Ozone as a Modulator of the Immune System

In order to clarify the immunomodulating properties of ozone, we have investigated: a) the effects of stimulation on isolated peripheral human blood mononuclear cells (PBMC) from normal donors with either ozone or ozonated serum; b) the range (in terms of O3 concentrations) of the therapeutic window; c) the stimulatory and toxic effects and d) the pattern, of both proinflammatory and immunosuppressive cytokine production up to 86 hours after exposure to O3. Results show that ozone can act as a weak inducer of cytokines producing IL-6, IL4, TNF-α, IFN-γ, IL-2 and IL-10 and, most importantly, there is a significant relationship between cytokine production and ozone concentration. Analysis of the proliferation index shows that progressively increasing O3 concentrations inhibit IP and therefore appear cytotoxic. Introduction Leukocytes comprise a heterogeneous cell population composed of lymphocytes (20-25%), monocytes (about 5%) and three type of granulocytes of which the neutrophils are about 70%. We have been the first to show that an appropriate ozone dose can induce a small release of interferon γ (IFNγ) from human blood (Bocci and Paulesu, 1990). Later on the number of cytokines has expanded to IFN-β, interleukin 2 (IL-2), IL-6, IL-8, tumor necrosis factor α (TNF-α), transforming grow factor β1 (TGF-β1) and granulocyte-monocyte colony stimulating factor (GM-CSF) (Paulesu et al., 1991; Bocci et al., 1993a, b; 1994; 1998a, b). Later on several authors (Beck et al., 1994; Arsalane et al., 1995; Jaspers et al., 1997) have confirmed that ozone can induce the production of cytokines after that epithelial cells of the respiratory mucosa have been in contact with ozone. Our results were obtained by ozoning blood directly and cytokines were detected in the plasma during the following 4-8 hours of incubations. These initial studies shed light on several aspects such as the protective effect of blood antioxidants, the dissimilar production of different cytokines and the progressive inhibitory activity of increasing ozone concentrations, particularly above 80 μg/ml per ml of blood. However they had limitations because firstly, whole blood can be incubated only for a limited time and, most importantly, we could not decide which cell type produced the cytokines. During the last year we decided to isolate from normal blood donors either peripheral blood mononuclear cells (lymphocytes and monocytes, PBMC) in order to investigate their viability and the production and type of cytokines released after two different ozonation modalities. The first is a direct ozonation of PBMC suspended in human serum, so that cells undergo the total action of ozone due to immediate effects by hydrogen peroxide (H2O2) and other unidentified reactive oxygen species (early ROS), with very short half-life, and late effects, provided by lipid oxidative products (LOPs), with fairly long half-life. The second approach has examined the effect of ozonated serum 20 min before addition to PBMC and therefore ozone activity is expressed only by “late LOPs”. Materials and Methods Ozone generation and measurement O3 was generated from medical-grade O2 using electrical corona arc discharge by the O3 generator (Model Ozonosan PM 100K, Hansler GmbH, Iffezheim, Germany), which allows the gas flow rate and O3 concentration to be controlled in real time by photometric determination at 253.7 nm, as recommended by the Standardisation Committee of the International O3 Association. Tygon polymer tubing and single-use silicon treated syringes were used throughout the reaction procedure to ensure containment of O3 and consistency in concentrations. O3 delivery to biological samples A predetermined volume of O2/O3 gas mixture at various O3 concentrations was collected with a syringe and immediately introduced into a second syringe containing an equivalent volume of human serum via a multidirectional stopcock. The final gas pressure remained at normal atmospheric pressure. In order to obtain reproducible results, it needs to be emphasised that O3 is a very reactive gas so that extremely rapid and precise handling is required. The sample is gently but continuously mixed with the gas for twenty minutes and afterwards dispensed into test tubes for various analyses. Control samples were either not treated or mixed with an equal volume of O2. It is worth mentioning that O2 represents at least 95% of the O2-O3 mixture. Collection of human blood samples and purification of PBMC After obtaining their informed consent, venous blood was withdrawn from healthy donors who had not been affected by any infection for at least one month and not taken any medication. Donors ranged in age from 24 to 67 years. Blood was collected in 0.14 ml citratephosphate-dextrose (CPD) per ml of blood. PBMC were isolated by Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) gradient centrifugation, washed twice in RPMI 1640 medium (Sigma Chemical Co.) supplemented with 20 mM HEPES, spun down at low speed to remove platelets, and resuspended in RPMI-1640 medium supplemented with 2 mM HEPES, 10% heat-inactivated fetal calf serum (FCS, Sigma Chemical Co.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Life Technologies, Gaithersburg, MD) at the final concentration of 1 x 10 6 viable cells/ml. The cells viability was assayed by the trypan blue exclusion technique and light microscope observation. Experimental approaches The aim was to examine two aspects of the ozonation process: (a) In the first case PBMC were suspended in sterile human serum (from male AB plasma, Sigma Co.) and directly treated with ozone at different concentrations, in order to evaluate the effect of early and late ROS including LOPs. (b) In the second case, PBMC were suspended in sterile human serum after that this has been ozonized for 20 min before cell addition. In this case PBMC could undergo only the effect of late ROS and LOPs, as early ROS decay in a few minutes during the ozonation process. In both cases all samples underwent successively the same procedure for incubation and related measurements. PBMC proliferation The various sample of PBMC suspension were added per well in triplicate wells to 96 well flat bottomed tissue culture plates (Costar, Cambridge, MA). PBMC were cultured without stimulation or stimulated with phytohemagglutinin (PHA, Sigma Chemical Co.) at final concentration of 5 μg/ml. Cell proliferation was evaluated by a colorimetric immunoassay (Boehringer Mannheim, Mannheim, Germany) based on BrdU incorporation. Briefly, after 48 h of incubation at 37°C in air-CO2 (5%) and 100% humidity, the cells were labelled with BrdU for 24 h (10 UI/well). The cells were then fixed, anti-BrdU-POD antibody was added and the immune complexes were detected by the subsequent substrate reaction. The proliferative index (PI) was obtained by calculating the ratio between PHA-stimulated cells and unstimulated ones, after subtraction of the corresponding blanks. Determination of cytokines Aliquots of all blood samples were layered on sterile tissue culture wells that were incubated in air-CO2 (5%) for 38, 62 and 86 hours. After 62 hours, 20 μl of sterile glucose solution were added to readjust glucose level at about 5 mM. At the end of each incubation period, samples were centrifuged at high speed and the plasma supernatants were kept at -70°C until determinations of cytokines were carried out. Immunoassays of either proinflammatory cytokines (IL-2; IFN-γ and TNF-α) or suppressive cytokines (IL-4; IL-6 and IL-10) were carried out using Cytoscreen immunoassay kits produced by Biosource Intern. All plasma samples were diluted 1:1 with the appropriate diluent. A three-cycle automatic washing was routinely performed. Negative plasma samples, in absence or presence of haemoglobin, were spiked with the cytokine’s standards to assess the reliability and precision of the various assays. Yields ranged between 93% and 105%. Biochemical determinations (a) Total antioxidant status (TAS) in plasma samples was carried out according to Rice-Evans and Miller (Rice-Evans and Miller, 1994). (b) Protein thiol groups (PTG) were measured in plasma according to Hu (Hu, 1994) using procedure 1 with 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) dissolved in absolute methanol. Values are expressed as mM. (c) The thiobarbituric acid (TBA) assay was carried out in plasma as described by Buege and Aust’s method (Buege and Aust, 1994). Values are reported (μM) as TBA reactive substances. Statistical analyses Results were expressed as the mean ± SD and the data were analysed using the Student’s ttest. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered significant. Results and Measurements First of all we examined the effect of ozonation performed directly on isolated human PBMC resuspended in human serum. The TAS value of the serum was 0.526 mM and this is a value markedly lower than the usual range (1.4-1.8 mM) found in fresh human plasma. In preliminary experiments we estimated the pattern of both the peroxidation (TBARS) and of thiol (PTG) oxidation that represent the usual markers indicating an effective ozonation. As it was expected, TBARS and PTG values increase and decrease, respectively, by progressively increasing ozone concentration (Fig. 1).