Alcohol affects the late differentiation of progenitor B cells.

AIMS: Previous studies show that alcohol exposure can affect the differentiation of progenitor B cells. Before final commitment to a B lineage, progenitor B cells usually undergo several important stages. However, it is still unclear whether alcohol alters B cell differentiation at which stages. The aim of this study was to determine which stage(s) of progenitor cell differentiation are affected by alcohol and to elucidate the mechanism(s) responsible for the effect of alcohol on B cell differentiation. METHODS: Oligoclonal-neonatal-progenitor (ONP) cells from bone marrow cells of 2-week-old mice were cultured under different conditions in vitro with or without the exposure of 100 mM alcohol. Phenotype analysis was performed at different time points and expression levels of transcription factors (TFs) and cytokine receptors were measured quantitatively and kinetically. RESULTS: After 3 days in vitro culture, ONP cells differentiated into two populations: B220(-)CD11b(-) and B220(-)CD11b(+) cells. B220(-)CD11b(-) cells can further differentiate into B lineage cells only with the support of B220(-)CD11b(+) cells. Cells exposed to 100 mM of alcohol during the first 3 days of culture showed no statistically significant difference in B cell formation after 12 days compared with the control group. However, cells exposed to alcohol from Day 4 till the end of culture yield very few B cells. Expression levels of TFs and cytokine receptors were down-regulated kinetically among ONP cells co-cultured with the addition of 100 mM alcohol. CONCLUSIONS: Alcohol affects the ONP cell differentiation into B lineage at a late stage. Alcohol also down-regulates the expression level of TFs and cytokine receptors resulting in the impairment of B cell differentiation.

CD4+ T cells are required for the priming of CD8+ T cells following infection with herpes simplex virus type 1.

The role of CD4(+) helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4(+) T cells are needed for the generation of the protective HSV-1-specific CD8(+)-T-cell response. This study examined the contribution of CD4(+) T cells in the generation of the primary CD8(+)-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8(+)-T-cell response generated in the draining lymph nodes of CD4(+)-T-cell-depleted C57BL/6 mice and B6-MHC-II(-/-) mice is quantitatively and qualitatively distinct from the CD8(+) T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8(+) T cells express comparable levels of the activation marker CD44 in mice lacking CD4(+) T cells and normal mice. In contrast, CD8(+) T cells generated in the absence of CD4(+) T cells express the interleukin 2 receptor alpha-chain (CD25) at lower levels. Importantly, the CD8(+) T cells in the CD4(+)-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8(+) T cells is diminished in the absence of CD4(+)-T-cell help. These results suggest that CD4(+)-T-cell help is essential for the generation of fully functional CD8(+) T cells during the primary response to HSV-1 infection.

Dendritic cells are required for optimal activation of natural killer functions following primary infection with herpes simplex virus type 1.

Natural killer (NK) cells play an important role in the optimal clearance of herpes simplex virus type 1 (HSV-1) infection in mice. Activated NK cells function via cytokine secretion or direct cytolysis of target cells; dendritic cells (DCs) are thought to make critical contributions in the activation of both of these functions. Yet, the magnitude and physiological relevance of DC-mediated NK cell activation in vivo is not completely understood. To examine the contribution of DC help in regulating NK cell functions after infection with HSV-1, we utilized a transgenic mouse model that allows the transient ablation of DCs. Using this approach, it was found that the gamma interferon (IFN-gamma) expression potential of NK cells is quantitatively and qualitatively impaired in the absence of DCs. With regard to priming of NK cytolytic functions, the ablation of DCs did not significantly affect cytotoxic protein expression by NK cells. An in vivo cytolytic assay did, however, reveal impairments in the magnitude of NK cell cytotoxicity. Overall, this study provides direct evidence that functional DCs are required for optimal IFN-gamma expression and cytolytic function by NK cells following infection with HSV-1.

Ethanol exhibits specificity in its effects on differentiation of hematopoietic progenitors.

Ethanol is a known teratogen but the mechanisms by which this simple compound affects fetal development remain unresolved. The goal of the current study was to determine the mechanism by which ethanol affects lymphoid differentiation using an in vitro model of ethanol exposure. Primitive hematopoietic oligoclonal-neonatal-progenitor cells (ONP), with the phenotype Lin(-)HSA(lo)CD43(lo)Sca-1(-)c-Kit(+) that are present in neonatal but not adult bone marrow were sorted from the bone marrow of 2-week-old C57BL/6J mice and cultured under conditions that favor either B cell or myeloid cell differentiation with or without addition of ethanol. The overall growth of the ONP cells was not significantly affected by inclusion of up to 100mM ethanol in the culture medium. However, the differentiation of the progenitor cells along the B-cell pathway was significantly impaired by ethanol in a dose-dependent manner. Exposure of ONP cells to 100mM ethanol resulted in greater than 95% inhibition of B cell differentiation. Conversely, ethanol concentrations up to and including 100mM had no significant effect on differentiation along the myeloid pathway. The effect of ethanol on transcription factor expression was consistent with the effects on differentiation. ONP cells grown in 100mM ethanol failed to upregulate Pax5 and EBF, transcriptional regulators that are necessary for B cell development. However, ethanol had no significant effect on the upregulation of PU.1, a transcription factor that, when expressed in high concentration, favors myeloid cell development. Taken together, these results suggest that ethanol has specificity in its effects on differentiation of hematopoietic progenitors.

TCRbeta enhancer activation in early and late lymphoid progenitors.

An earlier report from our laboratory indicates that the activation of the T cell receptor (TCR) beta enhancer (Ebeta) is not always an indicator of T lineage potential in bone marrow-resident pre-lymphocytes. In order to more precisely investigate the consequences of Ebeta activation in lymphopoiesis, a genetic reporter animal, in which the expression of green fluorescent protein (GFP) is controlled by Ebeta, was used to examine two well-defined lymphopotent populations. Adoptive transfer experiments suggest that primitive lymphoid precursor populations (specifically, hematopoietic stem cells) consist of two discrete-populations discernible by Ebeta-GFP activation, although the two populations display no overt differences in lineage potential. In contrast, subsets of more differentiated pre-lymphocytes (specifically, common lymphoid progenitors), while also discernible by Ebeta-GFP activation, display different capacities for reconstituting lymphoid compartments. Interestingly, late lymphoid progenitors containing inactive Ebeta elements generated both T and B cells in vivo, in accord with the original description of this population; however, progenitors containing active Ebeta elements displayed an unexpected bias toward the B lineage. Our findings suggest that Ebeta activation is an indicator of B lineage specification in late, but not early lymphoid precursors.

A cancer gene therapy approach through translational control of a suicide gene.

The translation initiation factor eIF4E is elevated in most solid tumors resulting in translation of mRNAs that are normally repressed by their structured 5' untranslated region. We have introduced a translational repressor element in a vector (BK-UTK) designed to express herpes thymidine kinase (HTK). This and a control vector (BK-TK) were used to treat experimental tumors of a murine breast cancer line. Both vectors were equally effective in reducing subcutaneous tumors and lung metastases following ganciclovir administration. However, the BK-TK vector was found to be highly toxic, resulting in severe weight loss, degeneration of various organs, and early death of mice following systemic vector delivery, whereas the BK-UTK increased mean survival without toxicity.

Regulatory T cells and Th17 cells in viral infections: implications for multiple sclerosis and myocarditis.

In immune-mediated diseases, Treg and proinflammatory Th17 cells have been suggested to play either suppressor (beneficial) or effector (detrimental) roles, respectively. Tissue damage in viral infections can be caused by direct viral replication or immunopathology. Viral replication can be enhanced by anti-inflammatory responses and suppressed by proinflammatory responses. However, Tregs could suppress proinflammatory responses, reducing immunopathology, while Th17 cell-induced inflammation may enhance immunopathology. Here, the roles of Treg and Th17 cells depend on whether tissue damage is caused by direct virus replication or immunopathology, which differ depending on the virus, disease stage and host immune background. Although the precise mechanisms of tissue damage in multiple sclerosis and myocarditis are unclear, both viral replication and immune effector cells have been proposed to cause pathogenesis. Personalized medicine that alters the balance between Treg and Th17 cells may ameliorate viral pathology during infections.

In vivo ablation of CD11c-positive dendritic cells increases susceptibility to herpes simplex virus type 1 infection and diminishes NK and T-cell responses.

The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.

In utero exposure to alcohol alters cell fate decisions by hematopoietic progenitors in the bone marrow of offspring mice during neonatal development.

Fetal alcohol syndrome and alcohol related birth defects represent a spectrum of disorders that can result from the consumption of alcohol during pregnancy. Previous studies from this laboratory have shown that alcohol exposure in utero adversely affects hematopoietic progenitors in the bone marrow. Neonatal mice that were exposed in utero to alcohol showed a marked delay in B lymphocyte development. Recent studies have focused on an oligopotential progenitor cell, with the phenotype of HSA(lo)CD43(lo)Lin(-), which yields both B cells and myeloid lineage cells at a high frequency when cultured in vitro with stromal cells and the appropriate cytokines. However, these progenitor cells isolated from neonatal offspring of alcohol fed dams showed a significant decrease in the frequency of B cell formation following in vitro culture. In order to understand the mechanism underlying this defect we examined the expression of key transcription factors (early B cell factor, EBF, and Pax5) in this progenitor pool. Here, we report that >95% of HSA(lo)CD43(lo)Lin(-) cells express EBF and 5% express Pax5. Following liquid culture in the presence of IL-7, these progenitor cells respond by up-regulating Pax5 and the surface expression of CD19 indicating that the cells have committed to the B lineage. By contrast 75% of HSA(lo)CD43(lo)Lin(-) cells isolated from the bone marrow of neonatal animals exposed in utero to alcohol expressed EBF but at a level that was less than 25% the level of cells isolated from control animals. Furthermore, these alcohol-exposed progenitor cells failed to up-regulate Pax5 in response to IL-7 indicating a greatly reduced capacity to expand and differentiate to B lineage cells in liquid cultures. However, the HSA(lo)CD43(lo)Lin(-) cells isolated from the alcohol exposed animals retained the capacity to differentiate to myeloid lineage cells. These results suggest that the interference with the sequential expression of transcription factors in early progenitor cells by in utero alcohol exposure is a potential mechanism for the observed decrease in B lymphocytes in neonatal mice.

TCRbeta enhancer activation occurs in some but not all cells with T cell lineage developmental potential.

Previous studies have shown that murine bone marrow contains a fraction of CD3(-)/B220(-)/Thy1(lo) cells that have pre T cell activity following adoptive transfer and produce sterile transcripts of the T cell receptor beta chain gene. The relationship between progenitors and TCRbeta transcription has not been examined. Transgenic mice were generated that express green fluorescent protein under the control of the TCRbeta enhancer (Ebeta). Phenotypic analysis of the founders revealed faithful expression of GFP in populations that express TCRbeta transcripts. Examination of the bone marrow showed two populations, CD3(-)/B220(-)/Thy1(-) and CD3(-)/B220(-)/Thy1(lo), which were GFP(+). Both populations were analyzed for their developmental potential following intrathymic transfer into recipient mice. Surprisingly, the GFP(+)/CD3(-)/B220(-)/Thy1(lo) cells failed to reconstitute; however, the GFP(+)/CD3(-)/B220(-)/Thy1(-) cells exhibited thymic repopulation. These data demonstrate that Ebeta is active pre-thymically; however, pre-thymic transcription of the TCRbeta chain gene is neither required for T cell development, nor is it limited to pre T cells.

Regulation of chronic colitis in athymic nu/nu (nude) mice.

The objective of this study was to assess the roles of NK cells, B cells and/or intraepithelial lymphocytes (IEL) in suppressing the development of colitis in nude mice reconstituted with CD4(+)CD45RB(high) T cells. BALB/c nude mice were lethally irradiated and reconstituted with bone marrow from different immunodeficient mice to generate athymic chimeras devoid of one or more lymphocyte populations. Transfer of CD4(+)C45RB(high) T cells into chimeric recipients devoid of B cells, T cells and IEL produced severe colitis within 6-8 weeks, whereas transfer of these same T cells into B cell- and T cell-deficient or T cell-deficient chimeras produced little to no gut inflammation. In addition, we found that nude mice depleted of NK cells or RAG-1(-/-) mice reconstituted with IEL failed to develop colitis following transfer of CD45RB(high) T cells. Severe colitis could, however, be induced in nude mice by transfer of activated/T(h)1 CD4(+)CD45RB(low) T cells. Taken together, our data suggest that IEL, but not B cells or NK cells, play an important role in suppressing the development of chronic colitis in this model. In addition, our data demonstrate that suppression of disease may be due to polarization of naive CD4(+) cells toward a non-pathogenic and/or regulatory phenotype.

The role of T-lymphocytes in hypercholesterolemia-induced leukocyte-endothelial interactions.

OBJECTIVE: Hypercholesterolemia promotes the adhesion of leukocytes to vascular endothelium in large and microscopic blood vessels. Lymphocytes that can modulate endothelial cell adhesion molecule expression have been implicated in the altered structure and function of large arterial vessels associated with chronic hypercholesterolemia. This study assesses the contribution of CD4(+) and CD8(+) T-cells to acute inflammatory responses observed in the microcirculation of hypercholesterolemic mice. METHODS: Intravital microscopy was used to quantify baseline leukocyte-endothelial cell interactions in cremasteric postcapillary venules of wild-type (WT) and severe combined immunodeficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. A group of SCID-HC mice received splenocytes from WT-HC mice (WT-->SCID). Separate WT-HC groups were depleted of neutrophils or CD4(+) and/or CD8(+) T-cells. RESULTS: WT-HC mice, compared with WT-ND, exhibited exaggerated leukocyte adherence and emigration. These leukocytes were predominantly granulocytes. These responses were absent in neutropenic WT-HC mice. SCID-HC mice also showed significantly less leukocyte adherence and emigration than WT-HC mice. Elevated leukocyte adherence and emigration were restored in WT-->SCID mice, despite a continued absence of circulating blood lymphocytes. Selective depletion of either CD4(+) or CD8(+) cell populations attenuated HC-induced leukocyte adhesion but not emigration. However, simultaneous depletion of both CD4(+) and CD8(+) cells attenuated both leukocyte adhesion and emigration to ND levels. DISCUSSION: These findings indicate that both CD4(+) and CD8(+) T-cells contribute to granulocyte adhesion and emigration elicited in postcapillary venules by hypercholesterolemia.