Keywords: local immune response, immune regulation, mannose receptor, MHC class II, CD80, CD86
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Copyright © 1998 Blackwell Science Ltd IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules Correspondence: Percy A. Knolle MD, Zentrum f. Molekulare Biologie Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. Accepted July 27, 1998.  This article has been cited by other articles in PMC. | |||||||||||||
Abstract Our study demonstrates that antigen-presenting liver sinusoidal endothelial cells (LSEC) induce production of interferon-gamma (IFN-γ) from cloned Th1 CD4+ T cells. We show that LSEC used the mannose receptor for antigen uptake, which further strengthened the role of LSEC as antigen-presenting cell (APC) population in the liver. The ability of LSEC to activate cloned CD4+ T cells antigen-specifically was down-regulated by exogenous prostaglandin E2(PGE2) and by IL-10. We identify two separate mechanisms by which IL-10 down-regulated T cell activation through LSEC. IL-10 decreased the constitutive surface expression of MHC class II as well as of the accessory molecules CD80 and CD86 on LSEC. Furthermore, IL-10 diminished mannose receptor activity in LSEC. Decreased antigen uptake via the mannose receptor and decreased expression of accessory molecules may explain the down-regulation of T cell activation through IL-10. Importantly, the expression of low numbers of antigen on MHC II in the absence of accessory signals on LSEC may lead to induction of anergy in T cells. Because PGE2 and IL-10 are released from LSEC or Kupffer cells (KC) in response to those concentrations of endotoxin found physiologically in portal venous blood, it is possible that the continuous presence of these mediators and their negative effect on the local APC may explain the inability of the liver to induce T cell activation and to clear chronic infections. Our results support the notion that antigen presentation by LSEC in the hepatic microenvironment contributes to the observed inability to mount an effective cell-mediated immune response in the liver. Keywords: local immune response, immune regulation, mannose receptor, MHC class II, CD80, CD86 | |||||||||||||
INTRODUCTION Presentation of antigen in the liver is believed to lead to an ineffective cell-mediated immune response [1–4] which can be due either to down-regulation of T cell activation or to induction of antigen-specific tolerance. The inability of many patients to eliminate hepatic infection of hepatitis B or C virus infection may also be due to a unique milieu for immune responses in the liver. The activation of CD4+ T cells is an important event in the induction of an antigen-specific immune response. So-called ‘professional’ antigen-presenting cells (APC) are believed to activate CD4+ T cells by presenting specific antigen on MHC class II molecules [5–8]. Little is known about the APC populations in the liver that can induce CD4+ T cell activation. We have recently shown that the liver has a unique population of APC, the sinusoidal endothelial cells (LSEC) [9]. In contrast to other vascular endothelial cells, LSEC express MHC class II constitutively and thus can present antigen without prior stimulation by interferon-gamma (IFN-γ) [10,11]. In addition, we have shown that LSEC express CD80 and CD86 as well as CD54 and produce IL-1 [9]. The apparent contradiction of the presence of an abundant population of effective APC on the one hand and lack of an effective cell-mediated immune response in the liver on the other hand might be resolved by the effect of IL-10, which can down-regulate antigen-specific T cell proliferation induced by LSEC [9]. Recently Morel et al. analysed the differential down-regulatory effect of IL-10 on antigen uptake and antigen presentation by dendritic cells (DC) [12]. It was therefore the aim of the present study to compare their findings in DC with the effect of IL-10 on the function of antigen-presenting LSEC. We studied the effect of IL-10 on surface expression of MHC class II and accessory molecules. Furthermore, we analysed the role of the mannose receptor, a known constituent of LSEC [13–15], in uptake of antigen as well as the modulation of mannose receptor activity by IL-10. | |||||||||||||
MATERIALS AND METHODS Materials Antibodies (R4A62 and AN17.18) used for IFN-γ sandwich ELISA were purified from hybridoma cell culture supernatant following ammonium sulphate precipitation and affinity chromatography on protein G. Recombinant mouse IFN-γ and IL-10 were purchased from Pharmingen (Hamburg, Germany). Prostaglandin E2(PGE2), transforming growth factor-beta (TGF-β), mannan and monensin were obtained from Sigma (München, Germany). Dextran-FITC (mol. wt 40 000 kD) was obtained from Molecular Probes (Mobitec, Göttingen, Germany).Cell isolation and culture Sinusoidal endothelial cells and Kupffer cells Isolation of LSEC and Kupffer cells (KC) from murine liver was performed as has been previously described [16–18]. Briefly, LSEC and KC were obtained from the livers of female 8–12-week-old female BALB/c mice by portal perfusion with 0.05% collagenase A in a calcium-free phosphate buffer. Liver tissue was mechanically separated using forceps and further singularization of cells was achieved through 30 min incubation in 0.05% collagenase A (Sigma) in a rotary water bath at 240 rev/min and 37°C. LSEC and KC were separated from parenchymal cells by density gradient centrifugation on a metrizamide (Nycomed, Oslo, Norway) gradient (1.089 g/cm3), followed by two washing steps to remove cell debris. Further separation of sinusoidal endothelial cells and KC was achieved by counterflow centrifugal elutriation using a J2-MC centrifuge (Beckman, München, Germany) equipped with a JE-6B rotor and a standard elutriation chamber. Rotor speed was kept constant at 2500 rev/min and cell populations were separated by increasing pump speed (LSEC 23 ml/min; KC 55 ml/min). Equilibration of the pump was performed prior to each cell separation. Elutriated cells were washed once in PBS at 4°C and seeded onto 96-well Primaria (Falcon, Heidelberg, Germany) flat-bottomed plates at a density of 1 × 105 cells/well or onto collagen type I-coated Petri dishes (Iwaki, Bibby Dunn, Karlsruhe, Germany) at a density of 1 × 107/well. Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS)/2% glutamine was used as culture medium. LSEC and KC were kept in culture for 4–5 days before experiments were performed. The purity of cell populations was routinely checked the day before the experiment by characteristic phagocytosis of opsonized sheep erythrocytes (for KC) and uptake of I-acetylated low-density lipoprotein (for LSEC). Cell populations were ≥ 95% pure as has been shown previously [17,19]. Contaminating cells were mainly fibroblasts which were identified by their typical microscopic appearance. Isolation of murine bone marrow macrophages Bone marrow macrophages were prepared from the femur of BALB/c mice and cultured in Petri dishes in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FCS, 10% horse serum and 15 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Pharmingen). After 3 weeks of in vitro culture, cells were harvested with versene and used for experiments. Antigen-specific T cells Antigen-specific CD4+ T cell clones were raised against purified protein derivative (PPD) as previously described [20]. T cell clones were restimulated with specific antigen every 2–3 months and kept in culture with low concentrations of IL-2 (2 U/ml). CD4+ T cells showed a Th1 phenotype secreting IFN-γ upon antigen-specific stimulation. Only T cells that had been in rest for > 2 weeks after in vitro restimulation were used in experiments. T cell activation Specific T cell activation by APC was measured by IFN-γ production or by methyl-3H-thymidine incorporation. T cells were added to co-culture experiments at a concentration of 4 × 104 cells/well and specific antigen (PPD) was used at 10 μg/ml. Where indicated, cytokines or PGE2 were added to the cultures. Cell culture supernatant was assayed after 48 h of co-culture for the concentration of IFN-γ by specific sandwich ELISA. In a parallel assay adherent antigen-presenting LSEC were treated with mitomycin C (10 μg/ml) to reduce background methyl-3H-thymidine incorporation prior to addition of specific T cells as described [9]. During the last 12 h of 72 h culture, cells were incubated with 1 μCi (37 kBq) of methyl-3H-thymidine (Amersham, Braunschweig, Germany) and proliferation was assessed by thymidine incorporation. ELISA Sandwich ELISA for IFN-γ determination in cell culture supernatant was performed according to standard procedures. Briefly, flat-bottomed microtitre plates (Nunc, Maxisorb, Wiesbaden, Germany) were coated with antibody specific for IFN-γ (R46A2) at a concentration of 3 μg/ml at 4°C. After 2 h of incubation post-coating was done with 1% bovine serum albumin (BSA)/PBS. Four washing steps with PBS–Tween 0.5% were carried out between all incubation steps. Different dilutions of cell culture supernatant were assayed in a total volume of 100 μl and incubated for 2 h at 4°C. The second specific antibody (AN17.18) was biotinylated according to standard protocol and used at a concentration of 2 μg/ml. Detection of bound biotinylated antibody was performed with avidin–horseradish peroxidase (HRP) (1:1500). After addition of substrate (ABTS) optical density (OD) was measured at 405 nm using a SpectraMAX Elisa-reader by Molecular Devices (München, Germany).Flow cytometry Sinusoidal endothelial cells were isolated from murine livers as described and plated onto collagen I-coated six-well dishes. Three days after isolation, sinusoidal endothelial cells were incubated with murine IL-10 (Pharmingen) for 18 h. Subsequently, cells were detached by gentle trypsin treatment for 3 min and washed twice in DMEM/10% FCS before staining with FITC-labelled antibodies to MHC class II, CD80 and CD86 (Pharmingen) at a concentration of 3 μg/ml at room temperature. After 15 min incubation, cells were washed thoroughly, fixed in PBS/1% formalin and analysed using a FACScan by Becton Dickinson (Heidelberg, Germany) equipped with Lysis II software (Becton Dickinson). | |||||||||||||
RESULTS LSEC are efficient APC and mediate T cell activation CD4+ Th1-like cells produce IFN-γ following antigen-specific activation. We tested whether LSEC can induce antigen-specific IFN-γ production as a measure of T cell activation in a Th1 CD4+ T cell clone specific for PPD (LNC.2.F1). Resting LNC.2.F1 and PPD (10 μg/ml) were incubated with LSEC and IFN-γ production was determined as measure of T cell activation. Time kinetic experiments revealed that IFN-γ release by LNC.2.F1 was maximal after 36–48 h. In the following experiments, IFN-γ was measured in co-culture supernatant after 48 h. To compare the capacity of LSEC to act as APC, bone marrow-derived macrophages (BM-macrophages) and KC were used as controls. It was observed that LSEC mediated IFN-γ release from activated T cells comparable to so-called professional APC (Fig. 1). Neither LSEC, KC nor BM-macrophages released IFN-γ spontaneously or upon contact with T cells in the absence of specific antigen (data not shown). The capacity of LSEC to serve as APC did not depend on duration of in vitro culture, i.e. LSEC tested on day 1 after isolation presented antigen as efficiently as on day 7 after isolation (data not shown).
Antigen-specific T cell activation by antigen-presenting LSEC is regulated by cytokines Activation of a T cell leads to clonal expansion and induces cytokine release. We examined whether LSEC induce both proliferation and cytokine release of CD4+ T cells. Resting LNC.2.F1 and PPD (10 μg/ml) were incubated with LSEC for 48 h before IFN-γ production and methyl-3H-thymidine incorporation were determined as measures of T cell activation. Figure 2 shows that LSEC activated LNC.2.F1 and induced IFN-γ production and release by these cells (Fig. 2a) as well as T cell proliferation (Fig. 2b). Antibodies to MHC class II completely abrogated IFN-γ production, which demonstrates that induction of T cell activation was antigen-specific (Fig. 2a,b).
The co-incubation of the cytokines IL-1β, IL-6 or IFN-γ with LSEC did not alter antigen-specific IFN-γ production or proliferation of LNC.2.F1 cells (Fig. 2a,b). However, preincubation of LSEC for 24 h with IFN-γ (10 U/ml) substantially increased subsequent IFN-γ production and T cell proliferation (Fig. 2c), probably as a result of increased surface expression of MHC class II. The stimulatory effect of preincubation with IFN-γ was dose-dependent, with a maximum reached at 10 U/ml (Fig. 2c). Down-regulation of LSEC-induced T cell activation by IL-10 and PGE2 To study whether antigen-specific T cell activation by LSEC can be down-regulated, we investigated the effect of anti-inflammatory cytokines and PGE2. We found that IL-10 and PGE2 markedly down-regulated T cell activation following antigen presentation through LSEC (Fig. 3a). The down-regulation of T cell activation by IL-10 or PGE2 was dose-dependent (Fig. 3b). IL-10 (10 ng/ml) reduced IFN-γ production by 46 ± 8% (results from three independent experiments). PGE2 (100 μm) reduced IFN-γ production by 62 ± 6% (results from three independent experiments). TGF-β1 (10 ng/ml) and TGF-β2 (10 ng/ml) alone or in combination did not show any modulatory effect on IFN-γ production by LNC.2.F1 cells (Fig. 3a). LSEC are known to produce PGE2 constitutively [21]. Inhibition of endogenous prostanoid production in LSEC by indomethacin led to increased T cell activation (data not shown), suggesting that antigen presentation is tightly controlled in LSEC.
The mannose receptor is involved in antigen uptake in LSEC LSEC have been described to express the mannose receptor [15], which is reported to be involved in antigen uptake in different APC populations [22]. We demonstrate that antigen-pulsed LSEC effectively activated PPD-specific CD4+ T cells (Fig. 4). The addition of EDTA or monensin during PPD pulsing of LSEC led to a dose-dependent reduction in subsequent antigen-specific T cell activation (Fig. 4a,b). These findings support the notion that PPD is taken up by receptor-mediated endocytosis in LSEC. We further investigated the effect of mannan, which is a competitive inhibitor of uptake via the mannose receptor. Figure 4c shows that antigen-specific T cell activation was decreased, if mannan was present during antigen pulsing of LSEC. These findings are consistent with the notion that PPD uptake occurred through the mannose receptor in LSEC.
The function of the mannose receptor in LSEC is down-regulated by IL-10 Dextran is a ligand of the mannose receptor and can be used to test for functional activity of the mannose receptor [22]. Uptake of dextran–FITC into LSEC was measured by flow cytometry after in vitro incubation with dextran–FITC. Figure 5a shows that dextran–FITC was taken up efficiently by LSEC. After only 15 min of incubation at 37°C almost every LSEC had taken up dextran–FITC and uptake further increased over time (Fig. 5a). Monensin clearly inhibited dextran–FITC uptake by LSEC, thereby demonstrating the mechanism of receptor-mediated uptake for dextran–FITC (Fig. 5b). The residual uptake of dextran–FITC in the presence of monensin may be accounted for by uptake of unbound FITC molecules into LSEC by macropinocytosis. We found that dextran–FITC uptake in LSEC was down-regulated by the addition of IL-10 (Fig. 5b), suggesting that antigen uptake via mannose receptor in LSEC may equally be down-regulated by IL-10.
Down-regulation of surface expression of CD80, CD86 and MHC class II on LSEC by IL-10 The surface expression of accessory molecules on the APC is important for T cell activation. We therefore studied whether IL-10 decreased surface expression of those molecules on LSEC that are important for T cell activation, i.e. MHC class II, CD80 and CD86. Table 1 shows that LSEC constitutively express MHC class II, CD86 and CD80. Treating LSEC with IL-10 (10 ng/ml) for 18 h decreased the surface expression of MHC class II and CD86; the surface expression of CD80 was only slightly decreased by IL-10. This result suggests that inhibition of T cell activation may be mediated in part by down-regulation of accessory molecules on antigen-presenting LSEC.
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DISCUSSION Macrovascular endothelial cells have been demonstrated to deliver costimulatory signals to CD4+ T cell subsets [23–26], which result in T cell proliferation and production of cytokines, e.g. IFN-γ. However, CD4+ T cell activation only was achieved when either T cells or endothelial cells were prestimulated. We have recently shown that sinusoidal endothelial cells isolated from the liver (LSEC) antigen-specifically activate CD4+ T cells without the requirement of prestimulation [9]. In the present study we show that LSEC induced not only proliferation of CD4+ T cells but also production of significant amounts of IFN-γ. Even higher concentrations of IFN-γ were produced from CD4+ T cells, when LSEC were pretreated with IFN-γ, which probably improved T cell activation through increased expression of MHC II. IFN-γ is a pivotal cytokine in the initiation of an effective immune response, activating macrophages, NK cells and T lymphocytes to exert effector functions, i.e. phagocytosis, cytotoxicity and release of mediators further amplifying the immune response [27,28]. Furthermore, IFN-γ has been shown to have a direct effect on the clearance of intracellular infection by parasites or viruses in the liver [29,30]. However, the regulation of IFN-γ production in the liver is critical, as uncontrolled up-regulation of cytokine production by CD4+ T cells in IL-10 knock-out mice during an infection leads to liver damage, organ failure and death [31]. In this study, we have shown that PGE2 and IL-10 decreased IFN-γ production by T cells activated through antigen-presenting LSEC. IL-10 or PGE2 are released in vitro by KC [32] or LSEC [21], respectively, and reach concentrations which are necessary for down-regulation of T cell activation if LSEC or KC are exposed to concentrations of endotoxin which are present physiologically in portal venous blood [33]. IL-12 is produced by APC and leads to IFN-γ production of activated CD4+ T cells [34,35]. It has been reported that IL-12 expression and concomitantly IFN-γ production are down-regulated by exogenous IL-4 or IL-10 [36,37]. However, murine LSEC did not express IL-12 but were still able to activate CD4+ T cells in the absence of IL-12; exogenous IL-12 markedly increased IFN-γ production of activated T cells (P. Knolle, submitted for publication). Therefore, we conclude that modulation of IL-12 expression does not play a role in IL-10-mediated down-regulation of antigen-presenting function in LSEC. IL-10 has been reported to act mainly at the level of the APC, although it has been shown to have a direct effect on CD4+ T cells, too [38–40]. We have shown that IL-10 decreased surface expression of MHC class II molecules as well as CD80 and CD86 on LSEC (Table 1). The down-regulation of MHC class II as well as accessory molecules may well account for the observed decrease in antigen-presenting function of LSEC, as a critical number of T cell receptors have to engage with MHC class II/peptide on the APC together with appropriate accessory signals in order to launch T cell activation [41,42]. By decreasing surface expression of both MHC class II molecules and the accessory molecules CD80/CD86 on LSEC, IL-10 might inhibit T cell activation with more certainty than by down-regulating just one or the other. The effective down-regulation of MHC class II molecules and CD80/CD86 on LSEC by IL-10 was not predictable, as other APC populations do not decrease surface expression of accessory molecules upon treatment with IL-10 [12]. We were further interested whether IL-10 decreased accessory function of LSEC in other ways. LSEC have long been known to express mannose receptors [13–15,43], which are implicated in antigen uptake by so called ‘professional’ APC populations [22,44,45]. In our assay system, we have used PPD—a glycosylated mycobacterial protein [46,47]—as model antigen and PPD-specific Th1 CD4+ T cells (LNC.2.F1) as responder T cells. We could demonstrate that T cell activation was decreased if LSEC were antigen-pulsed in the presence of a chelating agent (EDTA) or monensin (inhibitor of receptor-mediated endocytosis). This is consistent with PPD uptake by receptor-mediated endocytosis in LSEC. Importantly, decrease of T cell activation by the addition of mannan during antigen pulsing of LSEC points to the involvement of the mannose receptor in antigen uptake. Complete competition of mannan for PPD is not possible: first, mannan is toxic for primary cell cultures in higher concentrations, and second, mannose receptor activity is extremely high and therefore difficult to saturate [15,48,49]. Alternatively, uptake of PPD may occur in part through the scavenger receptors that are abundantly present on the surface of LSEC [11,50,51]. Taken together, the presence of the mannose receptor on LSEC as well as its involvement in antigen uptake underline the function of LSEC as APC. Using a fluorochrome-labelled ligand of the mannose receptor (dextran–FITC [22]) we demonstrated that LSEC take up dextran–FITC as efficiently as DC. Importantly, IL-10 decreased mannose receptor activity in LSEC by a factor of three, measured as dextran–FITC uptake into LSEC by flow cytometry (Fig. 5b). In stark contrast, Morel et al. demonstrated that mannose receptor activity in DC was increased by IL-10 [12]. The differential effect of IL-10 on antigen capture and antigen presentation may be relevant in the context of migration of DC from the peripheral site of antigen uptake to lymphoid organs where antigen presentation occurs [12]. However, LSEC are resident liver cells, i.e. antigen uptake and antigen presentation occur at the same site; the synergistic down-regulatory effect of IL-10 on antigen capture as well as on antigen presentation in resident LSEC may account for an effective regulation of the immune response by IL-10 in the liver. In conclusion, we found that LSEC use the mannose receptor for antigen uptake and therefore functionally behave like ‘professional’ APC. Down-regulation of T cell activation by antigen-presenting LSEC through IL-10 appeared to be mediated by at least two different mechanisms: decrease in LSEC surface expression of MHC class II/CD80/CD86, and decrease in antigen uptake via the mannose receptor. The induction of IL-10 and PGE2 production in KC or PGE2 in LSEC by physiological concentrations of endotoxin found in portal venous blood argues for a role of these inhibitory mediators in controlling the local immune response in the liver. | |||||||||||||
Acknowledgments This study was supported by grants from the Deutsche Forschungsgemeinschaft (Kn 437/1) and SFB-311/A13, C7. | |||||||||||||
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