Inter-laboratory study of the in vitro dendritic cell migration assay for identification of contact allergens

The aim of this study was to investigate the transferability of technology and reproducibility of MUTZ-3 derived Langerhans Cell (MUTZ-LC) migration assay. The protocol was transferred from the NL-lab to two Sens-it-iv project partners (UK-lab, Italy-lab). Intra- and inter-laboratory variation with regards to MUTZ-3 progenitor culture, differentiation to MUTZ-LC, maturation and migration assay were investigated. In the transwell-migration-assay, preferential migration of sensitizer-exposed MUTZ-LC towards CXCL12 was observed (three sensitizers), whereas non-sensitizer-exposed MUTZ-LC only migrated towards CCL5 (two non-sensitizers). Four pre-pro-haptens were also identified by UK-lab. When taking the arbitrary criteria of at least two of three independent repetitions per laboratory having to have a CXCL12/CCL5 ratio >1.1 for classification as a sensitizer, all sensitizers tested in all labs were easily distinguished from all non-sensitizers. The number of repetitions giving false negative or false positive was very low (only 7 out of a total of 54 repetitions), indicating that both intra- and inter-laboratory variation was extremely low. Even though only a few chemicals were tested in this study, we show clearly that the in vitro DC migration assay is transferable between laboratories. The results were consistent between the laboratories, and the dose response data were reproduced in the three laboratories.     

A potential in vitro epidermal equivalent assay to determine sensitizer potency

Most in vitro assays aim to distinguish sensitizers from non-sensitizers. Few aim to classify sensitizers according to potency. Here, we describe a potential method for classifying sensitizers according to their irritant potency with the aid of in house epidermal equivalents (EE). Sixteen sensitizers were applied topically in a dose response to EE for 24 h. The EE-EC₅₀ value (effective chemical concentration required to reduce cell viability by 50%) and the EE-IL-1α_10× value (chemical concentration which increases IL-1α secretion by 10-fold) were calculated. From 16 sensitizers, EE-EC₅₀ and/or EE-IL-1α_10× values were obtained from 12 skin sensitizers. EE-EC₅₀ and IL-1α_10× values decreased in proportion to increasing sensitizer potency. The in vitro assay correlated with existing in vivo mouse and human sensitization data (LLNA, HRIPT), and showed low intra- and inter-experimental variability. Additionally DNCB and resorcinol were correctly assessed as extreme and moderate sensitizers using commercial EE (EST1000™ and RHE™). In conclusion, our data supports the view that irritancy may in part be a factor determining sensitizer potency. Since this assay does not distinguish sensitizers from non-sensitizers, its potential application is in a tiered strategy, where Tier 1 identifies sensitizers which may then tested in Tier 2, this assay, which determines sensitizer potency.     

Identification of PDL-1 as a novel biomarker of sensitizer exposure in dendritic-like cells

The development of novel in vitro methods to assess risks of allergic sensitization are essential in reducing animal testing whilst maintaining consumer safety. The main research objectives of this study were to identify novel biomarkers to assess the sensitization predictability of chemicals. Phenotypic and cytokine responses of moDCs and MUTZ-3 cells were investigated following application of contact sensitizers; dinitrochlorobenzene (DNCB), cinnamaldehyde (Cin), eugenol (E), isoeugenol (IE), P-phenylenediamine (PPD) and non-sensitizers; salicyclic acid (SA) and sodium lauryl sulphate (SLS). CD86 was up-regulated on MUTZ-3 cells in response to DNCB, Cin and PPD, however, moDCs only modulated CD86 in response to DNCB and E. PDL-1 (Programmed death receptor ligand-1) proved a promising sensitization biomarker in MUTZ-3 cells where up-regulation occurred in response to DNCB, Cin, IE and PPD. Additionally, moDC-expressed PDL-1 was modulated in response to Cin, IE and E thus demonstrating improved sensitizer predictability when compared with CD86. MCP-1 and RANTES were identified as biomarkers of DNCB exposure but MCP-1 did not show any change in expression above controls for the other sensitizers investigated. However, RANTES was increased in MUTZ-3 cells by both DNCB and Cin. Our findings highlight novel biomarkers which, in MUTZ-3 cells, could be taken forward within a multiple biomarker in vitro assay ensuring strong and reliable predictability.     

Peptide reactivity associated with skin sensitization: The QSAR Toolbox and TIMES compared to the DPRA

The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies.Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in ‘no alert’ for 83%–100% of the non-sensitizers, but alerted only 55%–61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new “DPRA profilers” (QSAR Toolbox) provided unsatisfactory results.Additionally, the results were combined in the ‘2 out of 3’ prediction model with in vitro data derived from LuSens and h-CLAT. Using DPRA results, the model identified 90% of human sensitizers and non-sensitizers; using in silico results (including abiotic and biotic activations) instead of DPRA results led to a comparable high predictivity.     

Effects of beta-tricalcium phosphate particles on primary cultured murine dendritic cells and macrophages

Beta-tricalcium phosphate (β-TCP) is widely used for bone substitution in clinical practice. Particles of calcium phosphate ceramics including β-TCP act as an inflammation mediators, which is an unfavorable characteristic for a bone substituent or a prosthetic coating material. It is thought that the stimulatory effect of β-TCP on the immune system could be utilized as an immunomodulator.Here, in vitro effects of β-TCP on primary cultured murine dendritic cells (DCs) and macrophages were investigated. β-TCP particles enhanced expression of costimulatory surface molecules, including CD86, CD80, and CD40 in DCs, CD86 in macrophages, and MHC class II and class I molecules in DCs. DEC205 and CCR7 were up-regulated in β-TCP-treated DCs. Production of cytokines and chemokines, including CCL2, CCL3, CXCL2, and M-CSF, significantly increased in DCs; CCL2, CCL3, CCL4, CCL5, CXCL2, and IL-11ra were up-regulated in macrophages. The results of the functional assays revealed that β-TCP caused a prominent reduction in antigen uptake by DCs, and that conditioned medium from DCs treated with β-TCP facilitated the migration of splenocytes in the transwell migration assay. Thus, β-TCP induced phenotypical and functional maturation/activation of DCs and macrophages; these stimulating effects may contribute to the observed in vivo effect where β-TCP induced extensive migration of immune cells. When compared to lipopolysaccharide (LPS), an authentic TLR ligand, the stimulatory effect of β-TCP on the immune systems is mild to moderate; however, it may have some advantages as a novel immunomodulator. This is the first report on the direct in vitro effects of β-TCP against bone marrow-derived DCs and macrophages.     

Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis

Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6 h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.     

Neutrophil migration towards C5a and CXCL8 is prevented by non-steroidal anti-inflammatory drugs via inhibition of different pathways

BACKGROUND AND PURPOSE

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to induce PG-independent anti-inflammatory actions. Here, we investigated the role of three different NSAIDs (naproxen, ibuprofen and oxaprozin) on neutrophil responses to CXCL8 and C5a.

EXPERIMENTAL APPROACH

Human neutrophils were isolated from healthy volunteers by dextran and Ficoll-Hypaque density gradients. Neutrophils were pre-incubated with different concentrations (1–100 µM) of NSAIDs or kinase inhibitors. Neutrophil degranulation into supernatants was tested by elisa and zymography. Neutrophil chemotaxis was determined using Boyden chambers. F-actin polymerization was determined by Alexa-Fluor 488-conjugated phalloidin fluorescent assay. Integrin expression was assessed by flow cytometry. The phosphorylation of intracellular kinases was studied by Western blot.

KEY RESULTS

Pretreatment with NSAIDs did not affect neutrophil degranulation, but inhibited neutrophil migration and polymerization of F-actin, in response to CXCL8 and C5a. Pretreatment with different NSAIDs prevented C5a-induced integrin (CD11b) up-regulation, while only ibuprofen reduced CXCL8-induced CD11b up-regulation. Pre-incubation with naproxen or oxaprozin, but not ibuprofen, inhibited the PI3K/Akt-dependent chemotactic pathways. Both endogenous (released in cell supernatants) or exogenous (added to cell cultures) PGE₂ did not affect C5a- or CXCL8-induced activities. Short-term incubation with NSAIDs did not affect neutrophil PGE₂ release.

CONCLUSION AND IMPLICATIONS

Treatment with NSAIDs reduced C5a- and CXCL8-induced neutrophil migration and F-actin polymerization via different mechanisms. Inhibition by ibuprofen was associated with integrin down-regulation, while naproxen and oxaprozin blocked the PI3K/Akt pathway. Both NSAID actions were independent of COX inhibition and PGE₂ release.     

A novel phenylcyclohex-1-enecarbothioamide derivative inhibits CXCL8-mediated chemotaxis through selective regulation of CXCR2-mediated signalling

Background and Purpose

Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function.

Experimental Approach

We used the CXCR2 Tango™ assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338.

Key Results

CX4338 selectively inhibited CXCR2-mediated recruitment of β-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice.

Conclusions and Implications

A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated β-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis.     

Applicability of a keratinocyte gene signature to predict skin sensitizing potential

There is a need to replace animal tests for the identification of skin sensitizers and currently many alternative assays are being developed that have very promising results. In this study a gene signature capable of very accurate identification of sensitizers was established in the HaCaT human keratinocyte cell line. This signature was evaluated in a separate study using six chemicals that are either local lymph node (LLNA) false-positive or false-negative chemicals in addition to nine sensitizers and four non-sensitizers. Similar studies do not apply these more difficult to classify chemicals, which show the true potential for human predictions of an assay. Although the gene signature has improved prediction accuracy compared to the LLNA, the misclassified compounds were comparable between the two assays. Gene profiling also showed a sensitizer specific response of the Nrf2-keap1 and Toll-like receptor signaling pathways. After exposure to non-sensitizing chemicals that induce either of the pathways the signature misclassified all Nrf2-inducers, while the Toll-like receptor ligands were correctly classified. In conclusion, we confirm that keratinocyte based prediction assays may provide essential information on the properties of compounds. Furthermore, chemical selection is critical for assessment of the performance of in vitro alternative assays.     

N-acetylated Proline-Glycine-Proline induced G-protein dependent chemotaxis of neutrophils is independent of CXCL8 release

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline–Glycine–Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation.     

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.     

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.     

Comparison of flow cytometry and immunohistochemistry in non-radioisotopic murine lymph node assay using bromodeoxyuridine

Non-radioisotopic local lymph node assay (LLNA) employing 5-bromo-2′-deoxyuridine (BrdU) with flow cytometry (FACS) or immunohistochemistry (IHC) is gaining attention due to a regulatory issue of using radioisotope, ³H-thymidine, in vivo in traditional LLNA. In this study, to compare the performance of these non-radioisotopic endpoints, 7 chemicals with known sensitizing potencies were examined in LLNA. Mice were topically treated with chemicals or vehicle on both ears for 3 days. After intraperitoneal injection of BrdU, bilateral lymph nodes were isolated separately and undergone respectively, FACS or IHC to determine BrdU incorporated lymph node cells (LNCs). Weight and histology of treated ears were also examined to evaluate chemical-induced edema and irritation. Both FACS and IHC could successively identify the skin sensitizers from non-sensitizers. Comparison of FACS and IHC with traditional LLNA revealed that FACS has a higher sensitivity although both assays produced comparable sensitivity and performance to traditional LLNA. In conclusion, non-radioisotopic LLNA using FACS and IHC can successfully detect sensitizers with a good correlation to traditional LLNA. Notably, FACS showed almost equivalent sensitivity and accuracy to traditional LLNA.     

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM–H₂DCFDA), for 15 min, then incubated with test chemicals for 30 min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals.     

Oligomannurarate sulfate inhibits CXCL12/SDF-1-mediated proliferation and invasion of human tumor cells in vitro

Aim:

JG6 is a novel marine-derived oligosaccharide that has shown to inhibit angiogenesis and tumor metastasis. In this study, we sought to identify the potential target responsible for the anti-cancer activity of JG6.

Methods:

Human liver cancer cell line Bel-7402 and human cervical cancer cell line HeLa were examined. CXCL12-stimulated cell proliferation and migration were determined using a CCK-8 kit and a transwell assay, respectively. Western blotting was performed to examine the changes in CXCL12/CXCR4 axis. Molecular docking and surface plasmon resonance (SPR) were performed to characterize the possible interaction between JG6 and the CXCL12/CXCR4 axis.

Results:

Treatment with CXCL12 potently stimulated the proliferation and migration in both Bel-7402 and HeLa cells. Co-treatment of the cells with JG6 (10, 50 and 100 μg/mL) dose-dependently impeded the CXCL12-stimulated cell proliferation and migration. Furthermore, CXCL12 rapidly induced phosphorylation of AKT, ERK, FAK and Paxillin in Bel-7402 and HeLa cells, whereas pretreatment with JG6 dose-dependently inhibited the CXCL12-induced phosphorylation of these proteins. The SPR assay showed that JG6 bound to CXCL12 with a high affinity. In molecular docking study, JG6 appeared to interact with CXCL12 via multiple polar interactions, including 6 ionic bonds and 7 hydrogen bonds.

Conclusion:

Inhibition of the CXCL12/CXCR4 axis by JG6 may account for its anticancer activity.     

CXCL8 secretion by dendritic cells predicts contact allergens from irritants.

Monocyte-derived dendritic cell functions have been explored for identification of contact allergens in vitro. Current methods, including measurement of changes in cell surface marker expression (e.g. CD83, CD86) do not provide a sensitive method for detecting the sensitising potential of a chemical. In this study, we investigated whether chemokine production by monocyte-derived dendritic cells is increased upon maturation and whether chemokine production can provide methodology for the detection of allergens. Monocyte-derived dendritic cells were exposed to allergens (nickel sulphate, cobalt chloride, palladium chloride, copper sulphate, chrome-(III)-chloride, potassium dichromate, p-phenylenediamine and dinitrochlorobenzene) and irritants (sodium dodecyl sulphate, dimethylsulphoxide, benzalkoniumchloride and propane-1-ol). CD83 and CD86 expression was analysed by flow cytometry and chemokine production (CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20, CCL22) was determined by ELISA. Significant up regulation of CD83 and CD86 expression could only be induced by three out of seven and five out of seven allergens, respectively. In contrast, CXCL8 production was significantly increased after stimulation with all allergens tested, whereas irritant exposure led to decreased CXCL8 production. All other chemokines tested, failed in identifying contact allergens. In conclusion, CXCL8 production, next to CD83 and CD86 up regulation, by monocyte-derived dendritic cells provides a promising in vitro tool for discrimination between allergens and irritants.