Human neutrophil antigens: Nature, clinical significance and detection.

Granulocytes are an essential part of both the innate and adaptive immune systems. Human neutrophil antigens (HNAs) are a family of epitopes that are located on glycoproteins that are mostly expressed on human granulocytes. Antibodies that recognize these epitopes have been associated with neutropenia, transfusion complications, haematopoietic stem cell transplant nonengraftment and renal transplant rejection. Currently, there are fourteen recognized HNA alleles across five antigen systems (HNA-1 through HNA-5), the molecular basis of which are located on the genes FCGR3B, CD177, SLC44A2, ITGAM and ITGAL, respectively. Elucidation of the associated genes has permitted the development of testing strategies for HNA typing and aided understanding of the associated epitopes. This review will outline the associated clinical conditions that require HNA investigation and how these are performed in specialized laboratories. Investigations provided are both reactive for patients with a variety of existing or suspected neutropenias and proactive in the testing of blood component donors in order to reduce the potential risk to patients who require transfusion.

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies.

Therapeutic mAbs are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs. Here, using an array of in vitro assays, we explored the functional effects of Trx-mediated reduction on the mechanisms of action of six therapeutic mAbs. We found that Trx reduces the interchain disulfide bonds of the mAbs, after which they remain intact but have altered function. In general, this reduction increased antigen-binding capacity, resulting in, for example, enhanced tumor necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the antiproliferative activity of an anti-tyrosine kinase-type cell-surface receptor HER2 mAb. In all of the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced interchain disulfide bonds reoxidize, and ADCC activity is restored. In summary, Trx-mediated reduction has a substantial impact on the functional effects of an mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo.

Impact of a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic Monoclonal Antibodies.

PURPOSE: Anti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice. METHODS: Heat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice. RESULTS: rmHSP70 was shown to bind preferentially to aggregates of both mAbs, but not to the native, monomeric proteins. Aggregates supplemented with 0.1% rmHSP70 induced significantly enhanced IgG2a antibody responses compared with aggregates alone but the effect was not observed for monomeric mAbs. Dendritic cells pulsed with mAb aggregate showed enhanced IFNγ production on co-culture with T cells in the presence of rmHSP70. CONCLUSION: The results indicate a Th1-skewing of the immune response by aggregates and show that murine rmHSP70 selectively modulates the immune response to mAb aggregates, but not monomer. These data suggest that heat shock protein impurities can selectively accumulate by binding to mAb aggregates and thus influence immunogenic responses to therapeutic proteins.

T lymphocyte phenotype of contact-allergic patients: experience with nickel and p-phenylenediamine.

BACKGROUND: There is considerable interest in understanding the immunological variables that have the greatest influence on the effectiveness of sensitization by contact allergens, particularly in the context of developing new paradigms for risk assessment of novel compounds. OBJECTIVES: To examine the relationship between patch test score for three different contact allergens and the characteristics of T cell responses. METHODS: A total of 192 patients with confirmed nickel, p-phenylenediamine (PPD) or methylisothiazolinone (MI) allergy were recruited from the Contact Dermatitis Investigation Unit at Salford Royal Hospital. Severity of allergy was scored by the use of patch testing, peripheral blood lymphocytes were characterized for T cell phenotype by flow cytometry, and proliferative activity was characterized by radiolabelled thymidine incorporation. Comparisons were drawn with buffy coat samples from healthy volunteers. RESULTS: Patch test positivity for nickel, PPD and MI was associated with changes in the phenotype of peripheral blood T cells: increases in naïve cells, decreases in regulatory T cell frequency and the CD4+ /CD8hi ratio, and increased expression of the skin-homing marker cutaneous lymphocyte antigen (CLA), particularly for those patients with a +++ patch test score. CONCLUSIONS: This increased understanding of the characteristics of the T cell responses to contact allergens may provide parameters with which to better measure health risks associated with skin sensitization.

Lower levels of interleukin-1ß gene expression are associated with impaired Langerhans' cell migration in aged human skin.

Langerhans' cells (LC) play pivotal roles in skin immune responses, linking innate and adaptive immunity. In aged skin there are fewer LC and migration is impaired compared with young skin. These changes may contribute to declining skin immunity in the elderly, including increased skin infections and skin cancer. Interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) are mandatory signals for LC migration and previous studies suggest that IL-1ß signalling may be dysregulated in aged skin. Therefore, we sought to explore the mechanisms underlying these phenomena. In skin biopsies of photoprotected young (< 30 years) and aged (> 70 years) human skin ex vivo, we assessed the impact of trauma, and mandatory LC mobilizing signals on LC migration and gene expression. Biopsy-related trauma induced LC migration from young epidermis, whereas in aged skin, migration was greatly reduced. Interleukin-1ß treatment restored LC migration in aged epidermis whereas TNF-α was without effect. In uncultured, aged skin IL-1ß gene expression was lower compared with young skin; following culture, IL-1ßmRNA remained lower in aged skin under control and TNF-α conditions but was elevated after culture with IL-1ß. Interleukin-1 receptor type 2 (IL1R2) gene expression was significantly increased in aged, but not young skin, after cytokine treatment. Keratinocyte-derived factors secreted from young and aged primary cells did not restore or inhibit LC migration from aged and young epidermis, respectively. These data suggest that in aged skin, IL-1ß signalling is diminished due to altered expression of IL1B and decoy receptor gene IL1R2.

Influence of Escherichia coli chaperone DnaK on protein immunogenicity.

The production of anti-drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process-related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single-chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant-like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process-related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.

Investigating Liquid-Liquid Phase Separation of a Monoclonal Antibody Using Solution-State NMR Spectroscopy: Effect of Arg·Glu and Arg·HCl.

Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases and should be avoided in the final drug product. Understanding the factors leading to LLPS and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (mAb5), its solubility, and parameters extracted using 1H NMR spectroscopy, for various formulations. We show that the formulations demonstrating least LLPS lead to the largest mAb5 NMR signal intensities. In the formulations exhibiting the highest propensity to phase-separate the mAb NMR signal intensities are the lowest, even at higher temperatures without visible phase separation, suggesting a high degree of self-association prior to distinct phase separation. Addition of arginine glutamate prevented LLPS and led to a significant increase in the observed mAb signal intensity, whereas the effect of arginine hydrochloride was only marginal. Solution NMR spectroscopy was further used to characterize the protein-lean and protein-rich phases separately and demonstrated that protein self-association in the protein-rich phase can be significantly reduced by arginine glutamate. Solution NMR spectroscopy may be useful as a tool to assess the propensity of mAb solutions to phase-separate.

Interleukin-1ß Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin.

The secretion of IL-1ß is a central event in the initiation of inflammation. Unlike most other cytokines, the secretion of IL-1ß requires two signals: one signal to induce the intracellular up-regulation of pro-IL-1ß and a second signal to drive secretion of the bioactive molecule. The release of pro-IL-1ß is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here we sought to identify novel proteins involved in IL-1ß secretion and intracellular processing to gain further insights into the mechanism of IL-1 release. A human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1ß. Probes with a signal-to-noise ratio of >3 were defined as biologically relevant. In these analyses, calmodulin was identified as a particularly strong hit, with a signal-to-noise ratio of ∼ 11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1ß, but not mature IL-1ß, was confirmed and shown to be calcium-dependent. Finally, using small molecule inhibitors, it was demonstrated that both calcium and calmodulin were required for nigericin-induced IL-1ß secretion in THP-1 cells and primary human monocytes. Together, these data suggest that, following calcium influx into the cell, pro-IL-1ß interacts with calmodulin and that this interaction is important for IL-1ß processing and release.

T lymphocyte dynamics in methylisothiazolinone-allergic patients.

BACKGROUND: Methylisothiazolinone (MI), a preservative that is commonly used in personal care products, is now recognized as an important contact allergen in both cosmetic and occupational settings. OBJECTIVES: To analyse T lymphocyte responses to MI, in order to provide important information regarding the relationship between the nature of such responses and skin sensitization potency. METHODS: Proliferative responses to free MI and to an MI-human serum albumin (HSA) conjugate were measured according to [(3) H]thymidine incorporation (n = 56 donors; patch test scores of + in 20, ++ in 29, and +++ in 7). Peripheral blood mononuclear cells were cultured in the presence of MI (0.001-1 µg/ml) or MI-HSA (0.001-100 µg/ml). Proliferating CD4(+) and CD8(+) T lymphocytes were identified by flow cytometry with the intracellular marker Ki-67. RESULTS: For free MI, modest positive responses were recorded for 7 of 31 donors. In contrast, MI-HSA stimulated more marked responses in 17 of 31 donors. Characterization of positive proliferative responses showed variable patterns of proliferating CD4(+) and CD8(+) T lymphocytes from donors with the same patch test scores and similar maximal values. CONCLUSIONS: MI-HSA is able to induce secondary responses in lymphocytes drawn from sensitized subjects, and provides a more effective source of antigen than free MI. Furthermore, individual donors show differential activity profiles with respect to T lymphocyte subsets.

Anti-hapten antibodies in response to skin sensitization.

Whereas T lymphocyte (T cell) activation is the key event in the acquisition of skin sensitization and subsequent elicitation of allergic contact dermatitis, the humoral component of immune responses to organic contact allergens has received little consideration. There is evidence that, in experimental animals, topical exposure to potent contact allergens is associated with B cell activation and proliferation, and hapten-specific antibody production. However, there is very limited evidence available for anti-hapten antibody responses being induced following topical exposure of humans to contact allergens. Nevertheless, it is important to appreciate that there are almost no negative studies in which evidence for antibody production as the result of skin sensitization has been sought and not found. That is, there is absence of evidence rather than evidence of absence. Furthermore, exposure to chemical respiratory allergens, in which the skin has been implicated as a potential route of sensitization, results in anti-hapten antibody responses. It is proposed that skin sensitization to contact allergens will normally be accompanied by antibody production. The phenomenon is worthy of investigation, as anti-hapten antibodies could potentially influence and/or regulate the induction of skin sensitization. Moreover, such antibodies may provide an informative correlate of the extent to which sensitization has been acquired.

Dendritic cell IL-1α and IL-1ß are polyubiquitinated and degraded by the proteasome.

IL-1α and ß are key players in the innate immune system. The secretion of these cytokines by dendritic cells (DC) is integral to the development of proinflammatory responses. These cytokines are not secreted via the classical secretory pathway. Instead, 2 independent processes are required; an initial signal to induce up-regulation of the precursor pro-IL-1α and -ß, and a second signal to drive cleavage and consequent secretion. Pro-IL-1α and -ß are both cytosolic and thus, are potentially subject to post-translational modifications. These modifications may, in turn, have a functional outcome in the context of IL-1α and -ß secretion and hence inflammation. We report here that IL-1α and -ß were degraded intracellularly in murine bone marrow-derived DC and that this degradation was dependent on active cellular processes. In addition, we demonstrate that degradation was ablated when the proteasome was inhibited, whereas autophagy did not appear to play a major role. Furthermore, inhibition of the proteasome led to an accumulation of polyubiquitinated IL-1α and -ß, indicating that IL-1α and -ß were polyubiquitinated prior to proteasomal degradation. Finally, our investigations suggest that polyubiquitination and proteasomal degradation are not continuous processes but instead are up-regulated following DC activation. Overall, these data highlight that IL-1α and -ß polyubiquitination and proteasomal degradation are central mechanisms in the regulation of intracellular IL-1 levels in DC.

Skin sensitization induced Langerhans' cell mobilization: variable requirements for tumour necrosis factor-α.

Upon antigen/allergen recognition, epidermal Langerhans' cells (LC) are mobilized and migrate to the local lymph node where they play a major role in initiating or regulating immune responses. It had been proposed that all chemical allergens induce LC migration via common cytokine signals delivered by TNF-α and IL-1ß. Here the dependence of LC migration on TNF-α following treatment of mice with various chemical allergens has been investigated. It was found that under standard conditions the allergens oxazolone, paraphenylene diamine, and trimellitic anhydride, in addition to the skin irritant sodium lauryl sulfate, were unable to trigger LC mobilization in the absence of TNF-α signalling. In contrast, two members of the dinitrohalobenezene family (2,4-dinitrochlorobenzene [DNCB] and 2,4-dinitrofluorobenzene [DNFB]) promoted LC migration independently of TNF-R2 (the sole TNF-α receptor expressed by LC) and TNF-α although the presence of IL-1ß was still required. However, increasing doses of oxazolone overcame the requirement of TNF-α for LC mobilization, whereas lower doses of DNCB were still able to induce LC migration in a TNF-α-independent manner. These novel findings demonstrate unexpected heterogeneity among chemical allergens and furthermore that LC can be induced to migrate from the epidermis via different mechanisms that are either dependent or independent of TNF-α. Although the exact mechanisms with regard to the signals that activate LC have yet to be elucidated, these differences may translate into functional speciation that will likely impact on the extent and quality of allergic sensitization.

P2X7R activation drives distinct IL-1 responses in dendritic cells compared to macrophages.

The P2X(7)R is a functionally distinct member of the P2X family of non-selective cation channels associated with rapid activation of the inflammasome complex and signalling interleukin (IL)-1ß release in macrophages. The main focus of this investigation was to compare P2X(7)R-driven IL-1 production by primary murine bone marrow derived dendritic cells (BMDC) and macrophages (BMM). P2X(7)R expression in murine BMDC and BMM at both transcriptional (P2X(7)A variant) and protein levels was demonstrated. Priming with lipopolysaccharide (LPS) and receptor activation with adenosine triphosphate (ATP) resulted in markedly enhanced IL-1 (α and ß) secretion in BMDC compared with BMM. In both cell types IL-1 production was profoundly inhibited with a P2X(7)R-specific inhibitor (A-740003) demonstrating that this release is predominantly a P2X(7)R-dependent process. These data also suggest that P2X(7)R and caspase-1 activation drive IL-1α release from BMDC. Both cell types expressed constitutively the gain-of-function P2X(7)K as well as the full P2X(7)A variant at equivalent levels. LPS priming reduced significantly levels of P2X(7)A but not P2X(7)K transcripts in both BMDC and BMM. P2X(7)R-induced pore formation, assessed by YO-PRO-1 dye uptake, was greater in BMDC, and these cells were protected from cell death. These data demonstrate that DC and macrophages display distinct patterns of cytokine regulation, particularly with respect to IL-1, as a consequence of cell-type specific differences in the physicochemical properties of the P2X(7)R. Understanding the cell-specific regulation of these cytokines is essential for manipulating such responses in health and disease.

Respiratory allergenic potential of plant-derived proteins: Understanding the relationship between exposure and potency for risk assessments.

Botanical ingredients (ingredients derived from plants) are finding increasing application in personal care products and the public perceives these ingredients to be safe. However, some proteins in botanicals have the potential to cause immediate-type (IgE-mediated) respiratory allergic reactions. Although reports of such reactions are uncommon, when they do occur, they can be severe. Experience with soap containing wheat proteins illustrated that under certain specific conditions, consumers may be affected. Establishing safe exposure levels for botanical proteins has been challenging. Industrial enzymes provide a rich reference dataset based on their historical association with allergic reactions among workers, which includes robust dose-response information. In the absence of similar data on the potency of plant proteins, a conservative default approach has historically been applied based on information derived from allergenic enzymes. In this article we review the historical default approach and dataset for setting limits for plant proteins in botanical ingredients based on analogy to industrial enzymes followed by a synthesis of literature data on allergic reactions following inhalation exposure to plant-derived proteins. The aim is to share relevant background information and display the relationship between exposure and potency as a first step in the development of a strategy for the development of an improved approach to support the risk assessment of plant-derived proteins.

Differential immunogenicity and allergenicity of native and recombinant human lactoferrins: role of glycosylation.

Human native milk lactoferrin (LF) and recombinant forms of lactoferrin (rLF) are available with identical aa sequences, but different glycosylation patterns. Native lactoferrin (NLF) possesses the intrinsic ability to stimulate vigorous IgG and IgE antibody responses in BALB/c mice, whereas recombinant forms (Aspergillus or rice) are 40-fold less immunogenic and 200-fold less allergenic. Such differences are independent of endotoxin or iron content and the glycans do not contribute to epitope formation. A complex glycoprofile is observed for NLF, including sialic acid, fucose, mannose, and Lewis (Le)(x) structures, whereas both rLF species display a simpler glycoprofile rich in mannose. Although Le(x) type sugars play a Th2-type adjuvant role, endogenous expression of Le(x) on NLF did not completely account for the more vigorous IgE responses it provoked. Furthermore, coadminstration of rLF downregulated IgE and upregulated IgG2a antibody responses provoked by NLF, but was without effect on responses to unrelated peanut and chicken egg allergens. These results suggest glycans on rLF impact the induction phase to selectively inhibit IgE responses and that differential glycosylation patterns may impact on antigen uptake, processing and/or presentation, and the balance between Th1 and Th2 responses.

Diisocyanates, occupational asthma and IgE antibody: implications for hazard characterization.

Sensitization of the respiratory tract by chemicals resulting in rhinitis and asthma is an important occupational health issue. Occupational asthma is associated with significant morbidity and can be fatal. Tests for the identification and characterization of chemicals with the potential to cause sensitization of the respiratory tract are lacking. In spite of sustained interest there are no validated or widely accepted methods available, and this presents toxicologists with a considerable challenge. One important constraint on the development of appropriate testing strategies has been uncertainty and controversy about the immunological mechanisms through which chemicals may induce sensitization of the respiratory tract. By analogy with protein respiratory allergy it is legitimate to consider that IgE antibody-dependent mechanisms may play a pivotal role. However, although many aspects of chemical respiratory allergy are consistent with reactions caused by IgE antibody, uncertainty remains because among patients with occupational asthma caused by chemical respiratory allergens there are commonly a proportion, and sometimes a significant proportion, of subjects that lack detectable IgE antibody. Here we consider the relevance of IgE antibody responses for the development of a chemical respiratory allergy to diisocyanates. A case is made that IgE antibody responses are, either directly or indirectly, closely associated with occupational asthma to the diisocyanates (and to other chemical respiratory allergens). As such the argument is advanced here that IgE antibody represents an appropriate readout for the characterization of chemical respiratory allergens, and that uncertainty about mode of action should no longer represent a hurdle in the development of suitable test methods.

Characterization of the allergenic potential of proteins: an assessment of the kiwifruit allergen actinidin.

Assessment of the potential allergenicity (IgE-inducing properties) of novel proteins is an important challenge in the overall safety assessment of foods. Resistance to digestion with pepsin is commonly measured to characterize allergenicity, although the association is not absolute. We have previously shown that specific IgE antibody production induced by systemic [intraperitoneal (i.p.)] exposure of BALB/c strain mice to a range of proteins correlates with allergenic potential for known allergens. The purpose of the present study was to explore further the utility of these approaches using the food allergen, actinidin. Recently, kiwifruit has become an important allergenic foodstuff, coincident with its increased consumption, particularly as a weaning food. The ability of the kiwifruit allergen actinidin to stimulate antibody responses has been compared with the reference allergen ovalbumin, and with the non-allergen bovine haemoglobin. Haemoglobin was rapidly digested by pepsin whereas actinidin was resistant unless subjected to prior chemical reduction (reflecting intracellular digestion conditions). Haemoglobin stimulated detectable IgG antibody production at relatively high doses (10%), but failed to provoke detectable IgE. In contrast, actinidin was both immunogenic and allergenic at relatively low doses (0.25% to 1%). Vigorous IgG and IgG1 antibody and high titre IgE antibody responses were recorded, similar to those provoked by ovalbumin. Thus, actinidin displays a marked ability to provoke IgE, consistent with allergenic potential. These data provide further encouragement that in tandem with analysis of pepsin stability, the induction of IgE after systemic exposure of BALB/c strain mice provides a useful approach for the prospective identification of protein allergens.

The XS106 cell: a Langerhans' cell surrogate with a selective type 2 phenotype.

Epidermal Langerhans' cells (LC) play important roles in initiating and regulating cutaneous immune responses. However, LC comprise less than 3% of all epidermal cells and consequently are difficult to study ex vivo. In the current investigations, we have examined the utility of the XS106 cell line, a dendritic cell (DC) line derived from mouse epidermis, as a surrogate for LC. Membrane marker expression, type 1- and type 2-associated chemokine production, and migration patterns have been characterised following treatment of XS106 cells with a range of toll-like receptor (TLR) ligands. Comparisons have been made with mouse bone marrow-derived DC- and LC-derived ex vivo. Like BMDC, XS106 cells expressed generic DC markers, in addition to displaying higher levels of skin DC markers compared with BMDC. XS106 cells and LC-enriched epidermal fractions both displayed higher constitutive expression of type 2-associated chemokines than type 1 chemokines. Furthermore, although treatment with a range of TLR ligands induced cytokine secretion by XS106 cells, only type 2 TLR ligands increased membrane marker expression of major histocompatibility complex class II and co-stimulatory molecules. Moreover, type 1-associated TLR ligands failed to induce selective type 1 chemokine secretion by XS106 cells. XS106 cells also displayed functional similarity to LC, migrating in response to chemokines that are known to induce the migration of LC. On the basis of membrane marker expression and selective type 2 polarisation XS106 cells provide a useful surrogate for LC.

Dendritic cells and the assessment in vitro of skin sensitizing potential.

It is now well established that dendritic cells (DC) play pivotal roles in the initiation and orchestration of adaptive immune responses, including cutaneous immune responses to chemical allergens that drive the acquisition of skin sensitization. It is not unexpected, therefore, that a large number, and wide variety, of proposed approaches for the identification of skin sensitizing chemicals in vitro are based upon the use of cultured DC or DC-like cells. The use of DC in this context is legitimate. However, with our rapidly increasing understanding of the diversity of cutaneous DC with respect to both phenotype and function, it is timely now to review briefly the potential limitations and interpretive difficulties that are associated with the use of DC-based assays. Among the important considerations are the fact that chemical-induced changes in the characteristics and function of cultured DC will not necessarily reflect accurately the events that that support the development of skin sensitization in vivo. In addition, most DC-based assays are predicated on a view that cutaneous DC have as their primary function the initiation of adaptive immune responses. However, it is now appreciated that cutaneous DC, and in particular epidermal Langerhans cells (LC), may also play important immunoregulatory roles that serve to limit and contain skin immune responses. Notwithstanding these considerations there is reason to believe that at least some in vitro DC-based assays are of value, and indeed some are currently the subject of a formal validation process. However, it is appropriate that such assays are configured and interpreted carefully, and with an appreciation of the complexity of DC biology.

Impaired Langerhans cell migration in psoriasis.

We have examined whether psoriasis is associated with systemic effects on epidermal Langerhans cell (LC) function and, specifically, the migration of LCs from the skin. Compared with normal skin, the frequency and morphology of epidermal LCs in uninvolved skin from patients with psoriasis was normal. However, mobilization of these cells in response to stimuli that normally induce migration (chemical allergen, tumor necrosis factor alpha [TNF-alpha], and interleukin-1beta [IL-1beta]) was largely absent, despite the fact that treatment with TNF-alpha and IL-1beta was associated with comparable inflammatory reactions in patients and controls. The failure of LC migration from uninvolved skin was not attributable to altered expression of receptors for IL-1beta or TNF-alpha that are required for mobilization, nor was there an association with induced cutaneous cytokine expression. Although a role for altered dynamics of LC migration/turnover has not been formally excluded, these data reveal a very consistent decrement of LC function in psoriasis that may play a decisive role in disease pathogenesis.