Chibing Tan

Effects of Hepatic Zonal Oxygen Levels on Hepatocyte Stress Responses

BACKGROUND Hepatocytes spend their lifetimes in a gradient of oxygen, hormones, and enzymes. We used a three-dimensional Matrigel model to determine whether hepatocytes cultured at perivenous (zone 3) oxygen levels differed in susceptibility to anoxia-induced cell injury compared with hepatocytes cultured at periportal (zone 1) oxygen levels. MATERIALS AND METHODS Hepatocytes were harvested from Sprague Dawley rats and cultured at 9% oxygen (hepatic zone 1) or 5% oxygen (hepatic zone 3) and stressed at 0% oxygen. Microscopy, real-time reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to assess cell viability, mitochondrial potential, acute phase responses, and membrane blebbing. RESULTS Hepatocytes cultured in Matrigel with HepatoZyme medium at zone 1 and zone 3 oxygen conditions were viable for 1 wk and showed acute phase responses as measured by interleukin-6-induced fibrinogen production. In response to 3 h anoxia, cells maintained at the perivenous oxygen level showed increased membrane blebbing and increased loss of mitochondrial membrane potential in comparison to the periportal oxygen cultured cells. Cells at perivenous oxygen also showed a reduced ability to recover following reoxygenation. CONCLUSIONS Hepatocytes can remain viable and functional for extended periods in culture at low oxygen levels that mimic the hepatic perivenous environment, yet these cells are more susceptible to anoxia-induced damage than hepatocytes cultured at the periportal oxygen level. The small population of perivenous hepatocytes may be critical in determining the fate of the liver during ischemia/reperfusion since hepatocytes cultured at that concentration appear to be more labile in response to anoxia.

Disruption of thymopoiesis in ST6Gal I-deficient mice

Thymocyte development is accompanied by sequential changes in cell surface glycosylation. For example, medullary thymocytes have increased levels of alpha2,3-linked sialic acid and a loss of asialo core 1 O-glycans as compared to cortical thymocytes. Some of these changes have been linked to fine tuning of the T cell receptor avidity. We analyzed ST6Gal I transcript abundance and levels of alpha2,6-linked sialic acid across thymocyte subsets. We found that ST6Gal I transcript levels increased following T cell receptor beta-selection suggesting that this sialyltransferase may influence the development of early thymocyte populations. Indeed, low levels of alpha2,6-linked sialic acid were found in the earliest T lineage cells, and then increased in T cell receptor beta-selected cells. To determine whether ST6Gal I influences T cell development, we analyzed ST6Gal I-deficient mice for disruptions in thymocyte populations. We found reduced thymic cellularity in the ST6Gal I-deficient mice starting in the early thymocyte compartments.

Impaired Thymopoiesis in Interleukin-7 Receptor Transgenic Mice Is Not Corrected by Bcl-2

Murine thymocytes down-regulate IL-7 responsiveness following beta-selection and reacquire sensitivity after positive selection. To assess the potential consequences of IL-7 signaling during this phase of development, transgenic IL-7 receptor alpha (IL-7Ralpha) mice were evaluated for IL-7 responsiveness as gauged by STAT-5 phosphorylation. Transgenic IL-7Ralpha expression increased the percentage of thymocytes responsive to IL-7 yet resulted in a decrease in total thymic cellularity. Aberrant thymocyte development in transgenic mice was first manifested by a reduction of DN3 thymocytes that correlated with lower Bcl-2 expression. Surprisingly, transgenic restoration of Bcl-2 expression did not correct thymic hypocellularity induced by IL-7Ralpha overexpression. These findings demonstrate that failure to appropriately downregulate IL-7Ralpha expression interferes with thymocyte development past the pro-T stage resulting in significantly lower levels of mature thymocytes.

CD28 expression redefines thymocyte development during the pre-T to DP transition

CD27 and CD28 have emerged as indicators demarcating the transition of thymocytes through beta-selection. We found that CD28 exhibits a greater dynamic range of expression during this phase, thus it was employed to further parse the DN/CD44(-) compartment in order to assess IL-7 signaling during the beta-selection process. Plotting CD28 versus CD25 expression revealed six DN/CD44(-) populations. OP9-DL1 stromal cell co-culture was used to demonstrate a developmental linkage from DN3a (CD25(+)CD28(-/lo)) to DN3b (CD25(+)CD28(+)) to DN3c (CD25(int)CD28(+)) to DN4a (CD25(-)CD28(+)) to double positive (DP) and showed the DN4b (CD25(-)CD28(hi)) and DN4c (CD25(-)CD28(-/lo)) populations to be inefficient in producing DP cells. Using CD69 as an additional marker to further parse the DN4a population, we found the pre-DP cells to be the CD44(-)CD25(-)CD28(int)CD69(-)CD4(-/lo)CD8(-/lo) subset. Using this refined developmental scheme, IL-7R alpha expression was found to be transiently up-regulated post-beta-selection in the DN3b and DN3c subsets; however, this increase did not confer enhanced responsiveness over that observed in the DN3a population. CD28 messenger RNA expression was up-regulated in post-beta-selected cells, whereas transcripts for CD27, IL-7R alpha and Bcl-2 were lower than that observed in the DN3a population. This study refines the current thymocyte differentiation scheme to allow for more detailed evaluation of events controlling early T-cell development, specifically surrounding the beta-selection checkpoint.

Differential IL-7 responses in developing human thymocytes.

Interleukin (IL)-7 is a factor essential for mouse and human thymopoiesis. Mouse thymocytes have altered sensitivities to IL-7 at different developmental stages. CD4/CD8 double positive (DP) mouse thymocytes are shielded from the influence of IL-7 because of loss of CD127 (IL-7Ralpha). In this study, we assessed IL-7 receptor expression and IL-7 signaling in human thymocytes. We found human DP cells to be severely limited in their ability to phosphorylate STAT-5 in response to IL-7. The relative expression levels of the IL-7-inducible proteins Bcl-2 and Mcl-1 were also lower in human DP cells, consistent with a stage-specific decrease in IL-7 responsiveness. IL-7 responses were restored in a subset of cells that matured past the DP stage. Unlike the regulation of IL-7 signaling in mouse thymocytes, loss of IL-7 signaling in human DP cells was not due to absence of CD127, but instead correlated with downregulation of CD132 (common gamma chain).

Altered populations of natural killer cells, cytotoxic T lymphocytes, and regulatory T cells in major depressive disorder: Association with sleep disturbance

A subset of individuals with major depressive disorder (MDD) have impaired adaptive immunity characterized by a greater vulnerability to viral infection and a deficient response to vaccination along with a decrease in the number and/or activity of T cells and natural killer cells (NKC). Nevertheless, it remains unclear which specific subsets of lymphocytes are altered in MDD, a shortcoming we address here by utilizing an advanced fluorescence-activated cell sorting (FACS) method that allows for the differentiation of important functionally-distinct lymphocyte sub-populations. Furthermore, despite evidence that sleep disturbance, which is a core symptom of MDD, is itself associated with alterations in lymphocyte distributions, there is a paucity of studies examining the contribution of sleep disturbance on lymphocyte populations in MDD populations. Here, we measured differences in the percentages of 13 different lymphocytes and 6 different leukocytes in 54 unmedicated MDD patients (partially remitted to moderate) and 56 age and sex-matched healthy controls (HC). The relationship between self-reported sleep disturbance and cell counts was evaluated in the MDD group using the Pittsburgh Sleep Quality Index (PSQI). The MDD group showed a significantly increased percentage of CD127low/CCR4+ Treg cells, and memory Treg cells, as well as a reduction in CD56+CD16- (putative immunoregulatory) NKC counts, the latter, prior to correction for body mass index. There also was a trend for higher effector memory CD8+ cell counts in the MDD group versus the HC group. Further, within the MDD group, self-reported sleep disturbance was associated with an increased percentage of effector memory CD8+ cells but with a lower percentage of CD56+CD16- NKC. These results provide important new insights into the immune pathways involved in MDD, and provide novel evidence that MDD and associated sleep disturbance increase effector memory CD8+ and Treg pathways. Targeting sleep disturbance may have implications as a therapeutic strategy to normalize NKC and memory CD8+ cells in MDD.

IL-18 Acts in Synergy with IL-7 To Promote Ex Vivo Expansion of T Lymphoid Progenitor Cells

Although IL-18 has not previously been shown to promote T lymphopoiesis, results obtained via a novel data mining algorithm (global microarray meta-analysis) led us to explore a predicted role for this cytokine in T cell development. IL-18 is a member of the IL-1 cytokine family that has been extensively characterized as a mediator of inflammatory immune responses. To assess a potential role for IL-18 in T cell development, we sort-purified mouse bone marrow–derived common lymphoid progenitor cells, early thymic progenitors (ETPs), and double-negative 2 thymocytes and cultured these populations on OP9–Delta-like 4 stromal layers in the presence or absence of IL-18 and/or IL-7. After 1 wk of culture, IL-18 promoted proliferation and accelerated differentiation of ETPs to the double-negative 3 stage, similar in efficiency to IL-7. IL-18 showed synergy with IL-7 and enhanced proliferation of both the thymus-derived progenitor cells and the bone marrow–derived common lymphoid progenitor cells. The synergistic effect on the ETP population was further characterized and found to correlate with increased surface expression of c-Kit and IL-7 receptors on the IL-18–treated cells. In summary, we successfully validated the global microarray meta-analysis prediction that IL-18 affects T lymphopoiesis and demonstrated that IL-18 can positively impact bone marrow lymphopoiesis and T cell development, presumably via interaction with the c-Kit and IL-7 signaling axis.

Ten-Color flow cytometry reveals distinct patterns of expression of CD124 and CD126 by developing thymocytes

BackgroundWe have developed a 12-parameter/10-color flow cytometric staining method for the simultaneous detection and characterization of 21 mouse thymocyte subpopulations that represent discreet stages of T cell development. To demonstrate the utility of this method, we assessed cytokine receptor expression on mouse thymocyte subsets. These experiments revealed distinct patterns of surface expression of receptors for the cytokines IL-4 and IL-6.ResultsThe IL-4 receptor α chain (CD124) was highly expressed on the earliest thymocyte subsets, then downregulated prior to T cell receptor β-selection and finally upregulated in the CD4/CD8 double positive cells prior to positive selection. The IL-6 receptor α chain (CD126) showed a different pattern of expression. It was expressed on the most mature subsets within the CD4 and CD8 single positive (SP) compartments and was absent on all other thymocytes with the exception of a very small cKit-CD4-CD8- population. Intracellular staining of SP thymocytes for phosphorylated STAT-1 demonstrated that IL-6 signaling was confined to the most mature SP subsets.ConclusionsThis 12-parameter staining methodology uses only commercially available fluorochrome-coupled monoclonal antibodies and therefore could be employed by any investigator with access to a 4-laser flow cytometer. This novel staining scheme allowed us to easily phenotype thymocyte subpopulations that span across development, from the early thymic progenitors (ETPs) to the most mature subsets of the CD4 and CD8 single positive populations.