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Departments of
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Molecular Biology and Genetics and
Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
Department of Surgery, Medical University of South Carolina, Charleston, SC 29425;
Department of Surgery, University of Rochester School of Medicine, Rochester, NY 14642; and
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Laboratory of Clinical Investigation, National Institutes of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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40 distinct secreted proteins that
share 20 to 70% sequence identity. Their known activities include
regulation of immune cell trafficking and function. Within the
chemokine family, four distinct subfamilies have been defined based
upon the presence and positioning of conserved cysteine residues. The
two largest and best-characterized subgroups are the CXC and CC
chemokines. Members of the CXC subfamily, also termed 
-chemokines,
contain a variable residue between the first two invariant cysteines;
these cysteine residues are adjacent in the CC or 
-chemokine
subfamily. The CXC chemokines may be further subdivided based upon the
presence or absence of a glutamic acid, leucine, arginine (ELR) motif
near the N terminus. CXC chemokines with the ELR motif have potent
neutrophil chemotactic activity and bind to the CXCR1 and CXCR2
receptors, while non-ELR CXC chemokines do not bind to these receptors
and have greater chemotactic activity for NK cells, lymphocytes, and
monocytes (1, 2). Studies are beginning to define novel
activities for various chemokines beyond those involved in inflammatory
cell trafficking. For example a CXC chemokine, stroma-derived factor-1,
and its receptor, CXCR4, have been demonstrated to be essential for
normal heart, cerebellum, B cell, and vascular development
(3, 4, 5), while other members of the CXC chemokine family
have been demonstrated to possess angiogenic and angiostatic activity,
with some having associated endothelial growth-regulatory activity
(2, 5, 6, 7).
IFN-inducible protein-10
(IP-10)4 is a non-ELR
CXC chemokine. It is a chemoattractant for NK cells and T cells
(2, 8), and is believed to be an important regulator of
the Th1, IL-12-driven, inflammatory response as an inducer of cellular
infiltration, including perhaps induction of IFN-
(9).
A single physiological receptor for IP-10, termed CXCR3, has been
identified (10, 11). CXCR3 is a shared receptor for
monokine induced by IFN- (Mig) (10, 12) and
IFN-inducible T cell -chemoattractant (13), the two
chemokines most highly related to IP-10. CXCR3 is found on T, NK, and B
cells (10, 14). Recently, IP-10 has been demonstrated to
bind a second receptor, the Kaposi’s sarcoma-associated herpesvirus G
protein-coupled receptor, although on this receptor IP-10 functions as
an inverse agonist (15).
In addition to T cell and NK cell chemotaxis and activation, IP-10 has antiproliferative effects on endothelial cells (6), as well as angiostatic and antitumor activity (6, 7, 16, 17). IP-10 also inhibits fibroblast motility (18). The murine ortholog of IP-10 is cytokine-responsive gene-2 (CRG-2), and the two proteins are 68% identical (19). Elevated Crg-2/IP-10 expression has been noted in numerous infectious and autoimmune diseases (2, 20, 21, 22). In the liver, IP-10 is expressed in autoimmune liver disease (23), chronic hepatitis (24), and biliary atresia (25). The degree of IP-10 expression has been shown to correlate with the severity of inflammation in hepatitis, being most highly expressed in chronic active hepatitis (24), and in those patients with biliary atresia and secondary hepatic injury (25). IP-10 has been detected in most of these models late in the disease process or during active inflammatory cell recruitment. In this study, we describe that Crg-2 was expressed as an immediate early gene following multiple types of acute hepatic injury and resection, including models that involve hepatocyte, oval cell, and cholangiocyte regeneration. Furthermore, Crg-2 was expressed just before each known wave of hepatocyte proliferation following hepatectomy. These data suggest that IP-10 is a general marker of hepatic inflammation and injury, and furthermore that IP-10 may play a fundamental role in hepatic repair and regeneration.
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Materials and Methods |
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Unless otherwise specified, all reagents were purchased from Sigma (St. Louis, MO). Recombinant human IP-10, IL-6, and TNF were purchased from PeproTech (Rocky Hill, NJ).
Isolation of RNA from human liver samples
Freeze-clamped normal human liver was obtained at time of donor hepatectomy, from fatty liver obtained after hepatectomy and perfusion for transplantation, or from diseased liver obtained at time of transplant. All liver samples were obtained in accordance with institutional guidelines (Medical University of South Carolina). Frozen tissue was homogenized into RNAzol B (Tel-Test, Friendswood, TX), according to the manufacturer’s instructions.
In vivo injury models
The animal care and use committee of the Johns Hopkins University School of Medicine approved all animal studies. Both surgical and toxic injury models used 5- to 7-wk-old male mice. CD-1 mice were purchased from Charles River Breeding Laboratories (Wilmington, MA). B6;129S wild-type and B6;129S Tnfrsf1atm11 mx Tnfrsf1btm11 mx (TNFR superfamily member 1a null and 1b null) mice were purchased from The Jackson Laboratory (stock number 003243; Bar Harbor, ME).
All surgical procedures were performed between the hours of 10 a.m. and 2 p.m. to reduce diurnal variation. All surgical models were performed through a midline abdominal incision under methoxyflurane (Metaphane; Mallinckrodt Veterinary, Mundelein, IL) general anesthesia. Controls for all surgical experiments underwent general anesthesia, midline abdominal incision, gentle hepatic or renal manipulation, and closure. Partial hepatectomy was performed with ligature of the middle and left hepatic pedicles, as described (26). Bile duct ligation was performed with 4-0 silk ligation of the extrapancreatic common bile duct (27). Five-sixths nephrectomy was performed with hilar ligation and excision of the right kidney and resection of the upper and lower left kidney poles. Following the two-thirds left nephrectomy, 30-s finger pressure was applied to the remaining kidney parenchyma to tamponade bleeding.
For toxic injury models, mice were challenged by i.p. injection of the toxic agent in solubilizing carrier, as described below; control groups received injection of toxin-free carrier. CCl4 was administered by i.p. injection of 20 µl in 0.1 ml vegetable oil (Wesson, Fullerton, CA). D-Galactosamine (GalN) was injected i.p. in 0.1 ml saline at 0.7 g GalN per kg body weight. Methylene dianiline (DAPM) was administered by i.p. injection of 50 mg in 0.1 ml of a 50% ethanol/saline solution. LPS serotype 055:B5 (Sigma) was injected i.p. at a dose of 100 µg in 0.1 ml saline.
Implantable osmotic pumps (Alzet, Palo Alto, CA) were placed using standard sterile surgical technique under methoxyflurane anesthesia. A midline abdominal incision was performed, and the preloaded pump containing either 200 µg human IP-10 in saline or saline alone was placed in the peritoneal cavity. The abdominal incision was closed with a two-layer running closure to prevent subsequent fluid leakage. Following pump placement, daily i.p. injections of 1 ml cell-labeling reagent (bromodeoxyuridine (BrdU) and fluorodeoxyuridine; Amersham Pharmacia, Piscataway, NJ) were given at 10 a.m. on postoperative days 1 through 6. Animals were killed on postoperative day 7. BrdU-labeled nuclei were detected on frozen sections using the FLUOS in situ cell proliferation kit (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer’s instructions, counterstained with 4',6'-diamidino-2-phenylindole (Molecular Probes, Eugene, OR), and mounted in ProLong antifade mounting medium (Molecular Probes).
Cyclohexamide pretreatment of mice was performed as described (28). Mice were injected with 50 mg/kg cycloheximide in 1 ml saline i.p. 1 h before two-thirds hepatectomy or CCl4 injection. Animals were sacrificed, and RNA was isolated using RNAzol B.
Separation of liver cell components and primary hepatocyte culture
Following 18 h of GalN treatment, C57BL/6 mice were placed under methoxyflurane anesthesia, and hepatic cell compartments were separated, as previously described (29). Briefly, a midline abdominal incision was performed, then the portal vein was identified and cannulated with a 24-gauge angiocatheter. Following return of portal blood, the liver was perfused for 5 min with liver perfusion medium, followed by 10 min of liver digest medium (Life Technologies, Gaithersburg, MD). After excision, the cell suspension was passed twice through a 70-µm filter (Falcon Cell Strainer, Franklin Lakes, NJ) and placed on ice. Separation into parenchymal and nonparenchymal fractions was performed using multiple low-speed centrifugations.
For primary culture, hepatocytes were isolated from normal CD-1 male mice by the two-step collagenase perfusion method (30). Hepatocytes were plated on collagen-coated dishes in William’s E medium with 10% FBS, HEPES, penicillin/streptomycin, gentamicin, glutamine, 10 mM glucose, 10 nM insulin, and 1 nM dexamethasone, and maintained in the same medium without serum. Human IP-10 was added to the maintenance medium at concentrations ranging from 1 ng/ml to 1µg/ml, in the presence or absence of 20 ng/ml epidermal growth factor (Collaborative Biomedical Products, Bedford, MA). Medium was changed every 24 h. For measurement of DNA synthesis, hepatocytes were pulse labeled for 24 h with [3H]thymidine at 0-, 24-, 48-, 72-, and 96-h postseeding. Cells were lysed, then fixed with 10% TCA, and the amount of [3H]thymidine incorporation into glass filter-bound DNA was measured by liquid scintillation counting.
Cell culture
AML-12 cells (American Type Culture Collection (ATCC) CRL-2254, Manassas, VA) were grown to near confluence. CCl4 was added directly to each plate to a final concentration of 20 or 40 mM. Control flasks were treated identically, but received no addition of CCl4. Subsequently, all flasks were sealed and incubated at 37°C and 30 rpm on an orbital platform. RNA was harvested using RNAzol B.
MRC-5 cell lines (ATCC CCL-171) were grown in 10% FBS in DMEM until nearly confluent. Cells were washed with PBS and stimulated with human IP-10 for 48 h. Cells were harvested and RNA was isolated as described for the AML-12 cells. In a separate experiment, cells were treated with IP-10 in 0.1% FBS in Eagle’s medium for 24 h, and supernatants were collected and frozen. Hepatocyte growth factor (HGF) content was determined using the Quantikine human HGF immunoassay (R&D Systems, Minneapolis, MN).
CRG-2-secreting lines were generated as previously described (17). Control cell lines were transfected with the empty vector and similarly selected. Cell lines were grown to near confluence in MEM (Life Technologies) with 10% dialyzed FBS (Life Technologies), proline, glutamine, penicillin, streptomycin, and 1.6 µM methotrexate (amithopterin). Before injection, cells were washed in PBS and trypsinized. Following two PBS washes, cells were resuspended at 5 x 106 cells in 0.2 ml PBS and injected i.m. into the thighs of female athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN).
Northern blot analysis
RNA was isolated by homogenization in guanidinium thiocyanate or RNAzol B, as described (31). Where specified, once selected poly(A)+ RNA was generated using oligo(dT) cellulose, as described (31), or by using an Oligotex mRNA isolation kit (Qiagen, Valencia, CA). Total RNA (20 µg/lane) or poly(A)+ RNA (2 µg/lane) was subjected to Northern blotting analysis on GeneScreen Plus (NEN Life Science Products, Boston, MA), as described (31).
Western blot analysis
The right liver lobes were collected 3 h following treatment and snap frozen in liquid nitrogen. Liver samples were weighed, and equal amounts were homogenized in PBS. Protein concentration was determined by Coomassie Plus-200 Protein Assay Kit (Pierce, Rockford, IL). Equal amounts of protein from each sample were prepared in sample buffer and run on reducing SDS-PAGE. Samples were transferred to nitrocellulose (Schleicher & Schuell, Relliehausen, Germany) and blotted using a rabbit polyclonal anti-mouse CRG-2 antiserum (5171) diluted 1/20,000 in TBS with 0.03% Tween 20 and 0.5% nonfat dried milk (Bio-Rad, Richmond, CA). Bands were detected using HRP-conjugated goat anti-rabbit IgG (Pierce) and the SuperSignal West Pico detection kit (Pierce), according to instructions provided by the manufacturer.
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Results |
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Northern blots were prepared with RNA isolated from freeze-clamped
normal human liver obtained at time of donor hepatectomy, from fatty
liver obtained after hepatectomy and perfusion for transplantation, or
from diseased liver removed from the recipient at time of transplant.
As shown in Fig. 1
, markedly increased
expression of both IP-10 and the related chemokine gene,
MIG, was observed in several patients with end-stage liver
disease.
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Given the observed induction of IP-10 in livers of
patients with end-stage liver disease, we set out to examine whether
murine parenchymal cells could express Crg-2 directly
following injury. The differentiated, nontransformed hepatocyte cell
line, AML-12 (32), was treated with the potent hepatotoxin
CCl4 in vitro. A dose-responsive induction of
Crg-2 was seen following administration of 20 and 40 µM
CCl4, with peak expression at 3 h (Fig. 2). Of note, no such induction was seen
for the Mig gene (data not shown). These data show that
Crg-2 may be induced as a direct consequence of cellular
injury, occurring in the absence of inflammatory or other mediating
cells.
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Given the induction of IP-10 in end-stage cirrhosis,
and Crg-2 in AML-12 cells following
CCl4 exposure, we examined expression of
Crg-2 in mouse models of liver and bile duct injury that
preferentially injure and stimulate distinct liver cell populations.
Examination of Crg-2 expression following these models of
liver injury also demonstrated dramatic induction. Crg-2 was
induced following bile duct ligation and administration of DAPM (Fig. 3, A and B), two
interventions that preferentially injure bile ducts and induce
subsequent biliary epithelial cell proliferation (27, 33).
Administration of the RNA synthesis inhibitor, GalN, which is
hepatotoxic and requires the liver to recruit oval cells for subsequent
liver regeneration (34, 35), also potently induced
Crg-2 (Fig. 3C).
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Induction of Crg-2 in the AML-12 cell line suggested
that Crg-2 could be induced in hepatocytes in response to
injury. We next evaluated the expression of Crg-2 in hepatic
cell compartments following exposure to GalN. Parenchymal and
nonparenchymal cell fractions were obtained from mouse livers isolated
18 h after GalN injection when Crg-2 expression peaked.
By Northern analysis, Crg-2 expression was noted in both
parenchymal and nonparenchymal fractions. Excellent separation of the
parenchymal and nonparenchymal populations was achieved, as evaluated
by Northern analysis of the cell type-specific markers
albumin (parenchymal), and Hgf (nonparenchymal),
respectively (Fig. 4).
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Given the dramatic induction of Crg-2 following various
forms of hepatic injury, we expanded our studies to evaluate
Crg-2 in liver regeneration using two-thirds hepatectomy. In
contrast to the models examined above, partial hepatectomy produces
only minimal injury to the remnant liver around the time of surgery,
but results in a 70% loss of total hepatic mass, thereby eliciting a
vigorous regenerative response (26). This compensatory
hyperplasia results in a return to normal liver mass after two
coordinated cycles of hepatic cell division within days of surgery
(26). We performed Northern blot analysis of liver mRNA
isolated from CD-1 mice subjected to two-thirds hepatectomy.
Crg-2 was dramatically induced in a bimodal fashion
following two-thirds hepatectomy in the mouse (Fig. 5). The observed increased level of
Crg-2 expression began within 1 h of hepatic resection,
peaked at 3 h, and returned to baseline by 12 h. A second
peak of expression was noted at 4 days following hepatectomy.
Therefore, expression of Crg-2 is associated with both the
early injury and the later repair and precedes each of the two peaks of
DNA synthesis known to occur in the mouse during liver regeneration
(36). In contrast, Mig was induced late in the
regenerative response after hepatectomy, coinciding with only the
second observed peak of Crg-2 expression (Fig. 5).
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Given the dramatic induction of Crg-2 mRNA following
various forms of hepatic injury, we sought to determine whether CRG-2
protein levels were also elevated. Indeed, Western blot analysis using
a polyclonal antiserum generated against rCRG-2 revealed increased
CRG-2 protein in extracts from injured livers taken 3 h after
injury as compared with normal livers (Fig. 6). Injection of LPS, a known stimulant
of Crg-2 production, was used as a positive control.
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We considered whether the expression of Crg-2
immediately after partial hepatectomy might be part of a systemic
response to injury. Examination of nonhepatic tissues following
two-thirds hepatectomy demonstrated increased expression of
Crg-2 in kidney, small bowel, thymus, and spleen 1 h
after hepatectomy (Fig. 7), but not at 4
days during the second peak of Crg-2 expression in the
liver. Therefore, Crg-2 appears to be expressed in the liver
and other organs within 1 h of two-thirds hepatectomy in a time
course suggestive of a growth-regulatory or tissue-repair factor.
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), Crg-2 expression
following five-sixths nephrectomy was also examined. As shown in Fig. 8, dramatic up-regulation of
Crg-2 was observed in this model as well.
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The time course of Crg-2 induction after tissue or cell
injury suggested that Crg-2 is part of the acute response to
tissue damage or insufficiency. To investigate this possibility
further, CD-1 mice were pretreated with the protein synthesis inhibitor
cycloheximide and challenged with either two-thirds hepatectomy or
CCl4 injection. In both cases, expression of
Crg-2 was noted to be dramatically up-regulated in animals
pretreated with cycloheximide, characteristic of an immediate early
gene (Fig. 9, A and
B). As has been previously described (28),
c-myc superinduction was also observed following
cycloheximide pretreatment (Fig. 9A).
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A). IL-6, another known
regulator of liver regeneration, did not induce Crg-2 (Fig. 10A). To determine whether TNF signaling is required for
Crg-2 expression following liver resection or injury, TNFR
superfamily members Ia and Ib doubly null C57BL/6;129SVJ hybrid mice
were subjected to two-thirds hepatectomy, and total liver mRNA was
evaluated by Northern blot analysis. Crg-2 induction in
double knockouts and wild-type controls following
CCl4 toxic hepatic injury was similar (Fig. 10B), suggesting that although exogenous TNF induced an
elevation in Crg-2, the pathway for Crg-2
induction following liver injury could be TNF independent.
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The expression of Crg-2 before the peaks of liver DNA synthesis following hepatectomy led us to examine whether IP-10 may be functioning as a direct regulator of hepatic growth. We examined the ability of IP-10 to stimulate hepatocyte DNA synthesis and proliferation in primary murine hepatocyte cell cultures and in vivo. No increase in [3H]thymidine incorporation was observed in murine hepatocytes treated for 24–96 h with 1 ng/ml to 1 µg/ml IP-10 (data not shown). As well, no increase in liver mass or hepatocyte nuclear BrdU labeling was observed in mice treated for 1 wk with an i.p. pump releasing IP-10 at a rate of 1 µg/h (data not shown). Furthermore, nude mice bearing Chinese hamster ovary cell tumors secreting CRG-2 experienced only a slight increase in liver mass when compared with control mice bearing Chinese hamster ovary cell tumors not expressing CRG-2 (data not shown).
We next evaluated the possible role of IP-10 in regulating the
expression of the potent hepatic mitogen, HGF. Interestingly,
expression of Hgf, like that of Crg-2, occurs in
a bimodal fashion following partial hepatectomy, with each peak of
expression preceding a known peak of hepatocyte DNA synthesis
(40, 41, 42), with the described expression of Hgf
following that observed for Crg-2. Moreover, an
Hgf-inducing activity has been isolated from serum fractions
obtained from rats with injured and regenerating liver (42, 43). The human lung fibroblast cell line, MRC-5, can be used to
assay for this activity. Therefore, we used this cell line to evaluate
the ability of IP-10 to induce HGF protein expression by ELISA and
Hgf mRNA by Northern blotting. MRC-5 cells demonstrated
secretion of HGF in response to IP-10 in a dose-dependent fashion. The
EC50 of IP-10 for induction of HGF secretion was
300 ng/ml (Fig. 11B), but an
increase of Hgf mRNA was noted in response to 10 and 100
ng/ml IP-10 (Fig. 11A). These data suggest a potential role
for IP-10 in HGF production before the regenerative response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(45), IL-1
(2), and TNF (45). Systemic injection of LPS,
IFN-, or TNF has been noted to induce Crg-2 expression
potently in the liver (45, 46). In human liver disease,
increased expression of IP-10 has been demonstrated in
chronic hepatitis (24) and biliary atresia
(25). In both disorders, the extent of IP-10
expression correlates with the degree of inflammation. Rat studies have
demonstrated increased expression of rat IP-10 in severe,
chronic alcohol-induced hepatitis and have correlated degree of
expression with necrosis (47). Elevated Crg-2
expression has also been noted in the lung during the inflammatory
response following hepatic ischemia-reperfusion injury
(48). Our results demonstrating increased IP-10
expression in diseased human livers are consistent with these
observations and further demonstrate concomitantly elevated expression
of Mig, a related chemokine. Given the wide expression pattern of IP-10 and Crg-2 in chronic liver diseases, we investigated the expression of Crg-2 following hepatic injury. The hepatocyte cell line, AML-12, was injured with the known hepatotoxin, CCl4. A dose-responsive induction of Crg-2 was observed, suggesting that initial Crg-2 expression precedes the inflammatory response following liver injury, and further that Crg-2 expression may occur as a direct parenchymal response to cellular injury in the absence of inflammatory cells. To define Crg-2 expression following liver injury in vivo, we next examined the time course of Crg-2 expression in multiple models of liver injury. Three distinct models of liver injury were examined: bile duct ligation, DAPM injury, and GalN toxic injury. In contrast to CCl4, which induces hepatocellular injury and a hepatocyte proliferative response in vivo, bile duct ligation and DAPM injury induce extensive biliary tree injury and a resultant bile duct proliferative response (27, 33). Expression of Crg-2 was increased markedly and rapidly following the injury in both models, again in a fashion suggesting that Crg-2 induction preceded the inflammatory response. The third model examined exposure to the hepatotoxic RNA synthesis inhibitor GalN, which induces irreversible injury and widespread hepatocellular death and requires liver progenitor oval cells to undergo a subsequent proliferative response to reestablish hepatocellular mass (35). In this model also, increased expression of Crg-2 was observed. Interestingly, expression of Crg-2 is delayed in this model, consistent with the known onset of hepatotoxicity after GalN injury (34).
In situ hybridization studies have reported conflicting results for
hepatic Crg-2 expression following injection of LPS, TNF, or
IFN-, with both parenchymal and nonparenchymal sources having been
suggested (24, 46). Given the novel observation that the
transformed hepatocyte line, AML-12, expressed Crg-2 in
vitro following exposure to CCl4, we examined
Crg-2 expression in an in vivo injury model as well. The
GalN injury model was selected because it causes parenchymal injury as
well as a nonparenchymal proliferative response along with a prolonged
elevation of Crg-2 expression. Northern blot analysis of
mRNA isolated from these fractions revealed a signal for
Crg-2 in both parenchymal and nonparenchymal cells. Taken
together, these data suggest that both parenchymal and nonparenchymal
liver cells can induce Crg-2 expression as a direct response
to liver injury. This is reminiscent of Crg-2 expression
occurring as a direct response to viral infection through an NF-
B
pathway (20). Of note, NF-B activation, presumably
through TNF signaling, is a well-described phenomenon occurring within
30 min of liver injury, and thus may be involved in
Crg-2/IP-10 expression (36).
In the models of liver injury in which Crg-2 was induced,
death of hepatocytes or biliary epithelial cells is followed by a
potent proliferative response. Therefore, we wished to investigate the
possibility that Crg-2 might be functioning not only in
response to acute and chronic liver and biliary injury, but as a signal
of regeneration as well. Partial (two-thirds) hepatectomy is a model
that involves surgical removal of the left lateral and medial liver
lobes, with minimal, early hepatocellular injury to the remaining right
liver lobes that subsequently undergo a regenerative response
(26) (for review, see Refs. 36 and
49, 50, 51). In this model, rapid, highly reproducible
induction of hepatocyte cell division occurs with an initial wave of
DNA synthesis peaking at 40 h. In the mouse, a second peak of DNA
expression occurs at 4 days. Liver mass, through compensatory
hyperplasia, is reestablished within 1 wk of resection. Interestingly,
we found that Crg-2 RNA and protein, as in the previously
examined models of liver injury, were induced within 1 h of
two-thirds hepatectomy. Examination of other organs following
two-thirds hepatectomy also demonstrated increased expression of
Crg-2 in small bowel, kidney, thymus, and spleen,
demonstrating that the Crg-2 expression is initially a
systemic response to hepatic injury. A second peak of Crg-2
expression occurs at 4 days following two-thirds hepatectomy, a time
that corresponds with the second wave of hepatic mitosis during
regeneration. At this time, Mig was also expressed.
Incidentally, these results suggest that no IFN- is released
immediately post liver resection because no Mig expression
was observed at that time. However, it does suggest IFN- is present
at 4 days following two-thirds hepatectomy because Mig
induction is known to be highly IFN- specific.
The rapid induction of Crg-2 in the liver following systemic TNF administration is similar to that observed following two-thirds hepatectomy, suggesting that TNF, which is detectable in liver venous blood within 1.5 h of hepatic injury or resection (52), may be inducing its production. To examine whether Crg-2 may be induced as a TNF-dependent signal, TNFR superfamily member Ia and Ib doubly null mice were compared with colony controls following CCl4-induced toxic liver injury. Expression of Crg-2 appeared uninhibited in the livers of doubly null mice, demonstrating that Crg-2 expression, although TNF inducible, can occur independently of TNF. One may speculate that the known LPS release from the gut after hepatectomy (53) could be a distinct, redundant inducer of Crg-2 after liver resection.
Because Crg-2/IP-10 was induced in a pattern suggesting a
role in the regenerative response to hepatectomy, we investigated a
possible direct effect of CRG-2/IP-10 on liver growth. No direct
mitogenic effects upon hepatocytes could be demonstrated with either
IP-10 or CRG-2 in vivo or in vitro. However, CRG-2/IP-10 could be
having an indirect effect on regeneration through induction of liver
growth factors. In this regard, Matsumoto and coworkers
(42) have reported a partially purified activity that they
termed injurin, a serum protein fraction with potent HGF-inducing
activity produced following two-thirds hepatectomy or
CCl4 injury in the rat. Interestingly, CRG-2 has
many similarities to injurin. The time course for Crg-2
expression is similar to that reported for injurin and for HGF, even
demonstrating a bimodal expression pattern (40, 41).
Elevated injurin activity has been observed at 2 days after
hepatectomy in the rat, a time point that corresponds to the second
peak of DNA synthesis that occurs at 4 days in the mouse. Increased
renal Crg-2 expression was also observed acutely following
five-sixths nephrectomy, another injurin-inducing procedure. Moreover,
the partially purified activity noted by Matsumoto (42)
was a low molecular mass, stable protein.
To examine the possibility that CRG-2 may possess injurin activity, the HGF-secreting cell line, MRC-5, was treated with IP-10. Elevations of Hgf mRNA and increased HGF secretion by MRC-5 cells were noted following stimulation with IP-10. Unfortunately, neither injection of IP-10 into normal animals, nor growth of CRG-2-secreting tumors in nude mice elicited liver growth. Nor could we reproducibly demonstrate increased Hgf mRNA in vivo following injection of IP-10. Our inability to correlate the increase in HGF secretion in vitro with induction of liver growth in vivo may be due either to insufficient HGF stimulation by exogenously supplied CRG-2/IP-10 or to the requirement for additional, priming signals that are present following liver injury (see Ref. 50).
Given its known activities, another indirect role for CRG-2/IP-10 in
liver regeneration might be in recruitment of T cells and NK cells.
Injection of IP-10 is known to induce a local, predominantly
lymphocytic cellular infiltrate (54). T cells,
particularly after activation, as well as NK cells, are known to
respond to IP-10 (55, 56, 57). Thus, the early expression of
Crg-2 may function as a chemotactic signal to bring T cells
and NK cells into the liver. Interestingly, Tamaru and coworkers
(58) have recently reported that Crg-2 is
required for liver infiltration of T cells in the
Propionibacterium acnes/LPS-induced model of hepatitis.
Moreover, it is well known that a T cell infiltrate occurs during
normal hepatic regeneration. The importance of this T cell role has
been underscored by the observation that athymic, T cell-deficient nude
mice have a markedly delayed hepatic regenerative response and an
increased mortality rate following hepatic resection or injury
(59, 60). Similarly, C/EBP--deficient mice, which
possess an abnormal Thl response, also demonstrate abnormal liver
regeneration, although evidence for abnormal hepatocyte function in
these animals also exists (61). Finally, NK cells, another
CRG-2/IP-10 target, have recently been implicated in immune
surveillance during liver regeneration (62). If
CRG-2/IP-10 supports liver regeneration through the recruitment of T or
NK cells, then local production of CRG-2/IP-10 may be critical,
explaining our failure to demonstrate an effect with protein
administered systemically.
Taken together, these results imply that CRG-2/IP-10 may be present in a wider spectrum of conditions than have previously been recognized. As well, these data suggest a potential role for CRG-2/IP-10 in mediating not only the hepatic inflammatory response, but also liver regeneration following hepatocyte loss. CRG-2/IP-10 may well be a cytokine that links tissue injury and inflammation to repair and regeneration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leonidas G. Koniaris, Department of Surgery, University of Rochester School of Medicine and Dentistry, 601 Elmwood Street, Rochester, NY 14642. E-mail address: leonidas_koniaris{at}urmc.rochester.edu
3 Current address: Department of Surgery, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
4 Abbreviations used in this paper: IP-10, IFN-inducible protein-10; BrdU, bromodeoxyuridine; CRG-2, cytokine-responsive gene-2; DAPM, methylene dianiline; GalN, D-galactosamine; HGF, hepatocyte growth factor; ELR, glutamic acid, leucine, arginine; Mig, monokine induced by IFN-.
Received for publication June 19, 2000. Accepted for publication April 18, 2001.
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over IL-4 responses. FASEB J. 12:705. chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009.-inducible protein 10 (IP-10) inhibits constitutive signaling of Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor. J. Exp. Med. 188:405. in astrocytes and microglia. J. Immunol. 152:1411.[Abstract]
with patterns of tissue expression that suggest nonredundant roles in vivo. J. Immunol. 157:4511.[Abstract]
superfamily member induced following liver injury. Mol. Cell. Biol. 20:3742.C expression following partial hepatectomy. Biochem. Biophys. Acta 235:553.
. Proc. Natl. Acad. Sci. USA 91:674. inhibit liver regeneration after partial hepatectomy. Am. J. Physiol. 263:G579. contribute to liver repair following chemical-induced hepatotoxicity. Hepatology 25:133.[Medline]
expression during liver regeneration after partial hepatectomy and toxic injury, and potential interactions between transforming growth factor- and hepatocyte growth factor. Hepatology 18:1422.[Medline]
induces cell type and tissue-specific expression of chemoattractant cytokines in vivo. Am. J. Pathol. 142:861.[Abstract]
. J. Leukocyte Biol. 52:27.[Abstract]
B and cytokine imbalance in experimental alcoholic liver disease in the rat. Hepatology 30:934.[Medline]
and chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol. 155:3877.[Abstract]
is required for normal hepatocyte proliferation in mice after partial hepatectomy. J. Clin. Invest. 102:996.[Medline]
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