Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte...

Odette Vifias,' Ram& Bataller,2 Pau Sancho-Bru,2 Pere Cristina Berenguer,' Carlos Enrich,3 Josep M. NicolA~,~ Guadalupe Ercua,' Teresa Gallart,' Jordi Vives,' Vicente Arr~yo,~ and Juan Rod6s2

Following cell activation, hepatic stellate cells (HSCs) acquire proinflammatory and profi- brogenic properties. We investigated whether activated HSCs also display immune properties. Here we show that cultured human HSCs express membrane proteins involved in antigen presentation, including members of the HLA family (HLA-I and HLA-11), lipid- presenting molecules (CDlb and CDlc), and factors involved in T-cell activation (CD40 and CD80). Exposure of HSCs to proinflammatory cytokines markedly up-regulates these molecules. Importantly, cells freshly isolated from human cirrhotic livers (in vim activated HSCs) highly express HLA-I1 and CD40, suggesting that HSCs can act as antigen-presenting cells (APCs) in human fibrogenesis. We also explored whether human HSCs can efficiently process exogenous antigens. Activated HSCs internalize low- and high-molecular-weight dextran and transferrin, indicating that they can perform fluid-phase and receptor-mediated endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules because they internalize latex particles as well as bacteria. Interestingly, both culture-activated and in vivo activated HSCs express high levels of CD68, a protein involved in antigen trafficking. Finally, we studied whether HSCs modulate T-lymphocyte proliferation. In basal conditions, coculture of irradiated HSCs barely induces allogeneic T-lymphocyte proliferation. However, cytokine-stimulated HSCs stimulate the allogeneic T-lymphocyte response in an HLA-11- dependent manner. In conclusion, human activated HSCs express molecules for antigen presentation, internalize macromolecules, and modulate T-lymphocyte proliferation. These results suggest that HSCs may play a role in the immune function of the liver. (HEPATOLOGY 2003;38:919-929.)

ncreasing evidence indicates that hepatic stellate cells (HSCs) play an important role in the progression of I chronic liver diseases.' Following hepatic injury,

HSCs transdifferentiate into myofibroblast-like cells,

Abbreviations: HSC, hepatic stellate cell; APS, antigen-presenting cell; IL, interleukin; IFN, interferon; PBS, phosphate-buffered saline; FITC,juorescein isothiocyanate; PBMC, peripheral blood mononuclear cell.

From the 'Laboratory oflmmunology, 'Liver Unit, 3Department o f Cell Biology, and 4Department oflnternal Medicine, Hospital Clinic, University of Barcelona School of Medicine, Institut d'lnvestigacios Biom2diques August Pi-Sunyer (IDIBAPS), Barcelona, Catalonia, Spain.

Received December 23, 2002; accepted June 25, 2003. Supported by a grantfiom the Comisidn Interministerial de Ciencia y Tecnobgta

(SAF99/0014 and SAF2002-0369@, Instituto de Salud Carlos 111 (C03/2), and the Instituto Reina Soja de Investigacidn Nefiooldgica as well as a grant fiom the IDIBAPS (to P.S.-B.).

Adress reprint requests to: Pere Gin& M.D., Liver Unit, Hospital Clinic, Villarroel170, 08036 Barcelona, Spain. E-mail: gines@medicina. ub.es; fm: (34)

Copyright 0 2003 by the American Association for the Study of Liver Diseases.

doi:lO. 1053~hep.2003.50392

93-451 -5272.

0270-9 ~39/03/3804-0017$30.00/0

which display fibrogenic and contractile properties.2 Thus, HSCs are believed to be a major fibrogenic hepatic cell type and to regulate intrahepatic vascular resis- tance.3-5 Besides these actions, HSCs also play a role in promoting hepatic inflammation.6 Activated HSCs accumulate in injured hepatic areas, where they express cell adhesion molecules and secrete a number of proinflammatory cytokines and chemokines with chemoattractant properties toward white blood ~ells.7-l~ In clear contrast with these advances, little is known about the possible role of HSCs in the immune function of the liver. In particu- lar, it is unknown whether HSCs express the molecules involved in antigen presentation and modulate the growth of T lymphocytes, which are the main infiltrating blood cells in most types of chronic liver diseases.

The local regulation of the immune system in both normal and diseased liver is not totally understood. The liver is continuously involved in clearance of foreign antigens derived from the gastrointestinal tract; therefore, a

919

920 VINAS ETAL. HEPATOLOGY, October 2003

relative immune tolerance is mandatory to avoid damage to hepatocytes. 1 1 However, in chronic liver diseases, a number of antigens (i.e., viral particles, protein adducts) are processed by liver cells and presented to T lymphocytes, which become activated and accumulate, leading to progressive inflammation of the hepatic parenchyma. l2

Therefore, cells capable of presenting antigens to T lymphocytes could play an important role in the development of chronic liver diseases. A number of hepatic cell types express the molecules required for antigen presentation (mainly molecules of HLA class I1 [HLA-111) and are capable of internalizing macromolecules and modulating the growth of T lymphocytes. These cells include professional antigen-presenting cells (APCs), such as hepatic dendritic cells and Kupffer cells, and nonprofessional APCs such as sinusoidal endothelial cells.13-15 Although professional APCs exert immune properties in basal conditions, nonprofessional APCs predominantly acquire this hnction on stimulation with cytokines. Nonprofes- sional APCs can play a supporting role to the immune system in inflamed tissues, where there are increased levels of cytokines. In other tissues, resident pericytes such as astrocytes or mesangial cells display typical features of nonprofessional APCs under certain conditions.16J7 Because HSCs are considered liver-specific pericytes, we hypothesize that activated HSCs could act as nonprofessional APCs in the injured liver. This study was undertaken to test this hypothesis.

Materials and Methods Reagents and Monoclonal Antibodies

Interleukin lp (IL-lp) and interferon gamma (IFN-y) were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies against CDla, HLA-I1 DR-PE, HLA-I1 DP-PE, HLA DQ-FITC, and HLA-I W6-32 were purchased from Becton Dickinson (BD Biosciences, Erembodegern, Belgium). CD40-FITC antibody was purchased from Caltag Laboratories (Burlingame, CA). Antibodies against CD 1 c, CD80-FITC, CD86-PE, and CD83-PE were purchased from Coulter-Inmunotech (Izasa SA, Barcelona, Spain). Antibody against CD68 was obtained from DAKO Diagnostics (Sant Just Desvern, Spain). Antibodies against C D l b and HLA-I1 DR were kindly provided by Jordi Mild and Ramon Vilella (Hos- pital Clinic, Barcelona, Spain).

IsoEaEion and Culture of Human HSCs Human HSCs were isolated from fragments of normal

livers (n = 5) obtained from resections of liver metastases as described in detail p r e ~ i o u s l y . ~ ~ ~ ~ 9 Briefly, after a com- bined digestion of liver tissue with collagenase and pronase, HSCs were separated from other nonparenchymal cells by centrifugation over a gradient of Nycodenz (1 1 %

wt/vol; Sigma Chemical Co.). Cells recovered at this level showed a typical autofluorescence due to vitamin A-rich fat droplets and were highly viable and more than 90% pure. Experiments were performed after the second serial passage, when cells show phenotypic and immunocyto- chemical characteristics of activated HSCs. No contami- nation by hepatocytes, sinusoidal endothelial cells, dendritic cells, or Kupffer cells was noted. No noticeable differences were found between the 5 different cell prep- arations used. HSCs activated in vim were isolated from human cirrhotic livers (n = 4) obtained at the time of liver transplantation. The procedure of isolation was similar to that described for normal livers, except that the concentration of collagenase and pronase was 25% higher and the gradient of Nycodenz used was 13% wt/vol. HSCs isolated from cirrhotic livers were studied after 1 day in culture. At this time point, cells already showed phenotypic features of myofibroblasts, including positive immunostaining for vimentin and a-smooth muscle actin (Dakopatts, Glostrup, Denmark), marked up-regulation of procolla- gen al(1) and a-smooth muscle actin messenger RNA, increased cell proliferation, and contraction in response to vasoconstrictor factors such as endothelin 1. The protocol was approved by the investigational review board of the Hospital Clinic of Barcelona.

Flow Cytometric Analysis The expression of HLA molecules, CDl family mem-

bers, CD68, and T-cell costimulatory proteins was assessed by flow cytometry analysis as described in detail elsewhere.20 Briefly, human HSCs were cultured to confluence in 175-cm2 flasks (Nunc Brand Products, Den- mark). Medium was then removed and cells incubated in serum-free medium containing either cytokines (1 0 U/mL IL-lp + 100 U/mL IFN-y) or buffer for up to 6 days. This prolonged incubation time with cytokines has also been used in previous studies with nonprofessional APCs.16s17 Medium and agonists were changed every 2 days. At the end of the experiment, cells were rinsed twice with phosphate-buffered saline (PBS) and detached by incubating in PBS containing 2 mmol/L ethylenediami- netetraacetic acid. HSCs isolated from cirrhotic human livers (activated in vivo) were cultured on plastic for 24 hours in the absence of cytokine stimulation, and cells were detached using the same procedure. Aliquots containing 2 X lo5 cells were resuspended in PBS containing 2% fetal calf serum and 0.1% sodium azide, pH 7.4, in separate tubes containing the corresponding monoclonal antibodies. The optimal concentrations of antibodies were determined in preliminary experiments using white blood cells as a control. Isotype-matched control antibodies were used as a control in all experiments. After exten-

HEPATOLOGY, Vol. 38, No. 4, 2003 VIRAS ET AL. 921

sive washing, cells were incubated with fluorescein isothiocyanate (F1TC)-labeled goat anti-mouse immuno- globulin G (Research Diagnostics Inc.) and fixed in 3% paraformaldehyde. Viable cells were gated, and 5,000 cells were analyzed using a FACScan instrument and Consort 30 analysis software (Becton-Dickinson, Frank- lin Lakes, NJ).

Endocytosis Studies FLuid-Phase Endocytosis. T o investigate the capa-

bility of human HSCs to take up macromolecules by fluid-phase endocytosis, human HSCs were cultured in medium containing 0.5% bovine serum albumin and 10 mg/mL lysine-fixable FITC-dextran (molecular weight, 10 kd; Molecular Probes, Eugene, OR) for 10 minutes at 37°C. Cells were then washed twice with PBS (at 4°C) to stop dextran uptake and then incubated in culture medium for various periods of time at 37°C to allow the internalization of dextran. Cells were rinsed with ice-cold PBS and fixed in 3.7% paraformaldehyde for 15 minutes at 37°C. The presence of vesicular structures containing FITC-dextran in the cells was visualized using a confocal microscope (Leica TCS NT; Leica, Heidel- berg, Germany).

Receptor-Mediated Endocytosis. To assess the existence of transferrin and mannose receptor-mediated endocytosis, transferrin and high-molecular-weight dextran were used. For transferrin receptor assay, cells were cultured on a 12-mm-diameter glass coverslip until 80% confluence. Cells were then incubated with FITC-transferrin at 20 pglmL and 37°C for 5 , 10, and 20 minutes. Cells were washed twice with ice-cold PBS and fixed in 3.7% paraformaldehyde. FITC-transferrin internalized was visualized using a confocal microscope (Leica TCS NT). For the mannose receptor assay, cells were incubated with high-molecular-weight dextran (70 kd FITC- dextran; Molecular Probes) for 1 hour and studied as previously described.

Phagocytosis. The capability of human HSCs to perform phagocytosis was assessed by a bacteria phagocytic test and by incubating cells with latex particles. The bacteria phagocytic test (Phagotest; Orpegen Pharma, Heidelberg, Germany) was performed following the manufacturer’s instructions. In brief, HSCs were cultured until confluence in 12-mm-diameter glass coverslips. Medium was removed and cells incubated in medium containing AB serum and fluorescein-labeled opsonized Escherichia coli bacteria at 37°C for 3 hours. Parallel experiments incubated on ice were used as negative controls. Phagocytosis was stopped by placing the samples on ice and adding a quenching solution. This solution allows the discrimination between attachment and internalization of

bacteria by quenching the FITC fluorescence of surface- bound bacteria, leaving the fluorescence of internalized particles unaltered. After 2 washing steps, the existence of fluorescence bacteria inside the cells was assessed using a confocal microscope (Leica TCS NT). For the latex assay, cells were cultured on a 12-mm-diameter glass coverslip until 80% confluence. A l-hour pulse with either 1.01- pm-diameter latex beads or carboxylate-modified fluores- cent latex beads (both from Sigma Chemical Co.) was performed. Cells were then washed 3 times with PBS and left for 3 hours to allow the chase of the latex beads. Cells were then washed twice and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer for 1 hour. Cells were conventionally dehydrated and critical point dried. Internalized latex beads were visualized using an electron microscope (Zeiss DSM 940A; Carl Zeiss, Oberkochen, Germany) with an acceleration voltage of 10 kV.

Immunocytochemistry Culture-activated HSCs were trypsinized and cultured

on slide chambers (Falcon Slides; Becton Dickinson, Ce- dex, France) for 48 hours. HSCs isolated from human cirrhotic livers (activated in vim) were cultured for 24 hours. Cells were serum starved for 12 hours and then fixed with acetone at -20°C for 7 minutes. Fixed cells were immunostained for anti-CD68 monoclonal antibody (DAKO, Carpinteria, CA) using the DAKO Envi- sion System according to the manufacturer’s instructions. Briefly, endogenous peroxidase was blocked with peroxidase-blocking agent and slides were incubated with primary antibody for 30 minutes at room temperature in 1% bovine serum albumin in PBS. After two 3-minute washes in PBS, slides were incubated with labeled polymer for 10 minutes at room temperature. Cells were washed twice with PBS, incubated with 3,3-diaminobenzidine sub- strate chromogen for 8 minutes, washed with water, incubated with DAB enhancer (Innovex Biosciences, Richmond, CA) for 5 minutes, and washed with water before counterstaining with hematoxylin. As negative control, cells were incubated with an irrelevant isotype- matched control antibody under identical conditions.

T-Lymphocyte Proliferation S td ie s Human peripheral blood mononuclear cells (PBMCs)

were isolated from normal volunteers. Briefly, 30 mL of heparinized whole blood was diluted 1:2 with PBS. Two vol of diluted blood was overlaid on 1 vol of I .O80 g/mL FicolI Histopaque (Sigma Chemical Co.) and centrifuged at 20°C at 5009 for 30 minutes. The PBMC layer was obtained, washed with PBS, and centrifuged. Cells were genotyped and cultured in the presence of HSCs. Briefly,

922 VIRAS ET AL. HEPATOLOGY, October 2003

HLA-11-typed HSCs were stimulated with either buffer or cytokines (10 U/mL IL-1P + 100 U/mL IFN-y) for 3 days. HSCs were then washed twice with PBS, trypsinized, and irradiated with 6,000 rads, a dose shown to stop proliferation without affecting cell viability or membrane protein expression. A sample was analyzed by flow cytometry to confirm the expression of HLA-I1 molecules in cytokine-treated HSCs. Irradiated HSCs (1O,OOO cells/well) were seeded on flat-bottomed 96-well plates (TPP, Trasadingen, Switzerland) in W M I 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% AB human normal serum and 2 mmol/L L-glu- tamine 4 hours before adding human PBMCs. Both HLA-11-matched and -mismatched fresh human PB- MCs were then added (50,000 cells/well). Cultures were maintained at 37°C in a humidified atmosphere with 5% C O ~ for 7 days. In each experimental condition, a parallel experiment with irradiated (3,000 rads) nonproliferating PBMCs was performed to assess basal 3H-thymidine incorporation. To assess T-lymphocyte DNA synthesis, 3H- thymidine (1 pCi/well; Amersham, Arlington Heights, IL) was added 18 hours before harvesting the cells (LKB Wallac 1295-001; Perkin Elmer Inc., Boston, MA). In- corporation of 3H-thymidine was measured in a beta ra- diation counter (Wallac 1205 Betaplate; Perkin Elmer Inc.). Proliferation index was defined as cpm (nonirradi- ared T lymphocytes + HSCs)/cpm (irradiated T lymphocytes + HSCs).

Statistical Analysis The statistical analysis of the results was made using the

paired and unpaired Student's t test, the x2 test, ANOVA, and the Newman-Keuls test. Results are given as mean 2 SEM.

Results Expression of Molecubs Involved in Antigen Presentation in Human Activated HSCs

We first studied the expression of HLA molecules. HLA-I is constitutively expressed in nucleated cells and processes mainly internal antigens, whereas HLA-I1 molecules are typically expressed in APCs and process mainly external antigens to T ceIls.21,22 As expected, human HSCs strongly express HLA-I in basal conditions, whereas the expression of HLA-I1 (DR) was low (Fig. 1). Stimulation with IFN-y and IL- 1P and, to a lesser extent, prolonged cell culture resulted in up-regulation of both HLA-I and HLA-I1 (DR) molecules. HLA-I1 DQand DP were expressed neither in basal conditions nor afier stimulation with cytokines (data not shown). This pattern of expression is characteristic of nonprofessional APCs. l 6 ~ 7 We next investigated the expression of molecules belonging to

1901

B

d :I HLA-I1 OR)

n T

172

4 129

'U

'% 86 PI

V

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Fig. 1. Expression of HLA molecules in human activated HSCs. Cells were incubated in serum-free medium containing either cytokines (10 U/mL IL - lp + 100 U/mL IFN-7) or buffer for 1,3, and 6 days, and expression of (A) HLA-I and (6) HLA-II (DR, DP, and DQ) was assessed by flow cytometty. Only HLA-II (DR) is shown, because DP and OQ were not expressed. Results are shown as mean fluorescence ratio compared with cells incubated with isotype-matched antibody. Results are the mean of at least 5 experiments using 3 different cell isolations. *P < .01 vs. 1 day; #P < .05 vs. cells incubated with buffer. Representative experiments of HLA expression in basal conditions and after 6 days of cytokine stimulation are shown.


A *

l6 1 CDlb I -r

I

10'

CDlc B

" 1

3 clays

d9b IrotW 8 12*/ n

both CD 1 b and CDlc, whereas CD la was not detected (data not shown). Prolonged cell culture increased CDlb - expression, whereas CD 1 c remained unchanged. Interest- ingly, cytokine stimulation did not afFect CD1 expression. We also investigated the expression of costimulatory mole-

13 7

A CD40

B

B S

13

fi 101

Fig. 2. Expression of membrane molecules of the CD1 family in human activated HSCs. Cells were incubated in serum-free medium containing either cytokines (10 U/mL IL-1p + 100 U/mL IFN-y) or buffer for 1, 3, and 6 days, and expression of CDla, (A) CDlb, and (B) CDlc was assessed by flow cytometry. CDla was not expressed (not shown). Results are shown as fold expression compared with cells incubated with isotype-matched antibody. Results are the mean of at least 5 experiments using 3 different cell isolations. * P < .01 vs. 1 day and 3 days. Representative experiments of CDlb and CDlc expression in basal conditions and after 6 days of cytokine stimulation are shown.

the CDl family, which participate in antigen presentation in an HLA-independent manner. CD1 members are mainly expressed in professional APCs and mediate presentation of microbial lipid and glycolipid antigens to T cek23As shown in Fig. 2, human unstimulated HSCs expressed high levels of

cmo

10' 10'

Fig. 3. Expression of costimulatory molecules involved in T-cell activation in human activated HSCs. Cells were incubated in serum-free medium containing either cytokines (10 U/mL IL - lP + 100 U/mL IFN-y) or buffer for 1, 3, and 6 days, and expression of (A) CD40, (B) CD80, and CD86 was assessed by flow cytometry. CD86 was not expressed (not shown). Results are shown as fold expression compared with cells incubated with isotype-matched antibody. Results are the mean of at least 5 experiments using 3 different cell isolations. * P < .01 vs. 1 day; #P < .05 vs. cells incubated with buffer. Representative experiments of CD40 and CD80 expression in basal conditions and after 6 days of cytokine stimulation are shown.

924 VIRAS ET AL. HEPATOLOGY, October 2003

cules that bind to specific receptors ofT lymphocytes and are essential to induce specific T-cell responses. These molecules include CD40, CD80, and CD86. As shown in Fig. 3, HSCs express CD40 and low levels of CD80, whereas CD86 was not detected (not shown). Again, prolonged cell culture and stimulation with cytokines markedly up-regulated CD40, whereas CD80 expression only increased after prolonged exposure to cytokines. Collectively, these data indicate that human activated HSCs can express the elements required for antigen presentation to T cells. The fact that the expression of these proteins in culture-activated HSCs is mainly cytokine dependent supports our hypothesis that activated HSCs may act as nonprofessional APCs. To rule out that these findings are due to prolonged cell culture on plastic, we studied the expression of molecules involved in antigen presentation in HSCs freshly isolated from human cirrhotic livers (HSCs activated in vim). Flow cytometry analysis showed that in vim activated HSCs express high amounts of HLA-I1 DR and CD40, whereas CD80 was not detected (not shown).

Endocytosis Studies We investigated the capability of human HSCs to in-

ternalize extracellular material. This biological function can be achieved by fluid-phase endocytosis, receptor-mediated endocytosis, and phagocytosis. First, to address the

capability of HSCs to take up macromolecules by fluid- phase endocytosis, cells were incubated in the presence of low-molecular-weight dextran (dextran-FITC). As shown in Fig. 4A, human HSCs efficiently internalize low-molecular-weight dextran, which was observed after 10 minutes in cytoplasmic vesicles. To assess the existence of receptor-mediated endocytosis, we studied the ability of HSCs to internalize transferrin and high-molecular- weight dextran, which bind to transferrin and mannose receptors, respectively. As shown in Fig. 4B, cultured HSCs efficiently internalize transferrin, indicating that HSCs can perform receptor-mediated endocytosis. Simi- lar results were observed with 70 kd dextran (Fig. 4C). We next quantified the ability of HSCs to perform receptor- mediated endocytosis using human Kupffer cells as positive control. More than 90% of Kupffer cells showed positive endocytosis of 70 kd dextran (n = 67), with 78% of the cells showing intense cytoplasmic staining, indicating that these cells have a marked endocytic function (Fig. 4E). Under the same conditions, more than 60% of HSCs underwent receptor-mediated endocytosis (n = 44), with 30% of the cells showing intense cytoplasmic staining. These results indicate that HSCs have powerful endocytic activity. Moreover, cultured human HSCs express high amounts of CD68 or macrosialin (Fig. 5 ) , as assessed by

Fig. 4. (A) Demonstration of fluid-phase endocytosis by cultured HSCs. Cells were cultured in medium containing FITC/low-molecular-weight (10 kd) dextran for 10 minutes at 37'C, washed, and further incubated in culture medium to allow the internalization of dextran. Cells were fixed, and the presence of vesicles containing FITC-dextran was visualized using a confocal microscope. (B and C) Receptor-mediated endocytosis by HSCs. Cells were incubated with (6) FITC-transferrin and (C) high-molecular-weight (70 kd) dextran for 10 minutes at 37"C, fixed, and visualized using a confocal microscope (original magnification X400). Numerous vesicle-like structures containing both transferrin and/or 70 kd dextran were clearly observed in the cytoplasm. (D) As a negative control experiment, HSCs were incubated with 70 kd dextran for 1 hour at 4°C. No endocytosis was noted. (E) As a positive control experiment, human Kupffer cells were incubated with 70 kd dextran for 1 hour at 37°C. Numerous dextran vesicles were obsetved in the cytoplasm.

HEPATOLOGY, Vol. 38, No. 4, 2003 VINAS ET AL. 925

A In. vivo

activated

Cu!ture- achvated

B CD68 Isoty pe-control

f

I day 3 days 6 days

‘”1

Fig. 5. (A) Demonstration of CD68 (macrosialin) expression in in vivo activated and culture-activated human HSCs by immunocytochemistry. (B) Regulation of CD68 expression in HSCs by cytokines as assessed by flow cytometry. Cells were incubated in serum-free medium containing cytokines (10 U/mL IL-1p + 100 U/mL IFN-y) or buffer for 1,3, and 6 days. Results are shown as fold expression compared with cells incubated with isotype-matched antibody. Results are the mean of at least 5 experiments using 3 different cell isolations. * P < .01 vs. 1 day; #P < .05 vs. cells incubated with buffer. A representative experiment of CD68 expression in basal conditions and after 6 days of cytokine stimulation is shown.

flow cytometry and immunocytochemistry. CD68 is a protein involved in antigen trafficking that is commonly found in professional APCs such as dendritic cells and Kupffer cells. CD68 expression was markedly increased after stimulation with cytokines and, to a lesser extent, after prolonged cell culture. CD68 staining shows a dif- fuse intracellular staining similar to the pattern found in macrophages and different than the perinuclear characteristic distribution of mature dendritic cells. Importantly, HSCs isolated from human cirrhotic livers (HSCs activated in vivo) showed intense staining for CD68, indicating that this molecule is expressed in HSCs during liver fibrogenesis. Finally, we explored whether human HSCs

can perform phagocytosis of macromolecules and/or bacteria. This function was assessed using 2 different ap- proaches: an in vitro test of phagocytosis of E. coli and the internalization of latex particles. Figure 6A shows some E. coli inside cultured HSCs after an in vitro test with fluo- rescent bacteria. The capability of HSCs to internalize bacteria, however, was markedly lower than that observed with peripheral blood monocytes. Although 90% of monocytes showed intracellular bacterial products (n = 78), only 15% of HSCs show bacterial phagocytosis after prolonged periods of time (n = 42). Moreover, HSCs internalized latex particles (Fig. 6B and C), indicating that these cells can take up large material. Latex internalization was found in 20% of cultured HSCs (n = 53) compared with 95% ofmonocytes (n = 65). All together, these data indicate that human activated HSCs can take up extracellular macromolecules, mainly by endocytosis, further supporting their potential role as APCs.

Modulation of T-Lymphocyte Growth by Human HSCs

To investigate whether cultured human HSCs present antigens to T lymphocytes in a HLA-11-restricted manner, a modified mixed lymphocyte reaction was performed. The reaction consisted of inducing proliferation of T lymphocytes in a culture of PBMCs obtained from a subject, which were cocultured with HSCs from another subject wearing different HLA-I1 alleles. As a control, HSCs were cocultured with PBMCs from an HLA-identical subject. As shown in Fig. 7A, neither unstimulated nor cytokine-stimulated HSCs affect proliferation of “identical” (HLA-matched) T lymphocytes. Similarly, unstimulated HSCs, which express very low levels of HLA-I1 (DR), barely affect proliferation of “allogeneic” (HLA-mismatched) T lymphocytes. In contrast, cytokine-stimulated HSCs induced a more than 2-fold increase in 3H thymidine uptake by allogeneic T lymphocytes. The mitogenic effect of HSCs toward allogeneic T lymphocytes was only slightly blunted by HLA-I blocking antibodies and almost completely prevented by HLA-I1 blocking antibodies (Fig. 7B). Incubation with isotype-matched antibodies had no effect. These data indicate that, on stimulation with cytokines, HSCs induce T-lymphocyte proliferation in an HLA-11-restricted manner.

Discussion In the current study, we provide evidence that culture-

activated human HSCs express membrane proteins required for antigen presentation and modulate T-lymphocyte proliferation. Moreover, activated HSCs are able to internalize macromolecules and bacteria. Based on these data, we

926 VINAS E T AL. HEPATOLOGY, October 2003

propose that HSCs play a role in the immune function of the liver. Similar properties have been described in other organ-specific pericytes, such as mesangial cells and astrocytes,16,17,2*-26 indicating that under certain circum- stances these cell types could play a supporting role in the local regulation of the immune system.

We first investigated whether activated cultured HSCs express key elements for antigen presentation. It is well known that optimal T-cell activation by APCs requires at least 2 signals. One is provided by the interaction between antigen-specific T-cell receptors and antigen-HLA com- plexes.21,22 The other is provided by costimulatory signals driven by proteins such as CD40, CD80, and CD86.27,28 We show that HSCs express HLA-I and low levels of the remaining proteins in basal conditions, whereas their expression is markedly increased by cytokines. This pattern of expression has been previously described for other nonprofessional APCs.16,24~26,29-31 Like most nucleated mam- malian cells, unstimulated cultured HSCs markedly express HLA-I, which is up-regulated by cytokines. The immune function of HLA-I molecules is to transport and present peptides derived from intracellularly degraded proteins to cytotoxic CD8’ T cells.21 Notably, cytokine- stimulated HSCs express high amounts of HLA-I1 (DR), which is considered the gold-standard element in APCs. HLA-I1 binds peptides derived from antigens that access the endocytic route of APCs and display them on the

Fig. 6. (A) Phagocytosis of bacteria by cultured human HSCs. Cells were incubated in the presence of fluorescein-labeled opsonized E. coli at 37°C for 3 hours, and phagocytosis was stopped by placing the samples on ice and adding a quenching solution. The existence of fluorescence bacteria inside the cells was assessed using a confocal microscope. Several bacteria were clearly observed inside HSCs (ar- rows). (6) Demonstration of phagocytosis of macromolecules by HSCs. Cells were incubated with fluores- cent latex particles and visualized with a fluorescence microscope (arrow) (original magnification X400). (C) Phagocytosis of latex particles by cultured HSCs. Cells were incubated with 1-pm-diameter latex beads, washed, fixed, and visualized using a scanning microscope. Some beads were clearly seen inside the cultured cells (arrow) (original magnification X400).

plasma membrane for recognition by CD4+ T ~el l s .3~ Moreover, HSCs highly express C D l b and CDlc, which mediate a newly described pathway for presentation of lipids and glycolipids for specific recognition by T ~ e l l s . ~ 3 Although some of these lipids can derive from the same cell, this finding raises the possibility that HSCs would play a role in antigen presentation of interaction with bacterial lipid products such as lipopolysaccharide. Fi- nally, activated HSCs express the costimulatory proteins CD40 and CD80 in a cytokine-dependent manner. Al- though the characterization of HSCs as AI’Cs has not been previously reported, previous studies suggest that HSCs can interact with lymphocytes. First, cuitured activated HSCs express intracellular adhesion molecule 1, which plays an important role in the interaction between APCs and lymphocytes.33 Moreover, a recent study has shown that human activated HSCs express CD40, an important costimulatory molecule.^* This latter study confirms our data that CD40 expression is markedly up- regulated by cytokines such as IFN-?. Moreover, stimulation of CD40 by specific CD40 ligands activates intracellular pathways and induces chemokine secretion by HSCs. These data indicate that costimulatory molecules not only play a role in the development of CD40 T cell- dependent effector functions but also can exert biological functions in the same HSCs.


35 i

* 7

HLA-identical Allogeiieic

;i-;j , , * f $ 1 5

& - 05

0

Anb-isotyp Anh HLA-I Anb HLA-II

Fig. 7. T-lymphocyte proliferation by human activated HSCs in a coculture system. (A) HSCs were stimulated with either buffer or cytokines (10 U/mL IL-lP + 100 U/mL IFN-.)I) for 3 days, irradiated, and cocultured with HLA-compatible (HLA-identical) or incompatible (Alloge- neic) T lymphocytes. Cocultures were maintained for 7 days, and T- lymphocyte proliferation was assessed by 3H-thymidine incorporation. Results are expressed as fold stimulation of proliferative index (see Materials and Methods). * P < .05 vs. HLA-identical and vs. HSCs incubated with buffer. (6) Effect of blocking antibodies anti-HLA-l and anti-HLA-ll in the proliferative effect of HSCs toward allogeneic T lymphocytes. Irradiated cytokine-stimulated HSCs were cocultured with allogeneic T lymphocytes for 7 days in the presence of isotype-matched antibody, monoclonal antibody anti-HLA-I, or monoclonal antibody anti- HLA-II. Medium and antibodies were replaced evety 2 days. T-lymphocyte proliferation was assessed as previously described. * P < .05 vs. isotype antibody.

We next showed that human HSCs are capable of internalizing extracellular material by either endocytosis or phagocytosis. In fact, we have recently reported that liver cells express caveolin,35 which might be considered an additional port of entry for small molecules, macromolecules, and pathogens (or their pr0ducts).3~ The capacity to internalize extracellular material has also been described in other fibrogenic cell types (i.e., mesangial cells) as well as in hepatic nonprofessional APCs such as sinusoidal endothelial ~ells.3733~ Our results are consistent with the findings of a recent study showing that activated HSCs can phagocytose apoptotic hepatocytes.39 Intetest- ingly, in this latter study, it was found that the phagocytosis process was associated with increased fibrogenic potency of HSCs. An interesting finding of our study is that activated human HSCs express high amounts of

CD68 or macrosialin. CD68 is a 110-kd glycoprotein commonly expressed in dendritic cells and macrophages, including Kupffer cells.40 CD68 belongs to the family of lysosomal/plasma membrane shuttling proteins that play a role in endocytosis and lysosomal traffic of antigens.41 Therefore, the expression of this protein by activated HSCs further supports their role as potential APCs. In fact, CD68 immunostaining has been traditionally considered a marker of dendritic/Kupffer cells in the dam- aged l i ~ e r . ~ ~ , ~ 3 The finding that CD68 staining is commonly found in areas of active liver fibrogenesis sug- gests that activated HSCs could also account for this expression.

An important finding of the current study is that HSCs can modulate the proliferation of T lymphocytes in vitro. We show that, on stimulation with cytokines, HLA-II- expressing HSCs induce proliferation of allogeneic T lymphocytes. Because proinflammatory cytokines (i. e., IFN-y) are produced by infiltrating T h l cells in the dam- aged liver,44 it is conceivable that these cytokines activate HSCs, which in turn up-regulate HLA-I1 to induce lymphocyte proliferation. Therefore, a crosstalk between infiltrating cells and HSCs is likely to occur in chronic liver diseases. Our finding that in vivo activated HSCs contain key elements to interact with lymphocytes supports this hypothesis. Importantly, this effect was inhibited by blocking antibodies against HLA-11, indicating that HLA-I1 mediates the mitogenic effect. This novel function for HSCs has been previously described in other fibrogenic “nonprofessional” APCs, such as mesangial cells, vascular smooth muscle cells, or fibroblasts.25~31~45 As with HSCs, these cells also require pretreatment with cytokines to stimulate T-lymphocyte growth. By contrast, professional APCs such as glial cells, which highly express HLA-I1 in basal conditions, induce lymphocyte activation without previous stimulation with cytokine~.*~ In these cells, however, stimulation with cytokines further increases HLA-I1 expression and antigen presentation capacity. It is important to note that the level of expression of HLA-I1 and costimulation factors in cultured human HSCs, even on cytokine stimulation, is lower than in professional APCs. By contrast, HSCs express similar amounts of C D 1 b and C D l c as peripheral blood monocytes, suggesting that HLA-independent pathways of antigen presentation are likely involved in the interaction between HSCs and T lymphocytes.

The findings of this study that cultured HSCs display immune properties could be of pathophysiologic rele- vance. Activated HSCs are perisinusoidal cells that accumulate at the sites of liver injury, where inflammatory cells are also pre~ent.~7 Moreover, activated HSCs express cell adhesion molecules that can contribute to the recruit-

928 VINAS ETAL.

ment of inflammatory cells at the sites of tissue repair.*8 Therefore, a crosstalk between HSCs and infiltrating lymphocytes is likely to occur. Different cytokines secreted by T cells (ie. , IFNs, IL-6, and IL-10) can regulate important functions of H S C S . * ~ . ~ ~ In addition, HSCs secrete a number of chemoattractants that may participate in the recruitment of inflammatory Our data showing that HSCs affect T-lymphocyte growth further reinforces an active role of this cell type in regulating liver inflammation. Besides, immune properties of HSCs could contribute to their fibrogenic properties. In fact, the development of fibrosis in many types of liver diseases (ie. , chronic viral hepatitis, chronic rejection) is preceded by the inflammation of the hepatic parenchyma, and treat- ments inhibiting liver inflammation also attenuate the progression of liver fibrosis.51 Future studies should investigate the mechanisms by which HSCs modulate the growth of lymphocytes and its implication in liver fibrosis.

In summary, we provide evidence that human activated HSCs express the molecules necessary for antigen presentation and modulate lymphocyte proliferation. Therefore, HSCs may participate in the immune function of the liver.

Acknowledgment: The authors thank Anna Bosch and Elisenda Coll (Serveis Cientifico-Tkcnics, University of Barcelona) for their help in confocal and scanning electron microscopy images as well as Jordi Mild and Ramon Vilella (Hospital Clinic of Barcelona) for the kind gift of CDlb and HLA-I1 DR monoclonal antibodies.

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