Lipopolysaccharide-enhanced transcellular transport of HIV-1 across the blood-brain barrier is mediated by luminal microvessel IL-6 and GM-CSF
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Elevated levels of cytokines/chemokines contribute to increased neuroinvasion of human immunodeficiency virus type 1 (HIV-1). Previous work showed that lipopolysaccharide (LPS), which is present in the plasma of patients with HIV-1, enhanced transcellular transport of HIV-1 across the blood-brain barrier (BBB) through the activation of p38 mitogen-activated protein kinase (MAPK) signaling in brain microvascular endothelial cells (BMECs). Here, we found that LPS (100 μg/mL, 4 hr) selectively increased interleukin (IL)-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) release from BMECs. The enhancement of HIV-1 transport induced by luminal LPS was neutralized by treatment with luminal, but not with abluminal, antibodies to IL-6 and GM-CSF without affecting paracellular permeability as measured by transendothelial 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 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.
KeywordsBlood-brain barrier Human immunodeficiency virus type 1 Lipopolysaccharide Interleukin-6 Granulocyte-macrophage colony-stimulating factor Mitogen-activated protein kinase
Analysis of variance
Brain endothelial cells
Brain microvascular endothelial cells
Combination antiretroviral therapy
Granulocyte-macrophage colony-stimulating factor
Highly active antiretroviral therapy
Human immunodeficiency virus type 1
Mitogen-activated protein kinase
Transendothelial electrical resistance
Transforming growth factor
Tumor necrosis factor-α.
Human immunodeficiency virus type 1 (HIV-1) infection 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 increasing with the longer lifespan of HIV patients . Most antiretroviral drugs comprising HAART have a restricted entry into the brain because of blood-brain barrier (BBB) efflux transporters so that the brain serves as a reservoir for HIV-1  and a source for viral escape . 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 virus by an adsorptive endocytosis-like mechanism [4, 5, 6, 7] and trafficking of HIV-1-infected immune cells across the BBB . HIV-1 infection of brain endothelial cells (BECs) is not a productive infection  and penetration of HIV-1 is independent of the CD4 receptor . At the early stage, HIV-1 enters the brain through an intact, normally functioning BBB . At later stages of infection, elevated levels of proinflammatory cytokines/chemokines in the blood of patients with AIDS [12, 13, 14] are likely associated with the increase in HIV-1 infiltration [15, 16, 17], while HIV-1 gp120 and Tat induce the disruption of tight junctions in BECs [17, 18, 19, 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 . Peripheral LPS induces various inflammatory and immunological reactions including the production of cytokines/chemokines, such as tumor necrosis factor-α (TNF-αinterleukin (IL)-1, and IL-6 [23, 24, 25]. TNF-α enhances HIV-1 transport across the BBB  and LPS induces an increase in HIV-1-infected monocyte transport across the BBB . In our previous in vivo study, we found that the peripheral injection of LPS enhanced gp120 uptake by brain . These studies suggest that elevated levels of inflammatory mediators, 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  and are targets of LPS. The barrier function of the BBB is affected by various cytokines/chemokines in the blood compartment . Several studies using in vitro BBB models have shown that LPS increases the paracellular permeability of the BBB [29, 30, 31, 32, 33]. LPS induces or enhances the secretion of several cytokines by BECs . 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 the BBB through p38 mitogen-activated protein kinase (MAPK) . 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
HIV-1 (MN) CL4/CEMX174 (T1) prepared and rendered noninfective by aldrithiol-2 treatment as previously described  was a kind gift of the National Cancer Institute, NIH. The 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 μg of chloramine-T (Sigma) and 5.0 μg of the virus were incubated together for 60 sec. The radioactively labeled 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 Szabó et al.  and Nakagawa et al. . The animals were housed in clean cages in the laboratory with free access to food and water and were maintained on a 12-h dark, 12-h light cycle in a room with controlled temperature (24 ± 1°C) and humidity (55 ± 5%). All procedures involving experimental animals were approved by the local Animal Care and Use Committee and were performed in a facility approved by Association for Assessment and Accreditation of Laboratory Animal Care. Cerebral cortices harvested from 8-week-old male CD-1 mice from our in-house colony were homogenized, BMECs extracted, and cultured as previously performed . Cultures were treated with puromycin to remove pericytes.
Preparation of in vitro BBB models
BMECs (4 × 104 cells/well) were seeded on the inside of the fibronectin-collagen IV (0.1 and 0.5 mg/mL, respectively)-coated polyester membrane (0.33 cm2, 0.4 μm pore size) of a Transwell®-Clear insert (Costar, Corning, NY) placed in the well of a 24-well culture plate (Costar). Culture methods were the same as previously reported . Transendothelial electrical resistance (TEER in Ω × cm2) was measured before the experiments and after an exposure of LPS using an EVOM voltohmmeter equipped with STX-2 electrode (World Precision Instruments, Sarasota, FL). The TEER of cell-free Transwell®-Clear inserts were subtracted from the obtained values.
Lipopolysaccharide from Salmonella typhimurium (LPS; Sigma), monoclonal anti-mouse GM-CSF antibody, anti-mouse IL-6 antibody, mouse GM-CSF, and mouse IL-6 (all purchased from R&D systems, Minneapolis, MN) were dissolved in serum-free DMEM/F-12 (DMEM/F-12 containing 1 ng/mL bFGF and 500 nM hydrocortisone). The dose of LPS used in previous BMEC studies (100 μg/mL) was added to the luminal chamber of the Transwell® inserts, and anti-mouse GM-CSF antibody (10 μg/mL), anti-mouse IL-6 antibody (10 μg/mL), mouse GM-CSF (1-100 ng/mL), or mouse IL-6 (1-100 ng/mL) was loaded into the luminal or abluminal chamber. Then, the BMEC monolayers were incubated for 4 hr at 37°C with a humidified atmosphere of 5% CO2/95% air. In the experiments using antibodies, rat IgG (Sigma) was added to the control and LPS-treated group (10 μg/mL as final concentration).
U0126 (MEK inhibitor; Tocris Cookson Inc., Ellisville, MO), SB203850 (p38 MAPK inhibitor; Tocris) and SP600125 (Jun kinase (JNK) inhibitor; Sigma) were first dissolved in dimethyl sulfoxide (DMSO) and diluted with serum-free DMEM/F-12 (0.1% as the final DMSO concentration).
Transendothelial transport of 131I-HIV-1
The PSe values were divided by the surface area of the Transwell® inserts (0.33 cm2) to generate the endothelial permeability coefficient (Pe, in cm/min).
BMECs (4 × 104 cells/well) were seeded on the fibronectin/collagen I/collagen IV (0.05, 0.05, and 0.1 mg/mL, respectively)-coated 24-well culture plate (Costar). BMECs were washed with serum-free DMEM/F-12, and then exposed to 200 μL of LPS (100 μg/mL) with or without U0126 (10 μM), SB203580 (10 μM), and SP600125 (10 μM) for 4 hr at 37°C. Culture supernatant was collected and stored at -80°C until use. The cytokines (GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12 (p70), and TNF-α) were measured with the mouse cytokine/chemokine Lincoplex® kit (Linco Research, St. Charles, MO) by following the manufacturer's instructions.
Western blot analysis
LPS, GM-CSF, or IL-6-treated and control BMECs were washed three times with ice-cold phosphate buffered saline containing 1 mM sodium orthovanadate (Na3VO4) and 1 mM sodium fluoride (NaF). Cells were scraped and lysed in phosphoprotein lysis buffer (10 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 20 mM sodium pyrophosphate decahydrate, 2 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride) containing 1% protease inhibitor cocktail (Sigma) on ice. Cell lysates were centrifuged (15, 000 ×g at 4°C for 15 min) and the supernatants were stored at -80°C until use. The protein concentration of each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL). Twenty to thirty μg of the total protein was mixed with NuPAGE® LDS sample buffer (Invitrogen) and incubated for 3 min at 100°C. Proteins were separated on NuPAGE® Novex 4-12% Bis-Tris gel (Invitrogen) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen). After transfer, the blots were blocked with 5% BSA/Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.05% Tween 20 (TBS-T) for 1 hr at room temperature. The membrane was incubated with the primary antibody diluted in 5% BSA/TBS-T overnight at 4°C. The phosphorylation of p44/42 MAPK, p38 MAPK and JNK were detected using anti-phospho-p44/42 MAPK (1:1000), anti-phospho-p38 MAPK (1:500) and anti-phospho-JNK (1:500) rabbit monoclonal antibodies, respectively (all purchased from Cell Signaling Technology, Beverly, MA). Occludin, claudin-5, and ZO-1 were detected using anti-occludin, anti-claudin-5, and anti-ZO-1 mouse monoclonal antibodies (all purchased from Zymed, South San Francisco, CA). Blots were washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:10, 000; Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 5% BSA/TBS-T for 1 hr at room temperature. The immunoreactive bands were visualized on an X-ray film (Kodak) using SuperSignal® West Pico chemiluminescent substrate kit (Pierce). To reprobe total p44/42 MAPK, p38 MAPK, JNK, and actin, the membrane was incubated in stripping buffer (0.2 M glycine, 0.1% SDS and 1% Tween 20, pH 2.2) for 15 min twice and blocked with 5% non-fat dry milk/TBS-T. The total p44/42 MAPK, p38 MAPK and JNK were detected using anti-p44/42 MAPK (1:1000), p38 MAPK (1:1000), JNK (1:1000) (all purchased from Cell Signaling Technology), and actin (1:1000; Santa Cruz Biotechnology) antibodies, respectively. To quantify the relative levels of protein expression, the intensity of specific protein bands was quantified using ImageJ software (National Institute of Health, Bethesda, MD) and then normalized by that of each loading control protein.
Values are expressed as means ± SEM. One-way and two-way analysis of variances (ANOVAs) followed by Dunnett's or Tukey-Kramer's test were applied to multiple comparisons. Paired t-test was applied to the densitometry analysis. The differences between means were considered to be significant when P values were less than 0.05 using Prism 5.0 (GraphPad, San Diego, CA).
LPS stimulated release of GM-CSF and IL-6 by BMEC
Effect of LPS on the release of cytokines by BMECs.
LPS 100 μg/mL
4.8 ± 3.1
160.0 ± 21.7**
4.4 ± 1.1
1.6 ± 0.6*
1.1 ± 0.5
0.9 ± 0.2
0.5 ± 0.2
6.7 ± 1.1
16.3 ± 2.3**
2.3 ± 1.1
1.6 ± 0.6
0.3 ± 0.3
0.5 ± 0.3
0.2 ± 0.2
Polarized effect of antibodies to IL-6 and GM-CSF on LPS-induced increase in HIV-1 permeability and paracellular permeability of BMEC monolayer
Polarized response to IL-6 and GM-CSF in the permeability of BMEC monolayer
These results indicated that the effects of LPS on BMECs permeability to HIV-1 were mainly mediated by IL-6 and GM-CSF acting at the luminal surface of the BMEC. In all subsequent studies, therefore, we employed the luminal chamber as the loading chamber.
Effects of LPS, IL-6, and GM-CSF on the expression of tight junction proteins
Effect of MAPK inhibitors on the release of IL-6 and GM-CSF enhanced by LPS
Effects of IL-6 and GM-CSF on phosphorylation of p44/42 MAPK, p38, and JNK
BBB disruption can occur either  through the paracellular route (increased leakage between cells as measured by a decrease in TEER) or though the transcellular route (increased passage across a cell). Viral-sized particles , including HIV-1 , generally cross by the transcellular route. Our previous work found that LPS both increased the transcellular permeability of the BMEC monolayer to HIV-1 and decreased TEER . Here, we examined whether IL-6 and GM-CSF release from BMEC by LPS mediated these effects. The presence of LPS and antibodies to IL-6 or GM-CSF in the luminal chamber attenuated LPS-enhanced HIV-1 transport across the BMEC monolayer without a change in TEER (Figure 1A and 1B). BMECs secrete IL-6 and GM-CSF into both the luminal and abluminal chambers . To determine whether IL-6 and GM-CSF secreted by BMECs into the abluminal chamber are also involved in the LPS-induced increase in HIV-1 transport, we added antibodies to IL-6 or GM-CSF to the abluminal chamber. Neither antibody in the abluminal chamber inhibited the luminal LPS-induced changes in HIV-1 transport and TEER (Figure 1C and 1D). These results show that the IL-6 and GM-CSF secreted by BMECs in response to luminal exposure to LPS act at the luminal, but not the abluminal, endothelial surface to increase the transcellular permeability of BMECs to HIV-1. Furthermore, the results suggest that the LPS-induced increase in the paracellular permeability of the BMEC monolayer as measured by TEER is not mediated by extracellular IL-6 and GM-CSF.
We further investigated this functional polarity by adding IL-6 and GM-CSF to the luminal or abluminal chamber. Polarity of other cytokine actions has been investigated. We previously found that BMECs show no functional polarity in the reduction of paracellular permeability by transforming growth factor (TGF)-β1 . That is, either luminal or abluminal TGF-β1 has the same effect on the BBB paracellular permeability. In contrast, MCP-1 is only able to stimulate monocyte migration across BMECs when added to the abluminal surface . In the current study, only luminal IL-6 increased HIV-1 transport and was 10-100 fold more potent than abluminal IL-6 in decreasing TEER (Figure 2). Consistent with this, de Vries et al. reported that IL-6 increased paracellular permeability of BMECs . However, we found here that the IL-6-induced decrease in TEER was less than the LPS-induced decrease in TEER. Other soluble factors, such as other cytokines or chemokines, may be responsible for the remaining increase in the paracellular permeability induced by LPS. An IL-6-independent, P44/42-mediated phosphorylation of tight junction proteins may also be operational. The ability of IL-6 to decrease TEER but an inability of IL-6 antibody to block the effect of LPS on TEER suggests either that the LPS effect is not mediated through IL-6 or that IL-6 acts at a site not available to antibodies, such as inside the cell. Abluminal IL-6 (100 ng/mL) did not alter HIV-1 permeability despite the decrease in TEER. This finding is consistent with IL-6 promoting a transcellular or transcytotic mechanism for HIV-1 passage across the BBB that is independent of the paracellular pathway.
Luminal GM-CSF at the concentration of 100 ng/mL increased HIV-1 transport, whereas abluminal GM-CSF did not. Neither luminal nor abluminal GM-CSF changed TEER (Figure 3). This result further supports the idea that HIV-1 penetration across the BBB is through the transcellular route rather than the paracellular route. In addition, these results may suggest that the receptors for IL-6 and GM-CSF that affect HIV-1 permeability are mainly localized to the luminal membrane of BMECs. Therefore, enhanced invasion of HIV-1 into the brain may be mediated by BMEC-derived cytokines secreted into blood or by blood-borne cytokines. Consistent with this, IL-6 in the blood compartment induces BBB dysfunction [48, 49]. As summarized above, LPS, IL-6, and GM-CSF altered both HIV-1 permeability and TEER. The disparities discussed above between these two parameters of BBB function make it likely that they are separate events. Whereas the increased permeability to HIV-1 is likely mediated through transcytotic mechanisms, the decrease in TEER is caused by increased paracellular permeability resulting from altered tight junction function. LPS is known to alter the intensity and pattern of immunohistochemistry for the tight junction proteins claudin-5, ZO-1, and F-actin in BMECs [31, 33]. We examined whether LPS, IL-6, and GM-CSF affected the expression of these tight junction proteins in our models (Figure 4). The luminal treatment with LPS, IL-6, or GM-CSF did not induce significant changes in the expression of tight junction proteins in BMECs. Therefore, under the conditions of our model, LPS and IL-6 are likely increasing paracellular permeability of BMECs by altering tight junction function rather than expression of their proteins. For example, LPS and IL-6 may affect the localization of tight junction proteins in BMECs to increase the paracellular permeability.
Our previous work showed that LPS activated p44/42 MAPK and p38 MAPK in BMECs, and the activation of p38 MAPK resulted in the increase in HIV-1 transport . The activation of the p38 MAPK pathway leads to the production and release of inflammatory cytokines . Considering our present results, we hypothesized that either (i) LPS induced the production of IL-6 and GM-CSF through MAPKs or (ii) IL-6 and GM-CSF activated MAPKs. First, we determined whether the LPS-enhanced release of IL-6 and GM-CSF was mediated by MAPK signaling pathways as shown by the experiments using U0126 (MEK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) (Figure 5). U0126 and SB203580 inhibited the LPS-enhanced release of IL-6 and GM-CSF by BMECs. In the SP600125-treated group, inhibitory effects were not detected. This is reasonable as an LPS-induced increase in the phosphorylation of JNK has not been detected . These results indicated that LPS enhanced the release of IL-6 and GM-CSF from BMECs through the phosphorylation of p44/42 MAPK and p38 MAPK. Thus, the transcellular pathway taken by free virus differs from the JNK dependent, CD40-mediated pathway used by infected monocytes to cross the BBB .
Next, we determined whether IL-6 and GM-CSF increased the phosphorylation of MAPKs. IL-6 and GM-CSF did not increase the phosphorylation of p44/42 MAPK, p38 MAPK, or JNK (Figure 6). These results indicated that the IL-6- and GM-CSF-induced changes in the BMEC permeability for HIV-1 and paracellular permeability are downstream of the MAPK signaling pathways. Pathways downstream of the cytokines are likely COX-2 for IL-6-induced changes in TEER  and the JAK/STAT pathway for IL-6 and GM-CSF [51, 52] mediation of HIV-1 effects on immune cell migration . Thus, IL-6 and GM-CSF likely increase HIV-1 transport across the BBB through other intracellular signaling pathways. As for the mechanisms by which LPS could increase HIV-1 transport across the BBB, the following sequential events are proposed: (1) LPS activates p44/42 MAPK and p38 MAPK in BMECs; (2) this activation induces BMECs to release IL-6 and GM-CSF into the blood; (3) IL-6 and GM-CSF act at the luminal surface of the BMECs to enhance the transcellular transport of HIV-1 across the BBB.
In our previous study, we demonstrated that p38 MAPK mediated LPS-enhanced HIV-1 transport and p44/42 MAPK mediated the LPS-induced increase in paracellular permeability using each pathway inhibitor . U0126, the p44/42 MAPK inhibitor, did not attenuate LPS-enhanced HIV-1 transport. Here, U0126 as well as SB203580 decreased the release of IL-6 and GM-CSF (Figure 5). These findings suggest that the p38 MAPK signaling pathway directly leads to enhanced LPS-mediated transcellular transport of HIV-1.
In conclusion, we found that LPS potentiated the release of IL-6 and GM-CSF by BMECs through the activation of p44/42 MAPK and p38 MAPK. In addition to the p38 MAPK pathway, IL-6 and GM-CSF released from BECs acted at the luminal but not the abluminal surface to enhance HIV-1 transcellular transport. The p44/42 MAPK pathway and IL-6 likely acted at an intracellular site to increase paracellular permeability. Thus, LPS effects on HIV-permeation and on paracellular permeability were mediated through different cellular pathways. These results suggest that the release of cytokines by BECs plays an important role in the invasion of HIV-1 into the central nervous system. Preventing cytokine release by BECs through MAPK signaling pathways may be a therapeutic target in HIV-associated neurological dysfunction.
The authors thank Dr. Maria A. Deli (Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences) for technical advice on primary BMEC culture and comments on this study. The virus was a kind gift the National Cancer Institute, National Institutes of Health. Funded in part with Federal funds from National Cancer Institute, National Institutes of Health, under Contract No. N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of names, commercial products, or organization imply endorsement by the U.S. government. Supported by VA Merit Review (WAB), NIH R01NS050547 (WAB), and NIH R01AG029839 (WAB).
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