Interactions of Contact Allergens with Dendritic Cells: Opportunities and Challenges for the Development of Novel Approaches to Hazard Assessment

Cindy A. Ryan*,1, G. Frank Gerberick*, Lucy A. Gildea*, Ben C. Hulette*, Catherine J. Betts{dagger}, Marie Cumberbatch{dagger}, Rebecca J. Dearman{dagger} and Ian Kimber{dagger}

* Miami Valley Innovation Center, Central Product Safety Department, The Procter & Gamble Company, Cincinnati, Ohio 45253, and {dagger} Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK

1 To whom correspondence should be addressed at The Procter & Gamble Company, Miami Valley Innovation Center, P.O. Box 538707, Cincinnati, OH 45253–8707. Fax: (513) 627-0400. E-mail: ryan.ca{at}pg.com.

Received May 10, 2005; accepted June 22, 2005


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The identification of potential skin sensitizing chemicals is a key step in the overall skin safety risk assessment process. Traditionally, predictive testing has been conducted in guinea pigs. More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential. However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop nonanimal test methods for skin sensitization. Mechanistic understanding of allergic contact dermatitis has increased substantially in recent years. For example, a number of changes are known to occur in epidermal Langerhans cells, the principal antigen-presenting dendritic cell in the skin, as a result of exposure to chemical allergens, including the internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis, the induction of tyrosine phosphorylation, the modulation of cell surface markers, and cytokine expression. The application of this knowledge to the design of predictive in vitro alternative tests provides both unique opportunities and challenges. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells and the potential use of that information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.

Key Words: contact allergy; dendritic cells; in vitro method.


    SKIN SENSITIZATION TESTING: CURRENT STATE OF THE ART
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Allergic contact dermatitis (ACD) resulting from skin sensitization is a common occupational and environmental health problem. Therefore, prior to the market introduction of new products or product ingredients that contact the skin, it is necessary to conduct a thorough skin sensitization risk assessment to assure the product/ingredient will not induce ACD. The first step in this process is generally a determination of the skin sensitization hazard of the chemical of interest. Traditionally, sensitization hazard tests have been conducted in guinea pigs (Buehler, 1965Go; Magnusson and Kligman, 1969Go). More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential (Gerberick et al., 2000Go; ICCVAM, 1999Go; OECD, 2002Go). While still an in vivo test, the LLNA provides an alternative method for identifying skin-sensitizing chemicals that permits a reduction in the number of animals required as compared to guinea pig tests and offers a substantial refinement in the way in which the animals are used (Gerberick et al., 2000Go).

However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop nonanimal test methods for skin sensitization. The mechanistic understanding of ACD has increased substantially in recent years; the challenge is to apply this knowledge to the design of predictive in vitro alternative tests. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells (DC) and the potential application of such information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.


    IN VITRO TESTING FOR CONTACT SENSITIZATION: A NEW OPPORTUNITY IS RECOGNIZED
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Given the importance of epidermal Langerhans cells (LC) in the initiation of skin sensitization (Cumberbatch et al., 2003Go), it seems appropriate to explore whether there are opportunities to develop alternative approaches to hazard identification based upon chemical-induced changes in phenotype or function of these cells. The first indication that this could be a useful strategy came from work conducted by Enk and Katz (1992)Go characterizing changes in epidermal cytokine gene expression induced by topical exposure of mice to contact allergens or contact irritants. The salient observation was that some cytokines appeared to be induced or up-regulated only by skin sensitizers; one such cytokine was interleukin (IL)-1ß, which in mouse epidermis is produced exclusively by LC (Enk et al., 1993Go). Taken together with other data demonstrating that that IL-1ß plays an essential role for the induction of allergic contact dermatitis in the skin (Cumberbatch et al., 1997aGo,bGo; Shornick et al., 1996Go), it seems reasonable to suggest that measurement of induced IL-1ß expression could provide an alternative approach for assessing the sensitizing activity of chemicals. In April of 1995, the European Centre for the Validation of Alternative Methods (ECVAM) organized a workshop to review the then current state of the art of nonanimal methods for skin sensitization and to develop recommendations for approaches toward obtaining replacement alternatives (de Silva et al., 1996Go). As described above, one biological test system identified at the workshop as holding promise as a potential replacement was the production of IL-1ß by human LC-like DC.


    THE IMMUNOBIOLOGICAL ROLE OF DENDRITIC CELLS IN THE ACQUISITION OF CONTACT SENSITIZATION
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DC are a distinct group of leukocytes characterized by their unique morphology and their ability to initiate immune responses by processing and presenting antigens. DC are widely distributed throughout the body. Subpopulations of DC reside both in the epidermis and dermis of normal skin (Shortman and Liu, 2002Go). Langerhans cells (LC), the principal DC residing in the epidermis, typify the sentinel role of immature DC (Banchereau and Steinman, 1998Go). Located near the dermal–epidermal junction, the LC form a network designed to "trap" foreign antigens that have entered the skin, including chemical allergens.

Skin-sensitizing chemicals, typically lipophilic and low-molecular-weight (<500 kDa) molecules, act as haptens and need to bind to protein to form a hapten–protein complex in order to be recognized by LC (Dupuis and Benezra, 1982Go; Landsteiner and Jacobs, 1936Go). Following encounter with a chemical allergen, LC become activated and subsequently migrate from the skin to the draining lymph nodes. This migration is stimulated by changes in the cytokine microenvironment, including up-regulated expression of tumor necrosis factor (TNF)-{alpha} by epidermal keratinocytes and IL-1ß by LC (Cumberbatch et al., 1997aGo,bGo). During transit from the skin to the lymph nodes, LC undergo maturation and differentiate from antigen-capture and processing cells to potent immunostimulatory DC, able to present antigen effectively to responsive T cells. Among the changes reported to occur in LC as a result of exposure to chemical allergens are internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis (Becker et al., 1992Go; Girolomoni et al., 1990Go), induction of tyrosine phosphorylation (Kühn et al., 1998Go), the modulation of cell surface markers (Aiba and Katz, 1990Go; Verrier et al., 1999Go) and cytokine expression (Enk and Katz, 1992Go).


    IN VITRO GENERATION OF LANGERHANS CELL–LIKE DENDRITIC CELLS
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Given the importance of epidermal LC in the initiation of skin sensitization, an experimental approach for identifying allergens based upon their effects on LC appears scientifically sound. However, from a practical standpoint, obtaining sufficient numbers of these cells has been a limiting factor in the development of LC-based in vitro methods. LC constitute only 1–3% of all epidermal cells, and while many isolation techniques have been developed to obtain purified populations of these cells (Hanau et al., 1988Go; Teunissen et al., 1988Go), the cell yields are relatively low. Furthermore, following isolation LC are subject to rapid changes in phenotype such that they may no longer be representative of in situ LC. However, the discovery of methods for the generation of LC-like DC from other tissue sources has made this approach for the development of in vitro tests for identifying contact allergens more feasible. LC-like DC have been generated from CD34+ precursors obtained from bone marrow (Inaba et al., 1992Go), neonatal cord blood (Caux et al., 1996Go), and peripheral circulation (Strunk et al., 1996Go). However, bone marrow and cord blood samples are difficult to obtain, and CD34+ cells are rare in adult blood, so methods have also been developed to derive DC from peripheral blood mononuclear cells (PBMC) (Bender et al., 1996Go; Romani et al., 1996Go). These culture techniques provide sufficient quantities of LC-like antigen-presenting cells for the characterization of the effects of allergen exposure and permit exploration of their utility as an in vitro model system for the development of predictive tests for skin sensitization.


    FIRST-GENERATION APPROACHES USING DENDRITIC CELLS: CHANGES IN PHENOTYPE AND CYTOKINE EXPRESSION
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One of the first attempts to translate the in vivo observation of the relationship between IL-1ß mRNA expression in LC and allergen exposure into an in vitro system was by Reutter et al. (1997)Go. They investigated changes in IL-1ß gene expression in PBMC-derived DC following culture for 30 min in the presence of five contact sensitizers and one irritant using reverse transcriptase-polymerase chain reaction (RT-PCR). They reported that all contact sensitizers increased IL-1ß gene expression, whereas similar treatment with an irritant had no significant effect. However, the allergen-induced increases in expression were rather modest, ranging from approximately 1.5- to 3.5-fold. Pichowski et al. (2000)Go confirmed that potent contact sensitizers such as dinitrofluorobenzene (DNFB) could selectively up-regulate IL-1ß mRNA expression in PBMC-DC, but found that this increase was donor dependent. In vitro exposure to DNFB induced increases in IL-1ß mRNA expression in PBMC-derived DC from four out of nine donors, whereas exposure to the irritant SDS did not increase IL-1ß mRNA levels, even in DC derived from individuals that responded to DNFB. To determine whether or not this interindividual variability in IL-1ß mRNA expression was allergen specific, Pichowski et al. (2001)Go evaluated responses to additional contact allergens, paraphenylenediamine (PPD) and methylchloroisothiazolinone/methylisothiazolinone (CMIT), and to a second irritant, benzalkonium chloride. PBMC-derived DC from donors that had demonstrated increased IL-1ß mRNA expression in response to DNFB also responded to PPD and CMIT by up-regulating levels of IL-1ß mRNA. However, the changes in IL-1ß expression in this and the previous studies were modest, with no more than a two- or three-fold increase over vehicle control mRNA levels observed. PBMC-derived DC isolated from donors who failed to respond to DNFB also did not respond to treatment with either PPD or CMIT. Exposure to benzalkonium chloride did not induce changes in IL-1ß mRNA expression in DC from any donors. Based on the interindividual variability in response to allergen treatment and the modest changes observed in responding donors even to potent allergens, it was concluded that IL-1ß mRNA expression was of limited utility as predictive endpoint.

The potential for changes in expression by DC of mRNA for other cytokines or chemokines to serve as useful markers for sensitization testing has recently been explored by Verheyen et al. (2005)Go. Following exposure of CD34+-progenitor derived DC to allergens or irritants, mRNA expression for IL-1ß, IL-6 and IL-8, and the chemokines CCL2, CCL3, CCL3L1, and CCL4 was examined by real-time RT-PCR. Significant interindividual variations in mRNA expression in response to chemical treatment were observed. Based on their results, the authors concluded, as did Pichowski et al. (2001)Go, that allergen-induced IL-1ß mRNA expression in DC was not an appropriate indicator of sensitizing potential. Neither IL-6 nor IL-8 was able to discriminate clearly allergens from irritants. However, at the 24-h time point, mRNA levels for CCL2, CCL3, and CCL4 displayed a two-fold or greater increase relative to control for the allergens, but not for the irritants. The authors suggest that further investigation of these chemokine genes is warranted.

In addition to the up-regulation of cytokine or chemokine mRNA expression, a number of other changes known to occur in LC following allergen exposure have also been investigated in DC as potential markers for the assessment of skin sensitization potential in vitro. Phenotypic alterations in DC induced by hapten treatment have been explored as a possible method for identifying potential contact allergens. The cell surface markers that have received the most attention for this purpose are MHC Class II molecule HLA-DR, the costimulatory molecules CD86 and CD80, the adhesion molecule CD54 (intercellular adhesion molecule (ICAM)-1), and CD83, a marker of mature DC, and CD40, a molecule known to play a role in several DC functions. Early investigations conducted by Degwert et al. (1997)Go found that incubation of monocyte-derived DC with subtoxic concentrations of contact sensitizers for periods of less than 3 h resulted in decreased HLA-DR expression, whereas incubation with irritants for the same length of time was without effect. There were no allergen- or irritant-specific effects on CD54 expression. Using LC-like DC generated from CD34+ cord blood cells, Rougier et al. (2000)Go consistently observed increased expression of HLA-DR, CD83, and CD86, and decreased levels of E-cadherin following treatment with a strong allergen but not with an irritant. However, when weaker allergens were tested, changes in CD86 expression were relatively robust, whereas changes in HLA-DR or CD83 were observed only in a limited number of subjects (one or two out of eight). Other investigators have reported that 48-h treatment of PBMC-derived DC with strong contact allergens induced only modest up-regulation of HLA-DR in three out of five donors and no significant changes in CD86, CD54, or CD80 expression (Hulette et al., 2002Go). Staquet et al. (2004)Go examined changes in expression of a panel of cell surface markers including HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7, and E-cadherin and intracellular HLA-DR on monocyte-derived DC following exposure to noncytotoxic concentrations of allergens of varying potencies and of irritants. They found that prolonging the exposure time from 2 to 4 days increased the number of markers impacted by most of the allergens tested. However, they noted a high degree of donor-dependent variability both in the number of markers and in which specific markers were influenced by the allergens. On the other hand, the same conditions of exposure to the irritant, SDS, never modified more than one of the markers. Other investigators have attempted to correlate chemical-induced up-regulation of CD86 and HLA-DR with cytotoxicity (Straube et al., 2005Go). The strong contact allergens dinitrochlorobenzene (DNCB) and trinitrobenzene sulfonic acid (TNBS) increased CD86 and HLA-DR expression on monocyte-derived DC at dose levels that were just below the concentration that caused frank cytotoxicity. Irritants (SDS and benzalkonium chloride) were also found to up-regulate CD86 and HLA-DR expression, but only at concentrations that induced significant cytotoxicity.

Some investigators have examined cytokine secretion concurrently with cell surface marker expression. Aiba et al. (1997)Go reported that culture of monocyte-derived DC with chemical allergens induced a significant increase in the surface expression of HLA-DR, CD54, and CD86 compared with untreated or irritant-exposed DC. Although irritants did not provoke cytokine production, there was some induction by allergens, but the pattern of expression varied according to the particular allergen tested. Consistent with previously observed effects on IL-1ß mRNA expression (Pichowski et al., 2000Go, 2001Go), there were a number of nonresponders with respect to both cell surface marker expression and cytokine secretion. Coutant et al. (1999)Go found that after 48 h incubation with chemical haptens, monocyte-derived DC expressed higher levels of HLA-DR, CD86, CD40, and CD54 compared with DC incubated with irritants. In addition, they observed that incubation with haptens, but not with irritants, provoked the secretion of TNF-{alpha}. They reported only small differences in responses between donors. However, the DC preparations used in their studies were prescreened; those preparations expressing high levels of HLA-DR were discarded, as they were found to respond poorly or not at all to inflammatory stimuli, possibly reflecting a higher level of maturation. Tuschl and Kovac (2001)Go examined CD86, CD54, and HLA-DR cell surface expression in parallel with the induction of intracellular expression of IL-1ß in PBMC-derived DC. An up-regulation of these surface markers was observed in the majority of donors following culture with allergen but not with irritant. However, no clear results were obtained for the induction of intracellular IL-1ß. Degwert et al. (1997)Go and Becker et al. (1997)Go reported an increase in receptor-mediated endocytosis of HLA-DR in monocyte-derived DC exposed to several strong contact allergens. Less potent sensitizers and irritants failed to produce a significant effect on HLA-DR internalization (Becker et al., 1997Go). In common with previous investigations (Aiba et al., 1997Go; Pichowski et al., 2000Go, 2001Go), responder and nonresponder populations were identified.

On balance, the results generated to date by a number of different investigators demonstrate that measurement of allergen-induced changes in phenotype or induced cytokine expression in monocyte-, CD34+-, or PBMC-derived DC as a potential in vitro method for predicting sensitization potential has certain limitations. The parameters examined thus far apparently lack the sensitivity and dynamic range to provide a robust method for the identification of potential contact sensitizers, with the possible exception of very potent skin allergens. One other major limitation was the common finding that there was considerable donor-to-donor variation in responsiveness, such that populations could be divided into responders and nonresponders. Although it has been suggested that this problem could be circumvented by screening potential DC donors for activity, this type of approach would not lend itself easily to the development of a routine testing procedure.


    FIRST-GENERATION APPROACHES USING CELL LINES AS DC SURROGATES
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One approach to circumvent the interdonor variation in responsiveness that has been reported is to develop an appropriate cell line that could be used as a DC surrogate. Thus far, there has been little success in the establishment of stable, long-term LC or DC lines. A LC-like murine cell line that was developed from the epidermis of newborn BALB/c mice, XS52 cells, possesses many of the morphologic, phenotypic, and functional characteristics of freshly isolated LC (Xu et al., 1995Go). Analogous to studies with cultured LC-like DC, an increase in tyrosine phosphorylation in XS52 cells has been demonstrated following stimulation with strong haptens (Neisius et al., 1999Go). In addition, XS52 cells have been shown to up-regulate cell surface MHC class II expression after exposure to contact allergens but not irritants (Herouet et al., 2000Go). While not available commercially, the XS52 cell line, with its similarities to LC, holds some promise for the development of a wholly in vitro method for sensitization testing. The development of two stable, self-replicating human DC-lines has been reported also (Nunez et al., 1998Go). These adherent cell lines, RAN1 and LAS1, were derived from a promonocytic, histiocytic lymphoma cell line and express CD1a, HLA-DR, CD45RO, and CD23 (Fc{varepsilon}RII). Functionally, they are capable of processing and presenting soluble antigen and activating T cells in an allogenic mixed lymphocyte reaction. These cell lines are not widely available, and to date, the effects of chemical allergens on these cells have not been examined.

A few commercially available human cell lines have been explored as potential surrogates for DC. Cytokine treatment of the human acute myelogenous leukemia cell line, KG-1, has been shown to induce a DC-like phenotype and morphology (Hulette et al., 2001Go; St. Louis et al., 1999)Go. Exposure to chemical allergens had little or no effect on the expression of HLA-DR, CD54, CD80, and CD86 in these cytokine-induced cells (Hulette et al., 2002Go; Yoshida et al., 2003Go). However in the absence of cytokine, KG-1 cells demonstrated increased expression of CD86 and CD54 following treatment with a strong contact sensitizer (Yoshida et al., 2003Go). The THP-1 cell line, derived from human monocytic leukemia cells, has also been examined for its potential as a replacement for PBMC-derived DC in the development of an in vitro predictive test for contact sensitizers (Ashikaga et al., 2002Go; Yoshida et al., 2003Go). Exposure to a number of skin sensitizers has been shown to enhance cell surface expression of CD86 (Ashikaga et al., 2002Go; Yoshida et al., 2003Go) and CD54 (Yoshida et al., 2003Go). In addition, THP-1 cells demonstrated an increased internalization of MHC class II molecules in response to allergen exposure (Ashikaga et al., 2002Go).

The most judicious view at present is that, with further refinement, some of these cell lines show promise as surrogates for DC in vitro assays. However, it must be noted that, in common with freshly isolated DC, the parameters of activation examined to date have shown relatively modest changes with potent allergens, thus the question of sensitivity still remains.


    SECOND-GENERATION APPROACHES USING DENDRITIC CELLS: SEARCH FOR NOVEL MARKERS BY HOLISTIC EXPRESSION PROFILING
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One approach that has generated considerable interest is the potential use of holistic gene expression profiling to identify novel markers associated with DC biology and skin sensitization. Microarrays are the most frequently used technology for transcript profiling allowing detection of thousands of mRNAs in cellular or tissue They have been used to study various aspects of DC biology and function such as differentiation, maturation, and migration (Griffin et al., 2004Go; Richards et al., 2002Go; Ryan et al., 2004aGo), as well as to elucidate DC intracellular signaling pathways for application to potential immunomodulation strategies (Ricciardi-Castagnoli and Granucci, 2002Go; Tang and Saltzman, 2004Go; for review Ju and Zenke, 2004Go). While expression profiles induced by various stimuli such as viral and bacterial pathogens (Huang et al., 2001Go), lipopolysaccharide (Chen et al., 2002Go; Messmer et al., 2003Go), and cytokines (Dietz et al., 2000Go; Le Naour et al., 2001Go) have been monitored, there are limited reports of genome-wide analysis of the changes induced in human DC upon contact with skin sensitizers.

Ryan et al. (2004aGo,bGo) analyzed gene expression changes induced in DC following treatment with a low and high dose of the water-soluble analog of DNCB, dinitrobenzensulfonic acid (DNBS). Using the Affymetrix GeneChip® platform, many changes in specific transcripts were observed in both the 1 mM and 5 mM DNBS treatments. However, as expected, more genes were regulated significantly (p ≤ 0.001) by 5 mM treatment than was observed for the 1 mM dose and corresponded to approximately 10.9 and 1.6%, respectively, of the total number of genes represented on the Affymetrix U95A2 Genechip®. Many of the 118 genes that were regulated in both treatment groups at p ≤ 0.001 could be correlated with DC function and could be further validated by quantitative real-time PCR analysis. Examples include interleukin 8, CD86, CCL2, CCL4, and CD43. In addition genes not previously associated with skin sensitization or DC biology, such as AKR1C2, DUSP6, and QPCT, were identified, therefore widening the pool from which potential markers can be selected. It is anticipated that similar genomics studies using LC surrogates with different allergens and irritants may provide additional gene targets not previously discovered. However, genes that are selected as markers for skin sensitization must fit the criterion for dynamic range, robustness, sensitivity, and selectivity for a predictive model and cover a range of chemical classes (Kimber et al., 2001Go).


    GENERAL CONSIDERATIONS, CURRENT CHALLENGES, AND FUTURE OPPORTUNITIES
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There is currently some enthusiasm for the application of microarray technology in identifying new gene candidates as potential markers of allergen-induced changes in DC, and it remains to be seen whether there derive from such analyses novel genes that provide the transcriptional dynamic range and selectivity required for use in in vitro prediction models. The ideal candidate in this context would be a gene that displays dramatic increases in expression following encounter of DC with a chemical allergen, fails to show similar changes in expression in response to other stimuli, and codes for a protein that correlates mechanistically and quantitatively with the acquisition of skin sensitization.

If such a strategy is found to be sound, insofar as genes are identified that display selective responses to chemical allergens, then an interesting question emerges that can be framed as follows: in what ways are DC able to distinguish between encounter with a sensitizing chemical and other changes in the cellular microenvironment (such as contact with nonsensitizing skin irritants)? There is no clear answer to this question, although one possible explanation is that the changes induced, which appear to be selective for contact allergens, in fact result from the required property of sensitizing chemicals to form stable associations with proteins. The argument is that skin sensitizers might cross-link membrane proteins on target cells and that this in turn results in characteristic changes in cytokine production and/or the expression of membrane determinants. This issue is of more than theoretical importance, since resolution of the proposed selective effect of chemical allergens on the phenotype and function of DC might pave the way for a more precise approach to the identification of sensitizing potential.

The possible requirement for protein reactivity to effect changes in gene expression by DC illustrates an issue that impacts upon all proposed in vitro approaches to skin sensitization testing; the need for metabolic activation. For the acquisition of skin sensitization, there is a need for the inducing chemical allergen to form a stable association with protein. The consequence is that skin-sensitizing chemicals are either naturally protein reactive, or have to be metabolized to a protein-reactive species (i.e., pro-haptens) (Dearman and Kimber, 2003Go; Kimber and Dearman, 2003Go). It is generally believed that in excess of 20% of skin sensitizing chemicals can be described as pro-haptens, and if a proposed in vitro method is going to be able to detect these, then there must be endogenous metabolic activity within the cell or tissue matrix used, or provision of an exogenous source of appropriate metabolizing enzymes.

Another confounding factor (which impacts on all in vitro cell assays, including those incorporating DC or DC-like cells) is delivery of the test material. Many of the chemicals are organic in nature and insoluble in an aqueous matrix that is most suitable for delivery to culture systems. There are ways of addressing this by using aqueous/organic solvent mixtures, but this nevertheless represents a significant technical challenge.

The other major issue is related to cell viability. The paradigm commonly in cell-based in vitro assays is to deliver test chemical at the maximum nontoxic concentration. This practice raises two questions. The first of these is what level of viability is deemed to be indicative of lack of toxicity—and by what method should this be measured? Clearly this particular issue impacts on all types of in vitro assay systems. The second question is of greater interest, insofar as it relates more specifically to the stimulation of immune responses. This is whether there is a need for some low-level cell trauma in order for DC to respond optimally to a chemical allergen. This thinking is based upon a view that encounter with antigen is not itself sufficient to provoke a normal immune response, and that, in addition, there is a requirement for some local tissue damage or trauma to provide a necessary costimulus. This theory was first developed by Matzinger and later elaborated further by others in relation to skin sensitization (Matzinger, 2002Go; McFadden and Basketter, 2000Go). With respect to the response by DC to chemical allergens in vitro, the speculation is that DC perhaps need a second danger signal and/or to be subject to some small amount of cellular trauma in order to mount vigorous responses. A number of investigators are exploring this currently and questioning whether some low-level cell injury induced concomitantly with, or prior to, exposure to allergen would enhance transcriptional responses induced in DC.

The need for signals additional to that provided by the allergen itself, of course, raises questions about the requirement for the presence of additional cell types within the culture system and, in particular, keratinocytes. The latter are the major cellular constituents of the epidermis and effectively surround LC. Many epidermal chemokines and cytokines that orchestrate the movement and function of LC derive from keratinocytes, and it is therefore legitimate to question whether provision of these signals by keratinocytes cocultured with LC-like DC would enhance or modify responses induced by exposure to allergen. This again is a subject of current research.

Present investigations, linked with a certain amount of ingenuity, may allow us to resolve some of these issues and uncertainties in the near future, and this would undoubtedly provide a firmer foundation for DC-based assays for skin sensitization hazard. In addition, however, it is worthwhile speculating on how some recent insights into DC and LC biology may also pay dividends in exploiting this area of science. For instance, there is a growing appreciation of the increasingly complex chemokine receptor-ligand interactions that are required for regulation of the motility and directed movement of LC and LC-like DC in health and disease, and it might be that some of the novel proteins described are selectively up-regulated during activation of DC (Partida-Sanchez et al., 2004Go; Qu et al., 2004Go; Vermi et al., 2005Go).

In the future, it may be possible to exploit DC responses in vitro not only for the identification of chemical allergens, but also for characterizing the type of allergic response that they will selectively induce. While the majority of chemical allergens are associated with skin sensitization and the development of ACD, others (fewer in number) are implicated primarily as inducers of allergic sensitization of the respiratory tract and occupational asthma. The type of allergic response a chemical allergen will provoke is largely a function of the development of discrete functional subpopulations of T lymphocytes (Kimber and Dearman, 2005Go). There is increasing evidence that the development of T-lymphoctyte subpopulations is in fact driven by DC of different phenotypes (either inherent or acquired) (Fujita et al., 2005Go; Smits et al., 2005Go). Associated with this is intriguing evidence that contact allergens and chemical respiratory allergens display differential selectivity for association with protein and a variable potential to mobilize LC in vivo (Cumberbatch et al., 2005Go; Hopkins et al., 2005Go). It is tempting to speculate that a more detailed understanding of the ways in which chemical allergens of different classes interact with LC and DC may yield opportunities to model this in vitro and permit not only identification of chemical allergens but also prediction of the type of sensitization they are likely to induce.

A continued investment in this exciting branch of applied immunotoxicology should see significant progress made in resolving some of the pressing technical issues. This in turn will permit a rigorous appraisal of the opportunities for hazard assessment that may be afforded by characterization of DC responses in vitro.


    ACKNOWLEDGMENTS
 
Conflict of interest: none declared.


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 GENERAL CONSIDERATIONS, CURRENT...
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