Abstract
Previous studies from our laboratory have shown that osteopontin (OPN) mediates neutrophil infiltration into the liver in a rodent model of alcoholic steatohepatitis (ASH). The objective of this study was to investigate the temporal and spatial pattern of hepatic OPN mRNA and protein expression during the progression of alcoholic liver disease. OPN mRNA and protein expression were evaluated using real time PCR, in situ hybridization, Western blot and immunohistochemistry respectively. ASH was induced in male Sprague–Dawley rats by feeding EtOH-containing Lieber-DeCarli diet for 6 weeks, followed by a single injection of lipopolysaccharide (LPS, 10 mg/kg, ip). Rats were sacrificed 2-, 12- and 24-hour post LPS injection. A progressive induction of OPN mRNA was observed that preceded hepatic neutrophil infiltration and the increase in OPN mRNA correlated with increases in OPN protein expression. OPN mRNA was localized primarily to the biliary epithelium. The data indicates that OPN is transcribed and translated within the biliary epithelium. These findings suggest a potential role of OPN as an early biomarker in predicting inflammatory liver diseases such as ASH.
Introduction
Osteopontin (OPN) also known as secreted phosphoprotein 1 (SPP1) is involved in cell-to-cell, cell-to-matrix communication and in various pathophysiological conditions like cell binding, spreading, migration and tumor metastasis (Denhardt et al., 2001; Rittling and Denhardt, 1999). OPN is also known to play an important role in a variety of inflammatory diseases like glomerular nephritis (Denhardt et al., 2001; Giachelli and Steitz, 2000; O’Regan and Berman, 2000), inflammation during CCl4 induced hepatotoxicity (Kawashima et al., 1999), puromycin-induced toxicity (Denhardt et al., 2001) and in non-alcoholic steato-hepatitis (Sahai et al., 2004). The role of OPN in inflammation has been confirmed in in vivo studies, where rats treated with neutralizing OPN antibody following induction of experimental crescent glomerulonephritis had substantially decreased accumulation of renal interstitial macrophages as compared to the controls (Yu et al., 1998). OPN neutralizing antibody has also been shown to inhibit macrophage infiltration in response to the bacterial chemotactic peptide, formyl–methionine–leucine–phenylalanine (Giachelli et al., 1998). Furthermore, acute macrophage influx in obstructed kidneys was 3-to 5-fold lower in osteopontin-null mice as compared to the wild-type mice (Ophascharoensuk et al., 1999).
The precise mechanism by which OPN recruits inflammatory cells is currently unknown, however, evidence from the literature points to the role of OPN binding sites. OPN has been reported to bind to α5β3 integrin via RGD domain and mediate its cellular effects (Smith and Giachelli, 1998; Bayless and Davis, 2001; Denhardt et al., 2001). The α5β1, α5β5, and α8β1 integrins also have affinities for the RGD motif of OPN that are similar to that of α5β3 (Hu et al., 1995, Denda et al., 1998). OPN also has binding affinity for the leukocyte integrins αmβ2 and αxβ2 (Bayless and Davis, 2001, Johnson et al., 2003). It has been shown to be a chemoattractant for macrophages and neutrophils both in vitro and in vivo (Bayless et al., 1998, Apte et al., 2005). Recent studies from our laboratory have shown that OPN acts as a chemokine in attracting neutrophils to the liver, making it more susceptible to injury in alcoholic steatohepatitis (ASH) (Apte et al., 2005; Banerjee et al., 2006). We have also reported that neutralizing antibodies directed against OPN significantly decrease neutrophil infiltration into the hepatic parenchyma in our rodent model of ASH (unpublished data).
The progression of Alcoholic liver disease (ALD) is sequential, ultimately resulting in hepatic fibrosis/cirrhosis following chronic ethanol ingestion in human and animal models (Lieber and DeCarli, 1982; Tsukomoto et al., 1985, Badger et al., 1993; Ramaiah et al., 2004). One of the intermediary stages of ALD is alcoholic steatohepatitis (Ramaiah et al., 2004), which is reported to be rate-limiting step in the progression of ALD to fibrosis/cirrhosis (Diehl, 2002; Nanji, 2002). However, the occurrence of ASH is variable and does not occur in all alcoholics. Several biomarkers such as hyaluronic acid, cytokeratin 1, growth factor and procollagen have been used to detect advanced stages of ALD such as fibrosis/cirrhosis (Nojgaard et al., 2003; Ren et al., 2003). At present there are no effective biomarkers that can detect ASH. Based on the expression of OPN protein in the liver before the occurrence of ASH, we hypothesize that OPN has a prognostic significance during the course of ALD. Thus, the objective of this study was to investigate the localization of OPN mRNA and OPN protein expression in a temporal fashion in a rodent model of ASH.
Materials and Methods
Rodent Model
The ASH model was based on previous studies (Banerjee et al., 2006) from our laboratory. Male Sprague–Dawley rats (SD) about 220–250 g in weight were purchased from Harlan Sprague–Dawley, Houston, TX, USA, and were housed individually in cages in a temperature-controlled animal facility with a 12-hour light-dark cycle. Rats were utilized after a 1-week equilibration period. Age-and weight-matched male SD rats were divided into experimental and control (n = 12 each) groups. The experimental rats were fed with EtOH-containing (EtOH = 35.5% of total calories, Apte et al., 2005) Lieber-DeCarli liquid diet for a period of 6 weeks. The control rats were pair fed with isocaloric maltose-dextrin diet. After 6 weeks of feeding, the control and the experimental rats were divided into 2 groups each: Control (n = 3), Control+LPS (n = 9), EtOH (n = 3) and EtOH+LPS (n = 9) groups. The rats from the Control+LPS and EtOH+LPS groups were injected with a single dose of LPS (E. coli 0111:B4, 10 mg/kg, ip in saline, Sigma Diagnostics, St. Louis, MO, USA). The vehicle controls received an equal volume of saline. The rats were then sacrificed at 2, 12 and 24 hours after LPS injection by CO2 asphyxiation. All rats were weighed at the beginning of the study and weekly thereafter. All animals were provided humane care in compliance with the institutional guidelines (ULACC; University Laboratory Animal Care Committee) of Texas A&M University.
Sample Collection and Processing
Blood was collected in heparinized tubes, from the dorsal aorta. Twenty micro liters of this blood was submitted for estimation of blood alcohol content (Department of Human Anatomy and Medical Neurobiology at the Texas A&M Health Sciences Center, College Station, TX). Liver transaminase activities were estimated from a fraction of heparinized plasma (about 0.5 ml), and the remaining plasma was snap-frozen in liquid N2 and stored at −80°C. Livers were harvested, weighed and divided into 2 parts. Slices of the left and median lobes were fixed in 10% neutral buffered formalin, while remaining liver tissue was snap-frozen in liquid N2 and stored at −70°C for further analysis.
Evaluation of Liver Injury
Liver injury was estimated by plasma transferase activities (alanine aminotransferase; ALT and aspartate aminotransferase; AST) and confirmed by histopathology of H&E-stained liver sections as described previously (Apte et al., 2005).
Assessment of Steatohepatitis
Histochemical detection of neutrophils was performed on paraffin-embedded liver sections. The H&E staining was employed to identify the neutrophils, based on the segmented morphology of the nucleus followed by quantification with Naphthol AS-D Chloroacetate Esterase Staining (Sigma Diagnostics, St. Louis, MO, USA) as described previously (Banerjee et al., 2006). Briefly, 4μm thick formalin-fixed paraffin-embedded liver sections were stained in a mixture of Sodium nitrite, Fast Red Violet LB Base solution, prewarmed deionized water, Trizma buffer and Naphthol AS-D Chloroacetate solution. The sections were then incubated in a 37°C water bath for 30 minutes in dark and then counterstained with Gill’s hematoxylin. The red colored cytoplasmic staining was specific for neutrophils. To quantify the degree of neutrophilic inflammation, (inflammation score), the number of neutrophils per 5 high power fields (40X) was counted. The neutrophilic foci (defined as an aggregate of ≥4 neutrophils) were quantitated per 5-40X fields.
OPN Protein Expression
Western Blot Analysis
Liver cell lysates from control and ASH groups were prepared in lysis buffer (1% Triton-x-100, 50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 2 mM Na vanadate, 0.2 mM PMSF, 1 mM HEPES, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) and protein concentration was estimated using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. Briefly, 100 μg of cell lysate was resolved by electrophoresis on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel (100 v, 1.5 hours) in a running gel buffer containing 25 mM Tris, pH 8.3, 162 mM glycine, and 0.1% SDS. The samples were transferred to nylon membrane for 3 hours at 500 mA. The membranes were incubated overnight in a mixture of T-TBS with 0.1% tween and 2% milk and OPN antibody (Rabbit polyclonal to OPN, 1:1000 dilution, Abcam Inc. Cambridge, MA). Subsequently the membrane was incubated in goat anti-rabbit secondary antibody for 1 hour at room temperature. The OPN antibody recognizes both the native (uncleaved) form of OPN (~66 KD), and also the cleaved form of OPN (32 KD; Rittling and Feng, 1998). Visualization was carried out with the enhanced chemiluminescence kit (Pierce, Rockfor, IL). GAPDH was used as an internal control to ensure equal loading of proteins per well.
Immunohistochemistry
Hepatic OPN expression was studied by immunohistochemical analysis conducted on 4μm thick formalin-fixed paraffin-embedded liver sections. Deparaffinized 4 μm thick paraffin-embedded unstained liver sections were treated with 3% solution of hydrogen peroxide, to quench the endogenous peroxidase activity. Antigen retrieval was achieved by citric acid treatment (samples were heated in a microwave in 10 mM citric acid and sodium citrate buffer; pH 6), followed by incubating the sections with 10% horse serum in PBS to block nonspecific binding sites. A mouse anti-OPN antibody (American Research products, MA, dilution 1:100) was employed as a primary antibody. Sections were then treated with a goat anti-mouse secondary antibody followed by streptavidin (Vectastain Elite ABC Kit, Vector Laboratories, Burligame, CA). The color was developed by exposing the peroxidase to diaminobenzidine reagent (Vector Laboratories, Burligame, CA), which forms a brown reaction product. The sections were then counterstained with Gill’s hematoxylin. OPN expression was identified by the brown colored cytoplasmic staining.
OPN mRNA Localization and Expression
Total RNA Extraction and Real-Time RT-PCR Analysis of OPN mRNA
Total RNA was extracted from the frozen liver tissue after lysis and homogenization using the RNeasy midikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RNA purity was determined by spectrophotometry (260/280 > 1.7). Real-Time-PCR was carried out using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and Titanium One-Step RT-PCR kit (BD Biosciences Clontech, Palo Alto, CA). The software Primer Express (Applied Biosystems, Foster City, CA) was used to design the primers (OPN Forward: TCA CCT CCC GCA TGA AGA G; Reverse: TCA GAC GCT GGG CAA CTG; β-actin Forward: CCG TGA AAA GAT GAC CCA GAT C; Reverse: CAC AGC CTG GAT GGC TAC GT). The primers were commercially obtained from Integrated DNA Technologies, Inc. (Coralville, IA). One step Real-time PCR was conducted in a Gene Amp 5700 Sequence Detection system (Applied Biosystems) using a total volume of 25 μl containing 2 μg of the RNA, 900 nM forward and reverse primers (each), 0.5 μl of RNase inhibitor and 0.125 μl of Reverse Transcriptase. The optimal assay conditions were: initial activation of AmpliTaq Gold at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. To control for DNA contamination of the RNA extracts, a no-RT reaction was run for each extract. Expression of the OPN gene relative to the housekeeping gene (beta-actin) was determined by ΔCT = CT(OPN) − CT(Beta-actin), and the Fold Expression was determined to be (2)ΔCT, where the threshold cycle (C T ) is defined as the cycle at which the fluorescence was significantly higher than the average standard deviation of the earlier cycles and the sequence detection application began to detect the increase in signal associated with an exponential growth of the PCR product (Khare et al., 2004).
Insitu Hybridization Analysis
Osteopontin mRNA expression in liver sections were localized by in situ hybridization as previously described by Johnson et al., (1999). Deparaffinized, rehydrated and deproteinated liver cross-sections (5 μm) were hybridized with [35S]-radiolabelled antisense or sense OPN cRNA probes. The sense probe has been used as a negative control to define non-specific hybridization. Following washes and RNAaseA digestion, the slides were dipped in Kodak NTB-2 liquid photographic emulsion (Kodak, Rochester, NY), stored at 4°C for 5 days, developed in Kodak D-19 developer, counter-stained with Harris modified hematoxylin (Fisher Scientific, Farilawn, NJ), dehydrated, and protected with cover slips.
Statistics
Group comparisons were performed using Independent T-test. A 1-way analysis of variance was used to determine statistical significance that might exist between more than 2 distributions or sample groups. Statistical analyses were made using SPSS 10.0 software (SPSS Inc., Chicago, IL). Statistical significance was set at p ≤ 0.05.
Results
Alcoholic Steatohepatitis Model
Rats administered ethanol for 6 weeks followed by a single dose of LPS (10 mg/kg, ip) resulted in significant fat accumulation along with neutrophil infiltration and multifocal coagulative oncotic necrosis. No significant difference was noted in the body weight of the rats in the control and ASH groups, indicating no nutritional differences between the groups. Blood alcohol concentration was also similar between the EtOH and EtOH+LPS treated groups (data not shown).
Liver Injury
The rats in the ASH group had significantly higher ALT and AST values as compared to the controls indicating extensive hepatocellular injury. Plasma ALT and AST values in the ASH group increased significantly over 2 (1.3-fold), 12 (3.3-fold), and 24 hours (5-fold) post LPS injection (Figures 1A and B). These data were corroborated with H&E stained liver sections, which showed increase in neutrophilic infiltration, neutrophilic foci and necrosis over 12 and 24 hr post LPS injection (Figures 2D, 2F). Mild neutrophilic infiltration was noted in the control+LPS treated group at 12 and 24 hours post-LPS injection (Figure 2C, E).
Hepatic Neutrophilic Infiltration as a Marker for Inflammation
Neutrophil infiltration was confirmed by immunohistochemical chloroacetate esterase staining (histochemical staining not shown). Rats treated with LPS alone experienced mild neutrophilic infiltration. Significantly higher neutrophilic infiltration was observed in the rats administered EtOH+LPS. No neutrophilic foci were seen in the rats in the ASH group at 2 hr following LPS injection. However, an increase in neutrophilic foci was noted in the rats of the ASH group at 12 and 24 hours post-LPS injection (Figure 3). The greater neutrophilic infiltration correlated with higher liver injury as evidenced by transferase elevations and histopathology (Figures 1 and 2).
OPN Protein Expression in ASH
Western Blotting
Rats in the EtOH+LPS group at 12 hours showed ~1.5-fold higher expression of OPN protein as compared to the control+LPS at 12 hours post LPS injection. (Figure 4A, B). Expression of OPN increased significantly in a temporal fashion at 2, 12 and 24 hr post LPS injection in the ASH group (Figure 5A). Densitometric analysis of OPN protein revealed a 2- and 3.5-fold higher expression of OPN protein at 12 and 24 hours post-LPS injection in the ASH group as compared to the expression levels 2 hours post-EtOH+LPS (Figure 5B).
Immunohistochemistry
Induction of OPN protein in the ASH group was further confirmed by immunohistochemistry (Figure 6). The level of OPN protein expression increased significantly in a temporal fashion with maximum OPN expression at 24 hours post-LPS injection. Induction of OPN was localized predominantly in the biliary epithelium (Figures 6D–F). Few mononuclear cells also stained positive for OPN (data not shown). The rats in the control group showed minimal stain for OPN (Figures 6A, 6C, 6E).
OPN mRNA Expression in ASH
Real-time RT-PCR results showed that EtOH+LPS treated groups had more than 35-fold higher expression of OPN mRNA as compared to the control+LPS groups. When the OPN mRNA expression was evaluated over a time course, a progressive increase of OPN mRNA was noted which was significantly higher compared to the control+LPS at 24 hours post-LPS injection (Figure 7).
Localization of OPN mRNA by In Situ Hybridization
In situ hybridization was carried out to determine the precise hepatic source of the OPN mRNA expression during the course of ASH. OPN mRNA was localized predominantly in the biliary epithelium. No or minimal signal of OPN mRNA was detected in the control+LPS groups (Figures 8A and 8B). However, in the ASH group, OPN mRNA localization was evident as early as 2 hours post-LPS injection (Figures 9A and 9B). A marked increase in the intensity of the signal was noted at 12- and 24-hour time points post-LPS injection in the ASH group (Figures 9C, 9D, 9E, 9F). The higher expression of OPN mRNA correlated with the real-time RT-PCR data. Application of a RNA sense probe for OPN showed no signal, thus confirming the selectivity of the anti-sense OPN probe (Figures 8E and 8F).
Discussion
In our rat model of ASH, a temporal increase in the expression of both OPN protein and mRNA correlated with higher liver injury and neutrophil infiltration. In addition, OPN mRNA expression preceded the hepatic neutrophil infiltration and ensuing liver injury. These findings are significant in the context of the progression of alcoholic liver disease. In alcoholics, ASH is a rate-limiting step during the progression of pathology, finally leading to hepatic cirrhosis (Diehl, 2002; French, 2002; Nanji, 2002). Currently, there are no valid biomarkers that can detect early phase alcoholic liver disease such as ASH. At present, ASH is diagnosed mostly by clinical signs, serum transferase levels and by histopathology (Marsano et al., 2003). It is a well-known fact that serum transferases lack specificity and moreover, the values vary depending on the gender and ethnicity of patients (Manolio et al., 1992; Carter-Pokras et al., 1993). Although several biomarkers of alcohol exposure have been used in clinics, their accuracy, sensitivity and specificity needs to be improved. In addition, the majority of tests available are related either to alcohol consumption and chronic ALD such as fibrosis and cirrhosis. Thus, there is a need for more precise and importantly early biomarkers that can predict ASH.
Since OPN mRNA expression precedes neutrophil infiltration and histopathologic evidence of oncotic necrosis during ASH, it can be argued that the study is correlative and lacks cause and effect evidence. A recent study from our laboratory has clearly shown that administration of neutralizing antibody against OPN abolishes hepatic neutrophilic infiltration and prevents ethanol-induced liver injury (Banerjee et al., 2006).
Measurement of OPN protein in the liver to assess neutrophil infiltration has potential limitations. In the present study we noted a baseline expression of OPN in the control+LPS group, yet no neutrophil infiltration occurred. This discrepancy can be attributed to the lack of expression of cleaved OPN (cOPN) (Apte et al., 2005), or the amount of OPN induction. OPN has an RGD sequence that is flanked by a thrombin cleavage site. cOPN with its exposed RGD sequence, has chemotactic potential and is known to attract more neutrophils than the native form of OPN (Apte et al., 2005; Giachelli et al., 1998). Previous studies from our laboratory have shown higher expression of cOPN in the ASH group as compared to the control+LPS group (Apte et al., 2005; Banerjee et al., 2006). Thus measurement of cOPN in addition to uncleaved OPN may be of higher diagnostic value in predicting inflammatory liver disease such as ASH. In addition to cOPN, the attraction of neutrophils to the liver may also depend on the amount of OPN induction, suggesting the existence of “threshold” for neutrophil infiltration by OPN.
Based on the possible predictive value of hepatic OPN in ASH, a potential question from a diagnostic point-of-view is the level of OPN in easily accessible biological fluids such as urine or plasma. Though, we have not measured serum OPN in a temporal fashion in the present study, we have previously reported elevated serum OPN during the course of early phase ALD such as alcoholic steatosis and steatohepatitis (Apte et al., 2005). A potential limitation of serum OPN as a valid biomarker is the contribution of OPN by extrahepatic sources. It is known that OPN is also secreted within the kidney, colon, uterus and bone (Johnson et al., 1999; Ishijima et al., 2001; Masuda et al., 2003; Vernon et al., 2005). Clearly additional studies are needed to correlate serum OPN with hepatic OPN expression.
From a physiological point-of-view, it is important to understand the source of OPN mRNA expression since there is no regiospecificity of neutrophil infiltration within the liver in the ASH model. Interestingly, the OPN mRNA was induced primarily in the biliary epithelium. Although the precise mechanism for OPN-induced hepatic parenchymal neutrophil infiltration is not known at present, it can be hypothesized that OPN secreted from the biliary epithelium diffuses into hepatic interstitial matrix acting as a chemoattractant for neutrophils. Another mechanism for regional recruitment of neutrophils may be related to some unidentified factors secreted by osteopontin-induced peribiliary infiltrates.
Finally, the reason behind induction of OPN during the course of ALD is currently unknown and is worthy of speculation. The likely candidates for OPN induction are endotoxemia and associated inflammatory cytokines (Sahai et al., 2004; Zhang et al., 2004). Although there was base-line expression of OPN in the group treated with LPS (endotoxin) alone in the current study, the OPN expression in the EtOH+LPS group is significantly much higher suggesting that endotoxin alone is not playing a prominent role in OPN induction. Further studies aimed at investigating OPN expression with structurally and mechanistically different hepatobiliary toxicants (for example CCl4, acetaminophen, allyl alcohol and α-napthyl isothiocyanate) should provide additional insights into the role of OPN in inflammatory and toxic liver diseases.
In conclusion, a temporal increase in OPN and neutrophilic infiltration was observed in a rodent model of ASH. OPN mRNA expression preceded hepatic neutrophil infiltration, suggesting the potential role of OPN as a predictive biomarker of ASH. However, since cOPN is a more potent chemoattractant of neutrophils than the uncleaved OPN, the pattern of hepatic expression of cOPN is also worthy of investigation. Examination of the temporal relationship of changes in serum OPN levels with respect to the temporal progression of liver injury is needed before OPN is implicated as a biomarker in ALD.
Footnotes
Acknowledgments
This work was supported in part by National Institute of Health Grant P30ES0910607.