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Am J Physiol Gastrointest Liver Physiol 289: G571-G578, 2005. First published April 28, 2005; doi:10.1152/ajpgi.00537.2004
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LIVER AND BILIARY TRACT

Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic stellate cells

Paola Brun,1,2 Ignazio Castagliuolo,1 Massimo Pinzani,3 Giorgio Palù,1 and Diego Martines2

Departments of 1Histology, Microbiology and Medical Biotechnologies and 2Gastroenterological Sciences, University of Padua, Padua; and 3Institute of Internal Medicine, University of Florence, Florence, Italy

Submitted 3 December 2004 ; accepted in final form 11 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activated hepatic stellate cells (HSCs) secrete extracellular matrix components during hepatic fibrosis, but recent studies have shown that HSCs can also release a variety of proinflammatory cytokines. Moreover, bacterial endotoxemia is not only associated with systemic complications in the late stages of liver failure but is also a direct cause of liver damage, activating resident inflammatory cells. In this study, we investigated whether HSCs can respond directly to bacterial cell wall products acquiring a new phenotype. RT-PCR and immunocytochemistry assays were used to show that murine HSCs expressed specific mRNA transcripts and proteins for LPS and lipoteichoic acid (LTA) receptor systems and peptidoglycan recognition proteins. Exposing HSCs to bacterial endotoxins led to phosphorylation of mitogen-activated protein kinase ERK1 and the development of a proinflammatory phenotype. After exposure to LPS, LTA, or N-acetyl muramyl peptide, transforming growth factor-{beta}1, IL-6, and monocyte chemoattractant protein-1 (MCP-1) mRNA specific transcripts and proteins increased significantly in HSCs, as assayed by quantitative real-time RT-PCR and ELISA. These LPS-mediated effects in HSCs were receptor dependent, because LPS-induced ERK1 phosphorylation, IL-6, and MCP-1 mRNA and protein level upregulation were significantly less pronounced in HSCs isolated from C3H/HeJ mice lacking Toll-like receptor 4. In conclusion, our results show that murine HSCs express functional receptors for bacterial endotoxins, and HSCs exposed to bacterial products develop a strong proinflammatory phenotype. We speculate that high levels of bacterial endotoxins in the portal vein may directly induce a proinflammatory phenotype in HSCs that contributes to liver damage.

fibrosis; hepatitis; nonalcoholic liver disease; fibronectin; collagen; toll-like receptors

GUT-DERIVED BACTERIA HAVE been associated with complications such as endotoxemia, high circulating levels of proinflammatory cytokines, and spontaneous bacterial peritonitis in cirrhotic patients (18, 36). More recent evidences suggest, however, that products derived from intestinal Gram-negative and Gram-positive bacteria may contribute directly to liver injury in apparently unrelated hepatic diseases such as obstructive jaundice, cholestasis, or chronic alcoholic hepatitis (1, 16, 39). Intestinal absorption of endotoxins thus increases in cholestatic disorders (22) and following alcohol intake (7, 31), whereas mononuclear cells containing bacterial DNA have been detected in primary biliary cirrhosis patients (44). When hepatic clearance of enteric bacteria is exceeded, bacterial fragments spread into hepatic sinusoids, activating Kupffer cells and hepatocytes (10). Accordingly, administering LPS to normal rodents triggers TNF-{alpha} and IL-1 and -6 release by Kupffer cells, inducing hepatocyte necrosis and activating other hepatic cell populations (13).

Hepatic stellate cells (HSCs) undergo a process of activation and phenotypic modulation after acute or chronic liver tissue injury. In chronic fibrotic liver diseases, HSCs are the key cellular element in the excessive accumulation of extracellular matrix (12). Activated HSCs exposed to proinflammatory cytokines can secrete a variety of chemokines [macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1)] and cytokines [transforming growth factor-{beta}1 (TGF-{beta}1), IL-6] that eventually contribute to liver damage (34). Because activated human HSCs in the cirrhotic liver respond directly to LPS via a specific receptor complex (30), we speculated that HSCs could exacerbate the inflammatory cytokine-mediated hepatic damage in portal endotoxemia.

A broad range of microbial products is recognized by immune and nonimmune cells through the so-called Toll-like receptors (TLRs), a family of proteins strictly conserved from invertebrates to humans, involved in innate immunity (19). TLR4 interacts mainly with LPS bound to CD14, and TLR2 preferentially recognizes lipoproteins and peptidoglycan (PGN) fragments, whereas TLR9 is involved in the response against unmethylated CpG DNA common in the bacterial genome (2, 46). More recently, other membrane receptors recognizing PGN fragments, called PGN recognition proteins (PGRPs), have also been described (24). Activation of these receptors triggers stereotyped responses leading to NF-{kappa}B nuclear translocation and the transcription of genes encoding proinflammatory soluble factors (42).

Bacterial cell wall products, such as LPS, lipoteichoic acid (LTA), and PGN fragments (e.g., N-acetyl muramyl peptide), are known to activate monocytes directly (17), whereas their effects on hepatocytes are more controversial. We hypothesized that HSCs may also respond to these stimuli by developing an inflammatory phenotype and contributing to tissue damage. We report here that first-passage murine HSCs express functional LPS receptor complex, TLR2, PGRP long (PGRP-L), and PGRP short (PGRP-S) to recognize a broad range of bacterial products. Incubating murine HSCs with bacterial cell wall products significantly increased the expression and release of inflammatory mediators. In this setting, we speculate that HSCs may make a relevant contribution to the inflammatory network operating in the liver during endotoxemia.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Balb/c and C3H/HeJ mice (8–12 wk old, Harlan, Oderzo, Italy) were housed in groups of six per cage, kept under controlled temperature and humidity conditions, and given standard pelleted chow and tap water ad libitum. The animal studies were approved by the Institutional Animal Care and Use Committee of the University of Padua.

Isolation and culture of murine HSCs. HSCs were isolated from the livers of Balb/c and C3H/HeJ mice as described elsewhere, with minor modifications (32, 45). Briefly, aseptically removed livers were finely minced, digested with 0.5% (wt/vol) pronase E (Merck KGaA, Darmstadt, Germany), 0.05% (wt/vol) type IV collagenase (Sigma, Milan, Italy), and 10 µg/ml deoxyribonuclease I from bovine pancreas (Calbiochem, Milan, Italy), then filtered through a 100-µm wire mesh, and washed three times in HBSS (GIBCO, Milan, Italy). Cells were centrifuged through a two-step Percoll [Amersham Biosciences, Uppsala, Sweden; 50% and 35% (vol/vol)] gradient (at 1,200 rpm for 30 min at 4°C). HSCs were collected, washed twice in HBSS, and then cultured in Iscove's modified medium supplemented with 0.6 U/ml insulin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 1% (vol/vol) antibiotic solution, and 20% (vol/vol) heat-inactivated FBS (all provided by GIBCO). The experiments described in this study were always performed on cells after one serial subculture. HSC purity was confirmed, after the first subculture passage, by morphological light microscopic appearance, staining of fat droplets with oil red O, immunofluorescent staining for {alpha}-smooth muscle actin ({alpha}-SMA; Sigma), and vitamin A-specific ultraviolet fluorescence. In addition, any presence of contaminating Kupffer and endothelial cells was ruled out by the absence of nonspecific esterase activity and immunofluorescent staining for factor VIII (Dako, Milan, Italy), respectively.

Immunocytochemistry on cultured HSCs. Subconfluent HSCs were trypsinized and seeded on sterile coverslips. After 24 h, the cells were washed with ice-cold PBS (pH 7.4) and fixed in methanol (5 min at –20°C). After being washed, nonspecific binding was blocked by incubation with 2% donkey serum in PBS for 20 min. CD14 and TLR4 were probed with a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and detected by a donkey anti-rabbit IgG fluorescein (FITC)-conjugated antibody (Jackson Immunoresearch Laboratories, West Grove, PA). Coverslips were extensively washed, mounted, analyzed, and photographed using a Leica TCS-NT/SP2 confocal microscope (x40 objective). Images were digitally stored with Leica software. In control samples, cells were incubated with nonimmune rabbit IgG.

Incubation of HSCs with bacterial cell wall products. Subconfluent HSCs were trypsinized, seeded (105 cells/well) in six-well plates, and grown in complete media to reach ~80% of confluence, then the FBS was gradually reduced to 1% vol/vol. Cell monolayers were then incubated for 24 h in medium containing LPS (from Salmonella enteritidis, Sigma), LTA (from Streptococcus faecalis, Sigma), or N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (NAM; Sigma) at 10–0.01 µg/ml. In each experiment, at least one internal control was performed consisting of HSCs incubated with medium supplemented with 1% (vol/vol) FBS.

RNA extraction and RT-PCR analysis. Total RNA was isolated from the HSCs by a single-step acid guanidium phenol-chloroform extraction procedure using OMNIzol (Euroclone, Milan, Italy) (6). Contaminating DNA was removed with the DNA-free kit (Ambion). Two micrograms of total RNA were reverse transcribed using random primers and MulV RT (Applied Biosystems, Monza, Italy). Five microliters of the RT reaction were subjected to PCR to determine the presence of mRNA coding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CD14, TLR4, MD2, TLR2, PGRP-L, and PGRP-S. Amplification products were separated on 2% (wt/vol) agarose gel and visualized by ethidium bromide staining using an ultraviolet transilluminator. Real-time quantitative RT-PCR analysis was performed on the ABI Prism 7700 sequence detector (Applied Biosystems) using SYBR Green PCR core reagents kit (Applied Biosystems) to measure steady-state mRNA transcript levels for TGF-{beta}1, IL-6, fibronectin, collagen type I, monocyte chemoattractant protein (MCP)-1, and PDGF-BB. Primers and PCR conditions are listed in Table 1. Genes underwent absolute quantification against a standard curve generated by amplification of 10-fold serial dilutions of correspondent cDNAs subcloned into the pCR2.1 vector (Invitrogen, Milan, Italy). The coefficient of correlation was r = 0.99, and the slope was constant at each experiment. The expression of all target genes was normalized to GAPDH.


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  		Table 1. Primers used in the study

 
Cytokine ELISA assay. HSCs were cultured in medium containing 1% FBS alone or supplemented with LPS, LTA, or NAM for 24 h, as described in Incubation of HSCs with bacterial cell wall products. TGF-{beta}1 (Promega), IL-6 (Bender MedSystems, Milan, Italy), and MCP-1 (BioLegend, San Diego, CA) were measured in HSC-conditioned medium using commercially available ELISA kits. Optical densities were measured using an ELISA plate reader (Spectra I Tecan, Salzburg, Austria) at a wavelength of 450 nm. TGF-{beta}, IL-6, and MCP-1 levels were expressed as picograms per milliliter.

Immunoprecipitation and Western blotting. HSCs were cultured in medium containing 1% FBS alone or incubated with LPS, LTA, or NAM (10 µg/ml) for 30 min. Monolayers were washed twice with ice-cold PBS and then lysed (45 min on ice) using nondenaturing RIPA buffer (150 mM NaCl, 50 mM Tris·HCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 µM NaVO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Particulate material was removed by centrifugation (15,000 g for 5 min at 4°C), supernatants were collected, and protein concentrations were determined using the bicinchoninic acid method (Pierce). Lysates (2 mg/ml) were incubated with a rabbit anti-ERK1 polyclonal antibody (Santa Cruz Biotechnology; 10 µg/mg cell lysate) for 2 h at 4°C. Then, protein A-agarose (Santa Cruz Biotechnology) was added and incubated for 1 h at 4°C. Beads were washed twice by centrifugation (20 s, 12,000 g) with ice-cold RIPA buffer followed by one wash with ice-cold PBS and then boiled in 25 µl of sample loading buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 5% {beta}-mercaptoethanol, and 0.1% bromophenol blue). Immunoprecipitated proteins were fractionated on an SDS-PAGE gel and then transferred and immobilized on a nitrocellulose membrane. Membranes were blocked overnight at 4°C in 5% skim milk in PBS containing 0.05% Tween 20 and then incubated for 2 h with anti-phosphotyrosine antibody (PY99, Santa Cruz Biotechnology) to identify phosphorylated tyrosine residues. Bound antibody was detected by a horseradish peroxidase-conjugated donkey anti-mouse IgG antibody (Sigma). Immunocomplexes were visualized using the ECL Western blot analysis detection reagents (Pierce) and photographed using a VersaDoc imaging system (Bio-Rad). Images were digitally stored with Quantity One (Bio-Rad) software.

Proliferation assay. HSCs were seeded at 10 x 103 cells/ml in 12-well plates. After 24 h, FBS content was gradually reduced to 1%. Cells were then incubated with medium alone or supplemented with the indicated concentrations of LPS, LTA, or NAM. [3H]thymidine (1 µCi/well, Amersham) was added to each well, and, 24 h later, monolayers were extensively washed to remove unincorporated [3H]thymidine. Cells were then dissolved by adding 200 µl of SDS 10%, and incorporated [3H]thymidine was quantified in a liquid scintillation {beta}-counter (LKG Wallac).

Statistical analysis. Data from RT-PCR quantitative analysis are expressed as n-fold induction over the internal control in each experiment. All data are expressed as means ± SE. Statistical analysis was performed using ANOVA and Bonferroni's test. Statistical significance was considered for P values <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Murine HSCs express bacterial cell wall product receptors. We first attempted to determine whether murine HSCs express receptors for bacterial cell wall products, such as LPS, LTA, and NAM. Total RNA isolated from resting, unstimulated HSCs purified from Balb/c mice was subjected to RT-PCR. As shown in Fig. 1A, CD14, TLR4, PGRP-L, and PGRP-S mRNAs were clearly detectable in murine HSCs, whereas TLR2 and MD2 mRNA levels were barely visible. The presence of CD14 and TLR4 proteins was confirmed on HSC membranes by immunocytochemistry (Fig. 1B).



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  		Fig. 1. Murine hepatic stellate cells (mHSCs) express bacterial endotoxin receptors. A: total RNA was isolated from HSCs, and RT-PCR was performed using specific primers. Amplification products were separated through an agarose gel stained with ethidium bromide and visualized on a ultraviolet transilluminator. B: HSCs seeded on glass coverslips were fixed and incubated with anti-CD14 (I) or anti-TLR4 (II) polyclonal antibody. The presence of specific immunocomplexes was detected by confocal microscopy. Control cells were incubated with an irrelevant mouse antibody (III). PGRP-S, short peptidoglycan recognition proteins; PGRP-L, long peptidoglycan recognition proteins; TLR, Toll-like receptor; MW, molecular weight.

 
LPS, LTA, and NAM upregulate proinflammatory mRNA transcripts in HSCs. To evaluate the effect of bacterial cell wall products on HSC phenotype, cells purified from Balb/c mice were exposed for 24 h to LPS, LTA, or NAM at concentrations ranging from 10 to 0.01 µg/ml. Total RNA was reverse transcribed, and cDNA was analyzed by real-time quantitative PCR to determine TGF-{beta}1, IL-6, MCP-1, PDGF-BB, fibronectin, and collagen type I specific mRNA transcript levels.

Figure 2 shows that HSCs exposed to bacterial cell wall products for 24 h developed a strong inflammatory phenotype, as shown by TGF-{beta}1, IL-6, and MCP-1 mRNA upregulation. Ten micrograms per milliliter of LPS significantly increased steady-state IL-6 and MCP-1 mRNA levels, whereas TGF-{beta}1 mRNA transcripts only increased 1.4-fold (P = not significant). Indeed, 10 µg/ml of LTA induced a significant upregulation of TGF-{beta}1, IL-6, and MCP-1 mRNA levels, whereas NAM only upregulated MCP-1 and TGF-{beta}1 mRNAs. The most sensitive target of bacterial cell wall products in HSCs was MCP-1 because LPS and LTA were still active at 0.01 and 0.1 µg/ml, respectively (Fig. 2C).



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  		Fig. 2. Bacterial cell wall products enhance proinflammatory cytokine mRNA level. HSCs were incubated in medium alone or supplemented with either LPS, lipoteichoic acid (LTA), or N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (NAM; 10–0.01 µg/ml) for 24 h. Total RNA was isolated, and quantitative RT-PCR analysis was performed to quantify transforming growth factor (TGF)-{beta}1 (A), IL-6 (B), and monocyte chemoattractant protein (MCP)-1 (C) mRNA transcript levels. Data are given as the ratio of stimulated HSCs to control cells. Each experiment was performed 2–3 times with triplicate determinations for each condition (n = 6–9); samples were then assayed in duplicate for each quantitative RT-PCR. °P < 0.01 and *P < 0.05 vs. control.

 
None of the stimuli used, namely, LPS, LTA, and NAM, had any significant effect on PDGF-BB mRNA steady-state transcript level in murine HSCs (data not shown). So it was not surprising that we detected no significant increase in HSC proliferation in response to bacterial cell wall products (data not shown). Furthermore, 24-h exposure to 10 µg/ml of LPS, LTA, and NAM induced no profibrotic phenotype in HSCs because fibronectin and collagen type I steady-state mRNA levels were substantially unaffected (0.8 ± 0.19-, 1.22 ± 0.17-, and 1.49 ± 0.4-fold increase over control, respectively, for fibronectin; 0.6 ± 0.2-, 1.43 ± 0.6-, and 1.38 ± 0.3-fold increase over control, respectively, for collagen type I).

LPS, LTA and NAM stimulate TGF-{beta}1, IL-6 and MCP-1 release from HSCs. As shown in Fig. 3B, HSCs exposed to LPS and LTA, 10 and 1 µg/ml, secreted considerable amounts of IL-6 (with a 2.8- and 2.5-fold increase over control, respectively, for LPS and a 2.8- and 3.2-fold increase over control, respectively, for LTA). In accordance with the data obtained by quantitative RT-PCR analysis, NAM did not significantly alter IL-6 secretion. In addition, LPS and LTA significantly in-creased MCP-1 release from HSCs at concentrations ranging from 10 to 0.1 µg/ml, whereas NAM was effective only at the highest concentration tested (10 µg/ml, Fig. 3C).



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  		Fig. 3. Bacterial cell wall products enhance TGF-{beta}1, IL-6, and MCP-1 release. HSCs were incubated with medium alone or supplemented with either LPS, LTA, or NAM (10–0.01 µg/ml). Conditioned medium was collected after 24 h, and total TGF-{beta}1 (A), IL-6 (B), and MCP-1 (C) were measured by ELISA. Each experiment was performed 2–3 times with triplicate determinations for each condition (n = 6–9); samples were then assayed in duplicate. °P < 0.01 and *P < 0.05 vs. control (contr).

 
HSCs exposed to LPS, LTA, or NAM for 24 h increased TGF-{beta}1 release (Fig. 3A), although a statistically significant increase in total TGF-{beta}1 protein levels was detectable only for LTA and NAM.

Bacterial cell wall products induce ERK phosphorylation in HSCs. MAPKs are key molecules for converting extracellular stimuli to intracellular signals, such as the activation of transcriptional factors, which control the expression of many genes involved in inflammatory reactions. We therefore tested whether exposing HSCs to LPS, LTA, and NAM could trigger the phosphorylation of ERK1, a kinase involved in the trans-mission of endotoxin signaling (37). As shown in Fig. 4, different bacterial cell wall products, namely, LPS, LTA, and NAM, triggered ERK1 phosphorylation within 30 min of exposure. LPS was a more active stimulus than LTA or NAM.



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  		Fig. 4. Effect of bacterial cell wall products on ERK phosphorylation. HSCs were incubated with medium containing LPS (lane 2), LTA (lane 3), or NAM (lane 4; 10 µg/ml) for 30 min. Cells incubated with medium alone were used for control purposes (lane 1). Cell lysates were prepared and immunoprecipitated with anti-ERK polyclonal antibody and then underwent immunoblotting analysis with anti-phosphotyrosine (top) or anti-ERK (bottom) antibodies. Data are representative of 3 separate experiments.

 
Defective activation of TLR4 reduces the proinflammatory phenotype in HSCs. To examine whether the LPS-mediated proinflammatory effects in HSCs were receptor-mediated, we purified HSCs from C3H/HeJ mice (known to carry a mutation in Tlr4) (35). As shown in Fig. 5A, LPS- and LTA-induced ERK1 phosphorylation was drastically reduced in Tlr4–/– HSCs, whereas NAM-mediated ERK1 phosphorylation was substantially unaffected (compare Figs. 4 and 5). LPS-induced IL-6 and MCP-1 mRNA upregulation in C3H/HeJ-derived HSCs was significantly inhibited compared with LPS-mediated effects in Balb/c-derived HSCs (a 2.58 ± 0.49-fold increase over control as opposed to a 4.12 ± 0.81-fold increase over control for IL-6 and 1.44 ± 0.61-fold increase over control compared with 4.3 ± 1.08-fold increase over control for MCP-1). As shown in Fig. 5, similar results were obtained also at the protein level. Indeed, LTA-mediated effects on IL-6-specific mRNA transcripts levels and protein were also significantly reduced (P < 0.05), whereas NAM-mediated effects did not differ significantly in HSCs obtained from Balb/c and C3H/HeJ mice (Fig. 5).



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  		Fig. 5. LPS-mediated HSC activation requires a functional TLR4. HSCs purified from C3H/HeJ mice were incubated with medium containing 10 µg/ml of either LPS (lane 2), LTA (lane 3), or NAM (lane 4). HSCs incubated with medium alone were used as control (lane 1). Cell lysates were prepared and immunoprecipitated with anti-ERK polyclonal antibody and then underwent immunoblotting analysis with anti-phosphotyrosine antibody (A). HSCs obtained from C3H/HeJ mice were exposed to LPS, LTA, or NAM (10 µg/ml). After 24-h incubation, total RNA underwent quantitative RT-PCR analysis to quantify IL-6 (B) and MCP-1 (C) mRNA transcript levels. Results are reported as the ratio of stimulated HSCs to control cells. Conditioned medium was collected after 24 h and IL-6 (C) and MCP-1 (E) were measured by ELISA. Each experiment was performed twice with duplicate determination (n = 4 each condition). *P < 0.05 vs. Balb/c mice.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mammal's first unspecific defense against bacteria, viruses, or fungi relies on a large armamentarium of pattern recognition receptors (PRRs) to conserved microbial structures and expressed by a variety of cells located in key anatomical districts (19). Typically, immune cells such as monocytes, macrophages, immature dendritic cells, and other myeloid-lineage cells express regulable levels of PRRs to orchestrate immediate, unspecific inflammatory responses against bacterial derived products. Activation of immune cells eventually leads to severe tissue damage through uncontrolled cytokine release (18). Indeed, TLR2 upregulation in CD14+ PBMCs of cirrhotic patients is associated with increased serum TNF-{alpha} levels, a cytokine that, in turn, may promote liver damage (38). However, functional PRRs have recently been identified also in nonimmune cells including vascular endothelial cells, adipocytes, cardiac myocytes, and intestinal epithelial cells (9, 11). Here, we report also that murine HSCs express functional cell membrane PRRs and respond to bacterial cell wall products, developing a proinflammatory phenotype.

HSCs activation is generally considered an event secondary to liver injury. Hepatocytes and Kupffer cells damaged or activated by toxic substances, bacterial, and viral infections release soluble factors that activate HSCs, leading to liver fibrosis (15, 47). Mediators released by activated HSCs may induce leukocyte recruitment directly, however, thereby enhancing inflammation-mediated tissue damage (26, 27). Indeed, activated HSCs isolated from human cirrhotic livers express CD14 and TLR4 and secrete chemoattractant factors following LPS exposure (30). In this study, we further characterized the PRRs expressed by HSCs. We described that HSCs isolated from healthy mice express not only LPS-complex receptor, including CD14, TLR4, and MD2, but also TLR2 and PGRP-S and PGRP-L, to bind and recognize both Gram-negative- and Gram-positive-derived components (20, 24).

The expression of receptors for bacterial products is greatly influenced in vitro and in vivo by prior exposure to the ligands as well as by the cell activation state. Indeed, mCD14 is upregulated in monocytic cells after exposure to LPS (23), whereas TLR4 and TLR2 are overexpressed in circulating mononuclear cells of cirrhotic and intensive care unit patients, respectively (4, 38). CD14 expression in Kupffer cells is also enhanced after ethanol administration, a condition known to increase circulating LPS (21). The study of physiological responses in certain cell populations represents a challenging task, however, because both stabilized cell lines and primary cultures may have disadvantages. Specifically, HSCs are a nonparenchymal cell population that, after isolation and in vitro culture, spontaneously undergoes a differentiation process characterized by the acquisition of a myofibroblastlike phenotype and overexpression of membrane proteins, such as PDGF-BB and TGF-{beta}1 receptors (33). Similarly, PRRs expression in HSCs may be secondary to the activation process ensuing from cell culture as well as from proinflammatory cytokines and growth factors in vivo. Nevertheless, primed HSCs respond to bacterial endotoxins and can contribute to enhancing liver damage in a variety of conditions, such as nonalcoholic steatohepatitis, alcoholic hepatitis, and liver cirrhosis.

Although it is generally agreed that Kupffer cells are the main source of inflammatory cytokines and chemokines in the liver after systemic LPS administration, previous studies (40, 41) reported that HSCs exposed to LPS or inflammatory cytokines in vitro release IL-8, MCP-1, and MIP-2. We report here that pure murine HSCs cultured in the presence of LPS and LTA develop a strong proinflammatory phenotype, upregulating IL-6 and MCP-1 mRNA transcript level and peptide release. HSCs develop a proinflammatory phenotype only following exposure to at least 100 ng/ml of bacterial cell wall products, a dose higher than assayed in portal venous blood of normal Balb/c mice (0.51 ± 0.09 ng/ml) as well as in cirrhotic patients (25). Furthermore, HSCs do not develop a profibrogenetic phenotype within 24 h exposure to bacterial endotoxins, because fibronectin and collagen type I transcripts are not affected. We might speculate that this high-threshold proinflammatory response and lack of profibrogenetic activity in HSCs exposed to bacterial endotoxins may be a sort of protective mechanism to prevent any occasional exposure to microbial products (due to physiological portal blood endotoxemia) from triggering liver damage and fibrosis.

The binding of bacterial endotoxins to specific membrane receptors in monocytes, lymphocytes, and epithelial cells triggers stereotyped responses characterized by the activation of complex signal cascade pathways leading to cytokine release (5, 17). As shown in this study, HSCs also failed to develop a unique response profile to different cell wall products, with regard to protein tyrosine phosphorylation pattern (data not shown) and cytokine release (Fig. 3) as well as free radical productions (data not shown). Futhermore, LPS, LTA, and NAM induced a comparable ERK1 phosphorylation in HSCs (Fig. 4), part of a well-known signal transduction pathway leading to the activation of transcription factors, e.g., NF-kB, and the rapid induction of inflammatory cytokines (8). According to previous studies, we observed quantitative differences among different bacterial endotoxins with regard to the intensity of the immunostimulatory activity triggered (Figs. 2 and 3) (3, 29). As expected, LPS-mediated effects in HSCs are receptor mediated (30). Indeed, LPS-induced ERK1 phosphorylation, IL-6, and MCP-1 upregulation was almost completely abolished in HSCs isolated from C3H/HeJ mice carrying a missense mutation in Tlr4 gene (Fig. 5) (35). However, in Tlr4–/–-derived HSCs, we observed a small residual response to LPS as well as a blunted responses to LTA. Indeed, PRRs are quite promiscuous because each TLR may recognize multiple bacteria-derived products, although with different specificity (28). Moreover, several recent reports highlighted the existence of additional intracellular receptors for bacterial-derived products, such as NOD2 or NALPs, that may further contribute to HSC activation (43). In addition, commercial preparations of LTA or peptidoglycan, sold as "pure," are in fact contaminated by bacterial DNA and LPS (14, 29). Therefore it is possible that the responses observed in Tlr4–/–-derived HSCs are the effect of contaminants or cross-activation of different PRRs. Because our aim, however, was mainly to demonstrate that HSCs express functional receptors for bacterial endotoxins, we chose to use commercially available material rather than highly purified bacterial cell products.

Bacterial endotoxin clearance by the portal blood is one of the liver's most important functions (10). In the hepatic sinusoids, bacterial endotoxins absorbed through the gastrointestinal wall may reach parenchymal and nonparenchymal hepatic cells. It is generally assumed today that Kupffer cells respond to bacterial cell wall products by means of specific PRRs, but our data support the view that HSCs also respond to bacterial endotoxins, and these bacterial products induce a strong proinflammatory phenotype, triggering the release of soluble factors such as IL-6 and MCP-1 and thus contributing to tissue injury. In this view, HSCs should no longer be considered as the final target of proinflammatory mediators arising from other activated hepatic cell populations (47) but rather as part of the inflammatory network activated in the liver secondary to portal blood endotoxemia.

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   ACKNOWLEDGMENTS
 
This study was partially supported by a grant from the University of Padua.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Castagliuolo, Univ. of Padua, School of Pharmacy, Via Gabelli, 63, 35121 Padova, Italy (e-mail: ignazio.castagliuolo{at}unipd.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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