SINGLE-WALLED CARBON NANOTUBES AFFECT ONLY BLOOD GRANULOCYTES BUT NOT
HL-60 CELLS
BLANKA EMŐDY-KISS, EDINA TAKÁCS, JÁNOS FENT AND SUSAN LAKATOS DEPARTMENT OF PATHOPHYSIOLOGY, LABORATORY INSTITUTE FOR HEALTH PROTECTION, MEDICAL CENTRE OF HUNGARIAN DEFENSE FORCES H-1134, RÓBERT KÁROLY KRT. 44, BUDAPEST, HUNGARY E-MAIL: LATYAKOS24@GMAIL.COM
INTRODUCTION
METHODS
According to our previous study, single-walled carbon nanotubes activate platelets and promote the formation of platelet-granulocyte complexes. Furthermore, they induce a slight increase in the expression of some adhesion molecules (CD 11b, CD18) on the surface of granulocytes if measured in whole human blood. Interestingly enough, in spite of the increase in this adhesion molecule expression no changes of firm adhesion to the endothelium or transmigration were detected in the cremasteric microcirculation of mice. To reveal the direct effect of SWCNT exerted on granulocytes the undifferentiated and partially differentiated HL-60 promyelocytic cells modeling the immature and partially matured granulocytes were tested.
Samples Cultures of HL-60 cells were maintained in RPMI supplemented with 10% FCS, 37oC, in 5% CO2 humidified atmosphere and differentiated for 4-6 days with 1.25% DMSO Sodium citrate anticoagulated whole human blood was also tested
Treatment of cells with 0,1 mg/ml SWCNTs - HL-60 cells: 37oC, 5% CO2, 1, 4, 24 hours - whole blood: room temperature, 1 hour
Flow cytometry Samples were stained with anti-human CD18 (PE)/CD14 (PerCP) and CD11b (PE)/CD14 (PerCP) and with appropriate isotype controls. Samples were measured by FACSCalibur flow cytometer. HL-60 cell populations were identified according to their CD11b while whole blood granulocytes according to the low-intensity CD14 fluorescent signal and gated on their light-scatter dot-plot. CD11b and CD18 expressions were characterized and statistically evaluated by their MFI values.
RESULTS Matured granulocyte in whole blood
Partially differentiated HL-60 cell
Undifferentiated HL-60 cell
Detailed analysis Partially differentiated HL-60 cells can be divided into two subpopulations in terms of their light scattering properties.
400x
400x
400x
M A T U R A T I O N Flow cytometric FS vs.SS dot plots:
*
* Control SWCNT PMA *
Part.Diff
*
160
250
140 Control
150
SWCNT PMA
100 50 0 UnDiff
Part.Diff
*
Control SWCNT PMA
PMNL in Blood
Undifferentiated HL-60 cells cannot be stimulated. Partially differentiated HL-60 cells are responsive towards PMA but not towards SWCNTs. Only MATURED granulocytes IN WHOLE BLOOD are affected by SWCNTs.
CD11b
*
*
Control SWCNT PMA
1 4 24 Incubation time with SWCNT (hours)
CD18
CD18
300
*
*
250
120
* CD18 MFI
CD18 MFI
* 200
*
1000 900 800 700 600 500 400 300 200 100 0
1 4 24 Incubation time with SWCNT (hours)
PMNL in Blood
CD18
300
CD11b
100 80
Control
60
SWCNT
40
PMA
CD18 MFI
UnDiff
500 450 400 350 300 250 200 150 100 50 0
Gate B
CD11b MFI
CD11b
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
CD11b MFI
CD11b MFI
Gate A
200
*
150
50
0
0
Control SWCNT
100
20 1 4 24 Incubation time with SWCNT (hours)
*
PMA
1 4 24 Incubation time with SWCNT (hours)
Neither of the two subpopulations exhibits significant changes in the surface expressions of CD 11b and CD18 even if they are treated with SWCNTs for an extended time
DISCUSSION SWCNT does not affect the surface expressions of CD11b and CD18 adhesion molecules on partially differentiated but already functionally active (PMA responsive) HL-60 cells. SWCNT affects granulocytes only in whole blood suggesting that SWCNT induces a complex process with more participants resulting in changes of CD11b and CD18 expressions on granulocytes. MATERIALS NANOPARTICLES: SWCNT - purified single-walled carbon nanotubes - Sigma–Aldrich Nanoparticles were dispersed at a concentration of 200 mg/ml in physiological saline by sonication. The suspension was stabilized by human serum albumin
Cell line: HL-60 promyelocytic leukemia cell line - Passage No 21-27 - ECACC 98070106 Sigma-Aldrich RPMI – Lonza FCS – BioWest PMA- Sigma-Aldrich
SELECTED REFERENCES Holzer, M et al.: Carbon-based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape. J Appl Toxicol. 2014, 34:1167- 76 doi: 10.1002/jat.2996. Bihari, P. et al.: Optimized dispersion of nanoparticles for biological in vitro and in vivo studies. Particle and Fibre Toxicology2008, 5:14 doi:10.1186/1743-8977-5-14 Collins, S.J. et al.: Normal functional characteristics of cultured human promyelocytid leukemia cells (HL-60) after inductuion of differentiation by dimethylsulfoxide. The Journal of Experimental Medicine 1979, 149: 969-974 Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds Proc. Nati. Acad. Sci. USA 1978, 75:2458-2462