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.