Intrinsic Resistance of Hepatocytes to Complement-Mediated Injury1
http://www.100md.com
免疫学杂志 2005年第11期
Abstract
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
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, 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
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
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).
To determine whether hepatocytes actually express Gal1–3Gal, we studied the binding of Griffonia simplicifolia type I lectin, isolectin B4 (GS-IB4), a lectin that specifically recognizes Gal1–3Gal, 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 Gal1–3Gal expression on hepatocytes is developmentally regulated (Fig. 4).
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.
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.
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 (G?6976, 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).
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).
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
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).
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).
2 C.A.K. and A.K. contributed equally.
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
4 Abbreviations used in this paper: Gal1–3Gal, GS-IB4, Griffonia simplicifolia type I lectin isolectin B.
Received for publication February 8, 2005. Accepted for publication March 18, 2005.
References
Saadi, S., R. A. Holzknecht, C. P. Patte, D. M. Stern, J. L. Platt. 1995. Complement-mediated regulation of tissue factor activity in endothelium. J. Exp. Med. 182: 1807-1814.
Saadi, S., R. A. Holzknecht, C. P. Patte, J. L. Platt. 2000. Endothelial cell activation by pore forming structures: pivotal role for IL-1. Circulation 101: 1867-1873.
Platt, J. L., G. M. Vercellotti, B. J. Lindman, T. R. Oegema, Jr, F. H. Bach, A. P. Dalmasso. 1990. Release of heparan sulfate from endothelial cells: implications for pathogenesis of hyperacute rejection. J. Exp. Med. 171: 1363-1368.
Lovett, D. H., G. M. Haensch, M. Goppelt, K. Resch, D. Gemsa. 1987. Activation of glomerular mesangial cells by the terminal membrane attack complex of complement. J. Immunol. 138: 2473-2480.
Pippin, J. W., R. Durvasula, A. Petermann, K. Hiromura, W. G. Couser, S. J. Shankland. 2003. DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes. J. Clin. Invest. 111: 877-885.
Jacob, A. I., P. K. Goldberg, N. Bloom, G. A. Degenshein, P. J. Kozinn. 1977. Endotoxin and bacteria in portal blood. Gastroenterology 72: 1268-1270.(Cody A. Koch2,*,, Akiyosh)
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
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, 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
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
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).
To determine whether hepatocytes actually express Gal1–3Gal, we studied the binding of Griffonia simplicifolia type I lectin, isolectin B4 (GS-IB4), a lectin that specifically recognizes Gal1–3Gal, 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 Gal1–3Gal expression on hepatocytes is developmentally regulated (Fig. 4).
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.
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.
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 (G?6976, 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).
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).
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
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).
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).
2 C.A.K. and A.K. contributed equally.
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
4 Abbreviations used in this paper: Gal1–3Gal, GS-IB4, Griffonia simplicifolia type I lectin isolectin B.
Received for publication February 8, 2005. Accepted for publication March 18, 2005.
References
Saadi, S., R. A. Holzknecht, C. P. Patte, D. M. Stern, J. L. Platt. 1995. Complement-mediated regulation of tissue factor activity in endothelium. J. Exp. Med. 182: 1807-1814.
Saadi, S., R. A. Holzknecht, C. P. Patte, J. L. Platt. 2000. Endothelial cell activation by pore forming structures: pivotal role for IL-1. Circulation 101: 1867-1873.
Platt, J. L., G. M. Vercellotti, B. J. Lindman, T. R. Oegema, Jr, F. H. Bach, A. P. Dalmasso. 1990. Release of heparan sulfate from endothelial cells: implications for pathogenesis of hyperacute rejection. J. Exp. Med. 171: 1363-1368.
Lovett, D. H., G. M. Haensch, M. Goppelt, K. Resch, D. Gemsa. 1987. Activation of glomerular mesangial cells by the terminal membrane attack complex of complement. J. Immunol. 138: 2473-2480.
Pippin, J. W., R. Durvasula, A. Petermann, K. Hiromura, W. G. Couser, S. J. Shankland. 2003. DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes. J. Clin. Invest. 111: 877-885.
Jacob, A. I., P. K. Goldberg, N. Bloom, G. A. Degenshein, P. J. Kozinn. 1977. Endotoxin and bacteria in portal blood. Gastroenterology 72: 1268-1270.(Cody A. Koch2,*,, Akiyosh)