Nemani V. Prasadarao

Bovine brain microvascular endothelial cells transfected with SV40-large T antigen: Development of an immortalized cell line to study pathophysiology of CNS disease

Dear Editor: The blood brain barrier is formed by endothelial cells of cerebral capillaries and is highly selective by preventing specific substances, cells, and pathogens from passing and allowing other needed compounds through (3,16). Primary cultures have been used to study pathophysiology of the blood brain barrier in vitro (5,6,8,12,24,25). Isolation and characterization of primary cultures of brain endothelial cells are, however, time consuming; the resulting cultures differ in quality, contaminating cells, and their culture requirements are more fastidious than systemic and/or macrovascular endothelial cells. Previously, we reported the isolation, cultivation, and characterization of endothelial cells derived from bovine cerebral microvessels (23). These cerebral endothelial cells were relatively pure at early passages (e.g., Passage 3) and exhibited specific endothelial and brain characteristics. However, the exhibition of specific brain characteristics decreased with passage as also shown by other investigators (5,8,10,12,21). To facilitate the use of brain microvascular endothelial cells for studies of pathogenesis of CNS disease, we wanted to develop an in vitro model by extending the life span of brain microvascular endothelial cells. In this paper, we report the transfection and immortalization of bovine brain endothelial cells (BBEC) with SV40 large T (7) and demonstrate that these ceils maintain original morphologie and functional characteristics of brain endothelial cells. Cell culture and transfection. Bovine brain capillaries were isolated and cultured as described previously (23). SV40-1arge T, a pBR 322 based construct (kindly provided by A. Srinivasan, The Wistar Institute, Philadelphia, PA) (7) was transfected into Passage 3 or 4 of the brain microvessel cultures. BBEC monolayers were incubated with pSVT DNA (10-15 p,g/dish at approximately 40-50% confluence along with pSV2neo plasmid (2-4 lag, obtained from Dr. A. Srinivasan) for co-transfection, and thereafter selected using G418. Islands of transfected cells showing cobblestone appearance were transferred using cloning cylinders, expanded, and characterized as previously described (23) by indirect immunoperoxidase techniques. As shown in Fig. 1, the transfected cells (TBBEC) were positive for Factor VIII related antigen (F-VIII-Rag) and took up AcLDL-Dil, both markers for endothelial cells. Uptake of AcLDL is considered a halhnark for endothelial cells from various organs and species (13,18), including human (Dr. W. Carley, personal communication) and bovine brain (6,15, unpublished result). Moreover, the presence of carbonic anhydrase IV (CAIV) (9) and gamma glutamyl transpeptidase (GGTP) as histochemically assessed using the method of DeBault and Cancilla (4) supported the endothelial character of the isolated cells. No nonendothelial cells were present such as pericytes and glial cells, as determined by immunocytochemistry, respectively, for smooth muscle actin (24) and glial fibrillary acidic protein. Presence of large T antigen in nuclei of transfected cells was confirmed using monoclonal antibody 101 (1) (not shown). These characteristics were maintained up to Passage 60 and cultures are still ongoing. Selected endothelial cell cultures were used in further experiments. Cell adhesion molecules in Tumor Necrosis Factor alpha (TNF)a activated brain endothelial cells. In order to examine responses to cytokines, we incubated confluent cultures of primary (= nontransfected) (Passages 4-10) and transfected brain endothelial cells (Passages 18-22) in 24-wetl plates ~.ith TNFa (10 ng/ml) for 4 and 24 h and assessed expression of ELAM and VCAM on cytospin slides immunocytochemically as described by Hofman et al. (11). In short, the cells were lifted with EDTA, transferred to slides using a cytospin centrifuge, fixed with acetone/methanol (1:1), and air dried. Thereafter, the ceils were incubated sequentially with antibodies directed against ELAM-1 (1:1000) or VCAM-1 (1:1000) (AMAC, Westbrook, ME), biotinylated horse-anti-mouse (1:200) and avidin-biotin complexed to peroxidase (ABC-PO) and color developed with 3-amino9-ethylcarbazole (AEC) according to the manufacturers instructions (all from Vector Laboratories, Burlingame, CA). Thereafter, cells were counterstained with heInatoxylin, mounted, and viewed. No expression of ELAM and VCAM on nonactivated endothelial cells was found. After 4 h of treatment with TNFc~, approximately 50% of both primary and transfected BBEC began to show a slight increase in VCAM expression. Extending the stimulation to 24 h, resulted in a marked increase in the number of positive cells and the intensity of VCAM staining (Fig. 2). A similar response was found for nonbrain endothelial cells (e.g., HUVEC, data not shown). No clear-cut expression of ELAM was found on primary or transfected BBEC after 4 and 24 hours of stimulation with 10-100 ng/ml TNFc~ (Fig. 2), whereas under the same conditions HUVECs did express ELAM (not shown). This indicates that primary and transfected brain endothelial cells responded in a similar way to TNFa (i.e., they expressed VCAM) as did nonbrain endothelial cells (HUVEC), but ELAM expression was only evident on nonbrain endothelial cells. We previously found similar results (expression of VCAM but no ELAM) on primary and transfected human brain microvascular endothelial cells derived from children (Stins et al., submitted for publication). Various authors have reported differences between macroand microvascular endothelial cells within one organ (14) or even within one vascular bed, resulting in heterogeneous responses to cytokines, which are different from human umbilical vein endothelial cells (17). Similarly, Shatos et al. (22) have shown that brain microvascular endothelial cells respond differently to thrombin compared to systemic large and small vessel endothelial cells. Taken together, CNS endothelium seems to be unique and, as demonstrated here, selective expression of adhesion molecules can have important consequences

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation.

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C.u2003neoformans across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of C.u2003neoformans. Our previous studies have shown that the CPS1 gene is required for C.u2003neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C.u2003neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant‐negative form of PKCα in HBMEC. During C.u2003neoformans infection, phosphorylation of PKCα was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCα isoform might play a crucial role during C.u2003neoformans invasion. Immunofluorescence microscopic images showed that induced phospho‐PKCα colocalized with β‐actin on the membrane of HBMEC. In addition, cytochalasin D (an F‐filament‐disrupting agent) inhibited fungus invasion into HBMEC in a dose‐dependent manner. Furthermore, blockage of PKCα function attenuated actin filament activity during C.u2003neoformans invasion. These results suggest a significant role of PKCα and downstream actin filament activity during the fungal invasion into HBMEC.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis

Despite the fundamental function of neutrophils (PMNs) in innate immunity, their role in Escherichia coli K1 (EC-K1) induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by down regulating rac1, rac2 and gp91phox transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis.

Regulation of Toll‐like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β), is critical for the pathogenesis of Escherichia coliu2005K1 meningitis. Since Hsp90 chaperones Toll‐like receptors (TLRs), we examined the role of TLRs in E.u2009coliu2005K1 infection. Herein, we show that newborn TLR2−/− mice are resistant to E.u2009coliu2005K1 meningitis, while TLR4−/− mice succumb to infection sooner. In vitro, OmpA+ E.u2009coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA− E.u2009coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E.u2009coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C termini of Ecgp96 and TLR2 are critical for OmpA+ E.u2009coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 andTLR2 induces a bipartite signal, one from Ecgp96 through PKC‐α while the other from TLR2 through MyD88, ERK1/2 and NF‐κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E.u2009coliu2005K1 invasion of HBMEC.

Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Bacterial meningitis is a serious central nervous system infection and Escherichia coli K1 (E. coli K1) is one of the leading etiological agents that cause meningitis in neonates. Outer membrane protein A (OmpA) of E. coli K1 is a major virulence factor in the pathogenesis of meningitis, and interacts with human brain microvascular endothelial cells (HBMEC) to cross the blood-brain barrier. Using site-directed mutagenesis, we demonstrate that two N-glycosylation sites (NG1 and NG2) in the extracellular domain of OmpA receptor, Ecgp96 are critical for bacterial binding to HBMEC. E. coli K1 invasion assays using CHO-Lec1 cells that express truncated N-glycans, and sequential digestion of HBMEC surface N-glycans using specific glycosidases showed that GlcNAc1-4GlcNAc epitopes are sufficient for OmpA interaction with HBMEC. Lack of NG1 and NG2 sites in Ecgp96 inhibits E. coli K1 OmpA induced F-actin polymerization, phosphorylation of protein kinase C-α, and disruption of transendothelial electrical resistance required for efficient invasion of E. coli K1 in HBMEC. Furthermore, the microvessels of cortex and hippocampus of the brain sections of E. coli K1 infected mice showed increased expression of glycosylated Ecgp96. Therefore, the interface of OmpA and GlcNAc1-4GlcNAc epitope interaction would be a target for preventative strategies against E. coli K1 meningitis.

Escherichia coli K1 induces pterin production for enhanced expression of Fcγ receptor I to invade RAW 264.7 macrophages

Macrophages serve as permissive niches for Escherichia coli (E. coli) K1 to attain high grade bacteremia in the pathogenesis of meningitis in neonates. Although pterin levels are a diagnostic marker for immune activation, the role of macrophages in pterin production and in the establishment of meningitis is unknown. Here, we demonstrate that macrophages infected with E. coli K1 produce both neopterin and biopterin through increased expression of GTP-cyclohydrolase 1 (GCH1). Of note, increased production of biopterin enhances the expression of Fc-gamma receptor I (CD64), which in turn, aided the entry of E. coli K1 in macrophages while increased neopterin suppresses reactive oxygen species (ROS), thereby aiding bacterial survival. Inhibition of GCH1 by 2, 4-Diamino-6-hydroxypyrimidine (DAHP) prevented the E. coli K1 induced expression of CD64 in macrophages inxa0vitro and the development of bacteremia in a newborn mouse model of meningitis. These studies suggest that targeting GCH1 could be therapeutic strategy for preventing neonatal meningitis by E. coli K1.

Tuberculous meningitis: a roadmap for advancing basic and translational research

Tuberculous meningitis is a serious, life-threatening disease affecting vulnerable populations, including HIV-infected individuals and young children. The US National Institutes of Health convened a workshop to identify knowledge gaps in the molecular and immunopathogenic mechanisms of tuberculous meningitis and to develop a roadmap for basic and translational research that could guide clinical studies.