SWCNT (CNI, Houston, TX) produced by the high-pressure CO disproportionation (HiPco) process, employing CO in a continuous-flow gas phase as the carbon feedstock and Fe(CO)₅ as the iron-containing catalyst precursor and purified by acid treatment to remove metal contaminates were used in the study. Analysis performed by NMAM 5040 and ICP-AES revealed that SWCNT comprises 99.7% (wt) elemental carbon and 0.23% (wt) iron. For purity assessment of HiPco SWCNT, we used several standard analytical techniques, including thermo-gravimetric analysis with differential scanning colorimetry (TGA-DSC), thermo-programming oxidation (TPO), and Raman and near-infrared (NIR) spectroscopy. Comparative analytical data obtained by TGA-DSC, TPO, NIR, and Raman spectroscopy revealed that >99% of carbon content in the SWCNT HiPco product was accountable in a carbon nanotube morphology. Morphology of SWCNT is presented at Figure 1(A,B). The length of the individual SWCNT is approximately 1–3 μm as confirmed by transmission electron microscopy (TEM). Surface area was determined by Brunauer, Emmett, and Teller analysis. Zeta potential and particle size were determined on the Malvern Zetasizer Nano (Malvern Instruments, Westborough, MA). Zeta potential was measured at −42.3 ± 0.9 mV. The mean diameter and surface area of SWCNT were 1–4 nm and 1040 m²/g, respectively. Stock suspensions (1 mg/ml) were prepared before each experiment in PBS or culture medium, pH was adjusted to 7.0. To obtain a more homogenous and dispersed suspension, SWCNT were ultrasonicated (30 sec × 3 cycles). The dispersity of sonicated SWCNT assessed as previously described¹² was 83%.

The degree of inflammatory response induced by the aspirated particles was estimated by the number of total cells, macrophages, PMNs, and lymphocytes recruited into the mouse lungs and recovered in the broncho-alveolar lavage (BAL) fluid. Experiments were conducted under a protocol approved by the Animal Care and Use Committee of NIOSH. SWCNT were administered by pharyngeal aspiration (40 or 120 μg/mouse). At days 1 and 7 post exposure, mice were weighed, sacrificed and BAL fluid was collected in sterile centrifuge tubes. Cell counts were performed with an electronic cell counter. AM, PMNs and lymphocytes were identified by their typical cell shape and morphology in the cytospin preparations stained with a Hema-3 kit (Fisher Scientific, Pittsburgh, PA). Pulmonary DC were analyzed in non-lavaged lungs by flow cytometry. DC counts were normalized by the total numbers of lung cells upon digestion. Pulmonary exposure to SWCNT significantly increased the number of PMNs in BAL fluid on day 1 post exposure, with an almost 500-fold increase at the high dose of SWCNT, and up to a 37-fold increase in response to the lower dose as compared to controls (Figure 2B). On day 7 post exposure, PMNs, although markedly decreased as compared to day 1 post exposure, still remained at the elevated levels up to 22-fold and 6-fold over controls at the two respective doses (p<0.05). AM displayed a different homing pattern with significant increases observed only after day 7 post exposure (Figure 2A). Lymphocyte recruitment into the lung was not detected on day 1 post exposure. A relatively small number of lymphocytes were found later on day 7 post exposure in BAL fluid (p<0.05) (Figure 2C). Notably, elevated numbers of DC were found in the lung at 1d and 7d post exposure (p<0.05) (Figure 2D).

To investigate if pulmonary exposure to SWCNT (in the absence of specific antigen) altered systemic immunity, we tested the proliferative response of splenic T cells stimulated with Concavalin A. In SWCNT-exposed animals we observed the dose-dependent decrease of splenocyte proliferation, as evidenced by decrease of both proliferation and expansion indices (Figure 3). The proliferation index was decreased at all doses of SWCNT, reaching its maximum (~15% decrease) at 120 μg/mouse dose while the expansion index showed ~23% decrease at that dose, as compared to control.

Specific pathogen–free adult female BALB/c and C57BL/6 mice (7–8 wk old) were supplied by Jackson Labs (Bar Harbor, ME). Animals were individually housed in the National Institute for Occupational Safety and Health (NIOSH) facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were acclimated for 1 wk before use. Sterile Beta Chip bedding (Northeastern Products, Warrensburg, NY) was changed weekly. Animals were supplied with tap water and food (Harlan Teklad, 7913, Harlan Teklad, Madison, WI) ad libitum and housed under controlled light, temperature, and humidity conditions. Experiments were conducted under a protocol approved by the Animal Care and Use Committee of NIOSH.

Pharyngeal aspiration was used to introduce SWCNT to the mouse (BALB/c) lung. In brief, after anesthesia with a mixture of ketamine and xylazine (Phoenix, St. Joseph, MO) (62.5 and 2.5 mg/kg subcutaneous in the abdominal area), the mouse was placed on a board in a near-vertical position, and the tongue was gently extended with lined forceps. A suspension of SWCNT (40, 80 or 120 μg/mouse; 60 μl in PBS) was placed posterior in the throat, and the tongue was held until the suspension was aspirated into the lungs. Five or six animals per study group were used. Particles were sterilized before administration by autoclaving the particle suspension.

At days 1 and 7 post exposure, mice were weighed and anesthetized with intraperitoneal injection of sodium phenobarbital and exsanguinated. The trachea was cannulated with a blunted 22-gauge needle, and bronchoalveolar lavage (BAL) was performed with cold sterile Ca²⁺ and Mg ²⁺-free PBS at a volume of 0.7 ml for first lavage (kept separate) and 0.8 ml for subsequent lavages. Approximately 5 ml of BAL fluid per mouse were collected and pooled in sterile centrifuge tubes. Pooled BAL cells were washed in Ca²⁺ and Mg²⁺-free PBS by alternate centrifugation (800 × g for 10 min at 4°C) and resuspension. Cell-free first-fraction BAL aliquots were frozen and kept until processed.

The degree of inflammatory response induced by the aspirated particles was estimated by the total cells, macrophages, PMNs, and lymphocytes recruited into the mouse lungs and recovered in the BAL fluid. Cell counts were performed with an electronic cell counter equipped with a cell sizing attachment (Coulter model Multisizer II with a 256C channelizer; Coulter Electronics, Hialeah, FL). Alveolar macrophages, neutrophils, and lymphocytes were identified by their characteristic cell shape in cytospin preparations stained with a Hema-3 kit (Fisher Scientific, Pittsburgh, PA), and differential counts of BAL cells were performed. Three hundred cells per slide were counted.

For pulmonary DC analysis, non-lavaged lungs were dispersed using 2% collagenase A and 0.75% DNAse I in RPMI 1640 with 10% FBS at 37°C for 1h and labeled with anti-CD11b and anti-CD11c antibodies (BD Biosciences, CA) directly conjugated to FITC or PE. Percent of CD11c⁺/CD11b⁺ cells was obtained by flow cytometry. DC counts were normalized by total cell numbers in the digested lung.

Spleens from exposed or non-exposed BALB/c mice (day 7 post exposure) were aseptically harvested into 5 ml of sterile supplemented medium in a sterile culture dish. Harvested spleens were ground and suspension filtered through a cell strainer. Isolated splenocytes were centrifuged at 300 g for 10 min. Red blood cells were lysed with lysing buffer (155 mM NH4Cl in 10 mM Tris-HCl buffer (pH 7.5), 25°C) for 3 min. After lysis, splenocytes were washed with complete RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 [g/ml streptomycin, 10 mM HEPES, 10% heat-inactivated FBS, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (Invitrogen Life Technologies) (further referred as complete medium) and pellets were resuspended in 4 ml of complete medium. T cells (utilized in MLR assay) were obtained from spleen cell suspensions by passage through the nylon wool columns, counted using hemacytometer, diluted in the complete medium and used immediately for subsequent assays.

Murine DC were generated from hematopoietic progenitors isolated from bone marrow. Bone marrow cells were collected from tibias and femurs of C57BL/6 mice, passed through a nylon cell strainer to remove debris. Bone marrow cells were then depleted of red blood cells with lysing buffer for 3 min. The single-cell suspensions were then incubated with anti-mouse B220, CD4 and CD8 antibodies for 1 h at 4 °C followed by incubation with guinea pig complement for 30 min at 37 °C to deplete B and T lymphocytes. Cells were then cultured overnight (37°C, 5% CO₂) in six-well plates (Falcon) at the concentration of 1 × 10⁶ cells/ml in complete medium. The nonadherent cells were collected and seeded at a concentration of 2 × 10⁵ cells/ml in six-well plates in complete medium in the presence of recombinant mouse GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) (PeproTech, Rocky Hill, NJ).

Splenocytes were obtained from exposed (40, 80 or 120 μg/mouse) or non-exposed BALB/c mice (day 7 post exposure). Cells were labeled with 5-(and-6)-carboxyfluorescein diacetate (CFSE) at 5μM concentration for 5 min (Invitrogen, Carlsbad, CA), counted using a hemacytometer, diluted in complete medium (1×10⁶ cells/ml) and stimulated with 5 μg/ml Concavalin A (Sigma, St. Louis, MO) for 4 days in 24 well plates in triplicates. The proliferation response was measured using flow cytometry (BD FACSCalibur instrument, BD, NJ). Dead cells were excluded from the assay with propidium iodide staining preceding the flow cytometry. The background fluorescence readings were subtracted during the analysis. Proliferation index is the average number of cell divisions that the responding T cells underwent. Only responding T cells are reflected in the proliferation index. Expansion index determines the fold-expansion of the overall culture. This statistic is identical to that obtained by manual cell counting, and is predicted to be closely related to a 3H-T assay of the culture (http://www.flowjo.com/v9/html/proliferation.html). The proliferation and expansion indices were calculated from flow cytometry data using Flowjo software package (Tree Star Inc, Ashland, OR).

Cultured bone marrow-derived (C57BL/6 mice) DC were exposed to SWCNT (6.25 or 25 μg/ml), E. coli LPS (1 μg/ml, Sigma, St. Louis, MO), both (1 μg/ml LPS + 25 μg/ml SWCNT), or vehicle only for 48h on day 5 of DC culture. Non-cytotoxic doses of SWCNT were selected in the preliminary experiments (>95% viability after 48h exposure as determined by trypan blue exclusion). Sterile diluted suspensions of SWCNT in complete medium were sonicated and added to the cells (8 × 10⁵) to reach final concentrations of 6.25 or 25 [g/ml. The content of the wells was gently mixed and cultured for additional 48h. At day 7, exposed DC were collected, washed twice in complete medium, counted and aliquoted. Unexposed mouse (BALB/c) T lymphocytes were used as responder cells in MLR. MLR was set at a 1:30 DC:T cell ratio (after preliminary optimization). Following 72h of co-culture, exposed DC 16 (CD11c positive cells) were depleted utilizing magnetic separation. Co-cultured cells were collected, labeled with CD11c MicroBeads (MiltenyiBiotec GmbH, Germany) according to the manufacturer’s protocol and passed through LD columns mounted on a MidiMACS separator (MiltenyiBiotec GmbH, Germany). The effluent T cells were collected and labeled with 5-(and-6)-carboxyfluorescein diacetate (CFSE) at 5μM concentration for 5 min (Invitrogen, Carlsbad, CA). The labeled DC-depleted T cells were mixed with newly generated unexposed allogeneic DC (day 7 of DC culture) at a 1:30 DC:T cell ratio. The T cell proliferation response was measured on day 5 of MLR using flow cytometry (BD FACSCalibur instrument, BD, NJ). The proliferation and expansion indices were calculated from flow cytometry data using Flowjo software package (Tree Star Inc, Ashland, OR).

To determine DC phenotype, cells were exposed for 48h to SWCNT, washed in FACS medium (Hanks Balanced Salt Solution containing 0.1% BSA and 0.1% NaN₃) and stained with appropriately diluted antibodies (BD Biosciences, CA) directly conjugated with FITC or PE, followed by fixation in 2% paraformaldehyde. Control samples were labeled with isotype-matched antibodies conjugated with the same fluorochrome. Expression of CD11c, CD80, CD86, CD40 and MHCII (IAB) was measured using the BD FACS Calibur instrument.

Results were compared by one-way ANOVA and Student’s unpaired t test with Welch’s correction for unequal variances. All results are presented as means ± SEM. P values of < 0.05 were considered to be statistically significant.