Modulation of phenotype, cytokine production and stimulatory function of CD34+-derived DC by NiCl(2) and SDS.

One of the most promising alternatives to identify the sensitizing potency of new products is the in vitro culture of human dendritic cells (DC). In vivo, dendritic cells present in the skin are highly specialized antigen-presenting cells (APC) of the immune system, which play a crucial role in the induction of allergic reactions. The DC produce specific cytokines and upregulate specific co-stimulatory molecules in addition to the antigen-MHC complex in order to promote an optimal T-cell activation. The aim of our study is to assess the phenotype, cytokine production and autologous T-cell stimulatory capacity of the in vitro CD34+-derived dendritic cells after 24 hours of incubation with the metal allergen NiCl(2) (100-300 microM) and the irritant sodium dodecyl sulfate (SDS; 0.01%), in order to find a sensitive endpoint to discriminate between sensitizers and irritants. After exposure to Ni, a significant increase in the number of CD83+ and CD86+ cells was noticed. The intensity of CD86 as well as the intensity of the HLA-DR molecule on the DC also showed a significant increase. The expression of the co-stimulatory molecule CD80 was not changed after exposure. SDS was not able to increase the expression of any of the analysed markers. The production of IL-6 increased significantly after exposure of dendritic cells to Ni, but not after SDS exposure. Results on tumor necrosis factor-alpha (TNF-alpha) production are somewhat equivocal. Although not statistically significant, TNF-alpha was upregulated in one out of three experiments after 48 hours of exposure to the Ni allergen, but increases were also noticed after exposure to SDS (two out of three experiments). Both exposure to Ni and SDS caused an upregulation (not significant) in the IL-12 production by DC, but the production was higher in Ni-exposed DC compared to SDS-exposed cells. In none of the exposed DC cultures was it possible to detect IL-1 beta. The antigen-presenting capacity of the DC in autologous MLR could not be demonstrated. Nevertheless, T-cell proliferation after DC stimulation was noticed in allogenic MLR.

Use of in vitro assays to assess hematotoxic effects of environmental compounds.

The number of chemicals being introduced into the environment increases and many of these substances may pose a health risk to exposed individuals. Many environmental toxicants with a potential toxicity to the hematopoietic system have been identified by animal experiments. Owing to the risks of severe chronic hematopoietic disorders, it is important to screen chemicals for their hematotoxicity. The aim of this work was to identify, through the use of in vitro techniques, targets for hematotoxic effects. Our study focused on myeloid and erythroid hematopoietic progenitors and stromal stem cells as possible targets. The in vitro assays showed that various hematotoxic compounds exert different effects on these cell populations. In vitro exposure of murine bone marrow cells to various inorganic (cadmium, lead) and organic (benzene metabolites, lindane. benzo-[a]-pyrene (BaP), PCB (polychlorinated biphenyl) congeners) environmental chemicals indicated that hematopoietic or stromal bone marrow cells were targets for most of the chemicals. Stromal cells were more affected by lead, cadmium, and BaP compared to myeloid cells. Benzene and phenol gave no response, but the metabolites catechol and hydroquinone were equally toxic to the stromal and the myeloid progenitor cells. Among the PCBs tested, PCB126 was most toxic. Human progenitor cells derived from cord blood were exposed in vitro to catechol, hydroquinone, lead nitrate, and PCBs. Human hematopoietic cells were sensitive to the tested compounds. Human erythroid progenitors are more susceptible to lead exposure than are myeloid progenitors. Based on the in vitro tests, humans are more sensitive to lead, catechol, and PCB126 than are mice. In contrast to the murine data, humans responded with individual differences to lead and PCB126.

Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells.

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations.

Effects of in vitro alpha-particle irradiation on osteogenic bone marrow cultures.

Murine bone marrow contains osteogenic precursor cells that undergo differentiation during in vitro cultivation. In vitro these cells are potential target cells for alpha-irradiation-induced bone tumour formation. Under defined tissue culture conditions these differentiating cells were directly exposed to alpha-particle irradiation from the radon daughter 210Po. Po deposits in soft tissue and it was shown to be associated with marrow cells and with the extracellular marrow tissue formed in vitro. These differentiating marrow cultures showed high sensitivity to alpha-irradiation. Cell death was observed at 210Po concentrations in tissue culture medium (TCM) > 7 Bq 210Po/ml. At lower concentrations (between 1 and 5 Bq 210Po per ml TCM) proliferation was enhanced as measured by uptake of 3H-thymidine, also differentiation was stimulated as measured by alkaline phosphatase activity and incorporation of 3H-proline in newly synthesized collagen. At several times of culture, the association of 210Po with the extracellular matrix and cells was measured. These retention data enabled us to calculate the daily alpha-particle fluence. At 1 Bq 210Po present per ml tissue culture, a daily alpha-particle fluence as low as 3-6 per 1000 cells seemed very efficient in changing the expression of osteogenic differentiation of marrow cells.

High radiosensitivity of the mineralization capacity of adult murine bone marrow in vitro to continuous alpha-irradiation compared to acute X-irradiation.

Adult BALB/c mice, injected with osteosarcomogenic amounts of 241Am (between 40 and 500 Bq/g mouse) showed an impaired mineralization capacity of their femoral bone marrow. This effect persisted until at least 1 year after 241Am injection and was expressed after incubation of bone marrow cells in vitro in conditions allowing osteogenic differentiation. The mineralization capacity of marrow in vitro was evaluated by measurement of 85Sr uptake from the tissue culture medium. Two osteogenic assays were used: in marrow cultured as an intact organ (marrow organ cultures), reduced mineralization was observed in mice given 149 Bq 241Am/g mouse or more (skeletal dose rate of 25 mGy/day), in stromal marrow cells cultured from adherent cell layers and subsequently brought into a three-dimensional (3D) mineralizing condition (stromal 3D cultures), reduced 85Sr uptake was observed from the lowest dose level tested (42 Bq 241Am/g mouse, skeletal dose rate of 7 mGy/day). Taking into account that only a fraction of the skeletal alpha-dose reached the marrow of the femoral diaphyses, marrow organ cultures and stromal 3D cultures exhibited high radiosensitivity to alpha-irradiation in vivo. However, after acute X-irradiation of marrow in vivo or in vitro prior to initiation of the marrow organ cultures, X-ray doses of 4 Gy or higher were needed to significantly impair the mineralization capacity of marrow organ cultures in vitro. Our data demonstrated that the osteogenic cells from the bone marrow are subjected to long-term damage after low doses of continuous alpha-irradiation in vivo.

Bone marrow from Balb/c mice radiocontaminated with 241Am in utero shows a deficient in vitro haemopoiesis.

Radiation damage from 241Am to bone marrow cells was manifest in long-term bone marrow cultures (LTC) from offspring of mice radiocontaminated at the 14th day of gestation (119, 479, 803, 1754 kBq 241Am/kg). Offspring were reared by their own contaminated mother for 3 weeks postnatal. LTC from these offspring were less able to support in vitro CFC proliferation than control LTC from non-contaminated offspring. This radiation damage persisted 71 weeks after radiocontamination in utero. Using this in vitro culture system, damage was observed at lower doses if 241Am contamination occurred at foetal than at adult ages. Radiation damage was observed only using LTC, while the haemopoietic stem cell concentration (CFU-S, in vitro CFC) and the stromal stem cell concentration (CFU-F) from marrow in situ were not impaired after 241Am radiocontamination in utero. After culturing LTC in 25 per cent FCS and recharging the stromal adherent layer with bone marrow cell suspensions originating either from control offspring or from offspring contaminated with 241Am in utero, some evidence was found that the proliferation capacity of the haemopoietic cells was diminished. However, the nature of effects on the stromal elements is currently somewhat equivocal. Following in utero contamination the stromal adherent cells appeared to support better the production of in vitro CFC.

Haemopoiesis in long-term cultures of liver, spleen and bone marrow of pre- and postnatal mice: CFU-GM production.

The CFU-GM yield in confluent long-term cultures (LTC) derived from liver, spleen and bone marrow cells at different gestational and postnatal ages has been studied after the stromal adherent layer reached confluency. The stromal cell compartment of fetal and neonatal haemopoietic organs is able to sustain haemopoiesis in vitro. Moreover, the granulocyte-macrophage stem cell (CFU-GM) yield of these LTC reflects the CFU-GM content of the haemopoietic organs from which the cultures are originated. LTC from the liver produce high numbers (between 100 and 150 CFU-GM per well) of CFU-GM if the cultures are derived from fetal livers between 13 d of gestation and birth. Cultures from spleens just before and after birth, give maximal CFU-GM numbers (between 50 and 100 CFU-GM per well). The CFU-GM yield in long-term bone marrow cultures increases 10 times from 17-day-old fetus towards adult life (between 700 and 1000 CFU-GM per well.

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice.

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.

Stromal stem cells (CFU-f) in yolk sac, liver, spleen and bone marrow of pre- and postnatal mice.

Our results demonstrate that in yolk sac, liver, spleen and femoral bone marrow of mice at ages ranging between 11 d of gestation and adult life, important changes in the stromal stem cell population (CFU-f assay) occur which are correlated with haemopoiesis. In the liver, spleen and bone marrow, high numbers of CFU-f precede high haemopoietic stem cell values. As haemopoiesis starts in the spleen, CFU-f numbers in fetal liver are low. Similarly, CFU-f numbers decrease in the spleen as bone marrow haemopoiesis starts. This suggests the existence of a migration stream of stromal stem cells. In spleen and bone marrow, CFU-f numbers decrease towards adult life as these organs maintain a stable haemopoietic activity.