Joyce G. Slusser

Expression and Function of PDCD1 at the Human Maternal-Fetal Interface

Abstract The failure to reject the semiallogenic fetus by maternal T lymphocytes suggests that potent mechanisms regulate these cells. PDCD1 is a CD28 family receptor expressed by T cells, and its ligand CD274 is strongly expressed by trophoblast cells of the human placenta. In this study, we examined whether human maternal T cells express PDCD1. Immunofluorescence examination of uterine tissues revealed PDCD1 expression on CD3+ cells was low in nonpregnant endometrium but increased in first-trimester decidua and remained elevated in term decidua (P < 0.05). In addition, higher relative proportions of term decidual CD8bright, CD4+, and regulatory T cells expressed PDCD1 in comparison to autologous peripheral blood (P < 0.05). Term decidual T cells also expressed full-length and soluble PDCD1 mRNA isoforms more abundantly than their peripheral blood counterparts (P ≤ 0.05). We also examined the effects of PDCD1:CD274 interactions in decidual T cells. Jar choriocarcinoma cells were transfected with CD274 and cocultured with activated decidual CD4+ or CD8bright T cells for 72 h followed by analysis of cytokine concentration and decidual T cell apoptosis. Compared with empty vector-transfected cells, CD274-transfected Jar cells caused a significant suppression of interferon gamma and tumor necrosis factor alpha production by CD4+ (P < 0.05) but not CD8bright T cells, while having no effect on secretion of IL10 or T cell apoptosis. These results suggest that the PDCD1:CD274 pathway functions in modification of maternal decidual lymphocyte cytokine secretion during pregnancy.

NK Cells and Gamma Interferon Coordinate the Formation and Function of Hepatic Granulomas in Mice Infected with the Francisella tularensis Live Vaccine Strain

ABSTRACT Host innate immune responses to many intracellular pathogens include the formation of inflammatory granulomas that are thought to provide a physical barrier between the microbe and host. Because two common features of infections with the live vaccine strain (LVS) of Francisella tularensis within the mouse liver are the formation of granulomas and the production of gamma interferon (IFN-γ), we have asked what role IFN-γ plays in hepatic granuloma formation and function. Francisella antigens were predominantly localized within granulomas of the livers of mice infected with F. tularensis LVS 4 days postinfection. Hepatic granulomas also contained large numbers of dying cells, some of which coexpressed the F4/80 macrophage antigen and activated caspase-3. IFN-γ-deficient mice did not form normal numbers of hepatic granulomas and showed widely disseminated Francisella antigens within the liver. The incidence of cell death within hepatic granulomas also decreased significantly in the absence of IFN-γ. Inducible NO synthase (iNOS) expression was restricted to the granulomas of wild-type mice but was not seen for IFN-γ-deficient mice. Cell death within granulomas was also significantly decreased for iNOS-deficient mice. The predominant IFN-γ-expressing cells in the liver were NK cells. Depleting NK cells resulted in the expression of bacterial antigens and iNOS outside the granulomas and the appearance of extensive hepatic focal necrosis. These findings indicate that IFN-γ and hepatic NK cells that are activated during F. tularensis LVS infections regulate hepatic granuloma formation, the spatial containment of infection, the expression of iNOS, and the induction of cell death within the liver.

Development of a single-plasmid-based regulatable gene expression system for Borrelia burgdorferi.

ABSTRACT We developed a single-plasmid-based regulatable protein expression system for Borrelia burgdorferi. Expression of a target gene is driven by Post, a hybrid B. burgdorferi ospA-tetO promoter, from a recombinant B. burgdorferi plasmid constitutively expressing TetR. The system was tested using the green fluorescent protein (GFP) as a reporter. Under noninducing conditions, recombinant B. burgdorferi cells were nonfluorescent, no GFP protein was detected, and residual, small amounts of transcript were detectable only by reverse transcription-PCR but not by Northern blot hybridization. Upon induction with anhydrotetracycline, increasing levels of GFP transcript, protein, and fluorescence were observed. This tight and titratable promoter system will be invaluable for the study of essential borrelial proteins. Since target protein, operator, and repressor are carried by a single plasmid, the systems application is independent of a particular strain background.

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells

Summary Histamine is a well‐recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)‐induced production of prostaglandin I2 (PGI2), PGE2 and interleukin‐6 (IL‐6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine ‐induced expression of Toll‐like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10–1000 ng/ml) resulted in two‐fold to fourfold increases in H1R mRNA expression in a time‐dependent and concentration‐dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS‐induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20–24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS‐treated cells. The specific binding of [3H]pyrilamine to H1R in membrane proteins from LPS‐treated HCAEC was threefold higher than the untreated cells. The LPS‐induced H1R expression was mediated through TLR4 as gene silencing by TLR4‐siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre‐treated with LPS for 24 hr, washed and challenged with histamine, 17‐, 10‐ and 15‐fold increases in PGI2, PGE2 and IL‐6 production, respectively, were noted. Histamine‐induced enhancement of the synthesis of PGI2, PGE2 and IL‐6 by LPS‐primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up‐regulates the expression and function of H1R and amplifies histamine‐induced inflammatory responses in HCAEC.

Flow cytometry and laser scanning cytometry, a comparison of techniques

ObjectiveFlow and laser scanning cytometry are used extensively in research and clinical settings. These techniques provide clinicians and scientists information about cell functioning in a variety of health and disease states. An in-depth knowledge and understanding of cytometry techniques can enhance interpretation of current research findings. Our goal with this review is to reacquaint clinicians and scientists with information concerning differences between flow and laser scanning cytometry by comparing their capabilities and applications.MethodsA Pubmed abstract search was conducted for articles on research, reviews and current texts relating to origins and use of flow and laser scanning cytometry. Attention was given to studies describing application of these techniques in the clinical setting.ResultsBoth techniques exploit interactions between the physical properties of light. Data are immediately and automatically acquired; they are distinctly different. Flow cytometry provides valuable rapid information about a wide variety of cellular or particle characteristics. This technique does not provide the scanned high resolution image analysis needed for investigators to localize areas of interest within the cell for quantification. Flow cytometry requires that the sample contain a large amount disaggregated, single, suspended cells. Laser scanning cytometry is slide-based and does not require as large of a sample. The tissue sample is affixed to a slide allowing repeated sample analyses. These cytometry techniques are used in the clinical setting to understand pathophysiological derangements associated with many diseases; cardiovascular disease, diabetes, acute lung injury, hemorrhagic shock, surgery, cancer and Alzheimer’s disease.ConclusionsUnderstanding the dif- ferences between FCM and LSCM can assist investigators in planning and design of their research or clinical testing. Researchers and clinicians optimize these technique capa- bilities with the cellular characteristics they wish to measure delineating molecular and cellular events occurring in health and disease. Discovery of mechanisms in cells using FCM and LSCM provide evidence needed to guide future treatment and interventions.

Tissue factor contributes to neutrophil CD11b expression in alpha-naphthylisothiocyanate-treated mice

Cholestatic liver injury induced by alpha-naphthylisothiocyanate (ANIT) is provoked by injury to intrahepatic bile ducts and the progression of hepatic necrosis requires the procoagulant protein tissue factor (TF) and extrahepatic cells including neutrophils. Recent studies have shown that myeloid cell TF contributes to neutrophil activation. We tested the hypothesis that myeloid cell TF contributes to neutrophil activation in ANIT-treated mice. TF activity in liver homogenates increased significantly in TF(flox/flox) mice treated with ANIT, but not in TF(flox/flox)/LysMCre mice (TF(ΔMyeloid) mice), which have reduced TF expression in monocytes/macrophages and neutrophils. Myeloid cell-specific TF deficiency did not alter expression of the chemokines KC or MIP-2 but reduced hepatic neutrophil accumulation in ANIT-treated mice at 48 h as indicated by tissue myeloperoxidase (MPO) activity. Myeloid cell TF deficiency significantly reduced CD11b expression by blood neutrophils in ANIT-treated mice, and this was associated with reduced plasma MPO protein levels, an index of neutrophil degranulation. However, myeloid cell-specific TF deficiency had no effect on ANIT-induced coagulation cascade activation. The increase in serum ALT and ALP activities in ANIT-treated mice was reduced by myeloid cell TF deficiency (p<0.05), but the myeloid cell TF deficiency did not reduce hepatic necrosis at 48 h, as determined by histopathology and morphometry. The results suggest that myeloid cell TF contributes to neutrophil CD11b expression during cholestasis by a coagulation-independent pathway. However, the resultant reduction in neutrophil accumulation/activation is insufficient to substantially reduce ANIT hepatotoxicity, suggesting that myeloid cell TF is only one of many factors modulating hepatic necrosis during cholestasis.

Deferiprone modulates in vitro responses by peripheral blood T cells from control and relapsing–remitting multiple sclerosis subjects

T cells are important mediators of autoimmune inflammation in relapsing-remitting multiple sclerosis (RRMS). Previous studies found that deferiprone, an iron chelator, suppressed disease activity in a mouse model of multiple sclerosis, and inhibition of T cell proliferation was implicated as a putative mechanism. The objective of the present study was to examine the effects of deferiprone on suppressing in vitro responses of T cells from control and RRMS subjects. Peripheral blood T cells were co-stimulated with anti-CD3+anti-CD28 and cultured with or without interleukin 2 (IL-2). Proliferating CD4+ T cells from control and RRMS subjects, cultured with or without IL-2, decreased in response to 75 μM deferiprone, although the extent of decreased proliferation of CD4+ T cells from RRMS subjects was less than for control subjects. Proliferating CD8+ T cells from control subjects, cultured with or without IL-2, also decreased in response to 75 μM deferiprone, and this decrease was seen in proliferating CD8+ T cells from RRMS cultured with IL-2. CD4+CD25+ and CD8+CD25+ cells from control subjects, cultured with or without IL-2, declined in 75 μM deferiprone, but the decrease was smaller than for the CD4+ and CD8+ proliferative responses. CD4+CD25+ and CD8+CD25+ cells from RRMS subjects showed more variability than for control subjects, but CD4+CD25+ cultured with IL-2 and CD8+CD25+ cells cultured without IL-2 significantly declined in 75 μM deferiprone. CD4+FoxP3+ and CD4+CD25+FoxP3+ cells tended to remain constant or increase. In summary, deferiprone induced declines in proliferative responses at a dosage that is within peak serum pharmacological concentrations.

A flow cytometric assay for the study of E3 ubiquitin ligase activity

Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. For this study, a target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV‐1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML‐GFP fusion protein were selected by cell sorting and infected with an adenoviral vector expressing ICP0. In contrast to mock‐infected cells, only PML‐GFP‐expressing cells infected with the ICP0 adenoviral vector led to a significant decrease in the fluorescence signal of PML‐GFP when examined by fluorescence microscopy and FCM analysis. Our results suggest that it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture by FCM analysis.