p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.     

UVB irradiation of normal human skin favors the development of type-2 T-cells in vivo and in primary dermal cell cultures

To determine the effect of UVB exposure on the balance of type-1 or type-2 T-cells in skin, we examined the expression of key markers interferon (IFN)-gamma and interleukin (IL)-4 in cryostat sections. IFN-gamma mRNA was clearly detectable in nonirradiated control skin, and IFN-gamma protein was found in 2% of the dermal CD3pos T-cells, whereas IL-4 mRNA was hardly detectable, and no IL-4 protein was found. In contrast, IL-4 mRNA expression increased upon irradiation, and IL-4 was found in 2% of the T-cells at day 2 after UVB-exposure. Concomitantly, IFN-gamma mRNA expression decreased, and IFN-gamma protein became absent. We also analyzed T-cells present in primary dermal cell cultures, which were used as an in vitro equivalent of the in vivo situation. As compared with T-cells from control skin, T-cells in dermal cell cultures from UVB-exposed skin displayed an increased IL-4 and decreased IFN-gamma expression. No such skewing occurred when the T-cells from irradiated skin were cloned in the absence of a dermal microenvironment. Except for an occasional positive T-cell, type-1-associated cell-surface markers (CCR5, CXCR3) or type-2 markers (CCR3, CD30, CRTH2) were undetectable in situ. But these markers were expressed on cultured dermal T-cells from UVB-exposed and control skin at a comparable level, but did not correlate with the IFN-gamma and IL-4 production. Altogether, UVB-induced changes of the dermal microenvironment favor the development of type-2 T-cells.     

Ultraviolet-B irradiation induces differential regulations of hyaluronidase expression and activity in normal human keratinocytes

Skin aging is a complex process determined by genetic factors (intrinsic aging) and environmental factors (extrinsic aging). One of the most influential environmental factor is UV-B irradiation. Hyaluronic acid (HA) is an abundant component of skin extracellular matrix where it plays many roles such as hydration and architectural support. Downregulation of HA during photoaging was reported previously. Changes in expression and function of its degrading enzymes, the hyaluronidases (Hyals) might be involved in this decrease. In the present study, normal human keratinocytes were submitted to increasing doses of UV-B. The mRNA expression of HYAL1, HYAL2 and HYAL3 and the hyaluronidase enzymatic activity were quantified using real-time PCR and a microtiter-based assay, respectively. After UV-B irradiation, HYAL1 mRNA expression was upregulated whereas HYAL2 and HYAL3 mRNAs were downregulated and hyaluronidase enzymatic activity was increased in both cell layer and culture medium. In parallel, immunohistochemical studies performed on UV-B irradiated reconstructed epidermis confirmed that Hyal-1, Hyal-2 and Hyal-3 protein expression were differently regulated by UV-B. Taken together, our results demonstrate that UV-B irradiation induces differential regulations of hyaluronidase expression and enzymatic activity in human keratinocytes. These differential modulations of hyaluronidase expression and activity by UV-B could contribute to cutaneous photoaging.     

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.     

Constitutive expression of 4-1BB on T cells enhances CD4+ T cell responses

4-1BB, a transmembrane molecule, member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule in the immune response, plays a key role in the clonal expansion and survival of CD8(+) T cells. In this study, we investigated 4-1BB regulation of CD4(+) T cell responses using 4-1BB transgenic (TG) mice that constitutively expressed 4-1BB on mature T cells. We first showed that CD4(+) T cells of 4-1BB TG mice had more sustained proliferative capacity in response to TCR/4-1BB stimulation in vitro compared to WT mice. Secondly, 4-1BB TG mice exhibited a more elevated contact hypersensitivity (CHS) response mediated by CD4+ Th1 cells due to more vigorous expansion of and apoptotic inhibition of CD4(+) T cells. Finally, CD4(+) T cells of 4-1BB TG mice had a heightened capacity for T cell priming. Overall, our results demonstrate the involvement of 4-1BB in CD4(+) Th1 cell responses by regulating the clonal expansion and survival of CD4(+) T cells as seen in CD8(+) T cells.     

A comparative analysis of alterations in protein expression after activation or human immunodeficiency virus, type 1 infection of human CD4+ T cells

We have previously described an in vitro model for studying human immunodeficiency virus, type 1 (HIV-1) infection in CD4+ T cells [1]. This model employs the WE17/10 cell line, which loses expression of its T cell receptor/CD3 (TCR/CD3) after several months of productive infection. We have used this model to analyze the synthesis and posttranslational modification of viral and cellular proteins after HIV-1 infection and to determine the relationship of these changes to TCR/CD3 expression. Mainly we observe positive changes in protein expression after infection. A phosphoprotein, referred to as WH:1, appears in infected cells that still express their TCR/CD3 complex, and its persistence is linked to the presence of the complex. We examined whether loss of the TCR/CD3 complex could be associated with alterations in the T cell activation pathway as a result of infection. We used T cell activators and inhibitors to determine whether there were common elements between the two events. Quantitative enhancement in one spot, Cs:1, occurred after both Cyclosporin A treatment of uninfected cells and HIV-1 infection of untreated cells. Taken altogether, these data suggest that a correlation exists between negative regulation of late events in the T cell activation pathway and down regulation of the TCR/CD3 complex after HIV-1 infection.     

Alternation in Photosynthetic Characteristics of Anabaena doliolum Following Exposure to UVB and Pb

Abstract— Anabaena doliolum , when exposed to either ultraviolet-B (UVB) radiation or Pb, showed reduced growth rate, carbon fixation, O₂-evolution, photosynthetic electron transport activity and ATP pool size. The rate of respiration was found to increase in UVB-treated cells; this increase was more pronounced in the cells exposed to UVB and Pb simultaneously. The UVB-induced inhibition of 2,6-dichlorophenol indophenol (DCPIP) photoreduction and lowering of chlorophyll a fluorescence could not be reversed by artificial electron donors (diphenyl carbazide, NH₂OH and MnCl₂). These electron donors, however, substantially reversed the inhibition caused by Pb, thereby suggesting that UVB primarily inhibits the photosys-tem II (PS II) reaction center and Pb arrests the electron flow at the water splitting site. Nevertheless, the suppressed fluorescence intensity and the reduced emission and excitation peaks of phycobilisomes indicate the involvement of Pb in inhibition of PS II. All combinations of UVB and Pb inhibited the different metabolic processes in a synergistic manner.     

ICAM-1/LFA-1 interaction contributes to the induction of endothelial cell-cell separation: implication for enhanced leukocyte diapedesis

The basic route and mechanism for diapedesis has not yet to be fully defined. Here we present evidence that "cell-cell separation" between endothelial cells (ECs) may provide a route for leukocyte diapedesis. We unexpectedly found that extensive interaction between peripheral blood leukocytes and ECs that were activated by TNF-α induced the opening of EC contacts and, surprisingly, resulted in cell-cell separation. This event was specific to the intercellular adhesion molecules-1 (ICAM-1)/leukocyte function-associated antigen-1 interaction, as demonstrated by the following: (1) ICAM-1 expression correlated with increased EC contraction; and (2) the blocking of ICAM-1 selectively inhibited EC separation. Thus, we suggest that "cell-cell separation" could be a mechanism for diapedesis in situations that may require massive leukocyte infiltration.     

人外周血CD4+IL-21+记忆T细胞的特征

目的:探讨人外周血中白细胞介索21(IL-21)的产生细胞及其特征.方法:分离人外周血单个核细胞(PBMC),分为不刺激或anti-CD3(OKT3)、OKT3+anti-CD28、PMA+ionomycin刺激四个组,流式细胞术(FCM)检测产生IL-21的细胞亚群.PMA+ionomycin刺激PBMC、纯化CD4+、CD4+CD45RA-、CD4+CD45RA+细胞、脐带血单个核细胞(CB-MC),FCM分析产生IL-21细胞的表型特征和IL-21与Th1、Th2、Th17和Th22细胞因子之间的关系.结果:与OKT3、OKT3+anti-CD28相比,PMA+ionomycin能诱导最高量的IL-21产生.产生IL-21的主要细胞为CD4+T细胞,少数CD8+T细胞.CD4+IL-21+T细胞表达CD45RO,不表达CD45RA,其中部分细胞表达CCR6、CCR7或CXCR5.CD4+CD45RA-细胞表达IL-21远高于CIM+CD45RA+细胞.进一步研究表明,PBMC产生IL-21,而CBMC不产生.此外,大约24%的CD4+IL-21+细胞表达IFN-γ,小于10%CD4+IL21+细胞表达IL-4、IL-17或IL-22.结论:人PBMC在多克隆刺激的条件下,可以诱导IL-21的产生.产生IL-21的主要细胞亚群具有记忆CD4+T细胞的表型.其中一部分CD4+IL-21+T细胞的表型独立于Th1、Th2、Th17和Th22细胞亚群.     

Low and moderate irradiances of UVB and UVC irradiation are equally erythemogenic in human skin

Abstract —The effect of irradiance on ultraviolet-induced delayed erythema was studied in normal human skin. With both UVB and UVC radiation, the minimal erythema dose was the same whether exposure lasted several seconds or 5 h. A reciprocal relationship between exposure duration and irradiance has previously been shown to exist for exposures ranging from nanoseconds to minutes. The present study shows that even if exposures last over 4 h the minimal erythema dose is a function of exposure dose and not of irradiance or exposure duration.     

Evaluation of the protective effect of sunscreens on in vitro reconstructed human skin exposed to UVB or UVA irradiation

We have previously shown that skin reconstructed in vitro is a useful model to study the effects of UVB and UVA exposure. Wavelength-specific biological damage has been identified such as the formation of sunburn cells (SBC) and pyrimidine dimers after UVB irradiation and alterations of dermal fibroblasts after UVA exposure. These specific effects were selected to evaluate the protection afforded by two sunscreens after topical application on the skin surface. Simplified formulations having different absorption spectra but similar sun protection factors were used. One contained a classical UVB absorber, 2-ethylhexyl-p-methoxycinnamate. The other contained a broad-spectrum absorber called Mexoryl SX, characterized by its strong absorbing potency in the UVA range. Both filters were used at 5% in a simple water/oil vehicle. The evaluation of photoprotection on in vitro reconstructed skin revealed good efficiency for both preparations in preventing UVB-induced damage, as shown by SBC counting and pyrimidine dimer immunostaining. By contrast, only the Mexoryl SX-containing preparation was able to efficiently prevent UVA-specific damage such as dermal fibroblast disappearance. Our data further support the fact that skin reconstructed in vitro is a reliable system to evaluate the photoprotection provided by different sunscreens against specific UVB and UVA biological damage.     

Hypoxia induces Wee1 expression and attenuates hydrogen peroxide-induced endothelial damage in MS1 cells

In an oxygen-depleted environment, endothelial cells initiate an adaptive pattern of synthesis, which may enable them to survive hypoxic crises. Using high-resolution two-dimensional gel electrophoresis in conjunction with mass spectroscopy, we obtained a 24 differential display of proteins in the pancreatic endothelial cell line, MS-1, at four time points following induction of hypoxia. The induction of Wee1 under hypoxia was confirmed both at the mRNA and protein levels. The phosphorylation of cell division cycle 2, which is downstream of Wee1, was also increased after hypoxic exposure. In addition, pre-exposure to hypoxia attenuated a decrease in hydrogen peroxide-induced cell number. The induction of bax (a pro-apoptotic protein) and reduction of bcl (an anti-apoptotic protein) after hypoxia stimulus were also attenuated by hypoxic pre-exposure. Moreover, hydrogen peroxide-induced morphologic damage did not appear in the wild-type Wee1-expressing cells. Taken together, our results suggest that Wee1 may have important role in hypoxia- induced pathophysiological situations in endothelial cells.     

CD137 induces adhesion and cytokine production in human monocytic THP-1 cells

CD137, which is expressed on activated T cells, plays a critical role in inflammatory responses. However, the exact role that CD137 plays in monocytes is not fully known. Here we studied the expression and function of CD137 in human monocytic THP-1 cells, which we found constitutively expresses CD137 at the mRNA and protein level. Cross-linking of CD137 increased the secretion of IL-8 and TNF-alpha, promoted the expression of CD54 and CD11b, and increased adhesion to extracellular matrix (ECM) proteins. In particular CD137-induced adhesion of THP-1 cells was inhibited by an inhibitor of mitogen-activated protein kinase kinase (MEK), but not by a p38 kinase inhibitor. Taken together, these results show that the adhesion and cytokine production of THP-1 cells induced by CD137 occur via activation of MEK, which results in the activation of ERK-1/2 signaling pathways. Therefore, this study suggests that CD137 induces an activating and migrating signal during inflammatory processes.     

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.     

Treatment of cutaneous T cell lymphoma with extracorporeal photopheresis induces Fas-ligand expression on treated T cells,but does not suppress the expression of co-stimulatory molecules on monocytes

Following extracorporeal photopheresis (ECP), lymphocytes become apoptotic and upregulate class I MHC antigenic peptides. Conversely, ECP treated monocytes demonstrate activation markers and have an increased avidity for the phagocytosis of apoptotic T cells. Processing of apoptotic T cells by monocytes, following ECP, is thought to induce an immunomodulatory response, which targets untreated, but clonal T cells. Recently we detected apoptotic lymphocytes immediately post ECP. Although enhanced CD95 (Fas) expression has been observed 24 h post ECP, CD95 and Fas-ligand (Fas-L) expression have not been determined at this very early apoptotic stage. Exposure of monocytes to UV has previously suppressed expression of the co-stimulatory molecules required for the presentation of processed antigens to T cells. Our data demonstrate no increase in CD95 or Fas-L expression on T cells tested immediately following ECP. However, the number of T cells expressing Fas-L significantly increased 24 h post ECP (P<0.005). The expression of the co-stimulatory molecules, CD54, CD80 and CD86, remained unaltered on monocytes treated by ECP. Although the mechanism responsible for early induction of lymphocyte apoptosis remains unclear, the later apoptosis involves Fas-L expression. The maintenance of co-stimulatory molecules, on treated monocytes, indicates that they retain the ability to induce an immunomodulatory response.     

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3⁺CCR4^-CD4⁺ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-γ, TNF-α, and IL-2 in CXCR3⁺CCR4^-CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4⁺ or CXCR3⁺CCR4^-CD4⁺ T cells with DC (CD4⁺/DC or CXCR3⁺CD4⁺/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-α and LPS (mDC). Although there was no significant difference between the effects of the CXCR3⁺CCR4^-CD4⁺ and CD4⁺ T cells on DC phenotype expression, CXCR3⁺CD4⁺/DC in CTL culture were able to expand number of CD8⁺ T cells and increased frequencies of IFN-γ secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3⁺CCR4^-CD4⁺ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.