Robert Chervenak

CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice

To understand the cellular basis for recovery from HSV infection, it is critical to identify functional interactions between HSV-specific T lymphocyte subpopulations involved in the generation of the optimal response. To this end, the requirement for CD4+ (L3T4+) T lymphocytes in the development of the primary and secondary CD8+ (Lyt-2+) cytolytic T lymphocyte (CTL) response following HSV infection in C57BL/6 mice was investigated. It was found that chronic depletion of CD4+ cells in vivo by treatment with the mAb GK1.5, which resulted in greater than 95% depletion of peripheral CD4+ T lymphocytes in treated animals, caused a profound decrease in the levels of cytolytic activity obtained during the primary response in the draining popliteal lymph nodes of mice responding to infection in the hind footpads. However, treatment did not affect the levels of in vivo secondary CTL activity in the popliteal lymph nodes, nor the in vitro secondary response in the spleen. The decreased CTL activity observed during the primary response was not due to an inability to prime HSV-specific CTL precursors (CTLp), as full cytolytic activity was obtained following culture of lymphocytes in the presence of exogenous IL-2 and antigen, and the response could be reconstituted by treatment with recombinant IL-2 in vivo. Analysis of the secondary CTL response in the spleen indicated that CD4+ cells were not required for either the generation or maintenance of this aspect of the response. However, blockade of IL-2 utilization by CTL using anti-IL-2R antibodies indicated that this lymphokine was absolutely essential for secondary CTL expansion in vitro. Finally, mice that had been infected 12 months previously exhibited a decreased ability to generate secondary HSV-specific CTL in vitro following CD4-depletion in vivo. Taken together, these results suggest two distinct stages of CTL development during the response: an early primary stage dependent upon the presence of CD4+ cells, and a later, CD4-independent stage operative during the secondary response, which decays with time postinfection.

Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 μM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 μM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (ΔΨm) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of ΔΨm. This drop in ΔΨm was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation → excess ROS production → GSH depletion → oxidative stress → disruption of ΔΨm → release of cytochrome C and other apoptosis related proteins to cytosol → apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.

In Vivo Ablation of CD11c-Positive Dendritic Cells Increases Susceptibility to Herpes Simplex Virus Type 1 Infection and Diminishes NK and T-Cell Responses

ABSTRACT 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.

T-lymphocytes contribute to hepatic leukostasis and hypoxic stress induced by gut ischemia/reperfusion

Although neutrophils have been implicated in the hepatic injury elicited by gut ischemia/reperfusion (I/R), the contribution of other leukocyte populations to this injury process remains unclear. The objective of this study was to determine whether lymphocytes contribute to gut I/R‐induced microvascular dysfunction and inflammatory responses in the liver. Intravital videomicroscopy was used to monitor leukocyte recruitment, the number of nonperfused sinusoids and pyridine nucleotide (NADH) autofluorescence in livers of wild‐type, SCID, and interferon‐γ (IFN‐γ) knockout mice exposed to 15 min of gut ischemia and 1 h of reperfusion. In wild‐type mice, gut I/R elicited significant increases in the number of stationary leukocytes, nonperfused sinusoids, NADH autofluorescence (indicating hypoxia), and elevated plasma alanine aminotransferase (ALT) and TNF‐α levels. All of these responses were profoundly attenuated in SCID mice, while only some of the responses (in the midzonal region) were blunted in IFN‐γ knockout mice. Reconstitution (24 h before ischemia) of the circulating lymphocyte pool with T‐cell enriched splenocytes, but not T‐cell deficient (from nude mice), CD4+ T‐cell depleted splenocytes or splenocytes derived from IFN‐γ knockout mice, allowed the SCID mice to respond to gut I/R in a manner similar to wild‐type mice. Some of the responses were restored following reconstitution with CD8+ T‐cell depleted splenocytes. These findings implicate CD4+ Tlymphocytes and IFN‐γ in the hepatic microvascular dysfunction and inflammatory cell accumulation elicited by gut I/R.

Rapid Generation and Flow Cytometric Analysis of Stable GFP-Expressing Cells

Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably expression GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1lo CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation.

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

ABSTRACT 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 α-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.

Cytokine and adhesion molecule expression in SCID mice reconstituted with CD4+ T cells.

Summary: The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate‐buffered saline (PBS) developed clinical evidence of colitis at 6‐8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Thl and macrophage‐derived cytokines including interferon &ggr;, tumor necrosis factor‐&agr;, interleukin (IL)‐1&bgr;, IL‐6, IL‐12, and IL‐18 lymphotoxin‐&bgr;. In addition, message levels and vascular surface expression of ICAM‐1, VCAM‐1, and MAdCAM‐1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

Dendritic Cells Are Required for Optimal Activation of Natural Killer Functions following Primary Infection with Herpes Simplex Virus Type 1

ABSTRACT 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-γ) 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-γ expression and cytolytic function by NK cells following infection with HSV-1.

Coincident Nonlinear Changes in the Endocrine and Immune Systems due to Low-Frequency Magnetic Fields

Objective: The characteristic biological effects of low-frequency electromagnetic fields (EMFs) appear to be functional changes in the central nervous, endocrine and immune systems. For unapparent reasons, however, the results of similar studies have often differed markedly from one another. We recognized that it had generally been assumed, in the studies, that EMF effects would exhibit a dose-effect relationship, which is a basic property of linear systems. Prompted by recent developments in the theory on nonlinear systems, we hypothesized that there was a nonlinear relationship between EMFs and the effects they produced in the endocrine and immune systems. Methods: We developed a novel analytical method that could be used to distinguish between linear and nonlinear effects, and we employed it to examine the effect of EMFs on the endocrine and immune systems. Results: Mice exposed to 5 G, 60 Hz for 1–175 days in 7 independent experiments reliably exhibited changes in serum corticosterone and lymphoid phenotype when the data were analyzed while allowing that the field exposure and the resulting effects could be nonlinearly related. When the analysis was restricted to linear relationships, no effects due to the field were found. Conclusions: The results indicated that transduction of EMFs resulted in changes in both the endocrine and immune systems, and that the laws governing the changes in each system were not the type that govern conventional dose-effect relationships. Evidence based on mathematical modeling was found suggesting that the coincident changes could have been causally related.