Enhancement of antibody responses to native G protein-coupled receptors using E. coli GroEL as a molecular adjuvant in DNA immunization.

Antibody-based drug research involves the preparation of polyclonal and monoclonal antibodies, especially those that are reactive with native G protein-coupled receptors (GPCRs) on the cell membrane. Here, we report that DNA immunization of mice with a plasmid that encodes endothelin A receptor (ETAR) fused to Escherichia coli (E. coli) GroEL at its C-terminus (ETAR-GroEL) induced very strong and specific antibody responses to native ETAR. Co-injection of plasmids that expressed ETAR and GroEL (ETAR+GroEL) induced significantly lower antibody responses compared with the ETAR-GroEL plasmid. Monoclonal antibodies that are prepared by using GroEL as a molecular adjuvant could be used in immunoassays, such as flow cytometry, western blotting, and immunoprecipitation, to detect both exogenous and endogenous ETAR. The adjuvant activity of GroEL might involve inflammatory cytokine mediators via Toll-like receptor 4 in addition to the anticipated carrier effect. DNA immunization using GroEL might become a standard method for producing antibodies that are useful for the functional analysis of GPCRs.

A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA).

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential.

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test--human cell line activation test (h-CLAT).

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

Production of IL-8 in THP-1 cells following contact allergen stimulation via mitogen-activated protein kinase activation or tumor necrosis factor-alpha production.

Contact allergens induce in vitro and in vivo the activation of dendritic cells (DC) and Langerhans cells (LC), which includes the up-regulation of surface marker expression (e.g. CD86, CD54) and cytokine production (e.g. TNF-alpha, IL-1beta, IL-8). The mitogen-activated protein kinase (MAPK) pathway also has a crucial role in this activation. However, the extent of MAPK involvement in the IL-8 production during DC/LC activation is not well understood. Earlier, we reported that contact allergens activated THP-1 cells, human monocytic cell line, like LC/DC in vitro. In this study, we further characterize the mechanism of IL-8 production using THP-1 cells as surrogate DCs. First, we evaluated the potential of 23 chemicals with different skin sensitization potencies to predominantly induce IL-8 production in vitro. Next we investigated the role of MAPK signaling and TNF-alpha, which is known to have autocrine effects on DC activation (e.g., IL-8 production). Inhibition of extracellular signal-regulated kinase (ERK), one of the MAPK pathways, suppressed the IL-8 production induced by both 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO(4)), and inhibition of p38 MAPK, a second MAPK pathway, significantly suppressed IL-8 production induced by only DNCB. Additionally, neutralization of TNF-alpha activity suppressed IL-8 production in THP-1 cells exposed to DNCB and NiSO(4). In conclusion, IL-8 production was predominantly induced in THP-1 cells following allergen stimulation, and MAPK pathways and TNF-alpha were involved in the IL-8 production induced by DNCB and NiSO(4). A better understanding of the mechanism of DC activation in vitro might lead to the clarification of the in vivo skin sensitization mechanism.

Role of MAPK signaling pathway in the activation of dendritic type cell line, THP-1, induced by DNCB and NiSO4.

The activation of dendritic cells (DC), including Langerhans cells (LC) that reside within the epidermis, is a critical event in the induction phase of allergic contact hypersensitivity. Although recently, p38 mitogen-activated protein kinase (MAPK) has been reported to play a role in the activation of DC induced by allergens, the signaling pathways involved in this process have yet to be determined. We previously found that THP-1 cells have a high capacity to induce TNF-alpha release and CD86, CD54, and CD40 expression following allergen treatment; reflecting in vitro allergen-induced DC activation during skin sensitization. In this study, we investigated the signaling pathways in THP-1 cells activated by two representative allergens, DNCB and NiSO(4). We found that DNCB and NiSO(4) induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK activation selectively blocked DNCB-induced TNF-alpha release, but not NiSO(4)-induced release. In contrast, inhibition of ERK pathways selectively suppressed NiSO(4)-induced TNF-alpha release but not DNCB-induced release. In addition, we found that the inhibition of p38 MAPK and ERK pathways caused a selective inhibition of CD86, CD54, and/or CD40 expression following treatment with DNCB or NiSO(4). In particular, inhibition of p38 MAPK suppressed CD86, CD54, and CD40 expression induced by DNCB and CD86 expression induced by NiSO(4) while inhibition of ERK pathways suppressed CD86, CD54 and CD40 expression induced by DNCB and NiSO(4). These data indicate that both DNCB and NiSO(4) activate p38 MAPK and ERK, and thereby stimulate TNF-alpha release and phenotypic changes through the different signal transduction pathways.

Role of TNF-alpha and extracellular ATP in THP-1 cell activation following allergen exposure.

Dendritic cells (DCs), including Langerhans cells (LCs), play a critical role in the induction phase of allergic contact hypersensitivity. Following exposure to chemical allergens in the skin, LCs undergo a maturation process leading to the up-regulation of expression of co-stimulatory molecules, such as CD86, CD54 and CD40. Our previous study revealed that chemical allergens induce phenotype alterations (e.g., CD86, CD54 and CD40) and cytokine production (TNF-alpha and IL-8) in THP-1 cells that possibly reflect the maturation of dendritic cells during skin sensitization. However, the physiological signals for phenotypic alterations by chemical allergens are still not fully understood. Therefore, in this study, we investigated the effect of TNF-alpha and extracellular ATP on THP-1 cell activation induced by chemical allergens. Kinetic studies revealed that TNF-alpha and IL-8 release occurred in a time-dependent manner with release of two cytokines beginning at 3 hr post-exposure to well-known haptens, DNCB and NiSO(4). While recombinant human TNF-alpha augmented CD54 and CD40 expression in a dose-dependent manner, rhTNF-alpha did not increase CD86 expression. Furthermore, neutralization of TNF-alpha activity strongly inhibited CD54 and CD40 expression induced by allergens. On the contrary, extracellular ATP induced the up-regulation of both CD86 and CD54 expression. In the presence of the P2 receptor antagonist suramin, the up-regulation of CD86 and CD54 expression by allergens was in part suppressed. Therefore, we postulate that not only TNF-alpha but also extracellular ATP may contribute to cell activation following allergen stimulation, which might reflect the mechanism by which DCs respond to allergens.