Dennis Way

Microglial and Astrocyte Chemokines Regulate Monocyte Migration through the Blood-Brain Barrier in Human Immunodeficiency Virus-1 Encephalitis

The numbers of immune-activated brain mononuclear phagocytes (MPs) affect the progression of human immunodeficiency virus (HIV)-1-associated dementia (HAD). Such MPs originate, in measure, from a pool of circulating monocytes. To address the mechanism(s) for monocyte penetration across the blood-brain barrier (BBB), we performed cross-validating laboratory, animal model, and human brain tissue investigations into HAD pathogenesis. First, an artificial BBB was constructed in which human brain microvascular endothelial and glial cells-astrocytes, microglia, and/or monocyte-derived macrophages (MDM)-were placed on opposite sides of a matrix-coated porous membrane. Second, a SCID mouse model of HIV-1 encephalitis (HIVE) was used to determine in vivo monocyte blood-to-brain migration. Third, immunohistochemical analyses of human HIVE tissue defined the relationships between astrogliosis, activation of microglia, virus infection, monocyte brain infiltration, and beta-chemokine expression. The results, taken together, showed that HIV-1-infected microglia increased monocyte migration through an artificial BBB 2 to 3.5 times more than replicate numbers of MDM. In the HIVE SCID mice, a marked accumulation of murine MDM was found in areas surrounding virus-infected human microglia but not MDM. For human HIVE, microglial activation and virus infection correlated with astrogliosis, monocyte transendothelial migration, and beta-chemokine expression. Pure cultures of virus-infected and activated microglia or astrocytes exposed to microglial conditioned media produced significant quantities of beta-chemokines. We conclude that microglial activation alone and/or through its interactions with astrocytes induces beta-chemokine-mediated monocyte migration in HAD.

Scatter factor promotes motility of human glioma and neuromicrovascular endothelial cells

Malignant gliomas are characterized by rapid growth, infiltration of normal brain tissue, and high levels of tumor‐associated angiogenesis. The genetic and local environmental tissue factors responsible for the malignant progression from low to high grade gliomas and the highly malignant behavior of glioblastomas are not well understood. In a study of 77 human brain tissue extracts, high grade (III‐IV) tumors had significantly greater scatter factor (SF) content than did low grade tumors or non‐neoplastic tissue. To investigate the potential significance of SF accumulation in gliomas, we measured the effects of SF on DNA synthesis and motility of cultured human glioma cell lines. SF stimulated DNA synthesis in 7/10 glioma cell lines and in 3/3 neuromicrovascular endothelial cell (NMVEC) lines, consistent with our previous report that SF stimulated cell proliferation of a few human glioma cell lines. SF markedly stimulated the chemotactic migration of 10/10 glioma cell lines as well as 3/3 NMVEC lines. In addition, SF stimulated the 2‐dimensional migration of glioma cells on culture surfaces coated with specific extracellular matrix molecules (collagen IV, laminin, and fibronection). As expected based on these biologic responses to SF, 10/10 glioma lines and 4/4 NMVEC lines expressed mRNA for c‐met, the SF receptor. To assess the possible in vivo significance of these migration assays, we compared the chemotactic response of a glioma cell line to human brain cyst fluids and tumor extracts that contained high or low SF concentrations. Fluids and extracts with high SF content tended to induce higher levels of chemotactic migration than did fluids and extracts with low SF content. Addition of anti‐SF monoclonal antibody (MAb) inhibited migration induced by fluids and extracts with high SF content by about 30–50%. Int. J. Cancer 75:19–28, 1998.© 1998 Wiley‐Liss, Inc.

CXC and CC chemokine receptors on coronary and brain endothelia.

BackgroundChemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis.Materials and MethodsThe expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay.ResultsIn BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MlP-1α, and MIP-1β.ConclusionsThe endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.

Scatter factor expression and regulation in human glial tumors

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a cytokine that induces cell motility in vitro and angiogenesis in vivo. SF appears to be a determinant of the malignant phenotype in certain systemic cancers. We detected SF in extracts prepared from human gliomas, with the highest levels found in malignant tumors. Human glioblastoma cells expressed both SF and its receptor (c‐met protein) in vivo, as demonstrated by immunohistochemistry. Consistent with these observations, we found moderate to high levels of production of immunoreactive and biologically active SF by cultured human glioblastoma cells (3 of 8 lines) and by neural microvascular endothelial cells (NMVEC) (3 of 3 lines). SF stimulated the proliferation of glioblastoma and NMVEC cell lines by paracrine or autocrine mechanisms. Conditioned medium (CM) from both glioblastoma and NMVEC cells contained SF‐inducing factor (SF‐IF) activity, defined by its ability to stimulate SF production in an indicator cell line (MRC5 human fibroblasts). This activity consisted of a high‐molecular‐weight (>30 kDa), heat‐sensitive component and a low‐molecular weight (<30 kDa), heat‐stable component. Furthermore, glioblastoma CM stimulated NMVEC SF production, and NMVEC CM stimulated glioblastoma cell SF production, by 3‐ to 6‐fold in each case. Our findings demonstrate that SF‐dependent interactions between glioma cells, and between glioma cells and endothelium, can contribute to the heterogeneous proliferative and angiogenic phenotypes of malignant gliomas in vivo.

Cocaine opens the blood-brain barrier to HIV-1 invasion.

Cocaine abuse has been associated with vasculitis and stroke, and is suspected to influence the progression of AIDS dementia. Cocaine may enhance HIV-1 neuroinvasion by actions directed at the blood-brain barrier. HIV-1 appears to penetrate the human brain microvascular endothelial cell barrier by a paracellular route breached by tumor necrosis factor-alpha (TNF-alpha). Cocaines effects on the blood-brain barrier were investigated using human brain microvascular endothelial cells and peripheral blood monocytes. Cocaine (10(-5) M and 10(-6) M) increased molecular permeability of the barrier and viral invasion by the macrophage-tropic HIV-1(JR-FL) into the brain chamber. Cocaine also augmented apoptosis of brain endothelial cells and monocytes, increased secretion of four chemokines (interleukin-8, interferon-inducible protein-10, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1) and the cytokine, TNF-alpha, by human monocytes. TNF-alpha enhanced invasion of the brain compartment by macrophage-tropic, lymphotropic, and bitropic HIV-1 strains. These data indicate that HIV-1 neuroinvasion can be increased by (a) cocaines direct effects on brain microvascular endothelial cells and (b) paracrine effects of cocaine-induced pro-inflammatory cytokines and chemokines on the blood-brain barrier.

Discordance between flow cytometric abnormalities and dysplasia in Barrett's esophagus

Eighty-six specimens from 25 patients with Barretts epithelium were analyzed by both histology and flow cytometry. Of these, 73 were without dysplasia and 13 had dysplasia (7 low grade, 6 high grade). Eight of the nondysplastic specimens were aneuploid and another 15 had increased G2. Among the dysplastic specimens, two were aneuploid and two had increased G2. These data were grouped into four classifications: type 1 (65 specimens), specialized columnar epithelium (Barretts) without dysplasia and no aneuploidy; type 2 (8 specimens), Barretts epithelium without dysplasia and an aneuploid cell population; type 3 (11 specimens), Barretts epithelium with dysplasia and no aneuploidy; and type 4 (2 specimens), Barretts epithelium with dysplasia and aneuploidy. Distribution by type was 76%, 9%, 13%, and 2%, respectively. We conclude that histologic dysplasia and aneuploidy are often discordant. They may identify separate subgroups at risk, or when concordant, may reflect an increased cancer risk in that population. Further study will define the role of histology and flow cytometry in the screening and management of patients with Barretts esophagus.

[Ca2+]i and pHin Homeostasis in Kaposi Sarcoma Cells

Changes in intracellular pH (pHin) and intracellular calcium concentration [Ca2+]i play a major role in signal transduction leading to cell growth, differentiation and

Inhibition of normal and experimental angiotumor endothelial cell proliferation and cell cycle progression by 2-methoxyestradiol.

Abstract With rapid growth and metabolism, aggressive cancers require an extensive vascular network, termed tumor angiogenesis. The body produces a variety of natural angiogenic inhibitors, among which is the mammalian estrogen metabolite, 2-methoxyestradiol (2-MeOE2). In this study, we compared the effects of 2-MeOE2 on a human umbilical vein cell line (HUVEC-C) and on an immortal, angiotumor-producing rat sinusoidal endothelial cell line (RSE-1). In vitro, the effects of varying concentrations of 2-MeOE2 from 0.01-100.0 μM were measured with cell counts and compared to control cells. HUVEC-C had an ED50 ~3.5 μM with ~27% inhibition of cell growth whereas RSE-1 had an ED50 ~2.2 μM with ~50% inhibition of cell growth compared with controls. The lowest concentration with maximal effect was 10.0 μM2-MeOE2 for both cell lines. Using this concentration, flow cytometric analysis of cell cycles was performed with propidium iodide stained DNA of HUVEC-C and RSE-1 at 24 and 48 hr. Both demonstrated a significant (P < 0.0001) block at G2M of the cell cycle. At 48 hr, HUVEC-C had 32% of cells in G2M (control = 9% G2M), and RSE-1 had 36% of cells in G2M (control = 18% G2M). These findings demonstrate a strong in vitro antiproliferative effect of 2-MeOE2 on normal dividing endothelial as well as angiotumor cells mediated through a cell cycle-specific block at G2M. The antiendothelial, antiangiotumor effect of 2-MeOE2 supports its potential as a therapeutic agent against solid organ cancers, benign or malignant vascular growths, and other pathologic states dependent on angiogenesis.

Expression of multiple matrix metalloproteinases and urokinase type plasminogen activator in cultured Kaposi sarcoma cells

Kaposis sarcoma (KS) cells are considered to be of endothelial origin. KS lesions are characterized by hyperproliferation and an invasive phenotype. We have determined that KS cell cultures constitutively secrete multiple forms of several matrix metalloproteinases (MMPs) and an altered form of urokinase plasminogen activator (uPA) by zymogram and Western analysis of the culture media. MMPs are a family of secreted endoproteinases which degrade components of the extracellular matrix. Their enhanced expression and activity are strongly correlated with cellular processes involving tissue remodeling and invasion. The KS cells secrete increased levels of gelatinase A and B and a high molecular weight uPA in vitro when compared with non-KS endothelial or epithelial cells. Multiple forms of gelatinases A and B were observed on gelatin zymograms. Caseinolytic bands observed were confirmed by Western blot analysis to be due to stromelysin activity, whereas matrilysin was not detected by casein zymography. Western blot analysis also detected secretion of interstitial collagenase and high molecular weight uPA. Gelatinolytic activity with the mobility of gelatinase B was detected on gelatin zymograms, but not by Western analysis. This unusual constitutive expression pattern of MMPs and uPA by KS cells in vitro is characterized by elevated levels of gelatinase A, gelatinase B, interstitial collagenase, stromelysin and a high molecular weight form of uPA, and the lack of expression of matrilysin. These secreted MMPs, taken together, are capable of digesting a broad range of components of the extracellular matrix. This unusual pattern is likely to contribute to the characteristic hyperproliferative and invasive phenotype of KS lesions.

Divergent Effects of Cocaine on Cytokine Production by Lymphocytes and Monocyte/Macroophages

Cocaine-related immunosuppression is asumed to have serious consequences, but its evaluation in drug-addicted subjects is lacking. In this study performed with materials from addicted subjects receiving intravenous cocaine and normal control subjects, acute cocaine effects on cytokine production in vivo and in mononuclear cells in vitro were determined. Acute intravenous cocaine administration resulted in (a) increased white blood cell and lymphocyte counts, (b) decreased tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 serum levels; (c) depressed TNF-α, IL-10 and IL-12 production by unstimulated or LPSstimulated mononuclear cells; (d) increased TNF-a production by PHA-stimulated mononuclear cells. These observations suggest that cocaine has stimulatory effect on TNF-α production by lymphocytes but inhibitory action on TNF-α production by monocyte/macrophages. In vitro cocaine treatment of monocyte-enriched preparations of mononuclear cells from normal donors resulted in suppression of cytokine production. A blood-brain barrier model was constructed using human brain microvascular endothelial cells. In this model mononuclear cell transmigration was correctly regulated by Thl and Th2 cytokines and preferential migration of “memory” T cells was inhibited by cocaethylene. TNF-α and cocaethylene increased HIV-1 titers in the brain-side of the model.