The authors have declared that no competing interests exist.
The portfolio of cytokines is key to the function of macrophages as sentries of the innate immune system as well as being critical for the transition from innate to adaptive immunity. Cytokine bias is critical in the fate of macrophages into a continuum of inflammatory to anti-inflammatory macrophages. Due to advances in the field of toxicology, increasingly advanced multi-cellular
Macrophages are the sentinels of the immune system and in concert with neutrophils, serve as the first line of defence against viral, bacterial and parasitic infections [
A crucial characteristic of macrophages is their functional plasticity, predominantly with regards to their response based on the inflammatory microenvironment. This response can be generally categorised into “M1”—pro-inflammatory or “M2”—immunoregulatory. The role of macrophage polarization in the governance of a NM-induced immune response is not yet fully understood. As a further complication, macrophages can adapt to their tissue location, and their biological function can be heavily governed by their location within the specific tissue and in all reality the interactions of these cells with NMs are both tissue- and cell-state-specific [
Macrophages stand poised to rapidly produce large amounts of inflammatory cytokines in response to danger signals. The production of these cytokines can initiate a cascade of inflammatory mediator release that can lead to wholesale tissue destruction. Hence macrophages are implicated in a wide range of pathological conditions including but not limited to metabolic inflammation [
Interleukin (IL)6 is an important mediator of innate immunity. The protein is secreted by macrophages; and instigates various innate immune signalling cascades resulting in an augmentation of inflammatory responses and cell recruitment [
Tumour necrosis factor-α (TNF-α) is one of the main contributors to septic shock. They stimulate the release of corticotropic releasing hormone, which induces fever, and suppress appetite. Furthermore, it also stimulates the acute inflammatory response by promoting the synthesis of C-reactive protein and other mediators in the liver. TNF-α also induces vasodilation and loss of vascular permeability both critical for leukocyte infiltration. The cytokine is also important in the recruitment of inflammatory cells to the inflammation site [
IL1β is an extremely strong pro-inflammatory cytokine. Similarly to TNF-α, IL1β is an endogenous pyrogen that is produced at the early stages of the immune response to infections and physical stress. During an inflammatory response, IL1β stimulates the production of acute phase proteins from the liver and acts to induce fever and prostaglandin secretion [
IL10 is a multi-functional cytokine with diverse effects on a wide range of cells. The cytokine’s principle function is to terminate inflammatory responses [
Nanotechnology promises significant economic and societal benefits, but the commercialization of this technology is partially hindered by safety concerns. Due to the limitations of traditional mono-culture cellular models and the advances in the field of toxicology, increasingly advanced multi-cellular
In these experiments, the macrophage models were all exposed to a panel of NMs—namely—zinc oxide (ZnO), silver (Ag), titanium dioxide (TiO2) and quartz containing ~ 87% crystalline silica (DQ12). These NMs have been extensively studied previously both in
The NMs were sourced as follows: TiO2 (JRC Nanomaterials Repository—Italy, JRCNM01005a), ZnO (JRC Nanomaterials Repository—Italy, JRCNM01101a), Ag (Fraunhofer IME—Germany, NM300-K) and crystalline silica (Institute of Occupational Medicine—UK, DQ12). The NMs were sub-sampled under Good Laboratory Practice conditions and preserved under argon in the dark until use (with the exception of the DQ12).
The investigated particles were characterised using a combination of analytical techniques in order to infer primary physical and chemical properties important for understanding their toxicological behaviour. These measured physical and chemical properties have been described in detail previously [
NM code | NM type | Phase | Primary size (nm) | Surface area |
Known coating | Size in RPMI (nm) |
Size in KC medium (nm) |
---|---|---|---|---|---|---|---|
JRCNM01005a | TiO2 | Rutile-anatase | 15–24 | 46 | None | 337.47±8.27 | 427.5±39.2 |
JRCNM01101a | ZnO | Coated | 152 | 15 | Triethoxycaprl-silane | 261.1±6.45 | 287.4±8.72 |
NM300-K | Ag | - | 15 | - | 4% each of polyoxyethylene glycerol trioleate and Tween 20 | 35.1±1.05 | 56.57±3.83 |
DQ12 | Silica | - | - | - | None | 256.87±3.24 | 229.2±22.9 |
Ψ Size in biological media measured within 30 min of sonication.
Buffy coats (fraction of an anticoagulated blood sample that contains most of the white blood cells and platelets following density gradient centrifugation of the blood) were obtained from the Scottish National Blood Transfusion Service. Buffy coat was prepared to a concentration of 1/4 in PBS, before being distributed equally into four 25 ml falcon tubes. Next, 13 ml of Lymphoprep (Stem Cell Technologies, UK) was pipetted underneath the blood/PBS mixture. The tubes were centrifuged at 1000xg for 20 min at 25°C with slow deceleration. The PBMC layer was removed into two Falcon tubes and made up to 50 ml with PBS. The tubes were again centrifuged for 8 min at 500 g, and the supernatant removed. The cells were re-suspended with a small volume of PBS and pooled. The volume was made up to 50 ml using PBS and a cell count performed. Following a further centrifugation step for 8 min at 500 g, the cell pellet was re-suspended in MACS buffer (Miltenyi Biotech Limited, UK) (0.5% BSA, 2 mM EDTA, PBS and adjusted to pH 7.2) (80 μl—107 total cells) and human CD14 microbeads (Miltenyi Biotech Limited, UK) (10 μl per 107 cells) were mixed well and incubated for 15 min in the fridge. The tube was filled to 50 ml with MACS buffer and centrifuged for 8 min at 500 g. The supernatant was removed and the cell pellet was re-suspended in 500 μl of MACS buffer per 108 cells [
The cells were separated using a separation column fitted into a magnetic column (midiMACS separation system—Miltenyi Biotech Limited). The column was primed by flushing with 3 x 3 ml volumes of MACS buffer before the cell suspension was added. The column was washed with 3 x 3 ml volumes of MACS buffer and the unlabelled cells collected. The labelled cells (monocytes) were collected by removing the column from the magnet and flushed with 5ml of MACS buffer. The cells were washed as previously described and adjusted to the required number.
The THP-1 cells were was obtained from ATCC (USA—ATCC TIB-202D). The THP-1 monocytes were differentiated using 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, UK) for 24 hr. The cells were maintained in RPMI (Gibco, UK) medium supplemented with 10% Fetal Calf Serum (FCS) (Gibco, UK), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin (Gibco, UK) at 37°C and 5% CO2.
The primary human KCs were purchased from Axol Bioscience Limited (UK). The KCs were thawed in phenol red free Dulbecco's Modified Eagle Medium (DMEM) (Gibco, UK) supplemented with 5% FBS, 2 nM glutamax (Gibco, UK), 15 mM HEPES (Sigma, UK) and 100 U/ml penicillin/streptomycin. The KCs were seeded on type I collagen (Thermo Scientific, UK) coated plates. The liver macrophages were maintained in phenol red free DMEM supplemented with 10% FCS, 1% non-essential amino acids (Sigma, UK), 2 nM glutamax, 15 mM HEPES and 100 U/ml penicillin/streptomycin.
After isolation, the primary blood monocytes were plated in 6 well plates at a concentration of 106 cells /ml (3 ml per well) in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and incubated at 37°C and 5% CO2 for 7 days. After this differentiation step, the plates were placed on ice and washed using ice cold PBS. One millilitre of PBS containing 1 mM EDTA was added to each well and incubated on ice for 30 min. The cells were scraped from the wells using a cell scraper, re-suspended in complete RPMI medium and adjusted to the required concentration prior to plating in 96 well plates.
For the cytokine analysis experiments, all macrophages were utilised at 104 cells per well in a 96 well plate format. For subsequent experiments the macrophage models were either used in a non-activated form, or after activation utilising 20 ng/ml of IFN-ϒ plus 1 μg/ml lipopolysaccharide (LPS) (both Sigma, UK) for 12 hr [
NMs were prepared following the NANOGENOTOX dispersion protocol [
Following a 24 hr NM and ethanol treatment, the supernatants were collected and stored at -80°C. The levels of human IL1β, IL6, IL10 and TNF-α was determined in the cell supernatant using R&D Systems magnetic Luminex® Performance Assay multiplex kits (bead based immunoassay; Bio-techne, USA) according to the manufacturers instruction. The proteins were detected via a Bio-Rad Bio-Plex MAGPIX multiplex reader. The technology is constructed on the use of analyte-specific antibodies pre-coated onto magnetic microplates embedded with fluorophores at set ratios for each unique microparticle region.
For statistical analysis, the experimental results were compared to their corresponding control values using an ANOVA with Tukey’s multiple comparison. The statistical analysis was carried out utilizing Minitab 18. A p value of <0.05 was considered as significant. All experiments were repeated on three separate occasions.
The investigated NMs were extensively characterised by a combination of analytical techniques in order to infer their primary physiochemical properties. A comprehensive description of the measured physical properties for the NMs has been described previously [
The changes in cytokine production levels (IL1-β, IL6, IL10 and TNF-α) as a consequence of NM and ethanol exposure was assessed within the supernatant of non-activated and activated macrophage models. From the data, it is evident that the cytokine profile (both in terms of levels of cytokines produced and the protein itself) of the three macrophage models are markedly and significantly different from each other (Figs
The cells were cultured with cell medium (C) or exposed to the xenobiotic for a period of 24 hr
The cells were cultured with cell medium (C) or exposed to the xenobiotic for a period of 24 hr
The cells were cultured with cell medium (C) or exposed to the xenobiotic for a period of 24 hr
The cells were cultured with cell medium (C) or exposed to the xenobiotic for a period of 24 hr
Cytokines are powerful signalling molecules that are as crucial to everyday life. They are small proteins that mediate inter/intracellular communication and are produced by numerous cell types but predominately those belonging to the immune system. They coordinate a variety of important processes ranging from the regulation of local and systemic inflammation, chemotaxis, metabolism and tissue repair.
The binding of cytokines to specific cell surface receptors instigate a cell-signalling cascade that orchestrates cell function. This may result in secretion of other cytokines, as well as an increase in the number of surface receptors for other cytokines or in the suppression of the biological response. Cytokines are fundamental to the functions of macrophages, governing the unleashing of an effective immune response and most crucially link an innate and adaptive immune response [
In this study, we compared a range of
It was clear that the PMBCs produced the highest levels of pro-inflammatory cytokines (e.g. IL1β, IL6 and TNF-α), while the KCs secreted sizeable quantities of anti-inflammatory IL10. With respect to the THP-1 cells, these were not effective cytokine producers under the investigated conditions. These findings are similar to a previous examination showing THP-1 being poor cytokine producers as compared to primary macrophages following exposure to carbon nanotubes [
It might be important to state that a note of caution is required in the interpretation of findings in this study an increased cytokine secretion by a cell population
Interestingly, the pre-activation of the macrophages did not greatly influence the inflammatory response for the xenobiotic exposed macrophages. On occasion, the cytokine secretion was higher for the non-activated macrophages exposed to a given xenobiotic as compared to the activated macrophages of the same group. A final noteworthy observation and as touched upon, was the lack of clear patterns in NM or concentration dependant cytokine response under the experimental confounds. This could potentially be explained by the low concentrations of xenobiotic utilised or the specific time-point investigated. Finally, ethanol treatment was not associated with a dose response for any of the cytokines investigated although distinct variances between the different macrophage types was evident which is clearly indicative of the necessity for the appropriate selection of macrophage populations is not a particulate specific issue.
Overall, the findings from this investigation in addition to the complexities of cytokine biology discussed above, highlight the importance of the selection of appropriate macrophage cell population for a physiologically relevant
The authors are grateful to colleagues at Heriot Watt University.