- Email: [email protected]
Infiltrating neutrophils in bile duct-ligated livers do not promote hepatic fibrosis
Hepatology Research 25 (2003) 180 /191 www.elsevier.com/locate/ihepcom
Infiltrating neutrophils in bile duct-ligated livers do not promote hepatic fibrosis Jacqueline M. Saito a,b, Michelle K. Bostick b,c, Carson B. Campe b,c, Junquan Xu b,c, Jacquelyn J. Maher b,c,* a
Department of Surgery, University of California, San Francisco, CA, USA b UCSF Liver Center, University of California, San Francisco, CA, USA c Department of Medicine, University of California, San Francisco, CA, USA Received 7 May 2002; received in revised form 26 July 2002; accepted 27 August 2002
Abstract Bile duct obstruction causes rapid infiltration of neutrophils into the liver and leads ultimately to hepatic fibrosis. In this study, we assessed whether neutrophils play an active role in the pathogenesis of hepatic fibrosis under conditions of biliary obstruction. We performed bile duct ligation (BDL) on rats, some of which were depleted of neutrophils by means of an anti-neutrophil antiserum. Rats treated with the antiserum had 48% fewer neutrophils than control rats. Despite this, they showed no difference in either bile duct proliferation or hepatic fibrogenesis after BDL compared with control rats. In a second set of experiments, we performed BDL on mice with an underlying defect in neutrophil function due to transgenic expression of interleukin-8. Mice with neutrophil dysfunction deposited less ( /22%) collagen in their livers after BDL than wild-type mice, but the difference was not statistically significant. In summary, data from two independent rodent models indicate that infiltrating neutrophils do not influence hepatic fibrogenesis following bile duct obstruction. The findings suggest that neutrophils play little if any role in the immunomodulation of liver fibrosis. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cirrhosis; Cholestasis; Biliary; Leukocyte; Inflammation
1. Introduction
Abbreviations: ALT, alanine aminotransferase; BDL, bile duct ligation; CK19, cytokeratin-19. * Corresponding author. Present address: Liver Center Laboratory, San Francisco General Hospital, Building 40, Room 4102, 1001 Potrero Avenue, San Francisco, CA 94110, USA. Tel.: /1-415-206-4805; fax: /1-415-641-0517. E-mail address: [email protected] (J.J. Maher).
Obstruction of bile flow through the extrahepatic biliary system results in rapidly progressive liver disease [1,2]. The hallmarks of liver injury following biliary obstruction are periductal inflammation, bile duct proliferation and portal fibrosis. Prominent among the inflammatory cells that invade obstructed livers are neutrophils; studies in experimental animals show that neutrophils
1386-6346/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 2 ) 0 0 2 4 7 - 4
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
infiltrate the liver within 3 h of experimental bile duct occlusion [3], and remain there for days to weeks as fibrosis progresses [3,4]. Although in many types of tissue injury, inflammatory cells colocalize with fibrosis [5 /7], in the obstructed liver the proximity between neutrophils and collagen is unusually close. This, together with the large number of neutrophils that are found in both animal and human models of extrahepatic biliary obstruction [3,4,8], suggests that neutrophils are central to the pathogenesis of biliary fibrosis. In all forms of liver fibrosis, the principal collagen-producing cells are hepatic stellate cells [9]. In normal liver stellate cells are quiescent, but in the setting of liver injury they transform into myofibroblast-like cells that are proliferative and migratory and produce abundant fibrillar collagen [9]. Stellate cell transformation can be promoted by a number of factors including oxidant stress [10,11], exposure to fibrogenic cytokines [12,13] and contact with foreign extracellular matrix proteins [14]. Because neutrophils produce oxidants and proteinases as they transmigrate into tissues [15 /17], they may contribute to stellate cell transformation in vivo. A connection between neutrophils and liver fibrosis was first suggested by Parola et al. [4]. These authors quantitated hepatic neutrophils in an experimental model of bile duct obstruction and found that the number of infiltrating cells correlated directly with the degree of liver fibrosis. A similar association between neutrophil infiltration and hepatic fibrogenesis has been reported in dimethylnitrosamine-induced liver disease [18]. Casini et al. [19] offered direct evidence in favor of neutrophil-mediated regulation of hepatic stellate cells in a report demonstrating that activated neutrophils enhance stellate cell collagen synthesis in culture. Proof that neutrophils modulate hepatic fibrogenesis in vivo, however, is lacking. The objective of this study was to determine directly in an in vivo model of liver injury whether neutrophils promote hepatic fibrogenesis. We accomplished this by subjecting rodents with altered neutrophil numbers or neutrophil responses to bile duct ligation (BDL), and comparing the progression of fibrosis in these animals with that in rodents with normal neutrophils. The
181
results indicate that neutrophils have little impact on the progression of hepatic fibrosis in response to BDL.
2. Methods 2.1. BDL in rats and administration of antineutrophil antiserum Male Sprague/Dawley rats (225 /250 g) were chosen for BDL. Under inhalation anesthesia, rats underwent laparotomy with either complete ligation of the common bile duct or sham ligation. After the abdominal incision was closed, rats were turned prone and a subcutaneous pouch was created between the scapulae. In the pouch was placed an osmotic infusion pump (Alzet 2ML1, Alza Corporation, Palo Alto, CA) containing either rabbit anti-rat neutrophil antiserum (Accurate Chemical Company, Westbury, NY) or nonimmune rabbit serum (Accurate Chemical Company). After the operations were completed, rats were returned to standard housing and permitted free access to food and water for 7 days. During this interval, the infusion pumps discharged at a constant rate of 10 ml/h (total dose of 1 ml/(kg/d)). At the end of the experiment, rats were killed for collection of blood and liver tissue. 2.2. BDL in interleukin-8 transgenic mice BDF-1 mice that express a liver-specific transgene encoding human interleukin-8 (IL-8) [20] were obtained from Amgen, Inc. (Thousand Oaks, CA). Heterozygotes are characterized by the presence of nanogram quantities of IL-8 in the circulation [20]. A colony was maintained by breeding heterozygotes with wild-type BDF-1 mice (Jackson Laboratories, Bar Harbor, ME); offspring were screened for transgene expression at 6/8 weeks of age by quantitation of IL-8 in serum (Quantikine, R&D Systems, Minneapolis, MN). Serum IL-8 levels in positive offspring ranged from 38 to 65 ng/ml. Adult IL-8 transgenic and wild-type mice (approx. 25 g) were subjected to BDL under inhalation anesthesia. Postoperatively, mice were
182
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
maintained for 14 days with free access to food and water. At the end of the experiment, mice were killed for collection of blood and liver tissue. All animals received humane care based on guidelines set by the American Veterinary Association. All experimental protocols involving live animals were approved by the Committee on Animal Research at the University of California, San Francisco. 2.3. Blood counts and plasma chemistries Blood was collected from rats at the time of killing and mixed with heparin. Leukocytes and neutrophils were quantitated in whole blood using an automated cell counter (Technicon). Plasma was separated from cellular elements by centrifugation and assayed for liver chemistries on a BAX autoanalyzer (Bayer Corporation, Tokyo, Japan). Blood was collected from mice at the time of killing but not mixed with anticoagulants. Serum was separated by centrifugation and assayed for liver chemistries in the same fashion as described for rats. 2.4. Histology and immunohistochemistry The livers of experimental animals were removed and rinsed immediately in saline solution. A portion of each liver was fixed in 10% buffered formalin and embedded in paraffin. The remainder was snap-frozen in liquid nitrogen and stored at / 80 8C. Sections of formalin-fixed, paraffin-embedded liver were stained with hematoxylin and eosin. Serial sections were stained with picrosirius red to highlight connective tissue. Frozen unfixed liver was used for immunohistochemical staining. Neutrophils were identified with specific antigranulocyte antibodies (Gr-1 or Ly6-G; Pharmingen, San Diego, CA). Details of the staining protocol have been reported previously [3]. Neutrophils were counted manually under 10 / magnification in blinded liver sections; 3/5 microscopic fields were evaluated per liver, each centered on a portal tract. Counts represent the average number of Gr-positive cells per portal field in rats from each treatment group (n /3).
Bile ducts were identified with mouse antihuman cytokeratin-19 (anti-CK19, Novocastra, Newcastle-Upon-Tyne, UK). Briefly, 8 mm sections of frozen liver were adhered to glass slides, treated with ice-cold acetone for 10 min and airdried. After rehydration and blocking in PBS/1% BSA, sections were incubated with anti-CK19 (1:500) overnight at 4 8C. They were then washed with PBS/1% BSA and incubated for 30 min at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000; Molecular Probes, Eugene, OR). After further washes, sections were covered with Vectamount† mounting medium (Vector Laboratories, Burlingame, CA), coverslipped and sealed. Sections were viewed and photographed on a Nikon Microphot-FXA epifluorescence microscope (Nikon Corporation, Tokyo, Japan). 2.5. Image analysis Bile duct area was quantitated morphometrically in livers stained with anti-CK19. Photographs were taken with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI) and analyzed using Simple PCI software (Compix, Cranberry Township, PA). Bile duct area was measured in 10 fields per liver section (10 /), each centered on a portal tract. Data were expressed as a percent of total liver area. For morphometric measurement of liver collagen, sections stained with picrosirius red were photographed at 4 / magnification. Fifteen adjacent fields were photographed to limit sampling error from the localized fibrosis that follows BDL. Collagen staining was expressed as a percent of total liver area. 2.6. Analysis of collagen gene expression Livers were homogenized in TRI reagent (Molecular Research Center, Cincinnati, OH) for extraction of total RNA. mRNA encoding procollagen I was quantitated by RNase protection [21] using S14 mRNA as an internal control. cRNA probes for procollagen I and S14 were transcribed with a-32P-CTP (/800 Ci/mmol, Amersham Corp., Arlington Heights, IL). Ali-
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
quots of liver RNA (20 mg) were combined with both probes (each at 3 /105 Cerenkov cpm) and incubated at 55 8C for 16 h. Unhybridized probes and RNA were then digested with ribonuclease T2 (GIBCO BRL, Grand Island, NY). The remaining RNA /RNA hybrids were precipitated in isopropanol. After denaturation at 100 8C, hybrids were separated through 5% polyacrylamide urea. Radioactive bands corresponding to the protected probe were visualized by autoradiography (XOMat AR-5, Eastman Kodak, Rochester, NY) and quantitated by scanning densitometry (Hoefer Scientific Instruments, San Francisco, CA). Signals for procollagen I [22] were normalized to the control RNA signal encoding S14 [23].
2.7. Quantitation of hepatic hydroxyproline Hydroxyproline was quantitated in liver homogenates by the method of Jamall et al. [24]. A portion of liver was first homogenized in distilled water at a concentration of 1 mg/ml. The homogenate was then combined with an equal volume of 12 N HCl and heated to 110 8C for 20 h. The acid hydrolysate was clarified by centrifugation; 50-ml aliquots were then added to several test tubes and air-dried. Each group of test tubes corresponding to a single liver was supplemented with standards containing known amounts of 4-hydroxy-L-proline (Sigma Chemical Company, St. Louis, MO). Hydroxyproline was measured by adding p -dimethylaminobenzaldehyde (Ehrlich’s reagent; Fisher Scientific, Hanover Park, IL) and reading absorbance at 558 nm. The amount of hydroxyproline in each sample was determined by measuring the degree to which the sample displaced the standard curve from the origin.
2.8. Statistical analysis All quantitative data are reported as mean9/ S.E.M. Differences between means were calculated using analysis of variance with post hoc Bonferroni adjustment. P -values B/0.05 were considered significant.
183
3. Results
3.1. Anti-neutrophil antiserum does not impact BDL-induced cholestasis in rats but significantly reduces circulating and tissue neutrophils Rats developed typical cholestasis in response to BDL. On day 7 after surgery, plasma bilirubin was 10 times higher in BDL rats than sham rats; alanine aminotransferase (ALT) was also significantly increased (Table 1). This was true whether or not the animals received anti-neutrophil antiserum (Table 1). BDL also caused a significant increase in circulating neutrophil counts (Fig. 1, black bars), which confirms prior observations that BDL causes neutrophilia [3]. Treatment with anti-neutrophil antiserum reduced the number of circulating neutrophils in BDL rats by 48% (5.7 / 103 cells/ml vs. 11.0 /103 cells/ml, P B/0.005; Fig. 1). In sham-operated rats, the same dose of antiserum reduced the number of circulating neutrophils by 88% (3.4 /103 cells/ml vs. 0.4 / 103 cells/ml, P B/0.05). Higher doses of anti-neutrophil antiserum were tested in an effort to enhance neutrophil depletion in BDL rats, but these proved to be toxic (data not shown). Anti-neutrophil antiserum eliminated neutrophils from the livers as well as the circulation of BDL rats. This is illustrated in Fig. 2, in which panel a shows abundant periportal neutrophils in 7-day BDL rats and panel b demonstrates many fewer neutrophils in BDL rats treated with antineutrophil antiserum. Direct counting of stained Table 1 Serum chemistries in BDL and sham rats at 7 days Group
Bilirubin (mg/ dl)
ALT (IU/ ml)
BDL/control serum BDL/anti-neutrophil antiserum Sham/control serum Sham/anti-neutrophil antiserum
5.49/0.3a 5.29/1.0a
1749/9b 2209/47b
0.79/0.4 0.29/0
749/0 619/6
Values represent mean9/S.E.M. a P B/0.01 vs. sham rats. b P B/0.05 vs. sham rats.
184
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Fig. 1. Absolute neutrophil counts in BDL and sham rats. Graph depicts circulating neutrophil counts in BDL and sham rats treated with anti-neutrophil antiserum (hatched bars) or control antiserum (solid bars) for 7 days. Bars represent mean9/ S.E.M. for n ]/4. *P B/0.05, **P B/0.005 vs. control serum; §P B/0.05, §§P B/0.005 vs. sham rats.
cells showed that the antiserum caused a 50% reduction in tissue neutrophils, from 92.69/10.3 to 46.79/14.3 per portal field. This directly paralleled the level of depletion of circulating neutrophils. Sham rats had 17.89/1.6 neutrophils per portal field, which were reduced to 3.39/0.1 by antineutrophil antiserum. 3.2. Neutrophil depletion does not affect bile duct proliferation after BDL Biliary obstruction causes cholangiocyte proliferation and expansion of intrahepatic bile ducts. To determine whether neutrophils impact this process, rat livers were stained with an antibody that recognizes the biliary cell intermediate filament protein CK19 (Fig. 3). Bile duct area was measured in BDL and sham livers by morphometry. As expected, bile duct area was significantly greater in BDL liver compared with sham liver. Neutrophil depletion had no effect on bile duct
Fig. 2. Neutrophil immunohistochemistry in 7-day BDL liver. Photomicrographs illustrate immunoperoxidase staining for neutrophils using anti-Gr (see Section 2). (a) BDL liver with control serum for 7 days; (b) BDL liver with anti-neutrophil antiserum for 7 days. Neutrophil counts in portal fields from each treatment group measured 17.89/1.6 for sham/control antibody, 3.39/0.1 for sham/anti-neutrophil antibody, 46.79/ 14.3 for BDL/control antibody and 92.79/20.3 for BDL/antineutrophil antibody. Original magnification 10 /.
expansion after BDL (2.38% vs. 2.42% bile duct area in depleted vs. control BDL rats, n /6). 3.3. Neutrophil depletion does not affect collagen gene expression in BDL liver To determine whether neutrophil depletion attenuates hepatic fibrogenesis after BDL, procollagen I mRNA was measured in the livers of BDL
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
185
increase; Fig. 4). Neutrophil depletion, however, had no effect on procollagen I gene expression. Procollagen I mRNA levels in BDL rats treated with anti-neutrophil antiserum were no different than those measured in BDL rats treated with control serum (Fig. 4, lanes 5 /9 vs. lanes 10 /15). 3.4. Neutrophil depletion does not influence hepatic collagen deposition in BDL rats Although neutrophil depletion did not alter the amount of procollagen I mRNA in rat liver after BDL, we reasoned that it might modify the pattern or extent of collagen deposition in the liver. To investigate this, BDL and sham livers were stained with picrosirius red and examined by light microscopy (Fig. 5). Fig. 5a and b illustrate the sparse collagen in the portal tracts of sham liver. Fig. 5c / f show more abundant collagen at the basal surface of bile ducts in 7-day BDL liver. By image analysis, hepatic collagen was threefold greater in BDL liver than in sham liver (collagen area 2.68% vs. 0.75%, P B/0.005). There was no difference in total collagen between BDL rats treated with control serum (Fig. 5c and d) and BDL rats treated with anti-neutrophil antiserum (Fig. 5e and f; 2.85% vs. 2.34% collagen area, n /6). Nor was there any difference in hydroxyproline measured in liver homogenates (190.89/10.1 mg hydroxyproline/g liver vs. 213.79/33.6 mg hydroxyproline/g liver in neutrophil-depleted vs. control rats, n /6). 3.5. Neutrophil dysfunction does not alter biliary fibrosis in mice
Fig. 3. Bile duct proliferation in 7-day BDL liver. Photomicrographs illustrate bile ducts in BDL livers highlighted with antiCK19. (a) Sham liver with control serum for 7 days; (b) BDL liver with control serum for 7 days; (c) BDL liver with antineutrophil antiserum for 7 days. Original magnification 10 /.
and sham rats by RNase protection. Procollagen I mRNA was markedly induced in BDL livers compared with sham livers at 7 days (5.3-fold
IL-8 transgenic mice exhibit defective neutrophil migration in response to classical chemotactic stimuli in vivo [20]. Neutrophil dysfunction in these mice and others that overexpress CXC chemokines has been attributed to chemokine receptor desensitization [20,25]. When IL-8 transgenic mice were subjected to BDL, they exhibited less periportal infiltration of neutrophils than wildtype mice (Fig. 6). Biochemical measures of cholestasis, however, were comparable between the two groups (Table 2). Survival after BDL was much better in IL-8 transgenic mice than in
186
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Fig. 4. Steady-state expression of procollagen I mRNA in 7-day BDL liver. Autoradiogram illustrates procollagen I mRNA (Collagen I) 7 days after BDL or sham operation, in the presence of anti-neutrophil antiserum (/) or control serum ( /). BDL induces hepatic procollagen gene expression 5.3-fold over sham operation. Note that procollagen mRNA in BDL liver is not influenced by administration of anti-neutrophil antiserum. Hybridization signals were generated by RNase protection using 20 mg total liver RNA (see Section 2). S14 serves as an internal control signal for ribosomal RNA.
wild-type controls. 14-day mortality in the control group was 54% (7/13), whereas mortality in the IL8 transgenic group was only 8% (1/12). Liver fibrosis was evaluated in all mice that survived 14 days of BDL. Histologically, there was no discernible difference in collagen staining with picrosirius red (Fig. 7). Hepatic hydroxyproline levels after BDL were 22% lower in IL-8 transgenic mice than in wild-type controls (426.49/35.4 mg hydroxyproline/g liver vs. 548.09/80.8 mg hydroxyproline/g liver). This difference did not reach statistical significance.
4. Discussion Tissue injury often leads to inflammation, and if sustained, to fibrosis. This injury/inflammation / fibrosis sequence is observed in many solid organs, including heart [7], lung [6], kidney [5] and liver [4,18,26 /29]. Because of their central position in the pathway from injury to fibrosis, inflammatory cells are believed to play an important role in the process of tissue fibrogenesis. The influence of inflammatory cells is even more likely when one considers that they are an important source of profibrogenic oxidants and cytokines. In the liver, recent studies have focused on the potential for lymphocytes to modulate fibrosis [29]. The role of
neutrophils, however, has not been thoroughly addressed. BDL offered an excellent means for investigating a relationship between neutrophils and hepatic fibrosis, because of the significant influx of neutrophils [3] and the rapid time course to fibrosis [2] in this experimental model. We chose neutrophil depletion as an experimental strategy, based on prior success with this technique for assessing the role of neutrophils in acute tissue injury [30 /33]. Most studies involving neutrophil depletion have been carried out over very short time frames (24 /48 h); our experiments required more prolonged depletion, to coincide with the early phase of hepatic fibrogenesis after BDL. By administering anti-neutrophil antibody through an osmotic infusion pump, we achieved a 48% reduction in neutrophils in BDL rats that was sustained for 7 days. Contrary to our expectation, neutrophil depletion had no effect on the progression of biliary fibrosis. Neutrophil-depleted rats were indistinguishable from control rats in several responses to BDL, including biochemical cholestasis, bile duct proliferation, collagen gene expression and hepatic collagen deposition. Although the degree of neutrophil depletion achieved in BDL rats was significant, it was not complete. This was likely due to the strong neutrophilic stimulus provoked by BDL itself [3,4]. Whether the few neutrophils that remained
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
187
Fig. 5. Histochemical staining for collagen in rat liver. Photomicrographs illustrate hepatic collagen fibrils stained with picrosirius red. Sham liver (a, b) contains very little collagen. BDL liver (c /f) contains much more collagen in portal tracts and around bile ducts. BDL rats treated with control serum (c, d) have no more hepatic collagen than BDL rats treated with anti-neutrophil antiserum (e, f). By morphometry, total liver collagen was similar in both groups of BDL rats (see text).
in the liver despite depletion were sufficient to promote fibrosis could not be directly addressed, because more aggressive attempts to deplete them
were unsuccessful. To more fully examine the role of neutrophils in fibrosis, we pursued BDL in an alternative animal model characterized by neutro-
188
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Fig. 6. Neutrophilic infiltration of mouse liver after BDL. Photomicrographs illustrate livers stained with anti-neutrophil antibody (Ly6-G) 14 days after BDL. Liver from a wild-type mouse (a) shows marked neutrophilic inflammation in a periportal distribution, particularly around bile ducts. Liver from an IL-8 transgenic mouse (b) shows fewer periportal neutrophils. The transgenic liver also contains scattered neutrophils throughout the hepatic lobule. This is presumably related to neutrophilia, which results in high background neutrophil counts in many tissues [20]. Similar staining patterns were observed in IL-8 transgenic mice after sham operation (data not shown).
phil dysfunction rather than depletion. IL-8 transgenic mice offered a good complement to neutrophil-depleted rats for studies of BDL. They also had one advantage over rats, in that they could be
maintained for longer intervals after BDL (neutrophil depletion in BDL rats was restricted to 1 week due to the limited capacity of the osmotic infusion pumps). One group has recently argued that transgenic overexpression of IL-8 does not suppress neutrophil responses in vivo [34]. We found that IL-8 transgenic mice did exhibit impaired neutrophil recruitment to the liver after BDL (Fig. 6). This may be attributable to the liverspecific nature of the transgene [20], which could cause such a substantial chemotactic gradient from the liver to the circulation to effectively obscure any independent chemotactic signal induced by BDL. Despite the reduced hepatic inflammation in IL-8 transgenic mice after BDL, we were unable to detect any reduction in hepatic fibrosis compared with wild-type controls. Thus, our data show in two independent animal models that neutrophils exert little influence over the process of hepatic fibrogenesis in vivo. If neutrophils do not regulate hepatic fibrogenesis in vivo, one must reconcile this finding with evidence that neutrophils promote myofibroblastic transformation of hepatic stellate cells in culture [19]. One possible explanation for the discrepancy is that the cell culture experiments were performed with neutrophils from normal animals, whereas in vivo, neutrophil activity may be suppressed due to the presence of liver disease. Bile duct obstruction has been reported to impair certain neutrophil functions such as adhesion to endothelial cells and bacterial killing [35,36]. Interestingly, however, oxidant production is not influenced by cholestasis [37,38]. The latter is most relevant to fibrogenesis, because neutrophil-derived oxidants are the compounds believed to stimulate collagen synthesis by stellate cells [19]. If the fibrogenic activity of neutrophils toward stellate cells is not adversely affected by cholestasis in vivo, an alternative possibility is that their contribution to fibrosis is offset by the simultaneous release of matrixdegrading proteinases. Neutrophils release numerous proteinases during their activation and transmigration into tissues [16,17]. These could degrade newly synthesized collagen, and thus limit the net effect of neutrophils on the overall process of hepatic fibrogenesis. If neutrophils do indeed stimulate hepatic stellate cells but balance the
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Table 2 Serum chemistries in BDL mice at 14 daysa Group IL-8 transgenic (n /7) Wild-type (n /4)
Bilirubin (mg/dl) b
16.59/1.2 22.89/2.0
ALT (IU/ml) 4159/47b 2829/42
a
Cholestasis was more severe in mice than rats because mice were maintained for 14 days after BDL. b Differences between IL-8 transgenic and wild-type mice were not statistically significant.
Fig. 7. Histochemical staining for collagen in BDL mouse liver. Photomicrographs illustrate hepatic fibrosis in mice at 14 days after BDL, using picrosirius red to highlight collagen. Liver from an IL-8 transgenic mouse (a) appears similar to that of a wild-type mouse (b). Hepatic hydroxyproline levels were slightly lower in IL-8 transgenic mice than wild-type mice, but the difference was not significant (see text).
189
effect by promoting collagen degradation, then our experiments should have revealed some influence of neutrophil depletion on pretranslational events related to fibrosis such as hepatic collagen gene expression. This was not the case, and thus it remains uncertain why neutrophils do not promote hepatic fibrosis in vivo. Liver fibrosis develops rapidly after BDL despite the lack of a major influence by neutrophils. This suggests that in the setting of cholestasis, hepatic stellate cells are receiving fibrogenic signals from other cells in the local environment. These could emanate from hepatocytes [39], biliary cells [40,41] or Kupffer cells [39,42], or alternatively from other populations of infiltrating leukocytes. Together these cells produce a variety of oxidants and cytokines that can promote hepatic fibrogenesis. Dissecting which cells and which compounds are of the greatest influence to fibrosis represents a significant challenge. Work in this area is active, with genetically engineered mice being used to pursue the role of cytokines [43] and pharmacologic inhibitors being used to address the influence of oxidants [44,45]. It should be noted that biliary fibrosis is distinct from other forms of liver fibrosis associated with neutrophilic inflammation such as alcoholic fibrosis. Biliary obstruction provokes portal-based fibrosis, whereas alcoholic fibrosis begins pericentrally and extends along the hepatic sinusoids. This raises the question whether neutrophils have the same influence over fibrogenesis in all regions of the liver. Because our experiments focused only on biliary fibrosis, they cannot directly discount neutrophils as a stimulus to all forms of liver fibrosis. The effect of neutrophils may be unique when combined with disease-specific alterations in hepatocytes, Kupffer cells and endothelial cells. In summary, this study does not support the notion that infiltrating neutrophils promote liver fibrosis in the setting of biliary obstruction. This was unexpected, in view of the large numbers of neutrophils that invade the liver after BDL and the potential for neutrophils to stimulate hepatic stellate cells in cell culture. The primary purpose of neutrophil recruitment to the cholestatic liver may be to eliminate hepatocytes that have undergone bile salt-induced apoptosis [46]. Fibrogenesis
190
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
is likely mediated by other cells, resident or recruited, within the portal tracts of obstructed livers.
Acknowledgements This work was supported by grants from the United States Public Health Service (AA07810, AA00215, DK26743) and a gift from Mr. and Mrs. Robert H. Shepard. IL-8 transgenic mice were a gift from Dr. Scott Simonet (Amgen, Inc., Thousand Oaks, CA).
References [1] Scobie BA, Summerskill WHJ. Hepatic cirrhosis secondary to obstruction of the biliary system. Am J Dig Dis 1965;10:135 /46. [2] Kountouras J, Billing BH, Scheuer PJ. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 1984;65:305 /11. [3] Saito JM, Maher JJ. Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant. Gastroenterology 2000;118:1157 /68. [4] Parola M, Leonarduzzi G, Robino G, Albano E, Poli G, Dianzani MU. On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis. Free Radic Biol Med 1996;20:351 /9. [5] Diamond JR. Macrophages and progressive renal disease in experimental hydronephrosis. Am J Kidney Dis 1995;26:133 /40. [6] Neuringer IP, Mannon RB, Coffman TM, Parsons M, Burns K, Yankaskas JR, Aris RM. Immune cells in a mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998;19:379 /86. [7] Lee JK, Zaidi SH, Liu P, Dawood F, Cheah AY, Wen WH, Saiki Y, et al. A serine elastase inhibitor reduces inflammation and fibrosis and preserves cardiac function after experimentally induced murine myocarditis. Nat Med 1998;4:1383 /91. [8] Gulubova MV. Intercellular adhesion molecule-1 (ICAM1) expression in the liver of patients with extrahepatic cholestasis. Acta Histochem 1998;100:59 /74. [9] Friedman SL. The cellular basis of hepatic fibrosis. N Engl J Med 1993;328:1828 /35. [10] Parola M, Pinzani M, Casini A, Albano E, Poli G, Gentilini A, Gentilini P, et al. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Commun 1993;194:1044 /50.
[11] Nieto N, Friedman SL, Greenwel P, Cederbaum AI. CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells. Hepatology 1999;30:987 /96. [12] Matsuoka M, Pham NT, Tsukamoto H. Differential effects of interleukin-1 alpha, tumor necrosis factor alpha, and transforming growth factor beta 1 on cell proliferation and collagen formation by cultured fat-storing cells. Liver 1989;9:71 /8. [13] Bachem MG, Meyer D, Melchior R, Sell KM, Gressner AM. Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells: a potential mechanism of self-perpetuation in liver fibrogenesis. J Clin Invest 1992;89:19 /27. [14] Friedman SL, Roll FJ, Boyles J, Arenson DM, Bissell DM. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix. J Biol Chem 1989;264:10756 /62. [15] Crockett-Torabi E, Sulenbarger B, Smith CW, Fantone JC. Activation of human neutrophils through L-selectin and Mac-1 molecules. J Immunol 1995;154:2291 /302. [16] Tschesche H. Human neutrophil collagenase. Meth Enzymol 1995;248:431 /49. [17] Kafienah W, Buttle DJ, Burnett D, Hollander AP. Cleavage of native type I collagen by human neutrophil elastase. Biochem J 1998;330:897 /902. [18] Svegliati-Baroni G, Saccomanno S, van Goor H, Jansen P, Benedetti A, Moshage H. Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells. Liver 2001;21:1 /12. [19] Casini A, Ceni E, Salzano R, Biondi P, Parola M, Galli A, Foschi M, et al. Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide. Hepatology 1997;25:361 /7. [20] Simonet WS, Hughes TM, Nguyen HQ, Trebasky LD, Danilenko DM, Medlock ES. Long-term impaired neutrophil migration in mice overexpressing human interleukin-8. J Clin Invest 1994;94:1310 /9. [21] Gilman M. Ribonuclease protection assay. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, editors. Current Protocols in Molecular Biology, vol. 1. New York: Wiley, 1987:4.7.1 /8. [22] Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 1984;23:6210 /6. [23] Rhoads DD, Dixit A, Roufa DJ. Primary structure of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol 1986;6:2774 /83. [24] Jamall IS, Finelli VN, Que Hee SS. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Anal Biochem 1981;112:70 /5. [25] Wiekowski MT, Chen SC, Zalamea P, Wilburn BP, Kinsley DJ, Sharif WW, Jensen KK, et al. Disruption of neutrophil migration in a conditional transgenic model:
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
[26]
[27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35]
evidence for CXCR2 desensitization in vivo. J Immunol 2001;167:7102 /10. Harada K, Van de Water J, Leung PS, Coppel RL, Ansari A, Nakanuma Y, Gershwin ME. In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis: predominance of the Th1 subset. Hepatology 1997;25:791 /6. Baroni GS, Pastorelli A, Manzin A, Benedetti A, Marucci L, Solforosi L, Di Sario A, et al. Hepatic stellate cell activation and liver fibrosis are associated with necroinflammatory injury and Th1-like response in chronic hepatitis C. Liver 1999;19:212 /9. Vaillant B, Chiaramonte MG, Cheever AW, Soloway PD, Wynn TA. Regulation of hepatic fibrosis and extracellular matrix genes by the Th response: new insight into the role of tissue inhibitors of matrix metalloproteinases. J Immunol 2001;167:7017 /26. Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 2001;21:417 /26. Buell MG, Berin C. Neutrophil-independence of the initiation of colonic injury. Dig Dis Sci 1994;39:2575 / 2588. Schimmer RC, Schrier DJ, Flory CM, Dykens J, Tung DK, Jacobson PB, Friedl HP, et al. Streptococcal cell wallinduced arthritis: requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, and macrophage-inflammatory protein-2. J Immunol 1997;159:4103 /8. Molnar RG, Wang P, Ayala A, Ganey PE, Roth RA, Chaudry IH. The role of neutrophils in producing hepatocellular dysfunction during the hyperdynamic stage of sepsis in rats. J Surg Res 1997;73:117 /22. Iwahori Y, Ishiguro N, Shimizu T, Kondo S, Yabe Y, Oshima T, Iwata H, et al. Selective neutrophil depletion with monoclonal antibodies attenuates ischemia/reperfusion injury in skeletal muscle. J Reconstr Microsurg 1998;14:109 /16. Remick DG, Green LB, Newcomb DE, Garg SJ, Bolgos GL, Call DR. CXC chemokine redundancy ensures local neutrophil recruitment during acute inflammation. Am J Pathol 2001;159:1149 /57. Swain MG, Tjandra K, Kanwar S, Kubes P. Neutrophil adhesion is impaired in rat model of cholestasis. Gastroenterology 1995;109:923 /32.
191
[36] Tjandra K, Woodman RC, Swain MG. Impaired neutrophil microbicidal activity in rat cholestasis. Gastroenterology 1997;112:1692 /8. [37] Tsai LY, Lee KT, Liu TZ. Evidence for accelerated generation of hydroxyl radicals in experimental obstructive jaundice of rats. Free Radic Biol Med 1998;24:732 /7. [38] D’Odorico A, Melis A, Baragiotta A, Martines D, Naccarato R, Floreani A. Oxygen-derived free radical production by peripheral blood neutrophils in chronic cholestatic liver diseases. Hepatogastroenterology 1999;46:1831 /5. [39] Gressner AM, Lotfi S, Gressner G, Haltner E, Kropf J. Synergism between hepatocytes and Kupffer cells in the activation of fat storing cells (perisinusoidal lipocytes). J Hepatol 1993;19:117 /32. [40] Kinnman N, Hultcrantz R, Barbu V, Rey C, Wendum D, Poupon R, Housset C. PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury. Lab Invest 2000;80:697 /707. [41] Sedlaczek N, Jia JD, Bauer M, Herbst H, Ruehl M, Hahn EG, Schuppan D. Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am J Pathol 2001;158:1239 /44. [42] Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium: direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 1989;84:1780 /5. [43] Shi Z, Wakil AE, Rockey DC. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci USA 1997;94:10663 /8. [44] Baron V, Muriel P. Role of glutathione, lipid peroxidation and antioxidants on acute bile duct obstruction in the rat. Biochim Biophys Acta 1999;1472:173 /80. [45] Boigk G, Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 1997;26:643 /9. [46] Faubion WA, Guicciardi ME, Miyoshi H, Bronk SF, Roberts PJ, Svingen PA, Kaufmann SH, et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J Clin Invest 1999;103:137 /45.
Infiltrating neutrophils in bile duct-ligated livers do not promote hepatic fibrosis Jacqueline M. Saito a,b, Michelle K. Bostick b,c, Carson B. Campe b,c, Junquan Xu b,c, Jacquelyn J. Maher b,c,* a
Department of Surgery, University of California, San Francisco, CA, USA b UCSF Liver Center, University of California, San Francisco, CA, USA c Department of Medicine, University of California, San Francisco, CA, USA Received 7 May 2002; received in revised form 26 July 2002; accepted 27 August 2002
Abstract Bile duct obstruction causes rapid infiltration of neutrophils into the liver and leads ultimately to hepatic fibrosis. In this study, we assessed whether neutrophils play an active role in the pathogenesis of hepatic fibrosis under conditions of biliary obstruction. We performed bile duct ligation (BDL) on rats, some of which were depleted of neutrophils by means of an anti-neutrophil antiserum. Rats treated with the antiserum had 48% fewer neutrophils than control rats. Despite this, they showed no difference in either bile duct proliferation or hepatic fibrogenesis after BDL compared with control rats. In a second set of experiments, we performed BDL on mice with an underlying defect in neutrophil function due to transgenic expression of interleukin-8. Mice with neutrophil dysfunction deposited less ( /22%) collagen in their livers after BDL than wild-type mice, but the difference was not statistically significant. In summary, data from two independent rodent models indicate that infiltrating neutrophils do not influence hepatic fibrogenesis following bile duct obstruction. The findings suggest that neutrophils play little if any role in the immunomodulation of liver fibrosis. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cirrhosis; Cholestasis; Biliary; Leukocyte; Inflammation
1. Introduction
Abbreviations: ALT, alanine aminotransferase; BDL, bile duct ligation; CK19, cytokeratin-19. * Corresponding author. Present address: Liver Center Laboratory, San Francisco General Hospital, Building 40, Room 4102, 1001 Potrero Avenue, San Francisco, CA 94110, USA. Tel.: /1-415-206-4805; fax: /1-415-641-0517. E-mail address: [email protected] (J.J. Maher).
Obstruction of bile flow through the extrahepatic biliary system results in rapidly progressive liver disease [1,2]. The hallmarks of liver injury following biliary obstruction are periductal inflammation, bile duct proliferation and portal fibrosis. Prominent among the inflammatory cells that invade obstructed livers are neutrophils; studies in experimental animals show that neutrophils
1386-6346/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 2 ) 0 0 2 4 7 - 4
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
infiltrate the liver within 3 h of experimental bile duct occlusion [3], and remain there for days to weeks as fibrosis progresses [3,4]. Although in many types of tissue injury, inflammatory cells colocalize with fibrosis [5 /7], in the obstructed liver the proximity between neutrophils and collagen is unusually close. This, together with the large number of neutrophils that are found in both animal and human models of extrahepatic biliary obstruction [3,4,8], suggests that neutrophils are central to the pathogenesis of biliary fibrosis. In all forms of liver fibrosis, the principal collagen-producing cells are hepatic stellate cells [9]. In normal liver stellate cells are quiescent, but in the setting of liver injury they transform into myofibroblast-like cells that are proliferative and migratory and produce abundant fibrillar collagen [9]. Stellate cell transformation can be promoted by a number of factors including oxidant stress [10,11], exposure to fibrogenic cytokines [12,13] and contact with foreign extracellular matrix proteins [14]. Because neutrophils produce oxidants and proteinases as they transmigrate into tissues [15 /17], they may contribute to stellate cell transformation in vivo. A connection between neutrophils and liver fibrosis was first suggested by Parola et al. [4]. These authors quantitated hepatic neutrophils in an experimental model of bile duct obstruction and found that the number of infiltrating cells correlated directly with the degree of liver fibrosis. A similar association between neutrophil infiltration and hepatic fibrogenesis has been reported in dimethylnitrosamine-induced liver disease [18]. Casini et al. [19] offered direct evidence in favor of neutrophil-mediated regulation of hepatic stellate cells in a report demonstrating that activated neutrophils enhance stellate cell collagen synthesis in culture. Proof that neutrophils modulate hepatic fibrogenesis in vivo, however, is lacking. The objective of this study was to determine directly in an in vivo model of liver injury whether neutrophils promote hepatic fibrogenesis. We accomplished this by subjecting rodents with altered neutrophil numbers or neutrophil responses to bile duct ligation (BDL), and comparing the progression of fibrosis in these animals with that in rodents with normal neutrophils. The
181
results indicate that neutrophils have little impact on the progression of hepatic fibrosis in response to BDL.
2. Methods 2.1. BDL in rats and administration of antineutrophil antiserum Male Sprague/Dawley rats (225 /250 g) were chosen for BDL. Under inhalation anesthesia, rats underwent laparotomy with either complete ligation of the common bile duct or sham ligation. After the abdominal incision was closed, rats were turned prone and a subcutaneous pouch was created between the scapulae. In the pouch was placed an osmotic infusion pump (Alzet 2ML1, Alza Corporation, Palo Alto, CA) containing either rabbit anti-rat neutrophil antiserum (Accurate Chemical Company, Westbury, NY) or nonimmune rabbit serum (Accurate Chemical Company). After the operations were completed, rats were returned to standard housing and permitted free access to food and water for 7 days. During this interval, the infusion pumps discharged at a constant rate of 10 ml/h (total dose of 1 ml/(kg/d)). At the end of the experiment, rats were killed for collection of blood and liver tissue. 2.2. BDL in interleukin-8 transgenic mice BDF-1 mice that express a liver-specific transgene encoding human interleukin-8 (IL-8) [20] were obtained from Amgen, Inc. (Thousand Oaks, CA). Heterozygotes are characterized by the presence of nanogram quantities of IL-8 in the circulation [20]. A colony was maintained by breeding heterozygotes with wild-type BDF-1 mice (Jackson Laboratories, Bar Harbor, ME); offspring were screened for transgene expression at 6/8 weeks of age by quantitation of IL-8 in serum (Quantikine, R&D Systems, Minneapolis, MN). Serum IL-8 levels in positive offspring ranged from 38 to 65 ng/ml. Adult IL-8 transgenic and wild-type mice (approx. 25 g) were subjected to BDL under inhalation anesthesia. Postoperatively, mice were
182
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
maintained for 14 days with free access to food and water. At the end of the experiment, mice were killed for collection of blood and liver tissue. All animals received humane care based on guidelines set by the American Veterinary Association. All experimental protocols involving live animals were approved by the Committee on Animal Research at the University of California, San Francisco. 2.3. Blood counts and plasma chemistries Blood was collected from rats at the time of killing and mixed with heparin. Leukocytes and neutrophils were quantitated in whole blood using an automated cell counter (Technicon). Plasma was separated from cellular elements by centrifugation and assayed for liver chemistries on a BAX autoanalyzer (Bayer Corporation, Tokyo, Japan). Blood was collected from mice at the time of killing but not mixed with anticoagulants. Serum was separated by centrifugation and assayed for liver chemistries in the same fashion as described for rats. 2.4. Histology and immunohistochemistry The livers of experimental animals were removed and rinsed immediately in saline solution. A portion of each liver was fixed in 10% buffered formalin and embedded in paraffin. The remainder was snap-frozen in liquid nitrogen and stored at / 80 8C. Sections of formalin-fixed, paraffin-embedded liver were stained with hematoxylin and eosin. Serial sections were stained with picrosirius red to highlight connective tissue. Frozen unfixed liver was used for immunohistochemical staining. Neutrophils were identified with specific antigranulocyte antibodies (Gr-1 or Ly6-G; Pharmingen, San Diego, CA). Details of the staining protocol have been reported previously [3]. Neutrophils were counted manually under 10 / magnification in blinded liver sections; 3/5 microscopic fields were evaluated per liver, each centered on a portal tract. Counts represent the average number of Gr-positive cells per portal field in rats from each treatment group (n /3).
Bile ducts were identified with mouse antihuman cytokeratin-19 (anti-CK19, Novocastra, Newcastle-Upon-Tyne, UK). Briefly, 8 mm sections of frozen liver were adhered to glass slides, treated with ice-cold acetone for 10 min and airdried. After rehydration and blocking in PBS/1% BSA, sections were incubated with anti-CK19 (1:500) overnight at 4 8C. They were then washed with PBS/1% BSA and incubated for 30 min at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000; Molecular Probes, Eugene, OR). After further washes, sections were covered with Vectamount† mounting medium (Vector Laboratories, Burlingame, CA), coverslipped and sealed. Sections were viewed and photographed on a Nikon Microphot-FXA epifluorescence microscope (Nikon Corporation, Tokyo, Japan). 2.5. Image analysis Bile duct area was quantitated morphometrically in livers stained with anti-CK19. Photographs were taken with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI) and analyzed using Simple PCI software (Compix, Cranberry Township, PA). Bile duct area was measured in 10 fields per liver section (10 /), each centered on a portal tract. Data were expressed as a percent of total liver area. For morphometric measurement of liver collagen, sections stained with picrosirius red were photographed at 4 / magnification. Fifteen adjacent fields were photographed to limit sampling error from the localized fibrosis that follows BDL. Collagen staining was expressed as a percent of total liver area. 2.6. Analysis of collagen gene expression Livers were homogenized in TRI reagent (Molecular Research Center, Cincinnati, OH) for extraction of total RNA. mRNA encoding procollagen I was quantitated by RNase protection [21] using S14 mRNA as an internal control. cRNA probes for procollagen I and S14 were transcribed with a-32P-CTP (/800 Ci/mmol, Amersham Corp., Arlington Heights, IL). Ali-
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
quots of liver RNA (20 mg) were combined with both probes (each at 3 /105 Cerenkov cpm) and incubated at 55 8C for 16 h. Unhybridized probes and RNA were then digested with ribonuclease T2 (GIBCO BRL, Grand Island, NY). The remaining RNA /RNA hybrids were precipitated in isopropanol. After denaturation at 100 8C, hybrids were separated through 5% polyacrylamide urea. Radioactive bands corresponding to the protected probe were visualized by autoradiography (XOMat AR-5, Eastman Kodak, Rochester, NY) and quantitated by scanning densitometry (Hoefer Scientific Instruments, San Francisco, CA). Signals for procollagen I [22] were normalized to the control RNA signal encoding S14 [23].
2.7. Quantitation of hepatic hydroxyproline Hydroxyproline was quantitated in liver homogenates by the method of Jamall et al. [24]. A portion of liver was first homogenized in distilled water at a concentration of 1 mg/ml. The homogenate was then combined with an equal volume of 12 N HCl and heated to 110 8C for 20 h. The acid hydrolysate was clarified by centrifugation; 50-ml aliquots were then added to several test tubes and air-dried. Each group of test tubes corresponding to a single liver was supplemented with standards containing known amounts of 4-hydroxy-L-proline (Sigma Chemical Company, St. Louis, MO). Hydroxyproline was measured by adding p -dimethylaminobenzaldehyde (Ehrlich’s reagent; Fisher Scientific, Hanover Park, IL) and reading absorbance at 558 nm. The amount of hydroxyproline in each sample was determined by measuring the degree to which the sample displaced the standard curve from the origin.
2.8. Statistical analysis All quantitative data are reported as mean9/ S.E.M. Differences between means were calculated using analysis of variance with post hoc Bonferroni adjustment. P -values B/0.05 were considered significant.
183
3. Results
3.1. Anti-neutrophil antiserum does not impact BDL-induced cholestasis in rats but significantly reduces circulating and tissue neutrophils Rats developed typical cholestasis in response to BDL. On day 7 after surgery, plasma bilirubin was 10 times higher in BDL rats than sham rats; alanine aminotransferase (ALT) was also significantly increased (Table 1). This was true whether or not the animals received anti-neutrophil antiserum (Table 1). BDL also caused a significant increase in circulating neutrophil counts (Fig. 1, black bars), which confirms prior observations that BDL causes neutrophilia [3]. Treatment with anti-neutrophil antiserum reduced the number of circulating neutrophils in BDL rats by 48% (5.7 / 103 cells/ml vs. 11.0 /103 cells/ml, P B/0.005; Fig. 1). In sham-operated rats, the same dose of antiserum reduced the number of circulating neutrophils by 88% (3.4 /103 cells/ml vs. 0.4 / 103 cells/ml, P B/0.05). Higher doses of anti-neutrophil antiserum were tested in an effort to enhance neutrophil depletion in BDL rats, but these proved to be toxic (data not shown). Anti-neutrophil antiserum eliminated neutrophils from the livers as well as the circulation of BDL rats. This is illustrated in Fig. 2, in which panel a shows abundant periportal neutrophils in 7-day BDL rats and panel b demonstrates many fewer neutrophils in BDL rats treated with antineutrophil antiserum. Direct counting of stained Table 1 Serum chemistries in BDL and sham rats at 7 days Group
Bilirubin (mg/ dl)
ALT (IU/ ml)
BDL/control serum BDL/anti-neutrophil antiserum Sham/control serum Sham/anti-neutrophil antiserum
5.49/0.3a 5.29/1.0a
1749/9b 2209/47b
0.79/0.4 0.29/0
749/0 619/6
Values represent mean9/S.E.M. a P B/0.01 vs. sham rats. b P B/0.05 vs. sham rats.
184
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Fig. 1. Absolute neutrophil counts in BDL and sham rats. Graph depicts circulating neutrophil counts in BDL and sham rats treated with anti-neutrophil antiserum (hatched bars) or control antiserum (solid bars) for 7 days. Bars represent mean9/ S.E.M. for n ]/4. *P B/0.05, **P B/0.005 vs. control serum; §P B/0.05, §§P B/0.005 vs. sham rats.
cells showed that the antiserum caused a 50% reduction in tissue neutrophils, from 92.69/10.3 to 46.79/14.3 per portal field. This directly paralleled the level of depletion of circulating neutrophils. Sham rats had 17.89/1.6 neutrophils per portal field, which were reduced to 3.39/0.1 by antineutrophil antiserum. 3.2. Neutrophil depletion does not affect bile duct proliferation after BDL Biliary obstruction causes cholangiocyte proliferation and expansion of intrahepatic bile ducts. To determine whether neutrophils impact this process, rat livers were stained with an antibody that recognizes the biliary cell intermediate filament protein CK19 (Fig. 3). Bile duct area was measured in BDL and sham livers by morphometry. As expected, bile duct area was significantly greater in BDL liver compared with sham liver. Neutrophil depletion had no effect on bile duct
Fig. 2. Neutrophil immunohistochemistry in 7-day BDL liver. Photomicrographs illustrate immunoperoxidase staining for neutrophils using anti-Gr (see Section 2). (a) BDL liver with control serum for 7 days; (b) BDL liver with anti-neutrophil antiserum for 7 days. Neutrophil counts in portal fields from each treatment group measured 17.89/1.6 for sham/control antibody, 3.39/0.1 for sham/anti-neutrophil antibody, 46.79/ 14.3 for BDL/control antibody and 92.79/20.3 for BDL/antineutrophil antibody. Original magnification 10 /.
expansion after BDL (2.38% vs. 2.42% bile duct area in depleted vs. control BDL rats, n /6). 3.3. Neutrophil depletion does not affect collagen gene expression in BDL liver To determine whether neutrophil depletion attenuates hepatic fibrogenesis after BDL, procollagen I mRNA was measured in the livers of BDL
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
185
increase; Fig. 4). Neutrophil depletion, however, had no effect on procollagen I gene expression. Procollagen I mRNA levels in BDL rats treated with anti-neutrophil antiserum were no different than those measured in BDL rats treated with control serum (Fig. 4, lanes 5 /9 vs. lanes 10 /15). 3.4. Neutrophil depletion does not influence hepatic collagen deposition in BDL rats Although neutrophil depletion did not alter the amount of procollagen I mRNA in rat liver after BDL, we reasoned that it might modify the pattern or extent of collagen deposition in the liver. To investigate this, BDL and sham livers were stained with picrosirius red and examined by light microscopy (Fig. 5). Fig. 5a and b illustrate the sparse collagen in the portal tracts of sham liver. Fig. 5c / f show more abundant collagen at the basal surface of bile ducts in 7-day BDL liver. By image analysis, hepatic collagen was threefold greater in BDL liver than in sham liver (collagen area 2.68% vs. 0.75%, P B/0.005). There was no difference in total collagen between BDL rats treated with control serum (Fig. 5c and d) and BDL rats treated with anti-neutrophil antiserum (Fig. 5e and f; 2.85% vs. 2.34% collagen area, n /6). Nor was there any difference in hydroxyproline measured in liver homogenates (190.89/10.1 mg hydroxyproline/g liver vs. 213.79/33.6 mg hydroxyproline/g liver in neutrophil-depleted vs. control rats, n /6). 3.5. Neutrophil dysfunction does not alter biliary fibrosis in mice
Fig. 3. Bile duct proliferation in 7-day BDL liver. Photomicrographs illustrate bile ducts in BDL livers highlighted with antiCK19. (a) Sham liver with control serum for 7 days; (b) BDL liver with control serum for 7 days; (c) BDL liver with antineutrophil antiserum for 7 days. Original magnification 10 /.
and sham rats by RNase protection. Procollagen I mRNA was markedly induced in BDL livers compared with sham livers at 7 days (5.3-fold
IL-8 transgenic mice exhibit defective neutrophil migration in response to classical chemotactic stimuli in vivo [20]. Neutrophil dysfunction in these mice and others that overexpress CXC chemokines has been attributed to chemokine receptor desensitization [20,25]. When IL-8 transgenic mice were subjected to BDL, they exhibited less periportal infiltration of neutrophils than wildtype mice (Fig. 6). Biochemical measures of cholestasis, however, were comparable between the two groups (Table 2). Survival after BDL was much better in IL-8 transgenic mice than in
186
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Fig. 4. Steady-state expression of procollagen I mRNA in 7-day BDL liver. Autoradiogram illustrates procollagen I mRNA (Collagen I) 7 days after BDL or sham operation, in the presence of anti-neutrophil antiserum (/) or control serum ( /). BDL induces hepatic procollagen gene expression 5.3-fold over sham operation. Note that procollagen mRNA in BDL liver is not influenced by administration of anti-neutrophil antiserum. Hybridization signals were generated by RNase protection using 20 mg total liver RNA (see Section 2). S14 serves as an internal control signal for ribosomal RNA.
wild-type controls. 14-day mortality in the control group was 54% (7/13), whereas mortality in the IL8 transgenic group was only 8% (1/12). Liver fibrosis was evaluated in all mice that survived 14 days of BDL. Histologically, there was no discernible difference in collagen staining with picrosirius red (Fig. 7). Hepatic hydroxyproline levels after BDL were 22% lower in IL-8 transgenic mice than in wild-type controls (426.49/35.4 mg hydroxyproline/g liver vs. 548.09/80.8 mg hydroxyproline/g liver). This difference did not reach statistical significance.
4. Discussion Tissue injury often leads to inflammation, and if sustained, to fibrosis. This injury/inflammation / fibrosis sequence is observed in many solid organs, including heart [7], lung [6], kidney [5] and liver [4,18,26 /29]. Because of their central position in the pathway from injury to fibrosis, inflammatory cells are believed to play an important role in the process of tissue fibrogenesis. The influence of inflammatory cells is even more likely when one considers that they are an important source of profibrogenic oxidants and cytokines. In the liver, recent studies have focused on the potential for lymphocytes to modulate fibrosis [29]. The role of
neutrophils, however, has not been thoroughly addressed. BDL offered an excellent means for investigating a relationship between neutrophils and hepatic fibrosis, because of the significant influx of neutrophils [3] and the rapid time course to fibrosis [2] in this experimental model. We chose neutrophil depletion as an experimental strategy, based on prior success with this technique for assessing the role of neutrophils in acute tissue injury [30 /33]. Most studies involving neutrophil depletion have been carried out over very short time frames (24 /48 h); our experiments required more prolonged depletion, to coincide with the early phase of hepatic fibrogenesis after BDL. By administering anti-neutrophil antibody through an osmotic infusion pump, we achieved a 48% reduction in neutrophils in BDL rats that was sustained for 7 days. Contrary to our expectation, neutrophil depletion had no effect on the progression of biliary fibrosis. Neutrophil-depleted rats were indistinguishable from control rats in several responses to BDL, including biochemical cholestasis, bile duct proliferation, collagen gene expression and hepatic collagen deposition. Although the degree of neutrophil depletion achieved in BDL rats was significant, it was not complete. This was likely due to the strong neutrophilic stimulus provoked by BDL itself [3,4]. Whether the few neutrophils that remained
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
187
Fig. 5. Histochemical staining for collagen in rat liver. Photomicrographs illustrate hepatic collagen fibrils stained with picrosirius red. Sham liver (a, b) contains very little collagen. BDL liver (c /f) contains much more collagen in portal tracts and around bile ducts. BDL rats treated with control serum (c, d) have no more hepatic collagen than BDL rats treated with anti-neutrophil antiserum (e, f). By morphometry, total liver collagen was similar in both groups of BDL rats (see text).
in the liver despite depletion were sufficient to promote fibrosis could not be directly addressed, because more aggressive attempts to deplete them
were unsuccessful. To more fully examine the role of neutrophils in fibrosis, we pursued BDL in an alternative animal model characterized by neutro-
188
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Fig. 6. Neutrophilic infiltration of mouse liver after BDL. Photomicrographs illustrate livers stained with anti-neutrophil antibody (Ly6-G) 14 days after BDL. Liver from a wild-type mouse (a) shows marked neutrophilic inflammation in a periportal distribution, particularly around bile ducts. Liver from an IL-8 transgenic mouse (b) shows fewer periportal neutrophils. The transgenic liver also contains scattered neutrophils throughout the hepatic lobule. This is presumably related to neutrophilia, which results in high background neutrophil counts in many tissues [20]. Similar staining patterns were observed in IL-8 transgenic mice after sham operation (data not shown).
phil dysfunction rather than depletion. IL-8 transgenic mice offered a good complement to neutrophil-depleted rats for studies of BDL. They also had one advantage over rats, in that they could be
maintained for longer intervals after BDL (neutrophil depletion in BDL rats was restricted to 1 week due to the limited capacity of the osmotic infusion pumps). One group has recently argued that transgenic overexpression of IL-8 does not suppress neutrophil responses in vivo [34]. We found that IL-8 transgenic mice did exhibit impaired neutrophil recruitment to the liver after BDL (Fig. 6). This may be attributable to the liverspecific nature of the transgene [20], which could cause such a substantial chemotactic gradient from the liver to the circulation to effectively obscure any independent chemotactic signal induced by BDL. Despite the reduced hepatic inflammation in IL-8 transgenic mice after BDL, we were unable to detect any reduction in hepatic fibrosis compared with wild-type controls. Thus, our data show in two independent animal models that neutrophils exert little influence over the process of hepatic fibrogenesis in vivo. If neutrophils do not regulate hepatic fibrogenesis in vivo, one must reconcile this finding with evidence that neutrophils promote myofibroblastic transformation of hepatic stellate cells in culture [19]. One possible explanation for the discrepancy is that the cell culture experiments were performed with neutrophils from normal animals, whereas in vivo, neutrophil activity may be suppressed due to the presence of liver disease. Bile duct obstruction has been reported to impair certain neutrophil functions such as adhesion to endothelial cells and bacterial killing [35,36]. Interestingly, however, oxidant production is not influenced by cholestasis [37,38]. The latter is most relevant to fibrogenesis, because neutrophil-derived oxidants are the compounds believed to stimulate collagen synthesis by stellate cells [19]. If the fibrogenic activity of neutrophils toward stellate cells is not adversely affected by cholestasis in vivo, an alternative possibility is that their contribution to fibrosis is offset by the simultaneous release of matrixdegrading proteinases. Neutrophils release numerous proteinases during their activation and transmigration into tissues [16,17]. These could degrade newly synthesized collagen, and thus limit the net effect of neutrophils on the overall process of hepatic fibrogenesis. If neutrophils do indeed stimulate hepatic stellate cells but balance the
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
Table 2 Serum chemistries in BDL mice at 14 daysa Group IL-8 transgenic (n /7) Wild-type (n /4)
Bilirubin (mg/dl) b
16.59/1.2 22.89/2.0
ALT (IU/ml) 4159/47b 2829/42
a
Cholestasis was more severe in mice than rats because mice were maintained for 14 days after BDL. b Differences between IL-8 transgenic and wild-type mice were not statistically significant.
Fig. 7. Histochemical staining for collagen in BDL mouse liver. Photomicrographs illustrate hepatic fibrosis in mice at 14 days after BDL, using picrosirius red to highlight collagen. Liver from an IL-8 transgenic mouse (a) appears similar to that of a wild-type mouse (b). Hepatic hydroxyproline levels were slightly lower in IL-8 transgenic mice than wild-type mice, but the difference was not significant (see text).
189
effect by promoting collagen degradation, then our experiments should have revealed some influence of neutrophil depletion on pretranslational events related to fibrosis such as hepatic collagen gene expression. This was not the case, and thus it remains uncertain why neutrophils do not promote hepatic fibrosis in vivo. Liver fibrosis develops rapidly after BDL despite the lack of a major influence by neutrophils. This suggests that in the setting of cholestasis, hepatic stellate cells are receiving fibrogenic signals from other cells in the local environment. These could emanate from hepatocytes [39], biliary cells [40,41] or Kupffer cells [39,42], or alternatively from other populations of infiltrating leukocytes. Together these cells produce a variety of oxidants and cytokines that can promote hepatic fibrogenesis. Dissecting which cells and which compounds are of the greatest influence to fibrosis represents a significant challenge. Work in this area is active, with genetically engineered mice being used to pursue the role of cytokines [43] and pharmacologic inhibitors being used to address the influence of oxidants [44,45]. It should be noted that biliary fibrosis is distinct from other forms of liver fibrosis associated with neutrophilic inflammation such as alcoholic fibrosis. Biliary obstruction provokes portal-based fibrosis, whereas alcoholic fibrosis begins pericentrally and extends along the hepatic sinusoids. This raises the question whether neutrophils have the same influence over fibrogenesis in all regions of the liver. Because our experiments focused only on biliary fibrosis, they cannot directly discount neutrophils as a stimulus to all forms of liver fibrosis. The effect of neutrophils may be unique when combined with disease-specific alterations in hepatocytes, Kupffer cells and endothelial cells. In summary, this study does not support the notion that infiltrating neutrophils promote liver fibrosis in the setting of biliary obstruction. This was unexpected, in view of the large numbers of neutrophils that invade the liver after BDL and the potential for neutrophils to stimulate hepatic stellate cells in cell culture. The primary purpose of neutrophil recruitment to the cholestatic liver may be to eliminate hepatocytes that have undergone bile salt-induced apoptosis [46]. Fibrogenesis
190
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
is likely mediated by other cells, resident or recruited, within the portal tracts of obstructed livers.
Acknowledgements This work was supported by grants from the United States Public Health Service (AA07810, AA00215, DK26743) and a gift from Mr. and Mrs. Robert H. Shepard. IL-8 transgenic mice were a gift from Dr. Scott Simonet (Amgen, Inc., Thousand Oaks, CA).
References [1] Scobie BA, Summerskill WHJ. Hepatic cirrhosis secondary to obstruction of the biliary system. Am J Dig Dis 1965;10:135 /46. [2] Kountouras J, Billing BH, Scheuer PJ. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 1984;65:305 /11. [3] Saito JM, Maher JJ. Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant. Gastroenterology 2000;118:1157 /68. [4] Parola M, Leonarduzzi G, Robino G, Albano E, Poli G, Dianzani MU. On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis. Free Radic Biol Med 1996;20:351 /9. [5] Diamond JR. Macrophages and progressive renal disease in experimental hydronephrosis. Am J Kidney Dis 1995;26:133 /40. [6] Neuringer IP, Mannon RB, Coffman TM, Parsons M, Burns K, Yankaskas JR, Aris RM. Immune cells in a mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998;19:379 /86. [7] Lee JK, Zaidi SH, Liu P, Dawood F, Cheah AY, Wen WH, Saiki Y, et al. A serine elastase inhibitor reduces inflammation and fibrosis and preserves cardiac function after experimentally induced murine myocarditis. Nat Med 1998;4:1383 /91. [8] Gulubova MV. Intercellular adhesion molecule-1 (ICAM1) expression in the liver of patients with extrahepatic cholestasis. Acta Histochem 1998;100:59 /74. [9] Friedman SL. The cellular basis of hepatic fibrosis. N Engl J Med 1993;328:1828 /35. [10] Parola M, Pinzani M, Casini A, Albano E, Poli G, Gentilini A, Gentilini P, et al. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Commun 1993;194:1044 /50.
[11] Nieto N, Friedman SL, Greenwel P, Cederbaum AI. CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells. Hepatology 1999;30:987 /96. [12] Matsuoka M, Pham NT, Tsukamoto H. Differential effects of interleukin-1 alpha, tumor necrosis factor alpha, and transforming growth factor beta 1 on cell proliferation and collagen formation by cultured fat-storing cells. Liver 1989;9:71 /8. [13] Bachem MG, Meyer D, Melchior R, Sell KM, Gressner AM. Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells: a potential mechanism of self-perpetuation in liver fibrogenesis. J Clin Invest 1992;89:19 /27. [14] Friedman SL, Roll FJ, Boyles J, Arenson DM, Bissell DM. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix. J Biol Chem 1989;264:10756 /62. [15] Crockett-Torabi E, Sulenbarger B, Smith CW, Fantone JC. Activation of human neutrophils through L-selectin and Mac-1 molecules. J Immunol 1995;154:2291 /302. [16] Tschesche H. Human neutrophil collagenase. Meth Enzymol 1995;248:431 /49. [17] Kafienah W, Buttle DJ, Burnett D, Hollander AP. Cleavage of native type I collagen by human neutrophil elastase. Biochem J 1998;330:897 /902. [18] Svegliati-Baroni G, Saccomanno S, van Goor H, Jansen P, Benedetti A, Moshage H. Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells. Liver 2001;21:1 /12. [19] Casini A, Ceni E, Salzano R, Biondi P, Parola M, Galli A, Foschi M, et al. Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide. Hepatology 1997;25:361 /7. [20] Simonet WS, Hughes TM, Nguyen HQ, Trebasky LD, Danilenko DM, Medlock ES. Long-term impaired neutrophil migration in mice overexpressing human interleukin-8. J Clin Invest 1994;94:1310 /9. [21] Gilman M. Ribonuclease protection assay. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, editors. Current Protocols in Molecular Biology, vol. 1. New York: Wiley, 1987:4.7.1 /8. [22] Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 1984;23:6210 /6. [23] Rhoads DD, Dixit A, Roufa DJ. Primary structure of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol 1986;6:2774 /83. [24] Jamall IS, Finelli VN, Que Hee SS. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Anal Biochem 1981;112:70 /5. [25] Wiekowski MT, Chen SC, Zalamea P, Wilburn BP, Kinsley DJ, Sharif WW, Jensen KK, et al. Disruption of neutrophil migration in a conditional transgenic model:
J.M. Saito et al. / Hepatology Research 25 (2003) 180 /191
[26]
[27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35]
evidence for CXCR2 desensitization in vivo. J Immunol 2001;167:7102 /10. Harada K, Van de Water J, Leung PS, Coppel RL, Ansari A, Nakanuma Y, Gershwin ME. In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis: predominance of the Th1 subset. Hepatology 1997;25:791 /6. Baroni GS, Pastorelli A, Manzin A, Benedetti A, Marucci L, Solforosi L, Di Sario A, et al. Hepatic stellate cell activation and liver fibrosis are associated with necroinflammatory injury and Th1-like response in chronic hepatitis C. Liver 1999;19:212 /9. Vaillant B, Chiaramonte MG, Cheever AW, Soloway PD, Wynn TA. Regulation of hepatic fibrosis and extracellular matrix genes by the Th response: new insight into the role of tissue inhibitors of matrix metalloproteinases. J Immunol 2001;167:7017 /26. Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 2001;21:417 /26. Buell MG, Berin C. Neutrophil-independence of the initiation of colonic injury. Dig Dis Sci 1994;39:2575 / 2588. Schimmer RC, Schrier DJ, Flory CM, Dykens J, Tung DK, Jacobson PB, Friedl HP, et al. Streptococcal cell wallinduced arthritis: requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, and macrophage-inflammatory protein-2. J Immunol 1997;159:4103 /8. Molnar RG, Wang P, Ayala A, Ganey PE, Roth RA, Chaudry IH. The role of neutrophils in producing hepatocellular dysfunction during the hyperdynamic stage of sepsis in rats. J Surg Res 1997;73:117 /22. Iwahori Y, Ishiguro N, Shimizu T, Kondo S, Yabe Y, Oshima T, Iwata H, et al. Selective neutrophil depletion with monoclonal antibodies attenuates ischemia/reperfusion injury in skeletal muscle. J Reconstr Microsurg 1998;14:109 /16. Remick DG, Green LB, Newcomb DE, Garg SJ, Bolgos GL, Call DR. CXC chemokine redundancy ensures local neutrophil recruitment during acute inflammation. Am J Pathol 2001;159:1149 /57. Swain MG, Tjandra K, Kanwar S, Kubes P. Neutrophil adhesion is impaired in rat model of cholestasis. Gastroenterology 1995;109:923 /32.
191
[36] Tjandra K, Woodman RC, Swain MG. Impaired neutrophil microbicidal activity in rat cholestasis. Gastroenterology 1997;112:1692 /8. [37] Tsai LY, Lee KT, Liu TZ. Evidence for accelerated generation of hydroxyl radicals in experimental obstructive jaundice of rats. Free Radic Biol Med 1998;24:732 /7. [38] D’Odorico A, Melis A, Baragiotta A, Martines D, Naccarato R, Floreani A. Oxygen-derived free radical production by peripheral blood neutrophils in chronic cholestatic liver diseases. Hepatogastroenterology 1999;46:1831 /5. [39] Gressner AM, Lotfi S, Gressner G, Haltner E, Kropf J. Synergism between hepatocytes and Kupffer cells in the activation of fat storing cells (perisinusoidal lipocytes). J Hepatol 1993;19:117 /32. [40] Kinnman N, Hultcrantz R, Barbu V, Rey C, Wendum D, Poupon R, Housset C. PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury. Lab Invest 2000;80:697 /707. [41] Sedlaczek N, Jia JD, Bauer M, Herbst H, Ruehl M, Hahn EG, Schuppan D. Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am J Pathol 2001;158:1239 /44. [42] Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium: direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 1989;84:1780 /5. [43] Shi Z, Wakil AE, Rockey DC. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci USA 1997;94:10663 /8. [44] Baron V, Muriel P. Role of glutathione, lipid peroxidation and antioxidants on acute bile duct obstruction in the rat. Biochim Biophys Acta 1999;1472:173 /80. [45] Boigk G, Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 1997;26:643 /9. [46] Faubion WA, Guicciardi ME, Miyoshi H, Bronk SF, Roberts PJ, Svingen PA, Kaufmann SH, et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J Clin Invest 1999;103:137 /45.