Jiehao Zhou

Neutrophils promote mononuclear cell infiltration during viral-induced encephalitis

Neutrophils are the first infiltrating cell population to appear within the CNS during infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). To determine whether neutrophils play a role in limiting acute JHMV infection, mice were depleted of neutrophils. Infection of neutropenic animals resulted in increased levels of virus replication and mortality compared with control mice. Furthermore, neutropenia resulted in significantly reduced mononuclear leukocyte infiltration possibly due to reduced loss of blood brain barrier integrity during acute JHMV infection. These data suggest that infiltrating neutrophils are crucial for limiting virus replication during acute JHMV infection, contribute to the loss of blood brain barrier integrity and play a role in shaping adaptive immunity within the CNS.

Kinetics of virus-specific CD8+ -T-cell expansion and trafficking following central nervous system infection.

ABSTRACT CD8+ T cells control acute infection of the central nervous system (CNS) by neurotropic mouse hepatitis virus but do not suffice to achieve sterile immunity. To determine the lag between T-cell priming and optimal activity within the CNS, the accumulation of virus-specific CD8+ T cells in the CNS relative to that in peripheral lymphoid organs was assessed by using gamma interferon-specific ELISPOT assays and class I tetramer staining. Virus-specific CD8+ T cells were first detected in the cervical lymph nodes. Expansion in the spleen was delayed and less pronounced but also preceded accumulation in the CNS. The data further suggest peripheral acquisition of cytolytic function, thus enhancing CD8+-T-cell effector function upon cognate antigen recognition in the CNS.

Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization

Clinical observations suggest that genetic factors may influence heterogeneity of angiogenic responses in cardiovascular disease, proliferative diabetic retinopathy, and neoplasia. Experiments among mouse strains using a corneal micropocket assay indicate that extent of angiogenesis may be genetically determined. Here, we established the strain-dependence of hypoxia-induced retinal angiogenesis in multiple mouse strains which paralleled the rank order found for bFGF-induced corneal angiogenesis. Using quantitative real-time RT-PCR, strain-related gene expression differences in retina/choroid between C57BL/6J and 129S3/SvIM, inbred strains with relatively low and high levels of angiogenesis, respectively, after 0, 6, 12, 24, 48, and 96 h hypoxia were determined for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic ligands potently induced by hypoxia, and for pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1), endogenous broad-spectrum antiangiogenic factors. Indirect ELISA was used to correlate VEGF and PEDF protein levels with mRNA expression. At the onset of hypoxia, both PEDF and TSP-1 levels were increased over 15-fold and VEGF was increased over 10-fold compared to Ang-2 in both strains. At the onset of neovascularization (48 h), both VEGF and Ang-2 mRNA levels were increased in the more angiogenic 129S3/SvIM strain (P<0.02), which was not observed among developmental control animals. PEDF expression was higher in the less angiogenic C57BL/6J strain at 6, 12, 24, and 96 h hypoxia (P<0.03), while TSP-1 expression was higher in C57BL/6J throughout the entire time course of hypoxia (4 days) compared to 129S3/SvIM (P<0.02). Among developmental control animals, PEDF and TSP-1 expression was also increased at P14 and P16 in C57BL/6J strain compared to 129S3/SvIM (P<0.02). Strain-dependent expression of both pro- and antiangiogenic growth factors may determine heterogeneity in the angiogenic response and potentially, susceptibility to angiogenesis-dependent diseases.

Efficient Induction of Primary and Secondary T Cell-Dependent Immune Responses In Vivo in the Absence of Functional IL-2 and IL-15 Receptors

IL-2 and IL-15 are thought to be important cytokines for T cell-dependent immune responses. Mice deficient in IL-2, IL-2Rα, and IL-2Rβ are each characterized by a rapid lethal autoimmune lymphoproliferative disorder that complicates their use in studies aimed at investigating the role of these cytokines and receptors for immune responses in vivo. We have previously characterized a novel transgenic (Tg) mouse on the IL-2Rβ−/− genetic background (Tg−/− mice) that lacks autoimmune disease but still contains peripheral T cells that are nonresponsive to IL-2 and IL-15. In the present study, these mice were used to investigate the extent by which IL-2 and IL-15 are essential for T cell immunity in vivo. Tg−/− mice generated near normal primary and secondary Ab responses to OVA, readily mounted first and second set allogeneic skin graft rejection responses, and developed primary and recall CD8 T cell responses to vaccinia virus. However, Tg−/− mice generated a slightly lower level of IgG2a Abs to OVA, exhibited a somewhat delayed first set skin graft rejection response with lower allo-specific CTL, and developed a significantly lower number of IFN-γ-producing vaccinia-specific CD8+ T cells. Thus, although T effector function is somewhat impaired, T cell immunity is largely functional in the absence of IL-2- and IL-15-induced signaling through IL-2Rβ.

Expression of Matrix Metalloproteinases and Their Tissue Inhibitor during Viral Encephalitis

ABSTRACT Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.

Matrix Metalloproteinase Expression Correlates with Virulence following Neurotropic Mouse Hepatitis Virus Infection

ABSTRACT The relationship(s) between viral virulence and matrix metalloproteinase (MMP) expression in the central nervous system (CNS) of mice undergoing lethal and sublethal infections with neurotropic mouse hepatitis virus was investigated. Lethal infection induced increased levels of MMP-3 and MMP-12 mRNAs as well as that of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) compared to sublethal infection. Increased induction of MMP, TIMP, and chemokine expression correlated with increased virus replication but not with inflammatory cell infiltration. Infection of immunosuppressed mice suggested that expression of most MMP, TIMP, and chemokine mRNA was induced primarily in CNS-resident cells. By contrast, MMP-9 protein activity was associated with the infiltration of neutrophils into the CNS. These data indicate an association between the magnitude of inflammatory gene expression within the CNS and viral virulence.

N-(4-hydroxyphenyl) Retinamide Augments Laser-Induced Choroidal Neovascularization in Mice

PURPOSE To evaluate the effect of N-4-hydroxyphenyl retinamide (4-HPR) on experimental laser-induced choroidal neovascularization (CNV) and on the expression and secretion of relevant growth factors by cultured human retinal pigment epithelial (RPE) cells. METHODS CNV was induced by laser photocoagulation in C57BL/6 mice. 4-HPR (0.2 or 1 mg) or vehicle, was injected intraperitoneally twice daily for 14 days. Plasma and tissue levels of 4-HPR were measured by HPLC. CNV was evaluated by fluorescein angiography, histology, and quantitative confocal analysis of isolectin B4 histochemistry on days 7 and 14. Induction of apoptosis and expression and secretion of growth factors was studied in 4-HPR-treated RPE cultures. RESULTS Mice treated with 4-HPR exhibited time- and dose-dependent increases in plasma and tissue 4-HPR levels. CNV lesions showed increased volume with increased vascular leakage and contained fewer lesion-associated RPE in treated versus untreated mice. Treatment of nonpolarized RPE cultures with 4-HPR in the presence of serum resulted in RPE apoptosis; however, apoptosis was minimal in similarly treated highly polarized RPE. Treatment of RPE cells with 4-HPR resulted in the upregulation of VEGF-A and -C (P < 0.05) and Ang-1 (P < 0.01) mRNA and increased secretion of VEGF-A and -C (P < 0.05), whereas pigment epithelium-derived growth factor (PEDF) and thrombospondin (TSP)-1 mRNA expression and secretion were downregulated (P < 0.05). CONCLUSIONS 4-HPR increases lesion size and leakage in laser-induced CNV and is associated with the upregulation of key proangiogenic factors and the downregulation of antiangiogenic factors. Consistent with the preferential loss of RPE in CNV lesions in vivo, 4-HPR induces apoptosis of nonpolarized RPE in the presence of serum.

Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity

We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [3H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P < .05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P < .05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.

Regulation of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Matrix Metalloproteinase (TIMP) Genes During JHMV Infection of the Central Nervous System

The central nervous system (CNS) is refractive to many aspects of the immune system primarily due to its limited ability to repair damage induced by the cytopathic mechanisms deployed by most immune cells. A primary obstacle to CNS inflammation is the blood-brain-barrier (BBB), which limits entry of immune cells into the CNS. To pass the BBB, inflammatory cells release matrix metalloproteinases (MMPs); a growing family of proteases with overlapping substrate specificities for components of the extracellular matrix (reveiwed by Kieseier et al. 1999). MMPs break down the extracellular matrix surrounding the endothelial layer of BBB thereby permitting peripheral immune cells to traverse the BBB and migrate through the parenchyma of the CNS in response to inflammatory signals. To limit potential damage resulting from infiltration of inflammatory cells, MMP activity is tightly regulated at both the level of gene expression and proenzyme activation as well as by expression of a second gene family, the tissue inhibitors of MMPs (TIMPs). TIMPs act as competitive inhibitors for the active sites of MMPs and thus limit inflammatory infiltrates.

The Role of Metalloproteinases in Corona Virus Infection

Infection with neurotropic strains of mouse hepatitis virus (MHV) results in rapid leukocyte infiltration into the central nervous system (CNS). The inflammatory response controls virus replication but fails to mediate sterile clearance. The persistence of viral RNA and inflammatory cells within the CNS is associated with the development of ongoing demyelination. Matrix metalloproteinases (MMPs) are a family of proteases involved in degradation of the extracellular matrix (ECM). During inflammatory responses MMPs are thought to play a significant role in breaking down the basement membrane surrounding blood vessels as well as parenchymal ECM thereby facilitating leukocyte infiltration. MMPs have also been associated with activation of chemokines and perhaps more significantly the degradation of myelin proteins and generation of autoantigens. Recent examination of MMP expression during MHV infection suggests that MMP-3, -9 and -12 are involved in the inflammatory response. The proinflammatory effects of these MMPs are likely tempered by induction of tissue inhibiter of metalloproteinase-1 expression.