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The Journal of Immunology, 2005, 174: 7302-7309.
Copyright © 2005 by The American Association of Immunologists

Intrinsic Resistance of Hepatocytes to Complement-Mediated Injury1

Cody A. Koch2,*,{dagger}, Akiyoshi Kanazawa2,*, Ryuta Nishitai*, Bruce E. Knudsen*, Kiyoshi Ogata*, Timothy B. Plummer*, Kim Butters* and Jeffrey L. Platt3,*,{dagger},{ddagger},§

* Transplantation Biology and the Departments of {dagger} Immunology, {ddagger} Surgery, and § Pediatrics, Mayo Clinic College of Medicine, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Exposure of cells to complement and its activators usually leads to cellular dysfunction and sometimes to death. For example, activation of small amounts of complement on endothelial cells causes the cells to produce IL-1{alpha}, changes the function of the cells from anticoagulant to procoagulant (1) and from anti-inflammatory to proinflammatory (2), and in larger amounts causes lysis (3). Complement activation on podocytes leads to secretion of IL-1, PGE, prostacyclin, and thromboxane A2 (4). Moreover, podocytes respond to complement activation by synthesizing DNA but are unable to undergo mitosis due to complement-mediated DNA damage (5).

The liver is constantly exposed to activated complement and bacterial products capable of activating complement via the portal venous system. For example, Jacob et al. (6) detected endotoxin, a natural activator of complement (7) and/or bacteria in the portal venous system of 97% of patients undergoing elective abdominal surgery. Similarly, Prytz et al. (8) detected endotoxin in the portal venous circulation of 9 of 21 patients who had no evidence of liver disease. Not only is the liver exposed to complement activators, it clears immune complexes, anaphylatoxins, and activated complement components from the circulation, preventing dissemination to the systemic circulation without apparent detriment to hepatic function (9). Thus, patients suffering from liver failure exhibit increased levels of immune complexes in their systemic circulation (10).

Activated complement proteins and immune complexes in the portal circulation are thought to be cleared by cells of the reticuloendothelial system lining the sinusoids. For example, Muro et al. (11) found that both Kupffer cells and sinusoidal endothelial cells possess Fc receptors capable of efficiently clearing immune complexes from the portal blood of rats and humans. Kupffer cells also possess complement receptors which bind particles and organisms coated with C1q and/or C3b, clearing them from the circulation and preventing their systemic dissemination (12, 13). Besides clearing complement activators and complexes from the blood, the liver produces most complement components found in the blood except C1q, factor D, and properdin (9). Thus, those who suffer from hepatic failure exhibit striking deficiencies of complement in the blood and defective opsonization of Escherichia coli (14).

Whether in fact liver cells, other than sinusoidal lining cells, resist complement-mediated injury and how the function and viability of the liver are maintained when complement is activated in large amounts in the portal circulation is unknown. To address these questions, we tested the extent to which hepatocytes, which some have proposed for use in devices (15) or as transplants (16) for the treatment of hepatic failure, maintain integrity and function when exposed to complement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell isolation and culture

{smtexf}Hepatocytes were isolated from the livers of swine 1–2 mo of age and weighing 8–15 kg, unless otherwise specified, using a modified Seglen technique (17). The livers were sequentially perfused with 5.0 liters of perfusate I (140 mM NaCl, 6.7 mM KCl, 10 mM acid-free HEPES, 2.5 mM EGTA, pH 7.4) followed by 2.0 liters of perfusate II (67 mM NaCl, 6.7 mM KCl, 100 mM HEPES, 4.8 mM CaCl2 2H2O, 2 g collagenase D (Sigma-Aldrich), and 1% bovine serum albumin, pH 7.4), which was recirculated for 20 min as previously described (18). The viability of isolated hepatocytes was consistently >90% as assessed by exclusion of trypan blue. Endothelial cells were explanted from porcine aortae and cultured in DMEM containing 10% FCS, 2.0 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies) as described elsewhere (1, 19). Endothelial cells were grown to confluency in 3–7 days and were characterized by cobblestone morphology, uptake of acetylated low-density lipoprotein, and the absence of staining for smooth muscle {alpha}-actin (Accurate Chemical and Scientific) as described previously (20).

Complement-mediated cytotoxicity

Porcine hepatocytes and aortic endothelial cells, grown to confluency on 96-well tissue culture plates (BD Labware), were labeled with 1.0 µCi/ml [51Cr]sodium chromate (Amersham International) overnight at 37°C. The labeled cells were washed with DMEM and then tested for cytotoxicity as follows. Labeled cells were incubated sequentially with 25% heat-inactivated human serum as a source of anti-swine Abs followed by serial dilutions of normal human serum as a source of complement or melittin (Sigma-Aldrich) for 4 h at 37°C and 5% CO2 (21). Where indicated, cells were treated with 1.0 U/ml phosphatidylinositol-specific phospholipase C (Sigma-Aldrich), LY294002, Akt inhibitor, SB203580, SP600125 (Calbiochem), Gö6976, wortmannin, or PD-98059 (Biomol). The supernatant from cells treated with serum or melittin was collected and the radioactivity was measured (cpm test sample) using a Wallac scintillation counter (Wallac). Cells remaining in the well following collection of the supernatant were lysed with 1% Triton X-100 and the residual radioactivity was measured (cpm residual cell sample). The percent specific lysis was calculated as follows: ((cpm test sample/(cpm test sample + cpm residual cell sample)) – (cpm spontaneous release/(cpm spontaneous release + cpm spontaneous release residual))/((cpm Triton X-100 – cpm residual cells Triton X-100) – (cpm spontaneous release/(cpm spontaneous release + cpm spontaneous release residual)) x 100.

Binding of human xenoreactive Abs and deposition of complement components

The binding of human IgM or IgG to cells and the deposition of iC3b or the membrane attack complex were measured by ELISAs as previously described (19, 22). Porcine aortic endothelial cells and hepatocytes were grown to confluency in 96-well microtiter plates. For determination of Ab binding, confluent cells were fixed with 0.1% glutaraldehyde for 5 min at 4°C and then incubated for 1 h with 1% BSA/PBS to block nonspecific protein binding. Porcine IgG was used to block Fc receptors. Cells were incubated with heat-inactivated human serum for 1 h at room temperature. Bound Ab was then assayed using alkaline phosphatase-conjugated goat anti-human-IgM or -IgG (Sigma-Aldrich). For determination of complement deposition, confluent cells were exposed to 25% heat-inactivated human serum followed by serial dilutions of normal human serum for 1 h at 37°C. The cells were then fixed as described and incubated with mAbs specific for iC3b (Quidel) or for a neoantigen of the membrane attack complex (generous gift from A. Michael, University of Minnesota, Minneapolis, MN) diluted 1/1000 in 0.5% BSA/PBS. The amount of mAbs bound were measured by incubating the cells with alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma-Aldrich) diluted 1/5000 in 0.5% BSA/PBS. The immunochemical reactions were detected using p-nitrophenylphosphate in 0.1 M diethanolamine (Sigma-Aldrich), and absorbance of the reaction product was measured at 405 nm with an ELISA plate reader (Power wave; Bio-Tek Instruments).

To determine the relative contribution of anti-Gal{alpha}1–3Gal4 Abs to complement fixation on hepatocytes, two types of experiments were performed. In some experiments, anti-Gal{alpha}1–3Gal Abs were blocked by adding Gal{alpha}1–3Gal{beta}1–4GlcNAc (V-Labs) at a final concentration of 10 mM to 50% normal human serum and incubated at 4°C overnight. In other experiments, hepatocytes were fixed as described above and treated with 1.0 U of {alpha}-galactosidase (green coffee bean; Boehringer Mannheim) in 100 mM NaCl and 50 mM sodium acetate (pH 5.0) at 37°C for 3 h and washed with PBS before Ab binding was assayed as described previously (23).

Immunoblotting for total and phospho-Akt(Ser473)

Total Akt and phospho-Akt were measured as follows. Hepatocytes cultured to confluency in six-well plates were lysed with Beech’s lysis buffer. SDS-PAGE sample buffer was added to the cell lysates and 30 µg of protein was applied to each lane of 7.5% polyacrylamide gels (Bio-Rad). Protein was transferred to Immobilon-P membranes (Millipore) and blocked with 5% non-fat-dried milk/TBST for 1 h at room temperature. The membranes were incubated with Abs specific for total Akt or phospho-Akt(Ser473) (Cell Signaling Technology) at a dilution of 1/1000 in 5% milk/TBST overnight at 4°C. Anti-Akt Abs were detected using HRP-conjugated anti-rabbit IgG secondary Ab (Cell Signaling Technology) diluted 1/2000 in 5% milk/TBST. The conjugate was incubated for 1 h at room temperature and detected using the LumiGlo reagent and peroxide (Cell Signaling Technology),

Diazepam transformation analysis

Hepatocytes were grown to confluence in 10-well PRIMERA plates (BD Biosciences). After various treatments, the cells were incubated with diazepam (4.0 µg/ml) for various times, after which the medium was collected and analyzed using HPLC as previously described (24).

Determination of protein synthesis

Synthesis of protein by porcine hepatocytes was assayed by measuring incorporation of [3H]leucine. Porcine hepatocytes grown to confluence in six-well plates were treated by various means and then incubated with RPMI 1640 containing 1.0 µCi/ml [3H]leucine for 1 h. Incubation was terminated by removing the labeled medium and washing the cells twice with ice-cold 10 mM HEPES-buffered saline. The cells were then treated with 1% Triton X-100/PBS and collected. The samples were incubated for 15 min (4°C) and centrifuged at 5000 rpm for 5 min. Incorporated [3H]leucine was determined using a Wallac 1414 liquid scintillation counter (Wallac).

Analysis of albumin, cytochrome P450 1A1, and cytochrome P450 3A4 mRNA

The mRNA for albumin, cytochrome P450 1A1 and cytochrome P450 3A4 were assayed by real-time RT-PCR as follows. Total RNA was isolated from cultured cells using the acid guanidinium thiocyanate-phenol-chloroform extraction method (25). RNA was reverse transcribed using poly(dT) DNA primers (Amersham Pharmacia Biotech). The reaction used primer pAlb-S (GCT GTG ATA AGC CTC TGT TGG) and pAlb-AS(GGT GTA ACG AAC TAT GAG CGC) for albumin mRNA, primer pCYP1a-S(CAG TGC ATC ATC ACA GCC AAC ATC ATC TGC) and pCYP1a-AS(AGT GTG GTG CTG CTG GTC TCA G) for cytochrome P450 1A1 mRNA, and primer pCYP3a-S(GCA GAC AGA CAA GCA GAG ATG AAC) and pCYP3a-AS (ATC ACG CTC CAG TTA TGA CTG CAT C) for cytochrome P450 3A4 (26). Real-time PCR was performed on cDNA using a LightCycler rapid thermal cycler system (Roche Diagnostics) according to the manufacturer’s instructions. Reactions were performed in a 20-µl volume with 0.5 µM primers and a MgCl2 concentration optimized between 2 and 5 mM. Nucleotides, TaqDNA polymerase, and buffer were included in the LightCycler-DNA Master SYBR Green I mix (Roche Diagnostics). The protocol consisted of a 30-s denaturation step followed by 40 cycles with a 95°C denaturation for 0 s, 55°C annealing for 5 s, and 72°C extension for 5 s. Extension periods varied with specific primers depending on the length of the product (~1 s/25 bp). Detection of the fluorescent product was conducted at the end of the 72°C extension period. To confirm specificity, the PCR products from each primer pair were subjected to melting curve analysis and subsequent agarose gel electrophoresis. Data were analyzed with the LightCycler analysis software (Roche Diagnostics) as described previously (27). The baseline of each reaction was equalized by calculating the mean value of the five lowest measured data points for each sample and subtracting this value from each reading point. Background fluorescence was removed by setting a noise band. The number of cycles at which the best-fit line through the log-linear portion of each amplification curve intersects the noise band is inversely proportional to the log of the copy number (28).

Analysis of urea synthesis

The synthesis of urea by hepatocytes was measured as follows. Hepatocytes grown to confluency in six-well plates were treated by various means and then incubated with 1 ml of medium containing 1.0 mM NH4Cl for various times. Following incubation, the medium was removed and combined with 0.2 ml of trichloroacetic acid. The mixture was centrifuged at 13,000 x g for 1 min and the urea content was determined spectrophotometrically at 525 nm using diacetyl monoxime (Sigma-Aldrich).

Statistical analysis

Results are expressed as mean ± SEM unless otherwise specified. A two-sided unpaired Student’s t test was used to compare means with a p < 0.05 being considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Susceptibility of hepatocytes to complement-mediated lysis

We first asked to what extent are hepatocytes susceptible to complement-mediated lysis. To address this question, we incubated confluent cells sequentially with human serum (25%) known to contain anti-swine Abs and heated to 56°C to inactivate complement as a source of Ab and then with serial dilutions of normal human serum as a source of complement and measured cellular lysis as described above. Only 7.9 ± 2.5% of hepatocytes were lysed by the highest concentrations of human complement used (25%) while 70.8 ± 1.1% of aortic endothelial cells were lysed under these conditions (p < 0.0001; Fig. 1). The susceptibility of porcine aortic endothelial cells to complement is typical of most cells studied. For example, HUVECs, human aortic endothelial cells, and human monocytes exhibited similar susceptibility to complement-mediated lysis as porcine aortic endothelial cells (data not shown). Neither hepatocytes nor aortic endothelial cells were killed by heat-inactivated human serum (data not shown). Thus, porcine hepatocytes profoundly resisted complement-mediated lysis compared with porcine aortic endothelial cells. The sensitivity of hepatocytes to complement-mediated lysis did not increase with extended culture from 4 to 14 days (data not shown), suggesting that the resistance was not acquired from exposure to complement or other substances in vivo or during harvesting, but rather reflects a constitutive property of the cells. Nor were hepatocytes dying by apoptosis as hepatocytes exposed to anti-swine Abs and complement as described above for 4 or 8 h exhibited only low levels of apoptosis (3.1 ± 2.3% and 4.6 ± 3.3%), as measured by TUNEL, similar to hepatocytes treated with heat-inactivated serum (2.3 ± 3.7% and 3.3 ± 4.5%; data not shown).



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  		FIGURE 1. Sensitivity of hepatocytes to complement-mediated injury. Porcine hepatocytes and aortic endothelial cells, used as controls, were labeled with 1.0 µCi/ml [51Cr]sodium chromate and incubated with 25% heat-inactivated human serum as a source of anti-porcine Abs followed by serial dilutions of normal human serum as a source of complement. Specific lysis was determined as described in Materials and Methods. The results show a profound resistance of hepatocytes to complement-mediated lysis compared with endothelial cells. Results are expressed as mean ± SEM of five experiments. *, p < 0.005;**, p < 0.0001).

 
Measurement of Ab binding

We questioned whether the decreased susceptibility of hepatocytes to complement-mediated lysis could be due to decreased binding of complement-fixing Abs to the cells leading to decreased complement activation. To exclude this possibility, we compared binding of human IgM and IgG to hepatocytes and aortic endothelial cells. As Fig. 2A shows, human IgM bound to porcine hepatocytes to the same extent as to porcine aortic endothelial cells. Human IgG bound to both hepatocytes and aortic endothelial cells at a similar but low level (Fig. 2B). Thus, decreased binding of complement-fixing Abs did not cause decreased susceptibility of hepatocytes to complement-mediated lysis.



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  		FIGURE 2. Binding of human Abs to porcine hepatocytes. Porcine hepatocytes and aortic endothelial cells were exposed to serial dilutions of heat-inactivated human serum and the deposition of IgM (A) and IgG (B) measured by ELISA. Incubations were performed in triplicate and results are expressed as mean ± SD. Human xenoreactive Abs bound to porcine hepatocytes and endothelial cells at similar levels. Similar results obtained in five independent experiments.

 
Despite similar levels of Ab binding, we questioned whether the Ags recognized by the Abs might be different. The human Abs that bind to endothelial cells predominantly recognize Gal{alpha}1–3Gal (21, 29). However, Hayashi et al. (30) reported that hepatocytes do not express Gal{alpha}1–3Gal, while McKenzie et al. (31) reported that liver parenchyma does in fact express high levels of Gal{alpha}1–3Gal. To test whether anti-Gal{alpha}1–3Gal Abs bind to porcine hepatocytes, we measured the extent to which binding of human IgM was prevented by treatment of the cells with {alpha}-galactosidase, which cleaves Gal{alpha}1–3Gal. Treatment of hepatocytes with {alpha}-galactosidase eliminated binding of human IgM nearly completely, suggesting that Gal{alpha}1–3Gal is the major Ag to which human Abs bound (Fig. 3A). To confirm these results, we tested whether absorption of human sera with Gal{alpha}1–3Gal{beta}1–4GlcNAc would reduce binding (32). Absorption of human serum with Gal{alpha}1–3Gal{beta}1–4GlcNAc dramatically reduced but did not eliminate Ab binding to hepatocytes (Fig. 3B). The residual Ab binding could reflect recognition of other Ags; however, it is more likely that the soluble sugar does not completely block binding of all human anti-Gal{alpha}1–3Gal Abs (29, 33).



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  		FIGURE 3. Binding of human Abs to Gal{alpha}1–3Gal on porcine hepatocytes. The extent to which porcine hepatocytes express Gal{alpha}1–3Gal, a saccharide recognized by human natural Abs, is a matter of controversy. Hence, the level of binding of anti-Gal{alpha}1–3Gal Abs to porcine hepatocytes was tested using two methods. A, Abrogation of human Ab binding to porcine hepatocytes treated with {alpha}-galactosidase. Human IgM binding to hepatocytes treated with 1.0 U of {alpha}-galactosidase and untreated hepatocytes was measured by ELISA. B, Blocking by Gal{alpha}1–3GalGlcNAc of human IgM binding to porcine hepatocytes. Human serum was preabsorbed with 10 mM Gal{alpha}1–3GalGlcNAc sugar and the binding of human IgM to porcine hepatocytes was measured by ELISA. Incubations were performed in triplicate and results are expressed as mean ± SD. Similar results obtained in four independent experiments.

 
To determine whether hepatocytes actually express Gal{alpha}1–3Gal, we studied the binding of Griffonia simplicifolia type I lectin, isolectin B4 (GS-IB4), a lectin that specifically recognizes Gal{alpha}1–3Gal, to sections of liver obtained from pigs at varying ages. GS-IB4 bound only weakly to livers obtained from fetal pigs at 100 days gestation and from 3-day-old newborn pigs (binding was mainly to endothelium), but the lectin bound at high levels to livers obtained from pigs 1 mo of age and older (binding was both to endothelium and hepatocytes), suggesting that Gal{alpha}1–3Gal expression on hepatocytes is developmentally regulated (Fig. 4).



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  		FIGURE 4. Developmentally regulated expression of Gal{alpha}1–3Gal in porcine liver. Porcine livers were harvested at the indicated time points during gestation or after birth. Tissues from the livers were fixed in Formalin, embedded in paraffin, and sectioned. Tissue sections were deparaffinized and then incubated with GS-IB4, a lectin that specifically recognizes Gal{alpha}1–3Gal. Paraffin-embedded liver sections obtained from pigs at 100 days gestation (A), 12 h (B), 3 days (C), 1 mo, 4 mo (E), 8 mo (F), and 10 mo (G). Original magnification, x100.

 
Measurement of complement deposition

We next tested the extent to which complement is activated by Abs bound to the surface of porcine hepatocytes. For this purpose, hepatocytes and aortic endothelial cells were incubated sequentially with 25% heat-inactivated human serum and serial dilutions of human complement, as described above, and the deposition of iC3b and the membrane attack complex was measured by ELISA. Following incubation with human sera, very similar amounts of iC3b (Fig. 5A) and the membrane attack complex (Fig. 5B) were detected on hepatocytes and aortic endothelial cells. The finding of similar levels of bound complement, particularly the membrane attack complex, on hepatocytes and endothelial cells suggests the resistance of hepatocytes to lysis cannot be ascribed to a lesser amount of complement activated on the surface of the cells.



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  		FIGURE 5. Measurement of complement deposition on hepatocytes and endothelial cells. Porcine hepatocytes and aortic endothelial cells were incubated with heat-inactivated human serum as a source of anti-porcine Abs and serial dilutions of normal human serum as a source of complement. The deposition of the complement components iC3b (A) and the membrane attack complex (B) was measured by ELISA. Incubations were performed in triplicate and results are expressed as mean ± SD. The results demonstrate that iC3b and the membrane attack complex are deposited to the same extent on hepatocytes and aortic endothelial cells. Results are representative of three independent experiments.

 
Mechanism of resistance to complement

We next asked whether the resistance of hepatocytes to complement-mediated injury might be mediated by CD59 and/or CD55, complement regulatory proteins expressed both by endothelial cells and hepatocytes (34). We first ascertained that both the porcine hepatocytes and porcine endothelial cells we used contained mRNA for these proteins based on semiquantitative RT-PCR (data not shown). To determine whether CD59 and CD55 might protect hepatocytes from complement-mediated injury, we tested the extent to which release of these proteins, by phosphoinositide-specific phospholipase C, would increase sensitivity of the cells to lysis by human complement (Fig. 6A). Following treatment with phosphoinositide-specific phospholipase C, the sensitivity of hepatocytes to complement-mediated lysis did not change; whereas, treatment with the same enzyme caused aortic endothelial cells to become significantly more susceptible to lysis. This difference suggests the resistance of hepatocytes to lysis does not depend on CD59 or CD55.



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  		FIGURE 6. The role of complement regulatory proteins in the resistance of hepatocytes to complement-mediated injury. The extent to which complement regulatory proteins account for the apparent resistance of hepatocytes to lysis was determined by testing whether sensitivity to complement-mediated lysis was increased after complement regulatory proteins were removed from the cells (A) and whether hepatocytes resist lysis by pore-forming proteins not subject to control (B). A, Sensitivity of phosphoinositide-specific phospholipase C (PIPLC) treated (tx) hepatocytes to complement-mediated lysis. Porcine hepatocytes or aortic endothelial cells were treated with 1.0 U/ml phospholipase C for 1 h to release CD59 and CD55 from the cell surface. After treatment with phospholipase C, cells were exposed to 25% heat-inactivated human serum, as a source of anti-porcine Abs, and 12.5% normal human serum, as a source of complement, for 4 h. Lysis was measured as described in Materials and Methods. Results are expressed as mean ± SEM of at least three independent experiments. B, Sensitivity of hepatocytes to lysis by melittin. Hepatocytes or aortic endothelial cells were treated with 1.25 µM melittin, a pore-forming molecule from bee venom, for 4 h. Cell lysis was measured as described in Materials and Methods. The concentration of melittin was normalized for the amount of lysis obtained following exposure to 12.5% normal human serum as depicted in A. Results are expressed as mean ± SEM of at least three independent experiments.

 
Since resistance of hepatocytes to lysis did not depend on cell-associated complement regulatory proteins, we hypothesized it might reflect an intrinsic property of the cells rather than inhibition of complement. To test this possibility, we compared the sensitivity of hepatocytes and aortic endothelial cells to lysis by melittin, a pore-forming protein in bee venom, the lytic properties of which bypass inhibitors of complement. As shown in Fig. 6B, hepatocytes significantly resisted lysis by melittin compared with aortic endothelial cells (12.2 ± 2.0% vs 36.1 ± 3.6%; p < 0.01). These results suggest that hepatocytes utilize an intrinsic mechanism to resist lysis by pore-forming proteins.

Identification of protective pathway

If the resistance of hepatocytes to lysis was not mediated by cell surface complement regulatory proteins, we reasoned it might involve the activity of a "protective" signaling pathway. To test this possibility, we treated hepatocytes with inhibitors of pathways thought to modify cellular responses to injury and then determined whether sensitivity to lysis was changed. Treatment of hepatocytes with inhibitors of the MEK/ERK (PD98059, 50 µM), p38 MAPK (SB203580, 200 nM), protein kinase C (Gö6976, 25 nM), and JNK (SP600125, 25 µM), which have been implicated in protecting cells from cellular injury and stress (35, 36, 37), did not increase susceptibility of hepatocytes to complement-mediated injury compared with vehicle-treated cells (Fig. 7A). In contrast, treatment of hepatocytes with LY294002 (25 µM), a specific inhibitor of PI3K, which has also been implicated in cellular resistance to various types of injury (38), increased susceptibility to complement-mediated lysis (27.9 ± 2.3% vs 1.8 ± 0.9%; p < 0.01). The increase in lysis of treated cells was not caused by some toxic property of LY294002, as treatment with inhibitor alone did not cause significant lysis (data not shown). Wortmannin (500 nM), another inhibitor of PI3K, also heightened susceptibility (19.9 ± 3.7% vs 2.5 ± 0.5%; p < 0.01).



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  		FIGURE 7. The mechanism(s) underlying the resistance of hepatocytes to complement-mediated lysis. A, Pathway(s) protecting hepatocytes from lysis by complement. Hepatocytes were treated with inhibitors of pathways thought to protect cells from injury (–) or vehicle control (+) and then with anti-porcine Abs and complement. Lysis was measured as described in Materials and Methods. The pathways and corresponding inhibitors used are as follows: PI3K (LY294002 (Ly), 25 µM; wortmannin (Wort), 500 nM), Akt (25 µM), MEK1/2 (PD98059, 50 µM), p38 MAPK (SB203580, 200 nM), protein kinase C (Gö6976, 25 nM), or JNK (SP600125, 25 µM). The results are expressed as mean ± SEM of at least three independent experiments. Inhibition of the PI3K/Akt pathway abrogates the resistance of hepatocytes to lysis by complement. *, p < 0.05; **, p < 0.01). B, Activation of Akt by complement. To test whether complement activates Akt, hepatocytes were treated with 25% heat-inactivated human serum as a source of anti-porcine Abs and then with 10% normal human serum as a source of complement for the times indicated. Cells were lysed with Beech’s lysis buffer and extracted proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes and probed with Abs specific for total or phospho-Akt(Ser473). C, State of activation of Akt in normal liver. To determine whether Akt is in the "active" state in normal liver, tissue from normal porcine liver, heart, and kidney was ground with mortar and pestle, cells were lysed with Beech’s lysis buffer, and proteins were resolved, transferred, and detected as in B. The graph depicts the amount of activated Akt in porcine liver, heart, and kidney as a percentage of total Akt, determined by densitometry. The results are representative of three independent experiments.

 
Akt (protein kinase B), a downstream molecule in the PI3K pathway, has been implicated in protection of hepatocytes from hypoxia (39, 40). To determine whether resistance of hepatocytes to complement depends on Akt, we tested whether inhibition of Akt would similarly vitiate resistance to lysis. As Fig. 7A shows, inhibition of Akt in hepatocytes increased sensitivity of hepatocytes to complement-mediated lysis compared with controls (15.9 ± 2.8% vs 5.1 ± 1.7%; p < 0.01).

We next asked whether Akt is constitutively active in hepatocytes or whether it is activated in response to complement. Since activity of Akt depends on phosphorylation of the protein, we tested whether Akt is phosphorylated in response to complement. Toward that end, hepatocytes were incubated with human complement for various periods of time and then assayed for phospho-Akt (Ser473) by Western blotting. As Fig. 7B shows, exposure of hepatocytes to complement resulted in a dramatic increase in the amount of phospho-Akt (Ser473) in the cells. The increase in phospho-Akt was observed as early as 15 min after exposure to complement and returned to baseline within 2 h. The level of phospho-Akt did not change in hepatocytes incubated with heat-inactivated human serum, suggesting the response resulted from activated complement and not other constituents of the serum.

To determine whether sustained phosphorylation and activation of Akt might protect the liver in vivo from constant exposure to activated complement components, we measured the level of phospho-Akt (Ser473) in fresh porcine liver and compared it to levels of phospho-Akt in fresh heart and kidney tissue. Liver tissue contained substantially higher levels of phospho-Akt than either heart or kidney tissue (Fig. 7C).

Impact of complement on the functions of hepatocytes

When complement is activated on cell surfaces under conditions in which cytotoxicity does not occur, cellular functions can change dramatically. For example, complement activation on endothelial cells profoundly changes the function of the cells, causing them to become procoagulant and proinflammatory (1, 2). Complement activation on fibroblasts induces production of growth factors and proliferation (41). Complement activation on oligodendrocytes modifies their state of differentiation (42). Although hepatocytes clearly resist lysis by complement, at least in part, due to the function of the PI3K/Akt pathway, this pathway or other pathways triggered by complement might change the function of liver cells. To test this possibility, we activated complement on the surface of porcine hepatocytes, as described above, and then measured various metabolic functions of the treated cells.

We asked whether complement activation on the surface of hepatocytes compromises detoxification. Detoxification by hepatocytes was assessed by measuring the expression of cytochrome P450 1A1 and cytochrome P450 3A4 mRNA and elimination of diazepam. Surprisingly, the level of cytochrome P450 1A1 and cytochrome P450 3A4 mRNA in hepatocytes, as measured by real-time quantitative RT-PCR, was not changed when complement was activated on hepatocytes or when anaphylatoxins were generated by adding cobra venom factor to the human serum applied to the cells (data not shown). Consistent with this observation, diazepam, which is metabolized by cytochrome P450 3A4 (43), was metabolized as well by hepatocytes treated with anti-porcine Abs and complement as by hepatocytes treated with human serum heated to 56°C to inactivate complement, 3.3 ± 1.6% per h and 3.2 ± 1.1% per h, respectively (p > 0.05; Fig. 8A). Similar results were observed when hepatocytes were treated with complement activated by cobra venom factor or with normal porcine serum (data not shown).



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  		FIGURE 8. Impact of complement on the functions of hepatocytes. The extent to which complement perturbs the functions of hepatocytes was tested by measuring the ability of hepatocytes treated with complement to metabolize diazepam (A) and to synthesize urea (B). A, Effect of complement on the metabolism of diazepam. Hepatocytes were treated for 4 h with 10% human serum as a source of anti-porcine Abs and complement or 10% human serum heated to 56°C to inactivate complement. The hepatocytes were washed and incubated with medium containing diazepam (4.0 µg/ml) for various periods of time, after which the medium was collected and the concentration of diazepam measured by HPLC. The results represent the mean ± SEM of five independent experiments. B, Effect of complement on the synthesis of urea. Hepatocytes were treated as described in A and then incubated with medium containing NH4Cl (1.0 mM) for various periods of time. The concentration of urea was determined by adding trichloroacetic acid to the medium followed by diacetyl monoxime and measuring the absorbance of the product at 525 nm using a spectrophotometer. The results represent the mean ± SEM of three independent experiments.

 
We next asked whether complement activation on hepatocytes changes synthesis of proteins. To address this question, we measured incorporation of [3H]leucine following exposure to complement for various times. Protein synthesis was not significantly altered in hepatocytes treated with anti-porcine Abs and complement for 3 h (27,276 ± 4,545 cpm) or with human serum treated with cobra venom factor (26,477 ± 2,703 cpm) compared with hepatocytes treated with human serum heated to 56°C to inactivate complement (25,852 ± 5,548 cpm) or normal porcine serum (26,627 ± 4,267 cpm). Consistent with this observation, the synthesis of albumin, determined based on the level of mRNA, the level of which is directly related to albumin synthesis (44), measured by real-time quantitative RT-PCR, was unchanged after exposure to complement (data not shown).

We next asked whether complement hampers the ability of hepatocytes to synthesize urea. Hepatocytes were treated with anti-porcine Abs and complement or heat-inactivated human serum for 4 h followed by medium containing 1.0 mM NH4Cl for various times. Urea synthesis was determined by the diacetyl monoxime technique and measured with a spectrophotometer at 525 nm. Hepatocytes treated with anti-porcine Abs and complement synthesized urea at a rate of 1.14 ± 0.67 µg/ml per h while control hepatocytes (treated with heat-inactivated complement) synthesized urea at a rate of 1.43 ± 0.51 µg/ml per h (p > 0.05; Fig. 8B). Similar rates of urea synthesis were observed for hepatocytes exposed to anaphylatoxins (serum to which cobra venom factor was added) or normal porcine serum (data not shown).


   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
If any organ is threatened by unwanted activation of complement, it should be the liver. The liver produces the proteins of the complement cascade in large amounts (9), and indeed hepatic failure is marked by complement deficiency (14). The liver receives the portal circulation which contains the products of bacteria and sometimes intact bacteria that are the most potent activators of complement (6, 8). Thus, not surprisingly, failure of the liver is associated with spontaneous peritonitis and sepsis in which deficiency of complement is implicated (9, 14). The normal functions of the liver include an extraordinary series of metabolic processes; at least some of which should be subject to perturbation by toxic events, such as complement activation. Indeed, conditions such as sepsis, in which exogenous toxins circulate, are commonly associated with a range of abnormalities in hepatic function. Yet, as we show here, hepatocytes are nearly inured to complement and its activated products, and this resistance to injury requires integrity of the PI3K/Akt pathway. Yet, if our findings disclose an unappreciated and important property of hepatocytes, these findings also suggest new questions that we believe will merit consideration.

Perhaps the resilience of hepatocytes in the face of activated complement should have been expected, but it was not. The blood vessels of the liver are lined by cells of the reticuloendothelial system that efficiently clear complement complexes from the circulation. However, the efficiency of the reticuloendothelial system in clearing complement complexes probably does not suffice to protect the liver, since complement activators undoubtedly still pass through the fenestrated sinusoidal endothelial cells to confront newly synthesized components. Sodoyez-Goffaux et al. (45) found that while large immune complexes were efficiently cleared by the liver following i.v. injection, smaller complexes remained in the circulation and presumably passed through the sinusoidal fenestrae. As a response to bacterial infection, hepatocytes produce large amounts of C-reactive protein which is capable of binding bacteria entering the liver via the portal circulation and initiating the classical complement pathway (46, 47). One might expect that this combination of activators and complement proteins would threaten the function and viability of hepatocytes.

The resistance of hepatocytes to injury not only permits the liver to survive and function under physiologic conditions that would destroy other tissues, it protects the liver in disease as well. Loegering et al. (48) found that suppressing the ability of the reticuloendothelial system to clear immune complexes by saturating complement receptors with Ab-coated rat erythrocytes leads to heightened susceptibility to Pseudomonas aeruginosa infection and endotoxic shock but does not cause liver injury. As another example, when as a consequence of abdominal surgery bacteria enter the blood and activate complement, the liver is spared from complement-mediated necrosis, even as its function is impaired by bacterial toxins (6). Similarly, when the liver is transplanted into recipients who have complement-fixing Abs against Ags carried by the graft, hyperacute or acute vascular rejection is rarely observed (22, 49, 50) and, thus, the liver is said to "resist" humoral injury (51). Given these considerations, understanding the means by which the liver resists complement-mediated injury might allow the devising of strategies to protect other organs from complement or to reverse complement-mediated disease.

Our work provides at least initial insights into the mechanisms by which the liver resists injury from complement. We show that the mechanism does not depend on cell membrane-associated regulators of the complement cascade, such as CD55 and CD59, as complement is fully activated on hepatocyte cell surfaces and removing these proteins by enzymatic cleavage does not make the cells more sensitive to complement. Others have found that complement regulatory proteins contribute to resistance of hepatoma cells to complement-mediated injury (52). Presumably, the protection conferred on CD55 to hepatoma cells reflects the heightened susceptibility of those cells compared with hepatocytes to complement-mediated lysis. Nor is the resistance of hepatocytes to complement a condition of the cells acquired over time, as resistance is retained, without decrement, over a period of days in culture. Nevertheless, resistance is associated with and may depend on the phosphorylation of Akt. On this basis, we would conclude that resistance to complement is a basic property of differentiation of hepatocytes. How this resistance is achieved is at present incompletely known. We do show that activity of the PI3K/Akt pathways is required, but that is not to say that this pathway suffices. So many of the metabolic functions of hepatocytes resist impairment by complement; we believe other protective mechanisms are likely to be found.

Activation of the PI3K/Akt pathway in hepatocytes by complement, as we report here, suggests a possible mechanism by which complement might promote regeneration of the liver. Mice deficient in C3 and/or C5 exhibit impaired liver regeneration following partial hepatectomy or toxic liver injury (53, 54, 55). Although this defect in the complement cascade might have various consequences, the activation of the PI3K/Akt pathway may be especially important because it has a well-established role in both cell survival and proliferation. For example, Sautin et al. (36) found that lysophosphatidic acid enhances survival of a murine hepatocyte cell line by PI3K-dependent phosphorylation of Akt. Thus, activation of the PI3K/Akt pathway and its downstream mediators by complement could serve to stimulate the proliferation of hepatocytes required for regeneration.

Finally, our findings may have implications for the treatment of liver disease. Isolated hepatocytes are being explored as transplants for the treatment of metabolic diseases or even as a treatment for hepatic insufficiency. For example, Horslen et al. (56) observed temporary relief of hyperammonemia in a child with ornithine transcarbamylase deficiency following transplantation of allogeneic hepatocytes. Gunsalus et al. (57) showed that porcine hepatocytes introduced into the liver of Watanabe rabbits, which have a genetic defect in low-density lipoprotein receptors causing severe hypercholesterolemia, brings about a substantial lowering of blood cholesterol. Nagata et al. (58) found that transplantation of porcine hepatocytes into cirrhotic rats prolonged survival and reconstituted metabolic liver function. Transplantation of isolated hepatocytes might be preferred over transplantation of the intact liver because the former is far less invasive and because it does not require removal of the native liver (16). One impediment to using hepatocytes for transplantation is the possibility that immune responses might lend to activation of complement in the vicinity of the cells; however, if hepatocytes maintain resistance to complement they should not be threatened. Hepatocytes transplanted across broad phylogenetic distances do appear to maintain this resistance because they can survive for prolonged periods in recipients with high levels of Abs directed against the transplanted cells (58).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (HL52297 and A1049742). Back

2 C.A.K. and A.K. contributed equally. Back

3 Address correspondence and reprint requests to Dr. Jeffrey L. Platt, Mayo Clinic College of Medicine, 200 First Street SW, Medical Sciences 2-66, Rochester, MN 55905. E-mail address: platt.jeffrey{at}mayo.edu Back

4 Abbreviations used in this paper: Gal{alpha}1–3Gal, GS-IB4, Griffonia simplicifolia type I lectin isolectin B. Back

Received for publication February 8, 2005. Accepted for publication March 18, 2005.


   References
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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