In vitro approaches to pharmacological screening in the field of atopic dermatitis.

In vitro models are valuable for evaluating potential active ingredients and other molecules used in medications for atopic dermatitis (AD). However, finding appropriate in vitro models can be problematic. Our strategy was to set up different in vitro models that would mimic the pathomechanisms of AD. We describe five such models - the AD keratinocyte model, the AD reconstructed human epidermis model, the adaptive immunity model, the innate immunity model and the pruritus model - which we have used to evaluate a new ingredient for emollients derived from a biological extract. The models chosen provide useful data for the pharmacological characterization of active ingredients in adjunctive treatments for AD.

Expression of NADPH oxidase is induced by all-trans retinoic acid but not by phorbol myristate acetate and 1,25 dihydroxyvitamin D3 in the human promyelocytic cell line NB4.

Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O2- producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O2- after 2 days of differentiation, they remain unable to generate O2- when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O2- in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.

NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils.

In addition to the extracellular production of O2- by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b559, the redox component of the NADPH oxidase complex, is mainly associated with specific granule membrane in resting neutrophils. We examined whether these granules could be a site for intracellular production of O2-. Phorbol myristate acetate (PMA)-stimulated neutrophils were fractionated by differential centrifugation, and generation of O2- was detected in both the granule and the plasma membrane-enriched fractions, but more in the granules. Translocation of p47phox and p67phox, two cytosolic components of the NADPH oxidase, was also quantitatively more important in the granules than in the plasma membrane fraction. After separation of the specific from the azurophil granules, p47phox and p67phox were found to be present only in the specific granules of PMA-activated cells. As a control, the production of O2- was studied in retinoic acid-differentiated NB4 cells that lack specific granules. During stimulation of NB4 cells with PMA, only the plasma membrane-enriched fraction was the site of O2- production. Together, these results indicate that NADPH oxidase can be functionally assembled in specific granules.