Effect of trichloroacetaldehyde on the activation of CD4+T cells in occupational medicamentosa-like dermatitis: An in vivo and in vitro study.

Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is a hypersensitivity disease with autoimmune liver injury, which has increasingly become a serious occupational health problem in China. However, the pathogenesis of OMLDT remained undefined. In this study, 30 TCE-induced OMLDT patients, 58 exposure controls, and 40 non-exposure controls were recruited. We showed that the ratio of activated CD4+ T cells (downregulation of CD62 L) was dramatically increased in OMLDT patients compared to exposure and non-exposure control, suggesting that CD4+ T cells activation was a key cellular event in the development of OMLDT. In parallel, the expression of cytokine including IL-2, IFN-γ, TNF-α and IL-17A were increased obviously and IL-4 decreased in CD4+ T cells from OMLDT patients. in vitro assay, we found that trichloroethylene metabolites trichloroacetaldehyde (TCAH), not trichloroacetic acid (TCA) or Trichloroethanol (TCOH) could activate the naïve CD4+ T cells characterized by a rise in intracellular calcium, down-regulated CD62 L and subsequently trigger the secretion of IL-2, IFN-γ and TNF-α. Notably, the phosphorylation status of NF-κB and p38MAPK were elevated in OMLDT patients. Moreover, TCAH also could activate the p38MAPK and NF-κB, suggesting the role of p38MAPK and NF-κB pathways in the activation of CD4+ T cells. In addition, we found that the inhibition of Schiff base formation decreased the ability of TCAH to induce the activation of naïve CD4+ T cells and p38MAPK and NF-κB pathway. In conclusion, we revealed that the CD4+ T activation and increased the cytokines including IL-2, IFN-γ and TNF-α but decreased IL-4 in CD4+ T cells were associated with OMLDT. TCAH could activate naïve CD4+ T cells through NF-κB and p38MAPK activation induced by Schiff base formation, which might contribute to the development of OMLDT. These findings provide a new insight into the pathogenesis of OMLDT.

Chemical reactivity and skin sensitization potential for benzaldehydes: can Schiff base formation explain everything?

Skin sensitizers chemically modify skin proteins rendering them immunogenic. Sensitizing chemicals have been divided into applicability domains according to their suspected reaction mechanism. The widely accepted Schiff base applicability domain covers aldehydes and ketones, and detailed structure-activity-modeling for this chemical group was presented. While Schiff base formation is the obvious reaction pathway for these chemicals, the in silico work was followed up by limited experimental work. It remains unclear whether hydrolytically labile Schiff bases can form sufficiently stable epitopes to trigger an immune response in the living organism with an excess of water being present. Here, we performed experimental studies on benzaldehydes of highly differing skin sensitization potential. Schiff base formation toward butylamine was evaluated in acetonitrile, and a detailed SAR study is presented. o-Hydroxybenzaldehydes such as salicylaldehyde and the oakmoss allergens atranol and chloratranol have a high propensity to form Schiff bases. The reactivity is highly reduced in p-hydroxy benzaldehydes such as the nonsensitizing vanillin with an intermediate reactivity for p-alkyl and p-methoxy-benzaldehydes. The work was followed up under more physiological conditions in the peptide reactivity assay with a lysine-containing heptapeptide. Under these conditions, Schiff base formation was only observable for the strong sensitizers atranol and chloratranol and for salicylaldehyde. Trapping experiments with NaBH3CN showed that Schiff base formation occurred under these conditions also for some less sensitizing aldehydes, but the reaction is not favored in the absence of in situ reduction. Surprisingly, the Schiff bases of some weaker sensitizers apparently may react further to form stable peptide adducts. These were identified as the amides between the lysine residues and the corresponding acids. Adduct formation was paralleled by oxidative deamination of the parent peptide at the lysine residue to form the peptide aldehyde. Our results explain the high sensitization potential of the oakmoss allergens by stable Schiff base formation and at the same time indicate a novel pathway for stable peptide-adduct formation and peptide modifications by aldehydes. The results thus may lead to a better understanding of the Schiff base applicability domain.

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis.

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.

Schiff base-mediated co-stimulation primes the T-cell-receptor-dependent calcium signalling pathway in CD4 T cells.

In addition to macromolecular interactions that provide co-stimulation during antigen-presenting cell (APC) and CD4+ T-cell conjugation, covalent chemical events between specialized ligands have been implicated in T-cell co-stimulation. These take the form of transient Schiff base formation between carbonyls and amines expressed on APC and T-cell surfaces. Small Schiff base-forming molecules, such as tucaresol, can substitute for the physiological donor of carbonyl groups and provide co-stimulation to T cells, thereby functioning as orally active immunopotentiatory drugs. The Schiff base co-stimulatory pathway in T cells has been partially characterized in terms of changes in Na+ and K+ transport, and activation of the mitogen activated protein kinase (MAPK) ERK2. In the present study, the effects of Schiff base co-stimulation by tucaresol on the T-cell receptor (TCR)-dependent pathway leading to Ca2+ release were investigated. Schiff base co-stimulation by tucaresol was found to prime for enhanced TCR-dependent phospholipase C-gamma phosphorylation, inositol 1,4,5-triphosphate production, and Ca2+ mobilization that correlated with functional enhancement of interleukin-2 production in primary T cells. The effects on Ca2+ occurred comparably in Jurkat and primary CD4+ T cells responding to anti-CD3 monoclonal antibody. Enhancement of the Ca2+ response required a 10-min priming period and was prevented by prior covalent ligation of cell-surface free amino groups by sulpho-N-hydroxy succinimido-biotin; clofilium-mediated inhibition of tucaresol-induced changes in intracellular K+; and selective inhibition of the MAPK pathway. The data are consistent with a priming mechanism in which late co-stimulation-triggered events exert a positive influence on early TCR-triggered events. In additional studies of murine T cells expressing trans-gene TCRs, tucaresol was likewise shown to prime for enhanced Ca2+ mobilization in response to physiological TCR-engagement by MHC-peptide complexes.

Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway.

Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan-MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan-MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1,000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.