The control values of permeability coefficient for 131I-HIV-1 in panel A was 137 0

The control values of permeability coefficient for 131I-HIV-1 in panel A was 137 0.13 10-5 and 1.32 0.13 10-5 cm/min for the luminal and abluminal control, respectively. electrical resistance (TEER). Luminal, but not abluminal, IL-6 or GM-CSF also increased HIV-1 transport. U0126 (MAPK kinase (MEK)1/2 inhibitor) and SB203580 (p38 MAPK inhibitor) decreased the LPS-enhanced release of IL-6 and GM-CSF. These results show that p44/42 Rabbit Polyclonal to EPB41 (phospho-Tyr660/418) and p38 MAPK signaling pathways mediate the LPS-enhanced release of IL-6 and GM-CSF. These cytokines, in turn, act at the luminal surface of the BMEC to enhance the transcellular transport of HIV-1 independently of actions on paracellular permeability. strong class=”kwd-title” Keywords: Blood-brain barrier, Human immunodeficiency computer virus type 1, Lipopolysaccharide, Interleukin-6, Granulocyte-macrophage colony-stimulating factor, Mitogen-activated protein kinase Background Human immunodeficiency computer virus type 1 (HIV-1) contamination induces neurological dysfunctions known as the AIDS-dementia complex or HIV-associated dementia (HAD). Although highly active antiretroviral therapy (HAART) and combination antiretroviral therapy (cART) have dramatically decreased the incidence and severity of HAD, the prevalence of HAD, including minor cognitive and motor disorders, is usually increasing with the longer lifespan of HIV patients [1]. Zileuton Most antiretroviral drugs comprising HAART have a restricted entry into the brain because of blood-brain barrier (BBB) efflux transporters Zileuton so that the brain serves as a reservoir for HIV-1 [2] and a source for viral escape [3]. Therefore, HIV-1 in the brain can contribute to the incidence and development of HIV-associated neurological impairment in HIV-1 patients both prior to and after treatment with HAART/cART. HIV-1 can enter the brain by two routes: the passage of cell-free computer virus by an adsorptive endocytosis-like mechanism [4-7] and trafficking of HIV-1-infected immune cells across the BBB [8]. HIV-1 contamination of brain endothelial cells (BECs) is not a productive contamination [9] and penetration of Zileuton HIV-1 is usually independent of the CD4 receptor [10]. At the early stage, HIV-1 enters the brain through an intact, normally functioning BBB [11]. At later stages of contamination, elevated levels of proinflammatory cytokines/chemokines in the blood of patients with AIDS [12-14] are likely associated with the increase in HIV-1 infiltration [15-17], while HIV-1 gp120 and Tat induce the disruption of tight junctions in BECs [17-20]. As reported by Brenchley et al. and confirmed by others, plasma levels of lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, are higher in chronic HIV-infected patients with HAART than in the uninfected [3,21]. Bacterial infection in HIV patients influences the severity and rate of disease progression [22]. Peripheral LPS induces various inflammatory and immunological reactions including the production of cytokines/chemokines, such as tumor necrosis factor- (TNF-interleukin (IL)-1, and IL-6 [23-25]. TNF- enhances HIV-1 transport across the BBB [15] and LPS induces an increase in HIV-1-infected monocyte transport across the BBB [8]. In our previous in vivo study, we found that the peripheral injection of LPS enhanced gp120 uptake by brain [26]. These studies suggest that elevated levels of inflammatory mediators, Zileuton including cytokines/chemokines and LPS, regulate the permeability of the BBB to HIV-1. BECs express LPS receptors, such as Toll-like receptor (TLR)-2, TLR-4, and CD14 [27] and are targets of LPS. The barrier function of the BBB is usually affected by various cytokines/chemokines in the blood compartment [28]. Several studies using in vitro BBB models have shown that LPS increases the paracellular permeability of the BBB [29-33]. LPS induces or enhances the secretion of several cytokines by BECs [34]. Thus, bacterial infection and the accompanying inflammatory state could be involved in the enhancement of HIV-1 entry into the brain. We recently reported that LPS increased transcellular transport of HIV-1 across Zileuton the BBB through p38 mitogen-activated protein kinase (MAPK) [35]. Here, we examined whether LPS-enhanced release of cytokines by BMECs mediated the transcellular transport of HIV-1 and was regulated by MAPK signaling pathways. Materials and methods Radioactive labeling HIV-1 (MN) CL4/CEMX174 (T1) prepared and rendered noninfective by aldrithiol-2 treatment as previously described [36] was a kind gift of the National Malignancy Institute, NIH. The computer virus was radioactively labeled by the chloramine-T method, a method which preserves vial coat glycoprotein activity [37,38]. Two mCi of 131I-Na (Perkin Elmer, Boston, MA), 10 g of chloramine-T (Sigma) and 5.0 g of the computer virus were incubated together for 60 sec. The radioactively labeled computer virus was purified on a column of Sephadex G-10 (Sigma). Primary culture of mouse brain microvascular endothelial cells (BMECs) BMECs were isolated by a modified method of Szab et al. [39] and Nakagawa et al. [38]. The animals were housed in clean cages in the laboratory with free access.