 10% autologous human serum. A 25 μl aliquot of fluorescently labeled platelets (250,000/μl) was added to each well of macrophages. Platelets and macrophages were co-incubated for 45 min.
For SEM analysis, the co-cultures were washed once with PBS and fixed in 2% paraformaldehyde and 0.5% glutaraldehyde. The samples were then processed as previously described [
33] and examined using a Cambridge S200 scanning electron microscope at 20 kV. For TEM analysis, the co-cultures were washed 5 times with PBS and fixed in 2% paraformaldehyde, processed as previously described [
34], and examined using a Leo EM 910 transmission electron microscope. For flow cytometric analysis, warm trypsin/EDTA was then added to the macrophages to remove adherent platelets (confirmed by microscopy) and cells were incubated 15 min at 37°C. Macrophages were then collected and fixed in 1% cold paraformaldehyde and analyzed using a CyAn flow cytometer (Beckman Coulter) and the Summit analysis software. Data is expressed as the percentage of FL1-positive macrophages in a given collection of 10,000 macrophages. Data shown represent the average of at least 3 independent experiments and for each experiment 10,000 macrophages were analyzed.
Latrunculin (Sigma, 1 μg/ml final concentration), used as a pan-phagocytosis inhibitor, was added to a portion of macrophage-containing wells 30 min prior to addition of platelets. Fucoidan (Sigma) was added to macrophages at a final concentration of 250 μg/ml 30 min prior to addition of platelets.
Cytokine Experiments
Each well of MDMs was washed 3 times with PBS and incubated with fresh RPMI + 10% autologous human serum. Activated, degranulated, or dexamethasone-loaded platelets (5 × 106) were added to each well in addition to LPS (100 ng/ml). Some samples also received dexamethasone alone at a final concentration of 1 μM. After 24 hrs, supernatants were collected, spun 10 min at 14,000 g, and frozen at -80°C. Cytokines were measured by ELISA using capture and detection antibodies (eBioscience) per the manufacturer's instructions. Cytokines were measured in duplicate and averaged. The amount of protein secreted was normalized to the amount secreted by macrophages treated with LPS alone. Each experiment was performed at least 3 times using 3 different MDM donors. In each experiment, the platelets added were from the same donor as the MDMs. Treatment groups were compared using an unpaired t-test.
Discussion
Phagocytosis is an important means of clearing both immunologically compromised and apoptotic cells. Monocyte-derived macrophages are efficient phagocytes in organs of the reticuloendothelial system and within injured tissues; however, the process of platelet clearance by macrophages is poorly understood. This work has demonstrated phagocytosis of fresh autologous activated platelets (AAPs) by monocyte-derived macrophages using an entirely human-derived in vitro system. In this system, uptake of freshly isolated platelets is dependent on actin polymerization, but occurs independently of any soluble serum factors. Additionally, phagocytosis of platelets is enhanced with platelet activation.
Previous studies on platelet phagocytosis have focused on modified platelets such as chilled platelets, opsonized platelets, and aged platelets [
39‐
46], each of which involves distinct changes to the platelet surface. Aged platelets most closely resemble freshly activated platelets because during aging platelets increase expression of phosphatidylserine and P-selectin [
20]. Interestingly, Brown et al. have also shown
in vitro that phagocytosis of aged platelets is mediated by scavenger receptors [
20]. Therefore, macrophages may recognize freshly activated platelets in the same way that they clear aged platelets. The finding that phosphatidylserine exposure is not a requirement for phagocytosis of AAPs in the present study was somewhat unexpected, considering that phosphatidylserine is a well documented 'eat me' signal for the phagocytosis of many different types of cells undergoing apoptosis [
47‐
50].
Because phagocytosis correlates with platelet activation, we would expect no phagocytosis to occur in the presence of quiescent platelets. However, RPMI media alone causes platelet activation (Table
1), probably due to the presence of low levels of calcium and phosphate. Additionally, the use of platelet inhibitors such as aspirin, EDTA, or prostaglandin, which may have maintained the platelets in a resting state, could not be used because they directly affect macrophage function [
51,
52]. Thus, one disadvantage of our system is that the platelet-macrophage interaction using activated platelets could not be compared to an interaction in which the platelets were in a truly resting state. Nonetheless, the interaction involving activated platelets is relevant because platelets are most likely activated at sites of tissue injury and perhaps during removal in the spleen. Thus, the interaction involving activated platelets was the focus of this work.
The macrophage response following phagocytosis of cells expressing surface phosphatidylserine is usually immunosuppressive [
2,
26,
27,
53,
54]. In the present study, co-culture with apoptotic cells inhibited production of pro-inflammatory cytokines by LPS-activated macrophages. These results are in agreement with previous findings for TNF-α, IL-1β, IL-8, IL-12 [
2,
4,
55,
56], and are extended to now include IL-6 and IL-23.
In contrast to the effect of apoptotic cells, activated platelets enhanced pro-inflammatory cytokine secretion from LPS-activated macrophages. The cytokines measured in the current study, TNF-α, IL-6 and IL-23, are significant readouts because they are known to be secreted by macrophages, but not platelets, and they play important roles in mediating pro-inflammatory responses. Interestingly, the pro-inflammatory cytokine secretion was also enhanced by platelets with surface phosphatidylserine exposure. The finding that degranulated platelets, washed free from their secreted proteins, also enhanced LPS-induced macrophage cytokine secretion suggests that a secreted platelet factor is not likely to be responsible for this effect. However, a secreted platelet factor could exert the observed effect if it remained bound to the platelet surface after secretion from the platelet. Because the platelets remained in the co-incubation for the entire experiment (24 hrs), the possibility also exists that the inflammatory consequences of platelet-macrophage interactions occur independently of phagocytosis. Cell contact itself could be responsible for the observed effects. Nonetheless, we have shown the pro-inflammatory effect of platelets does occur in conditions which favor platelet uptake (Figure
1).
Recent studies have highlighted additional roles of platelets beyond hemostasis, particularly with respect to platelet-mediated effects on inflammation [
28,
57,
58]. These results are particularly relevant to chronic inflammatory diseases, during which macrophages may interact with apoptotic or necrotic cells, as well as platelets, for prolonged periods of time. Studies in mice have demonstrated that depletion of platelets or platelet proteins affects macrophage infiltration and inflammation in lesions of the skin, joints, gut, and vasculature [
59‐
63]. Although the precise mechanisms by which platelets impact macrophage activation remain unclear, the current study provides direct evidence, using human cells, of specific macrophage cytokines that are enhanced by activated platelets.
Pro-inflammatory cytokines secreted by macrophages can also exert effects on surrounding cells and tissues. For example, IL-6 and IL-23 stimulate T-cells for induction of Th17 immune responses, which are operant in autoimmune diseases such as inflammatory bowel disease, lupus, psoriasis and arthritis [
64‐
67]. We speculate, therefore, that in addition to amplifying general pro-inflammatory responses, platelet-macrophage interactions might also play a role in Th17-mediated autoimmune diseases.
Glucocorticoids such as dexamethasone can exert powerful immunosuppressive effects on leukocytes and are thus an attractive therapy for modulating inflammation [
68]. After steroid binding to glucocorticoid receptors, which occurs within the cytoplasm, activated glucocorticoid receptors translocate to the nucleus and inhibit transcription of a variety of pro-inflammatory cytokines [
68]. We speculate, therefore, that the immunosuppressive action of dexamethasone-loaded platelets occurs by facilitating delivery of dexamethasone to macrophage glucocorticoid receptors. Because macrophage glucocorticoid receptors are cytoplasmic, we further speculate that the immunosuppressive effect of dex-platelets is a result of phagocytosis. The use of dexamethasone-loaded platelets for modulating macrophage action may prove useful in treating diseases characterized by excessive and unresolving inflammation. Our results demonstrating similar levels of immunosuppression with both free dexamethasone and dexamethasone bound to platelets suggests that tethering glucocorticoids to platelets may increase drug targeting and reduce the need for high systemic doses of glucocorticoids, which can have unwanted side effects [
69]. Furthermore, given the role of IL-6 and IL-23 in Th17-mediated inflammatory responses, the platelet-macrophage interaction is therefore a rational pharmacological target for inhibiting some Th17-related diseases.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CMS conceived of the study, participated in its design and coordination, performed phagocytosis assays and cytokine measurements, and drafted the manuscript. WDH participated in the design of the study, coordinated the selection and participation of blood donors, and processed the blood samples. THF was the principal investigator on this project and provided guidance and advice on the experiments and manuscript. All authors read and approved the final manuscript.