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Hepatobiliary Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
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Abstract |
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Rs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4+ or CD8+ TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liver sinusoidal endothelial cells (LSEC) 2 constitute the predominant cell type within the liver sinusoids. LSEC form a fenestrated barrier between the blood and hepatocytes and are therefore strategically situated to interact with leukocytes as they traverse the liver. In addition to anatomic and rheologic factors favoring interaction of LSEC with lymphocytes, LSEC have high Ag uptake and processing capacity, a property associated with professional APC (5). LSEC are also believed to express the costimulatory moieties CD80 and CD86 and stimulate T cells through peptide presentation in the context of MHC class I and II molecules (6, 7). LSEC have been shown to stimulate CD4+ T cell cytokine production and to stimulate CD8+ T cells via cross-presentation of soluble Ag (7, 8). However, LSEC have also been found to express CD4, CD11b, and CD11c, markers generally associated with T cells, myeloid cells, and dendritic cells (DC) (7, 9, 10).
Because many of the phenotypic and functional features attributed to LSEC are also properties of DC, we speculated that previous studies of LSEC may have been confounded by contamination with DC. Previous isolation techniques, such as counterflow elutriation, may not have adequately discriminated DC from LSEC. In addition, given that DC take up a wide variety of macromolecules, we hypothesized that the methods used to confirm LSEC purity, such as uptake of acetylated low-density lipoprotein (AcLDL), may not be specific (7, 11, 12, 13, 14, 15, 16, 17). To test our hypotheses, we designed a more specific LSEC isolation protocol based on surface marker expression and then re-evaluated the phenotype and function of pure LSEC. We isolated LSEC from nonparenchymal cells (NPC) using immunomagnetic beads to exclude CD45+ cells. We showed that this isolation process resulted in <1% contamination by CD45+ NPC and that nearly all CD45– NPC were von Willebrand factor positive (vWF+) and CD31+. In contrast to previous reports, we found LSEC to express negligible levels of MHC class II, CD80, CD86, and CD11c. Consistent with this phenotype, LSEC induced minimal allogeneic and Ag-specific T cell stimulation when compared with DC. Therefore, although LSEC capture and process Ag, they are not sufficient for activation of naive T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult 6- to 10-wk-old male C57BL/6 (B6, H-2Kb) and BALB/c (H-2Kd) mice were purchased from Taconic Farms (Germantown, NY). OT-I (B6.Cg-RAG2tm1Fwa-TgN) and OT-II (B6.Cg-RAG2tm1Alt-TgN) TCR-transgenic Rag-2–/– mice on a B6 background were also obtained from Taconic Farms. All animals were maintained in a pathogen-free animal housing facility at Memorial Sloan-Kettering Cancer Center. All procedures were approved by the Institutional Animal Care and Use Committee.
LSEC and DC preparation
Liver NPC were isolated as previously described, with modifications (18). Briefly, animals were euthanized and the portal vein was isolated, cannulated with a 24-gauge catheter (BD Biosciences, Franklin Lakes, NJ), and injected with 2 ml of 1% (w/v) collagenase D (Sigma-Aldrich, St. Louis, MO) in HBSS. We then mechanically disrupted the liver before a 20-min incubation in 20 ml of 1% collagenase at 37°C. The resulting cell suspension was then passed through a sterile 100-µm nylon mesh filter (Falcon; BD Biosciences) and spun three times at 30 x g for 5 min to remove hepatocytes. The supernatant was centrifuged (300 x g for 10 min) and the pellet containing NPC was resuspended in 1.5 ml of complete RPMI 1640 medium (10% FCS, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100 U/ml streptomycin) and added to 2.8 ml of 30% (w/v) metrizamide (Sigma-Aldrich; product recently discontinued) or 3.0 ml of 40% (w/v) Optiprep (Sigma-Aldrich) to remove debris and enrich the NPC. With metrizamide, this suspension was layered under 5 ml of HBSS in a 15-ml centrifuge tube (Falcon) and spun at 1500 x g for 20 min. When Optiprep was used, the cell suspension was layered under 4 ml of HBSS and spun at 400 x g for 15 min. The layer of low-density cells at the interface was harvested. The cells were incubated with 1 µg of anti-FcRIII/II (FcR) mAb 2.4G2 (Fc block; mAb Core Facility, Sloan-Kettering Institute, New York, NY) per 1 x 106 cells and then fractionated based on CD45 expression using immunomagnetic beads (Miltenyi Biotec, Auburn, CA) as follows. Enriched NPC were incubated with anti-CD45 immunomagnetic beads and then passed through a single-positive selection column (Miltenyi Biotec). The effluent (CD45– NPC) was then further purified by passing it through a high-affinity depletion column (Miltenyi Biotec) which has a slower column flow rate and removes residual (CD45+) cells with greater efficiency. Liver DC were isolated by incubation of NPC with anti-CD11c immunomagnetic beads and passage through two consecutive positive selection columns. Splenic DC were isolated similarly with anti-CD11c beads following mechanical disruption of the organ and erythrocyte lysis (18).
Flow cytometry
Four-color flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences). Voltages were based on unstained cells and compensation was set using single-stained positive controls for each color. All samples were incubated with Fc block before staining. We labeled 1 x 106 cells with 0.1 µg FITC-, PE-, allophycocyanin-, or biotin-conjugated Ab (BD PharMingen, San Diego, CA, except as indicated). Biotinylated Abs were secondarily stained with streptavidin PerCP. Cells were stained for endothelial markers (CD31 (WM-59), vWF (Serotec, Raleigh, NC)); CD4 (RM4-4); FcR (2.4G2), Mac-3 (M3/84); CD11c (HL-3); MHC class II (AF6-120.1, I-Ab); MHC class I (AF6-88.5, H-2Kb); CD54 (3-E2); and costimulatory molecules (CD40 (3/23), CD80 (16-10A1), and CD86 (GL-1)).
Electron microscopy
For transmission electron microscopy (TEM), LSEC were pelleted and fixed in 2.5% glutaraldehyde/2% paraformaldehyde in 0.075 M cacodylate buffer for 1 h followed by postfixation in 2% OsO4 for 1 h. The samples were rinsed in distilled water and serially dehydrated in ethanol (50, 75, 95, and then 100%), followed by propylene oxide. The cells were placed in propylene oxide/PolyBed 812 (1/1) overnight (Polysciences, Warrington, PA). The samples were embedded in PolyBed 812 in a 60°C oven. Ultrathin sections were obtained with a Reichert Ultracut S-microtome (Leica, Vienna, Austria). The thin sections were photographed using a JEOL 1200-EX electron microscope (Peabody, MA). For scanning electron microscopy, the cells were placed on poly-L-lysine-coated Thermanox (Nunc, Rochester, NY) plastic coverslips. The cells were fixed in 2.5% glutaraldehyde/2% paraformaldehyde in 0.075 M cacodylate buffer for 1 h, rinsed in buffer, and dehydrated in a graded series of ethanol as above. The samples were then freeze dried in a Pearce-Edwards dryer (BOC Edwards, Wilmington, MA). The samples were subsequently coated with gold/palladium in a Technics Hummer VI sputtering system. The samples were photographed using a JEOL JSM 35 scanning electron microscope.
Ag uptake
To measure in vitro Ag uptake, we incubated purified LSEC or CD45+ NPC with 100 µg of FITC-conjugated dextran, OVA, DQ-OVA, or BODIPY AcLDL in duplicate wells at 37°C (Molecular Probes, Eugene, OR). At various time points, the reactions were stopped and the cells washed with cold PBS and then stained with anti-CD31 (LSEC) or anti-CD11c (CD45+ NPC) before measurement of mean fluorescence of the captured FITC-labeled Ags. To measure in vivo Ag uptake, we injected 100 µg of FITC-dextran, FITC-OVA, or FITC-DQ-OVA or 50 µg BODIPY-AcLDL into the lateral tail vein of mice and the livers were harvested 5 or 30 min later. CD45+ and CD45– NPC were purified as above. Ag uptake was then assessed by measuring mean fluorescence on a flow cytometer and LSEC were identified as CD45–CD31+ cells while DC were CD45+CD11c+.
T cell assays
MLR was performed as previously described (19). Briefly, LSEC and DC from C57BL/6 mice were added in various numbers to 1 x 105 BALB/c T lymphocytes purified using Thy1.2 (CD90.2) immunomagnetic beads according to the manufacturer’s protocol (Miltenyi Biotec) in 96-well U-bottom plates (Falcon). Assays were performed in complete RPMI 1640 and CpG oligonucleotide (oligodeoxynucleotide 1826, 10 µg/ml; Oligos Etc., Wilsonville, OR) or LPS (1 µg/ml; Sigma-Aldrich) were added as needed. Wells were pulsed with [3H]thymidine (1 µCi/well) on day 3 and radioactive uptake was measured 20 h later. In vitro Ag-specific CD8+ T cell activation was assayed with OT-I CD8+-transgenic T cells specific for OVA257–264 (20). In vitro Ag-specific CD4+ T cell activation was assayed with OT-II CD4+ transgenic T cells specific for OVA323–339 (20). OT-I and OT-II T cells were isolated with Thy 1.2 immunomagnetic beads (Miltenyi Biotec) from splenocytes. The Trp-2181–188 peptide was used as a negative control. All peptides were obtained from the Peptide Synthesis Facility (Sloan-Kettering Institute). LSEC or DC were plated at various concentrations with OVA-TCR-transgenic CD8+ (2 x 104) or CD4+ (1 x 104) T cells and OVA peptide (1 µg/ml) in a 96-well U-bottom plate (Falcon) for 3 days. [3H]Thymidine incorporation was measured as above. A stimulating anti-CD28 mAb (37.51; Southern Biotechnology Associates, Birmingham, AL) was added at a concentration of 20 µg/ml to some wells, in which case LSEC or DC were preincubated with OVA257–264 for 1 h and then washed before culture with T cells. T cell proliferation was determined as for the MLR experiments and cell culture supernatant was tested by cytometric bead array for IL-2 and IFN- according to the manufacturer’s protocols (BD PharMingen).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much of the published data on LSEC derives from cells isolated with elutriation. We sought to develop a more specific method of LSEC purification based on cell surface marker expression. We started by examining bulk liver NPC from mice. To remove cellular debris that results from collagenase digestion of the liver, we used density gradient centrifugation. Because LSEC are not of bone marrow origin (8), we used the common leukocyte marker CD45 to distinguish LSEC from other NPC, including Kupffer cells, B cells, T cells, NK cells, and DC. We found that 77% of enriched NPC lacked expression of CD45 (Fig. 1A). Furthermore, there was considerable overlap in the forward and side scatter profiles of CD45– and CD45+ NPC (Fig. 1B). To determine what proportion of CD45–-enriched NPC were actually LSEC, we tested markers of endothelial cell origin. Nearly all CD45– cells expressed CD31 and vWF (Fig. 2), which have been reported to be expressed by LSEC (21, 22, 23). As expected, CD45+ cells, which we have previously shown to contain liver DC (Fig. 2) (18), had only slight expression of vWF and CD31. Furthermore, LSEC demonstrated high levels of FcR expression (Fig. 2), which distinguishes LSEC from other NPC types including endothelial cells from central veins and portal vessels of the liver (24). In contrast to findings from previous reports (7, 25), we found that LSEC did not express the murine DC marker CD11c or the T cell marker CD4, but did express the macrophage marker Mac-3 (Fig. 2). Taken together, these data demonstrated that enriched CD45– NPC were predominantly comprised of LSEC. Furthermore, previous LSEC preparations may have been contaminated by DC and other leukocytes because LSEC and CD45+ NPC are similar in size as approximated by forward light scatter and are recovered from the same layer in a density gradient.
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Based on our finding that enriched CD45– NPC were predominantly LSEC, we used anti-CD45 immunomagnetic beads to isolate LSEC to study their function. To eliminate contaminating CD45+ NPC and DC, in particular, we developed a two-step procedure for LSEC isolation. First, we labeled enriched NPC with anti-CD45 immunomagnetic beads and passed them through a positive selection column. The effluent was then run through a high-affinity depletion column and was found to be 99% pure by lack of CD45 staining. The mean yield of LSEC from a mouse liver was 2.5 x 106 ± 6.2 x 105 cells. To further confirm that we had in fact purified LSEC, we performed scanning electron microscopy. At x 10,000 magnification, the cells were uniform in size and morphology. At higher power (Fig. 4A) or by TEM (Fig. 4B), freshly isolated LSEC consistently demonstrated characteristic fenestrated membranes (26, 27).
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Once we developed a reliable method of LSEC purification, we then tested their Ag uptake capacity, an integral component of in vivo T cell stimulation by APC. To provide a context in which to interpret the results, we compared LSEC directly to DC. We found that both LSEC and DC took up comparable levels of dextran and OVA in vitro, both of which are captured by macropinocytosis (Fig. 5A). In fact, LSEC took up more OVA in vivo than did DC (Fig. 5B), a finding which may be due to their relatively greater contact exposure to blood in the sinusoids. In addition to Ag uptake, we measured a component of Ag processing with DQ-OVA, which becomes fluorescent only after lysosomal acidification and proteolytic cleavage. By this measure, LSEC and DC functioned similarly in vitro and the results were comparable in vivo when normalized to the amount of captured OVA. Finally, we tested AcLDL uptake because it is purported to be a specific function of LSEC. As we expected, we found that DC were also able to capture AcLDL in vitro, although to a lesser extent than LSEC (Fig. 5A). More importantly, DC accomplished equivalent AcLDL uptake in vivo (Fig. 5B). B cells (CD45+CD19+) were unable to take up AcLDL, demonstrating that nonspecific uptake did not account for the ability of LSEC and DC to capture this molecule (data not shown). Thus, LSEC are efficient at Ag capture although their uptake of AcLDL is not unique.
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Despite their high Ag uptake capacity, we postulated that LSEC would induce minimal T cell proliferation because of their negligible expression of MHC class II and the costimulatory molecules CD40, CD80, and CD86. Again, to assess the potential of LSEC to function as APC, we compared them to DC. In a MLR, we found that LSEC consistently failed to stimulate allogeneic T cells (Fig. 6). In contrast, DC induced considerable alloproliferation in a dose-dependent fashion. The inability of LSEC to stimulate allogeneic T cells was not altered by the presence of CpG or LPS, two molecules that are known to activate DC (data not shown). To further assess their interaction with T cells, we tested the ability of LSEC to stimulate TCR-transgenic T cells. Varying numbers of LSEC or DC were loaded with OVA257–264 and then incubated with OT-I CD8+ OVA-restricted T cells. Despite the fact that LSEC express MHC class I, but in keeping with their lack of costimulatory molecules, we found that LSEC induced minimal CD8+ T cell proliferation (Fig. 7A). Similarly, we found that LSEC loaded with OVA323–339 could not stimulate OT-II CD4+ OVA-restricted T cells (Fig. 7B). Consistent with these findings, supernatant from LSEC cultured with CD8+ OT-I T cells had much lower concentrations of IFN- and IL-2 than supernatant from wells containing DC and T cells (Fig. 7C). To determine whether costimulation could enable LSEC to activate naive T cells, especially given their ability to capture Ag efficiently and their expression of MHC class I, we repeated the OT-1 proliferation assay following addition of a stimulating anti-CD28 Ab. Surprisingly, the presence of the anti-CD28 Ab markedly enhanced the ability of LSEC to stimulate proliferation of naive CD8+ T cells (Fig. 7D). Finally, given that IFN- has been shown to activate other endothelial cell types (28, 29, 30), we exposed LSEC to IFN- (20 ng/ml; R&D Systems, Minneapolis, MN) for 16, 36, or 60 h. IFN- did not induce MHC class II or costimulatory molecule expression on LSEC, while these moieties were up-regulated on splenic macrophages under the same experimental conditions. Moreover, pretreatment with IFN- for 60 h did not enable LSEC to stimulate allogeneic T cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production was induced from CD8+ T cells by LSEC (Fig. 7C), which conflicts with a previous report (13). Nevertheless, the failure of LSEC to activate CD8+ T cells was rescued by the addition of exogenous costimulation or, to a lesser extent, IL-2 (data not shown). In fact, in the presence of an activating Ab to CD28, the ability of LSEC to induce T cell proliferation was markedly enhanced (Fig. 7D), demonstrating that a lack of costimulatory molecule expression prevented LSEC from activating naive CD8+ T cells. Whether other endothelial cell types are able to stimulate T cells is unclear given the variable data that have been reported (29, 30). There are a number of potential explanations for why our data on LSEC phenotype and function differ from previous reports. Probably of greatest importance is that we used a more specific method of LSEC isolation and thereby minimized contamination with other cell types. Most investigators have used counterflow elutriation to isolate LSEC. However, we found that LSEC had comparable density to other types of NPC since they were situated in the same layer on a density gradient. Furthermore, size, as approximated by forward light scatter on flow cytometry, also did not reliably discriminate LSEC from other NPC (Fig. 1). We concluded that isolating LSEC on the basis of their physical properties does not adequately exclude other populations of NPC, including liver DC. It is especially important to remove DC from LSEC preparations since we and others have previously shown liver DC to be potent inducers of immunity (18, 19, 32). Instead of using cell density or size, we purified LSEC by their lack of CD45 expression. Using negative selection columns to deplete CD45+ cells, we consistently isolated CD45– NPC with <1% contamination by CD45+ cells. We showed by flow cytometry that nearly all CD45– cells from enriched NPC were LSEC (Fig. 2). We further validated our isolation method with transmission and scanning electron microscopy (Fig. 4) by identifying the characteristic membrane fenestrae of LSEC (26).
In addition to our method of LSEC isolation, there are other factors that may account for our discrepant data. LSEC are highly autofluorescent, which can confound the interpretation of flow cytometry analysis. We tailored our analyses of surface marker expression by choosing appropriate voltages according to the level of autofluorescence. Furthermore, we routinely blocked FcR during bead sorting and flow cytometric phenotype analysis. It is unclear whether anti-FcR was used previously. Because LSEC are known to express high levels of FcR, considerable nonspecific binding of Abs would be expected if these receptors were not blocked. Lastly, we used freshly isolated cells exclusively, while others often assessed LSEC phenotype and function after 3 days of culture (13, 25). However, we did not detect any noteworthy phenotypic changes by flow cytometry when LSEC were analyzed after culture for 3 days, even when treated with IFN- (data not shown).
Taken together, our findings that LSEC do not express MHC class II, costimulatory molecules, or CD11c, and do not activate T cells raises the possibility that LSEC preparations used in previous studies were contaminated with DC. In fact, we found that even low levels (i.e., <10%) of contamination by CD45+ cells resulted in T cell stimulation (data not shown). Furthermore, not only are the physical properties of LSEC inadequate to separate them from other NPC populations, we have shown that the uptake of AcLDL, an indicator of LSEC purity used widely by others, is not a function unique to LSEC (Fig. 5). Again, we believe that distinguishing LSEC from other NPC populations is more reliably accomplished by surface marker expression.
Although our results show that LSEC are unlikely to stimulate T cells directly in vivo through presentation of peptides in the context of MHC molecules, LSEC may still contribute to the induction of immunity or tolerance within the liver. Given the factors favoring cellular contact within the hepatic sinusoids, a neighboring cell may provide costimulation or a necessary cytokine during CD8+ TCR binding to Ag in the context of LSEC MHC class I molecules. Furthermore, the absence of MHC class II expression does not prohibit presentation of exogenous protein to T cells because LSEC may be able to cross-present soluble Ag (8). Moreover, LSEC may affect T cells in ways other than through presentation of Ag, such as PGE2 release to mediate T cell anergy (25) or by CD8+ T cell deletion through constitutive expression of ICAM-1 (33). Similar to endothelial cells from other organs, LSEC may play a role in recruitment of T cells to specific regions within the liver through presentation of Ag in the context of MHC class I molecules (29). Thus, LSEC are likely to possess important immunologic functions despite their inability to function as professional APC in a manner analogous to DC. In addition, although we did not detect distinct LSEC subsets, it is possible that certain LSEC subpopulations may possess unique functional characteristics (27).
We have shown that LSEC have a distinct phenotypic profile that is consistent with their inability to stimulate T cells. Nearly all CD45– cells from enriched NPC are LSEC and they can be isolated with a high degree of purity using immunomagnetic beads to exclude other NPC populations. Confirmation of LSEC purity is most reliably accomplished using phenotypic markers, as opposed to AcLDL uptake, which is nonspecific. Analysis of freshly isolated LSEC demonstrates minimal to no CD11c, MHC class II, CD40, CD80, or CD86 expression. LSEC are poor stimulators of T cells, but other properties, such as their high capacity for Ag uptake and direct access to circulating lymphocytes, may enable them to contribute to the unique immunologic function of the liver.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LSEC, liver sinusoidal endothelial cell; DC, dendritic cell; AcLDL, acetylated low-density lipoprotein; NPC, nonparenchymal cell; vWF, von Willebrand factor; TEM, transmission electron microscopy.
Received for publication January 21, 2004. Accepted for publication April 22, 2004.
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- and 
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interferon-induced major histocompatibility complex class II gene expression by Porphyromonas gingivalis membrane vesicles. Infect. Immun. 70:1185.This article has been cited by other articles:
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