Uptake of poly-dispersed single-walled carbon nanotubes and decline of functions in mouse NK cells undergoing activation

Figures & data

Figure 1. Effect of AF-SWCNT on generation of NK cell responses in vitro and in vivo. NK cells were activated in vitro by culturing spleen cells with IL-2/ml in absence or presence of AF-SWCNT/ml as described in the “Materials and methods” section. Cells were washed and their cytotoxic activity against YAC target cells were determined at E:T ratios of 100, 50, 25 and 12.5 in a 4-h chromium release assay. Values shown from a representative experiment are means [±SEM] of percent target lysis at different E:T ratios for NK cells activated with IL-2 in the absence (O = 0 μg/ml) or presence (▾ =10 μg, Δ = 25 μg, ▪ = 50 μg/ml) of AF-SWCNT [main Panel A]. Lytic units of cytotoxic activity were calculated for NK cells activated with IL-2 in presence of various concentrations of AF-SWCNT. Inset figure in Panel A shows dose-related decline in lytic units of NK cell activity generated in presence of different concentrations of AF-SWCNT. *p < 0.05 vs. AF-SWCNT untreated (ANOVA). Similar results for in vivo activation of NK cells by Poly(I:C) are shown in Panel B. Different groups of C57BL/6 mice were administered Poly(I:C) along with AF-SWCNT as described in “Materials and methods” section. In main Panel B, % target lysis at different E:T ratios of cells from Poly(I:C)-treated mice injected without (O = 0 μg/mouse) or with (▾ =10 μg, Δ = 50 μg, ▪ = 100 μg/mouse) AF-SWCNT are shown. Inset figure in Panel B reflects a dose-related inhibition of NK activation by AF-SWCNT. *p < 0.05, **p < 0.01 vs. AF-SWCNT untreated (ANOVA).

Table 1. Suppression of NK cell generation and activation in response to IL-2 (in vitro) and Poly(I:C) (in vivo) by AF-SWCNT.

	Treatment	Total NK cells (A)		Activated NK cells (B)		Apoptotic NK cells (C)
		NK1.1^+ cells (%)	Decline	NK1.1^+ CD69^+ cells (%)	Decline	NK1.1^+ Annexin V^+ cells (%)	Change
In vitro	+ IL-2	30.6 ± 1.1	–	53.2 ± 4.5	–	11.0 ± 2.2	–
	+ IL-2 + 50 μg AF-SWCNT/ml	25.3 ± 1.3*	17.3%*	41.5 ± 2.4*	22.0%*	22.4 ± 2.4**	**+103%
In vivo	+ Poly(I:C)	19.6 ± 0.9	–	21.7 ± 2.1	–	9.1 ± 1.8	–
	+ Poly(I:C) + 100 μg AF-SWCNT/mouse	16.5 ± 1.1*	16.8%*	16.6 ± 1.9*	23.5%*	8.5 ± 1.5	−12.1%

NK cell activation in vitro and in vivo was carried out as described in the “Materials and methods” section. Spleen cells activated by IL-2 in presence or absence of AF-SWCNT were stained with antibodies against NK1.1, CD69, and/or Annexin V and analyzed on a flow cytometer.

Values represent NK1.1⁺, NK1.1⁺CD69⁺ and NK1.1⁺Annexin V⁺ cell populations as percentages of all NK1.1⁺ cells (mean ± SEM) of data from four observations (four separate spleen cell cultures) for in vitro effect, and mean ± SEM of data from six observations (six individual mice) for in vivo effect. *p < 0.05, **p < 0.01 (Student’s t-test) to demonstrate any significant AF-SWCNT-induced inhibition.

Figure 2. Effect of AF-SWCNT on IL-2-activated NK cell sub-populations in vitro. Mouse spleen cells (2 × 10⁶/ml) were cultured with 500 U IL-2/ml in absence (□) or presence (▪) of 50 μg AF-SWCNT/ml. After 72 h, cells were washed and double-stained with a combination of antibodies against NK1.1 and CD107a markers or NK1.1 and FasL markers. Values shown are means ± SEM from four independent experiments. **p < 0.01 vs. AF-SWCNT untreated (Student’s t-test).

Table 2. Intracellular expression of IFNγ and TNFα by NK cells derived from mice treated with poly I:C with or without AF-SWCNT.

In vivo treatment	Ex vivo exposure to YAC cells	NK1.1^+ cells expressing cytoplasmic IFNγ (%)	NK1.1^+ cells expressing cytoplasmic TNFα (%)
A. None	No	2.4 ± 0.6	3.3 ± 0.7
B. None	Yes	2.8 ± 0.5	3.9 ± 0.5
C. + Poly(I:C), 200 μg IP	No	14.0 ± 1.4	11.0 ± 1.2
D. + Poly(I:C), 200 μg IP	Yes	41.3 ± 2.3^a	19.6 ± 1.8^a
E. + Poly(I:C), 200 μg IP + AF-SWCNT, 100 μg IV	No	12.8 ± 1.1	11.9 ± 1.3
F. + Poly(I:C), 200 μg IP + AF-SWCNT, 100 μg IV	Yes	27.9 ± 2.4^b,c	10.9 ± 1.4^b,c

C57BL/6 mice were administered 200 μg poly I:C IP with (or without) a single IV dose of 100 μg AF-SWCNT as described in the “Materials and methods” section. After 3 days, splenocytes were harvested and then cultured with or without YAC-1 cells for 5 h. The cells were then washed and stained for cytoplasmic IFNγ or TNFα and counterstained with anti-mouse NK1.1 mAb, and then analyzed by flow cytometry. Values shown are the mean [±SEM] percentages of NK1.1⁺ cells expressing cytoplasmic IFNγ and TNFα (from four replicate assay wells). ^ap < 0.05 vs. Treatment C; ^bp < 0.05 vs. Treatment E; ^cp < 0.05 vs. Treatment D.

Figure 3. Uptake of FAF-SWCNT by control and IL-2-induced NK1.1 cells in culture. Mouse spleen cells (2 × 10⁶/ml) were cultured in the absence (control) or presence of 500 U IL-2/ml along with 2 μg FAF-SWCNT/ml. After 12, 24, 48 or 72 h, cells were washed, stained with NK1.1 mAb and analyzed on a flow cytometer using NK 1.1 and FAF-SWCNTs [colors on X and Y axis, respectively]. Representative histograms for control and IL-2-activated cells at 24 h time-point are shown in Panels A and B, respectively. Values shown in each quadrangle indicate are the means [±SEM] fractions of cells in that quadrangle, from three replicate experiments. At all four timepoints, the percentages of NK 1.1⁺FAF-SWCNT⁺ cells as a percentage of all NK1.1⁺ cells were computed from flow histograms and plotted vs. time (Panel C). Values shown are mean [± SEM] percentages of NK cells positive for FAF-SWCNT at all timepoints, from three replicate experiments (Panel C). *p < 0.05, **p < 0.01 vs. control (Student’s t-test).

Figure 4. Intracellular distribution of FAF-SWCNT internalized by IL-2-activated NK1.1⁺ cells. Mouse spleen cells (2 × 10^6/ml) were cultured in presence of 500 U IL-2/ml and 2μg FAF-SWCNT/ml. After 24 h, cells were washed, stained with anti-mouse NK1.1 mAb, and NK1.1⁺ cells were sorted on a fluorescence activated cell sorter. Purified activated NK cells were then stained with DAPI and examined under a confocal microscope. Representative confocal images show combined DAPI/FAF-SWCNT fluorescent image at different depths (Z: -3.24 to 4.26 μm) (Magnification =60×).