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Endotoxin Down-Regulates T Cell Activation by Antigen-Presenting Liver Sinusoidal Endothelial Cells¹

Percy A. Knolle^2,*,, Tieno Germann, Ulrich Treichel^*, Anja Uhrig^*, Edgar Schmitt, Silke Hegenbarth^*,, Ansgar W. Lohse^* and Guido Gerken^*

^* First Medizinische Klinik und Poliklinik, Johannes Gutenberg Universität, Mainz, Germany; Zentrum f. Molekulare Biologie, Ruprecht Karls Universität, Heidelberg, Germany; and Institut für Immunologie, Johannes Gutenberg Universität, Mainz, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endotoxin is physiologically present in portal venous blood at concentrations of 100 pg/ml to 1 ng/ml. Clearance of endotoxin from portal blood occurs through sinusoidal lining cells, i.e., Kupffer cells, and liver sinusoidal endothelial cells (LSEC). We have recently shown that LSEC are fully efficient APCs. Here, we studied the influence of endotoxin on the accessory function of LSEC. Incubation of Ag-presenting LSEC with physiological concentrations of endotoxin lead to 80% reduction of the accessory function, measured by release of IFN- from CD4⁺ T cells. In contrast, conventional APC populations rather showed an increase of the accessory function after endotoxin treatment. Inhibition of the accessory function in LSEC by endotoxin was not due to lack of soluble costimulatory signals, because neither supplemental IL-1ß, IL-2, IFN-, or IL-12 could rescue the accessory function. Ag uptake was not influenced by endotoxin in LSEC. However, we found that endotoxin led to alkalinization of the endosomal/lysomal compartment specifically in LSEC but not in bone marrow macrophages, which indicated that Ag processing, i.e., proteolytic cleavage of protein Ags into peptide fragments, was affected by endotoxin. Furthermore, endotoxin treatment down-regulated surface expression of constitutively expressed MHC class II, CD80, and CD86. In conclusion, it is conceivable that endotoxin does not alter the clearance function of LSEC to remove gut-derived Ags from portal blood but specifically affects Ag processing and expression of the accessory molecules in these cells. Consequently, Ag-specific immune responses by CD4⁺ T cells are efficiently down-regulated in the hepatic microenvironment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endotoxins are LPS constituents of the outer membrane of Gram-negative bacteria (1). The presence of larger amounts of endotoxin in the blood stream, which is observed during severe infection by Gram-negative bacteria or as a result of massive bacterial translocation from the gut, leads to development of sepsis, which is characterized by fever, leukopenia, coagulation disorders, and finally multiorgan failure (1). Endotoxin exerts its profound effects in an indirect manner. The response to endotoxin (LPS) has been shown to involve activation of many different cell populations (monocytes/macrophages, endothelial cells, smooth muscle cells, neutrophils) and lead to expression of cytokines, arachidonic acid metabolites, and reactive oxygen and nitrogen intermediates that are responsible for the observed pathophysiological reactions (1). However, low concentrations of endotoxin are believed to have beneficial effects for the immune response of the host (2). Several studies have shown that cells of the immune system (T cells, B cells, monocytes, macrophages, dendritic cells) are activated by endotoxin and that their functional activity is increased (3, 4, 5, 6).

Portal venous endotoxemia, in contrast to systemic endotoxemia, has been demonstrated to be a physiologic event (7, 8). Endotoxin is constantly being produced in the terminal ileum and large intestine as a result of the death of Gram-negative bacteria and is regularly absorbed into the blood stream (9). Consequently, portal venous blood entering the liver was found to contain endotoxin and bacteria (7). The sinusoidal lining cells of the liver, Kupffer cells, and liver sinusoidal endothelial cells (LSEC)³ are the first cells to come in contact with portal venous blood and efficiently clear endotoxin from the portal circulation so that virtually no endotoxin is detected in hepatic venous blood draining into the systemic circulation (10). Although in vitro Kupffer cells as well as LSEC release proinflammatory mediators after contact with endotoxin (11, 12), there is little evidence that these cells induce a local inflammatory reaction in vivo in response to endotoxin (13). The rapid endotoxin-mediated release of anti-inflammatory mediators by Kupffer cells and LSEC may account for the absence of hepatic inflammation in vivo (14, 15, 16).

We have recently reported, that LSEC are an efficient resident APC population in the liver (17). In view of the observations that endotoxin leads to activation and maturation of APCs (3, 4, 5, 6, 18), we addressed the question whether endotoxin, which is cleared by LSEC from portal venous blood, influences the accessory function of LSEC. We found that physiological concentrations of endotoxin efficiently down-regulated the accessory function (T cell activation) of LSEC but not of conventional APC populations. The specific effect of endotoxin on Ag-presenting LSEC was found to be mediated by two mechanisms: first, alkalinization of the endosomal/lysosomal pH that may consecutively inhibit Ag degradation in the lysosomal compartment, and second, a decreased surface expression of MHC class II molecules as well as of the costimulatory molecules CD80 and CD86.

	Materials and Methods

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Material

Endotoxin (Escherichia coli 055:B5) and indomethacin were obtained from Sigma (München, Germany). mAb against IL-10 as well as recombinant proteins IL-1ß, IL-2, granulocyte-macrophage CSF, and IL-12 were purchased from PharMingen (Hamburg, Germany). Polyclonal goat anti-serum to murine TNF- was kindly provided by A. Wendel and T. Hartung (University of Konstanz, Germany). L-N-monomethyl arginine (L-NMMA) for inhibition of nitric oxide synthase was obtained from Calbiochem (Bad Soden, Germany). Dextran-FITC and LysoSensor Blue were obtained from Molecular Probes (Eugene, OR).

LSEC

BALB/c mice were bred in the animal facility of the university and received adequate care according to good laboratory practice guidelines. Isolation of LSEC and Kupffer cells from murine liver was performed as has been previously described (15, 19, 20). Briefly, LSEC and Kupffer cells were obtained from the livers of female 12- to 16-wk-old BALB/c mice by portal perfusion with 0.05% collagenase A in a calcium-free phosphate buffer. Liver tissue was mechanically separated using forceps followed by 30-min incubation in 0.05% collagenase A (Sigma) in a rotatory water bath at 240 rpm and 37°C. LSEC and Kupffer cells 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 from Kupffer cells 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 (both Beckman). Rotor speed was kept constant at 2500 rpm, and cell populations were separated by increasing counterflow speed (LSEC, 23 ml/min; Kupffer cells, 55 ml/min). Elutriated cells were washed once in PBS at 4°C and seeded onto 96-well Primaria flat-bottom plates (Falcon, Becton Dickinson, Heidelberg, Germany) at a density of 1 x 10⁵ cells/well or onto collagen type I-coated petri dishes at a density of 1 x 10⁷/dish. DMEM supplemented with 10% FCS/2% glutamine was used as culture medium. LSEC and Kupffer cells were kept in culture for 3 days before experiments were performed. The purity of cell populations was routinely controlled by characteristic phagocytosis of opsonized SRBC (for Kupffer cells) and uptake of acetylated low-density lipoprotein (Paesel & Lorei, Frankfurt, Germany) (for LSEC). The microvascular origin of LSEC was demonstrated by flow cytometry showing the expression of CD4 and VCAM-1 but the absence of CD31 or CD45. Furthermore, electron microscopy revealed a typical presence of fenestrae in the isolated LSEC. By these methods, LSEC were found to be 98% pure. Contaminating cells were fibroblasts that were identified by their typical microscopic appearance.

Generation of bone marrow macrophages (BM-M)

Bone marrow stem cells were prepared from the femur of BALB/c mice and cultured in petri dishes in Iscove’s modified Dulbecco’s medium supplemented with 10% FCS, 10% horse serum, and 15 ng/ml granulocyte-macrophage CSF. Adherent cells were cultured for 3 wk to allow maturation into BM-M.

Ag-specific Th₁ CD4⁺ T cell clone

An Ag-specific Th₁ CD4⁺ T cell clone (LNC.2.F1) raised against purified protein derivative (PPD) as previously described (21) was used in the experiments. PPD was kindly provided by Behring AG (Marburg, Germany). CD4⁺ T cell clones were restimulated with specific Ag every 2–3 mo and kept in culture with low concentration of IL-2 (2 ng/ml). Only T cells that had been in rest for >2 wk after in vitro restimulation were used for experiments.

TCR-transgenic mice

TCR-transgenic mice (6.5⁺) (22) express an ß-TCR specific for peptide 111–119 from influenza hemagglutinin presented by I-E^d. CD4⁺ T cells from transgenic mice were isolated by immunomagnetic separation (magnetic cell starting (MACS); Miltenyi, Braunschweig, Germany) using microbeads directly conjugated with anti-CD4 (L3T4) from Miltenyi. Isolated CD4⁺(6.5⁺) T cells were 95% pure by FACS analysis (data not shown). CD4⁺(6.5⁺) T cells were stimulated with hemophilus influenza vaccine (A/Singapore/6/86,H 09/1-3/95 D) (kindly provided by Behring Werke AG, Marburg, Germany) at a concentration of 2 µg/ml.

T cell activation

CD4⁺ T cells (LNC.2.F1) (4 x 10⁴ cells/well) were added to isolated LSEC or BM-M together with specific Ag (PPD at 10 µg/ml). CD4⁺(6.5⁺) T cells were used at a concentration of 1 x 10⁵/well for coculture experiments with LSEC and BM-M. Specific T cell activation by APCs was measured by IFN- production. Cell culture supernatant was tested after 48 h of coculture for the concentration of IFN- by specific sandwich ELISA.

ELISA for determination of IFN- concentration

Sandwich ELISA for IFN- determination in cell culture supernatant was performed according to standard procedure. Briefly, flat-bottom microtiter plates (Nunc, Maxisorb, Roskilde, Denmark) were coated with Ab specific for IFN- (clone R46A2) at a concentration of 3 µg/ml at 4°C. After 2 h of incubation, postcoating was done with 1% BSA/PBS. Four washing steps with PBS/Tween 0.5% were conducted 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 Ab (clone AN17.18) was biotinylated according to standard protocol and used at a concentration of 2 µg/ml. Detection of bound biotinylated Ab was performed with avidin-horseradish peroxidase (1:1500). After addition of substrate 2,2'-Azinobis (3-ethylbenzthiazoline sulfonic acid) (ABTS) optical density was measured at 405 nm using an ELISA reader from Molecular Devices (München, Germany).

Flow cytometry

LSEC were seeded onto collagen type I-coated six-well plates. After 3 days in culture, LSEC were used for experiments. LSEC were detached from the surface of six-well plates by gentle trypsin/EDTA treatment for 2 min. For study of surface expression of the accessory molecules, LSEC were stained with FITC-labeled mAbs (3 µg/ml) with specificity for CD80, CD86, or MHC class II (PharMingen, Hamburg, Germany) at 4°C for 30 min. Isotype-matched FITC labeled control Abs (3 µg/ml) were used. Flow cytometry was performed using a FACScan from Becton Dickinson (Heidelberg, Germany). A total of 2 x 10⁴ cells were analyzed in each sample using Lysis II software (Becton Dickinson).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Down-regulation of T cell activation in Ag-presenting LSEC by preincubation but not coincubation with endotoxin

Ag-presenting LSEC line the sinusoidal lumen in the liver and are exposed both to endotoxin present in portal venous blood as well as to passenger leukocytes. We examined whether endotoxin influenced the accessory function of LSEC for CD4⁺ T cells. LSEC Ag-specifically activated Th₁ CD4⁺ T cells and induced the production of IFN- in T cells (Fig. 1, lane 2). We demonstrate that preincubation of Ag-presenting LSEC with endotoxin for 16 h down-regulated the capacity of LSEC to activate Th₁ CD4⁺ T cells (LNC.2.F1) measured as IFN- release (Fig. 1, lane 4). The presence of endotoxin during Ag-specific activation of Th₁ CD4⁺ T cells did not result in reduced accessory function of LSEC (Fig. 1, lane 3), suggesting that endotoxin acted on Ag-presenting LSEC and that time was required for completion of the endotoxin effect. Time course experiments revealed that preincubation for 2 h with endotoxin (1 ng/ml) was as sufficient as 16 h of preincubation to down-regulate the accessory function of LSEC (not shown). Preincubation with endotoxin equally inhibited the capacity of LSEC to induce clonal expansion, i.e., proliferation, of Th₁ CD4⁺ T cells (not shown). Reduction of the accessory function by endotoxin in Ag-presenting LSEC was dose-dependent with even low concentrations of endotoxin (1 ng/ml), yielding significantly reduced accessory function (80% reduction) (Fig. 2A). The effect of endotoxin on the accessory function in LSEC was cell-specific as BM-M showed an increased capacity to present Ag to Th₁ CD4⁺ T cells following preincubation with endotoxin (Fig. 2A). Endotoxin equally increased the accessory function of spleen cells (data not shown).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Preincubation of LSEC but not coincubation with endotoxin down-regulates Ag presentation and subsequent T cell activation. LSEC were cultured in 96-well flat-bottom Primaria plates (1 x 105/well) for 3 days before experiments were performed. Endotoxin (E. coli 055:B5) was added either together with LNC.2. F1 (3 x 104/well) and specific Ag (PPD; 10 µg/ml) or was preincubated on LSEC for 18 h followed by extensive washing to remove unbound endotoxin from LSEC. Two days after the addition of LNC.2.F1, the supernatant was analyzed by specific ELISA for the concentration of IFN- as a measure of T cell activation. Experiments were always conducted in triplicates. The experiment shown is representative of six independent experiments.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Physiologic concentrations of endotoxin down-regulate Ag-specific T cell activation by LSEC but not by other APCs. LSEC were cultured as described in Fig. 1 and preincubated with different concentrations of endotoxin. A, BM-M were treated the same way. LNC.2.F1 and PPD were added as mentioned in Fig. 1 and cell culture supernatant was assayed after 2 days for the concentration of IFN-. Experiments were conducted in triplicates. Results are shown ± SD. The experiments shown are representative of five independent experiments. B, LSEC were preincubated with decreasing concentrations of endotoxin for 18 h. Naive CD4+(6.5+) T cells were isolated by positive immunomagnetic separation (MACS, Miltenyi) from animals transgenic for an MHC class II-restricted TCR specific for hemagglutinin of influenza virus (22). CD4+(6.5+) T cells were 95% pure by flow cytometry and 85% positive for the transgenic TCR (by staining with a clonotypic mAb). A total of 1 x 105 naive CD4+(6.5+) T cells were added to LSEC together with Ag and incubated for 5 days. CD4+(6.5+) T cells were thoroughly washed and restimulated for a period of 2 days with -CD3 (3 µg/ml) cross-linked by mouse anti-rat Ig (5 µg/ml). Concentrations of IFN- in the cell culture supernatants was determined by specific ELISA. Experiments always were conducted in triplicates. The experiment shown is representative of three separate experiments.

Exogenous cytokines cannot rescue endotoxin-mediated down-regulation of Ag presentation in LSEC.

We wondered whether supplementation of cocultures of LSEC and Th₁ CD4⁺ T cells with exogenous cytokines could reverse the negative immunomodulatory effect of endotoxin. However, neither IFN- (Fig. 3A) nor IL-2 (Fig. 3B) could alter the negative effect of endotoxin on the accessory function of LSEC. Supplementation with IL-1ß or IL-12 was equally ineffective (Fig. 3C, lane 3 and 4). We next addressed the question of whether a negative immunomodulatory mediator was induced in LSEC cultures by endotoxin-treatment. But neither neutralizing Abs to TNF- or IL-10 (not shown) nor blockade of prostanoid synthesis through indomethacin (Fig. 3C, lane 5) or blockade of nitric oxide synthase with L-NMMA (Fig. 3C, lane 6) modified down-regulation of the accessory function through endotoxin.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Endotoxin mediated down-regulation of Ag presentation in LSEC cannot be reverted by supplemental exogenous cytokines. LSEC were cultured as described in Fig. 1. IL-1ß, IL-2, IL-12, or IFN- (10 U/ml) were added to LSEC cultures as indicated. Indomethacin (10-5 M) or L-NMMA were added together with endotoxin to LSEC cultures. Subsequently, LSEC were thoroughly washed, and LNC.2.F1 together with specific Ag was added. Two days after the addition of LNC.2.F1, the cell culture supernatant was assayed for the concentration of IFN-. Experiments were conducted in triplicates. The experiments shown are representative of three separate experiments.

Ag uptake in "professional" APCs occurs by the help of receptor-mediated endocytosis or macropinocytosis in addition to endocytosis, and this process can be influenced by soluble mediators (23). We investigated the possibility that endotoxin may influence the uptake of Ag by LSEC. Endocytic activity of LSEC as measured by lucifer yellow uptake was not influenced by endotoxin (not shown). The mannose receptor is a well-known receptor for Ag uptake in, for example, dendritic cells (23), is expressed on LSEC (24), and is involved in Ag uptake by LSEC (25). The activity of the mannose receptor can be quantitatively measured by flow cytometry using a fluorochrome-labeled ligand (i.e., Dextran-FITC). We demonstrate here that Dextran-FITC uptake into LSEC was not influenced by preincubation with endotoxin (Fig. 4). Monensin as an inhibitor of receptor-mediated endocytosis significantly decreased uptake of Dextran-FITC into LSEC (Fig. 4). Monensin was equally effective to down-regulate Ag uptake and subsequent T cell activation by LSEC (25). Our experiments suggest that decreased Ag uptake was not responsible for the endotoxin-mediated decrease of the accessory function in Ag-presenting LSEC.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Endocytosis is not effected by endotoxin in LSEC. LSEC were cultured on collagen type I-coated 12-well plates and used 3 days after seeding in experiments. LSEC were incubated with Dextran-FITC (1 mg/ml) for 2 h in the presence of endotoxin (10 ng/ml), monensin (10 µM), or normal medium (DMEM/10% FCS/2% Glutamine). LSEC were detached from the plates with trypsin/EDTA and analyzed for uptake of Dextran-FITC by flow cytometry. A total of 1 x 104 cells were analyzed by FACScan using Lysis II software. One of three representative and independent experiments is depicted.

Endotoxin leads to alkalinization of the endosomal/lysosomal pH specifically in LSEC but not in BM-M

Following the uptake of Ag into an APC, proteolytic processing of Ag in acidic endosomal/lysosomal compartments is necessary to generate peptide fragments that in turn can bind to MHC class II molecules (for review see 26 . We show that Ag processing in LSEC required an acidic compartment because alkalinization of LSEC with chloroquine (Fig. 5) or NH₄Cl (not shown) resulted in a decreased ability to induce T cell activation. These agents are known to inhibit Ag degradation in an endosomal compartment and block Ag processing for MHC class II-restricted presentation (for review see Refs. 27 and 28). Therefore, we examined whether endotoxin interferes with Ag processing in LSEC. To this end, LSEC were incubated for 16 h with Dextran-FITC, which is known to accumulate in the endosomal/lysosomal compartment after uptake via the mannose receptor. Dextran-FITC is further known to change fluorescence intensity depending on pH: acidic pH yielding lowered fluorescence intensity and alkaline pH yielding increased fluorescence intensity. Table I shows that the addition of endotoxin to Dextran-FITC-loaded LSEC led to an increase in fluorescence intensity, which can be explained as the result of a change from an acidic pH to a more alkaline pH in the lysosomal compartment. Endotoxin was as efficient in increasing fluorescence intensity as the lysosomotropic agent chloroquine (Table I). Similar results were obtained using a different pH-sensitive dye (LysoSensor; Molecular Probes) (data not shown). The effect of endotoxin on lysosomal pH was specific for LSEC because Dextran-FITC-loaded BM-M did not show increased fluorescence intensity following exposure to endotoxin (Table I). BM-M were not generally insensitive to a change in intralysosomal pH as chloroquine clearly led to an increased fluorescence intensity, demonstrating that intralysosomal pH could be increased in this cell population (Table I). Our results demonstrate that endotoxin specifically leads to alkalinization of the endosomal/lysosomal compartment in LSEC but not in BM-M.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Ag processing in LSEC occurs in an acidic compartment that is sensitive to chloroquine treatment. LSEC were isolated and cultured as described in Fig. 1. Chloroquine was added together with LNC.2.F1/specific Ag to LSEC cultures, and the cell culture supernatant was assayed for the concentration of IFN- by ELISA. Experiments were conducted in triplicates. The experiment shown is representative of three independent experiments. Results are shown ± SD.

View this table: [in this window] [in a new window]   Table I. Endotoxin-induced alkalinization of the endosomal/lysosomal compartment in LSEC but not in BM-Ma

The expression of MHC class II molecules and of accessory molecules on the APC correlates with the ability to induce activation of CD4⁺ T cells. We show here that endotoxin down-regulated surface expression of MHC class II molecules in LSEC (Fig. 6A). Furthermore, surface expression of the accessory molecules CD80 (Fig. 6B) and CD86 (Fig. 6C) on LSEC was equally lowered by endotoxin. However, endotoxin did not generally down-regulate the surface expression of the accessory molecules on LSEC, because the surface expression of both CD54 and CD106 was up-regulated by endotoxin (A. Uhrig and P. A. Knolle, manuscript in preparation).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Down-regulation of MHC class II and CD80/CD86 surface expression on LSEC by endotoxin. LSEC cultured on 6-well plates coated with collagen type I were treated with endotoxin (10 ng/ml) for 18 h. LSEC were detached from the plates with trypsin/EDTA stained with -I-Ad-FITC, -CD80-FITC, -CD86-FITC, or isotype-matched FITC-conjugated control Abs (all at 5 µg/ml) for 30 min at 4°C, thoroughly washed, and analyzed by flow cytometry using a FACScan and Lysis II software. A total of 1 x 104 cells were analyzed; one representative experiment of three independent experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The induction of an Ag-specific immune response is critically regulated by the APCs. Endotoxin increases the ability of different APC populations to induce an immune response (4, 5, 31, 32). However, under physiological conditions only APC from the gastrointestinal tract and from the liver are likely to encounter endotoxin. We have recently reported that LSEC are mature and efficient APCs that can efficiently present protein as well as peptide Ags to T cells (17). The present study addressed the question of whether LSEC by virtue of their dual role (removal of endotoxin and Ag presentation) are modulated in their accessory function by endotoxin.

Many reports have described that endotoxin increases immune responses in vitro and in vivo (3, 4, 33). Dendritic cells, in particular, are sensitive to endotoxin treatment and show improved capacity to induce Ag-specific T cell activation, an effect that has been linked with a maturation step of dendritic cells from Ag capturing to APCs (34). Here we show that LSEC are APC that capture and present protein Ag to CD4⁺ T cells without the need for maturation. Importantly, our study has demonstrated that treatment of LSEC with endotoxin leads to an almost complete down-regulation of their accessory function. In sharp contrast, other APC populations, such as BM-M or spleen cells (data not shown), were not down-regulated in their accessory function but even showed an increase in their capacity to activate CD4⁺ T cells. Our experiments further demonstrate that those concentrations of endotoxin that are physiologically found in portal venous blood (35) reduced the accessory function of LSEC by 80% compared with Ag-presenting LSEC that were not in contact with endotoxin. The Ag-specific activation of naive CD4⁺ T cells in contrast to memory CD4⁺ T cells requires more costimulatory signals from the APC and is restricted to "professional" APC (36). We have shown that Ag-presenting LSEC can activate both naive and memory CD4⁺ T cells⁵ and that LSEC, therefore, have the functional characteristics of the so-called "professional" APC. In this study, we report that endotoxin treatment of LSEC prevents activation of naive CD4⁺ T cells. The physiologic presence of endotoxin in sinusoidal blood and the specific endotoxin-mediated down-regulation of accessory function of LSEC may in part explain why Ags that are presented to the immune system in the liver do not induce an immune response. This concept is supported by the observation that endotoxin-unresponsive mice (C3H/HeJ) show an immune response to oral Ags that enter the liver via the portal blood (37).

The striking difference between LSEC and other APC led us to investigate the mechanism of the endotoxin-induced decrease of the accessory function in LSEC. Two lines of evidence argue against the explanation that the endotoxin effect on Ag-presenting LSEC is due to release of an inhibitory mediator or due to lack of soluble accessory mediators. First, PGE₂ and IL-10 are produced by sinusoidal cells in response to endotoxin (14, 15), but neither inhibition of prostanoid synthesis nor neutralizing -IL-10 Ab restored the accessory function of endotoxin-treated LSEC. Furthermore, we do not have evidence for the involvement of nitric oxide because inhibition of nitric oxide synthase activity in LSEC by L-NMMA did not antagonise the endotoxin effect. Second, supplementation with IL-2, IL-1ß, IFN-, or IL-12, which are cytokines known to support T cell activation by APC, did not improve the accessory function of endotoxin-treated LSEC.

Endotoxin and inflammatory cytokines have been reported to lower the uptake of Ag in dendritic cells by decreasing receptor-mediated as well as fluid-phase endocytosis (23). LSEC are capable of pinocytosis (20, 38) as well as macropinocytosis (39) and can further take up Ag via the mannose receptor (25). However, in our experiments we did not observe that endocytic activity in LSEC was decreased by endotoxin. From this, we conclude that it is unlikely that decreased Ag uptake was responsible for endotoxin-mediated inhibition of the accessory function in LSEC.

Following Ag uptake into APC, the processing of protein Ags into peptide fragments by proteases occurs in acidic endosomal/lysosomal compartments (for review see 26 . A prerequisite for peptide loading onto MHC class II molecules is efficient processing of protein Ags into peptide fragments (40). Our experiments demonstrate that endotoxin treatment of LSEC leads to alkalinization of the endosomal/lysosomal compartment. This effect was specific for LSEC, because in BM-M endotoxin treatment did not affect pH of the endosomal/lysosomal compartment (Fig. 6). This is in line with our observation that endotoxin selectively down-regulates the Ag-presenting function in LSEC but not in other APC populations. It is likely that alkalinization of the endosomal/lysosomal compartment will interfere with proteolytic cleavage of endocytosed protein Ags into peptide fragments because lysosomotropic agents, e.g., chloroquine, that lead to alkalinization of the endosomal/lysosomal pH have been shown to inhibit the correct processing of protein Ags (28, 41, 42) (Fig. 5). Furthermore, peptide loading onto MHC class II molecules is optimal at a low pH (43). Therefore, endotoxin-mediated alkalinization of the endosomal/lysosomal compartment in LSEC is likely not only to lower Ag processing but may as well impede peptide loading onto MHC class II molecules.

Endotoxin is a potent inducer of surface expression of MHC class II molecules (5, 44) and accessory molecules on different APC populations in vitro and in vivo (23, 45). We demonstrate here that endotoxin treatment of LSEC reduced the surface expression of the important accessory molecules CD80 and CD86 as well as MHC class II molecules. Endotoxin acted at the transcriptional level in LSEC to down-regulate expression of CD80 and CD86, but increased expression of CD54 (A. Uhrig and P. Knolle, manuscript in preparation). This demonstrates that endotoxin differentially regulates the expression of surface molecules on LSEC. Viola et al. reported that a decrease in the expression of accessory molecules increases the need for efficient stimulation via the TCR if T cells are to be activated efficiently (36). The down-regulation of CD80 and CD86 on LSEC by endotoxin may provide further assurance, in combination with decreased MHC class II-restricted Ag presentation, that CD4⁺ T cells are not activated by LSEC in the liver sinusoid.

In conclusion, our observations point to an important role of physiologically present endotoxin in the liver sinusoid for the local immune response. Ags derived from the gastrointestinal tract can be efficiently cleared by LSEC from sinusoidal blood because endocytosis is not influenced by endotoxin. However, proteolytic processing of gut-derived protein Ags into peptide fragments, which can be presented to the immune system on MHC class II molecules, is prevented through endotoxin-mediated alkalinization of the endosomal/lysosomal compartment in LSEC. In consequence, a lower number of peptide-loaded MHC class II molecules may be expressed on LSEC treated with endotoxin, and T cell activation by Ag-presenting LSEC is reduced. Endotoxin further lowers the surface expression of costimulatory molecules on LSEC, which decreases the likelihood that sufficient costimulatory signals for T cell activation are generated. The described mechanisms may account for the physiological need of the liver to effectively clear gut-derived Ags from the portal circulation without inducing a specific immune response.

	Acknowledgments

 
We thank Ms. E. Löser for excellent technical assistance.

	Footnotes

 
¹ This research was supported by grants from the Deutsche Forschungsgemeinschaft (Kn 437/1 and SFB 311, Project A13, A15, and C7), Germany.

² Address correspondence and reprint requests to Dr. Percy A. Knolle, Zentrum f. Molekulare Biologie Heidelberg, Ruprecht-Karls-Universität, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. E-mail address:

³ Abbreviations used in this paper: LSEC, liver sinusoidal endothelial cells; BM-M, bone marrow macrophage; L-NMMA, L-N-monomethyl arginine; PPD, purified protein derivative.

⁴ P. Knolle. Submitted for publication.

⁵ P. Knolle. Submitted for publication.

Received for publication June 16, 1998. Accepted for publication October 26, 1998.

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