Modulation of Hepatocyte Protein Synthesis by ...

Modulation of Hepatocyte Protein Synthesis by Endotoxinactivated Kupffer Cells Evidence for the Role of a Monokine Similar to but not Identical with Interleukin-1 Ill.

GARY A. KELLER, M.D., MICHAEL A. WEST, M.D., JOHN T. HARTY, LOREN A. WILKES, FRANK B. CERRA, M.D., RICHARD L. SIMMONS, M.D.

From the Veterans of Foreign Wars Cancer Research Center and The Department of Surgery, University of Minnesota, Minneapolis, Minnesota

The authors previously reported that unstimulated peritoneal macrophages and LPS stimulated Kupffer cell-rich nonparenchymal liver cells (NPC) can inhibit protein synthesis in cultured rat hepatocytes. Hepatocyte function was similarly altered by supernatants from LPS-triggered NPC. Secretory products of macrophages and Kupffer cells (monokines) are possible mediators in this model of cell-mediated modulation of hepatocyte function. In this article, supernatants from NPC capable of altering hepatocyte protein synthesis were found to contain significant amounts of one monokine, interleukin-1 (IL-1). The kinetics of the generation of the ability to inhibit protein synthesis and the appearance of IL1 activity were roughly parallel for the first 24 hours. Exposure of hepatocytes to NPC supernatant and commercially available highly purified IL-1 resulted in similar response patterns with regard to onset of inhibition and progression of suppression after removal. Certain discrepancies cast doubt, however, on the likelihood that IL-1 is the mediator in this model of cell-mediated inhibition of hepatocyte protein synthesis. Commercial human IL-1 preparations did not always suppress protein synthesis in cultured rat hepatocytes. Furthermore, IL-1 activity was stable for several days but hepatocyte inhibition was lost. Unstimulated peritoneal macrophage supernatants containing IL-1 activity could not inhibit protein synthesis in hepatocytes. The evidence supports the idea that appropnrately stimulated cells of monocyte-macrophage lineage (including Kupffer cells that lie in direct apposition to hepatocytes) could mediate hepatocyte malfunction by means of secreted monokines similar to, but not necessarily identical to, IL-1.

(NPC)2 to inhibit total protein synthesis by isolated rat hepatocytes in culture. These studies, in concert with numerous reports of leukocyte-mediated cytotoxicity,39 suggest the possibility that leukocyte secretory products could mediate alterations in hepatocyte function in vivo. It is also possible that such a mechanism could play a role in the alterations of hepatic function seen in patients during the evolution of the multiple system organ failure syndrome. This syndrome, characterized by progressive pulmonary, renal, hepatic, or cardiovascular failure, has been recognized with increasing frequency in surgical patients following sepsis, trauma, and periods of hypoperfusion. In spite of its prevalence, the etiology of hepatic insufficiency in this setting is unclear. Under proper conditions, macrophages and Kupffer cells are known to secrete monokines capable of inducing effects on adjacent cells in coculture ranging from subtle alterations in enzyme kinetics8 to cellular lysis and death.38 In the accompanying article,9 we demonstrated that soluble products of NPC stimulation were capable of inhibiting protein synthesis in isolated hepatocyte culture. In this article we present evidence pertaining to the possible role of monokines similar to interleukin-1 (IL-1) in mediating these changes.

E HAVE previously demonstrated the ability of peritoneal cells (MRPC)1 and lipopolysaccharide-triggered nonparenchymal liver cells

ki imacrophage-rich

Supported by NIH Grant AI-14032. Dr. Keller and Dr. West hold National Research Service Awards AM-07182 and AI-06602, respectively. Reprint requests: Gary A. Keller, M.D., Department of Surgery, University of Minnesota Hospital, 516 Delaware St. SE, Box 309, Minneapolis, MN 55455. Submitted for publication: October 31, 1984.

Materials and Methods

Animals Male Sprague-Dawley rats, 4-6 weeks old, weighing 250-300 g, were obtained from the Charles River Breed-

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HEPATOCYTE PROTEIN SYNTHESIS

ing Laboratories, Wilmington, Massachusetts. C3H/HeJ female and C57B16 female mice were obtained from the immunobiology research center at the University of Minnesota. DBA/2J female mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. The animals were housed for at least 1 week prior to use with free access to water and Purina Lab Chow #500. Animals were not fasted prior to use. Hepatocyte Isolation

Rat hepatocyte suspensions were prepared by a modification of Seglen's collagenase perfusion technique.'0 Following intraperitoneal nembutal anesthesia, the portal vein was cannulated and perfusion was begun with the calcium-free solution.10 The hepatic artery, hepatic vein, hepatic duct, and retroperitoneal attachments were severed and the liver removed. Ex-vivo perfusion with the calcium-free solution was continued for a total of 7 minutes, followed by a 10-minute perfusion with 0.05% collagenase (clostridiopeptidase A) (Sigma #C-0 130 Type 1, 200 units/mg) in Hanks balanced salt solution (HBSS). Both perfusates were maintained at 37 C. After perfusion, the liver was gently combed in HBSS to produce a hepatocyte suspension. The hepatocytes were washed three times in HBSS at 4C and centrifuged at 10 X G. The procedure yielded 2.0-3.0 X 108 cells/animal with an 80-90% viability as determined by trypan-blue exclusion.

Hepatocyte Culture After counting, the rat hepatocytes were suspended in minimal essential medium (MEM) containing the following supplements: fetal calf serum 7 ml/100 ml, penicillin 10,000 units and streptomycin 10 mg/100 ml, L-glutamine 30 mg/100 ml, insulin 10 ,u/100 ml (Regular Ilentin .1 100 g/ml Eli Lilly Co.), and HEPES Buffer 1.5 ml/100 ml. A final cell concentration was adjusted to 5.0 X 104 viable hepatocytes/ml, and 1 ml aliquots were plated in 15 X 16-mm flat-bottom LinbroO tissue culture wells. Hepatocytes were incubated in 5% CO2 and air at 37C and allowed to adhere for 90 minutes. The medium was changed and replaced with identical medium except that 2% FCS was used for the remainder of the culture period. All hepatocytes were precultured for a 24-hour period before they were exposed to supernatants, commercial IL- 1, or NPC in coculture. Protein Synthesis Assay Following experimental manipulation (periods of exposure to supernatant, IL- 1, or NPC in coculture) the medium was removed and replaced with the medium described above without FCS. Next 7 gCi of L-4, 5 3H leucine, 5.0 Ci/mmol (New England Nuclear) was added

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in a 50-lambda volume of normal saline. Four hours after the label was added, incorporation of 3H leucine into protein was stopped by addition of 2 ml of cold 10% trichloroacetic acid (TCA) to each well. Wells were scraped by a rubber policeman and well contents transferred to 4-ml glass test tubes. Samples were washed three times in cold 10% TCA and centrifuged at 700 X G to remove unincorporated 3H leucine. The protein pellet was then solubilized with 0.5 ml of 0.5 mol/L ProtosolO tissue solubilizer to which 4 ml of scintillation cocktail was added. 3H leucine incorporation into the solubilized protein was then measured using a Beckman LS 2800 Scintillation counter. Specific activity of the radiolabel was measured throughout the course of the 4-hour labeling period and did not change.

Nonparenchymal Liver Cell Preparation Nonparenchymal liver cells (NPC) were obtained from rats by the method described by Knook and Sleyster." The liver was perfused at 37C via the cannulated portal vein with Gey's balanced salt solution (137 mM NaCl, 5 mM KCG, 1.6 mM CaCl2-2H20, 0.9 mM MgCl2. 6H20, 0.3 mM MgSO4. 7H20, 1.7 mM Na2HPO4 * 2H20, 0.2 mM KH2PO4, 2.7 mM Na HCO3, 5.5 mM glucose; pH 7.4; 275 mOsm). The rate of perfusion was maintained at 10 ml/min. After 2 minutes of perfusion, pronase 0.2% (Sigma #5147) was added to the perfusate. The liver was perfused with pronase for 1 minute, removed, and diced into small pieces in 10 ml of GBS. The pieces were then incubated in 90 ml of GBS solution plus 0.2% pronase at 37C for 1 hour while the pH was maintained at 7.4 by the addition of NaOH. Following filtration through wide-mesh gauze, NPC were washed in HBSS and centrifuged at 250 X G for 5 minutes. This procedure yielded 1.2-1.6 X 108 cells per rat with viability in excess of 95%. This cell preparation was comprised of 30-40% Kupffer cells by latex bead uptake.'2 Latex beads (0.8 gm) were washed and suspended in HBSS and added to freshly isolated NPC to achieve a latex bead/NPC ratio of 10/1. After 4 hours of incubation, the percentage of cells containing latex beads was determined by light microscopy. NPC were plated at 1.0 X 106 cells/ml alone or in coculture with 5.0 X 104 hepatocytes in the medium described above with 2% FCS. Their viability following 24-hour culture remained in excess of 95%. Where indicated, lipopolysaccharide (LPS), from Salmonella typhimurium (Ribi Immunochem Research Inc.), was added to NPC at 20 yg/ml. Macrophage-rich Peritoneal Cell (MRPC) Preparation Rats were killed by ether overdose. Twenty milliliters of HBSS supplemented by 7% FCS, penicillin 10,000

Ann. Surg. * April 1985 KELLER AND OTHERS 438 units, streptomycin 10 mg/ 100 ml, and beef lung heparin on the 14th day after stimulation in allogeneic mixed 500 M/ 100 ml were injected intraperitoneally as soon lymphocyte culture (C57B1/6 X DBA/2J), were suspended after death as possible. Following 1 minute of gentle in MEM with 2% fetal calf serum, and 2.0 X 104 cells

agitation, the peritoneal cavity was opened and the fluid removed by pipetting. Pooled MRPC were washed three times in HBSS solution and centrifuged at 250 X G for 5 minutes. During the washing procedure, RBCs were removed by hypotonic lysis; 1.5-2.0 X 107 cells were obtained per rat, with a viability rate of 95-98%. Differential cell counts using Wright's stain revealed 65% macrophages, 8% lymphocytes, and 27% granulocytes. The granulocytes consisted primarily of mast cells and eosinophils with very few neutrophils. Following enumeration, MRPC were suspended in the medium described above containing 2% FCS and were plated alone or added to existing hepatocyte cultures to establish a coculture. Supernatant Transfer The supernatants were removed from 5.0 X 104 hepatocytes, 1.0 X 106 NPC, or cocultures from these two cell types in the presence or absence of LPS following 24 hours of culture. Supernatants were also obtained from 1.0 X 106 unstimulated macrophage-rich peritoneal cells. The supernatants were centrifuged to remove cellular particles and stored at -70C until use. Hepatocytes were exposed to supernatants by removing the 1 ml of medium in which the hepatocytes were cultured and replacing it with 1 ml of supernatant. The IL- 1 and IL-2 content of these supernatants were determined by adding increasing increments of the supernatants to the assays as described below.

Lymphocyte Activation Factor (IL-I) Assay Assay of LAF activity was performed using a modification of Oppenheim's thymocyte proliferation assay.'3 C3H/HeJ murine thymocytes harvested from 3-5-weekold animals were suspended in RPMI 1640 containing the following supplements: fetal calf serum, 5 ml/100 ml; penicillin, 10,000 units; streptomycin 10 mg/100 ml; and 2 mercaptoethanol 5.0 X 10-5 M. Then 1.0 X 106 cells per well were cultured with various dilutions of supernatant in the presence or absence of 1 ,ug/ml phytohemagglutinin (PHA) (Difco #3110-57) at 37C for 72 hours in Linbro flat-bottom microtiter plates. 1 ,Ci 3HTdr was added to each well in 50 lambda of medium over the last 24 hours of culture. Plates were harvested onto glass fiber filter paper and 3HTdr incorporation was determined by liquid scintillation. IL-2 Assay

Supernatants were assayed for IL-2 activity by the method described by Ryser et al.'4 Mouse lymphocytes,

per well were plated in groups of four with serial dilutions of supernatant in flat-bottom microtiter plates (#3596, Costar). Plates were incubated 48 hours at 37C in 10% C02, and wells were pulsed with 2 ,MCi 3HTdr for 61/2 hours. Plates were harvested onto glass-fiber filters and 3HTdr incorporation was determined by liquid scintillation. Reagents Purified human interleukin-1 (IL-1) was purchased from Genzyme, Boston, Mass. This IL-1 was generated from human monocytes stimulated in vitro for 24 hours with Staphylococcus albus. The IL-1 was then purified by immunoabsorbence and column chromatography. Since IL-I was received in 0.15 M NaCl and 5% FCS, all control groups were exposed to equal volumes of the same vehicle. The IL-I was stored at -70C until it was used. The activity of IL-1 was assessed by Genzyme, and 1 unit was defined as that amount necessary to double the rate of 3HTdr incorporation by mouse thymocytes suboptimally stimulated by PHA. Although we always confirmed that each lot of IL-1 did activate mouse thymocytes in our laboratory, we recorded the units assigned to the IL-1 preparation by Genzyme.

Statistical Analysis Three wells of cultured cells constituted a group in each experiment. Means from at least three experiments were compared. Variance was expressed as standard deviation. The paired Student's t-test was used throughout for statistical analysis. Results Effect of Nonparenchymal Liver Cell Supernatants on Rat Hepatocyte Protein Synthesis and Mouse Thymocyte

Proliferation Supernatants were removed from 24-hour cultures of 5.0 X I04 hepatocytes, 1.0 X 106 NPC, or a coculture of both cells in the presence or absence of LPS (20 ug/ ml). The supernatants were stored at -70C until use. The effect of the supernatants on hepatocyte protein synthesis and on suboptimally PHA stimulated thymocytes is shown in Fig. 1. Supernatants from cultured hepatocytes, in the presence or absence of LPS, and NPC in the absence of LPS did not alter the rate of hepatocyte protein synthesis. Furthermore, the supernatant from cocultures of hepatocytes and NPC in the absence of LPS did not inhibit hepatocyte protein synthesis. However, supernatants from LPS-triggered

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HEPATOCYTE PROTEIN SYNTHESIS

vol. 201 * No. 4

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FIG. 1. Comparison of supernatant from various cell preparations: ability to inhibit hepatocyte protein synthesis versus ability to stimulate thymocytes.

FIG. 2. Comparative time course of ability of LPS-triggered NPC supernatant to inhibit hepatocyte protein synthesis versus ability to

NPC and LPS-triggered coculture of hepatocytes and NPC significantly inhibited hepatocyte protein synthesis. The ability to inhibit hepatocyte protein synthesis was roughly proportional to the supernatant content of IL- 1, as assayed by the ability of the supernatant to stimulate 3HTdr incorporation in suboptimally PHA stimulated mouse thymocytes.

concentration of supernatant (50%), there is some IL-2 activity, but no activity is noted at 12.5% and 25% concentrations. Significant IL-1 activity is present in this supernatant at all concentrations, however. The thymocyte-activating assay is therefore a valid tool for the determination of thymocyte-proliferating activity at the lower concentration (25%) of supernatant used throughout this report.

Kinetics of the Generation of Hepatocyte Inhibitory Activity and Thymocyte Activating Property by LPSstimulated NPC 1.0 X 106 NPC were exposed to LPS (20 Ag/ml) for periods of time ranging from 2 to 72 hours before the supernatant was removed. The supernatant was then assayed for its ability to inhibit hepatocyte protein synthesis and to stimulate the thymidine uptake by thymocytes. The time courses for the generation of the factor(s) responsible for these two effects were indistinguishable between 2 and 24 hours (Fig. 2). Supernatant from NPC exposed to LPS for 48 and 72 hours continued to stimulate thymocytes to a maximal degree, but the ability of such supernatant to inhibit hepatocyte protein synthesis was diminished with respect to the 24-hour supematant, suggesting that the factor responsible for the effect on hepatocytes may be more labile than the factor capable of stimulating thymocytes. Release of IL-2 by LPS-triggered NPC To interpret the thymocytes activation assay as a of IL-1 content in our supernatant, we measured the content of IL-2 in the supernatant. This is important because the endpoint for the IL-I assay is thought to be mediated by IL-2 generated within the assay. For this reason, exogenous IL-2 could cause a false-positive result. The amount of IL-2 contained in the LPStriggered supernatant is shown in Table 1. At the highest measure

stimulate thymocytes.

Effect of Commercial Human Interleukin-J Protein Synthesis

on Hepatocyte

Commercially available highly purified human IL-1 studied for its effect on cultured hepatocytes. Although there was lot variation, three of the five lots tested demonstrated an ability to reduce significantly the rate of protein synthesis by hepatocytes following a 24-hour period of exposure at a concentration of 20 ,u/ml (Fig. 3). All five lots had significant IL-1 lymphocyte-activating ability as determined in our laboratory. The time course of this inhibition of protein synthesis by IL-l is shown in Table 2. Only lots 2 and 3, which had been previously shown to inhibit protein synthesis at 24 hours, were used in these experiments. Inhibition was

TABLE 1. Release of IL-I and IL-2 by LPS-stimulated Nonparenchymal Cells (NPC) Concentration of Supernatant (PHA control) 0 12.5% 25% 50% *

IL-I Content: 3HTdr Incorporation by Thymocytes (cpm + SD) 2,765 30,893 34,251 45,791

± 603 ± 4.941* ± 2,919* ± 2,068*

1L-2 Content: 3HTdr Incorporation by MLC Cells (cpm + SD) (LPS control)

p < 0.001 compared with PHA control.

t p < 0.05 compared with LPS control.

2,218 ± 1,045 ± 2,747 ± 7,114 ±

894 323 481

1,29 It

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KELLER AND OTHERS O

Is

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Lot

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Differential Effect on IL-I on Hepatocyte Protein Synthesis and Thymocyte Proliferation While some lots of IL-1 failed to inhibit hepatocyte protein synthesis, all lots stimulated thymocyte proliferation, but to variable degrees in our assay (Table 2). This observation raised the possibility that the portion

= Hepatocyte Control = Hepatocyte + IL-I

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FIG. 3. Effect of commercial human IL-1 protein synthesis.

on

Ann. Surg. * April 1985

isolated hepatocyte

of the IL-1 molecule responsible for its suppression of hepatocyte protein synthesis might be unstable and susceptible to alterations induced by the way in which it was handled prior to addition to the hepatocyte culture. To evaluate this discrepancy of function, we added commercial lots of IL-1 that had been previously shown to inhibit protein synthesis to hepatocyte cultures following two different means of preparation (Table 5). One group of hepatocytes was exposed to freshly thawed human IL- 1, and a second group was exposed to IL- 1 that had been maintained at 37C for 24 hours after

was noted after only 8 hours of exposure and was directly related to the period of exposure. There was no significant change is viability following 24 and 48 hours of exposure. The time course was similar to that induced by supernatants of LPS-triggered NPC.9 The effect of commercial human IL- 1 preparations on hepatocyte protein synthesis at varying concentrations is depicted in Table 3. Inhibition of protein synthesis was first observed at 1.0 A/ml of IL-1; the effect was maximal at 20 ,/ml, with no further increase following exposure to 40 ,u/ml. To compare the effects of LPS-stimulated NPC supernatant and commercially available IL-1 on hepatocytes further, we compared the reversibility of the effect of each on hepatocyte protein synthesis. LPS-stimulated NPC supernatant and IL-1 both reduced hepatocyte protein synthesis to 70% of controls at the end of the 24-hour exposure period (Table 4). In addition, the inhibition induced by both preparations was irreversible and almost equally progressive 24 hours after they were removed from the culture medium.

TABLE 2. Commercial Human Interleukin-l

thawing. Following 24 hours of incubation at 37C, IL1 no longer inhibited hepatocyte protein synthesis but was still capable of stimulating thymocyte proliferation. In fact, an apparent increase in capacity to stimulate thymocytes was seen.

Effect of Macrophage-rich Peritoneal Cell (MRPC) Suon Rat Hepatocyte Protein Synthesis and Mouse Thymocyte Proliferation 5.0 X 104 precultured hepatocytes were cocultured with 1.0 X 106 MRPC or LPS-triggered NPC for 24 hours. Equal numbers of hepatocytes were also exposed to 24-hour-old supernatant from 1.06 unstimulated MRPC or LPS-triggered NPC for 24 hours. Each supernatant was assayed for its IL-1 activity. Unstimulated MRPC and LPS-triggered NPC exerted equivalent inhibitory effects on hepatocyte protein synthesis in coculture (28% and 30% of control, respectively). However, the supernatant from MRPC and LPS-triggered NPC differed in their effect on cultured hepatocytes. The supernatant of LPS-triggered NPC was capable of transferring hepatocyte inhibitory activity. By contrast, MRPC

pernatant

(IL-J)-mediated Inhibition of Hepatocyte Protein Synthesis: Time Course Period of Exposure

8 Hours

3H leucine incorporation into protein (cpm X 103 ± S.D.) Viability (trypan blue

exclusion) *

p