XS Cells Research Articles

Stratification of yeast cells during chronological aging by size points to the role of trehalose in cell vitality.

Cells of the budding yeast Saccharomyces cerevisiae undergo a process akin to differentiation during prolonged culture without medium replenishment. Various methods have been used to separate and determine the potential role and fate of the different cell species. We have stratified chronologically-aged yeast cultures into cells of different sizes, using centrifugal elutriation, and characterized these subpopulations physiologically. We distinguish two extreme cell types, very small (XS) and very large (L) cells. L cells display higher viability based on two separate criteria. They respire much more actively, but produce lower levels of reactive oxygen species (ROS). L cells are capable of dividing, albeit slowly, giving rise to XS cells which do not divide. L cells are more resistant to osmotic stress and they have higher trehalose content, a storage carbohydrate often connected to stress resistance. Depletion of trehalose by deletion of TPS2 does not affect the vital characteristics of L cells, but it improves some of these characteristics in XS cells. Therefore, we propose that the response of L and XS cells to the trehalose produced in the former differs in a way that lowers the vitality of the latter. We compare our XS- and L-fraction cell characteristics with those of cells isolated from stationary cultures by others based on density. This comparison suggests that the cells have some similarities but also differences that may prove useful in addressing whether it is the segregation or the response to trehalose that may play the predominant role in cell division from stationary culture.

Live-cell imaging of compartment-specific redox changes in menkes disease fibroblasts

Background Copper is an essential micronutrient and its misbalance in the body is associated with severe neurodegenerative disorders. While the importance of copper in the body is evident, mechanisms by which copper misbalance induces pathologic changes and disease symptoms are poorly understood. In this study, using fibroblasts from Menkes disease patient, as a cellular model of copper accumulation, we examined whether excess copper triggers specific and distinct changes in the redox environment of different cellular compartments. Subjects and Methods Skin fibroblasts from Menkes disease patient (YS cells, ATP7A -/- ) and his heterozygous mother (ATP7A +/- ) were used as an experimental system. Glutathione mediated redox environment and levels of H2O2 were investigated in nuclei, cytosol, and mitochondria of live cells by tagging the respective ratiometric sensors (GRX-roGFP and HyPer) with a compartment-specific localization signal. Results Under basal conditions, the YS and XS cells show similar glutathione mediated redox environment in the nucleus and cytosol. However, the mitochondria are oxidizing in YS cells. YS cells were observed to accumulate higher level of peroxide in the cytosol and mitochondria. We also found that copper accumulation in cytosol and nuclei of YS cells sensitize cells to glutathione depletion suggesting importance of glutathione in protection of cells against copper overload Conclusion Our experiments revealed differential response of cellular compartments to excess copper in cells. Nuclei, in spite of being a site of copper accumulation, do not show marked redox changes, suggesting presence of robust protective mechanisms operating in this compartment. H2O2 accumulation in cytosol in YS cells does not change glutathione balance, whereas mitochondria appear most affected, since both H2O2 levels and glutathione balance are altered. We propose that mitochondria could be a primary site of copper toxicity in Menkes fibroblasts. Molecular mechanisms underlying differential redox responses are presently being investigated.

Nitric oxide is toxic to melanocytes in vitro.

Nitric oxide is a diffusible gaseous mediator generated from l-arginine by inducible and constitutive nitric oxide synthases. It has been associated with cytotoxic effects. Inflammatory cells and Langerhans cells can express the inducible form of nitric oxide synthase and produce large quantities of nitric oxide. The proximity of these cells to melanocytes could result in melanocyte cell death. We studied melanocyte susceptibility to nitric oxide using the nitric oxide donor compound sodium nitroprusside and nitric oxide released by the Langerhans like cell-line XS-52 following stimulation with lipopolysaccharide (LPS). Melanocyte lysis, quantified by chromium release in the presence of sodium nitroprusside was both time and concentration dependent. Co-culture of LPS-stimulated XS cells with melanocytes also resulted in melanocyte cell death. No cell death was observed when melanocytes alone were exposed to LPS. Melanocytes were killed even when the co-cultures were performed across Transwells in which there was no direct contact between XS cells and melanocytes. XS-induced melanocyte death was thus dependent on a diffusible factor consistent with nitric oxide. Cell death was markedly decreased in co-cultures performed in the presence of hemoglobin, a nitric oxide quencher. The possible role that nitric oxide may play in disorders associated with loss of pigmentation is discussed.

Epidermal Langerhans Cell-Targeted Gene Expression by a Dectin-2 Promoter

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5'-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

Granulocyte–Macrophage Colony-Stimulating Factor Gene Transfer to Dendritic Cells or Epidermal Cells Augments Their Antigen-Presenting Function Including Induction of Anti-Tumor Immunity

Dendritic antigen-presenting cells derived from epidermis (Langerhans cells), bone marrow, and peripheral blood can present a wide variety of antigens, including tumor-associated antigens, for various immune responses. The development and function of dendritic cells is dependent upon a number of cytokines including granulocyte-macrophage-colony-stimulating factor. For example, Langerhans cells can present tumor-associated antigens for the induction of substantial in vivo anti-tumor immunity but only after activation in vitro by granulocyte-macrophage-colony-stimulating factor. Thus, we reasoned that insertion of a cDNA for granulocyte-macrophage-colony-stimulating factor into dendritic antigen-presenting cells may allow for autocrine stimulation and increased antigen-presenting capability. To test this possibility, we utilized an adenovirus vector to insert a cDNA for murine granulocyte-macrophage-colony-stimulating factor into the dendritic cell lines XS52-4D and XS106 (derived from neonatal mouse epidermis), bone marrow-derived dendritic cells, and epidermal cells that contain Langerhans cells. Infection of each of these cell types resulted in release of abundant quantities of granulocyte-macrophage-colony-stimulating factor. XS52-4D and XS106 cells infected with adenovirus granulocyte-macrophage-colony-stimulating factor exhibited prolonged dendrites and greater expression of major histocompatibility complex class II molecules and CD86 compared with cells infected with a null vector. Granulocyte-macrophage-colony-stimulating factor cDNA-containing XS cells, bone marrow-derived dendritic cells, and epidermal cells had more potent alloantigen presenting capability than cells infected with a null vector. Most importantly, granulocyte-macrophage-colony-stimulating factor gene-transferred epidermal cells were able to present tumor-associated antigens for in vivo anti-tumor immunity against challenge with the S1509a spindle-cell tumor whereas null vector-infected cells were unable to prime for immunity. These results suggest that introduction of a cDNA for granulocyte-macrophage-colony-stimulating factor into dendritic cells may be an effective means to augment their antigen-presenting capability and that granulocyte-macrophage-colony-stimulating factor gene-transfer- red epidermal cells may be useful in tumor vaccination strategies.

T cell-mediated terminal maturation of dendritic cells.

Numerous investigators have supported, with compelling evidence, the concept that the activation of naive T cells occurs primarily through antigens (Ag) presented by a distinct class of leukocytes known as dendritic cells (DC). As skin biologists, our own efforts have focused on Langerhans cells (LC), a unique population of DC that resides normally within the epidermis of skin and whose identification even predates knowledge about DC by more than one century. Three years ago, we initiated a program to develop long-term DC lines from the epidermis of mice, largely to accommodate the cellular homogeneity that is required for molecular biologic techniques, but also to facilitate the economic use of laboratory animals. Two concepts guided these efforts: 1) cells derived during the perinatal period would possess a highest proliferative potential, and 2) GM-CSF, which had promoted the survival and maturation of LC cultured by other investigators, would be essential. Our efforts were ultimately rewarded by the development of stable DC lines, termed the “XS series”, from specimens of epidermis in newborn BALB/c mice (1). These XS DC lines resemble resident LC in many respects, including: a) their dendritic morphology (1), b) Ag presenting capacity (1,2), and c) cytokine and cytokine receptor mRNA profiles (3–6). Importantly, our original XS cell cultures were “contaminated” by fibroblastoid cells, the “NS series,” which could be removed through their selective sensitivity to treatment with trypsin. Once this was accomplished, however, proliferation by the XS cells diminished substantially. We have determined subsequently that XS52 cells require for their optimal growth not only GM-CSF, which was added continuously to XS cell cultures, but also CSF-1, which was secreted in relatively large amounts by the NS cells (4). These XS lines and similar DC lines developed since that time have proven to be useful tools in our efforts to characterize the function of DC.KeywordsDendritic CellDendritic Cell MaturationProliferative ResponsivenessTerminal MaturationMurine EpidermisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

UVB Radiation Interrupts Cytokine-mediated Support of an Epidermal-derived Dendritic Cell Line (XS52) By a Dual Mechanism

We have established long-term dendritic cell lines from the epidermis of newborn mice. These cell lines (XS series) proliferate maximally in response to granulocyte/macrophage-colony stimulating factor, as well as to CSF-1, which is produced by skin-derived NS fibroblast lines and by keratinocytes (albeit in smaller amounts). The purpose of this study was to examine the impact of UVB radiation on CSF-1-mediated interaction of dendritic cells with fibroblasts and keratinocytes. Exposure of NS cells to UVB radiation (unfiltered FS20 sunlamp) decreased CSF-1 production at mRNA and protein levels. Both changes occurred in a dose-dependent fashion, with 50 J/m2 causing a significant reduction. UVB radiation also downregulated CSF-1 mRNA expression by Pam 212 keratinocytes. UVB exposure of XS cells diminished the surface expression of CSF-1 receptors, with 50 J/m2 causing a significant reduction. Thus, UVB radiation interrupts CSF-1-mediated cell-cell interaction by a dual mechanism: downregulating CSF-1 production and abrogating CSF-1 receptor expression. Importantly, granulocyte/macrophage-colony stimulating factor receptor expression by XS cells was also inhibited by UVB radiation, once again, with 50 J/m2 producing significant inhibition. We propose that the resulting CSF-1 deficiency in epidermal microenvironment and unresponsiveness by dendritic cells to relevant growth factors may contribute to UVB-mediated loss of resident epidermal dendritic cells (i.e., Langerhans cells) in skin.

Phenotypic and Functional Heterogeneity Among Murine Epidermal-Derived Dendritic Cell Clones

We have established recently long-term dendritic cell lines from the epidermis of newborn BALB/c mice. These lines, termed XS series, resembled epidermal resident Langerhans cells or their progenitors in terms of surface phenotype, antigen-presenting capacity, and growth factor requirement. We examined in this study the degree of clonal heterogeneity among XS cells with respect to each of these features. Twelve stable clones were established by limiting dilution microculture from 8-10-week-old cultures of the XS52 or XS20 line. Despite the uniform expression of CD45, these clones varied substantially in their expression of Ia, B7-1, and B7-2 molecules. They also varied significantly in their relative efficiency in activating T cells. Finally, remarkable clone-to-clone heterogeneity was also observed in their growth factor responsiveness; some clones responded equally well to granulocyte macrophage-colony-stimulating factor-1, whereas others responded preferentially to one or the other of these factors. We propose that the observed clonal heterogeneity in XS cells reflects possible heterogeneity in the state of maturation and mitotic potential among the starting populations, i.e., skin-associated dendritic cells in newborn mice.

Identity and Functional Properties of Novel Skin-Derived Fibroblast Lines (NS Series) That Support the Growth of Epidermal-Derived Dendritic Cell Lines

We have established recently a series of unique cell lines (NS series) from dispase-separated mouse epidermis that promote the growth of epidermal-derived antigen-presenting cell lines (XS series). The purposes of this study were to determine the identity of NS cells and to characterize their functional properties. NS cells were distinguishable from leukocytes by the lack of typical surface markers and by the failure to respond to leukocyte growth factors. Despite their epidermal derivation, NS cells were distinct from keratinocytes by the absence of cytokeratins. On the other hand, NS cells were indistinguishable from lines of dermal fibroblasts by their a) morphology, b) surface phenotype, and c) intracellular deposits of type I collagen. Growth of the XS antigen-presenting lines has been promoted by co-culturing with gamma-irradiated NS cells, and this activity could be replaced with NS cell-conditioned media alone, but not with paraformaldehyde-fixed NS cells. Each clone derived from the NS01 line secreted XS cell-growth-promoting activity, and this activity was blocked by monoclonal antibodies against colony-stimulating factor-1 receptors. Dermal fibroblasts also promoted the growth of XS cells in a colony-stimulating factor-1-dependent manner. By contrast, culture supernatants from other cell lines derived from skin (e.g., Pam 212 keratinocytes, 7-17 dendritic epidermal T cells, or XS lines) failed to promote XS cell growth. These results indicate that NS cells belong to the fibroblast lineage and that they share the intrinsic property to secrete large amounts of colony-stimulating factor-1 with dermal fibroblasts. Dermal cells may support the growth of skin-associated antigen-presenting cells in vivo.

Interleukin‐1β converting enzyme in murine Langerhans cells and epidermal‐derived dendritic cell lines

Interleukin (IL)-1 beta plays an essential role in the induction of T cell-mediated immune responses in skin. Langerhans cells (LC), which constitutively express IL-1 beta mRNA, have been assumed to be the primary source of IL-1 beta in murine epidermis. The purpose of this study was to determine whether LC express mRNA for the IL-1 beta converting enzyme (ICE), a protease that is required for processing pro-IL-1 beta into an active form. Here, we report that both IL-1 beta and ICE mRNA are expressed by the Ia+ population (i.e. LC) in murine epidermis. Moreover, murine epidermal-derived DC lines (XS series) also express both IL-1 beta and ICE mRNA, and they secrete relatively large amounts of IL-1 beta following lipopolysaccharide (LPS) stimulation. Finally, LPS-triggered IL-1 beta secretion by XS cells is blocked almost completely by the ICE inhibitor acetyl-Tyr-Val-Ala-Asp-CH2OC(O)-[2,6-(CF3)2]Ph. These results demonstrate that LC are the primary source of IL-1 beta within the epidermis, and suggest that the proinflammatory role of IL-1 beta may be regulated pharmacologically by ICE inhibitors in vivo.

Colony-stimulating factor-1 secreted by fibroblasts promotes the growth of dendritic cell lines (XS series) derived from murine epidermis.

We have established recently from mouse epidermis long-term dendritic cell lines (XS series) that resemble epidermal Langerhans cells (LC) by their surface phenotype, Ag-presenting profile and cytokine mRNA profile. The growth of XS lines was promoted maximally by granulocyte-macrophage-CSF or by a factor secreted by NS lines, which are fibroblastic cell lines established from dispase-separated specimens of mouse epidermis. The purpose of this study was to determine the identity of XS cell growth factor secreted by NS cells. We report the following: 1) NS cells express constitutively mRNA for CSF-1; 2) XS cells express the CSF-1R at mRNA and protein levels; 3) rCSF-1 mimics NS culture supernatant in its ability to promote XS cell growth; 4) NS supernatant-dependent XS cell growth is blocked completely by each of two Abs against the CSF-1R. We conclude that CSF-1 is responsible for the XS growth-promoting activity secreted by NS lines. We also report the following: 5) LC freshly isolated from skin express CSF-1R mRNA; and 6) fibroblasts derived from specimens of dermis also express mRNA and secrete large amounts (50-100 ng/ml) of CSF-1. These observations give rise to a new concept that dermal fibroblasts may support the survival and growth of LC (and their precursors) through the paracrine effect of elaborated CSF-1.