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The Journal of Immunology, 1998, 160: 6056-6061.
Copyright © 1998 by The American Association of Immunologists

IL-6 Produced by Kupffer Cells Induces STAT Protein Activation in Hepatocytes Early During the Course of Systemic Listerial Infections1

Stephen H. Gregory2,*, Edward J. Wing*, Kristine L. Danowski*, Nico van Rooijen{ddagger}, Kevin F. Dyer*,{dagger} and David J. Tweardy*,{dagger}

* Department of Medicine and the {dagger} University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and {ddagger} Department of Cell Biology, Free University, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. inoculation of Listeria monocytogenes. IL-6 mRNA expression and the production of IL-6 were reduced drastically within the nonparenchymal liver cell population derived from mice rendered Kupffer cell depleted by pretreatment with liposome-encapsulated dichloromethylene diphosphonate. A sharp increase in the appearance of activated STAT3 occurred in extracts of purified hepatocytes derived from normal mice infected i.v. with Listeria. Remarkably, the kinetics of this increase overlapped IL-6 mRNA expression by Kupffer cells; each peaked at approximately 30 min postinfection. No increase in STAT3 activation was observed in IL-6-deficient or Kupffer cell-depleted animals. The results of these experiments indicate that the synthesis of IL-6 and the activation of STAT3 within hepatocytes are critical functions of Kupffer cells occurring very early during the course of systemic listerial infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-6 is a pleiotropic cytokine produced by a variety of cell types, including hepatocytes (1), mononuclear phagocytes (2, 3), neutrophils (4, 5, 6), and endothelial cells (2). Among the many effects of IL-6 are rapid induction of peripheral blood neutrophilia (7), proliferation of T cells (8, 9, 10), and stimulation of acute phase protein synthesis by hepatocytes (11, 12). Activation of these cell populations is dependent upon the binding of IL-6 to cell surface receptors. Ligand-cytokine receptor interaction results in tyrosine phosphorylation and the activation of STAT proteins 3 and 1. Homodimers and heterodimers composed of activated STAT3 and STAT1 subsequently translocate from the cytoplasm to the nucleus where they bind characteristic enhancer elements and initiate gene transcription (13, 14, 15).

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium capable of causing severe and occasionally fatal infections in humans (16, 17). Listeriosis in mice is used extensively as a model to study the response to systemic infections and the factors that effect resistance to intracellular pathogens (18, 19). Following i.v. injection, the bulk of Listeria is cleared rapidly by the liver. Sixty percent is recovered in the liver at 10 min postinfection and is associated with the parenchymal (hepatocytes) as well as the nonparenchymal (NPC)3 cell populations (20). Between 10 min and 6 h postinfection, the amount of Listeria in the liver declines by 0.5 to 1.0 log10 (18, 20). This initial decrease in liver Listeria correlates with the massive influx of neutrophils. In contrast to normal animals, mice rendered neutrophil-deficient by pretreatment with monoclonal anti-granulocyte Ab (RB6-8C5) exhibit a marked increase, rather than a decrease, in liver Listeria during the same time frame (20). Cell separation experiments indicate that >90% of the organisms recovered in the liver at >=2 h postinfection are located within hepatocytes, which serve as the principal site of listerial replication in the liver (20, 21).

IL-6 mRNA is expressed in the livers of mice very early during the course of listerial infection (22). The rapid induction of IL-6 mRNA expression has led others to suggest that Kupffer cells are the probable source (22). Indeed, using in situ hybridization, one study localized IL-6 message to cells located within the liver sinusoids at 3 days, but not at 1 day, postinfection (23). Inasmuch as hepatic endothelial cells (2), hepatocytes (1), and immigrating neutrophils (4, 5, 6) can also produce IL-6, the cell source of cytokine produced early during the course of infection remains unclear.

It is generally assumed that most bacteria that are cleared from the bloodstream and taken up in the liver are internalized and killed by Kupffer cells (24, 25, 26, 27). Since neutrophils, not Kupffer cells, kill the vast majority of organisms trapped in the liver (20), this study was undertaken to re-examine the function of Kupffer cells in host defenses to bacterial pathogens in the blood. Our results indicate that Kupffer cells are the principal source of IL-6 produced in the livers of mice early during the course of listerial infection, i.e., within 30 min postinfection i.v. The IL-6-dependent activation of STAT3 within hepatocytes occurred concomitantly in control, but not Kupffer cell-depleted, animals. Taken together, these results are the first to demonstrate directly the role of Kupffer cells in the production of IL-6 and the activation of STAT proteins in hepatocytes during the course of systemic bacterial infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria

The EGD strain of L. monocytogenes was cultured and maintained in accordance with the U.S. Department of Health and Human Services booklet entitled "Biosafety in Microbiologic and Biomedical Laboratories" as previously described (28). The bacterium was passed periodically in mice to sustain its virulence; 1 x 105 organisms inoculated i.v. represents 1 LD50 for C57BL/6J mice. Bacteria derived from broth cultures growing exponentially were used in the experiments described. Heat-killed Listeria (HKL) was prepared by incubating bacterial suspensions at 60°C for 1 h. Killed preparations were washed, suspended in saline, and stored at -70°C until use.

Animals

Normal female C57BL/6J mice and C57BL/6 mice expressing a targeted mutation in the IL-6 gene (IL6; The Jackson Laboratory, Bar Harbor, ME) were housed and cared for in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee, University of Pittsburgh. Animals between 6 and 16 wk of age were used in the experiments described.

Preparation of hepatic cells

The parenchymal and nonparenchymal cells were obtained following perfusion of the liver with collagenase initiated at the times indicated in the text using the two-step method we reported previously (20, 21). Dendritic cells and mononuclear phagocytes represent 0 and <=2%, respectively, of the purified hepatocyte population (29). The Kupffer cells (>85% purity) were separated from other NPCs by a 20-min period of attachment (3). NPCs (1 x 105/ml) or the Kupffer cells derived from 1 x 105 NPCs were cultured in HEPES-buffered RPMI 1640 supplemented with 10% heat-inactivated FBS (Sterile System, Logan, UT), 1 mM L-glutamine, 5 x 10-5 M 2-ME, and 5 µg/ml gentamicin with or without 106 HKL/ml.

Kupffer cell depletion

Multilamellar liposomes containing dichloromethylene diphosphonate (Cl2MDP-L) were prepared as previously described (30). Cl2MDP was a gift from Boehringer Mannheim GmbH (Mannheim, Germany). Mice were rendered Kupffer cell deficient by the inoculation i.v. of 200 µl of Cl2MDP-L (containing 1 mg/ml of Cl2MDP) suspended in normal saline on day 3 before experimental use. Mice administered 200 µl of saline or liposome-encapsulated PBS (PBS-L) served as controls. In agreement with other investigators (31, 32), Cl2MDP-L did not adversely affect hepatocytes in vivo; serum aspartate aminotransferase levels on day 3 postinoculation were comparable in both control and Kupffer cell-depleted mice. Moreover, Cl2MDP-L had no effect on the viability of cultured hepatocytes or the intracellular replication of Listeria within hepatocytes treated in vitro.

Isolation of total RNA

Total cellular RNA was isolated from the total liver homogenate, perfused total liver cell, hepatocyte, and NPC populations using TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Residual DNA was removed with DNase I (amplification grade, Life Technologies) treatment using the manufacturer’s protocol.

Reverse transcription

One microliter of oligo(dT)12–18 primer (Life Technologies) and 1.0 or 2.5 µg total RNA were combined and heated to 70°C for 10 min. After quickly chilling on ice and microcentrifugation, 5x first strand buffer, 0.1 M DTT (both from Life Technologies), and 10 mM dNTP mixture (Pharmacia Biotech, Piscataway, NJ) were added, and the samples were incubated at 42°C for 2 min. Three hundred units of SuperScript II RT (Life Technologies) and 15 U of ribonuclease inhibitor (Life Technologies) were added, and the samples were incubated at 42°C for 50 min, followed by 70°C for 15 min.

Purification of competitor fragments

The plasmid containing IL-6 and housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) cDNA sequences modified to contain 75 additional bp was provided by Dr. Steven L. Reiner (University of Chicago, Chicago, IL) (33). Following isolation from Escherichia coli strain GM2163 using a modified alkaline lysis method (34), the plasmid was digested with SfiI (Life Technologies). The approximately 11-kb fragment was then gel-purified using GlassMax (Life Technologies) and quantified with DNA DipSticks (Invitrogen, San Diego, CA) according to the manufacturers’ instructions.

Competitive-quantitative PCR (CQ-PCR)

An aliquot of cDNA was amplified using PCR containing 1.25 U of Taq DNA polymerase; 1x PCR buffer; 1.5 mM MgCl2 (all from Life Technologies); 0.5 nM each of the upstream and the downstream primers (custom synthesis, Life Technologies); 0.2 mM each of dATP, dTTP, dCTP, and dGTP (Pharmacia); 5.0 µl of competitor diluted to concentrations ranging from 0.4 to 0.04 ng/ml; and nuclease-free water to 50 µl. The thermal profile was initial denaturation at 94°C for 1 min, followed by 35 cycles of 94°C for 20 s, 60°C annealing for 40 s, 72°C elongation for 20 s, and a final elongation at 72°C for 10 min in a Perkin-Elmer N801–0150 DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT). Ten microliters of the PCR products were analyzed on a 3% native agarose gel. PCR products were quantified using the method of Reiner et al. (33) and are expressed as the ratio of IL-6 product to HPRT product. The amount of product obtained in reactions that contained competitor IL-6 or HPRT sequences only correlated directly with the concentration of competitor initially added to the reaction mixture (data not shown).

Quantitation of IL-6 by ELISA

The concentration of IL-6 in culture supernatants was determined by ELISA according to a protocol obtained from PharMingen (San Diego, CA). Monoclonal rat IgG1 anti-mouse IL-6 capture Ab and biotinylated rat IgG2a anti-mouse IL-6 detection Ab were purchased from PharMingen; avidin-conjugated peroxidase was purchased from Sigma (St. Louis, MO). The concentrations of IL-6 were calculated from standard curves generated with each assay.

Electrophoretic mobility shift assay (EMSA) of STAT proteins

EMSA was performed using whole cell protein extracts prepared as described previously (35). Protein extracts (20 µg) were incubated with radiolabeled high affinity serum inducible element (hSIE) duplex oligonucleotide. Bound and unbound duplex oligonucleotides were separated on 4% native polyacrylamide gels. The gels were dried and exposed to film. Bound hSIE duplex oligonucleotide forms three complexes detected as bands on film: SIF-A (STAT3 homodimer), SIF-B (STAT3 and STAT1 heterodimer), and SIF-C (STAT1 homodimer). Where indicated in the text, extracts were preincubated with rabbit Ab specific for STAT3{alpha} (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) or with chicken IgY specific for STAT3ß (36) for 30 min at 4°C before the addition of radiolabeled-hSIE.

Statistical analysis

The results were analyzed using the SigmaStat statistics program (Jandel Scientific, San Rafael, CA). Individual means were compared using a nonpaired Student’s t test. Data derived from three or more groups were compared by one-way ANOVA followed by a Student-Newman-Keuls test to identify groups that differed significantly (p<0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-6 message expression is elevated in the livers of Listeria-infected mice

Experiments were undertaken to determine the kinetics of IL-6 mRNA expression in the livers of mice early during the course of listerial infection. Total RNA was purified from representative sections of livers, and IL-6 message expression was assessed by RT-PCR (Fig. 1GoA) and quantified using CQ-PCR (Fig. 1GoB). Relatively little IL-6 mRNA was present in the livers of uninfected mice (i.e., time zero). Listerial infection caused a marked (~25-fold) increase in the level of IL-6 mRNA, which peaked between 30 min and 2 h, then declined thereafter.



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  		FIGURE 1. IL-6 message expression in the livers of Listeria-infected mice. Total RNA was purified from the livers of mice sacrificed at the times indicated postinfection with 2 x 107Listeria; IL-6 (1/10 dilution of the total RT products amplified) and HPRT (1/1000 dilution amplified) message expression was assessed by RT-PCR (A). IL-6 mRNA was quantified by CQ-PCR and normalized in terms of the quantity of HPRT message expressed according to the method of Reiner et al. (33) (B). The data presented in A are derived from single representative mice at each time point; those presented in B are the mean ± SD IL-6/HPRT mRNA expressed in the livers of three mice sacrificed at each time point.

 
Listerial infection induces IL-6 mRNA expression by NPCs

To determine the cell source of IL-6 mRNA expressed in the livers of Listeria-infected mice, the total liver cell, hepatocyte, and NPC populations were isolated from the livers of uninfected mice and of mice infected 30 min previously. The total RNA purified from these populations was then subjected to RT-CQ-PCR. As shown in Figure 2Go, the NPC population accounted for most of the IL-6 mRNA expressed constitutively in the livers of uninfected mice and for the increased level of IL-6 message expressed at 30 min postinfection. At 30 min postinfection, the amount of mRNA expressed by the NPCs was approximately 20- and 90-fold more than that expressed by the total liver cell and purified hepatocyte populations, respectively.



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  		FIGURE 2. Listeria infection stimulates IL-6 mRNA expression by NPCs. Total RNA was isolated from the total liver cell, hepatocyte, and NPC populations obtained at time zero (uninfected control) and 30 min postinfection i.v. IL-6 mRNA (wild-type) expression was quantified by CQ-PCR (A) and standardized in terms of the quantity of HPRT mRNA (B). To quantify IL-6 mRNA, the RT products derived from the total liver cell and NPC populations at 30 min were diluted 1/10 and 1/100, respectively, before CQ-PCR; the cDNA products generated at all other time points were not diluted. To quantify HPRT message expression, the RT products of all samples were diluted 1/1000 before amplification. The data presented in A are derived from single representative mice; those presented in B are the mean ± SD for the cell populations obtained from three mice sacrificed at time zero and 30 min postinfection.

 
Kupffer cell depletion diminishes IL-6 message expression by the NPCs of Listeria-infected mice

In previous studies by other investigators, it was presumed that Kupffer cells constituted the principal source of IL-6 produced in the livers of mice during the course of listerial infection (2, 22, 37). To test this assumption, mice were rendered Kupffer cell deficient by the administration of Cl2MDP-L before infection. Subsequently, the NPC populations were isolated from Cl2MDP-L-pretreated and control animals at 0 and 30 min postinfection; total RNA was purified from these NPC populations, and IL-6 mRNA was quantified by RT-CQ-PCR. As shown in Figure 3Go, the constitutive expression of IL-6 message by NPCs derived from Cl2MDP-L-treated animals was reduced approximately fivefold relative to that expressed by NPCs obtained from animals pretreated with PBS-L or saline. Similarly, at 30 min postinfection, IL-6 mRNA expression by the NPCs obtained from Kupffer cell-depleted animals was significantly less (>90%) than that expressed by NPCs derived from the controls.



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  		FIGURE 3. Kupffer cell depletion abrogates IL-6 mRNA expression by NPCs. Mice were pretreated with saline, PBS-L, or Cl2MDP-L. IL-6 message expression by the NPCs derived from control and Kupffer cell-depleted mice at time zero and 30 min postinfection with Listeria was quantified by CQ-PCR and normalized in terms HPRT mRNA expression. Data are the mean ± SD obtained for the NPC populations derived from three mice treated identically.

 
IL-6 production by NPCs derived from Kupffer cell-depleted mice is diminished

IL-6 production correlated to the level of IL-6 message expressed by the NPC populations derived from control and Kupffer cell-depleted mice (Table IGo). The supernatants obtained from the culture of cells derived from Kupffer cell-depleted mice contained significantly less IL-6 than the supernatants obtained from comparable cultures derived from control animals regardless of the presence of HKL added to stimulate macrophage activity.


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  		Table I. IL-6 production is diminished in cell cultures derived from Kupffer cell-depleted mice1

 
STAT protein activation within hepatocytes of Listeria-infected mice

Activated STAT3 and STAT1 have been implicated in IL-6 signal transduction and in the acute phase response of hepatocytes (13, 38). EMSA of total liver cell extracts using radiolabeled hSIE duplex oligonucleotide demonstrated a marked increase in STAT3 homodimer (SIF-A complex) formation in the livers of Listeria-infected mice. STAT3 activity peaked between 30 min and 2 h postinfection i.v. and declined dramatically thereafter (Fig. 4Go). Supershift analysis using Abs specific for STAT3{alpha} (p92) and STAT3ß (p83, a truncated isoform of STAT3{alpha}) demonstrated the presence of both isoforms in the SIF-A complex (data not shown). In contrast to the results obtained using wild-type mice, EMSA of extracts derived from the liver cells of IL-6-deficient mice failed to exhibit a detectable increase in hSIE binding activity following listerial infection. This latter finding demonstrates conclusively the role of IL-6 in the activation of STAT3 in the livers of mice early during the course of listerial infection.



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  		FIGURE 4. STAT3 activation in the livers of Listeria-infected mice. Total liver cell and purified hepatocyte populations were obtained from wild-type (WT) and IL-6-deficient (KO) mice at the indicated times postinfection i.v. with 2 x 107 Listeria. EMSA was performed on whole cell extracts (20 µg) derived from duplicate mice at each time point (lanes 1 and 2) using radiolabeled hSIE duplex oligonucleotide. The positions of the SIF-A (STAT3 homodimer), SIF-B (STAT3 and STAT1 heterodimer), and SIF-C (STAT1 homodimer) complexes are indicated on the right.

 
To establish definitively that IL-6-dependent STAT protein activation occurred within the hepatocyte population, purified hepatocytes were prepared from wild-type and IL-6-deficient mice at periodic intervals postinfection. EMSA of hepatocyte extracts revealed levels of hSIE binding activity similar to those found in extracts of the total liver cell population from which the hepatocytes originated. That is, a sharp increase in the hSIE binding activity was detected in extracts derived from wild-type, but not IL-6-deficient, mice at 30 min postinfection. In addition to establishing the IL-6-dependent nature of STAT protein activation within hepatocytes, these findings indicate that the level of IL-6 sufficient for maximum activation of hepatocyte STAT proteins occurs with kinetics that overlap those of IL-6 mRNA production by Kupffer cells.

Kupffer cell depletion negates STAT protein activation in hepatocytes

Experiments were undertaken to demonstrate directly the role of Kupffer cells and the production of IL-6 in STAT protein activation within hepatocytes. The results clearly indicate that the activation of STAT proteins within the hepatocytes of Listeria-infected mice occurred as an indirect function of Kupffer cells. The purified hepatocytes derived from mice pretreated with Cl2MDP-L to eliminate Kupffer cells exhibited a marked decrease in STAT3 homodimer formation at 30 min postinfection i.v. with Listeria (Fig. 5Go).



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  		FIGURE 5. Kupffer cell depletion abrogates STAT3 activation in the hepatocytes of Listeria-infected mice. Hepatocytes were purified from control (pretreated with saline or PBS-L) or Kupffer cell-depleted (Cl2MDP-L-pretreated) mice at time zero and 30 min postinfection i.v. with 2 x 107 Listeria. EMSA was performed on whole cell extracts (20 µg) derived from duplicate mice (lanes 1 and 2) using radiolabeled hSIE duplex oligonucleotides. The positions of the SIF-A, -B, and -C complexes are indicated on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The majority of bacterial pathogens that enter the bloodstream are cleared by the liver (18, 24, 25, 39, 40). The fundamental mechanisms that contribute to clearance of the blood and eventual sterilization of the liver remain to be fully resolved. To date, studies have focused primarily on the role of the resident tissue macrophages, i.e., Kupffer cells, which line the liver sinusoids and partially obstruct the vascular channel. Indeed, it is generally assumed that Kupffer cells ingest and kill most of the organisms taken up in the liver (24, 25, 26, 27). Using listeriosis in mice as an experimental model, however, we recently reported that the bulk of L. monocytogenes injected i.v. and cleared by the liver was initially bound extracellularly by hepatic cells and subsequently killed by immigrating neutrophils (20). Thus, neutrophils and not Kupffer cells were principally responsible for killing Listeria early during the course of systemic infection.

While there is little evidence to indicate that Kupffer cells are directly involved in killing bacteria in vivo, it has been reported that Kupffer cells play a critical role in host defenses to Listeria (41, 42). Mice rendered Kupffer cell deficient by pretreatment with Cl2MDP-L exhibited a marked increase in susceptibility to both primary and secondary listerial infections. Although the role of Kupffer cells in host resistance has yet to be defined, the results of the experiments reported here suggest that cytokine synthesis may be a major function. Indeed, cytokine production in the liver during periods of infection or inflammation is frequently attributed to Kupffer cells. The rapid induction of IL-1ß, IL-6, IL-12, and TNF-{alpha} messages in the livers of mice following i.v. inoculation of Listeria, for example, led investigators to suggest that Kupffer cells were the probable source (22).

Previously, we reported the elevated production of IL-6 by purified mouse Kupffer cells cultured in the presence of heat-killed Listeria (3). Our results demonstrating a significant reduction in the expression of IL-6 mRNA and the production of IL-6 by NPCs derived from Cl2MDP-L-pretreated (Kupffer cell-depleted) mice indicate that Kupffer cells are the principal source of IL-6 produced in the livers of mice early during the course of listerial infection. Salkowski et al. (43) obtained similar results in mice inoculated i.v. with bacterial endotoxin. That is, IL-6 mRNA expression following the administration of endotoxin was reduced >95% in the livers of animals pretreated with Cl2MDP-L.

IL-6 is a proinflammatory cytokine found in tissue extracts, serum, and other bodily fluids during periods of severe infection, inflammation, and general trauma. In the case of Listeria-infected mice, the quantity of IL-6 assessed in the organs correlates directly with the number of bacteria recovered (9, 37, 44, 45). In accordance with the published results of other investigators (22), we found that IL-6 mRNA expression was elevated significantly in the livers of mice at 30 min postinfection i.v. with Listeria. IL-6 is an essential factor in host defenses to Listeria. Mice administered polyclonal rabbit anti-mouse IL-6 Ab exhibited a significant increase in the number of bacteria recovered in their organs at >=4 days postinfection. Likewise, IL-6-deficient mice exhibited a marked (>100-fold) increase in Listeria/liver relative to control animals (8, 46). Increased susceptibility to listerial infections correlated with inefficient neutrophilia (46). Similarly, the inability to induce neutrophilia immediately following challenge correlated with the increased susceptibility of IL-6-deficient mice to E. coli infections (47). On the other hand, both wild-type and IL-6-deficient mice administered rIL-6 exhibited enhanced neutrophilia and elevated resistance to bacteria (10, 44, 46).

In addition to stimulating neutrophilia, IL-6 can exert a number of effects that potentially affect host defenses to Listeria. It has been suggested, for example, that IL-6 promotes resistance to listerial infections by stimulating IFN-{gamma} production by activated T lymphocytes (10). Indeed, while rIL-6 enhanced host resistance to Listeria in normal mice, it had no effect on the course of infections in SCID mice that lacked T lymphocytes or in normal mice administered monoclonal anti-IFN-{gamma} Ab. Similarly, it was concluded that the reduced infiltration of CD4+ T lymphocytes and the diminished production of IFN-{gamma} accounted for the increased susceptibility to toxoplasma encephalitis exhibited by IL-6-deficient mice (48).

Hepatocytes are the major source of acute phase proteins synthesized during periods of infection and inflammation (49). These proteins, e.g., C reactive protein, fibrinogen, {alpha}1 proteinase inhibitor, and {alpha}1 acid glycoprotein, have a physiologic role in innate host defenses and in the preservation of homeostasis by limiting tissue injury and enhancing wound repair (12). IL-6 is the principal cytokine implicated in the acute phase response. The synthesis of acute phase proteins subsequent to listerial infections, for example, is reduced significantly in IL-6-deficient mice (8). In vitro, the increased expression of genes encoding acute phase proteins by hepatocytes cultured in the presence of IL-6 correlates with STAT3 activation (38). Here, we demonstrated the activation of STAT3 in hepatocytes early during the course of listerial infection. The kinetics of STAT3 activation paralleled IL-6 mRNA expression in the livers of infected mice; remarkably, both peaked between 30 min and 2 h postinoculation i.v. The absence of activated STAT3 in hepatocyte extracts derived from IL-6-deficient mice confirms that IL-6 is required for STAT protein activation in the livers of normal mice infected with Listeria. This finding also indicates that the time required to achieve local levels of IL-6 sufficient for maximum STAT3 activation is substantially shorter than the 1–2 days required to achieve peak IL-6 levels in the serum of animals inoculated i.v. with Listeria (9, 37).

Recent evidence presented in the literature documents the production of acute phase proteins by purified rat hepatocytes cultured in the presence of bacterial endotoxin (50). Protein synthesis occurred in the absence of contaminating Kupffer cells or endothelial cells and was blocked by the addition of polyclonal anti-IL-6 Ab. The researchers concluded, therefore, that IL-6 produced by endotoxin-stimulated hepatocytes could elicit acute phase protein synthesis in an autocrine or paracrine fashion (50). The marked reduction in IL-6 mRNA expression in the livers of Kupffer cell-depleted mice infected with Listeria (shown herein) suggests that Kupffer cells are the principal source of IL-6 synthesized in the liver early during the course of listerial infection. It is relevant to note that IL-6-deficient mice inoculated with bacterial endotoxin, in contrast to those infected with Listeria, exhibited a significant increase in acute phase protein synthesis (8, 51). This has led others to suggest that IL-6 is a critical mediator of the acute phase response to intracellular Gram-positive, but not to Gram-negative, bacteria (8). Our findings demonstrating the absence of activated STAT proteins in hepatocyte extracts derived from Listeria-infected, IL-6-deficient mice support this hypothesis. Taken together, our results indicate that Kupffer cells and the production of IL-6 are obligate factors in the acute phase response of hepatocytes to systemic, Gram-positive bacterial pathogens.


   Acknowledgments
 
We thank Athanasia J. Sagnimeni and Annemarie Sanders for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by U.S. Public Health Service Grants DK44367 (to S.H.G.) and GM53789 (to D.J.T.) obtained from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Stephen H. Gregory, Department of Medicine, UPMC Montefiore, 200 Lothrop St., Pittsburgh, PA 15213-2582. E-mail address: Back

3 Abbreviations used in this paper: NPC, nonparenchymal liver cells; HKL, heat-killed listeriae; Cl2MDP-L, liposome-encapsulated dichloromethylene diphosphonate; PBS-L, liposome-encapsulated PBS; HPRT, hypoxanthine-guanine phosphoribosyl transferase; CQ-PCR, competitive-quantitative PCR; EMSA, electrophoretic mobility shift assay; hSIE, high affinity serum inducible element. Back

Received for publication October 27, 1997. Accepted for publication February 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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