(1/132) Quantitative polymerase chain reaction using an external control mRNA for determination of gene expression in a heterogeneous cell population.

Gene expression can be evaluated quantitatively by conventional RT-PCR or Northern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantitating gene expression in a group of heterogeneous cell subsets, because the internal control gene expression may vary among the subsets. Therefore, we developed a new method for quantitative PCR using rat poly(A)+ RNA as an external control. We used this method to investigate cytokine gene expression in lymph node cells from mice during the induction of contact hypersensitivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAPDH) gene, a candidate internal control, was not constant in cells from trinitrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during induction of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitative PCR and was corrected based on external rat GAPDH cDNA. The reliability of this quantitative PCR was superior to that of the conventional method with an internal control.  (+info)

(2/132) Impaired contact hypersensitivity to trinitrochlorobenzene in interleukin-4-deficient mice.

We have examined the role of endogenously produced interleukin-4 (IL-4) in the contact hypersensitivity (CH) reaction to the haptene trinitrochlorobenzene (TNCB). The CH reaction was abolished in IL-4 genetically deficient mice (IL-4 KO), when compared to wild-type (wt) mice. The CH reaction was restored by treatment with IL-4 and further analysis revealed that IL-4 exerted its action both at the induction and effector stages of the CH reaction. Despite failure to develop a CH reaction, IL-4 KO mice developed a T helper type 1 (Th1) response to TNCB, in terms of lymphokine production in vitro. Furthermore, the number of Vgamma3+ cells accumulating in the lymph nodes of TNCB-immune IL-4 KO mice was normal. The recruitment of mononuclear cells and vascular leakage at the challenge site were consistently reduced in IL-4 KO mice and were restored by injection of IL-4. This suggests that IL-4 acts as a proinflammatory mediator in CH, perhaps favouring the accumulation of mononuclear cells at the site of inflammation. Among Th2-type cytokines, IL-13, but not IL-10, was shown to restore the CH reaction to TNCB in IL-4 KO mice. However, IL-4 KO mice developed a normal CH response to oxazolone, indicating that IL-4 was required for the CH reaction to TNCB, but not for that to oxazolone.  (+info)

(3/132) Simple chemicals can induce maturation and apoptosis of dendritic cells.

As is well known in the case of Langerhans cells, dendritic cells (DCs) play a crucial role in the initiation of immunity to simple chemicals such as noted in the contact hypersensitivity. Because DCs are scattered in non-lymphoid organs as immature cells, they must be activated to initiate primary antigen-specific immune reactions. Therefore, we hypothesized that some simple chemicals must affect the function of DCs. In this paper, we first demonstrated that human monocyte-derived DCs responded to such simple chemicals as 2, 4-dinitrochlorobenzene (DNCB), 2,4,6-trinitrochlorobenzene (TNCB), 2, 4-dinitrofluorobenzene (DNFB), NiCl2, MnCl2, CoCl2, SnCl2, and CdSO4 by augmenting their expression of CD86 or human leucocyte antigen-DR (HLA-DR), down-regulating c-Fms expression or increasing their production of tumour necrosis factor-alpha (TNF-alpha). In addition, the DCs stimulated with the chemicals demonstrated increased allogeneic T-cell stimulatory function. Next, we found that, among these chemicals, only NiCl2 and CoCl2 induced apoptosis in them. Finally, we examined the effects of these chemicals on CD86 expression by three different macrophage subsets and DCs induced from the cultures of human peripheral blood monocytes in the presence of macrophage colony-stimulating factor (M-CSF), M-CSF + interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and GM-CSF + IL-4, respectively. Among them, only DCs dramatically augmented their expression of CD86. These observations have revealed unique characteristics of DCs, which convert chemical stimuli to augmentation of their antigen presenting function, although their responses to different chemicals were not necessarily uniform in the phenotypic changes, cytokine production or in the induction of apoptosis.  (+info)

(4/132) IL-12 is produced by antigen-presenting cells stimulated with soluble alphabeta TCR and restores impaired T(h)1 responses.

Contact sensitivity (CS) is a cutaneous T(h)1 response that is induced by skin painting with reactive hapten. In prior in vivo studies of CS, we showed that recombinant soluble alphabetaTCR (sTCR) acted non-specifically to protect CS-effector T cells from suppression, but no molecular mechanism was determined. In the current study, we employed an in vitro system to investigate the mechanism of how sTCR protect CS-effector T cells from suppression. Immune CS-effector cells and appropriate hapten-conjugated antigen-presenting cells (APC) were incubated together with down-regulatory culture supernatant produced by suppressive spleen cells from mice tolerized i.v. with specific hapten, which produced strong inhibition of IFN-gamma production by the CS-effector cells. Importantly, addition of two different sTCR, of unrelated specificity, reversed this down-regulation and thus restored IFN-gamma production. We found that the APC, and not the CS-effector T cells, were the locus of the sTCR-mediated protection and showed direct binding of sTCR to APC by flow cytometry. Further, addition of anti-IL-12 showed that sTCR protection was due to IL-12 induced by sTCR and released by the APC, and was confirmed by ELISA measurement of IL-12 induced in APC supernatants by sTCR incubation. These results indicated a possible new regulatory loop in which suppression was reversed by IL-12 derived from APC, following direct surface binding of sTCR, and enhanced by IFN-gamma production from the T(h)1 CS-effector cells.  (+info)

(5/132) Ultraviolet B-induced suppression of immune responses in interleukin-4-/- mice: relationship to dermal mast cells.

Ultraviolet B radiation is immunosuppressive by multiple mechanisms. In interleukin-4-/- mice, ultraviolet B radiation was not able to suppress delayed-type hypersensitivity or contact hypersensitivity responses when the sensitizing antigen was applied to nonirradiated sites. In contrast, ultraviolet B significantly suppressed contact hypersensitivity responses to haptens applied to irradiated sites in interleukin-4-/- mice. In mast cell depleted Wf/Wf mice, ultraviolet B radiation also significantly suppressed contact hypersensitivity responses to sensitizing antigens applied to irradiated but not to unirradiated sites. In both interleukin-4-/- mice and Wf/Wf mice, the mast cell product, histamine, was immunosuppressive implicating mast cells as the dysfunctional cell in interleukin-4-/- mice. The prevalence of dermal mast cells was similar in wild-type and interleukin-4-/- mice. Dermal mast cells of interleukin-4-/- mice, however, express very low levels of c-kit and did not significantly degranulate in response to ultraviolet B. Ultraviolet radiation induced significant and similar levels of serum interleukin-10 in wild-type and interleukin-4-/- mice. We conclude that interleukin-4 indirectly affects ultraviolet B suppression of contact hypersensitivity and delayed-type hypersensitivity responses to sensitizing antigens applied at sites other than those irradiated by providing a critical differentiative signal for dermal mast cells. This study further emphasizes the central role of mast cells in the initial processes by which ultraviolet B radiation is immunomodulatory for immune responses to sensitizing antigens applied to nonirradiated sites.  (+info)

(6/132) Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity.

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6(-/)-) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6(-/)- mice compared with wt mice.A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6(-/)- mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate-modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6(-/)- mice, whereas the expression of interferon gamma was the same in STAT6(-/)- and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6(-/)- mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.  (+info)

(7/132) Transcription-coupled and global genome repair differentially influence UV-B-induced acute skin effects and systemic immunosuppression.

Exposure to UV-B radiation impairs immune responses in mammals by inhibiting especially Th1-mediated contact hypersensitivity and delayed-type hypersensitivity. Immunomodulation is not restricted to the exposed skin, but is also observed at distant sites, indicating the existence of mediating factors such as products from exposed skin cells or photoactivated factors present in the superficial layers. DNA damage appears to play a key role, because enhanced nucleotide excision repair (NER) strongly counteracts immunosuppression. To determine the effects of the type and genomic location of UV-induced DNA damage on immunosuppression and acute skin reactions (edema and erythema) four congenic mouse strains carrying different defects in NER were compared: CSB and XPC mice lacking transcription-coupled or global genome NER, respectively, as well as XPA and TTD/XPD mice carrying complete or partial defects in both NER subpathways, respectively. The major conclusions are that 1) transcription-coupled DNA repair is the dominant determinant in protection against acute skin effects; 2) systemic immunomodulation is only affected when both NER subpathways are compromised; and 3) sunburn is not related to UV-B-induced immunosuppression.  (+info)

(8/132) The effect of cyclophosphamide and irradiation on cells which suppress contact sensitivity in the mouse.

Contact sensitivity was produced in mice by painting the skin with picryl chloride and was assessed by the increase in ear thickness following local challenge. Contact sensitivity was passively transferred by immune lymph node and spleen cells taken at 4 days. The mice were then challenged immediately and the reactions read at 24 and 48 hr. Immune lymph node and spleen cells taken at day 8 virtually fail to transfer. Experiment showed that they contain cells which suppress passive transfer. These are demonstrated by mixing approximately equal numbers of 4-day cells, which transfer contact sensitivity, and cells taken at later times and injecting them intravenously into recipients. These 'suppressor cells' can be demonstrated by day 6 and are still present at day 11 after immunization. The precursors of the suppressor cells are sensitive to cyclophosphamide. Irradiation of immune mice 2 days before taking cells also selectively inactivates the suppressor cells. When mice are pretreated with cyclophosphamide before immunization or irradiated 2 days before transfer, the lymph node and spleen cells taken on day 9 after immunization transfer contact sensitivity. In contrast the same number of cells from untreated mice were inactive. This suggests that the cells which mediate passive transfer or their precursors may occur in an inhibited form in lymph nodes and spleen at later times after immunization. These suppressor cells in immune mice differ from the T suppressor cells produced by the injection of picryl sulphonic acid--an agent which causes unresponsiveness: (1) the precursors of the T suppressor cells resist cyclophosphamide; (2) the T suppressor cells are found in mice treated so as to produce unresponsiveness while the other type of suppressor cells occurs in mice immunized for contact sensitivity. However, both types of suppressor cells are selectively inactivated by irradiation as compared with the cells which mediate contact sensitivity and both are able to act on the effector stage of contact sensitivity.  (+info)