Gene expression studies in cultured dendritic cells: new indicators for the discrimination of skin sensitizers and irritants in vitro.

BACKGROUND: The replacement of animal tests for the detection of the sensitizing potential of chemicals is of great importance due to current legislation. One promising approach for the development of an in vitro assay is the exposure of immature dendritic cells (iDCs) to contact sensitizers and irritants, followed by an analysis of the maturation status of the cells. OBJECTIVE: The aim of this study was to further investigate the performance of our previously developed targeted microarray, the immune toxicity chip. In addition, we aimed to identify new marker genes for the discrimination of allergens and irritants using whole-genome microarrays. METHODS: Monocyte-derived iDCs were exposed to contact sensitizers and irritants in concentrations resulting in 10-20% cytotoxicity, as determined by dose-response curves. Changes in gene expression were analysed using the immune toxicity chip and a commercially available whole-genome microarray. RESULTS: Using the immune toxicity chip, we could identify a panel of marker genes suitable to discriminate strong allergens and irritants. Analysis with the whole-genome array revealed additional genes that are differentially expressed after allergen exposure, but not after irritant exposure. Hierarchical clustering of these genes showed distinct groups representing the different chemicals. CONCLUSION: Here we show that our test system based on an immune-specific microarray is suitable for the discrimination of strong allergens and irritants. Genes detected as differentially expressed with the whole-genome array and previously not connected to the maturation process of DCs might be suitable candidate genes for the identification of weaker sensitizers.

Cytotoxic effects of the herbicide 2,4-dichlorophenoxyacetic acid in HepG2 cells.

2,4-Dichlorophenoxyacetic acid (2,4-D) and its derivatives are herbicides widely used to control the growth of broadleaf and woody plants. Although 2,4-D is well known to be moderately toxic, little information is available on the mechanisms of its toxicity. Results on carcinogenicity, genotoxicity and mutagenicity are contradictory, but neurotoxic, immunosuppressive and hepatotoxic effects have been defined. The aim of the present study was to investigate the cytotoxic effects of 2,4-D on a human hepatoma cell line. HepG2 cells were treated with different concentrations of 2,4-D, and cell viability, induction of apoptosis/necrosis and cell cycle phases were determined. Apoptosis was detected in flow cytometric light scatter histograms, the annexin V assay, the determination of DNA strand breaks with the TUNEL assay and the occurrence of a sub G(0) peak after propidium iodide (PI) staining. The induction of apoptosis by 2,4-D was accompanied by a disruption of the mitochondrial membrane potential as verified by staining with the cationic JC-1 probe. In addition, 2,4-D affected the cell cycle in a concentration-dependent manner. Our investigation suggested that 2,4-D exerts its cytotoxic effects by the induction of apoptosis via a direct effect on the mitochondrial membrane potential.

Phenotypic alterations and IL-1beta production in CD34(+) progenitor- and monocyte-derived dendritic cells after exposure to allergens: a comparative analysis.

Dendritic cells (DC) have been shown to capture and process antigens and play an initiating role in contact sensitization. Cells with dendritic morphology can be generated in vitro either from CD34(+) cord blood cells or from CD14(+) peripheral monocytes. The aim of this study was to determine the state of maturation/activation of both populations after exposure to several concentrations of four well-established model allergens (nickel sulfate, eugenol, alpha-hexylcinnamaldehyde and 2,4,6-trinitrobenzene sulfonic acid) or the irritant sodium dodecyl sulfate. We analyzed the surface expression of CD86, CD83 and HLA-DR and the production of IL-1beta. DC from the two sources were generated separately in two laboratories, but challenged using identical test protocols. Using both DC populations it was possible to detect the allergens under investigation, though minor differences regarding effective concentrations were noted. The non-responsiveness of CD34-DC to CIN was probably due to non-optimal concentrations. Ni(2+), known as a moderate allergen in vivo, showed the most prominent effect in both cell systems. CD86 expression was the most reliable phenotypic marker for the in vitro identification of allergens. Due to substantial individual variations it was difficult to draw any definite conclusions as to the relevance of IL-1beta production as an activation endpoint. We conclude that both test systems are able to respond to allergens, but CD34-DC must be exposed to higher concentrations to demonstrate significant phenotypic changes. On the other hand, Mo-DC from only some of the donors reacted to allergens, in contrast to CD34-DC, which responded to allergens irrespective of the donor, thus necessitating the use of Mo-DC cultures from several blood donors.

Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers.

Langerhans cells are the most potent antigen-presenting cells in the skin and play a critical role in the induction of contact allergy. Research on the phenotypical and functional changes of LCs occurring after application of skin sensitizers indicated their use as an in vitro model for the screening of chemicals. In the present investigations, LCs from human skin explants served as the test system. The application of this cell system has been aggravated by the difficulty in isolating sufficient numbers of live LCs from skin. This disadvantage was overcome by the culture of immature dendritic cells from peripheral mononuclear blood cells. These cells can serve as a replacement for LCs as they bind haptens and show phenotypical and functional changes similar to LCs. The sensitizers NiSO(4), dinitrochlorobenzene, 2,4,6-trinitrobenzene sulfonic acid, alpha-hexylcinnamaldehyde and eugenol were applied. Both the expression of surface markers and the induction of intracellular interleukin-1 beta (IL-1 beta) were analyzed. No clear-cut results could be established for intracellular cytokine production, only NiSO(4) induced a remarkable number of IL-1 beta-positive cells. However, all skin sensitizers caused an up-regulation of the co-stimulatory molecule CD86, of intercellular adhesion molecule CD54 and of the HLA-DR antigen. The irritant sodium dodecyl sulfate (SDS) and the vehicle dimethyl sulfoxide (DMSO) had no effect.

The expression of surface markers on dendritic cells as indicators for the sensitizing potential of chemicals.

Novel approaches to testing of skin sensitizing chemicals have made use of immature dendritic cells (DCs) cultured from different hematopoietic progenitors. These cells resemble Langerhans cells (LCs), which are the most potent antigen presenting cells in the skin. Former research has focused on the phenotypic and functional changes of LCs after application of skin sensitizers. But it has proven difficult to isolate sufficient numbers of LCs from skin. This disadvantage is overcome by cultures of immature DCs providing high numbers of reactive cells. The aim of the present investigation was to test the response of DC cultures established from different blood donors to known sensitizers, an irritant and a vehicle. The sensitizers NiSO(4), dinitrochlorobenzene (DNCB), 2,4,6 trinitrobenzene sulfonic acid (TNBS), alpha-hexylcinnamaldehyde (Cinn) and eugenol (Eu) induced the up-regulation of the co-stimulatory molecule CD86, of intercellular adhesion molecule CD54 and of the HLA-DR antigen. The irritant sodium dodecyl sulfate (SDS) and the vehicle dimethyl sulfoxide (DMSO) had no effect. A high rate of responders within blood donors was found for NiSO(4), TNBS, Cinn and Eu, while DNCB was less effective. The augmentation of surface marker expression in dendritic cells obtained from peripheral human blood seems to be a promising readout in prescreening for strong and moderate sensitizers. This test could thus help to reduce animal numbers for in vivo testing.

Occupational exposure to static, ELF, VF and VLF magnetic fields and immune parameters.

Despite the important role of the immune system in defending the body against infections and cancer, very few investigations have been undertaken to study possible effects of electromagnetic fields on human immunity. The aim of the present study was to examine the effect of occupational exposure on hospital personnel operating magnetic resonance tomographs and on industrial workers at induction heaters. In both categories of workplaces, magnetic flux densities exceeding Austrian exposure standards have been registered. Because of the complexity and high redundancy of the immune system, an extensive range of assay systems was applied: relative and absolute numbers of lymphocytic subsets were counted, the proliferative activity of T and B cells determined, the production of interleukin 2, interferon gamma and tumour necrosis factor alpha analysed, serum immunoglobulins evaluated, as well as non-specific immunity of monocytes and granulocytes measured by their oxidative burst. The number of natural killer cells and oxidative burst in monocytes showed statistically significant differences in workers at induction heaters and controls. The observed effect on oxidative burst was counteracted by a higher number of active cells in the exposed group, indicating normal non-specific immunity. The high number of natural killer cells, observed in some of the study subjects working at induction heaters, was reconfirmed in another investigation and deserves a further follow-up.

Occupational exposure to high frequency electromagnetic fields and its effect on human immune parameters.

The present study recorded a considerable excess of recommended exposure limits in the vicinity of shortwave diathermy devices used for medical treatment of patients. Different kinds of field probes were used to measure electric and magnetic field strength and the whole body exposure of medical personnel operating shortwave, decimeter wave and microwave units was calculated. To investigate the influence of chronic exposure on the immune system of operators, blood was sampled from physiotherapists working at the above mentioned devices. Eighteen exposed and thirteen control persons, matched by sex and age, were examined. Total leucocyte and lymphocyte counts were performed and leucocytic subpopulations determined by flow cytometry and monoclonal antibodies against surface antigens. In addition, to quantify subpopulations of immunocompetent cells, the activity of lymphocytes was measured. Lymphocytes were stimulated by mitogen phytohemagglutinin and their proliferation measured by a flow cytometric method. No statistically significant differences between the control and exposed persons were found. In both study groups all immune parameters were within normal ranges.

Immunological investigations in a group of workers exposed to various levels of polycyclic aromatic hydrocarbons.

To evaluate a possible immunotoxic effect of chronic exposure to polycyclic aromatic hydrocarbons (PAHs), several immune parameters were investigated in two groups of coke-oven workers. Exposure levels were different for both groups, one group being employed in a new coking facility of sophisticated technical standard, and the other in a facility with rather high emission of PAH-containing dust, built during the late 1960s. Immunomodulating effects of exposure were found on three levels of immunity: a reduced mitogenic response of T cells to phytohaemagglutinin observed by a reduced rate of DNA synthesis and a retardation in the expression of interleukin 2 receptor; an impairment of B cell activity, observed by a reduced proliferation of B cells and a low synthesis of immunoglobulin M after Staphylococcus aureus stimulation; a reduced oxidative burst in monocytes after challenge with E. coli. The differences in numbers of lymphocytic subpopulations in peripheral blood and in immunoglobulin levels of serum were statistically insignificant. The data of the present investigations point to significant, albeit slight, changes in immune function by PAH exposure. But these changes could not yet be related to health impacts of the exposure.

Investigations on immune parameters in welders.

The aim of the present investigation was to study the effects of welding fumes on the human immune system. Thirty male subjects who had regularly welded and 16 control persons without occupational exposure were examined. Cellular immunity was evaluated by phenotyping of peripheral leucocytes, measurement of mitogenic T cell response and T cell stimulation in a heterologous mixed lymphocyte reaction. Non-specific immune reactions were quantified by oxidative burst of granulocytes and monocytes and the cytotoxicity of lymphokine-activated killer (LAK) cells. Serum immunoglobulin levels and immunoglobulin production by stimulated B cells served to demonstrate humoral immune reactions. Welding fumes retarded the kinetics of DNA synthesis after phytohaemagglutinin stimulation of T cells and reduced the cytotoxic activity of LAK cells. No effects on lymphocytic subpopulations, mixed lymphocyte reaction, the phagocytosis of leucocytes or the production of immunoglobulins were observed. Several welders reported on recurrent respiratory infections or bronchitis, a few on allergic skin reactions and one worker was affected by asthmatic symptoms. With the exception of a reduced activity of LAK cells, these effects could not be related to any impairment of immune reactions as they were measured by the immunotoxicity tests applied.

Occupational exposure and its effect on some immune parameters.

Some immunological parameters were investigated in a group of workers exposed to external radiation (1.4 to 9.8 mSv) and inhalation of tritium (comm. eff. dose equiv. 1.2 to 2.8 mSv). The present investigations indicate the differential radiosensitivity of lymphocytic subsets: CD8 positive suppressor T cells were found to be the most sensitive subpopulation in the peripheral blood of radiation exposed workers. CD4/CD8 ratios were increased mainly due to an increase in absolute numbers of CD4 positive helper T cells indicating a selective cell renewal after irradiation. Results obtained after phytohaemugglutinin stimulation of lymphocytes showed individual variation, though there seems to be a trend towards an inverse correlation between absolute T cell numbers in peripheral blood and the number of S phases observed after stimulation, low T cell numbers leading to a high rate of stimulation. The calculation of the committed effective dose equivalents show that radiation protection against internal tritium contamination in power plants should not be neglected.

[Genetic and immunologic studies after space flight]. / Genetische und immunologische Untersuchungen nach einem Raumflug.

The effect of spaceflight on lymphocytes of the peripheral blood was investigated in the Austrian cosmonaut flying the Austro-Soviet mission Austromir. Blood was sampled before and after flight, and the following parameters studied: quantities of lymphocytic subsets (T cells, helper cells, suppressor cells, B cells and natural killer cells); mitogenic stimulation of lymphocytes--DNA (= deoxyribonucleic acid) synthesis and expression of Interleukin-2-receptor; structural modification of DNA, unscheduled DNA synthesis and sister chromatid exchanges. Besides reducing the number of natural killer cells, spaceflight caused on impairment of lymphocyte activity and a slight modification of DNA structure. 4 weeks after flight, control values were reestablished, indicating efficient repair mechanisms.

T-lymphocyte subsets in occupationally exposed persons.

The percentage of CD2+, CD4+, CD8+ and HNK-1+ cells in peripheral blood was investigated in persons occupationally exposed to very low doses of ionizing radiation. Investigations were carried out by monoclonal antibodies and flow cytometry. While significant effects of age and smoking habits on the relative number of CD8 cells and CD4/CD8 ratios could be established, no influence of the very low radiation exposure on the profile of lymphocytic T cells in blood was found, except a very slight effect on the relative number of CD2+ cells.

Sister chromatid exchanges (SCEs) in lymphocytes of persons working at Shlobin (USSR), 150 km north of Chernobyl.

Workers at a building plot in Shlobin (USSR), 150 km north of Chernobyl, were examined for the occurrence of spontaneous and mitomycin C induced SCEs in peripheral lymphocytes. Personnel being present during and after the Chernobyl accident exhibited a decreased number of inducible SCEs compared with test-persons starting work at Shlobin by 1st of June, 1986. Since effective dose equivalents were rather low (about 2 m Sv till the end of August 1986), induced SCEs proved to be a very sensitive test for the demonstration of low dose exposure.

Indications of an adaptive response in C57BL mice pre-exposed in vivo to low doses of ionizing radiation.

C57BL/6 mice were whole-body irradiated with 5 cGy/day ('adapting dose') on 4 consecutive days and their spleens removed on day 1, 3, 7, 12, 19 or 26 after the last irradiation. In vitro UV-light-induced unscheduled DNA synthesis (UDS) and mitomycin C (MMC)-induced sister-chromatid exchanges (SCEs) were scored in lymphocytes (UV-light and MMC being the 'challenging agents'), yielding higher UDS values and lower frequencies of induced SCEs than cells of non-adapted animals. On day 12 this effect could only be seen in half, on days 19 and 26 in none of the performed experiments. The results support those published by Tuschl et al. (1980, 1983) and Liu et al. (1987), showing that it is possible to induce the adaptive response in vivo.

SCE frequencies in lymphocytes of systemic lupus erythematosus patients.

The frequency of sister-chromatid exchanges (SCEs) was investigated in peripheral lymphocytes of lupus erythematosus patients and compared with values obtained for healthy controls. Irrespective of the kind of medical treatment, an increased level of spontaneously occurring SCEs could be demonstrated in lupus patients. In addition to spontaneously occurring SCEs, mitomycin C (MMC)-induced SCEs were evaluated. No difference between patients and controls was found with respect to MMC-induced SCEs.

Investigations on the effect of repeated hair dyeing on sister chromatid exchanges.

The numbers of sister chromatid exchanges (SCE) were determined in the lymphocytes of ten volunteers (males and females) whose hair was dyed 13 times at intervals of 3-5 wk. Each volunteer used throughout the study a single commercial preparation containing a mixture of aminotoluenes, aminophenols and hydroxybenzenes, and in some cases naphthol, as the active ingredients. The findings were compared with those in a control group matched for age and sex. SCE were determined in blood samples taken before the first exposure, after a sham dyeing and after the first three and the last three actual dyeing procedures. Volunteers were carefully screened for disease, for use of medicines and for radiation exposure. Consumption of alcohol was the same in both groups, but there were more smokers in the treated group. No evidence was found of any effect of repeated hair dyeing on the frequency of SCE. In both the controls and in the hair-dyed subjects a slight decrease in SCE was detected during the course of the experiment; this was independent of sex as well as of the dyeing procedure.

UDS and SCE in lymphocytes of persons occupationally exposed to low levels of ionizing radiation.

Unscheduled DNA synthesis induced by in vitro UV irradiation was investigated in lymphocytes of persons occupationally exposed to low doses of ionizing radiation (maximum registered radiation dose: 98 mrad/month). For radiation exposures greater than 14 mrad/month above background level, increased rates of UDS after in vitro UV irradiation of lymphocytes were found. The bromodeoxyuridine differential chromatid labeling technique was applied to the examination of spontaneous and Mitomycin C (MMC)-induced sister chromatid exchanges (SCE) in the same population. No statistically significant difference could be determined in spontaneously occurring SCEs, while MMC induced SCEs were significantly reduced in persons exposed to radiation doses greater than 14 mrad/month, thus indicating increased repair capability for DNA lesions inflicted by a second insult after protracted low-dose irradiation.