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Keywords:

  • blood–brain barrier;
  • choroid plexus;
  • cytokine;
  • neuroinflammation;
  • prostaglandin

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The choroid plexus epithelium forms the interface between the blood and the CSF. In conjunction with the tight junctions restricting the paracellular pathway, polarized specific transport systems in the choroidal epithelium allow a fine regulation of CSF-borne biologically active mediators. The highly vascularized stroma delimited by the choroidal epithelium can be a reservoir for retrovirus-infected or activated immune cells. In this work, new insight in the implication of the blood–CSF barrier in neuroinfectious and inflammatory diseases is provided by using a differentiated cellular model of the choroidal epithelium, exposed to infected T lymphocytes. We demonstrate that T cells activated by a retroviral infection, but not non-infected cells, reduce the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of the potent pro-inflammatory prostaglandin PGE2, via the release of soluble factors. A moderate alteration of the paracellular permeability also occurs. We identified the viral protein Tax, oxygenated free radicals, matrix-metalloproteinases and pro-inflammatory cytokines as active molecules released during the exposure of the epithelium to infected T cells. Among them, tumour necrosis factor and interleukin 1 are directly involved in the mechanism underlying the decrease in some choroidal organic anion efflux. Given the strong involvement of CSF-borne PGE2 in sickness behaviour syndrome, these data suggest that the blood–CSF barrier plays an important role in the pathophysiology of neuroinflammation and neuroinfection, via changes in the transport processes controlling the CSF biodisposition of PGE2.

Abbreviations used
BCSFB

blood–CSF barrier

CP

choroid plexus

HTLV-1

human T cell lymphotropic virus type-1

IL-1

interleukin-1

MMP

matrix metalloproteinase

PBMC

peripheral blood mononuclear cells

PR

phenol red

SFM

serum-free medium

SOD

superoxide dismutase

TJ

tight junctions

TNF

tumour necrosis factor

The CSF participates in maintaining the proper cerebral microenvironment needed for normal brain functions. Secreted by the choroid plexuses (CPs), the CSF circulates within the lateral, third and fourth ventricles and into the basal cisterns and the subarachnoid spaces surrounding the brain and the spinal cord. Its functions range from the mechanical protection of the brain and ‘sink’ action, to pH and inorganic ion buffering of extracellular fluid, to the more recently recognized participation in signalling to the brain (Davson and Segal 1996; Nicholson 1999; Strazielle and Ghersi-Egea 2000). By secreting the CSF and controlling its composition, CPs contribute to the homeostasis of the CNS. The CPs are constituted by a single layer of highly specialized epithelial cells sealed by tight junctions (TJ), which lies on a basal membrane and delimits a highly vascularized conjunctive stroma containing myeloid and lymphoid cells. The choroidal epithelium strongly restrains the paracellular pathway between the blood and the CSF, hence is responsible for the so-called blood–CSF barrier (BCSFB) (Strazielle and Ghersi-Egea 2000). Polarized specific transport systems in these epithelial cells allow a fine regulation of blood-to-CSF influx of micronutrients, and of CSF-to-blood efflux of bioactive and potentially harmful endogenous compounds (Davson and Segal 1996). In particular, the CPs appear to be involved in the elimination out of the brain of pro-inflammatory leukotriens and prostaglandins such as PGE2 which is a key mediator of sickness behaviour syndrome, including the development of fever (Konsman et al. 2002). This hypothesis is based on results showing that both types of compounds are taken up by isolated CP tissue, via a saturable process (Spector and Goetzl 1985; DiBenedetto and Bito 1986; Krunic et al. 2000), a mechanism which is likely to involve different organic anion transport proteins (Strazielle et al. 2004).

Different pathogens and retrovirus-infected leucocytes are present in the choroidal stroma in the course of infectious diseases (Levine 1987; Strazielle and Ghersi-Egea 2000). For instance, in HIV-infected patients, infected immune cells have been identified in the CP stroma, suggesting that CP is a route of neuroinvasion for the virus present in these cells (Falangola et al. 1995; Petito 2004). Similar hypotheses have been formulated from studies on animals infected with retroviruses such as the simian immunodeficiency and feline immunodeficiency viruses (Lane et al. 1996; Bragg et al. 2002). While (i) CPs represent a potential gate of entry into the CNS for infected leucocytes and possibly, in the more general context of neuroinflammatory diseases, for activated lymphocytes, and (ii) CPs appear involved in the regulation of CNS pro-inflammatory mediators such as PGE2, the difficulty to develop suitable experimental models so far prevented investigations of the direct effects of infected and/or activated immune cells on the neuroprotective efflux and barrier properties of the CPs.

In this work, we addressed whether T lymphocytes subjected to a a retroviral infection can impair the tightness and the organic anion efflux capacity of the choroidal epithelium, with the efflux of PGE2 as a special concern for the following reasons. This cyclo-oxygenase product of arachidonic acid metabolism, whose synthesis is up-regulated in the brain by pro-inflammatory cytokines, is involved in the development of the clinical symptoms associated with inflammation and infections, collectively referred to as the sickness behaviour syndrome (Konsman et al. 2002). In particular, CNS-produced PGE2 is a central player in the development of fever, and injection of PGE2 into the CSF induces hyperthermia (Engblom et al. 2003). Intracerebroventricularly administrated PGE2 also activates the transcription of the corticotropin-releasing factor in the endocrine hypothalamus, suggesting that this prostaglandin is involved in the hypothalamic-pituitary-adrenal axis neuroendocrine responses (Lacroix et al. 1996). Following experimental peripheral inflammation, an increased PGE2 level occurs in CSF, which is paralleled by the appearance of fever, and other inflammation-associated symptomes (Guay et al. 2004). Elevated CSF levels of this prostanoid is also observed clinically in patients with HIV-associated dementia (Griffin et al. 1994) and bacterial meningitis (Mustafa et al. 1990), as well as in experimental models of bacterial meningitis (Boje et al. 2003).

Our study was conducted in an in vitro cellular model of the BCSFB constituted by a monolayer of choroidal epithelial cells that reproduces the polarity, and the phenotypic, restrictive and transport properties of the in vivo choroidal epithelium (Strazielle and Ghersi-Egea 1999; Strazielle et al. 2003a). The reconstituted epithelium was exposed to either T lymphocytes infected with human T cell lymphotropic virus type-1 (HTLV-1), or non-infected cells. HTLV1-infected cells were chosen because (i), in addition to leukaemia, HTLV-1 is associated with a range of chronic inflammatory conditions which include the CNS disease HTLV-1-associated myelopathy/tropical spastic paraparesis (Gessain et al. 1985; Osame et al. 1986), and (ii) HTLV-1-infected human T cells represent a relevant model for retrovirus-activated immune cells and can be used in rodent-derived experimental models (Hollsberg 1999; Szymocha et al. 2000). We used T cells isolated from HTLV-1-infected patients as well as a T cell line chronically activated by HTLV-1 infection to demonstrate that retrovirally activated T cells induce, in addition to a moderate structural alteration of the BCSFB integrity, a reduction in the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of PGE2. Then, in an attempt to identify the soluble factors involved in this process, we analyzed the potential role of the viral protein Tax, free radicals, matrix metalloproteinases (MMPs), and pro-inflammatory cytokines, that we all demonstrated to be secreted during exposure of the CP epithelium to infected T cells.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell isolation and cultures

Animal care and procedures have been conducted according to the guidelines approved by the French Ethical Committee (decree 87–848) and by the European Community directive 86–609-EEC. OFA-timed pregnant rats (200–240 g) were obtained from Harlan (Gannat, France). One- or two-day-old rat CPs, sampled in sterile conditions and kept in 10% fetal calf serum-containing Dulbecco's modified Eagle's medium (DMEM)/F12 (1/1) medium were used to prepare primary cultures of epithelial cells. Choroidal epithelial cells from the lateral ventricles were isolated and cultured on Transwell-Clear insert filters coated with laminin (6.5-mm diameter, 0.33-cm2 surface, 0.4-μm pore size; Costar Plastics, Cambridge, MA, USA) as described in detail elsewhere (Strazielle and Ghersi-Egea 1999). Experiments were performed 5 days after confluence. In some experiments, epithelial cells were rinsed twice and fed in both compartments with serum-free medium (SFM) containing 10 or 100 ng/mL human recombinant MMP-9 (active rh-MMP-9; PF024, Oncogene, San Diego, CA, USA). Human recombinant cytokine treatment by 25 ng/mL tumour necrosis factor-α (TNF-α) and 5 ng/mL interleukin-1 (IL-1) (RnD Systems, Abingdon, UK) was performed using 250 μL and 500 μL SFM in the apical and basolateral compartments, respectively.

Peripheral blood mononuclear cells (PBMC) from a healthy donor were freshly isolated from whole blood by red blood cell lysis in a NH4Cl (150 mm), KHCO3 (10 mm) and EDTA (10 μm) solution, pH 7.3, for 10 min, followed by a 7-min centrifugation at 120 g. PBMC were washed once in culture medium, their viability was assessed by trypan blue exclusion assay, and the cells were used immediately. Infectious CD4+ and CD8+ primary T lymphocytes were obtained by culturing PBMC from HTLV-1-infected patients (Cib and Rom) in RPMI-1640 (Invitrogen, Paisley, Scotland) supplemented with 10% human AB serum and 20 U/mL IL-2, without additional activation. (gift of Dr C Pique, CNRS UMR8104, Paris, France). As a result of HTLV-1 infection, these cells could undergo short-term culture while retaining the phenotype and morphology of infected and activated primary T lymphocytes (Wucherpfennig et al. 1992). The human non-infected T lymphocyte cell line CEM, and the HTLV-1-infected non-productive human T-cell line C8166/45 (Popovic et al. 1983), were cultured in suspension in RPMI (Invitrogen, Cergy-Pontoise, France), supplemented with 10% fetal calf serum until use. The expression of the viral protein Tax was assessed in all HTLV-1-infected T cells (not shown).

Transient co-culture of epithelial cells and T lymphocytes

T cells were re-suspended at a density of 1 million/mL in SFM (∼30–40% of normal blood T cell density) and 750 μL of the cell suspension were distributed into wells of 24-well plates. Inserts with epithelial cells, filled with 250 or 300 μL of SFM, were then transferred into the wells containing the T cell suspension. This conformation prevents direct contact between cells but enables soluble factors to diffuse through the microporous filter. All T-cells remained viable at least for 48 h in co-culture (not shown).

When required, two cytokine antagonizing agents, the human recombinant chimeric soluble construct containing two TNF-α soluble receptors sTNFRII-Fc and the rat recombinant IL-1 receptor antagonist rrIL-1-ra (R & D Systems, Abingdon, UK), were added to both compartments at a final concentration of 5 and 1 μg/mL, respectively. The free radical scavengers superoxide dismutase (SOD, Sigma, St Louis, MO, USA) and catalase (Sigma) were added in the basolateral compartment at a final dose of 300 and 400 U per 750 μL, respectively. The Tax blocking monoclonal antibody (ascite NIH 1314) was added in the basolateral compartment at a dilution of 1/100 or 1/500.

Transport and paracellular permeability studies

Phenol red active transport

The active transport of phenol red (PR), was assayed by measuring the ability of the epithelial cells to generate an imbalance in PR concentration over a 30 h-period between the apical and basolateral compartments. The initial PR concentration was of 23 μm in both compartments. PR concentrations were analyzed, and active clearance (μL/cm2) calculated, as previously described (Strazielle et al. 2003b).

Efflux of prostaglandin E2 (PGE2) and paracellular permeability measurement

The CSF-to-blood transfer of 0.4 μCi/mL of [3H]PGE2 (200 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), supplemented with unlabelled PGE2 (Sigma) to reach a final concentration of 100 nm, was measured in the presence of 0.2 μCi/mL [14C]sucrose (350 mCi/mmol; Amersham, Little Chalfont, UK) as a marker of paracellular permeability. Purity of the tritated compound was checked by HPLC with radiochemical analysis of the eluate and found higher than 90%. Inserts were rinsed once on both sides before starting the permeability study. All incubations were performed in Ringer-HEPES buffer on a rotating platform (200 r.p.m.) at 37°C, as previously described (Strazielle and Ghersi-Egea 1999).

Calculation of flux.  The flux of radiolabelled compounds across the cell monolayer was estimated as the amount cleared from the donor compartment. As the clearance volume increased linearly with time for both compounds during the course of the experiment, their respective permeability-surface area (PS) product (in microliters per minute per filter) were calculated. PGE2 transport across the epithelium involves a major transporter-mediated component (see Results), and its apical uptake is concentrative (Krunic et al. 2000). Hence the (low) passive resistance of the filter does not influence the overall permeability of the compound. The results were therefore expressed as permeability coefficient across the cell-filter system (Pt, in cm/min). For sucrose experiment, whose paracellular transfer is by passive diffusion, the permeability coefficient of the epithelial monolayer (Pe, in cm/min) was calculated by correcting for the resistance associated to the filter alone. Details of calculations have been reported elsewhere (Strazielle and Ghersi-Egea 1999; Strazielle and Preston 2003).

HPLC analysis.  Incubation media issued from PGE2 transfer experiments were analysed by reverse-phase HPLC performed on a LC10 Shimadzu system (Duisburg, Germany) as follows: samples (20 or 40 μL) were applied onto an Ultrasphere ODS RP-18 analytical column (5 μm, 46 mm × 150 mm, Beckman, Fullerton, CA, USA), and eluted using a mobile phase of 35% acetonitrile/0.1% acetic acid/water pumped at 1 mL/min. Absorbance of the effluent was monitored at 210 nm. The effluent was collected for radiochemical analysis by liquid scintillation counting. Retention times of PGE2 and its two main metabolites, i.e. 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2 are 8.3, 11 and 15.5 min, respectively. The purity of radiolabelled PGE2 was estimated for the working solutions as the ratio of radioactivity associated with PGE2 to the total radioactivity injected. These purity values (90–95%) were taken into account to calculate the percentage of radioactivity co-eluting with PGE2 in the incubation media sampled from the apical and basolateral compartments. For basolateral analysis, the working solution have been diluted 50 times prior injection to allow comparison of chromatograms with similar signal–noise ratio.

Immmunocytochemical analysis of tight junction proteins distribution

Rabbit polyclonal antibodies raised against occludin and claudin-2 (respectively no. 71–1500, diluted 1/400, and no. 51–6100, diluted 1/200, Zymed Laboratories Inc., South San Francisco, CA, USA) were used. The cell-covered filters were rapidly washed with ice-cold Dulbecco's phosphate-buffered saline (PBS) containing Ca++ and Mg++ (Invitrogen). The cell monolayers were fixed by methanol/acetone (1 : 1), cooled at −20°C, for 90 s, washed and stored at +4°C until immunocytochemical analysis was performed, as previously described (Strazielle et al. 2003b).

Measurement of superoxide anion (inline image) production

The measurement of inline image production was based on its ability to specifically reduce oxidized acetyl–cytochrome c (Azzi et al. 1975). Epithelial and T cells were rinsed twice and subsequently cultured, in solo or co-culture, in PR-free medium (Invitrogen) to avoid spectrophotometric interference with the pH indicator, at 37°C, 5% CO2. At 24 or 48 h of culture, acetyl–cytochrome c (Sigma) was added to the medium at a final concentration of 60 μm and allowed to react for 80 min. The amount of inline image that diffused in the medium was assayed by measuring the relative amount of acetyl–cytochrome c reduced by second derivative spectrophotometry, as previously described (Daval et al. 1995), with a Cary 100 dual spectrophotometer (Varian). Using this measurement procedure, the inline image trapper SOD (300 U per 750 μL) added to the medium completely abolished the signal. Results are expressed as nmoles/80 min.

Zymographic analysis

The secretion of MMP-2 and MMP-9 by the epithelial and T cells, in solo and in co-culture was analyzed by gelatin–zymography (Strazielle et al. 2003b). Cells were cultured for 48 h to allow sufficient accumulation of the enzymes in the medium. Samples of apical medium were diluted with SFM before loading on gel, to account for the volume difference between the two compartments. The technique allows to visualize two bands for MMP-2 (corresponding to the 72-kDa pro-enzyme and the 65-kDa enzyme), and two bands for MMP-9 (corresponding to the 92-kDa pro-enzyme and the 85-kDa enzyme). Treating the gel with 10 mm EDTA, a metal chelator that inhibits the catalytic activity of MMPs, completely abolished all signals (not shown). A relative quantification was obtained using standard curves run in similar conditions, as previously described (Strazielle et al. 2003b).

Measurement of rh-MMP-9 activity

The proteolytic activity of rh-MMP-9 was assessed in our experimental culture conditions using a gelatinolytic assay as follows: 100 ng of rh-MMP-9 were incubated with 320 μg gelatin in a final volume of 200 μL SFM, for 20 h at 37°C under gentle agitation. After incubation, 10 mm EDTA was added to stop the reaction and samples were resolved in 10% polyacrylamide gel by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli 1970). Proteins were stained for 15 min with 0.1% Coomassie blue R250 in 30% methanol−10% acetic acid aqueous solution.

Tax detection using immunoprecipitation

The culture medium (1 mL) was pre-cleared twice by incubation with 25 μL of protein G sepharose beads 17-0618-02, Amersham), for 1 h at 4°C. Samples were then centrifuged (17 900 g, 5 min, 4°C). The supernatant was collected and immunoprecipitated overnight at 4°C under rocking agitation, using a goat polyclonal antibody raised against Tax (AIDS Research and Reference Reagent Program, NIAID, NIH; Tax antiserum from Dr P. Szecsi, H. Halgreen and J. Tang, no. 1189) diluted at 1/500. The medium was then incubated with 25 μL of protein G sepharose beads for 1 h at 4°C under gentle agitation.

After supernatant removal, the sepharose beads were washed three times by centrifugation at 4°C in a buffer containing in mm: 50 Hepes, 150 NaCl; 1.5 MgCl2; 1 EDTA, 10% glycerol and 1% Triton X100. Bound proteins were resolved by 10% SDS–PAGE and electrophoretically transferred onto nitrocellulose. The blots were blocked in 5% non-fat dry milk in TBST (10 mm Tris, 150 mm NaCl, 0.1% Tween 20, pH 7.4) for 1 h at 20°C, and incubated overnight at 4°C with the immunoprecipitating Ab (1/400), in TBST containing 1% milk. After three washes with 1% milk-containing TBST, the blots were incubated with peroxidase-conjugated anti-goat IgG antibody (1/800, Jackson Laboratories, West Grove, PA, USA) and the proteins revealed by enhanced chemoluminescence (Covalight, Dako, France). C8166/45 cell lysates and culture medium from either epithelial cells in solo culture or CEM cells were used as positive and negative controls, respectively.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Functional and structural alterations of the choroidal epithelial monolayer are induced during co-culture with retrovirus-infected T cells

To analyze the paracellular permeability and the transport properties of the CP epithelium exposed to HTLV-1-infected T cells, we incubated the epithelium reconstituted on a porous filter with primary T cells (Cib and Rom) isolated from two HTLV-1-infected patients, both cell preparations showing the ability to be transiently maintained in culture as a result of the infection. Alternatively, T cells established in cell line (C8166/45 cells) also as a direct result of HTLV-1 infection (Popovic et al. 1983) were used. The latter cells are not virion productive. PBMC isolated from a healthy donor, and the non-infected T-cell line CEM were used as controls. T cells were added to the basolateral compartment of the cellular model (Fig. 1a). Incubating epithelial monolayers with non-infected PBMC or CEM cells did not lead to any modification of the parameters investigated (Fig. 1).

image

Figure 1. Alteration in the transport and permeability properties of the BCSFB upon exposure to HTLV-1-infected T cells. (a) Schematic representation of the cellular model used for the study. At confluence, the epithelial choroidal cells form a barrier delimiting an apical compartment (Ap), representing the CSF and a basolateral compartment (Bl) corresponding to the stroma/blood. T cells are placed in the basolateral compartment for 30 h. l represents the distance separating the microporous membrane from the bottom of the plate well, and is of 1 mm. The cumulated PR clearance from the apical to the basolateral compartment against its concentration gradient was assessed as an index of organic anion active efflux over the incubation period (b). Apical-to-basolateral transport of PGE2 was thereafter measured (c), and the paracellular permeability was assessed using sucrose as a polar, membrane-non-permeant marker (d). In (c), the dark bar represents the apparent apical-to-basolateral diffusion coefficient for PGE2, Kdiff calculated by non-linear regression analysis of Pt over five PGE2 concentrations ranging from 100 nm to 250 μm (r = 0.989), and illustrates the saturability of the transport. In (d), the effect of a complete tight junction dissociation upon calcium removal on the paracellular permeability is illustrated by the dark bar, for comparative purpose. For clarity, values are expressed as percentage of the control value (epithelial cells in solo culture) from the corresponding experiments and represents mean ± SD, n = 4, except in (d) (C8166/45 T cell-exposed epithelium) for which n = 11 from three separate experiments. Average control values from three experiments carried out on different batches of CP epithelial cells are (mean ± SD, n = 12): 581 ± 64 μL/cm2 for the active clearance of PR measured on a 30 h-period, 1.42 ± 0.14 10−3 cm/min for PGE2 Pt, and 0.37 ± 0.04 10−3 cm/min for sucrose Pe. *** and **, different from control, p < 0.001 and 0.01, respectively. Statistical analysis was performed by one-way anova followed by a posteriori Dunnett's test. PR, phenol red; PBMC, peripheral blood mononuclear cells from a healthy donor; Cib and Rom, infectious CD4+ and CD8+ primary T lymphocytes obtained from HTLV-1-infected patients; CEM, human non-infected T lymphocyte cell line; C8166/45, HTLV-1-infected, non-productive human T cell line.

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By contrast, co-culture with HTLV-1-infected T cells led to a reduction in the ability of epithelial cells to clear PR, from the apical compartment into the basolateral compartment. PR is used as a model substrate for unidirectional, energy-dependent choroidal efflux transport of organic anions. The 30-h-cumulated PR clearance was reduced by 20% during exposure to the infected primary T cells, and by 50% during exposure to the infected cell line (Fig. 1b). These results indicate that the active choroidal CSF-to-blood transport of organic anions becomes less efficient in the course of the 30-h-co-culture period. The effect could be already seen at 24 h and increased at 48 h (not shown). A 30-h-exposure time was chosen for all experiments thereafter as it allows both glucose and glutamine to remain present in the medium at concentrations high enough (> 16 and > 44%, respectively, of fresh medium) to preclude a non-specific alteration as a result of nutrient shortage.

PGE2 is a potent pro-inflammatory organic anion which is taken up by isolated CP tissue. We therefore investigated whether the CP epithelium is a site of PGE2 elimination from CSF into the blood, and whether this function is altered upon exposure to retrovirus-infected T cells. CP epithelial cells in solo culture efficiently transported PGE2 in the apical-to-basolateral direction. The permeability value of 1.42 ± 0.14 × 10−3 cm/min was much higher than that of sucrose (0.37 ± 0.04 10−3 cm/min) which represents the paracellular permeability. PGE2 transepithelial transport is saturable. Increasing PGE2 concentration in the apical compartment led to a strong decrease in its clearance rate, reaching a minimal value of 0.45 × 10−3 cm/min which represents the apparent non-saturable component of the transfer (Kdiff, Fig. 1c). When the epithelial cells were exposed to retrovirus-infected T cells for 30 h prior to PGE2 efflux measurement, PGE2 transport was consistently and significantly decreased (Fig. 1c). With C8166/45 cells, this decrease represented 35% of the total permeability, or 52% of the saturable component of the transport (above the dashed line, Fig. 1c), and occurred despite the increase in the paracellular permeability which was also observed following the 30 h-co-culture period, as illustrated by the average 3.4-fold increase in [14C]sucrose permeability coefficient (Fig. 1d). This increase was moderate, and represented only 11% of the maximal (30-fold) increase in sucrose Pe observed following the complete disassembly of TJs by removal of calcium into the medium (Fig. 1d). The retrovirus-infected primary T cells also showed a tendency to increase the BCSFB paracellular permeability, but these effects did not reach statistical significance (Fig. 1d). To appreciate the structural alterations of the TJs which are responsible for the establishment of a low paracellular permeability, we analyzed the distribution of the TJ-associated proteins occludin and claudin-2 known to participate to the choroidal TJ (Lippoldt et al. 2000). Co-culture with CEM cells did not induce any clear modification in TJ protein distribution which remained pericellular and continuous and resembled that of an epithelial monolayer in solo culture (as illustrated for claudin-2 and occludin in Figs 2a and c). By contrast, in epithelial cells exposed to C8166/45 cells, disrupted staining was observed for claudin-2 and occludin in several fields of observation such as those presented in Figs 2(b and d). This result indicates that the increased paracellular permeability of the BCSFB is paralleled by a concomitant alteration of its structural integrity. Co-culture of epithelial cells with Cib, and to a lesser extent with Rom T cells, also led to discrete alterations in TJ protein distribution (not shown).

image

Figure 2. Immunocytochemical localisation of the TJ-associated protein claudin-2 (a, b) and occludin (c, d) in confluent epithelial cell monolayers. When cultured alone or with non-infected T cells (a, c) the localisation of claudin-2 and occludin appeared pericellular and continuous. Example of fields of fainter or discontinuous circumferential labelling frequently displayed by epithelial cells co-cultured with HTLV-1-infected C8166/45 cells are shown in (b) and (d). Scale bar: 25 μm.

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Mechanisms of BCSFB functional impairment upon exposure to infected T cells

The design of the co-culture system prevents the direct contact of choroidal epithelial cells with T cells (Fig. 1a). This indicates that the functional alterations induced by infected T cells involve one or several soluble factors secreted by these lymphocytes. C8166/45 cells are non-virion productive and produced the most significant changes. Thus, the release of virions appears not to be involved in the BCSFB alteration. For this reason, and because the supply and lifespan of Cib and Rom cells were limited, C8166/45 cells were henceforth used to investigate the mechanisms of interaction between infected T cells and the choroidal epithelial cells that led to changes in the BCSFB properties. Several factors from viral or cellular origins, known to be specifically produced by infected T cells, and/or likely to be involved in the alteration mechanisms, have been investigated.

Viral protein Tax

The viral protein Tax is an HTLV-1 transcription transactivator protein which can be secreted and act on neighbouring cells (Lindholm et al. 1992). The involvement of extracellular Tax is suspected in different pathogenic mechanisms (Grant et al. 2002), for example a decreased glial transport of glutamate (Szymocha et al. 2000). Immunoprecipitation experiments followed by western blot analysis using a polyclonal anti-Tax antibody, or a monoclonal anti-Tax antibody (ascite NIH 1314), showed that Tax was released in the culture medium of C8166/45 cells, both in solo culture (not shown) or in co-culture (Fig. 3a). The neutralizing mouse monoclonal anti-Tax antibody (ascite NIH 1314), previously shown by our laboratory to block the Tax-induced decrease in glutamate uptake by astrocytes (Szymocha et al. 2000), did not revert the effects of C8166/45 cells on either PR cumulated clearance, PGE2 efflux or the paracellular permeability (Fig. 3b). These results suggest that, although released in the cell environment, the viral protein Tax per se is not involved in the HTLV-1-infected cell-induced alteration of the choroidal epithelium properties.

image

Figure 3. Secretion of the viral transactivator Tax in the medium of HTLV-1-infected T cells co-cultured with choroidal epithelial cells, and implication in the BCSFB alteration induced by infected cells. (a) Western blot analysis of culture medium immunoprecipitated by a polyclonal antibody raised against Tax. The band corresponding to Tax, with an apparent Mr of 40 kDa, is present in lane 3 corresponding to the co-culture medium, and is absent in lane 2 corresponding to the epithelial cell solo culture medium. Other bands include the immunoglobulins used to immunoprecipitate Tax (lane 1). (b) Adding a mouse monoclonal Tax-neutralizing antibody (ascite NIH 1314, diluted 1/100) to the basolateral compartment of the epithelial cells in co-culture with C8166/45 cells does not prevent the alteration induced by HTLV-1-infected T cells. Active clearance of PR, apical-to-basolateral transport of PGE2 and the paracellular permeability are measured as described in Fig. 1, mean ± SD (n = 4–5). All data obtained using epithelial cells exposed to C8166/45 cells were statistically different from the data obtained in epithelial cells in solo culture (control), p < 0.001, one-way anova followed by a posteriori Dunnett's test. No alteration was observed when the anti-Tax neutralizing antibody was added in the medium of the epithelial cells in solo culture (not shown).

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Matrix metalloproteinases

MMPs are endopeptidases with extracellular matrix-degrading and sheddase activity and are suspected to have a role in tight cellular barrier breakdown (Rosenberg et al. 1992; Mun-Bryce and Rosenberg 1998). MMP-2 and especially MMP-9 have been associated with inflammatory processes, and their secretion can be induced in both choroidal epithelial cells and T lymphocytes (Biddison et al. 1997; Strazielle et al. 2003b). Media of solo and co-cultures were analysed for MMP-2 and MMP-9 accumulation by gelatin–zymography. (example shown in Fig. 4a). MMP-2 was secreted at both apical and basolateral sides of the choroidal epithelial cells in solo culture, and traces of MMP-9 could also be detected in the apical medium. Co-incubating the epithelial cells with CEM or C8166/45 cells led to higher amounts of both MMP-2 and MMP-9 in the basolateral medium. Quantitative analysis of zymograms from different experiments (Fig. 4b) did not reveal a difference in the amount of MMP-2 released in the basolateral medium of CEM co-culture by comparison with C8166/45 cell co-culture. By contrast, MMP-9 amount in the basolateral compartment was greater in the presence of infected T cells than in the presence of CEM cells. The intrinsic secretion of MMP-9 by the infected cells (Fig. 4a, C8166/45 cells solo) accounts for most of this difference. To investigate whether this higher level of MMP-9, secreted by infected T cells, could be involved in choroidal transport and barrier dysfunctions, we tested the effect of the recombinant enzyme (rh-MMP-9) on the choroidal epithelium. After assessing the proteolytic activity of rh-MMP-9 in our cell culture conditions (Fig. 5a), we exposed the monolayers of epithelial cells to 10 ng/mL rh-MMP-9 for 30 h. This amount of MMP-9 was comparable with the amount of enzyme secreted by the infected cells over the total exposure period (Fig. 4a). Whereas rh-MMP-9 was not degraded and the integrity of its catalytic site was still maintained at the end of this incubation period (Fig. 5c), no significant change in organic anion transport activities was observed (Fig. 5b). The paracellular permeability was not altered either (Fig. 5b). Repeating the experiments with a dose of rh-MMP-9 (100 ng/mL) which was evaluated by zymography as much higher than the amount of MMP-9 released in our culture system (Fig. 5c), led to similar results. This indicates that, while the choroidal epithelium co-cultured with HTLV-1-infected T cells is exposed to high levels of MMP-9 at their basolateral membrane, this protease per se is not involved in either the structural or the functional BCSFB alterations induced by infected T cells.

image

Figure 4. MMP-2 and MMP-9 secretion by choroid epithelial cells and T cells in solo and co-culture. Apical and basolateral media from the choroidal monolayer culture, exposed or not exposed for 48 h to T cells, were subjected to zymographic analysis. (a) Example of zymogram showing MMP-2 and MMP-9 secreted in the medium of epithelial cells in solo culture (CTR), or in co-culture with non-infected (CEM), or infected (C8166/45) T cells. Enzymes appear as two bands, the pro- and cleaved forms. The epithelial cells in solo culture secrete MMP-2 in both compartments, and MMP-9 is detectable in the apical medium. CEM and C8166/45 cells both in solo culture and in co-culture with epithelial cells secrete the two gelatinases. MMP-9 was clearly visible in the medium of C8166/45 cells in solo and co-culture. SFM, serum-free medium. (b) Densitometric analysis of zymograms from different cell preparations showing the relative amounts of total MMP-2 and MMP-9 in the basolateral compartment. Values in arbitrary densitometric units are mean ± SD (n = 4, two different cell preparations). NQ, not quantifiable. *Statistically different from CEM co-culture, p < 0.05 (two-tailed student's t-test for equal variance).

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image

Figure 5. Effect of MMP-9 on the paracellular permeability and transport properties of the choroidal epithelial monolayer. (a) The efficient gelatinolytic activity of the recombinant MMP-9 was assessed in our experimental culture conditions. The gelatin digestion is reflected by the loss of the protein electrophoretic profile (left lane), and is inhibited by the Ca++ chelator EDTA (central lane). (b) Epithelial cell exposure to 10 ng/mL (this figure) or 100 ng/mL (not shown) rh-MMP-9 in both compartments does not lead to any statistically significant changes in PR active clearance, PGE2 transport, or paracellular permeability. The data are generated as described in Fig. 1, and for clarity are expressed as percentage of control values (without rh-MMP-9) (mean ± SD, n = 4). (c) The active MMP remaining at the end of the 30 h-incubation period was evaluated by conventional zymography analysis of an aliquote of the culture medium. This ascertained the absence of significant degradation of the recombinant enzymes at both concentrations. Arrow shows the active rh-MMP-9. The lower band, also present in the control epithelial cell medium, corresponds to MMP-2 secreted by the epithelial monolayer in the course of the incubation.

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Oxygen free radicals

inline image is an oxygen-derived radical intermediate involved in the cascade production of more reactive oxygen species, which occurs in biological systems through Fenton reaction and peroxinitrite formation (Liochev and Fridovich 1999). inline image can diffuse from the cells, be trapped and spectrophotometrically detected, and thus was used as an index of reactive oxygen species production (Daval et al. 1995). An inline image production by T cells was detected. This production, observed both after 24 and 48 h of culture, appears to be a continuous process (Fig. 6a). HTLV-1-infected C8166/45 cells produce significantly higher amounts of inline image than CEM cells (Fig. 6a), and this production was maintained in co-culture conditions (not shown). To address the role of free radicals in the alteration of the BCSFB induced by C8166/45 cells, we co-cultured the epithelial cells with these T cells in the presence or absence of both SOD, which catalyses the formation of hydrogen peroxide from inline image, and catalase, which catalyses the inactivation of hydrogen peroxide and prevents the formation of the highly toxic hydroxyl radical. This combination of antioxidant enzymes resulted in a significant but limited reduction of PR transport inhibition only (Fig. 6b), suggesting that oxygen free radicals do not initiate, but may favour, the changes in BCSFB properties observed upon exposure to the infected cells.

image

Figure 6. Involvement of oxygen-derived reactive species in the BCSFB alterations induced by HTLV-1-infected T cells. inline image production by T cells. At 24 or 48 h of culture, acetyl–cytochrome c was added to the medium and allowed to react for 80 min with inline image(a) diffusing from the cells, before second derivative spectroscopy analysis. Values are mean ± SD (n = 3–4). inline image production by choroidal epithelial cells in solo culture was low (< 1.5 nmoles/80 min). *p < 0.05 statistically different from CEM value, two-tailed student's t-test for unequal variance. (b) The antioxidant enzymes SOD and catalase, were added to the basolateral compartment of the epithelial cells in co-culture with C8166/45 cells, and PR active clearance, PGE2 and paracellular permeability data (mean ± SD, n = 4–5) were generated as described in Fig. 1. All data obtained using epithelial cells exposed to C8166/45 cells were statistically different from the data obtained for epithelial cells in solo culture, p < 0.001, one-way anova followed by a posteriori Dunnett's test. *Statistically different from co-culture in the absence of SOD/catalase, p = 0.036, one-tailed student's t-test. Adding antioxidant enzymes to the epithelial cells in solo culture had no effect on their BCSFB properties (not shown).

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Pro-inflammatory cytokines TNF-α and IL-1

We have shown that HTLV-1-infected T-lymphocytic cells such as C8166/45 cells secrete the pro-inflammatory cytokines TNF-α and β, and IL-1 (Giraudon et al. 2000). A soluble TNF receptor chimeric construct with potent affinity for both TNF-α and TNF-β (sTNFRII-Fc) and the-non-transducing recombinant rat ligand for IL-1 receptor I, rrIL-1-ra, were used in combination to trap TNF-α (and potentially TNF-β) and block IL-1RI-dependent IL-1 activities. The concentration of these agents have been chosen as to exceed the secretion of cytokines estimated for C8166/45 cells (Giraudon et al. 2000), but remained limited so as to avoid a toxicity on both cell types in presence. Adding sTNFRII-Fc and rrIL-1-ra in the co-culture medium increased by 43% the active transport of PR measured in the presence of C8166/45 cells for 30 h, without preventing the inhibition of PGE2 efflux measured at the end of the 30 h (Fig. 7a). These data suggest that sTNFRII-Fc and rrIL-1-ra can retard the alteration of organic anion transport. By contrast, the increase in paracellular permeability was not changed.

image

Figure 7. Implication of pro-inflammatory cytokines in the choroidal transport alterations induced by HTLV-1-infected cells. (a) A combination of sTNFRII-Fc (5 μg/mL) and rrIL-1-ra (1 μg/mL) was added to the epithelial cells in co-culture with C8166/45 cells, and PR active clearance, PGE2 transport and paracellular permeability data (mean ± SD, n = 4) were generated and expressed as described in Fig. 1. All data obtained using epithelial cells exposed to C8166/45 cells were statistically different from the data obtained in epithelial cells in solo culture (p < 0.001, one-way anova followed by a posteriori Dunnett's test). **Statistically different from C8166/45, p < 0.01, one-tail student's t-test for equal variance. sTNFRII-Fc and rrIL-1-ra had no deleterious effect on the transport properties of epithelial cells in solo culture (not shown). (b) Epithelial cells in solo culture were exposed to TNF-α (25 ng/mL) and IL-1β (5 ng/mL) combination and parameters measured as above. Values are expressed as percentage of control value (mean ± SD, n = 3). ***Statistically different from control, p < 0.001, two-tailed Student's t-test for equal variance.

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To further demonstrate the involvement of the pro-inflammatory cytokines on the alteration of the CP epithelium properties, we exposed the reconstituted epithelium to recombinant TNF-α/IL-1β for up to 48 h. This treatment strongly reduced both the active clearance of PR and the efflux of PGE2 by 50 and 65%, respectively, while the paracellular permeability of the monolayer was not affected (Fig. 7b). Both cytokines were required to induce a maximal inhibitory effect (not shown). The inhibition of organic anion transport processes was already observed after 24 h of treatment, yet to a lower extent (not shown). These data indicate that pro-inflammatory cytokines play a major role in the organic anion transport inhibition induced by infected T lymphocytes, without contributing per se to the increase in the paracellular permeability. Finally, the possibility that an increase in PGE2 metabolism in the choroidal cells could participate in the apparent reduction of PGE2 transfer across the cells was ruled out by analyzing the cell incubation media by HPLC with radioactive detection, following apical exposure to PGE2(Fig. 8). In both compartments, the radioactive signal was associated with the retention time of PGE2, rather than the retention times of its two potential metabolites. More importantly, the percentage of radioactivity associated with PGE2 at the end of the incubation period in the donor (upper) compartment of cytokine-treated cells was strictly identical to the percentage measured for control cells (Fig. 8b, insert), while an 8% decrease would be expected, if the reduction in the apparent efflux of PGE2 following cytokine treatment was to be accounted for by an increase in metabolism and release of the radiolabelled metabolites back in the donor compartment. In the basolateral compartment, a 14% decrease in the radioactivity associated with PGE2 was observed for treated cells versus control cells (Fig. 8c, insert), which is accounted for by the small increase in the amount of radioactivity eluting with a retention time corresponding to the dihydroketo metabolite of PGE2 (Fig. 8c).

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Figure 8. UV and radiochemical reversed phase HPLC analysis of epithelial cell culture media following apical exposure to [3H]-PGE2. (a) UV HPLC profile obtained from a 50-μm PGE2 solution in incubation buffer. (b, c) [3H]-PGE2 was added at a concentration of either 6 or 100 nm to the upper compartment and the cell monolayers incubated for 45 min. Radiochemical HPLC profiles were then obtained from the upper (b) and lower (c) compartments. Solid chromatograms are typical examples obtained using control cells. Dashed chromatograms are obtained from cytokine-treated cells, and shows the lower amount of PGE2 reaching the basolateral compartment (c) (solid and dashed chromatograms from the donor compartments are strictly overlapping). There was no apparent difference in HPLC profiles between experiments realized at the two different concentrations of PGE2. Data in the inserts are mean ± SD, n = 4. *: statistically different from control, p < 0.05, one-tailed student's t-test for unequal variance.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Retrovirus-infected T lymphocytes alter transport properties of the choroidal epithelium

Using a differentiated cellular model of the BCSFB validated for transepithelial transport studies, we demonstrate that soluble factors released by HTLV-1-infected T lymphocytes impaired an important function of the blood–CSF barrier, namely the carrier-mediated efflux process for organic anions. This efflux was evaluated by two different indices. The ability of the cells to generate an imbalance in PR concentration between the apical and basolateral compartment allowed appreciation of the cumulative efflux over the entire period of co-incubation. The saturable apical-to-basolateral transcellular transport of PGE2 that we demonstrated in the choroidal epithelial cells was used as end-point measurement of transporter-mediated CSF efflux of organic anions involved in inflammatory processes. Both PR active transport and PGE2 efflux across the epithelium were decreased upon exposure to retrovirus-activated T lymphocytes, irrespective of their infectiosity. This decrease is not dependent on the disorganization of the epithelial intercellular TJ network, for the following reasons. First, while the higher paracellular permeability induced upon exposure to C8166/45 cells may be responsible for an apparent decrease in PR active clearance by favouring a backflux process downhill of the newly formed concentration gradient, this cannot explain the inhibition of PR clearance observed upon exposure to T lymphocytes isolated from patients, as the latter did not increase the paracellular permeability of the epithelium. Second, in PGE2 efflux experiments, the substrate being added in the apical compartment only, the alteration of the TJ integrity would lead to an increase in the clearance of the compound by opening a paracellular pathway parallel to the transporter-mediated pathway. Thus, the inhibition of PGE2 transport may rather be underestimated in C8166/45 experiments in which the sucrose permeability was increased. Overall the results indicate that the impairment in efflux functions of the BCSFB induced by retrovirus-activated T lymphocytes affect the carrier-mediated transport process itself, and that the biological compounds whose elimination out of the CSF is impaired include PGE2.

Because exacerbated central biological actions of PGE2 (quoted in the Introduction) are expected from its decreased elimination out of the brain, our results point out choroidal transporters as an element involved in the physiopathology of neuro-inflammation and infection. In line with our findings, in the course of experimental lipopolysaccharide fever, a down-regulation of transporters and enzymes responsible for the inactivation of PGE2 in lung and liver has also been reported (Ivanov et al. 2003). This suggests a synergic regulation of PGE2 efflux/inactivation in the central and peripheral compartments, ultimately leading to increased brain levels.

The relative contribution of the different choroidal transporters to the efflux of PGE2 and RP is not known. Our data indicate that PGE2 not only is taken up by the choroidal cells, as previously reported (DiBenedetto and Bito 1986; Krunic et al. 2000), but actually undergoes a complete transcellular transport from the CSF side to the stromal/blood side, hence crossing both apical and basolateral membranes of the choroidal epithelium. PGE2 is a good substrate (affinity constant around 100 nm) for the rat prostaglandin transporter PGT (Kanai et al. 1995; Schuster 2002), and also for several organic anion transporters of both the SLC22A (OAT) family, and the SLC21A (oatp) family (Sekine et al. 1997; Cattori et al. 2001; Kimura et al. 2002). In the rat CP, the presence of PGT has not been searched, and SLC21A7 (oatp3) and SLC22A6/8 (OAT1/3) are the main organic anion uptake transporters described at the apical membrane so far (Strazielle et al. 2004). The outwardly directed basolateral extrusion of PGE2 can occur via potential-driven diffusion (Schuster 2002), or through members of the ATP binding cassette transporter family, such as ABCC4 (Reid et al. 2003), whose expression and basolateral localization has been recently demonstrated in the CP epithelium (Leggas et al. 2004). As for PR, its choroidal transport is competitively inhibited by benzylpenicilin, suggesting the involvement of SLC22A8 (Hakvoort et al. 1998), but is also partially inhibited by taurocholate, a typical SLC21 substrate (unpublished data). Therefore, identifying the subtypes of choroidal transport protein(s), the function of which is reduced as a result of either a down-regulation of the protein or a reduced transport capacity in inflammatory contexts, will require a better knowledge of the relative expression and cellular localization of choroidal transporters, and further studies using a panel of model substrates specific for the different transport proteins. Finally, CPs exert organic anion transport activity toward endogenous biologically active compounds other than PGE2, such as the neurotransmitter metabolites 5-hydroxyindolacetic acid and homovanillic acid (Davson and Segal 1996; Alebouyeh et al. 2003), whose excessive accumulation in the CSF may also have deleterious consequences. Whether and how the transport of these metabolites is regulated during CNS inflammatory/infectious diseases deserves to be investigated.

Pro-inflammatory cytokines are involved in the blood–CSF barrier functional changes induced by retrovirus-activated T lymphocytes

In an attempt to characterise the soluble factor(s) which are responsible for the functional changes observed at the BCSFB upon exposure to infected T lymphocytes, we first identified different biologically active or reactive molecules secreted by these cells in solo culture, or in co-culture with epithelial cells, and known to be involved in cell alteration processes. They included the viral protein Tax, as well as non-viral factors such as MMPs, oxygen-derived free radicals, and pro-inflammatory cytokines.

Tax is the main multifunctional viral protein which can be released from HTLV-1-infected cells. Yet an anti-Tax antibody previously shown to neutralize the Tax-induced alteration of astrocytic transporters (Szymocha et al. 2000) did not block any of BCSFB alterations induced upon exposition to infected T cells. This suggests that Tax indirectly modulates the barrier functions via the host cell production of active effectors, rather than acting directly as an extracellular cytokine on neighbouring cells (in our case the BCSFB cells), which is currently debated as an alternative mechanism leading to CNS dysfunction following HTLV-1 infection (Grant et al. 2002).

Among non-viral factors, the increased production of oxygenated free radicals by activated T lymphocytes, together with a limited blocking effect of antioxidant enzymes on PR transport inhibition, led us to conclude that a pro-oxidant environment is favourable to the appearance of transport alterations, but without being the major effectors in the process.

Our data also indicate that epithelial cells in co-culture with infected T cells are exposed to higher concentration of MMP-9 than in solo culture. MMPs, and especially MMP-9, have been associated with blood–brain barrier breakdown in different experimentally induced neuroinflammation processes (Rosenberg et al. 1995; Mun-Bryce and Rosenberg 1998). However, the treatment of CP epithelial cells in solo culture with an active recombinant MMP-9, even at a high dose, did not elicit any significant change in either the integrity or the transport properties of the BCSFB. These data indicate that MMP-9 is not a major factor responsible for the alteration of organic anion transport induced at the BCSFB by the infected T lymphocytes, and corroborate previous findings showing that secretion of MMP-2 and -9 by the CP exposed to an inflammatory environment is not paralleled by an alteration of the paracellular permeability of the epithelium (Strazielle et al. 2003b). Yet, our data are not inconsistent with an indirect role for this enzyme in the permeation of brain barriers in response to activated immune cells. While MMP-9 is not expected to directly alter the molecular organization of the TJs, as occludin and claudins which form the extracellular core of the junctions are not MMP-9 substrates, this enzyme can facilitate immune cell migration processes by locally degrading the surrounding extracellular matrix or the basement membrane of endothelia or epithelia. The released MMP-9 may also be involved in the activation of chemoattractants such as interleukin 8 (Van den Steen et al. 2000).

Pro-inflammatory cytokines appear potent effectors of organic anion transport alteration. First, HTLV-1-infected T cells such as C8166/45 cells secrete TNF-α and IL-1 (Giraudon et al. 2000). Second, treatment by sTNFRII-Fc and rrIL-1-ra attenuated the decrease in PR efflux observed following exposure of the epithelium to C8166/45 cells, without affecting the increased paracellular permeability of the monolayer. Third, exposing epithelial cells to a combination of TNF-α and IL-1 consistently led to a decrease in the CSF-to-blood flux of both PR and PGE2, and left the paracellular barrier unaffected, indicating again that the decrease in PGE2 efflux is independent on tight junction integrity. Since the choroidal epithelium in co-culture with activated T lymphocytes is under cumulative continuous exposure to deleterious factors secreted by the latter cells, it is conceivable that the cytokine antagonizing agents could only retard the appearance of transport alteration, hence explaining the partial reversal effect on the 30-h cumulated PR efflux and the lack of effect on PGE2 transport measured as an end-point. Alternatively, IL-1 may exert some activity through a mechanism which is independent from the rrIL-1-ra-sensitive IL-1 receptor, as previously reported in other experimental models (Touzani et al. 2002; Diem et al. 2003).

The identification of pro-inflammatory cytokines as major mediators in the decrease in choroidal organic anion transport following exposure to retrovirus-activated T cells implies that similar alteration could be achieved in the context of a broad range of infectious diseases, or of hyperimmunity leading to the presence of activated immune cells secreting pro-inflammatory cytokines in the choroidal stroma. Our data present some similarities with the down-regulation of hepatic organic anion transporters involved in bile acid transport, which occurs in the course of endotoxin-mediated cholestasis. Both in vivo and in vitro studies pointed out the role of IL-1 and TNF-α, secreted at least in part by locally activated macrophages, in this hepatic effect. The transporters involved were mainly the liver-specific ABCC2 (cMOAT) and members of the SLC21 families (Nakamura et al. 1999; Hartmann et al. 2002).

Treatment of the choroidal epithelial cells with the pro-inflammatory cytokines TNF-α and IL-1 did not elicit an alteration of TJ integrity. Thus, the mechanism by which the paracellular permeability of the monolayer is increased upon exposure to C8166/45 cells, involves additional unidentified effectors. TJs are size- and ion-selective paracellular gates (Nitta et al. 2003). Consistent with the accepted concept that the paracellular barrier efficiency is linked to a precise cellular distribution of the different TJ proteins, the increased paracellular permeability observed upon C8166/45 cells exposure is concomitant to an alteration of both occludin and claudin organization. The effect on the choroidal TJ structure also observed following exposure to the infected primary T cells, without a significant increase in paracellular permeability, may reflect a gradation in the sequence of events which ultimately leads to a functional impairment of the structural barrier.

Because, in our experimental design, T lymphocytes are distant from the choroidal epithelium (Fig. 1), secreted factors need to build up in the medium before generating a general response from the epithelium. In vivo, electron microscopy studies show that the epithelium is only separated from the stromal cells by a few microns (Peters et al. 1991; personal observations). It is, therefore, conceivable that the concentration of factors secreted by activated cells will quickly build up locally, thus initiating a focalized TJ alteration which, coupled to MMP-induced degradation of basal membranes, may favour the transepithelial migration of immune cells into the CSF. This latter step remains to be formally established.

In summary, the mechanism underlying the BCSFB transport alteration upon exposure to retrovirus-infected T lymphocytes is independent of the production of virion, and involves soluble factors, among which TNF-α and IL-1 are key effectors. The reported decrease in the CSF-to-blood elimination of PGE2 could substantially contribute to the CNS pathophysiology of both viral and inflammatory disorders by increasing the central biological action of this prostaglandin. Further molecular identification of the choroidal transporters involved in these processes are a prerequisite to the development of pharmacological strategies aiming at controlling CNS levels of pro-inflammatory eicosanoids in the course of inflammatory diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by Association pour la Recherche sur la Sclérose En Plaques, and Agence Nationale de Recherche sur le Sida (ANRS). STK is a recipient from ANRS. We are grateful to Dr Claudine Pique for providing two primary T cell preparations for the study, Marylin Batisson for her help in the preparation and viability control of PBMC, Eudeline Alix for HPLC radiochemical analysis and David Cheillan, Hôpital Debrousse, for glucose and glutamine concentration analysis.

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  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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