Ben C. Hulette

Approaches for the development of cell-based in vitro methods for contact sensitization

Allergic contact dermatitis (ACD) is a cell-mediated immune response to small molecular weight chemicals that contact and penetrate the skin. There are a variety of characteristics that determine whether a chemical can function as a contact sensitizer (or allergen) including the ability to penetrate into the skin, react with protein and be recognized as antigenic by immune cells. The ultimate challenge for developing non-animal test methods for skin sensitization testing will be applying our mechanistic understanding of ACD to the design of predictive in vitro alternative test methods. Specifically, the in vitro approach should be designed so that a chemicals potential to penetrate the skin, react with protein/peptide (biotransformation may be required) and initiate an antigen-specific immune response is incorporated in the test methods developed. In this review, we have focused on cellular-based assays that have been developed or proposed for assessing a chemicals skin sensitization potential in vitro. All of the promising leads to date are based on observations made from in vivo studies conducted in animals and humans, and therefore have a strong mechanistic foundation. However, it remains to be demonstrated whether a single in vitro test, or several in vitro tests in combination, which model the critical steps in sensitization, can replace animal experiments for predicting contact allergic reactions in humans. Regardless, the future looks promising with continued development of our understanding of the chemical and biological aspects of allergic contact dermatitis, and most importantly, with the application of genomics/proteomics to this field on the immediate horizon.

Cytokine induction of a human acute myelogenous leukemia cell line (KG-1) to a CD1a+ dendritic cell phenotype.

Abstract Dendritic cells (DC) are highly specialized antigen-presenting cells located in many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC as antigen-presenting cells play a critical role in the induction of allergic contact dermatitis. LC research is difficult because few LCs can be isolated from human skin, so efforts have focused on obtaining DCs from alternative sources. Mononuclear cells from peripheral blood and CD34 + stem cells from human cord blood and marrow can be induced to form phenotypic and functional DCs, but experiments of this type are expensive and the DC yield is low. We report here the induction of the myeloid leukemia cell line (KG-1) to a DC morphology and phenotype by culturing the cells in a defined cytokine cocktail. Morphologically, the KG-1-derived DCs are large irregularly shaped cells with prominent dendritic processes and hair-like cytoplasmic projections. Phenotypically, the KG-1-derived DCs lack lineage-specific markers, and express MHC class II, costimulatory molecules CD80 and CD86, and CD83. Functionally, KG-1-derived DCs are capable of phagocytosing latex microspheres and are able to induce a potent allogeneic T-cell response. Within the KG-1-derived DCs, a subpopulation maintains the DC phenotype and morphology described above but further develops CD1a + marker expression similar to that of resident skin-derived LCs. These findings illustrate that phenotypic, morphologic and functional DCs can be derived from the KG-1 cell line.

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

Examination of Phenotypic Changes in Peripheral Blood‐Derived Dendritic Cells Following Exposure to a Contact Allergen: Cell Surface Marker and Gene Expression

The evaluation of the potential to induce allergic contact dermatitis is an important component of the overall safety assessment process of a new chemical entity that may be encountered through the skin. Currently, the murine local lymph node assay (LLNA) is the method of choice for assessing skin sensitization potential. However, despite the success of the LLNA as an alternative test method that results in a reduction in and refinement of animal usage, it does require the continued use of animals. Therefore, the development of an in vitro predictive test method for skin sensitization is still necessary. A number of different approaches have been taken to develop such a method including the examination of changes in cell surface marker expression in cultured human dendritic cells (DC). The purpose of this current study was to examine the effect of allergen exposure on peripheral blood mononuclear cell (PBMC)‐derived DC using a panel of cell surface markers known to be important in the development of allergic contact dermatitis; CD86, CD80, CD83, CD54, CD40, and CD1a. Changes in the expression of these markers were measured at the level of transcription and cell surface expression. The DC derived from the adherent cell fraction of human PBMC were exposed to either 1 mM or 5 mM dinitrobenzene sulfonic acid (DNBS), the water‐soluble analog of the strong contact allergen 2,4‐dinitrochlorobenzene for 24 hr. After treatment, surface markers expression was measured by flow cytometry, and total RNA was obtained for microarray analysis using Affymetrix U95Av2 Genechips®. Increases in both the cell surface expression and transcript levels of CD86 were observed for PBMC‐DC exposed to DNBS, with 5 mM DNBS inducing the greater change. Cell surface expression of CD80 and CD54 was similar for 1 mM DNBS treated and control cells, and no significant changes were seen at the transcript level. Treatment with 5 mM DNBS resulted in a decrease in cell surface expression for both of these markers. Although a significant allergen‐induced decrease in the transcript level for CD54 was noted, CD80 transcripts were increased. No marked effects on CD83 cell surface or gene expression were observed for either the 1 mM or 5 mM DNBS‐treated cells. Cell surface expression of CD40 and CD1a was somewhat variable in DC preparations treated with 1 mM DNBS, and no significant changes in transcript levels were induced. However, treatment with 5 mM DNBS induced a more marked decrease in cell surface expression of CD40 and CD1a and a significant decrease in the transcript level for CD1a. In summary, we found that changes in cell surface expression in PBMC‐DC following 24‐hr exposure to a contact allergen were reflective of the transcript levels for each of the markers examined with the exception of CD80 in cells treated with 5 mM DNBS.

Transcript Profiling of T Lymphocytes and Dendritic Cells in a Co–culture System Using Anti‐CD3 and Allergen Activation

The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water soluble analog of DNCB), or media alone for 15 minutes. Total RNA was prepared and changes in gene expression were analyzed using Affymetrix U95Av2 GeneChips®. Comparative analysis of Affymetrix mean signal values from triplicate control cultures with those from anti‐CD3‐treated samples revealed highly significant (p ≤ 0.001) changes in the expression of 344 transcripts of the total of approximately 12,000 represented on the chip. However, mean signal values for T cells cocultured with allergen‐treated DC compared to vehicle‐treated DC‐T cell co–cultures identified only 17 significant gene changes (p ≤ 0.001), 11 of which were also identified as having changed significantly in response to stimulation with anti‐CD3. In parallel assays, antigen‐specific T cell proliferative responses were assessed as a function of tritiated thymidine incorporation. Increased T cell proliferative responses were observed in the cultures that contained both DNBS‐treated DC and T cells as well as the anti‐CD3 treated cultures compared with their respective controls. These data suggest that this approach can be used to identify genes that might serve as indicators of contact allergy and may be used in an in vitro predictive assay for skin sensitization.