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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Nat Nanotechnol. 2009 Jun 14;4(7):451–456. doi: 10.1038/nnano.2009.151

Mechanisms for how Inhaled Multiwalled Carbon Nanotubes Suppress Systemic Immune Function in Mice

L A Mitchell 1,2, F T Lauer 2, S W Burchiel 2, J D McDonald 1,*
PMCID: PMC3641180  NIHMSID: NIHMS464365  PMID: 19581899

Abstract

The potential health effects of inhaling carbon nanotubes are important because of possible exposures in an occupational setting. Previously, we showed that mice inhaling multiwalled carbon nanotubes (MWCNT) showed suppressed systemic immune function. Here we show the mechanisms for this immune suppression. Mice were exposed to 0, 0.3, or 1 mg/m3 MWCNT for 6h/day for 14 consecutive days in whole-body inhalation chambers. Those exposed to 1 mg/m3 showed compromised systemic immune function. Spleen cells from exposed animals increased gene expression of prostaglandin synthase enzymes and were rescued from immunosuppression when treated with ibuprofen. Cyclooxygenase-2 knockout mice were resistant to MWCNT-induced suppression. Proteins isolated from the lungs of exposed mice contained transforming growth factor-beta, which suppressed immune function of wild-type splenocytes but not those from knockout mice in vitro. This suggests that signals from the lung can activate signals in the spleen to suppress the immune function of exposed mice.

Many products are already on the market that use or claim to use nanotechnology in their manufacturing processes (www.nanotechproject.org). Although these products in their final form may not pose a risk to the general public, individuals are likely to be exposed to nanocomponents in an occupational setting during production and processing. Of these manufactured nanomaterials, carbon nanotubes have been studied in recent years because of their enormous potential to improve existing products in industrial sectors. Materials that have unique characteristics such as those of nanotubes, which are lightweight yet exhibit great strength, are very desirable to the fields of technology, engineering, and medicine. However, there is increasing concern in the toxicology community regarding the health hazards these materials may pose.

The potential health effects of carbon nanotubes following inhalation is of great interest because of their commercial applications and demonstrated potential for pulmonary exposure in the workplace. Some have shown that nanotubes cause pulmonary damage following intratracheal instillation, including granuloma formation, inflammation, and cellular damage2-5. However, others have reported that MWCNT inhalation exposures produce a different spectrum of toxicities than that reported following intratracheal instillation exposures. Studies conducted in our lab6 found that MWCNTs dispersed evenly throughout the lung by inhalation and caused little pulmonary effects. Furthermore, Li et al.7 performed a direct comparison of intratracheal instillation and inhalation exposures and found that intratracheally instilled MWCNTs caused lung lesions in mice that were not observed after inhalation exposures with the same material at similar doses.

While we did not observe overt lung toxicity in response to MWCNT inhalation, we discovered systemic immune effects. These results are novel to the field and suggest that in order to study the biocompatibility of nanomaterials we needed to study extrapulmonary sites for damage and dysfunction. Interestingly, recent studies by Deng et al.8 demonstrated that MWCNTs do not enter systemic circulation following pulmonary exposure. Therefore, we are interested in mechanisms of lung signaling to systemic tissues that would explain the immunosuppression that has been measured in the spleen.

The purpose of this work was to elucidate the mechanism of systemic immunosuppression produced by MWCNT inhalation. Our results reveal that activation of cyclooxygenase enzymes by MWCNTs following inhalation exposures is a critical element in suppression of systemic humoral immunity. The mechanism of action appears to involve activation and release of TGFß following alveolar macrophage phagocytosis of MWCNT particles. This data shows for the first time that inhaled MWCNT can activate release of TGFß in the lung that can have a direct effect on prostaglandin production in spleen cells leading to immune suppression.

RESULTS

Inhaled MWCNTs decrease T-cell dependent antibody response

T-cell-dependent antibody response was conducted to confirm previous observations that inhalation exposures up to 1 mg/m3 cause systemic immune suppression. As seen in Supplementary Figure 1a, inhaled MWCNTs caused a dose-dependent decrease in antibody formation in response to antigen challenge with sheep red blood cells, with 1 mg/m3 being the most sensitive exposure concentration. In order to determine if this suppression was due to decreases in lymphocyte subpopulations, we used antibodies to known cell surface markers for cytotoxic T cells (CD8), T helper cells (CD4), natural killer cells (NK), macrophages (MAC), B cells (CD19), and total T cells (CD3). Supplementary Figure 1b shows that lymphocyte subpopulations were not altered with exposure to inhaled MWCNTs. An additional set of animals were exposed for 14 days to control air or 1mg/m3 MWCNT then held for a 30 day washout period. T cell dependent antibody formation and T cell proliferation in response to mitogen continued to be suppressed at 30 days post exposure (Supplementary Figure 2a and b).

Ibuprofen partially rescues MWCNT-induced immunosuppression

Gene expression for enzymes involved in the metabolism of arachidonic acid to prostaglandin H (PTGS2 or COX-2) and conversion of prostaglandin H to prostaglandin E (PTGES2) were measured by real-time RT-PCR in spleen cells from animals exposed to 1 mg/m3 MWCNTs or control air. Both prostaglandin synthase enzymes were upregulated in spleen cells with MWCNT exposure (Figure 1a). Conversely, gene expression in lung tissue from MWCNT exposed animals was not induced (Supplementary Figure 3). After measuring the induction of prostaglandin-associated enzymes by real time RT–PCR, we decided to test the hypothesis that systemic activation of the cyclooxygenase pathway was involved in the observed immunosuppression by blocking COX-2 (PTGS2) using the pharmacologic inhibitor ibuprofen. Ibuprofen was successful in partially reversing MWCNT-induced T-cell-dependent antibody suppression (Figure 1b) and reducing T-cell proliferation (Figure 1c). Animals in control chambers that received ibuprofen also had partially suppressed T-cell-dependent antibodies and T-cell proliferation, which led us to use the COX-2 knockout mouse to confirm these findings.

Figure 1. Immunosuppression is partially rescued by treatment with ibuprofen.

Figure 1

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a. Gene expression of Prostaglandin synthase 2 (PTGS2 or COX-2) and Prostaglandin E synthase 2 (PTGES2) was upregulated in spleen cells from MWCNT exposed animals. b. Mice that received the cyclooxygenase antagonist ibuprofen in their drinking water exhibited partial rescue from MWCNT-induced T-cell dysfunction. T-cell-dependent antibody response was decreased with 1 mg/m3 MWCNT exposure. This suppression was attenuated with ibuprofen dosing. c. Mitogen-induced T-cell proliferation was decreased with 1 mg/m3 MWCNT exposure. This decrease was partially rescued when mice were treated with ibuprofen. Error bars represent standard error of the mean for a given exposure group.

COX-2 knockout mice are unaffected by MWCNT inhalation

When exposed to atmospheres containing 1 mg/m3 MWCNT, COX-2 knockout mice do not exhibit decreased T-cell proliferation in response to mitogen nor do they have a decreased antibody production when challenged with antigen. As shown in Figure 2a and 2b, wild-type mice respond to MWCNTs with suppressed T-cell proliferation and antibody response as seen before. In fact, immune function was slightly enhanced in COX-2 knockout mice. B-cell proliferation remained unaffected by these exposures.

Figure 2. MWCNT-induced immunosuppression is dependent on activation of the cyclooxygenase pathway.

Figure 2

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a. Unlike wild-type littermates (COX-2+/+), COX-2-/- mice did not exhibit decreased antibody production in response to antigen after exposure to 1 mg/m3 MWCNT. b. Following 1 mg/m3 MWCNT exposure, COX-2-/- mice did not express decreased T-cell proliferation as did COX-2+/+ littermates. c. BALF protein was collected from COX-2+/+ mice exposed to 1 mg/m3 MWCNTs or control air. BALF protein from MWCNT-exposed animals was capable of causing decreased antibody production in response to antigen in splenocytes collected from COX-2+/+ mice. Antibody formation in splenocytes from COX-2-/-mice was not compromised with treatment with BALF protein from MWCNT-exposed animals. d. T-cell proliferation was decreased in splenocytes from COX-2+/+ mice following exposure to BALF protein from MWCNT-exposed animals. Splenocytes from COX-2-/- mice were not suppressed by BALF protein from MWCNT-exposed mice. Error bars represent standard error of the mean for a given exposure group. *Signifies statistically significant difference from control exposed wild-type mice (p<0.05).

BALF protein from exposed mice affects immune function of splenocytes in vitro

Protein isolated from BALF of MWCNT-exposed and control air-exposed mice was used to dose naïve spleen cells in vitro. The naïve spleen cells were collected from nonexposed wild-type and COX-2 knockout mice. T-cell-dependent antibody response was suppressed with exposure to BALF protein from MWCNT-exposed animals compared to BALF protein from controls in spleen cells from wild-type but not COX-2 knockout mice (Figure 2c). Furthermore, BALF protein from MWCNT-exposed animals also caused significant decreases in T-cell proliferation in wild-type spleen cells, whereas T cells from COX-2 knockout mice were unaffected (Figure 2d).

In order to establish a role for TGFß signaling from the lung we continued to use protein collected from BALF from exposed and unexposed mice to dose naïve splenocytes in culture. Additionally recombinant TGFß was used as a positive control for splenocyte immunosuppression and a treatment group was added that contained MWCNT BALF protein as well as Anti-TGFß (4 μg/ml) antibody to neutralize the signal. The neutralizing antibody was added at a high concentration to ensure that an adequate quantity was available to block the TGFß that was present in the lavage fluid. When splenocytes were cultured with MWCNT BALF protein or TGFß they exhibited decreased T cell dependent antibody formation and T cell proliferation (Figure 3a and b) compared to splenocytes receiving BALF protein from animals exposed to control air. Suppressed T cell dependent antibody formation was partially rescued by adding Anti-TGFß to MWCNT BALF protein cultures (Figure 3a) and decreased T cell proliferation was completely attenuated by adding the neutralizing TGFß antibody (Figure 3b).

Figure 3. Lavage fluid protein collected from MWCNT exposed (1mg/m3) animals is capable of inducing immune dysfunction.

Figure 3

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a. MWCNT BALF protein and TGFß were effective in inducing decreased antibody production in naïve splenocytes similar to in vivo exposures. Neutralizing anti-TGFß added to MWCNT BALF protein cultures lead to less antibody suppression, albeit not complete rescue. b. T cell proliferation was completely restored when anti-TGFß was added to MWCNT BALF protein treated media.

BALF from MWCNT exposed animals was analyzed for TGFß protein. Active TGFß was present at a significantly higher concentration (approximately 26 pg/ml compared to approximately 16 pg/ml in control lavage fluid) in BALF from 1mg/m3 MWCNT exposed animals compared to animals that received control air (Figure 4a). Plasma from exposed animals was also analyzed for TGFß levels. Although a trend was observed showing an increase in TGFß in the plasma of exposed mice, plasma levels were on the lower end of detection of the assay and so high background prevented detection of a statistically significant increase in this medium (Data not shown). After 24 hour exposures of naïve splenocytes to exposed or unexposed BALF protein spleen cell supernatants were analyzed for the prostaglandin E2 metabolite (13, 14-dihydro-15-keto prostaglandin A2) and IL-10. The metabolite of PGE2 was significantly increased with exposure to MWCNT BALF protein and TGFß as was IL-10. Addition of TGFß antibody to MWCNT BALF protein cultures completely attenuated the increase in PGE2 metabolite and IL-10 in spleen cell cultures (Figure 4b and c).

Figure 4. MWCNTs induce TGFß release in the lung that activates prostaglandin E2 and IL-10 expression in splenocytes.

Figure 4

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a. TGFß was analyzed by ELISA in BALF from MWCNT exposed and unexposed animals. BALF from animals that received 1 mg/m3 MWCNT contained higher levels of TGFß than control animal BALF. b. After 24 hour treatment with Control or MWCNT BALF protein, TGFß, or MWCNT BALF protein + Anti-TGFß antibody cells that were treated with MWCNT BALF protein or TGFß secreted higher levels of Prostaglandin E2 (as measured by its metabolite 13,14-dihydro-15-keto prostaglandin A2) and c. the immunomodulatory cytokine IL-10. MWCNT BALF protein + Anti-TGFß did not exhibit significant increases in Prostaglandin metabolite or IL-10 in culture supernatants. *Signifies statistically significant difference from control BALF (p<0.05), n=7 mice exposed for BALF collection and n=7 naïve mouse spleens per group.

DISCUSSION

Maynard et al.13 conducted a risk assessment study that determined that a concentration of 53 μg/m3 of carbon nanotubes could be detected when a simulated occupational exposure scenario was carried out. The average human weighing 70 kg with a RMV of 15.5 L/min working in this environment would have approximately 0.039mg of MWCNT deposited in the lung in an 8 hour work day and a total of 0.55mg deposited over 14 days. 0.55mg of material in the human lung (approx 70m2 surface area) is equal to approximately 0.008 mg per m2 lung surface area over 14 days. Mice only have approximately 600cm2 of lung surface area; over 14 days at the 1mg/m3 exposure concentration approximately 0.06 mg per m2 lung surface area deposited. Therefore, it should be noted that based on a risk assessment model, the human calculated lung burden is 7.5 fold less than that which mice in this study received. With scaled up production as a result of increasing demand as well as occupational exposures that persist for much longer than the 14 day paradigm used here; immune dysfunction is a concern for those that work in this industry.

This work shows that inhalation exposure to MWCNTs can cause systemic immune suppression. These effects persist up to 30 days post exposure and are unrelated to lymphocyte population changes due to cell death. After initial findings that gene expression of prostaglandin synthase enzymes PTGS2 and PTGES2 were upregulated in the spleens of MWCNT-exposed mice, follow-on studies were conducted to characterize this pathway. First, mice were exposed to MWCNTs or control air while simultaneously receiving ibuprofen or vehicle in their drinking water. Ibuprofen, a nonsteroidal anti-inflammatory drug, is a global cyclooxygenase antagonist, meaning it blocks COX-1 and COX-2. The ibuprofen study was successful in that dosing with ibuprofen partially rescued MWCNT-induced immune suppression. Interestingly, control animals that received ibuprofen in their drinking water were also moderately suppressed. Because ibuprofen is a global COX inhibitor, we believe this suppression is due to modulation of COX-1. COX-1 is constitutively expressed while COX-2 is inducible. Without the induction of COX-2 by MWCNTs, we hypothesize that ibuprofen is acting more on COX-1 and causing a detrimental effect that is unrelated to that seen in MWCNT-induced suppression. To confirm the involvement of the COX-2 pathway in MWCNT-induced immune suppression, we utilized COX-2 mice in our inhalation exposure system. COX-2 knockout or wild-type littermates were exposed to control air or 1 mg/m3 MWCNT for 14 days. As expected, COX-2 knockout mice were not suppressed in response to MWCNT exposure. Wild-type littermates exhibited decreased T-cell proliferation and antibody responsiveness from MWCNT exposure. Previous data6 has shown that IL-10 gene and protein expression is upregulated with MWCNT inhalation. While IL-10 is a known immunomodulatory cytokine that keeps the immune system in homeostasis, it is also induced by prostaglandin E2 (PGE2). Furthermore, PGE2 can affect the immune system without the help of IL-10 by blocking T-cell IL-2 autocrine activity14-17.

A recent publication on the biodistribution of MWCNTs showed that translocation from the lung to the circulation is unlikely 8. Additionally, histological analysis of the spleen following inhalation exposures did not reveal any signs of foreign material whereas the lung was laden with black particulate matter. In vitro exposures of MWCNT with freshly isolated murine splenocytes shows that very high concentrations of MWCNT are required to induce immune alterations in culture. This indicates that the observed immunosuppressive effects seen in vivo are likely not due to MWCNT entering the circulation and acting directly on the spleen cells since concentrations that were efficacious in these in vitro studies were far higher than that which was calculated to deposit in the lung during inhalation studies. Also, the immunosuppressive profile of the in vitro work was not similar to that seen in vivo and was likely due to nonspecific cell death from MWCNT lying on top of cell monolayers (supplemental figure 5). We believe that a signal, likely TGFß, is being released by the lung upon MWCNT inhalation that results in systemic immunosuppression (see Figure 5). To test this, we isolated protein from BALF from MWCNT-exposed and unexposed mice and treated naïve splenocytes (harvested from COX-2 wild-type or knockout mice) in culture. These studies showed that BALF protein from MWCNT-exposed animals is capable of inducing suppressed T-cell proliferation and antibody formation in spleen cells isolated from COX-2 wild-type but not COX-2 knockout mice. BALF protein from control animals was not capable of inducing immunosuppression. The in vitro immunosuppression data indicates that a signaling mechanism exists in the lung that activates the cyclooxygenase pathway in the spleen, leading to T-cell dysfunction and decreased T cell dependent antibody formation.

Figure 5.

Figure 5

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Schematic of the proposed mechanism by which inhaled MWCNTs induce systemic immune suppression.

TGFß has been shown to have an immunoregulatory role in macrophage “house cleaning”. During macrophage phagocytosis of apoptotic cells (efferocytosis) and other “garbage” found in biological systems, macrophages will release anti-inflammatory mediators. Macrophages do this in order to counteract any potential release of products from dieing cells and to prevent an unnecessary immunologic response. The main anti-inflammatory mediator released is TGFβ. TGFβ functions to prevent T cell proliferation and suppress activation of macrophages. Furthermore, it has been shown that the anti-inflammatory cytokine, TGFβ, not only upregulates COX-2 but also increases arachidonic acid release 18.

Since TGFß was suspect as the signaling mechanism from the lung to the spleen we measured TGFß concentrations in BALF from exposed and unexposed animals. Active TGFß was upregulated in BALF from MWCNT exposed mice. Additionally, upon in vitro treatment of naïve splenocytes with MWCNT exposed BALF protein or recombinant TGFß, splenocytes exhibited suppressed T cell dependent antibody formation and T cell proliferation as well as increased prostaglandin E2 metabolite and IL-10 in culture supernatants compared to those that received control air exposed BALF protein. Addition of a monoclonal antibody against TGFß to media treated with MWCNT BALF protein was protective against PGE2 and IL-10 release and partially so against subsequent immunosuppression. One limitation of this work is the necessity for the development and use of a lung specific TGFß knockout mouse to fully elucidate a role for TGFß signaling in MWCNT-induced systemic immunosuppression.

The current studies have shown a role for TGFß secretion from the lung in signaling induction of the COX pathway and subsequent immunosuppression. We used a highly relevant inhalation exposure system to expose mice to inhaled MWCNTs. Additionally we used this exposure system to prime mouse lungs for isolation of MWCNT-induced proteins that were used to directly expose naïve splenocytes in vitro. The in vitro studies showed that TGFß is indispensable in PGE2 and IL-10 expression but does not act alone in the induction of immunosuppression as evidenced by only partial attenuation with TGFß blockage. Because COX-2 is necessary for MWCNT-induced immune suppression, as seen in studies utilizing COX-2 knockout mice, at least one additional signaling mechanism from the lung is inducing the COX pathway and resulting in arachidonic acid products other than PGE2 that are immunomodulatory. 15-deoxy-Δ14-Prostaglandin J2 is a product of the cyclooxygenase pathway that is known to inhibit LPS-induced TNFα, macrophage inflammatory protein 2, and nitric oxide production19,20. Prostaglandin J2 acts through peroxisome proliferator activator receptor γ dependent or independent mechanisms to block transcription of inflammatory genes. Prostaglandin J2 can also block IL-2 expression in T lymphocytes21. PGJ2 is downstream of the Prostaglandin D2 synthase that converts Prostaglandin H2 to Prostaglandin D2. Prostaglandin D2 is then metabolized to Prostaglandin J2. Prostaglandin D2 is also known to induce lipoxygenase expression resulting in the anti-inflammatory molecule, Lipoxin A4 22.

These novel results suggest that TGFß is released from the lung upon inhalation exposure to low levels of MWCNT. This cytokine activates the cyclooxygenase pathway in the spleen leading to prostaglandin and IL-10 production and release ultimately causing T cell dysfunction and altered systemic immunity.

MATERIALS AND METHODS

Animals

Male C57Bl/6 mice were purchased from Harlan Laboratories (Indianapolis, IN) at approximately 8 weeks of age. Male wild-type B6;129P2 (COX-2+/+) and B6;129P2-PTGS2tm1Unc(referred to as COX-2 knockout or COX-2-/- mice) were purchased from Taconic (Hudson, NY) at approximately 9 weeks of age. Mice were exposed for 6 h/day for 14 consecutive days to atmospheres containing 0.3 or 1 mg/m3 MWCNTs or control air (n=7 unless otherwise stated). We estimated the total amount of MWCNTs deposited in the lung to be 0.15 mg/kg at 0.3 mg/m3 and 0.5 mg/kg at 1 mg/m3 ( approximately 3.75 and 12.5 μg total, respectively). Some animals were treated with ibuprofen as part of these studies. This was accomplished by diluting ibuprofen in drinking water at a concentration of 0.05 mg/ml along with 0.01 mg/ml cyclodextrin, approximately 8 mg/kg/day ibuprofen.

Exposure System and Characterization

Dispersible MWCNTs were purchased from Shenzhen Nanotech Port Co. (Shenzhen, China). Inhalation exposure atmospheres were produced by mechanical agitation/resuspension of MWCNTs using a jet mill (Fluid Energy, Hatfield, PA) coupled to a dry powder screw feeder (Scheck AccuRate, Whitewater, WI)12. Aerodynamic particle size distribution was determined using a Mercer cascade impactor (In-Tox Products, Moriarty, NM) operated at 2 l/min. Particle number size distribution was determined by a Fast Mobility Particle Sizer (FMPS; TSI Corp., Shoreview, MN).

Spleen Harvest and Cell Isolation

Spleens were harvested into sterile HBSS in sterile 15-ml centrifuge tubes on ice. Using sterile instruments, spleens were homogenized. Counts and viabilities were recorded and used for normalizing lymphocyte number during cell plating.

Flow cytometry

All reagents for this analysis were obtained from BD Biosciences (San Diego, CA) unless otherwise indicated. Subsets of lymphocytes were characterized using antibodies against cell surface markers to specific cell types. Three custom cocktails (all rat anti-mouse) were ordered from BD Biosciences (San Diego, CA) to identify six lymphocyte subpopulations: CD3 (all T cells), CD4 (T-helper cells), CD8 (cytotoxic T cells), CD19 (B cells), CD16 (natural killer cells), and Mac-1 (macrophages). Samples were analyzed using a FACScalibur Flow Cytometer (Beckton Dickson, San Jose CA). CD45 positive cells were gated and 10,000 gated events were acquired for each sample. CellQuest software was used to collect the data.

Jerne-Nordin Plaque Assay

Each sample was immunized with Sheep red blood cells (SRBCs) in duplicate in 48-well tissue culture plates (Corning, corning, NY). SRBC-free media was used for nonimmunized control wells for each sample. Cells were incubated for 4 days in an incubator. An 0.8% solution of agarose (SeaPlaque®, Cambrex, Rockland, ME) in 2X RPMI medium (Gibco), SRBCs, and spleen cells were incubated face down on custom slide trays at 37°C for 1 h. Guinea pig complement (Colorado Serum) was diluted 1:20 in Dulbecco's PBS containing Ca2+ and Mg2+ (Sigma) was added and then incubated an additional 2 h. SRBC lysis was quantified by counting plaques in the SRBC/agar lawn.

Mitogenesis

Concanavalin A (Sigma), a T-cell mitogen, was added to wells at a final concentration of 1 μg/ml. Lipopolysaccharide (Alexis, San Diego, CA), a B-cell mitogen, was added to wells at a final concentration of 10 μg/ml. Supplemented RPMI media was added to “no mitogen” control wells. Plates were placed in a 5% CO2, 37°C incubator for 48 h. Following incubation, the cells were pulsed with 1 μCi/well of 3H-thymidine (MP Biomedicals, Irvine, CA) and incubated for an additional 18 h. Cells were lysed and harvested using a Brandel Model 24V cell harvester and collected on filter paper (Whatman, Maidstone, England) and counted on a liquid scintillation beta counter.

Real-time RT-PCR

RNA from spleen samples was isolated using RNeasy Qiagen Kit (Valencia, CA), and RNase-free sterile pellet pestles (Kimble/Kontes, Vineland, NJ). A reverse transcription step was performed on total RNA at a concentration of 8 ng/μl using cDNA archive kit (Applied Biosystems Foster City, CA). CDNA was detected using Universal PCR master mix (Applied Biosystems) and TaqMan primer/probe sets (Applied Biosystems) for indicated genes.

ELISAs and EIA

TGFß and IL-10 ELISA kits were purchased from ebiosciences (San Diego, CA). Prostaglandin E2 metabolite EIA was purchased from Cayman Chemical (Ann Arbor, MI).

Experiments Using BALF to Dose Splenocytes In Vitro

Naïve splenocytes were then exposed to BALF protein in vitro from exposed and unexposed animals. Total protein was quantified using BioRad protein assay (BioRad Hercules, CA) Total BALF protein was then added to in vitro treatment media at a concentration of 1 μg/ml and naïve splenocytes were assayed for T- and B-cell proliferation as well as T-cell-dependent antibody response as described above. Recombinant TGFß was used as a positive control (10ng/ml) (R&D systems, Germany). Anti-TGFß1 antibody (Sigma) was used (4 μg/ml) to neutralize MWCNT BALF protein induced immunosuppression. Additionally splenocytes were incubated for 24 hours with BALF protein, TGFß, or MWCNT BALF protein + Anti-TGFß and culture supernatants were harvested for analysis of Prostaglandin metabolite (Prostaglandin A2) and IL-10 by ELISA. Detailed methods are provided in the supplementary section.

Table 1.

Particle size data collected during animal exposures to MWCNT aerosol

dN/dlogDp (#/cm3) dM/dlogDp (μg/m3) Surface (nm2/cm3)
Median (nm) 86.4 590 134.2
Mean (nm) 94.5 579 146.3
Geometric Standard Deviation 1.73 2.39 1.57
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Particle number size distribution (dN/dlogDp [#/cm3]) as well as particle diameter based on particle mass (dM/dlogDp [μg/m3]) is described.

ACKNOWLEDGEMENTS

This work was supported by NIEHS (P30 ES-012072) and EPA (RD-83252701).

Footnotes

L.M. conceived and designed the experiments and wrote the article. L.M. and F.L. conducted the experiments. S.B. and J.M. contributed materials/analysis tools. All authors discussed the results and commented on the manuscript.

Supplementary information accompanies this paper at www.nature.com/naturenanotechnology. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/.

COMPETING FINANCIAL INTERESTS STATEMENT

The authors declare no competing financial interests.

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