EBM Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stefanovic, L.
Right arrow Articles by Stefanovic, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stefanovic, L.
Right arrow Articles by Stefanovic, B.
Experimental Biology and Medicine 230:573-586 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Direct Hepatotoxic Effect of KC Chemokine in the Liver Without Infiltration of Neutrophils

Lela Stefanovic*, David A. Brenner{dagger} and Branko Stefanovic*,1

* Department of Biomedical Science, College of Medicine, Florida State University, Tallahassee, Florida 32306; and {dagger} Department of Medicine, Columbia University, New York, New York 10032

1To whom requests for reprints should be addressed at Department of Biomedical Science, Florida State University College of Medicine, Tallahassee, FL 32306. E-mail: branko.stefanovic{at}med.fsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
KC is a mouse homolog of human chemokine gro-{alpha} (CXCL1), expression of which is increased in liver diseases. We show that activated, but not quiescent, hepatic stellate cells (HSCs) express KC. Hepatic stellate cells constitutively express the KC receptor, CXCR2. Addition of recombinant KC to HSCs undergoing activation in culture increases secretion and processing of Type I collagen. Overexpression of endogenous KC in the mouse liver could be achieved by an intraperitoneal injection of CCl4, followed after 24 hrs by an injection of recombinant KC into circulation. This protocol resulted in about a 14-fold increase in concentration of KC protein in the liver. Overexpression of KC was associated with upregulation of the mRNA for CXCR2 and MIP-2 and with necrosis and increased synthesis of Type I collagen. This suggests that KC has a direct hepatotoxic effect, which led to a massive liver necrosis after 48 hrs. No accumulation of neutrophils was seen in the livers as judged by histology and reverse transcriptase-polymerase chain reaction analysis of myeloperoxidase mRNA. Autostimulation of KC and CXCR2 expression by recombinant KC protein in the mice with preexisting liver injury indicates a positive feedback regulation. Such regulation and direct hepatotoxicity of KC with increased collagen synthesis represent novel findings about the role of KC/ gro-{alpha} in liver pathology.

Key Words: KC • chemokine • liver • fibrosis


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Liver fibrosis is characterized by the accumulation of extracellular matrix proteins in the liver, including Type I collagen (1, 2). Hepatic stellate cells (HSCs; also called Ito cells, lipocytes, or fat-storing cells) are the major cell type responsible for collagen synthesis in the fibrotic liver (3). In normal liver, quiescent HSCs store vitamin A, but only express trace amounts of Type I collagen. On fibrogenic stimulus, HSCs become activated, a process in which they lose retinoid droplets, proliferate, change morphologically into myofibroblasts, and increase their synthesis of extra-cellular matrix proteins (4–6). The most common causative agents of liver fibrosis induce chronic inflammation, and persistent inflammation leads to fibrosis (7, 8). Therefore, one of the goals of antifibrotic therapy is to control chronic inflammation. The molecular events linking inflammation and fibrosis are still poorly understood. Profibrogenic agents in the liver cause destruction of hepatocytes and activation of Kupffer cells. Damaged hepatocytes and activated Kupffer cells secrete proinflammatory cytokines and chemokines, which recruit immune cells and increase inflammation (9–13). Initially, this response is aimed to heal the damaged tissue, but after a prolonged inflammation, fibrosis becomes persistent and progressive, resulting in loss of the liver function. Activated Kupffer cells produce interleukin (IL)-1, IL-6, tumor necrosis factor {alpha} (TNF{alpha}), and transforming growth factor ß (TGFß) (14–16). Hepatocytes (17, 18) and endothelial cells (19, 20) can also be a source of IL-1, IL-6, and TNF{alpha} in the injured liver.

Gro-{alpha} chemokine (CXCL1) was first described as a growth factor for human melanoma cell line 294T and was termed melanoma growth–stimulating activity (MGSA) (21). Subsequent studies have shown that a physiological role of gro-{alpha} is chemotaxis of neutrophils to the site of tissue injury (22, 23). Two studies have shown that gro-{alpha} can also be chemotactic to monocytes and T lymphocytes (24, 25). No other functions of this chemokine have been described. Gro-{alpha} is expressed by most cell types; in the liver, this includes endothelial cells, (26) Kupffer cells, (27) HSCs (28), and hepatocytes (18). There are three genes for gro proteins in humans; gro-{alpha}, gro-ß, and gro-{gamma}, encoding proteins that share 85% identity at the amino acid level (29). Gro-{alpha} belongs to the CXC family of chemokines, with the best studied member of this family being IL-8 (30, 31). Functional differences have been described between IL-8 and gro-{alpha}, although they share 44% identity including the ELR motif at the amino terminus followed by the CXC motif (where X can be any amino acid) (32). Both IL-8 and gro-{alpha} bind with high affinity to the same receptor, CXCR2; however IL-8 also binds with high affinity to the CXCR1 receptor, whereas binding of gro-{alpha} to CXCR1 is of low affinity (33, 34). The CXCR2 receptor is expressed on neutrophils, monocytes, and T cells, but in contrast to the CXCR1 receptor, also on Purkinje cells, neurons, and astrocytes (35–37). There is only one gro gene in mouse encoding for the protein named KC. KC is 68% identical to human gro-{alpha} and 88% identical to rat cytokine-induced neutrophil attractant (CINC1) protein (38). Two more CXC chemokines have been described in rats, CINC2 and MIP-2, whereas mice have only MIP-2 (39–41). These chemokines share about 60%–65% identity to KC and about 40% identity to IL-8.

Liver diseases that are associated with increased infiltration of neutrophils include alcoholic hepatitis (42), ischemia/reperfusion after liver transplantation or resection (43), and endotoxemia (44). Maltby et al. (45) have described increased gro-{alpha} levels in liver homogenates of patients with alcoholic hepatitis, which correlated with infiltration of neutrophils in the liver. However, IL-8 was similarly increased in these samples, so it was not possible to address which chemokine was the main chemotactic factor. Another study has confirmed increased IL-8 in livers of patients with alcoholic hepatitis (46). Mice overexpressing human IL-8 did not have extravasation of neutrophils into the liver parenchyma (47), although mouse neutrophils show chemotaxis to human IL-8 in vitro (33). Other animal studies have suggested a role of KC in liver injury. When mice were injected with acetaminophen, KC mRNA was induced severalfold in their livers. The increase was evident 12 hrs after the acetaminophen injection (48). Lipopolysaccharide (LPS) is implicated in liver injury caused by alcohol in humans (49, 50). Perfusion of rat livers with LPS resulted in a 5- to 10-fold increase in CINC1 protein in the perfusate at 150 mins after the start of perfusion (51). Thus, these experiments have shown that liver injury is associated with increased production of KC.

In this paper we show that KC is upregulated in acute CCl4-mediated liver injury and that recombinant KC has a profibrogenic effect on isolated HSCs. Overexpression of KC had a proinflamatory and prefibrotic effect on the liver in vivo, which resulted in greatly increased acute liver injury. Increased infiltration of neutrophils into the liver, however, was not observed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of HSCs.
Hepatic stellate cells were isolated from normal rat livers by perfusion with collagenase and pronase, followed by centrifugation over stractan gradient, as described (5), and RNA was extracted from freshly isolated quiescent HSCs. For in vitro activation, HSCs were cultured in uncoated plastic dishes in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Two days after isolation, recombinant KC (Cell Sciences, Canton, MA) was added at 50 ng/ml. From Day 3 to Day 8, medium was changed every day, and fresh recombinant KC was added. Cells were collected for RNA and protein extraction after the indicated time-points, and the cell medium was collected for protein analysis. For treatment with cycloheximide and puromycin, activated rat HSCs (8 days in culture) were incubated with 50 µg/ml of cycloheximide or 20 µg/ml of puromycin for 3 hrs, when total RNA was extracted for analysis.

Injection of CCl4 and Recombinant KC into Mice.
BALB/c male mice (25 g) were injected intraperitoneally with a single dose of 2 µl/g of CCl4 in 50% mineral oil. Twenty four hours after the CCl4 injection, 200 µl of recombinant KC (1395-KC/CF; R&D Systems, Minneapolis, MN) at 5 µg/ml was injected through the tail vein (total dose, 40 ng/g). Control animals received saline instead of KC. Twenty four and 48 hrs after the KC or saline injections, the livers were harvested for histology (hematoxylin and eosin staining) and RNA and protein analysis. For the experiment shown in Figure 1Go, mice received only CCl4 or mineral oil, and livers were harvested after 3 days.



View larger version (71K):
[in this window]
[in a new window]
  		Figure 1. Upregulation of KC mRNA in acute liver injury. Total liver RNA was isolated 3 days after a single injection of CCl4 (2 µl/g) into mice (lanes 3 and 4). Control mice received mineral oil (lanes 1 and 2). RNA was analyzed by RT-PCR with primers specific for KC mRNA (top panel) and for EST-AL024215 mRNA and GAPDH mRNA (controls; bottom panels). Migration of specific PCR products is indicated.

 
RNA Analysis.
Total RNA was extracted from HSCs or mouse livers according to standard protocol (52). Poly-A+ RNA was isolated from HSCs or whole livers using Qiagen Oligotex mRNA kit. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the rTth kit from Perkin Elmer (Foster City, CA) in the presence of 32P-dCTP, as described (53). One hundred nanograms of total RNA and 20 ng of poly-A+RNA were used in the RT-PCR reactions. The following primers were employed: KC, 5'-TCGCCAAT-GAGCTGCGCTGTC and 3'-GCTTCAGGGTCAAGG-CAAGCC, which amplify the 160-nt product; CXCR2, 5'-GCCTGTCTGGGCTGCATCTA and 3'-GTGTCTC-TTCTGGATCAGTG, which amplify the 180-nt product; EST-AL024215, 5'-ACAAGCCATGAAGCATGTGG and 3'-GGCTGCTATATAAGGAGATC, which amplify the 226-nt product; myeloperoxidase, 5'-CCGAATGACAAG-TATCGCAC and 3'-GGCACCTTGAAGCCATTGCG, which amplify the 170-nt product; IL-1{alpha}, 5'-GCAACGG-GAAGATTCTGAAG and 3'-TGACAAACTTCTGCCT-GACG, which amplify the 177-nt product; IL-1ß, 5'-GCCCATCCTCTGTGACTCAT and 3'-AGGCCACAGG-TATTTTGTCG, which amplify the 230-nt product; TNF{alpha}, 5'-CGTCAGCCGATTTGCTATCT and 3'-CGGACTCCG-CAAAGTCTAAG, which amplify the 206-nt product; and MIP2, 5'-AAGTTTGCCTTGACCCTGAA and 3'-AGG-CACATCAGGTACGATCC, which amplify the 180-nt product. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were described before (53). Annealing and extension temperature was 50°C for all primers, and number of cycles was 20 for GAPDH, 30 for KC, IL-1{alpha}, IL-1ß, TNF{alpha}, EST-AL024215, MIP2, and CXCR2, and 50 for myeloperoxidase. When poly-A+ RNA was used, 25 cycles were employed for determination of KC expression and 18 cycles for GAPDH expression. Polymerase chain reaction products were resolved on sequencing gels and visualized by autoradiography. The identity of PCR products was confirmed by sequencing.

RNase protection assays were done with templates for mouse collagen and GAPDH riboprobes as described (54). Templates for riboprobes for mouse cytokines and cytokine receptors were from Pharmingen (mCK-3b and mCR-6, respectively; San Diego, CA).

Western Blots.
Western blots were performed using anticollagen antibody from Rockland (600-401-103; Gilbertsville, PA), as described (55). This antibody has been characterized in a previous paper (55). Fifty micrograms of total protein from HSCs and 200 µg of total liver proteins were loaded on the gel. As control, the samples were reprobed with anti-{alpha} tubulin antibody (Zymed, San Francisco, CA). For analysis of the cell medium of HSCs, 40 µl of medium, representing the equivalent number of cells, was loaded without previous concentration.

Liver Enzymes Measurement.
Alanine amino-transferase (ALT) and aspartate aminotransferase (AST) were determined in sera of three animals per group by the standard UV method using commercial reagents (Stanbio Laboratory, Boerne, TX). Results were expressed as units per liter, and the mean ± SEM is indicated.

KC Measurement by ELISA.
Concentration of KC protein in sera and liver extracts was determined by ELISA (R&D Systems), according to manufacturer’s protocol. Liver extracts were prepared by homogenizing liver in phosphate-buffered saline, following by removal of debris by centrifugation. Total protein concentration was estimated in each sample, and 200–500 µg was used for assay. Results for liver extracts were normalized per gram of protein and calculated per gram of liver tissue assuming that proteins represent 28% of wet liver weight (56, 57). Each sample was assayed in duplicate, and the average is shown in Figure 4CGo.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 4. Autostimulation of KC expression in injured liver by recombinant KC. (A) Injection of KC protein stimulates expression of KC mRNA in CCl4-treated mouse liver. Mice were injected with a single dose of CCl4, and after 24 hrs, were injected with recombinant KC (RKC, lanes 5 and 6) or saline (C, lanes 3 and 4). Livers were harvested at 24 hrs after KC or saline injections (lanes 5 and 3) or at 48 hrs after KC or saline injections (lanes 4 and 6). Expression of KC was analyzed by RT-PCR using poly-A+RNA, as in Fig. 2AGo. GAPDH signal was equal in all lanes (data not shown). Lane 2 is normal liver (N) and lane 1 is size marker (M). (B) Recombinant KC does not upregulate its mRNA in the absence of liver injury. Mice received only an injection of recombinant KC, and after 48 hrs, expression of KC mRNA was analyzed in the liver, as in A (lane 3). Lane 2 is normal liver (N) and lane 1 is size marker (M). (C) Expression of KC protein in the liver correlates with expression of its mRNA. Two mice were injected with CCl4 for 24 hrs, followed by saline (C-48h; lanes 1 and 2), and two mice were injected with CCl4 followed by recombinant KC (RKC-48h; lanes 3 and 4). Forty-eight hours after the saline or KC injections, KC mRNA in the liver was determined by RT-PCR (top panel), and KC protein was determined in sera and livers by ELISA (bottom panel). KC concentration in the liver was expressed as nanograms per gram of total protein and as nanograms per gram of liver tissue, assuming that liver proteins correspond to 28% of wet liver weight (56, 57). ELISA determinations were done in duplicate, and the average value is shown.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
KC mRNA Is Induced in Acute Liver Injury.
To assess if KC expression is induced in acute liver injury, we injected mice with a single dose of CCl4 (2 µl/g), and after 3 days, analyzed expression of KC mRNA in total liver by RT-PCR. As shown in Figure 1Go, top panel, there was a several-fold increase in the steady-state level of KC mRNA in two livers treated with CCl4 (lanes 3 and 4) compared with control livers (lanes 1 and 2). A similar increase was seen in multiple experiments (see Fig. 4Go). As a control for RNA integrity, we amplified GAPDH mRNA and transcripts representing the expressed sequence tag AL024215. The latter encodes for a novel protein (designated as RIKEN cDNA 1810055D05). Both mRNAs were unchanged with CCl4 treatment (Fig. 1Go, bottom panels). From these experiments, we concluded that KC mRNA is upregulated in acute liver necrosis.

Activated HSCs Express KC mRNA.
Liver injury is associated with activation of HSCs, and activation of HSCs can be reproduced in vitro by culturing freshly isolated HSCs on plastic (5). To investigate if expression of KC increases during culture activation of HSCs, we analyzed the level of KC mRNA in freshly isolated rat HSCs (quiescent) and in HSCs cultured for 8 days, when they were fully activated. We used the primers for mouse KC, which are 100% homologous to the rat CINC1 sequence, and therefore designated the PCR product as KC. There was an increase in expression of KC mRNA in activated HSCs (Fig. 2AGo, lane 2), compared to quiescent HSCs (Fig. 2AGo, lane 1). GAPDH mRNA was equally expressed in these samples, suggesting a comparable quality of both RNA preparations. Based on these results, we concluded that there is no expression of KC in quiescent HSCs, whereas activated HSCs express KC chemokine. A similar finding was previously published (28).



View larger version (53K):
[in this window]
[in a new window]
  		Figure 2. Expression of KC mRNA and CXCR2 receptor mRNA in isolated HSCs. (A) Expression of KC mRNA in HSCs. Poly-A+ RNA was extracted from freshly isolated rat HSCs (Q, lane 1) or HSCs cultured for 8 days (A, lane 2) and analyzed by RT-PCR specific for KC mRNA (top panel) or GAPDH mRNA (bottom panel) as an internal control. (B) Induction of KC mRNA by inhibitors of protein synthesis in HSCs. Total RNA from activated rat HSCs (A, lane 1) or activated HSCs treated with cycloheximide for 3 hrs (A/CHX, lane 2) or puromycin for 3 hrs (A/PUR, lane 3) was analyzed by RT-PCR with primers specific for KC. Migration of the specific PCR product is indicated. (C) Expression of CXCR2 mRNA in HSCs. Rat HSCs were cultured for the indicated time-points when total RNA was extracted and analyzed by RT-PCR with primers specific for CXCR2 mRNA (lanes 2–7). Lane 1 is total RNA from rat spleen analyzed with the same primers as a positive control.

 
KC mRNA contains multiple AUUUA motifs in its 3' untranslated region (UTR), which are often found in the 3' UTRs of transcripts of immediate early genes and which regulate their stability. One of the characteristics of this class of mRNAs is that they can be superinduced by inhibitors of protein synthesis (58). To investigate if KC mRNA can be regulated by a similar mechanism, we treated activated HSCs with two inhibitors of protein synthesis and estimated the steady-state level of KC mRNA (Fig. 2BGo). Both cycloheximide (lane 2) and puromycin (lane 3) induced KC mRNA above the levels seen in untreated activated HSCs (lane 1) after 3 hrs. Thus, KC mRNA can be superinduced by inhibitors of protein synthesis in HSCs, suggesting that in HSCs, KC mRNA may be regulated at the level of mRNA stability.

The only known receptor that binds KC with high affinity is CXCR2 (34). Thus far, there has been no report about expression of this receptor in HSCs. To see if CXCR2 is expressed during activation of HSCs, we cultured rat HSCs and extracted RNA every day starting from Day 3 until Day 8 after isolation. These samples were analyzed for expression of CXCR2 mRNA by RT-PCR (Fig. 2CGo). The mRNA of CXCR2 was expressed in HSCs at Day 3 after isolation, while the cells still have the quiescent phenotype (Fig. 3AGo), and remained unchanged even when the HSCs became fully activated (Day 8). From these experiments, we concluded that HSCs constitutively express the CXCR2 receptor and therefore may respond to KC stimulation.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 3. Effect of recombinant KC on HSCs activated in culture. (A) Effect of KC on cellular levels of procollagen {alpha}1(I) (pro-COLL) and {alpha} smooth muscle actin ({alpha}-SMA). KC was added at 50 ng/ml to HSCs after 2 days in culture, and cellular proteins were analyzed by Western blot at the indicated time-points (left panel). Right panel are the same cells without KC. Expression of {alpha}-tubulin ({alpha}-TUB) was used as internal control. Migration of the specific bands is indicated to the right. (B) Effects of KC on procollagen secreted into cell medium. Cell medium from the cells in A was analyzed for accumulation of procollagen by Western blot. Migration of 180-kda procollagen {alpha}1(I) (pro-COLL) and proteolitically processed 120-kDa collagen {alpha}1(I) (COLL) is indicated to the right.

 
KC Increases Procollagen Secretion by HSCs.
Having established that HSCs express the KC receptor even in the quiescent state, we wanted to assess the effect of KC chemokine on rat HSCs activated in culture. To this goal we added recombinant KC to rat HSCs that had been cultured for only 2 days. At this time-point, HSCs still have the quiescent phenotype and do not express Type I collagen or {alpha}-smooth muscle actin ({alpha}-SMA) as markers of activation (Fig. 3Go). Fresh medium supplemented with 50 ng/ml of KC was added daily until Day 7. Cellular proteins and cell medium were collected every day starting from Day 3 until Day 8 after isolation. The samples were analyzed for procollagen {alpha}1(I) protein expression by Western blot (Fig. 3Go). Expression of procollagen {alpha}1(I) was first detected in cellular extracts of HSCs at Day 5 in culture (Fig. 3AGo, right panel, lane 3). Addition of KC did not change the temporal appearance of cellular procollagen {alpha}1(I) (Fig. 3AGo, left panel, lane 3). Cellular level of procollagen {alpha}1(I) increased until Day 8, but was unaffected by treatment with KC (Fig. 3AGo, lanes 4, 5 and 6). Another marker of HSC activation, {alpha}-SMA, become detectable at Day 6 in control cells and at day 5 in KC-stimulated cells. Analysis of the cellular medium revealed that KC induced a dramatic increase in collagen {alpha}1(I) secretion from HSCs (Fig. 3BGo). In control cells, accumulation of procollagen {alpha}1(I) in the cellular medium started at Day 4 (Fig 3BGo, right panel, lane 2), and procollagen {alpha}1(I) accumulated at low levels at the subsequent time-points. The only detected molecular form was the unprocessed {alpha}1(I) procollagen chain of 180 kDa. Hepatic stellate cells stimulated with KC started secretion of procollagen {alpha}1(I) also at Day 4 (Fig. 3BGo, left panel, lane 2), but accumulated a much larger total amount of {alpha}1(I) chain in the medium at the subsequent time-points (lanes 4, 5, and 6). Most of the collagen was in the form of mature collagen {alpha}1(I) polypeptide of 120 kDa, with lesser amounts of the procollagen {alpha}1(I) form of 180 kDa (Fig. 3BGo, left panel, lanes 4 and 5). Appearance of the mature collagen {alpha}1(I) chain suggested not only that KC stimulated secretion, but also the activation of Type I collagen processing enzymes. From these experiments, we concluded that KC stimulates HSCs to secrete and process Type I collagen in vitro.

Recombinant KC Increases Expression of Its mRNA in Injured Liver.
Because KC stimulated secretion of collagen {alpha}1(I) from HSCs, we wanted to see if KC was fibrogenic in an animal model of liver injury. We first induced acute liver injury by injecting a single dose of CCl4 into mice. Twenty four hours after CCl4 injection, the mice were injected through the tail vein with recombinant KC or saline, as a control. As another control, some mice were injected with mineral oil, followed by recombinant KC after 24 hrs. Recombinant KC was an active form of KC containing amino acids 29–96 and was free of carriers and endotoxins. The livers were harvested 24 or 48 hrs after the KC or saline injections and analyzed. Figure 4Go shows expression of KC mRNA in these livers. Injection of CCl4 followed by saline injection resulted in a several-fold upregulation of KC mRNA at 24 and 48 hrs after the saline injection (Fig. 4AGo, lanes 3 and 4) compared with normal liver (Fig. 4AGo, lane 2). This increase was similar to that shown in Figure 1Go. However, injection of CCl4 followed by injection of recombinant KC resulted in about a 100-fold overexpression of KC mRNA, which was sustained at 24 and 48 hrs after KC administration (lanes 5 and 6). Both RT-PCR products resolved in these lanes (indicated by arrows) were sequenced, and they both represent KC. They appeared because of denaturation of the abundant double-stranded RT-PCR product in the sequencing gel used and different migration of the two DNA strands. Therefore, total KC expression is the sum of these bands. Injection of oil followed by KC did not produce a high induction of KC mRNA (Fig. 4BGo, lane 3), suggesting that preexisting liver damage is a prerequisite for autostimulation of KC mRNA expression by the KC protein. This control also excludes the possibility that KC preparation was contaminated with endotoxin, a known inducer of chemokine mRNA (50). The experiment was repeated (Fig. 4CGo), with two additional animals receiving CCl4 followed by saline (C-48h) and two receiving CCL4 followed by recombinant KC (RKC-48h), and the samples were collected 48 hrs after the saline or KC injections. We analyzed expression of KC mRNA by RT-PCR (top panel) and KC protein by ELISA in sera and liver homogenates (bottom panel). The concentration in the liver was expressed as nanograms per gram of liver protein and as nanograms per gram of liver tissue, which was calculated assuming that proteins represent 28% of wet liver weight (56, 57). There was a 14-fold increase in KC protein in the livers of test animals, which correlated well with the expression of the mRNA. There was also increase in KC concentration in the sera of test animals, but the levels in the sera (nanograms per milliliter) remained about 6-fold lower that the levels in the liver (nanograms per gram; Fig. 4CGo). From these results, we concluded that a single injection of KC protein in the circulation potentates expression of KC gene in the liver, but only if there is preexisting acute necrosis.

KC Induces Expression of MIP2 in the Liver.
The high degree of induction of KC gene expression by recombinant KC in the injured liver prompted us to investigate what other genes are affected by overexpression of KC. To this goal, we analyzed expression of cytokines and chemokine receptors by RNase protection assay. Figure 5AGo shows expression of three chemokine receptors, CXCR2, CXCR4, and CXCR5, mRNA. We could detect increased expression of CXCR2 receptor mRNA, but only in the livers of mice that received CCl4 for 24 hrs followed by recombinant KC (lanes 6 and 7). This receptor was not induced by CCl4 treatment alone (lanes 3 and 4) nor was it expressed in normal liver (lane 2). The CXCR4 receptor was expressed at a low level in all the livers that received CCl4, whereas the CXCR5 receptor could not be detected in any sample. From this we concluded that overexpression of KC induces expression of its receptor (CXCR2) in the liver.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 5. Effects of KC on expression of chemokine receptors and cytokines in injured liver. (A) Expression of chemokine receptor mRNA. RNase protection assay for expression of CXCR2, CXCR4, and CXCR5 mRNAs in normal liver (N, lane 3) and in livers of mice 24 hrs after injection of CCl4 that was followed by saline for 24 (C-24h; lane 4) or 48 hrs (C-48h; lane 5) or by recombinant KC for 24 (RKC-24h; lane 6) or 48 hrs (RKC-48h; lane 7). Lane 2 is tRNA control (tRNA) and lane 1 is size marker (M). Expression of ribosomal protein L22 and GAPDH served as an internal control. Migration of specific bands is indicated. (B) Expression of cytokine mRNA. The same experiment as in A except a multi-riboprobe set specific for MIF, LT{alpha}, LTß, IL-6, INF{gamma}, INFß, TGFß1, TGFß2, TGFß3, and TNF{alpha} was used. (C) Expression of MIP2 and IL-1 mRNA. Two additional livers, described in Figure 4CGo, were analyzed by RT-PCR for expression of MIP2, IL-1{alpha}, IL-1ß, and TNF{alpha}.

 
Expression of various cytokines is shown in Figure 5BGo. Overexpression of KC resulted in minimal up-regulation of mRNA for TNF{alpha} and TGFß1. This was seen only at 48 hrs after KC administration (lane 7). No expression was detected in the other samples. Expression of MIF was unchanged in all samples compared with normal liver, and we did not detect expression of lymphotoxin (LT) {alpha}, LTß, IL-6, interferon (INF) {gamma}, INFß, TGFß2, and TGFß3 by RNase protection assay (RPA). To further analyze changes in expression of proinflammatory genes, we employed a more sensitive RT-PCR assay and assessed the expression of MIP2, IL-1{alpha}, IL-1ß, and TNF{alpha} in two additional test and control animals (Fig. 5CGo). Expression of MIP2 was strongly induced by KC, whereas there was no significant change in expression of IL-1{alpha}, IL-1ß, and TNF{alpha}. Thus, slight upregulation of TNF{alpha} seen in the RPA assay (Fig. 5BGo) was probably caused by variation among the animals, and we concluded that only MIP2 was strongly induced by KC.

KC Increases Collagen Synthesis in Injured Liver.
Transforming growth factor-ß induces Type I collagen synthesis in the liver (59). Because TGFß1 was induced by KC, we wanted to know if this was also associated with increased Type I collagen expression. We analyzed expression of collagen {alpha}1(I) mRNA by RPA (Fig. 6AGo) and collagen protein expression by Western blot (Fig. 6BGo). Injection of a single dose of CCl4 followed by saline resulted in upregulation of collagen {alpha}1(I) mRNA starting 2 days after the CCl4 injection (lane 4), followed by a severalfold increase at day 3 (lane 5). Injection of CCl4 followed by KC resulted in a delayed upregulation of collagen {alpha}1(I) mRNA. No mRNA was detected at Day 2 after CCl4 injection (lane 6), but an increased level was seen at Day 3 (lane 7). We could not take additional time-points because the animals had to be killed because of failing livers. Analysis of collagen content in the liver by Western blot revealed that injection of CCl4 increased procollagen {alpha}1(I) deposition in the liver at Day 2 (Fig. 6BGo, lane 2), with a subsequent increase by Day 3 (lane 3). This was in parallel with increase of collagen {alpha}1(I) mRNA in these samples (Fig. 6AGo, lanes 4 and 5). When animals received CCl4 followed by recombinant KC, we could not detect procollagen {alpha}1(I) at Day 2 after CCl4 (lane 4). However, 1 day later, there was a dramatic increase in procollagen {alpha}1(I) deposition in the liver (Fig. 6BGo, lane 5). This was concomitant with the increase in collagen {alpha}1(I) mRNA (Fig. 6AGo, lane 7). The predominant form was 120-kDa mature collagen {alpha}1(I) chain, and the total level was several-fold higher than in livers of mice without KC injection (cf. lanes 5 and 3). Appearance of the mature collagen {alpha}1(I) chain is of significance, because the terminal peptides must be cleaved off from procollagen for fibrils to polymerize. Therefore, appearance of the 120-kDa mature collagen {alpha}1(I) chain may indicate the initiation of fibrillogenesis. We concluded from these experiments that overexpression of KC increases the prefibrotic response in the liver, consistent with the results with culture-activated HSCs.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 6. Effects of KC on expression of collagen {alpha}1(I) mRNA and protein in injured liver. (A) Expression of collagen {alpha}1(I) mRNA. RNase protection assay with the samples described in Figure 5AGo. GAPDH served as an internal control, and migration of a nonspecific band is indicated by asterisk. (B) Expression of collagen {alpha}1(I) protein. Western blot with total liver proteins extracted from samples described in Figure 5AGo. Migration of 180-kDa pro-collagen {alpha}1(I) and mature 120-kDa collagen {alpha}1(I) chain is indicated. Asterisk indicates a nonspecific band, which can serve as an internal standard.

 
KC Increases Necrosis of the Liver Without Infiltration of Neutrophils.
The only known function of KC is chemotaxis of neutrophils to the site of tissue injury (22, 23, 60). To assess if proinflamatory changes (Fig. 5Go) and increased collagen synthesis (Fig. 6Go) were associated with accumulation of neutrophils in the liver, we analyzed liver sections by histology (Fig. 7AGo). In the two representative control animals, a single injection of CCl4 resulted in pericentral necrosis after 72 hrs, which was limited to discrete areas covering about 40%–50% of the sections (top panels). The mice that received KC after CCl4 showed larger areas of necrosis (covering about 80% of the sections), with the remaining parenchyma showing prenecrotic changes (bottom panels). Serum aminotransferase levels measured in three CCl4 + saline– or three CCl4 + KC–treated animals are shown in Figure 7BGo. Injection of CCl4 + recombinant KC increased serum AST and ALT levels by 3- to 5-fold compared with animals injected with CCl4 alone. From these results, we concluded that KC greatly enhanced liver injury.



View larger version (71K):
[in this window]
[in a new window]
  		Figure 7. Enhanced necrosis of the liver after overexpression of KC. (A) Histology of liver samples. (Top panels) Mice were injected with a single dose of CCl4 followed by saline injection 24 hrs later (CCl4 + CON). The livers were harvested 48 hrs after the saline injection. (Bottom panels) Mice were injected with a single dose of CCl4 followed by KC injection 24 hrs later (CCl4 + RKC). The livers were harvested 48 hrs after the KC injection. Liver section were stained with hematoxylin/eosin, and images were taken under x10 magnification. Expression of KC in these livers is shown in Figure 4CGo. (B) Serum aminotransferase levels. Serum AST and ALT levels were measured in three mice treated with CCl4 followed by saline (CCl4 + CON) or with CCl4 followed by recombinant KC (CCl4 + RKC). The blood samples were taken 48 hrs after the saline or RKC injections. The results are shown as units per liter, and the mean ± SEM is indicated.

 
Careful inspection of the independent liver histology samples by pathologists did not reveal infiltration of neutrophils. To confirm the absence of neutrophils found in the histological examination, we analyzed expression of myeloperoxidase mRNA as a marker of myeloid cells (61, 62). We employed sensitive RT-PCR (Fig. 8AGo), but despite using 50 cycles, which clearly detected myeloperoxidase mRNA in total RNA of blood cells of a normal mouse (Fig. 8AGo, lane 6), no expression of myeloperoxidase mRNA could be detected in the liver 24 hrs after KC (lane 4), with barely detectable expression after 48 hrs (lane 5). Myeloperoxidase mRNA also could not be detected in animals treated with CCl4 only or in a normal liver (lanes 1, 2, and 3). GAPDH expression, as determined by RT-PCR, was similar in all samples (data not shown). We repeated measurement of myeloperoxidase mRNA in two additional control and test animals at 48 hrs after saline or KC injections and did not see a difference in expression (Fig. 8BGo), despite a 14-fold higher KC concentration in the livers of KC-injected animals compared with control animals (Fig. 4CGo). We concluded that overexpression of KC did not result in significant accumulation of neutrophils within the liver and that the catastrophic liver failure associated with overexpression of KC must have been a result of direct hepatotoxicity of KC and MIP2.



View larger version (58K):
[in this window]
[in a new window]
  		Figure 8. Expression of myeloperoxidase mRNA. (A) Samples described in Figure 5AGo were analyzed by RT-PCR with primers specific for myeloperoxidase mRNA. B in lane 6 is total RNA extracted from blood cells that served as a positive control. Migration of the specific band is shown. (B) Two additional livers, described in Figure 4CGo, were analyzed by RT-PCR for expression of myeloperoxidase mRNA by RT-PCR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
KC, the mouse homolog of human gro-{alpha}, belongs to the CXC family of chemokines. The only well-documented function of this group of chemokines is chemotaxis and extravasation of neutrophils (30, 31). We now describe some novel effects of KC on isolated HSCs and whole liver. To specifically overexpress endogenous KC in the liver, we first induced acute liver injury by CCl4. Twenty-four hours after the CCl4, we intravenously injected a single dose of recombinant KC. This combination resulted in about a 14-fold increase in KC expression within the liver, which was sustained for at least 2 days (Fig. 4CGo). Because injected chemokines are cleared form the circulation within 2 hrs (63), a single KC injection most likely only served to activate the KC gene expression in the preinjured liver. Injection of KC without preexisting liver injury did not result in activation of endogenous KC gene expression (Fig. 4BGo). Autostimulation of KC gene expression by the KC protein was previously shown in endothelial cells (64) and in melanoma cells (65). Autocrine stimulation may be a common feature of CXC chemokines, because expression of the IL-8 gene can also be induced in monocytes by IL-8 protein (66). In our system, KC also induced expression of another CXC chemokine, MIP2 (Fig. 5CGo).

Using this approach, we showed that (1) KC expression is upregulated in acute liver injury; (2) HSCs express the receptor for KC, CXCR2, suggesting that they can be stimulated by KC; (3) addition of KC to HSCs undergoing activation in culture results in increased secretion of Type I collagen, indicating that KC may act as prefibrotic cytokine; (4) injection of KC into circulation of mice with preexisting acute liver necrosis results in dramatic upregulation of KC mRNA and protein; thus, KC may perpetuate its own synthesis in an injured liver by a positive feedback mechanism; (5) KC induces expression of its receptor, CXCR2, which may further enhance the downstream effects; and (6) overexpression of KC in the injured liver greatly augments liver damage without infiltration of neutrophils. Increased hepatotoxicity associated with high expression of KC was probably mediated by direct effects of KC on liver cells.

Activated HSCs express mRNA for KC at a low level (Fig. 2BGo, lane 1); however, hepatocytes are the main source of KC in the liver (67–70). Hepatocytes also express CXCR2 (71). Increased levels of gro-{alpha} and IL-8 in the liver are associated with increased neutrophil sequestration within the liver in alcoholic hepatitis (45, 46, 72) and in a rat model of alcohol-induced liver injury (73). These studies suggested that CXC chemokines are sufficient for chemotaxis of neutrophils into the liver. However, several studies have shown no correlation between high level of chemokines and infiltration of neutrophils. For example, in alcoholic cirrhosis, there is a marked increase in IL-8 expression in the liver without sequestration of neutrophils (72). Thyroid glands affected with Graves disease express gro-{alpha} at a high level, but there is no infiltration of neutrophils in the glands (74). Overexpression of human IL-8 in the liver of transgenic mice did not result in accumulation of neutrophils within the liver (47), although human IL-8 can cause chemotaxis of mouse neutrophils in vitro (33). Likewise, injection of recombinant KC into circulation of mice did not result in recruitment of neutrophils into the liver (75). In our model, despite about a 14-fold increased amount of KC in the liver (Fig. 4CGo), we could not see significant accumulation of neutrophils either by histology or by expression of myeloperoxidase mRNA (61, 62). The concentration of KC in the liver, expressed as nanograms per gram, was about 6-fold higher than concentration in plasma, expressed as nanograms per milliliter (Fig. 4CGo). Because chemokines injected into circulation have a half-life of 2 hrs (63) and high KC expression was sustained in the liver for 2 days, it is not likely that a lack of concentration gradient of KC between the liver and the plasma was responsible for the absence of chemotaxis (76). A more likely explanation is that chemotaxis of neutrophils into the liver is a complex process that requires, in addition to KC, some other factors. In a wound healing model, elevated levels of gro-{alpha} in the wound persisted for 2 weeks after the number of neutrophils in the wound has returned to normal, suggesting that an increased level of gro-{alpha} was not sufficient to maintain high number neutrophils at the site of injury (60). The rat homolog of KC, CINC1, overexpressed in rat livers by adenovirus delivery, increased neutrophil accumulation within the liver 4 days after viral injections (77). However, the number of neutrophils in the liver overexpressing CINC1 was only twice that of the control liver. At the same time, the overexpression of CINC1 caused liver damage, which was in excess of what could be attributed to the increased number of neutrophils. In fact, depletion of rat neutrophils did not have any influence on liver damage and fibrosis in a bile duct ligation model of liver fibrosis (78). Therefore, the authors concluded that CINC1 must have a direct hepatotoxic effect (77). Although we cannot exclude that KC contributes to chemotaxis of neutrophils into the liver, it is clear that direct effects of KC (and its rat homolog CINC1) on the liver are far more prominent than chemotaxis of neutrophils. These effects include massive hepatic necrosis (Fig. 7Go) and (77) and induction of expression of MIP2 and TGFß1. Bautista et al. (79) have reported that MIP2 is cytotoxic in alcoholic hepatitis, additional evidence that CXC chemokines have a direct hepatotoxic effect. Up-regulation of the CXCR2 receptor by KC suggests a positive feedback loop, which may have accelerated the liver failure. Involvement of other proinflammatory cytokines in this process could not be confirmed, because we did not see an increase in expression of TNF{alpha}, IL-1{alpha}, IL-1ß, LT{alpha}, LTß, IL-6, INF{gamma}, and INFß (Fig. 5Go). We are currently studying the change in expression of other genes resulting from high KC expression in the liver.

Activated HSCs express KC mRNA, whereas this mRNA could not be detected in quiescent HSCs (Fig. 2Go). A similar result was reported for rat CINC1 (28). KC mRNA can be induced in HSCs by inhibitors of protein synthesis (Fig. 2BGo), suggesting that KC mRNA may be regulated in HSCs at the level of mRNA stability, as reported for the other cell types (38). However, this is the first report that HSCs can express the receptor for KC, CXCR2 (Fig. 2Go). The fact that HSCs constitutively express CXCR2 suggests that quiescent HSCs can be stimulated by KC released by hepatocytes, endothelial cells, Kupffer cells, or by activated HSCs. Addition of KC to HSCs undergoing activation in culture resulted in increased extracellular accumulation of Type I collagen (Fig. 3Go). Nonstimulated HSCs secreted smaller amounts of procollagen {alpha}(I) chain of 180 kDa, whereas the KC-stimulated cells secreted much larger amounts of mature {alpha}1(I) chain of 120 kd (Fig. 3BGo). Because accumulation of 120-kDa collagen {alpha}1(I) chain indicates processing of procollagen and fibrillogenesis (80), the effect of KC on HSCs in vitro is prefibrotic. The cellular levels of procollagen protein were unchanged with KC treatment (Fig. 3AGo), suggesting that the prefibrotic effect is to increase the efficiency of export and processing of Type I collagen and/or to inhibit its degradation. A prefibrotic effect of KC was observed in vivo, as well, where overexpression of KC was associated with increased accumulation of 120-kDa collagen {alpha}1(I) chain in the liver (Fig. 6BGo). Expression of Type I collagen is regulated at the translational level by the 5' stem-loop RNA element (53). Binding of 5' stem-loop RNA binding proteins is required for synthesis of triple helical Type I collagen (55); therefore, the small increase in mRNA level may result in large increase in protein level (Fig. 6Go) because of activation of translation by increased activity of the 5' stem-loop binding proteins.

In summary, overexpression of KC in the liver greatly augments acute liver necrosis. KC also increases expression of MIP2. Together, these chemokines may exhibit direct hepatotoxic effect (77, 79). This effect is not associated with chemotaxis of neutrophils. KC acts as a prefibrotic cytokine on isolated HSCs; in the whole liver, it triggers massive necrosis that is associated with up-regulation of prefibrotic genes. Autostimulation of KC gene expression by the KC protein, which is also associated with upregulation of its receptor, indicates a positive feedback regulation. Such regulation indicates that any uncontrolled expression of KC may rapidly lead to catastrophic liver failure. Positive feedback regulation of KC and its role in direct liver damage is an important novel finding for liver pathology.


   Footnotes
 
This study was supported in part by NIH Grant 1R01DK59466-01A1 to B.S.

Received for publication January 26, 2005. Accepted for publication May 5, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Aycock RS, Seyer JM. Collagens of normal and cirrhotic human liver. Connect Tissue Res 23:19–31, 1989.[Medline]
  2. Milani S, Herbst H, Schuppan D, Surrenti C, Riecken EO, Stein H. Cellular localization of type I III and IV procollagen gene transcripts in normal and fibrotic human liver. Am J Pathol 137:59–70, 1990.[Abstract]
  3. Friedman SL. Hepatic stellate cells. Prog Liver Dis 14:101–130, 1996.[Medline]
  4. Friedman SL. Stellate cell activation in alcoholic fibrosis—an overview. Alcohol Clin Exp Res 23:904–910, 1999.[Medline]
  5. Friedman SL, Rockey DC, McGuire RF, Maher JJ, Boyles JK, Yamasaki G. Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture. Hepatology 15:234–243, 1992.[Medline]
  6. Geerts A, Vrijsen R, Rauterberg J, Burt A, Schellinck P, Wisse E. In vitro differentiation of fat-storing cells parallels marked increase of collagen synthesis and secretion. J Hepatol 9:59–68, 1989.[Medline]
  7. Nejjari M, Couvelard A, Mosnier JF, Moreau A, Feldmann G, Degott C, Marcellin P, Scoazec JY. Integrin up-regulation in chronic liver disease: relationship with inflammation and fibrosis in chronic hepatitis C. J Pathol 195:473–481, 2001.[Medline]
  8. Keegan A, Martini R, Batey R. Ethanol-related liver injury in the rat: a model of steatosis, inflammation and pericentral fibrosis. J Hepatol 23:591–600, 1995.[Medline]
  9. Marra F. Hepatic stellate cells and the regulation of liver inflammation. J Hepatol 31:1120–1130, 1999.[Medline]
  10. Marra F. Chemokines in liver inflammation and fibrosis. Front Biosci 7:1899–1914, 2002.
  11. Gant TW, Baus PR, Clothier B, Riley J, Davies R, Judah DJ, Edwards RE, George E, Greaves P, Smith AG. Gene expression profiles associated with inflammation, fibrosis, and cholestasis in mouse liver after griseofulvin. EHP Toxicogenomics 111:37–43, 2003.[Medline]
  12. Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, Luster MI. The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 177:112–120, 2001.[Medline]
  13. Thompson K, Maltby J, Fallowfield J, McAulay M, Millward-Sadler H, Sheron N. Interleukin-10 expression and function in experimental murine liver inflammation and fibrosis. Hepatology 28:1597–1606, 1998.[Medline]
  14. Koo DJ, Chaudry IH, Wang P. Kupffer cells are responsible for producing inflammatory cytokines and hepatocellular dysfunction during early sepsis. J Surg Res 83:151–157, 1999.[Medline]
  15. Kamimura S, Tsukamoto H. Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease. Hepatology 22:1304–1309, 1995.[Medline]
  16. Tsukamoto H. Redox regulation of cytokine expression in Kupffer cells. Antioxid Redox Signal 4:741–748, 2002.[Medline]
  17. Dong W, Simeonova PP, Gallucci R, Matheson J, Flood L, Wang S, Hubbs A, Luster MI. Toxic metals stimulate inflammatory cytokines in hepatocytes through oxidative stress mechanisms. Toxicol Appl Pharmacol 151:359–366, 1998.[Medline]
  18. Rowell DL, Eckmann L, Dwinell MB, Carpenter SP, Raucy JL, Yang SK, Kagnoff MF. Human hepatocytes express an array of proinflammatory cytokines after agonist stimulation or bacterial invasion. Am J Physiol 273:G322–G332, 1997.
  19. Knolle PA, Loser E, Protzer U, Duchmann R, Schmitt E, zum Buschenfelde KH, Rose-John S, Gerken G. Regulation of endotoxin-induced IL-6 production in liver sinusoidal endothelial cells and Kupffer cells by IL-10. Clin Exp Immunol 107:555–561, 1997.[Medline]
  20. Rieder H, Armbrust T, Meyer zum Buschenfelde KH, Ramadori G. Contribution of sinusoidal endothelial liver cells to liver fibrosis: expression of transforming growth factor-beta 1 receptors and modulation of plasmin-generating enzymes by transforming growth factor-beta 1. Hepatology 18:937–944, 1993.[Medline]
  21. Richmond A, Balentien E, Thomas HG, Flaggs G, Barton DE, Spiess J, Bordoni R, Francke U, Derynck R. Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin. Embo J 7:2025–2033, 1988.[Medline]
  22. Luster AD. Chemokines—chemotactic cytokines that mediate inflammation. N Engl J Med 338:436–445, 1998.[Free Full Text]
  23. Kunkel SL, Lukacs N, Strieter RM. Chemokines and their role in human disease. Agents Actions Suppl 46:11–22, 1995.[Medline]
  24. Huo Y, Weber C, Forlow SB, Sperandio M, Thatte J, Mack M, Jung S, Littman DR, Ley K. The chemokine KC, but not monocyte chemo-attractant protein-1, triggers monocyte arrest on early atherosclerotic endothelium. J Clin Invest 108:1307–1314, 2001.[Medline]
  25. Jinquan T, Frydenberg J, Mukaida N, Bonde J, Larsen CG, Matsushima K, Thestrup-Pedersen K. Recombinant human growth-regulated oncogene-alpha induces T lymphocyte chemotaxis. A process regulated via IL-8 receptors by IFN-gamma, TNF-alpha, IL-4, IL-10, and IL-13. J Immunol 155:5359–5368, 1995.[Abstract]
  26. Ohkubo K, Masumoto T, Horiike N, Onji M. Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells. J Gastroenterol Hepatol 13:696–702, 1998.[Medline]
  27. Maher JJ. Rat hepatocytes and Kupffer cells interact to produce interleukin-8 (CINC) in the setting of ethanol. Am J Physiol 269:G518–G523, 1995.
  28. Maher JJ, Lozier JS, Scott MK. Rat hepatic stellate cells produce cytokine-induced neutrophil chemoattractant in culture and in vivo. Am J Physiol 275:G847–G853, 1998.
  29. Haskill S, Peace A, Morris J, Sporn SA, Anisowicz A, Lee SW, Smith T, Martin G, Ralph P, Sager R. Identification of three related human GRO genes encoding cytokine functions. Proc Natl Acad Sci U S A 87:7732–7736, 1990.[Abstract/Free Full Text]
  30. Ono SJ, Nakamura T, Miyazaki D, Ohbayashi M, Dawson M, Toda M. Chemokines: roles in leukocyte development, trafficking, and effector function. J Allergy Clin Immunol 111:1185–1200, 2003.[Medline]
  31. Cameron MJ, Kelvin DJ. Cytokines and chemokines—their receptors and their genes: an overview. Adv Exp Med Biol 520:8–32, 2003.[Medline]
  32. Fujiwara K, Matsukawa A, Ohkawara S, Takagi K, Yoshinaga M. Functional distinction between CXC chemokines, interleukin-8 (IL-8), and growth related oncogene (GRO)alpha in neutrophil infiltration. Lab Invest 82:15–23, 2002.[Medline]
  33. Bozic CR, Gerard NP, von Uexkull-Guldenband C, Kolakowski LF, Jr., Conklyn MJ, Breslow R, Showell HJ, Gerard C. The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. J Biol Chem 269:29355–29358, 1994.[Abstract/Free Full Text]
  34. Lee J, Cacalano G, Camerato T, Toy K, Moore MW, Wood WI. Chemokine binding and activities mediated by the mouse IL-8 receptor. J Immunol 155:2158–2164, 1995.[Abstract]
  35. D’Ambrosio D, Panina-Bordignon P, Sinigaglia F. Chemokine receptors in inflammation: an overview. J Immunol Methods 273:3–13, 2003.[Medline]
  36. Olson TS, Ley K. Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol 283:R7–R28, 2002.[Abstract/Free Full Text]
  37. Horuk R. Chemokine receptors. Cytokine Growth Factor Rev 12:313–335, 2001.[Medline]
  38. Ryseck RP, MacDonald-Bravo H, Mattei MG, Bravo R. Cloning and sequence of a secretory protein induced by growth factors in mouse fibroblasts. Exp Cell Res 180:266–275, 1989.[Medline]
  39. Shibata F, Konishi K, Nakagawa H. Gene structure, cDNA cloning, and expression of the rat cytokine-induced neutrophil chemoattractant-2 (CINC-2) gene. Cytokine 10:169–174, 1998.[Medline]
  40. Nakagawa H, Komorita N, Shibata F, Ikesue A, Konishi K, Fujioka M, Kato H. Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization. Biochem J 301:545–550, 1994.
  41. Modi WS, Yoshimura T. Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals. Mol Biol Evol 16:180–193, 1999.[Abstract]
  42. Bautista AP 2002 Neutrophilic infiltration in alcoholic hepatitis. Alcohol 27:17–21, 1988.
  43. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia/ reperfusion injury in rat liver in vivo. Faseb J 4:3355–3359, 1990.[Abstract]
  44. Han DW. Intestinal endotoxemia as a pathogenetic mechanism in liver failure. World J Gastroenterol 8:961–965, 2002.[Medline]
  45. Maltby J, Wright S, Bird G, Sheron N. Chemokine levels in human liver homogenates: associations between GRO alpha and histopathological evidence of alcoholic hepatitis. Hepatology 24:1156–1160, 1996.[Medline]
  46. Sheron N, Bird G, Koskinas J, Portmann B, Ceska M, Lindley I, Williams R. Circulating and tissue levels of the neutrophil chemotaxin interleukin-8 are elevated in severe acute alcoholic hepatitis, and tissue levels correlate with neutrophil infiltration. Hepatology 18:41–46, 1993.[Medline]
  47. 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 94:1310–1319, 1994.
  48. Gardner CR, Laskin JD, Dambach DM, Sacco M, Durham SK, Bruno MK, Cohen SD, Gordon MK, Gerecke DR, Zhou P, Laskin DL. Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-alpha and interleukin-10. Toxicol Appl Pharmacol 184:27–36, 2002.[Medline]
  49. Enomoto N, Ikejima K, Yamashina S, Hirose M, Shimizu H, Kitamura T, Takei Y, Sato, Thurman RG. Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin. Alcohol Clin Exp Res 25(6 Suppl):51S–54S, 2001.[Medline]
  50. Thurman RG. Alcoholic liver injury involves activation of Kupffer cells by endotoxin. Am J Physiol 275:G605–G611, 1998.
  51. Hasegawa T, Sakurai K, Kambayashi Y, Saniabadi AR, Nagamoto H, Tsukada K, Takahashi A, Kuwano H, Nakano M. Effects of OPC-6535 on lipopolysaccharide-induced acute liver injury in the rat: involvement of superoxide and tumor necrosis factor-alpha from hepatic macrophages. Surgery 134:818–826, 2003.[Medline]
  52. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 162:156–159, 1987.[Medline]
  53. Stefanovic B, Hellerbrand C, Brenner DA. Regulatory role of the conserved stem-loop structure at the 5' end of collagen alpha1(I) mRNA. Mol Cell Biol 19:4334–4342, 1999.[Abstract/Free Full Text]
  54. Stefanovic B, Hellerbrand C, Brenner DA. Post-transcriptional regulation of collagen alpha 1(I) mRNA in hepatic stellate cells. Nucleic Acids Symp Ser 33:212–214, 1995.
  55. Stefanovic B, Brenner DA. 5' stem-loop of collagen alpha 1(I) mRNA inhibits translation in vitro but is required for triple helical collagen synthesis in vivo. J Biol Chem 278:927–933, 2003.[Abstract/Free Full Text]
  56. Noziere P, Attaix D, Bocquier F, Doreau M. Effects of underfeeding and refeeding on weight and cellularity of splanchnic organs in ewes. J Anim Sci 77:2279–2290, 1999.[Abstract/Free Full Text]
  57. Scheaffer AN, Caton JS, Bauer ML, Redmer DA, Reynolds LP. The effect of pregnancy on visceral growth and energy use in beef heifers. J Anim Sci 81:1853–1861, 2003.[Abstract/Free Full Text]
  58. Ross J. Control of messenger RNA stability in higher eukaryotes. Trends Genet 12:171–175, 1996.[Medline]
  59. Hellerbrand C, Stefanovic B, Giordano F, Burchardt ER, Brenner DA. The role of TGFbeta1 in initiating hepatic stellate cell activation in vivo. J Hepatol 30:77–87, 1999.[Medline]
  60. Engelhardt E, Toksoy A, Goebeler M, Debus S, Brocker EB, Gillitzer R. Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am J Pathol 153:1849–1860, 1998.[Abstract/Free Full Text]
  61. Crisan D, David D, DiCarlo R. Use of myeloperoxidase mRNA as a marker for myeloid lineage in acute leukemias. Arch Pathol Lab Med 120:828–834, 1996.[Medline]
  62. Pinkus GS, Pinkus JL. Myeloperoxidase: a specific marker for myeloid cells in paraffin sections. Mod Pathol 4:733–741, 1991.[Medline]
  63. Rennen HJ, Boerman OC, Oyen WJ, Corstens FH. Kinetics of 99mTc-labeled interleukin-8 in experimental inflammation and infection. J Nucl Med 44:1502–1509, 2003.[Abstract/Free Full Text]
  64. Wen DZ, Rowland A, Derynck R. Expression and secretion of gro/ MGSA by stimulated human endothelial cells. Embo J 8:1761–1766, 1989.[Medline]
  65. Norgauer J, Metzner B, Schraufstatter I. Expression and growth-promoting function of the IL-8 receptor beta in human melanoma cells. J Immunol 156:1132–1137, 1996.[Abstract]
  66. Browning DD, Diehl WC, Hsu MH, Schraufstatter IU, Ye RD. Autocrine regulation of interleukin-8 production in human monocytes. Am J Physiol Lung Cell Mol Physiol 279:L1129–L1136, 2000.[Abstract/Free Full Text]
  67. Shiratori Y, Takada H, Hikiba Y, Okano K, Niwa Y, Matsumura M, Komatsu Y, Omata M. Increased release of KC/gro protein, intercrine cytokine family, from hepatocytes of the chronically ethanol fed rats. Biochem Biophys Res Commun 197:319–325, 1993.[Medline]
  68. Shiratori Y, Takada H, Hai K, Kiriyama H, Mawet E, Komatsu Y, Niwa Y, Matsumura M, Shiina S, Kawase T, Kamii K, Omata M. Effect of anti-allergic agents on chemotaxis of neutrophils by stimulation of chemotactic factor released from hepatocytes exposed to ethanol. Dig Dis Sci 39:1569–1575, 1994.[Medline]
  69. Mawet E, Shiratori Y, Hikiba Y, Takada H, Yoshida H, Okano K, Komatsu Y, Matsumura M, Niwa Y, Omata M. Cytokine-induced neutrophil chemoattractant release from hepatocytes is modulated by Kupffer cells. Hepatology 23:353–358, 1996.[Medline]
  70. Horbach M, Gerber E, Kahl R. Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-alpha from rat hepatocyte cultures. Toxicology 121:117–126, 1997.[Medline]
  71. Bone-Larson CL, Hogaboam CM, Evanhoff H, Strieter RM, Kunkel SL. IFN-gamma-inducible protein-10 (CXCL10) is hepatoprotective during acute liver injury through the induction of CXCR2 on hepatocytes. J Immunol 167:7077–7083, 2001.[Abstract/Free Full Text]
  72. Afford SC, Fisher NC, Neil DA, Fear J, Brun P, Hubscher SG, Adams DH. Distinct patterns of chemokine expression are associated with leukocyte recruitment in alcoholic hepatitis and alcoholic cirrhosis. J Pathol 186:82–89, 1998.[Medline]
  73. Bautista AP. Chronic alcohol intoxication enhances the expression of CD18 adhesion molecules on rat neutrophils and release of a chemotactic factor by Kupffer cells. Alcohol Clin Exp Res 19:285–290, 1995.[Medline]
  74. Aust G, Steinert M, Boltze C, Kiessling S, Simchen C. GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells. J Endocrinol 170:513–520, 2001.[Abstract]
  75. Bajt ML, Farhood A, Jaeschke H. Effects of CXC chemokines on neutrophil activation and sequestration in hepatic vasculature. Am J Physiol Gastrointest Liver Physiol 281:G1188–G1195, 2001.[Abstract/Free Full Text]
  76. Wiekowski MT, Chen SC, Zalamea P, Wilburn BP, Kinsley DJ, Sharif WW, Jensen KK, Hedrick JA, Manfra D, Lira SA. Disruption of neutrophil migration in a conditional transgenic model: evidence for CXCR2 desensitization in vivo. J Immunol 167:7102–7110, 2001.[Abstract/Free Full Text]
  77. Maher JJ, Scott MK, Saito JM, Burton MC. Adenovirus-mediated expression of cytokine-induced neutrophil chemoattractant in rat liver induces a neutrophilic hepatitis. Hepatology 25:624–630, 1997.[Medline]
  78. Saito JM, Bostick MK, Campe CB, Xu J, Maher JJ. Infiltrating neutrophils in bile ductligated livers do not promote hepatic fibrosis. Hepatol Res 25:180–191, 2003.[Medline]
  79. Bautista AP. Chronic alcohol intoxication induces hepatic injury through enhanced macrophage inflammatory protein-2 production and intercellular adhesion molecule-1 expression in the liver. Hepatology 25:335–342, 1997.[Medline]
  80. Myllyharju J, Kivirikko KI. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet 20:33–43, 2004.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
E. Galloway, T. Shin, N. Huber, T. Eismann, S. Kuboki, R. Schuster, J. Blanchard, H. R. Wong, and A. B. Lentsch
Activation of hepatocytes by extracellular heat shock protein 72