Hepatic Stellate Cells Express the Low Affinity Nerve Growth Factor Receptor p75 and Undergo Apoptosis in Response to Nerve Growth Factor Stimulation
2000; Elsevier BV; Volume: 156; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64994-2
ISSN1525-2191
AutoresNathan Trim, Sue Morgan, Martyn L. Evans, Razao Issa, David Fine, Simon C. Afford, Bridget S. Wilkins, John P. Iredale,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoWe have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were α-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC. We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were α-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC. 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Following the observations by Cattoretti et al and Wilkins and Jones that antibodies reactive with p75 identified dendritic fibroblast-like cells in bone marrow stroma,30Cattoretti G Schiro R Orazi O Soligo D Columbo MP Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro.Blood. 1993; 81: 1726-1735PubMed Google Scholar, 31Wilkins B Jones D Immunohistochemical characterization of intact stromal layers in long term cultures of human bone marrow.Br J Haematol. 1995; 90: 757-766Crossref PubMed Scopus (28) Google Scholar we determined to examine p75 expression in HSC with the aim of examining the hypothesis that HSC express p75 and that stimulation of p75 by NGF is associated with HSC apoptosis. We have also examined the recently described pancreatic stellate cells (PSC)31Wilkins B Jones D Immunohistochemical characterization of intact stromal layers in long term cultures of human bone marrow.Br J Haematol. 1995; 90: 757-766Crossref PubMed Scopus (28) Google Scholar, 32Pinzani M New kids on the block: pancreatic stellate cells enter the fibrogenesis world.Gut. 1999; 44: 451-452Crossref PubMed Scopus (8) Google Scholar, 33Bachem MG Schneider E Gross H Weidenbach H Schmid RM Menke A Siech M Beger H Grunert A Adler G Identification, culture, and characterization of pancreatic stellate cells in rats and humans.Gastroenterology. 1998; 115: 421-432Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar for expression of p75, to determine whether expression of this cell receptor is potentially a more general feature of fibrogenic cells. Hepatic stellate cells were isolated as previously described3Arthur MJP Friedman SL Roll FJ Bissell DM Lipocytes from normal rat liver release a neutral metalloproteinase that degrades basement membrane (type IV) collagen.J Clin Invest. 1989; 84: 1076-1085Crossref PubMed Scopus (161) Google Scholar and activated in primary culture on plastic in the presence of 16% fetal calf serum (FCS). Highly activated cells were obtained by passage of primary cultures of HSC and used for experiments between the first and fourth passage. Human HSC were isolated from the margin of an hepatic resection and cultured as described previously.7Iredale JP Goddard S Murphy G Benyon RC Arthur MJP Tissue inhibitor of metalloproteinase-1 and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes.Clin Sci. 1995; 89: 75-81Crossref PubMed Scopus (114) Google Scholar Human HSC were used after the fourth passage. Pancreatic stellate cells were extracted and cultured as described.32Pinzani M New kids on the block: pancreatic stellate cells enter the fibrogenesis world.Gut. 1999; 44: 451-452Crossref PubMed Scopus (8) Google Scholar, 33Bachem MG Schneider E Gross H Weidenbach H Schmid RM Menke A Siech M Beger H Grunert A Adler G Identification, culture, and characterization of pancreatic stellate cells in rats and humans.Gastroenterology. 1998; 115: 421-432Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar Cells were cultured in Dulbecco's modified Eagle's medium in the presence of 16% FCS and subjected to serial passage. PSC were used for experiments after the fourth passage. Six formalin-fixed, wax-embedded biopsies of fibrotic and cirrhotic human liver tissue were immunostained for p75. These consisted of three examples of early and three of advanced micronodular cirrhosis. In all six the pathology was alcoholic liver disease. Normal liver biopsy samples were obtained from the margins of two resection specimens (one for colonic cancer metastasis and the second for a simple vascular cyst.) and from four samples of unused normal donor liver that had been perfused with University of Wisconsin solution. In addition, three examples of fibrotic human pancreas were obtained at resection for chronic pancreatitis. These were formalin-fixed and wax-embedded for p75 immunostaining. Finally, fibrotic rat liver obtained after 4 weeks of CCl4 intoxication (as previously described16Iredale JP Benyon RC Pickering J McCullen M Northrop M Pawley S Hovell C Arthur MJP Mechanisms of spontaneous resolution of rat liver fibrosis: hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (954) Google Scholar) was also used for immunohistochemistry studies. Immunostaining of rat HSC and liver tissue and Western blot analysis of rat HSC were undertaken using a monoclonal antibody reactive with rat p75 (Boehringer Mannheim, Lewes, UK). Human liver and pancreatic biopsies were immunostained using a monoclonal antibody reactive with human p75 (Dako, Kidlington, UK). The same antibody was used for Western blot analysis of human HSC. Monoclonal anti α-smooth muscle action (α-SMA, Sigma, Poole, UK) was used to identify activated HSC in cultured HSC and tissue sections. Polyclonal rabbit anti-Fas was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Immunostaining of histological sections of liver and pancreas was undertaken using a streptavidin-biotin complex immunoperoxidase technique. Briefly, sections were deparaffinized, treated to inhibit endogenous peroxidase, and subjected to wet-heating antigen retrieval as previously described.16Iredale JP Benyon RC Pickering J McCullen M Northrop M Pawley S Hovell C Arthur MJP Mechanisms of spontaneous resolution of rat liver fibrosis: hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (954) Google Scholar Sections were then washed in Tris-buffered saline (TBS), pH 7.6, before addition of the primary antibodies at optimal dilutions as determined by prior titration for 18 to 24 hours at 4°C. For each liver or pancreas sample negative controls were performed on adjacent sections, replacing the primary antibody with nonimmune IgG, omitting the primary antibody and replacing the primary antibody with TBS. For each biopsy, sequential adjacent sections were stained for p75 and α-SMA. After incubation with the primary antibody, sections were allowed to warm to room temperature before washing in TBS (3 × 5 minutes. and incubation with the biotinylated anti-mouse antiserum (Boehringer Mannheim), diluted in TBS for 30 minutes at room temperature. Sections were then washed in TBS (3 × 5 minutes) before addition of streptavidin complexed with biotinylated horseradish peroxidase (Dako. for 30 minutes. After 30 minutes slides were washed in TBS (3 × 5 minutes) before adding 3′- 3′-diaminobenzidine (DAB, Sigma. for 8 minutes, then rinsed in TBS followed by running water. Finally, the sections were counterstained in Harris' hematoxylin, dehydrated through graded alcohols, and mounted. In a further experiment, colocalization of α-SMA and p75 was undertaken by incubating a representative cirrhotic biopsy sequentially with the antibodies reactive with α-SMA and p75, detected using immunoperoxidase, DAB, immunoalkaline phosphatase, and fast blue B, respectively. Appropriate parallel single immunostains and negative controls were undertaken concurrently. Representative sections of the cirrhotic liver biopsies were also immunostained to detect Fas expression as previously described.34Apte MV Haber PS Darby SJ Rodgers SC McCaughan GW Korsten MA Pirola RC Wilson JS Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis.Gut. 1999; 44: 534-541Crossref PubMed Scopus (505) Google Scholar Human and rat HSC and rat PSC were harvested and the extracted protein subjected to electrophoresis on a 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel as described35Afford SC Randham S Eliopoulis AG Hubscher SG Young LS Adams DH CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface Fas ligand expresion and amplifies Fas-mediated hepatocyte death during allograft rejection.J Exp Med. 1999; 189: 441-446Crossref PubMed Scopus (170) Google Scholar after normalization for protein content. After resolution, the protein samples were electrotransferred onto Polyvinylidene Fluoride using the semidry method as described.35Afford SC Randham S Eliopoulis AG Hubscher SG Young LS Adams DH CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface Fas ligand expresion and amplifies Fas-mediated hepatocyte death during allograft rejection.J Exp Med. 1999; 189: 441-446Crossref PubMed Scopus (170) Google Scholar The membrane was blocked for 1 hour in 5% nonfat dry milk in TBS. Membranes were incubated overnight at room temperature with the primary antibody or with nonimmune IgG (as negative control) in TBS. Membranes were washed twice for 5 minutes in TBS before the addition of the secondary antibody (rabbit anti-mouse IgG HRP in a final dilution of 1:1000) in TBS containing 5% nonfat dry milk for 1 hour. The membranes were then washed in TBS for 5 minutes followed by water for 5 minutes. Reactive bands were identified using enhanced chemiluminescence (ECL, Amersham, Poole, UK) and autoradiography according to the manufacturer's instructions. Parallel Western blot analysis for α-SMA was undertaken in an identical manner. Following the observation that p75 is expressed by the HSC in vivo, we determined to characterize further the HSC response to a ligand known to stimulate the receptor. The pro-apoptotic effect of p75 stimulation by NGF in a number of neural cell types has now been clearly defined. We therefore investigated the potential effect of NGF stimulation on HSC. HSC apoptosis in response to recombinant NGF was quantified by in vitro counting of HSC under fluorescent illumination in tissue culture wells after addition of acridine orange. Using this method, apoptotic cells can be identified by virtue of their enhanced fluorescence and characteristic nuclear morphology (bright green fluorescence, condensed chromatin, cytoplasmic shrinkage, nuclear and cytoplasmic blebbing, and detachment from the monolayer) and counted in situ together with cells of the attached monolayer. Quantification of HSC apoptosis in response to NGF was carried out using 80% confluent passaged cells (passage 2–4) on 24-well plates. Cells were washed twice and incubated for 1 hour in serum-free media. Thereafter cells were returned to serum-containing media together with varying concentrations of recombinant human NGF (Cambridge Bioscience, Cambridge, UK). Parallel control wells containing serum were also prepared. NGF was added to final concentrations of 0.1, 1, 10, and 100 ng/ml. Each concentration was added to 2 parallel wells and incubated for 6 or 24 hours. After this time, 0.5 μl of acridine orange (final concentration 1 μg/ml) was added per well and left for 15 minutes before viewing the wells with an inverted fluorescent microscope. Normal and apoptotic cells were counted by an observer blind to the treatment conditions in 3 high power fields per well and 2 wells per condition. Cells that had detached from the monolayer and were floating in the media supernatant were also counted by racking the objective lens up after assessing the monolayer. To confirm that condensed cells on the surface of the monolayer were apoptotic, loosely adherent and detached cells were harvested from representative HSC cultures by gentle washing followed by centrifugation. Total DNA was extracted from these cells, subjected to electrophoresis on an agarose gel containing ethidium bromide, and visualized under UV light to detect 200 bp laddering as previously described.36Gaca MDA Pickering JA Arthur MJP Benyon RC Human and rat hepatic stellate cells produce stem cell factor: a possible mechanism for mast cell recruitment in liver fibrosis.J Hepatol. 1999; 30: 850-858Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar To determine whether NGF affects the proliferation rate of HSC and to estimate the comparative cell number after incubation with NGF, a series of experiments to analyze cell proliferation (by [3H]-thymidine incorporation) and DNA content (by PicoGreen binding, Molecular Probes, Eugene, OR) was undertaken on three cell preparations in parallel. For each series of three experiments, three preparations of passaged cells were washed three times in serum-free media then incubated in serum-free media or serum-containing media with or without 100 ng/ml NGF. For the last 6 hours of incubation with NGF, 0.5 μCi of [3H]-thymidine were added to triplicate wells at a concentration of 1:1000 (0.5 μl/500 μl/well). Thymidine incorporation was then determined as previously described.37Baker AJ Mooney A Hughes J Lombardi D Johnson RJ Savill J Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis.J Clin Invest. 1994; 94: 2105-2116Crossref PubMed Scopus (405) Google Scholar For DNA analysis, the supernatant was discarded and 150μl of 1× TE (10 mmol/L Tris-HCl, 1 mmol/EDTA, pH 8.0) was added to duplicate wells. Adherent cells were removed with a cell scraper, transferred to a microcentrifuge tube, and sonicated for 15 minutes. Each sample (50 μl) was diluted with 50 μl TE and added to individual wells of a 96-well plate. Standard concentrations (0 to 2 ng/ml) of DNA were diluted from herring sperm DNA stock. PicoGreen (1:200 dilution with 1× TE to a final volume of 100 μl) was added to all samples and left to incubate in light-free conditions at room temperature for 5 minutes. Fluorescence was measured using a Cytofluor II Microwell Fluorescence reader (Perseptive Biosystems, Framingham, MA) at standard fluorescein wavelengths (excitation 485 nm, emission 530 nm). A standard curve was generated using known concentrations of herring sperm DNA. Concentrations of double-stranded DNA in the samples were subsequently calculated. Western blot analysis of total HSC protein derived from rat HSC during activation in culture on plastic using an antibody to rat p75 demonstrated that at 14 days (highly activated, α-SMA-expressing HSC. a single band of appropriate molecular weight was detected, indicating that activated HSC express p75 (Figure 1A). In contrast, quiescent (freshly isolated) HSC did not express p75; indeed, expression of this protein first became detectable in activated HSC after 7 days of culture (Figure 1A). In a further experiment, 14 day activated human HSC protein extracts were subjected to Western blot analysis using the anti-human p75 antibody. Comparable immunoreactivity was demonstrated in human HSC (Figure 1B). Protein extracts from PSC were also demonstrated to contain p75 by Western blot analysis (Figure 2).Figure 2Protein extract from passaged (fourth passage) pancreatic stellate cells was subjected to Western blot analysis for p75 as described in Materials and Methods (left panel). Parallel negative control incubated with nonimmune IgG is presented in the right panel. The results indicate that PSC express p75.View Large Image Figure ViewerDownload Hi-res image Download (PPT) p75 was detected in perisinusoidal cells in sections of normal donor livers (one child and three adults) and normal liver tissue from the margins of hepatic resections (n = 2). Representative examples are shown in Figures 3A (low power) and 3C (high power). Staining was also visible in the cells with a fibroblastic phenotype within portal tracts. Staining of these normal liver samples demonstrated perisinusoidal α-SMA positivity in an identical distribution (Figure 3B). In samples of fibrotic and cirrhotic liver (n = 6) intense staining for p75 was observed in cells with a myofibroblastic appearance within and surrounding areas of fibrosis and fibrotic bands. In addition, p75-positive cells could be observed in a perisinusoidal position extending from fibrotic bands into regenerative nodular parenchyma. No p75 expression was observed in hepatocytes (Figure 4A). Positive immuno
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