347
J. Photochem. Photobiol. B: Biol., 16 (1992) 347-355
Cytokine production after helium-neon laser irradiation in cultures of human peripheral blood mononuclear cells Jens
Oliver
Funk+,
Andrea
Kruse
and Holger
Kirchner
Institute of Immunology and Transficsion Medicine, University of Liibeck Medical School, Liibeck (Germany) (Received
March 23, 1992; accepted
June 5, 1992)
Abstract The effects of laser light on the immune system have not been extensively characterized. Low-power laser sources, such as the helium-neon (He-Ne) laser with a wavelength of 632.8 nm, have been found to produce photobiological effects with evidence of interference with immunological functions. We have investigated the effects of He-Ne laser irradiation on Ficoll-Hypaque-isolated human peripheral blood mononuclear cells (PBMC). Cultured cells were irradiated for various times at two selected intensities and then stimulated with different mitogens. The rate of incorporation of ‘H-thymidine into the DNA of stimulated cells decreased with increasing energy density. The levels of interleukin-lcu (IL-la), interleukin-2 (IL-2), tumour necrosis factor-a (TNF-a) and interferon-y (IFN--y) in supernatants of the cultures were determined (irradiated either before or after stimulation). When stimulating cells after irradiation, significantly increased levels of all cytokines were detected after 30 min of irradiation (18.9 J cm-‘), whereas after 60 min of irradiation (37.8 J cm-‘) cytokine levels were found to be significantly decreased.
Keywords: Cytokines, proliferation.
helium-neon
laser,
irradiation,
lymphocytes,
monocytes,
1. Introduction There have been many studies of the photobiological effects in vitro and in vivo following low-power laser irradiation (mW err-“) of unsensitized cells or tissues [l-3]. The most commonly used laser in all of these studies was the He-Ne laser with a wavelength of 632.8 nm. It is generally assumed that the resulting cellular changes depend mainly on the irradiation intensity and the irradiation time of the laser. The results concerning the effects of low-power lasers on cell cultures are inconsistent; in many cases either stimulating or inhibitory effects have been observed [3-6] and in others no effects have been noted at all [2, 71. The basic mechanisms of these effects are poorly understood [8]. Thermal effects can be ruled out as temperature changes due to laser irradiation do not occur in the irradiated cells [3, +Author to whom correspondence
loll-1344/92/$5.00
should be addressed.
0 1992 - Elsevier Sequoia. All rights reserved
348
91. Efforts to identify the subcellular components of energy absorption and transduction are currently under way. In several studies with animal models, interference with immunological functions, such as wound healing, cell-mediated hypersensitivity reaction and inflammatory processes, has been found [lO-121, but there are very few detailed descriptions of such effects on the cellular level. Effects on proliferation, cell interaction and calcium influx of immunocompetent cells have been reported [4, 13, 141. These processes include major cytokine action [15]. Despite the higher photochemical potential of UV radiation compared with visible light and therefore the possibly different mechanisms of light-cell interaction, there are parallels to low-power laser irradiation regarding the in vitro effects on immunocompetent cells. For instance, influences on the proliferation of lymphocytes, regulation of expression of membrane proteins and induction of cytokines in cultures have been reported [16-181. The stimulation of cultures of mononuclear cells with lectins or bacterial lipopolysaccharides (LPS) in an established method for the investigation of functional properties of the cells. Lymphocytes show increased mitotic activity [19], and all cells produce cytokines in picomolar concentrations, which are of major importance for communication and regulation in immune processes [15, 201. The aim of the present study was to evaluate the effects of He-Ne laser irradiation on human peripheral blood mononuclear cells (PBMC) in culture. In a first step, proliferation of the cells and the production of the four cytokines interleukin-la (ILla), tumour necrosis factor-a (TNF-cu), interleukin-2 (IL-2) and interferon-y (IFN-y), which are of central importance in the cytokine network, were measured after various experimental irradiation settings. In a second step, relevant irradiation parameters were selected and examined for significance.
2. Materials
and methods
2. I. Cell preparation Peripheral blood cells were obtained from the huffy coats of healthy blood donors of our blood bank. Mononuclear blood cells were separated by density centrifugation over Ficoll-Hypaque. The cells were washed twice in cold phosphate-buffered saline (PBS) and resuspended in RPM1 1640 medium without phenol red (Biochrom KG, Berlin, Germany) supplemented with 10% inactivated foetal calf serum (FCS), 2 mM L-glutamine, 100 units (U) ml-’ penicillin and 100 pg ml-’ streptomycin (Gibco GmbH, Karlsruhe, Germany, 5 ml minimum essential medium (MEM)-vitamin (100 x ) (Biochrom KG, Berlin, Germany) and 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) buffer (pH 7.2; Merck, Darmstadt, Germany). 2.2. Culture of mononuclear cells For proliferation studies, mononuclear cells obtained from butfy coats were seeded at a concentration of 1 X lo6 cells ml-’ into flat-bottomed 96-well plates (Falcon, Becton Dickinson, Heidelberg, Germany) and cultured in the presence of 5 pg ml-’ phytohaemagglutinin (PHA; Serva, Heidelberg, Germany) at 37 “C in a 5% humidified CO2 atmosphere for 48 h. For cytokine studies, cells were seeded at a concentration of 2~ lo6 cells ml-’ into 5 ml culture tubes (Greiner, Niirtingen, Germany) and cultured in the presence of 5 pg ml-’ PHA, 10 pg ml-’ concanavalin A (Con A) or 1 pg ml-’ LPS (Sigma, Deisenhofen, Germany) under identical conditions for 5
349 days without medium change. Supernatant fluids (less than 100 ~1) for measurement of cytokine concentrations were harvested daily in parallel cultures. The trypan-blue exclusion method was used to assess the viability of the cells before and after laser treatment. 2.3. Absorption measurements Prior to the irradiation experiments, absorption measurements were performed to rule out the presence of chromophores in the medium and to determine the absorption of the cells in the 500-800 nm band of the spectrum (UVICON 720 LC spectrophotometer, Kontron Analytic, Zurich, Switzerland). No significant absorption difference between phenol red-free RPM1 1640 medium and water existed, and the absorption did not change under the culture conditions. The light absorption of the unsensitized mononuclear cells in this band was very small. 2.4. Irradiation procedures The cultures were irradiated with a continuous wave (cw) He-Ne gas laser with a wavelength of 632.8 nm and a maximum power output of 25 f 1.2 mW (GLG 5700, NEC, Japan). The beam, originally of 1.2 mm in diameter defined at e-’ decrease, was enlarged by a convex lens and diverted by a mirror. For the irradiation of 96well plates the diameter of the bundle was adjusted to 3.2 mm at the medium surface of the well (0.32 cm2); for irradiation of the culture tubes the diameter was 7.5 mm at the medium surface (1.77 cm’). At each location the intensity was determined using an optical power meter (model 44XL, Photodyne Inc., CA, USA) and found to be 40 mW cm-2 for a well and 10.5 mW crn2 for a tube. In order to obtain different energy densities for the target cells the laser beam of constant intensity was applied for different times ranging from 10 s to 90 min. Relevant energy densities were chosen as specified for the experiments. Irradiation was carried out under sterile conditions in a temperature-controlled box at 37 “C. Non-irradiated, but otherwise equally treated, cultures were used as controls. 2.5. Proliferation test The effects of laser irradiation on lymphocyte transformation were studied by measuring the incorporation of 3H-thymidine into DNA at two time intervals after the irradiation period. One after the other the individual wells were placed under the laser. Radiolabelling was performed simultaneously for the last 4 h of the 48 h culture time by adding 20 ~1 of medium containing 3H-thymidine (specific activity, 540 GBq mmol-I; Amersham, Braunschweig, Germany) to each well (final activity, 18.5 kBq per 200 ~1 of medium). Thereafter, the reaction was stopped by lysing the cells with cold deionized water. Cell remnants were harvested onto paper filter discs using a PHDTM cell harvester (Cambridge Technology Inc., Watertown, MA, USA). After rinsing with absolute ethanol, the filter discs were dried in an incubator. Subsequently, the radioactivity was measured in aliquots by liquid scintillation (Scintillator 299TM, United Technologies Packard, Groningen, Netherlands). 2.6. Quantitative determination of qtokhe production The concentrations of IL-la, IL-2, IFN-y and TNF-cu were determined in a quantitative enzyme-linked immunosorbent assay (ELISA). Test kits were kindly provided by Hoffmann LaRoche (Basel, Switzerland). Separately for each cytokine, a microtitre plate was coated with a highly specific monoclonal mouse antihuman antibody against the human cytokine, i.e. IL-la, IL-2, IFN-y or TNF-(Y. Detection of the natural and recombinant forms of the human cytokine was possible in each case. No measurable
350
cross-reactivity with high levels of other cytokines was demonstrated for each ELISA. The ELISA kits were extremely sensitive; the minimal detectable dose was 5 pg ml-‘. Samples of supernatant fluids were pipetted into the wells and, after washing, an enzyme-linked specific murine monoclonal antibody was added. Following a wash to remove unbound antibody-enzyme reagent, a substrate solution was added. The intensity of the colour that developed was proportional to the cytokine concentration bound in the initial step. Absorption was measured at 450 nm in an ELISA plate reader (Anthos Labtec, Salzburg, Austria). Recombinant human cytokines (Hoffmann LaRoche, Basel, Switzerland) were used as standards. 2.7. Statistical analysis The results are expressed as the mean f standard deviation (SD). The Wilcoxon statistic for distribution-independent pair-groups was applied to compare two group means for significance of difference. P was considered significant at the
J. Photochem. Photobiol. B: Biol., 16 (1992) 347-355
Cytokine production after helium-neon laser irradiation in cultures of human peripheral blood mononuclear cells Jens
Oliver
Funk+,
Andrea
Kruse
and Holger
Kirchner
Institute of Immunology and Transficsion Medicine, University of Liibeck Medical School, Liibeck (Germany) (Received
March 23, 1992; accepted
June 5, 1992)
Abstract The effects of laser light on the immune system have not been extensively characterized. Low-power laser sources, such as the helium-neon (He-Ne) laser with a wavelength of 632.8 nm, have been found to produce photobiological effects with evidence of interference with immunological functions. We have investigated the effects of He-Ne laser irradiation on Ficoll-Hypaque-isolated human peripheral blood mononuclear cells (PBMC). Cultured cells were irradiated for various times at two selected intensities and then stimulated with different mitogens. The rate of incorporation of ‘H-thymidine into the DNA of stimulated cells decreased with increasing energy density. The levels of interleukin-lcu (IL-la), interleukin-2 (IL-2), tumour necrosis factor-a (TNF-a) and interferon-y (IFN--y) in supernatants of the cultures were determined (irradiated either before or after stimulation). When stimulating cells after irradiation, significantly increased levels of all cytokines were detected after 30 min of irradiation (18.9 J cm-‘), whereas after 60 min of irradiation (37.8 J cm-‘) cytokine levels were found to be significantly decreased.
Keywords: Cytokines, proliferation.
helium-neon
laser,
irradiation,
lymphocytes,
monocytes,
1. Introduction There have been many studies of the photobiological effects in vitro and in vivo following low-power laser irradiation (mW err-“) of unsensitized cells or tissues [l-3]. The most commonly used laser in all of these studies was the He-Ne laser with a wavelength of 632.8 nm. It is generally assumed that the resulting cellular changes depend mainly on the irradiation intensity and the irradiation time of the laser. The results concerning the effects of low-power lasers on cell cultures are inconsistent; in many cases either stimulating or inhibitory effects have been observed [3-6] and in others no effects have been noted at all [2, 71. The basic mechanisms of these effects are poorly understood [8]. Thermal effects can be ruled out as temperature changes due to laser irradiation do not occur in the irradiated cells [3, +Author to whom correspondence
loll-1344/92/$5.00
should be addressed.
0 1992 - Elsevier Sequoia. All rights reserved
348
91. Efforts to identify the subcellular components of energy absorption and transduction are currently under way. In several studies with animal models, interference with immunological functions, such as wound healing, cell-mediated hypersensitivity reaction and inflammatory processes, has been found [lO-121, but there are very few detailed descriptions of such effects on the cellular level. Effects on proliferation, cell interaction and calcium influx of immunocompetent cells have been reported [4, 13, 141. These processes include major cytokine action [15]. Despite the higher photochemical potential of UV radiation compared with visible light and therefore the possibly different mechanisms of light-cell interaction, there are parallels to low-power laser irradiation regarding the in vitro effects on immunocompetent cells. For instance, influences on the proliferation of lymphocytes, regulation of expression of membrane proteins and induction of cytokines in cultures have been reported [16-181. The stimulation of cultures of mononuclear cells with lectins or bacterial lipopolysaccharides (LPS) in an established method for the investigation of functional properties of the cells. Lymphocytes show increased mitotic activity [19], and all cells produce cytokines in picomolar concentrations, which are of major importance for communication and regulation in immune processes [15, 201. The aim of the present study was to evaluate the effects of He-Ne laser irradiation on human peripheral blood mononuclear cells (PBMC) in culture. In a first step, proliferation of the cells and the production of the four cytokines interleukin-la (ILla), tumour necrosis factor-a (TNF-cu), interleukin-2 (IL-2) and interferon-y (IFN-y), which are of central importance in the cytokine network, were measured after various experimental irradiation settings. In a second step, relevant irradiation parameters were selected and examined for significance.
2. Materials
and methods
2. I. Cell preparation Peripheral blood cells were obtained from the huffy coats of healthy blood donors of our blood bank. Mononuclear blood cells were separated by density centrifugation over Ficoll-Hypaque. The cells were washed twice in cold phosphate-buffered saline (PBS) and resuspended in RPM1 1640 medium without phenol red (Biochrom KG, Berlin, Germany) supplemented with 10% inactivated foetal calf serum (FCS), 2 mM L-glutamine, 100 units (U) ml-’ penicillin and 100 pg ml-’ streptomycin (Gibco GmbH, Karlsruhe, Germany, 5 ml minimum essential medium (MEM)-vitamin (100 x ) (Biochrom KG, Berlin, Germany) and 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) buffer (pH 7.2; Merck, Darmstadt, Germany). 2.2. Culture of mononuclear cells For proliferation studies, mononuclear cells obtained from butfy coats were seeded at a concentration of 1 X lo6 cells ml-’ into flat-bottomed 96-well plates (Falcon, Becton Dickinson, Heidelberg, Germany) and cultured in the presence of 5 pg ml-’ phytohaemagglutinin (PHA; Serva, Heidelberg, Germany) at 37 “C in a 5% humidified CO2 atmosphere for 48 h. For cytokine studies, cells were seeded at a concentration of 2~ lo6 cells ml-’ into 5 ml culture tubes (Greiner, Niirtingen, Germany) and cultured in the presence of 5 pg ml-’ PHA, 10 pg ml-’ concanavalin A (Con A) or 1 pg ml-’ LPS (Sigma, Deisenhofen, Germany) under identical conditions for 5
349 days without medium change. Supernatant fluids (less than 100 ~1) for measurement of cytokine concentrations were harvested daily in parallel cultures. The trypan-blue exclusion method was used to assess the viability of the cells before and after laser treatment. 2.3. Absorption measurements Prior to the irradiation experiments, absorption measurements were performed to rule out the presence of chromophores in the medium and to determine the absorption of the cells in the 500-800 nm band of the spectrum (UVICON 720 LC spectrophotometer, Kontron Analytic, Zurich, Switzerland). No significant absorption difference between phenol red-free RPM1 1640 medium and water existed, and the absorption did not change under the culture conditions. The light absorption of the unsensitized mononuclear cells in this band was very small. 2.4. Irradiation procedures The cultures were irradiated with a continuous wave (cw) He-Ne gas laser with a wavelength of 632.8 nm and a maximum power output of 25 f 1.2 mW (GLG 5700, NEC, Japan). The beam, originally of 1.2 mm in diameter defined at e-’ decrease, was enlarged by a convex lens and diverted by a mirror. For the irradiation of 96well plates the diameter of the bundle was adjusted to 3.2 mm at the medium surface of the well (0.32 cm2); for irradiation of the culture tubes the diameter was 7.5 mm at the medium surface (1.77 cm’). At each location the intensity was determined using an optical power meter (model 44XL, Photodyne Inc., CA, USA) and found to be 40 mW cm-2 for a well and 10.5 mW crn2 for a tube. In order to obtain different energy densities for the target cells the laser beam of constant intensity was applied for different times ranging from 10 s to 90 min. Relevant energy densities were chosen as specified for the experiments. Irradiation was carried out under sterile conditions in a temperature-controlled box at 37 “C. Non-irradiated, but otherwise equally treated, cultures were used as controls. 2.5. Proliferation test The effects of laser irradiation on lymphocyte transformation were studied by measuring the incorporation of 3H-thymidine into DNA at two time intervals after the irradiation period. One after the other the individual wells were placed under the laser. Radiolabelling was performed simultaneously for the last 4 h of the 48 h culture time by adding 20 ~1 of medium containing 3H-thymidine (specific activity, 540 GBq mmol-I; Amersham, Braunschweig, Germany) to each well (final activity, 18.5 kBq per 200 ~1 of medium). Thereafter, the reaction was stopped by lysing the cells with cold deionized water. Cell remnants were harvested onto paper filter discs using a PHDTM cell harvester (Cambridge Technology Inc., Watertown, MA, USA). After rinsing with absolute ethanol, the filter discs were dried in an incubator. Subsequently, the radioactivity was measured in aliquots by liquid scintillation (Scintillator 299TM, United Technologies Packard, Groningen, Netherlands). 2.6. Quantitative determination of qtokhe production The concentrations of IL-la, IL-2, IFN-y and TNF-cu were determined in a quantitative enzyme-linked immunosorbent assay (ELISA). Test kits were kindly provided by Hoffmann LaRoche (Basel, Switzerland). Separately for each cytokine, a microtitre plate was coated with a highly specific monoclonal mouse antihuman antibody against the human cytokine, i.e. IL-la, IL-2, IFN-y or TNF-(Y. Detection of the natural and recombinant forms of the human cytokine was possible in each case. No measurable
350
cross-reactivity with high levels of other cytokines was demonstrated for each ELISA. The ELISA kits were extremely sensitive; the minimal detectable dose was 5 pg ml-‘. Samples of supernatant fluids were pipetted into the wells and, after washing, an enzyme-linked specific murine monoclonal antibody was added. Following a wash to remove unbound antibody-enzyme reagent, a substrate solution was added. The intensity of the colour that developed was proportional to the cytokine concentration bound in the initial step. Absorption was measured at 450 nm in an ELISA plate reader (Anthos Labtec, Salzburg, Austria). Recombinant human cytokines (Hoffmann LaRoche, Basel, Switzerland) were used as standards. 2.7. Statistical analysis The results are expressed as the mean f standard deviation (SD). The Wilcoxon statistic for distribution-independent pair-groups was applied to compare two group means for significance of difference. P was considered significant at the
