apyrase and Dermatitis--Contact

Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.

Topics: Adenine Nucleotides; Animals; Antigens, CD; Apyrase; Croton Oil; Dermatitis, Contact; Dermatitis, Irritant; Disease Models, Animal; Humans; Hydrolysis; Keratinocytes; Mice; Nucleotide Deaminases; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7

Topics: Adoptive Transfer; Animals; Antigens, CD; Apyrase; Biomarkers; CD4-Positive T-Lymphocytes; Cell Growth Processes; Cells, Cultured; Coculture Techniques; Dermatitis, Contact; Disease Models, Animal; Interleukin-10; Mice; Mice, Knockout; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes, Regulatory

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) produce immunosuppressive adenosine by degradation of adenosine triphosphate (ATP) by the ectonucleotidases CD39 and CD73. In this sequence of events, ATP is not only the substrate for generation of adenosine but it also activates the immunosuppressive functions of Tregs. To compare the effects of ATP on IL-10-deficient (IL-10(-/-)) Tregs with wild-type (wt) Tregs, we incubated both types of Tregs with ATP and assessed their phenotype and function. We show that IL-10(-/-) Tregs failed to become activated by ATP and were impaired in adenosine production. As a consequence, IL-10(-/-) Tregs were unable to block adherence of effector T cells to the endothelium in vitro. When testing the signaling of the ATP receptor P2X(7) in IL-10(-/-) Tregs, we recorded no elevation of intracellular calcium after engagement of P2X(7) receptors, as compared with wt Tregs, thus indicating that IL-10(-/-) Tregs fail to react normally to ATP and display impaired adenosine production, which explains their inability to suppress contact hypersensitivity responses. Therefore, when using IL-10(-/-) Tregs in different disease models, one has to take into account that adenosine production is abrogated and reduced suppressive effects may not be exclusively attributable to the lack of IL-10 production.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Calcium; Dermatitis, Contact; Interleukin-10; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Receptors, Purinergic P2X7; T-Lymphocytes, Regulatory

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) require activation to develop their full suppressive capacity. Similar to conventional T cells, Tregs can be activated via their TCRs; however, other means may be in place. We injected naive and nonactivated Tregs, being CD69(-)CD44(low)CD62L(+) into mice, and analyzed their phenotype after sensitization or challenge with the contact sensitizer 2,4,6-trinitro-1-chlorobenzene. We found that Tregs acquired an activated phenotype (CD69(+)CD44(high)CD62L(-)) in the draining lymph node after sensitization. In contrast, Ag challenge activated Tregs in the blood. This tissue-specific activation was induced by ATP, which was released at the respective tissue sites after sensitization or challenge, respectively. To demonstrate that activation was also essential for the induction of the suppressive function of Tregs, Tregs were treated with ATP receptor antagonists. In this study, we show that ATP receptor antagonists abrogated the suppressive effects of injected naive Tregs in contact hypersensitivity reactions. Thus, these data indicate that activation of Tregs via ATP in vivo provides a novel pathway of stimulating the suppressive function of Tregs.

Topics: 5'-Nucleotidase; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Dermatitis, Contact; Immune Tolerance; Lymphocyte Activation; Mice; Mice, Inbred BALB C; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Injection of regulatory T (Treg) cells into sensitized mice abrogates the elicitation phase of contact hypersensitivity (CHS) reactions by blocking the adherence of leukocytes to vascular endothelium.. We set out to analyze whether adenosine, a suppressive factor recently described as produced by Treg cells, can account for the suppression of the effector T-cell-endothelial cell (EC) interaction.. T cells and ECs were cultured in the presence of adenosine, and expression of adhesion molecules and adhesion of T cells to ECs under shear stress were assessed. Furthermore, we injected Treg cells derived from ectonucleotidase-deficient (CD39-/-) mice into sensitized mice and analyzed the sticking and rolling of leukocytes during a CHS response using intravital microscopy.. Adenosine or Treg cells, respectively, abrogated the adherence of effector T cells to ECs in vitro. Likewise, injection of adenosine and Treg cells abrogated the ear-swelling reaction, indicating a role of adenosine during Treg cell-induced suppression of CHS responses. As a source for Treg cell-derived adenosine, we identified the ectonucleotidase CD39 because CD39-deficient Treg cells did not prevent adhesion of leukocytes to the endothelium. Furthermore, we show that the impaired adhesion of effector T cells to inflamed endothelium was induced by adenosine-mediated downregulation of expression of E- and P-selectin on the vascular endothelium.. Adenosine release by Treg cells is essential to block leukocyte adhesion to endothelium, providing a novel mechanism by which Treg cells mediate immune suppression in vivo.

Topics: Adenosine; Animals; Antigens, CD; Apyrase; CD4 Antigens; Cell Communication; Cell Proliferation; Dermatitis, Contact; E-Selectin; Endothelial Cells; Immune Tolerance; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; T-Lymphocytes, Regulatory

Xeroderma pigmentosum group A (XPA) gene-deficient mice easily developed skin cancers by the application of topical chemical carcinogens as well as by UV irradiation. As certain chemical carcinogens have been shown to be immunosuppressive, we examined the inflammatory and immunosuppressive effects of dimethylbenz(a)anthracene (DMBA) on XPA mice. Compared with wild-type mice, XPA mice showed greater ear swelling and reduction of epidermal Langerhans cells after DMBA application. Topical application of DMBA impaired the induction of contact hypersensitivity, initiated either locally or at distant sites. These DMBA-induced local and systemic immunosuppressions were more greatly enhanced in XPA mice than in wild-type mice. DMBA application induced pronounced production of PGE(2), IL-10, and TNF-alpha in the skin of XPA mice. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited DMBA-induced inflammation and local immunosuppression. In XPA mice, increased serum IL-10 was detected after DMBA treatment. Excess production of PGE(2), TNF-alpha, and IL-10 after DMBA application may be involved in the enhanced local and systemic immunosuppression in DMBA-treated XPA mice. Susceptibility to DMBA-induced skin tumors in XPA mice may be due to easy impairment of the immune system by DMBA in addition to a defect in the repair of DMBA-DNA adduct. Enhanced immunosuppression by chemical carcinogens as well as the mutagenicity of these mutagens might be associated with the high incidence of internal malignancies seen in XP patients. Moreover, these results supported the hypothesis that persistent DNA damage is a trigger for the production of immunoregulatory cytokines.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adjuvants, Immunologic; Administration, Cutaneous; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apyrase; Carcinogens; Dermatitis, Contact; Dinitrofluorobenzene; Dinoprostone; Disease Models, Animal; DNA-Binding Proteins; Ear; Edema; Female; Immunosuppressive Agents; Indomethacin; Inflammation; Interleukin-10; Langerhans Cells; Mice; Mice, Hairless; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Skin; Tumor Necrosis Factor-alpha; Up-Regulation; Xeroderma Pigmentosum; Xeroderma Pigmentosum Group A Protein

Xeroderma pigmentosum group A (XPA) gene-deficient mice cannot repair UV-induced DNA damage and easily develop skin cancers by UV irradiation. Just like human XP patients, homozygous (-/-) mice developed stronger longer-lasting acute inflammation than did wild-type mice after a single irradiation with UVB. Moreover, the model mice showed more severe UV-induced damage of keratinocytes and Langerhans cells than did the control mice. UVB-induced local and systemic immunosuppression was greatly enhanced in the (-/-) mice. Treatment with indomethacin, an inhibitor of prostaglandin (PG) synthesis, inhibited UV-induced inflammation and abrogated immunosuppression. In XPA-deficient mice, the amount of PGE2 and the expression level of COX-2 mRNA greatly increased after UVB irradiation compared with wild-type mice. These results suggest that the excess DNA photoproducts remaining in XPA-deficient cells after UV radiation induce COX-2 expression and subsequently produce a high amount of PGE2, which causes the enhancement of inflammation and immunosuppression. In XPA-deficient mice, the natural killer cell activity significantly decreased after repeated exposures to UVB. Our experimental data indicate that cancer development in XP patients involves not only mutagenesis due to the defect in DNA repair, but also the enhanced UV-immunosuppression and intensified impairment of natural killer function.

Topics: Animals; Apyrase; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dermatitis, Contact; DNA-Binding Proteins; Immunosuppression Therapy; Indomethacin; Isoenzymes; Killer Cells, Natural; Langerhans Cells; Lymphocyte Count; Mice; Mice, Knockout; Photobiology; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Radiodermatitis; RNA, Messenger; Sunburn; Ultraviolet Rays; Xeroderma Pigmentosum Group A Protein

Xeroderma pigmentosum group A (XPA) gene-deficient mice were developed by gene targeting in mouse embryonic stem cells. To examine whether these XPA-model mice display photodermatologic abnormalities similar to those in human xeroderma pigmentosum, we investigated the effects of acute ultraviolet radiation on the homozygous (-/-) mice compared to the wild type (+/+) and heterozygous (+/-) mice. A single irradiation with ultraviolet B or topical psoralen plus ultraviolet A treatment induced stronger and longer lasting ear swelling in the (-/-) mice than in the (+/+) and (+/-) mice. Histologic changes including epidermal necrosis, cell infiltration, and sunburn cell formation after ultraviolet B radiation were more prominent in the (-/-) model mice than in the control mice. The (-/-) model mice showed damage of ADPase(+)Langerhans cells at a lower ultraviolet B dose than did the control mice. Moreover, the reappearance of ADPase(+)Langerhans cells after ultraviolet B radiation was delayed in the (-/-) mice compared to the control mice. Although contact hypersensitivity was induced equally in all mice, ultraviolet B-induced local and systemic immunosuppression were greatly enhanced in the (-/-) model mice. The data suggest that the XPA gene-deficient mice may be a useful model of human XPA, because the responses to UV radiation in the mice were very similar to those in the patients with XPA. Moreover, it is possible that enhanced ultraviolet immunosuppression is involved in the development of skin cancers in xeroderma pigmentosum.

Topics: Animals; Apyrase; Dermatitis, Contact; Immunosuppression Therapy; Langerhans Cells; Mice; Mice, Mutant Strains; PUVA Therapy; Skin; Sunburn; Ultraviolet Rays; Xeroderma Pigmentosum

Topics: Animals; Apyrase; Cell Count; Cryosurgery; Dermatitis, Contact; Dinitrofluorobenzene; Epidermis; Histocompatibility Antigens Class II; Immune Tolerance; Immunization; Langerhans Cells; Mice; Mice, Inbred BALB C