THE EFFECT OF POLARIZED LIGHT ON THE RELEASE OF GROWTH FACTORS FROM THE U-937 MACROPHAGE-LIKE CELL LINE Peter Bolton, Mary Dyson and Steve Young Tissue Repair Research Unit, Division of Anatomy and Cell Biology. United Medical and Dental Schools of Guy's and ‘51 Thomas's, Guy's Hospital Campus, London SEV 9RT, U.K Macrophages are a source of many important mediators of wound repair. Cells of an established ‘macrophage-like cell line (U:937) were exposed in viiro to one of two light sources with different levels of polarization (95% and 14%). The irradiation times of 60 and 120 s were such that the energy densities to which the cells were exposed were 144 and 2.88 Jlem? for each of the light sources. Other groups of cells were sham-irradiated as controls. Twelve h after exptsure the macrophage-conditioned medium was and placed on 373 fibroblast monolayers. Fibroblast proliferation was assessed over a four-day erative response was greatest in the cultures exposed to supernatants from macrophages period. The pre cited with the 95% polarized light source, A treatment tine of 120 s was more effective than the of EY woRDs U-997 cell ling Polarized light Cell growth factor Introduction Growth factors synthesized and secreted by macro- phages can affect the activity of many types of cells.“ It has been shown that exposure to light of different wavelengths and energy densities can either encourage macrophage-like U-937 cells to release soluble factors that either stimulate or inhibit fibroblast proliferation.*? The aim of this study was to compare the effect of light at different degrees of polarization on these activities’ of macrophages. The macrophage-like cell line U-937* has been shown to produce soluble factors which stimulate mitogenic activity in cultured fibroblasts,” it shows many characteristics of the normal human macro- phage and it was considered appropriate to use it as a model in these investigations. The line has been used in earlier studies showing the effect of differerit parameters of light on factor release: Materials and Methods The phototherapeutic devices used were Bioptron™ polarized light sources. Both Jamps were calibrated using an Oriel spectroradiometer with a double monochromator containing a photomultiplier having, extended UV activity and with a ground quartz, diffuser of 2.5 cm in diameter placed in front of it (0898-5901/92/010033-05805.00 © 1992 by John Wiley & Sons, Led. Physical Characteristics of the Light Source Light Source A. (a) Wavelength: 400-2000 nm (b)Average power output: 5.772 W. (c) Degree of polarization: 95% (a) Wavelength: 400-2000 nm. (b) Light Source B. 17.316 W. {c) Degree of Average power output polarization: 14%. Dosimetry Because the output from the polarized and non- polarized light sources were different, it was necess- ary to alter the irradiation distance in order to ensure that the cells were exposed to the same energy density for both lamps. In this case, the more polarized lamp was placed at a distance of 25 cm from the cell cultures whilst the less polarized lamp was placed at a distance of 145 em. This gave an energy density of 1.44 J/cm? for both lamps for an irradiation time of 60 s and an energy density of 2.88 J/cm? for an irradiation time of 120 s. In both cases, the area of cells irradiated was 25 cm?. Cell Culture U-937 Cells. 1640 (Life ‘The U-937-cells were grown in RPMI Technologies) [LT] medium sup 3plemented with 1% penicillin streptomycin (LT] and 10% heat-activated fetal calf serum [FCS] [LT]. The cells were subcultured approximately every 2 days. They were grown at 37°C with a5% CO, atmosphere in 50 ml culture flasks, each flask containing 9 ml of medium plus cells. When confluent, each flask contained approximately 9 x 10* cells. Fibroblasts. The fibroblasts used were Swiss 373, supplied by the European Collection of Animal Cell Cultures (ECACC No 85022108] Porton Down They were grown in Dulbecco’s Minimum Essential Medium (DMEM [LT]), supplemented with 10% fetal calf serum (FCS [LT]) and 1% penicillin/strep- tomycin at 37°C in a 5% CO, atmosphere. The cultures were confluent by three days after subcul- ture. When confluent, each culture flask contained approximately 3-4 x 10* cells. The excess medium was poured out of each flask, and the cells were rinsed with phosphate buffered saline (PBS). Five ml of 0.25% trypsin was added to cach flask to cover the layer of cells and left for 30 s. The excess trypsin was then poured off and the flasks replaced in the \cubator in an inverted position so that the cell- covered surface was uppermost and any surplus trypsin remaining in the flask was not in contact with the cells. This prevented excess damage being caused to the cells by the trypsin remaining in the flasks. After 10 min the cells detached. ‘Three ml of DMEM were added to each flask and the resulting, cell suspension was poured into a universal container and mixed thoroughly. The cells were pipetted in 50 pl volumes into the wells of a 96-well microplate. Each well contained approximately 1000 cells. Fif- ty pl of fresh DMEM was then added to each well to give a final volume of 100 pl per well U-937 Irradiation When confluent, the U-937 cells were suspended and transferred to sterile 50 ml flasks (Life Technologies) in 10-ml volumes, at a cell density of 10'ml. The dilutions were made up with fresh RPMI. Each flask was then exposed to either (a) sham irradiation or (b) 95% polarized light irradiation or (c) 14% polarized light irradiation (Figure 1). Immediately after irradiation, the cells were transferred to a 37°C incubator for 12 h. The cells were then centrifuged for 5 min at 500 rpm. The macrophage-conditioned supernatant was removed and stored at ~20°C. Macrophage Viability Assay Twelve h after treatment, samples of macrophages from each group were removed and transferred to a glass vial, and 0.5 ml of trypan blue (0.4% wiv) was added. The samples were then placed in a Neubauer counting chamber and their viability tested." 34 Fibroblast Cell Proliferation Bioassay All the supernatants were. assayed for their effect on fibroblast proliferation using a methylene blue assay described by Martin er al."" and reviewed by Oliver et al.!2 One microplate was set up for each of the following time periods: time 0, 24h, 48h, 72h and 96 h post-plating. Each microplate was set up as in Figure 2. At the end of each time period one of the five plates was prepared for reading on a spectrophotometer as follows: (1) The cell medium was removed. (2) The cells were rinsed three times in borate buffer (pH 8.6, 0.01 m) (3) One hundred pil of 100% methanol was added to each well and left for 4 min to fix the cells. (4) The excess methanol was removed and the wells allowed to dry (5) One hundred pl of 1% methylene blue was added to each well and left for 30 min. (6) The excess methylene blue was removed and each well was rinsed three times with borate buffer. (7) One hundred j1! of acid alcohol was added to each well and the microplate agitated to ensure complete mixing of the eluted stain and the acid alcohol The microplates were then placed in an eight channel Anthos spectrophotometer (TechGen) and the absorbance of the contents of each well was read at 650 nm. ‘After the microplates had been read on the spectrophotometer, the absorbance was compared against a standard curve to determine the cell number per well Results U-937 Cell Viability The results of the trypan blue test for cell viability showed that by 12 h after treatment the number of U-937 cells remaining in all groups ranged from 93-95% of the initial number per well. There was no statistically significant difference between the cells exposed to either 95% polarized, 14% polarized light or the sham-irradiated controls, for both exposure periods. Fibroblast Proliferation Assay Table 1 shows fibroblast cell numbers per time period, and Table 2 shows statistical analyses of these data. The effect on fibroblast proliferation of, the macrophage-conditioned supernatant obtained from U-937 cells treated int vitro with the 95% and 14% polarized light sources (LA and LB) is illustrated in Figure 3. Fibroblast number is plotted P. BOLTON. St DYSON AND S_ YOUNG.TISSUE CULTURE FLASK. Figure 1. Experiment therapy against time after the addition of the supernatant By 96 h post-plating, cell“number was greatest in those populations exposed to supernatants from macrophages irradiated for 60 and 120s with the 95% polarized light source than with the 14% polarized light source for the same time periods. so7 CELLS PLUS MEDUM arrangement for the exposure of U-937 cells in suspension to light The greatest difference between the effects of 95% polarized and 14% polarized light was observed following irradiation for 120 s. Fibroblast prolifer- ation was increased by supernatants from macro- phages treated with 95% polarized light when the irradiation time was increased from 60 to 120 s. In couumn 1 a4 5 a 7 tot ROW 4 3 c ! al x sls] el el alas z|s| 3} 3] 3] 3] 3} 3] 3] als 2] 4] 2) 2] 3] 3] 4] a] a] 3] 31 4/3 6 4 MICROTEST PLAT: Figure 2, Diagram of the 96.well microtest plate showing the arrangement of the different test groups POLARIZED LIGHT AND FIBROBLAST PROLIFERATION 35Table 1. Fibroblast number per treat error of mean [SEM] Hours after plating Sham LA6O 0 1000 1000 24 (3184+ 35 4087 * 134 48 3734+ 89 4618 + 100 4364 £ 277 S116 + 151 96 4880 + 147 6945 + 180 Table 2. Results of the statistical analysis (Student's /-test) 96h post-plasting Sham LA6O—LAI20. LB60—LBI20 LAs P<005 - Ns NS P<0.05 LAI20 P< 0.05 NS = NS P< 0.05 LB6O NS. NS NS - NS LBI20 NS P< 0.05 P<0.05 NS = NS = No statistical difference LA = 95% polarized lamp. 14% polarized lamp, 120 = Irradiation time in s contrast, when the irradiation time with the 14% polarized light source was. increased from 60 to 120 s, there was a decrease in fibroblast proliferation (Figure 3); it was, however, significantly greater than the sham-icradiated control cells. Discussion ‘The results obtained suggest that the exposure of U-937 cells to polarized light is followed by the release of substances, presumably growth factors; which stimulate the proliferative activity of fibro- blasts, Furthermore, the level of polarization affects ment per time period (value Lai20 LB60 1000 37+ 72 4715 © 145 4913 = 74 8996 + 623 1000 2953+ 44 4619 + 286 4490 = 98 6352 * 342 1000 2683 + 53 3481 © 122 3763 5571 the level of stimulation. Of the two time periods examined, the best time for irradiation with the 95% polarized light was 120s; with the 14% polarized light source it was 60s. It has been suggested that the property of polarization induces a quantitative increase of negative surface charges on the irradiated cell surface of human embryo fibroblasts.' In contrast, non-polarized light is responsible for many biological effects,'**" its effects on the number of negatively charged binding sites on cells was only increased slightly compared to that of the sham irradiated cells in a recent study. This alteration of the plasma membrane induced by polarized light was significantly higher than that induced by non-polarized light. These observations suggest that the active participation of the cell membrane in the process of biostimulation by light is of significance. Evidence of increase in membrane permeability to calcium ions following the exposure of macrophage-like cells to low level laser therapy (LLLT) supports the view that membrane changes are of significance.’ Treatment of U-937 cells in vitro with polarized light appears to be an effective means of either (1) stimulating the release of fibroblast stimulating growth factor(s), or (2) modifying the irradiated culture medium in a stimulatory manner, or (3) a combination of both. Over-exposure to light 4.000 soo ou se 000 ws. « see 8 3“ ~s 7 12120 d 2.000 2 TIME 2 96 (HOURS) Figure 3-,Graph plotting fibroblast cell aumiter against time post-plating for each test group 36 P BOLTON, M. DYSON AND S. YOUNGirradiation may be possible, since in this in vitro iodel, increasing the irradiation time with 14% jolarized light reduced’ the stimulatory effect, though it remained greater than the effect of ham-irradiation. 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