In vitro assessment of sensitizing activity of low molecular weight compounds.

Predictive tests to identify the sensitizing properties of chemicals are carried out using animals. There is as yet, no accepted in vitro method for the identification of skin sensitizing chemicals. Such in vitro tests should encompass (parts of) the sensitization phase of contact hypersensitivity. Two cell types are predominantly involved in this process, keratinocytes (KC) and Langerhans cells, the latter being a specialized type of skin dendritic cells (DC). Low molecular weight chemicals act as haptens; KC respond to allergen contact by, among others, producing proinflammatory cytokines, while DC take up the haptenized protein, migrate, and present antigen to T-cells. During migration, DC mature, resulting in a loss of antigen uptake capacity, thereby increasing expression of certain surface molecules. Thus, both cytokine production by KC and surface marker expression by DC may be used as in vitro models for the identification of sensitizers. Several reports have shown that intracellular IL-1 alpha is a promising candidate to identify sensitizers using KC. We have recently shown that the potency of sensitizers may be determined by dose-response analysis of intracellular IL-1 alpha and IL-18 using a murine KC cell line. The ranking of potency using this in vitro method was similar to the ranking previously established using the local lymph node assay. Using DC, effects on the expression of various cell surface markers, cytokines, and molecules involved in antigen uptake have been shown to identify sensitizers. One study showed that also the potency of sensitizers may be determined using DC. Additional studies are required to establish whether KC or DC, or combinations thereof, are most suitable for in vitro identification and potency assessment of sensitizers.

Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1alpha and IL-18 production by a murine and human keratinocyte cell line.

Assessment of allergenic potency of low molecular weight compounds is generally performed using animal models, such as the guinea pig maximisation test and the murine local lymph node assay (LLNA). Progress in unravelling the mechanisms of skin sensitisation, including effects on the production of cytokines by the different cell types of the skin, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitisation testing. The aim of the present study was to establish an in vitro method to assess the potency of allergens, on the basis of their induction of cytokine production by murine and human keratinocytes. In the present study we used test systems comprised of the murine epidermal keratinocyte cell line HEL-30 and the human keratinocyte cell line HaCaT. We exposed these cell lines to the allergens ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), and phthalic anhydride (PA). IL-1alpha and IL-18 dose-response data were evaluated by non-linear regression analysis and at a stimulation index of 3 of cytokine production of treatment versus control, the corresponding allergen concentration was calculated. For HEL-30, for both cytokines DNCB showed the strongest potency followed in this order by PA, benzocaine, and DEA. This classification was similar to our previous findings obtained in the LLNA. For HaCaT, unfortunately, such ranking proved to be much less feasible. In conclusion, to assess the potency of allergens the murine keratinocyte cell line HEL-30 may be a useful in vitro test system, alternative to in vivo models, although this requires further testing using a much wider range of compounds.

Effect of prolonged exposure to low antigen concentration for sensitization.

The local lymph node assay (LLNA) is an assay in mice to identify potential allergens. Compounds that do not induce a stimulation index (SI)>or=3 are not considered sensitizers. Of the chemicals that do, the SI of 3 is used as a benchmark, and indicates the sensitizing potency of a chemical. Compared to the exposure duration of the LLNA (3 days), real life exposure often lasts for months or years. We therefore investigated whether prolonged exposure to sensitizers at concentrations that do not induce a SI>or=3 in the LLNA, were able to surpass this threshold. Mice were treated for 2 months at 7-day intervals with a range of concentrations of the known allergens ethyl-p-aminobenzoate (benzocaine, BENZ), 2,4-dinitrochlorobenzene (DNCB), and tetramethyl thiuram disulfide (TMTD). Both proliferative activity and cytokine production were established at day 60. Neither BENZ nor TMTD showed a significant increase in the proliferation rate compared to vehicle controls. Only DNCB at concentrations originally above the EC(3) a significant increase in proliferation was seen after prolonged exposure. No significant effect on IFN-gamma and IL-4 production was observed for all three compounds compared. These findings indicate that for classification of sensitizers the shorter exposure period employed in the standard LLNA is sufficient, and longer periods of exposure have no bearing on this classification.

Cytokine production induced by low-molecular-weight chemicals as a function of the stimulation index in a modified local lymph node assay: an approach to discriminate contact sensitizers from respiratory sensitizers.

In general, contact sensitizers have been shown to selectively induce Th1 immune responses, such as interferon-gamma (IFN-gamma) production, whereas Th2 responses, such as interleukin (IL)-4 production, were seen after exposure to respiratory allergens. However, these features may be dependent on the dose of the particular allergen. Therefore, the aim of the present study was to investigate the distinction between contact sensitizers and respiratory allergens, by establishing dose-dependent cytokine profiles. The contact allergens 2,4- dinitrochlorobenzene (DNCB), hexyl cinnamic aldehyde (HCA), and oxazolone (OXA, 4-ethoxymethylene 2-phenyloxazol-5-one) as well as the respiratory allergens fluorescein 5-isothiocyanate, phthalic anhydride, toluene diisocyanate, and trimellitic anhydride were tested. For a range of concentrations, both proliferative responses and cytokine production were established. Estimated concentrations were derived at several stimulation indices (SIs). From the estimated concentrations, IFN-gamma, IL-4, and IL-10 production as a function of stimulation indices were plotted. All four respiratory allergens showed significantly higher IL-4 and IL-10 production patterns compared to the contact allergens. Positive identification of DNCB, HCA, and OXA as contact allergens on the basis of IFN-gamma production was observed only at very high stimulation indices (SI >or= 35) for DNCB and OXA and at low SIs (SI

Ranking of allergenic potency of rubber chemicals in a modified local lymph node assay.

A modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5-95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy induction.