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Immune response to lipopolysaccharide in primary biliary cirrhosis and autoimmune diseases
Journal of Autoimmunity 22 (2004) 153–158 www.elsevier.com/locate/issn/08968411
Immune response to lipopolysaccharide in primary biliary cirrhosis and autoimmune diseases Eric Ballot a, Olivia Bandin a,b, Olivier Chazouilleres c, Catherine Johanet a,*, Raoul Poupon c a
Service d’Immunologie, Hoˆpital Saint-Antoine, AP-HP, 184 rue du faubourg Saint-Antoine, 75012 Paris, France b Laboratoire de Biologie, Hoˆpital Sainte-Camille, Bry-sur-Marne, France c Service d’He´pato-gastroente´rologie, Hoˆpital Saint-Antoine, AP-HP, Paris, France Received 13 August 2003; revised 29 October 2003; accepted 10 November 2003
Abstract A bacteriological aetiology is suspected to be the triggering factor in primary biliary cirrhosis. We studied lipid A, the toxic and immunogenic moiety of gram-negative bacteria lipopolysaccharide, which accumulates abnormally in Kupffer cells, hepatocytes, and biliary epithelial cells in primary biliary cirrhosis patients. Anti-lipid A antibody levels from serum samples from 36 primary biliary cirrhosis patients, drawn before and after ursodeoxycholic acid treatment, were compared to those from patients with other liver diseases (n=236), non-hepatic diseases (n=249), and healthy subjects (n=75). In primary biliary cirrhosis patients, the prevalence of IgM anti-lipid A antibodies was higher before than after ursodeoxycholic acid therapy (64% vs 22%, respectively; P<0.001). Patients with anti-lipid A antibodies had significantly higher IgM levels than those without antibodies (8.71.1 g/l vs 4.40.8 g/l, P<0.02). Total IgM levels were correlated with anti-lipid A antibody levels (r=0.65, P<0.02). After therapy, the serum IgM levels decreased significantly (P<0.03). These results indicate that bacterial antigens may participate in the observed increase of serum IgM levels, and support an aetiological role of a gut-derived endotoxin antigen in the pathogenesis of primary biliary cirrhosis. 2004 Elsevier Ltd. All rights reserved. Keywords: Anti-lipid A antibody; Endotoxin; Primary biliary cirrhosis; Ursodeoxycholic acid; Innate immunity
1. Introduction Primary biliary cirrhosis (PBC) is a chronic liver disease resulting from a T-cell mediated immunological attack of intrahepatic small bile ducts and characterized by autoantibodies directed against mitochondrial autoantigens, particularly the E2 component of the pyruvate dehydrogenase complex (PDC-E2) [1]. The aetiology of PBC remains largely speculative. Infectious agents including bacteria are suspected of being involved in this pathology [2–6]. Under normal circumstances, endotoxins relargued from gut bacterial flora are absorbed by the intestine, reach the portal circulation, * Corresponding author. Tel.: +33-1-49282011; fax: +33-1-49283046. E-mail address: [email protected] (J. Catherine). 0896-8411/04/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.11.002
then undergo clearance by Kupffer cells and hepatocytes [7,8]. The presence of abnormal deposits of lipid A, the immunogenic and toxic component of the lipopolysaccharide (LPS), in the cytoplasm of hepatocytes, in Kupffer cells and in biliary epithelial cells was reported in PBC patients [9–11]. This fact supports the hypothesis that intestinal colonization by Rough mutants of Escherichia coli may in some way play a role in the aetiology of PBC [9], or in the occurrence of antimitochondrial antibodies by molecular mimicry [12]. Years ago, attention was focused on the mechanism of innate immune system. This system is activated by receptors which recognize conserved molecular structures expressed on pathogens, including Toll-like receptor (TLR) which is stimulated by fixation of LPS [13]. This stimulation leads to recruitment of several molecules and to NF-kB translocation which in turn
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causes transcriptional activation of cytokine genes [14]. TLR activation play also a role in the induction of adaptive immune response. Ide et al. [15] have reported that IgM anti-lipid A levels were increased in PBC patients and were correlated with the total serum IgM level. In this study we investigated whether the presence of IgM anti-lipid A antibodies is characteristic of PBC or is also found in other autoimmune liver diseases or in conditions due to immune dysfunction. The prevalence of anti-lipid A antibodies in patients receiving long-term treatment with ursodeoxycholic acid (UDCA) was also evaluated.
2. Patients and methods 2.1. Patients The study population included 36 patients with PBC (3 men, 33 women; mean age, 52 years; age range, 31–77 years). Serum samples were obtained in all patients before and after (4 years on average) UDCA treatment (13–15 mg/kg/day), for a total of 72 serum samples. Before UDCA treatment, 35 patients had antimitochondrial antibodies type 2 (AMA2) detected by indirect immunofluorescence on a composite block of rat liver, kidney and stomach. Baseline liver biopsies included 10 stage I (portal inflammation confined to the portal tracts), 15 stage II (portal and periportal inflammation without septal fibrosis or bridging necrosis), 4 stage III (lobular fibrosis and/or bridging necrosis), and 7 stage IV (cirrhosis) according to Ludwig classification [16]. Controls consisted of 236 patients with other liver diseases, 249 patients with nonhepatic diseases, and 75 healthy subjects. Patients with other liver diseases included 14 primary sclerosing cholangitis, 67 autoimmune hepatitis (AIH) type I, 43 AIH type II, 60 chronic viral hepatitis C, 30 chronic viral hepatitis B, and 22 alcoholic liver disease. Serum samples from 92 patients with systemic lupus erythematosus (nonhepatic autoimmune disease) and 40 patients with inflammatory bowel diseases (Crohn’s disease, 20; ulcerative colitis, 20) were also evaluated. In order to assess the nonspecific binding in the ELISA test, we also studied serum samples from 22 patients without liver disease who had elevated serum gamma-globulin concentrations (15 with elevated IgG, 4 with elevated IgM, and 3 with both elevated IgG and IgM levels) and serum samples from 20 patients with rheumatoid arthritis and high rhumatoid factor levels. Because several bacterial membrane components (lipid A and cardiolipin) are candidates for antiphospholipid antibodies targeting, we also tested serum samples from 35 patients with primary antiphospholipid syndrome.
Furthermore, we included serum samples from 40 patients with gram-negative septicemia and high levels of endotoxins (including E. coli, 19; Pseudomonas aeruginosa, 11; Proteus mirabilis, 5) as well as serum samples from 75 healthy blood donors. All serum samples were stored at 80(C until assayed. 2.2. Enzyme-linked immunosorbent assay (ELISA) Monophosphoryl lipid A extracted from Salmonella minnesota R595 LPS (List Biological Laboratories, Campbell, CA, USA), containing less than 0.2% ketodeoxyoctonate, was reconstituted at 1 mg/ml in 0.5% triethylamine in sterile pyrogen-free water, and 250 µl aliquots were stored at 80(C until used. ELISA assays were carried out as follows: wells of flat-bottom polystyrene microtiter plates (Microwell, Nunc, Denmark) were coated with 100 µl of a 20 µg/ml solution of lipid A dissolved in ethanol. The dried plates (4 (C overnight) were washed 3 times with phosphatebuffered saline (PBS) at pH 7.4 containing 0.5 ml/l of Tween -20 (PBS-Tween). One hundred microliters of 1:100 PBS diluted serum sample was added to each well and incubated for 1 h at room temperature. Unbound antibodies were removed by washing three times in PBS–Tween. Bound antibodies were detected by the addition to each well of 100 µl of an alkaline phosphatase-conjugated goat anti-human IgM (Biosys, Compie`gne, France) diluted 1:1000 in PBS. After 1 h incubation, wells were washed twice in PBS–Tween and once in 0.9% NaCl. One hundred microliters of a 1 mg/ml p-nitropenylphosphate substrate (Sigma, St Louis, MO, USA) in glycine buffer at pH 10.4 were added to each well and incubated for 20 min at 37 (C. Optical density (OD) at 405 nm was read with a Dynatech MR 5000 automatic plate reader (Dynatech Laboratories, Chantilly, VA, USA). Serum samples with an OD value above 0.9 were considered to be positive for IgM to lipid A (cut-off: mean for healthy individuals+two standard deviations). 2.3. Quantitation of serum immunoglobulins Serum concentrations of IgM were determined by nephelemetry (BNA, Behring). 2.4. Liver biopsies Baseline liver biopsies and repeated liver biopsies were staged according to Ludwig classification [16]. The lesions of cholangitis and the ductopenia were also noted. Cholangitis was defined by the presence of a mononuclear inflammatory infiltrate surrounding and penetrating the bile duct epithelium with associated epithelial damage and/or destruction of the basement
E. Ballot et al. / Journal of Autoimmunity 22 (2004) 153–158
155
Table 1 Prevalence of IgM anti-lipid A antibody in 36 PBC patients before UDCA administration and in 560 controls
The prevalence of anti-lipid A antibody was very low in the other controls.
Diseases
3.2. Baseline characteristics of PBC patients with or without anti-lipid A antibody
N
IgM anti-lipid A N
%
Primary biliary cirrhosis
36
23
64
Liver diseases Primary sclerosing cholangitis Autoimmune hepatitis type I Autoimmune hepatitis type II Chronic viral hepatitis C Chronic viral hepatitis B Alcoholic liver disease
14 67 43 60 30 22
0 15 9 1 0 0
0 22 21 2 0 0
Non-hepatic diseases Systemic lupus erythematosus Inflammatory bowel diseases Elevated serum globulin Rheumatoid arthritis Primary antiphospholipid syndrome Septicemia (gram negative bacteria)
92 40 22 20 35 40
5 0 0 1 3 0
6 0 0 5 8 0
Healthy individuals
75
3
4
membrane. Ductopenia was defined by the absence of bile ducts in at least 50% of the portal tracts. 2.5. Quantitative measurement of serum soluble human CD-14 The quantitative measurement of serum soluble human CD-14 was done using an immunoenzymometric assay (sCD-14 EASIA, Biosource, Europe, SA; normal range: 1.4–4.5 µg/ml). 2.6. Statistical analysis The means of the two independent groups were compared using Student’s t-test or the Mann–Whitney test. The Wilcoxon signed rank test was used to compare nonparametric variables in paired groups. Fisher’s exact test was used to compare frequencies of dichotomous variables. The minimum level of statistical significance was set at P<0.05. Data are presented as meanstandard error (SE).
3. Results
The biochemical and histological data did not differ significantly between these two groups except for total serum IgM levels (Table 2) and the presence of cholangitis lesions and/or ductopenia. In fact, patients with anti-lipid A had significantly higher IgM levels than those without this antibody (8.71.1 g/l vs 4.40.8 g/l, P<0.02). Total serum IgM levels correlated with IgM anti-lipid A antibody levels estimated by OD of the ELISA test (r=0.65, P<0.02) (Fig. 1). Similarly, antilipid A-positive patients had significantly more lesions of cholangitis and/or ductopenia than anti-lipid A-negative patients (100% vs 50%, Fisher exact test, P<0.02). There is no correlation between the titer of antimitochondrial antibody and IgM anti-lipid A antibody (Fig. 2). 3.3. Biological data of PBC patients after UDCA administration After UDCA therapy, a significant decrease in prevalence of anti-lipid A antibody was observed [22% (8/36) after UDCA vs 64% (23/36) before (P<0.001)]. Serum total IgM levels decreased significantly after drug administration in patients with anti-lipid A antibody before UDCA (Wilcoxon test, P<0.03). Mean IgM levels were found to be significantly higher in the group anti-lipid A-positive both before and after UDCA than in the group anti-lipid A-positive before and anti-lipid A-negative after UDCA (8.11 g/l vs 4.80.6 g/l, Mann–Whitney test, P<0.02). In patients anti-lipid A-negative prior to treatment, IgM levels did not differ significantly after UDCA therapy (Wilcoxon test). Under UDCA treatment, normalization of serum alanine and serum aspartate aminotransferases and alkaline phosphatase levels was obtained in 27% antilipid A-positive patients compared to 45% anti-lipid A-negative patients (NS). 3.4. Detection of soluble CD-14 Soluble CD-14 were assayed in serum samples from 21 healthy subjects, 27 PBC patients, and 64 patients with various liver diseases. PBC patients had significantly higher soluble CD-14 levels than healthy individuals (6.80.3 vs 4.60.3 µg/ml, Mann–Whitney test, P<0.001). There was no significant difference between CD-14 levels in PBC patients and those with others liver diseases.
3.1. Detection of IgM anti-lipid A antibody As shown in Table 1, the prevalence of anti-lipid A antibody was 64% (23/36) in PBC patients before UDCA therapy and 22% (24/110) in patients with AIH.
4. Discussion As with other autoimmune diseases, bacterial infection has been incriminated in the pathogenesis of PBC,
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Table 2 Baseline characteristics of PBC patients with or without anti-lipid A antibody
Anti-lipid A positive (n=23) Anti-lipid A negative (n=13)
AP N<100 IU/l
-GT* N<33 IU/l Bilirubin* N<17 µmol/l
Total IgM** N 0.8–2.2 g/l
Liver biopsy* Stage 1–2
41360 452108
46963 36767
8.71.1 4.40.8
67% 60%
25.44.3 28.710.3
AP=serum alkaline phosphatase; -GT=serum -glutamyl transpeptidase. * Not significantly different. ** Significantly different (P<0.02).
Fig. 1. Correlation between serum total IgM and anti-lipid A antibody in 36 patients with PBC before UDCA therapy.
,J0 DQWLOLSLG $ E\ RSWLFDO GHQVLW\
U S 16
0
$QWLPLWRFKRQGULDO DQWLERG\ WLWHUV
Fig. 2. Relation between IgM anti-lipid A antibody and antimitochondrial antibody titer in patients with PBC before UDCA therapy.
the main argument being the existence of a crossreactivity between anti-mitochondrial antibodies and antigens of E. coli or other mycobacteria [12,17]. No definite proof of a bacterial aetiology has been identified to date [18]. In our study, we focused on lipid A, a substance that is the carrier of the main biological activities of LPS (a component of gram-negative bacteria). We confirmed the high prevalence (64%) of IgM anti-lipid A in PBC patients and the significant correlation between the level
of this antibody and total IgM (P<0.02). This data could explain the hyper IgM-emia observed in PBC patients. This anti-lipid A hyper IgM-emia appears specific of this disease because no anti-lipid A antibody was found in patients with hyper IgM-emia without liver disease. These results are in agreement with a previous report by Ide et al. [15]. A valid comparison between our study and the latter report [15] is difficult, however, because the authors did not use the prevalence of anti-lipid A antibody, but rather a ratio of OD in patients serum samples to normal controls serum samples. Futhermore, the number of healthy subjects that served as controls was small (n=19) and did not allow determination of a threshold. In our study, which involved a large population of controls (n=560), anti-lipid A antibody prevalence was very low, except in the group of AIH (24/110, 22%). This prevalence is not easily interpretable because we have little information on histological activity and treatment in this group. Further studies are required to determine the prevalence of anti-lipid A in patients with AIH according to treatment. The role of LPS, a potent immunostimulant which is a polyclonal B cell activator, has been suggested, although it has not been possible to design an experimental animal model for PBC. Masanaga et al. [19], however, induced autoimmune cholangitis by immunizing neonatally thymectomized mice with both recombinant protein containing the major mitochondrial autoantigens and LPS. Their results suggest that endotoxins may increase the immunopathogenicity of this disease. Some authors have shown that NK cells and NK1.Ag+T cells in mice are stimulated by superantigens like LPS through IL-12 production from Kupffer cells [20,21]. At the interface between innate and adaptive immunity, the NK1.Ag+T cells have cytotoxic activities in the absence of prior antigenic stimulation and are able to regulate the secondary adaptive immune response through cytokine production [22]. Interestingly, these cells are able to recognize the nonclassical antigen-presenting molecule CD1d and to respond to lipid or glycolipid components of the bacterial cell wall [23]. In PBC liver, Tsuneyama et al. have reported aberrant expression of CD1d on bile duct epithelium
E. Ballot et al. / Journal of Autoimmunity 22 (2004) 153–158
and epithelioid granuloma around lipid A [24]. Sakisaka et al. also found a significant retention of endotoxin and an increase in apoptosis in the biliary epithelial cells of PBC patients [10]. In our study, the lesions of cholangitis were more frequent and more severe in the group of PBC patients with antibodies to lipid A. This result supports the hypothesis that LPS may contribute to the pathogenesis of cholangitis. Furthermore, the inactivation by dephosphorylation of lipid A under the influence of alkaline phosphatase is pH-dependent [25]. It has been shown that the secretion and alkalinization of the bile are deficient in PBC because of the markedly reduced activity of the AE2 exchanger in the biliary tract [26]. Thus, the LPS present in the biliary tract may be partly responsible for the inflammation and destruction of lesser biliary ducts. The liver plays a central role in eliminating the endotoxins from the portal blood. The cell surface receptors responsible for binding LPS include the CD14/ Toll-like receptor 4, but also the Fc receptor for the complexes LPS-anti-LPS [27]. Thus, accelerated internalization and detoxification of endotoxins by anti-LPS appear to be a Fc receptor mediated process. Indeed, profound disturbances of Fc receptor mediated immune clearance by anti-LPS have been reported in PBC [28]. CD14 is present as both a membrane-bound and a soluble form [29,30]. Soluble CD14 is found in human serum under normal conditions and mediates LPS activation of non-CD14-bearing cells [31]. A recent report has shown that human hepatocytes produce CD14. This local production may have important biological consequences for the liver’s response to LPS [32]. In our study, we have shown a significant increase of the soluble CD14 level in PBC patients but also in other various liver diseases. UDCA is currently the only drug approved specifically for the treatment of PBC [33,34]. The mechanisms underlying the beneficial effects of UDCA in PBC are still ill-defined. It has been reported that UDCA can decrease the translocation of endotoxin from the gut [35] and restore the activity of the AE2 exchanger [26]. Therefore, UDCA may participate in inactivating LPS present in the lesser biliary ducts. Recently, Sasatomi et al. demonstrated the abnormal accumulation of endotoxin in biliary epithelial cells and the possible beneficial effects of UDCA on the intra-hepatic metabolism of endotoxin [11]. Ide et al. have shown that the serum level of IgM anti-lipid A antibody decreased with UDCA therapy [15]. In the present study, according to Ide et al. [15], we have demonstrated a significant decrease of the prevalence of anti-lipid A antibody after UDCA therapy, associated to a decrease of serum total IgM level. In conclusion, our data confirm that a large part of IgM antibodies in PBC patients’ serum samples are directed against lipid A and suggest that disturbance to
157
innate immunity in response to an infection may be involved in some cases of PBC.
Acknowledgements We wish to thank the Laboratoire d’immunochimie (Dr Dubois, Hopital Saint Antoine) for the serum total IgM determination and Y. Chretien for the statistical analysis of the lesions of cholangitis. References [1] Fussey SPM, Guest JR, James OFW, Bassendine MF, Yeaman SJ. Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis. Proc Natl Acad Sci U S A 1988; 85:8654–8. [2] Harada K, Koichi T, Sudo Y, Masuda S, Nakanuma Y. Molecular identification of bacterial 16S ribosomal RNA gene in liver tissue of primary biliary cirrhosis. Is propioni-bacterium acnes involved in granuloma formation? Hepatology 2001;33:530–6. [3] Kreisel W, Siegel A, Bahler A, Spamer C, Schietz E, Kist M et al. Hight prevalence of autoantibodies to calreticulin of the IgA class in primary biliary cirrhosis: a possible role of gut-derived bacterial antigens in its aetiology? Scand J Gastroenterol 1999;34:623–8. [4] Butler P, Valle F, Hamilton Miller JM, Brumfitt W, Baum H, Burroughs AK. M2 mitochondrial antibodies and urinary rough mutant bacteria in patients with primary biliary cirrhosis and in patients with recurrent bacteria. J Hepatol 1993;17:408–14. [5] Butler P, Hamilton Miller JM, McIntyre N, Burroughs AK. Natural history of bacteriuria in women with primary biliary cirrhosis and the effect of antimicrobial therapy in symptomatic and asymptomatic groups. Gut 1995;36:931–4. [6] Vilagut L, Vila J, Vinos O, Pares A, Gines A, Jimenez de Anta MT et al. Cross reactivity of anti-mycobacterium gordonae antibodies with the major mitochondrial autoantigens in primary biliary cirrhosis. J Hepatol 1994;21:673–7. [7] Nolan JP. Intestinal endotoxins as mediators of hepatic injury: an idea whose time has come again. Hepatology 1989;10:887–91. [8] Mimura Y, Sakisaka S, Harada M, Sata M, Tanikawa K. Role of hepatocytes in direct clearance of lipopolysaccharide in rats. Gastroenterology 1995;109:1969–76. [9] Hopf U, Mo¨ller B, Stemerowicz R, Lobeck H, Rodloff A, Freudenberg M et al. Relation between Escherichia coli R (Rough)-forms in gut, lipid A in liver, and primary biliary cirrhosis. Lancet 1989;16:1419–21. [10] Sakisaka S, Koga H, Sasatomi K, Mimura Y, Kawaguchi T, Tanikawa K. Biliary secretion of endotoxin and pathogenesis of primary biliary cirrhosis. Yale J Biol Med 1997;70:403–8. [11] Sasatomi K, Noguchi K, Sakisaka S, Sata M, Tanikawa K. Abnormal accumulation of endotoxin in biliary epithelial cells in primary biliary cirrhosis and primary sclerosing cholangitis. J Hepatol 1998;29:409–16. [12] Fussey SPM, Ali ST, Guest JR, James OFW, Bassendine MF, Yeaman SJ. Reactivity of primary biliary cirrhosis sera with E. coli dihydrolipoamine acetyl transferase (E2p); characterization of the main immunogenic region. Proc Natl Acad Sci U S A 1990; 87:3987–91. [13] Poltorak A, Ricciardi-Castagnoli P, Citterio A, Beutler B. Physical contact between LPS and Tlr4 revealed by genetic complementation. Proc Natl Acad Sci U S A 2000;97:2163–7. [14] Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate response. Nature 2000;405:782–7.
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[15] Ide T, Sata M, Nakano H, Suzuki H, Tanikawa K. Increased serum IgM class anti-lipid A antibody and therapeutic effect of ursodeoxycholic acid in primary biliary cirrhosis. Hepatogastroenterology 1997;44:409–16. [16] Ludwig J, Dickson ER, McDonald GS. Staging of chronic nonsuppurative destructive cholangitis (syndrome of primary biliary cirrhosis). Virchows Arch A Pathol Anat Histol 1978; 379:103–12. [17] Flannery GR, Burroughs AK, Butler P, Chelliah J, Hamilton-Miller JMT, Brumfitt W et al. Antimitochondrial antibodies in primary biliary cirrhosis recognize both specific peptides and shared epitopes of the M2 family of antigens. Hepatology 1989;10:370–4. [18] Poupon R, Chazouille`res O, Poupon RE. Chronic cholestatic diseases. J Hepatol 2000;32:129–40. [19] Masanaga T, Watanabe Y, Van de Water J, Leung PSC, Nakanishi T, Kajiyama G et al. Induction and persistence of immune-mediated cholangiohepatitis in neonatally thymectomized mice. Clin Immunol Immunopathol 1998;89:141–9. [20] Takahashi M, Ogasawara K, Takeda K, Hashimoto W, Sakihara H, Kumagai K et al. LPS induces NK1.1+alpha beta T cell with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells. J Immunol 1996;156:2436–42. [21] Dobashi H, Seki S, Habu Y, Ohkawa T, Takeshita S, Hiraide H et al. Activation of mouse liver natural killer cells and NK1.1+ T cells by bacterial superantigen-primed Kupffer cells. Hepatology 1999;30:430–6. [22] Doherty DG, O’Farrelly. Innate and adaptive lymphoid cells in the human liver. Immunol Rev 2000;174:5–20. [23] Spada FM, Koezuka Y, Porcelli SA. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J Exp Med 1998;188:1529–34. [24] Tsuneyama K, Yasoshima M, Harada K, Hiramatsu K, Gershwin E, Nakanuma Y. Increased CD1d expression on small bile duct epithelium and epithelioid granuloma in livers in primary biliary cirrhosis. Hepatology 1998;28:620–3. [25] Poelstra K, Bakker WW, Klok PA, Kamps JAAM, Hardonk MJ, Meijer DKF. Dephosphorylation of endotoxin by alkaline phosphatase in vivo. Am J Pathol 1997;151:1163–9.
[26] Medina JF, Martinez-Anso E, Vazquez JJ, Prieto J. Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis. Hepatology 1997;25:12–7. [27] Lazaron V, Leslie DB, Wasiluk KR, Dunn DL. Accelerated internalization and detoxification of endotoxin by antilipopolysaccharide antibody is an Fc receptor-mediated process. Surgery 2001;130:192–7. [28] Ekdahl KN, Lo¨o¨f L, Nyberg A, Nilsson UR, Nilsson B. Defective Fc receptor-mediated clearance in patients with primary biliary cirrhosis. Gastroenterology 1991;101:1076–82. [29] Ingalls RR, Heine H, Lien E, Yoshimura A, Golenbock D. Lipopolysaccharide recognition, CD14, and lipopolysaccharide receptors. Infect Dis Clin North Am 1999;13:341–53. [30] Su GL, Klein RD, Aminlari A, Zhang HY, Steinstraesser L, Alarcon WH et al. Kupffer cell activation by lipopolysaccharide binding protein and toll-like receptor 4. Hepatology 2000; 31:932–6. [31] Pugin J, Schurer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ, Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci U S A 1993; 90:2744–8. [32] Su GL, Dorko K, Strom SC, Nu¨ssler AK, Wang SC. CD14 expression and production by human hepatocytes. J Hepatol 1999;31:435–42. [33] Poupon RE, Lindor KD, Cauch-Dudek K, Dickson ER, Poupon R, Heathcote EJ. Combined analysis of randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. Gastroenterology 1997;113:884–90. [34] Poupon RE, Bonnand AM, Chretien Y, Poupon R, UDCA-PBC Study Group. Ten-year survival in ursodeoxycholic acid-treated patients with primary biliary cirrhosis. Hepatology 1999; 29:1668–71. [35] Schwarzenberg SJ, Bundy M. Ursodeoxycholic acid modifies gut-derived endotoxemia in neonatal rats. Pediatr Res 1994; 35:214–7.
Immune response to lipopolysaccharide in primary biliary cirrhosis and autoimmune diseases Eric Ballot a, Olivia Bandin a,b, Olivier Chazouilleres c, Catherine Johanet a,*, Raoul Poupon c a
Service d’Immunologie, Hoˆpital Saint-Antoine, AP-HP, 184 rue du faubourg Saint-Antoine, 75012 Paris, France b Laboratoire de Biologie, Hoˆpital Sainte-Camille, Bry-sur-Marne, France c Service d’He´pato-gastroente´rologie, Hoˆpital Saint-Antoine, AP-HP, Paris, France Received 13 August 2003; revised 29 October 2003; accepted 10 November 2003
Abstract A bacteriological aetiology is suspected to be the triggering factor in primary biliary cirrhosis. We studied lipid A, the toxic and immunogenic moiety of gram-negative bacteria lipopolysaccharide, which accumulates abnormally in Kupffer cells, hepatocytes, and biliary epithelial cells in primary biliary cirrhosis patients. Anti-lipid A antibody levels from serum samples from 36 primary biliary cirrhosis patients, drawn before and after ursodeoxycholic acid treatment, were compared to those from patients with other liver diseases (n=236), non-hepatic diseases (n=249), and healthy subjects (n=75). In primary biliary cirrhosis patients, the prevalence of IgM anti-lipid A antibodies was higher before than after ursodeoxycholic acid therapy (64% vs 22%, respectively; P<0.001). Patients with anti-lipid A antibodies had significantly higher IgM levels than those without antibodies (8.71.1 g/l vs 4.40.8 g/l, P<0.02). Total IgM levels were correlated with anti-lipid A antibody levels (r=0.65, P<0.02). After therapy, the serum IgM levels decreased significantly (P<0.03). These results indicate that bacterial antigens may participate in the observed increase of serum IgM levels, and support an aetiological role of a gut-derived endotoxin antigen in the pathogenesis of primary biliary cirrhosis. 2004 Elsevier Ltd. All rights reserved. Keywords: Anti-lipid A antibody; Endotoxin; Primary biliary cirrhosis; Ursodeoxycholic acid; Innate immunity
1. Introduction Primary biliary cirrhosis (PBC) is a chronic liver disease resulting from a T-cell mediated immunological attack of intrahepatic small bile ducts and characterized by autoantibodies directed against mitochondrial autoantigens, particularly the E2 component of the pyruvate dehydrogenase complex (PDC-E2) [1]. The aetiology of PBC remains largely speculative. Infectious agents including bacteria are suspected of being involved in this pathology [2–6]. Under normal circumstances, endotoxins relargued from gut bacterial flora are absorbed by the intestine, reach the portal circulation, * Corresponding author. Tel.: +33-1-49282011; fax: +33-1-49283046. E-mail address: [email protected] (J. Catherine). 0896-8411/04/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.11.002
then undergo clearance by Kupffer cells and hepatocytes [7,8]. The presence of abnormal deposits of lipid A, the immunogenic and toxic component of the lipopolysaccharide (LPS), in the cytoplasm of hepatocytes, in Kupffer cells and in biliary epithelial cells was reported in PBC patients [9–11]. This fact supports the hypothesis that intestinal colonization by Rough mutants of Escherichia coli may in some way play a role in the aetiology of PBC [9], or in the occurrence of antimitochondrial antibodies by molecular mimicry [12]. Years ago, attention was focused on the mechanism of innate immune system. This system is activated by receptors which recognize conserved molecular structures expressed on pathogens, including Toll-like receptor (TLR) which is stimulated by fixation of LPS [13]. This stimulation leads to recruitment of several molecules and to NF-kB translocation which in turn
154
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causes transcriptional activation of cytokine genes [14]. TLR activation play also a role in the induction of adaptive immune response. Ide et al. [15] have reported that IgM anti-lipid A levels were increased in PBC patients and were correlated with the total serum IgM level. In this study we investigated whether the presence of IgM anti-lipid A antibodies is characteristic of PBC or is also found in other autoimmune liver diseases or in conditions due to immune dysfunction. The prevalence of anti-lipid A antibodies in patients receiving long-term treatment with ursodeoxycholic acid (UDCA) was also evaluated.
2. Patients and methods 2.1. Patients The study population included 36 patients with PBC (3 men, 33 women; mean age, 52 years; age range, 31–77 years). Serum samples were obtained in all patients before and after (4 years on average) UDCA treatment (13–15 mg/kg/day), for a total of 72 serum samples. Before UDCA treatment, 35 patients had antimitochondrial antibodies type 2 (AMA2) detected by indirect immunofluorescence on a composite block of rat liver, kidney and stomach. Baseline liver biopsies included 10 stage I (portal inflammation confined to the portal tracts), 15 stage II (portal and periportal inflammation without septal fibrosis or bridging necrosis), 4 stage III (lobular fibrosis and/or bridging necrosis), and 7 stage IV (cirrhosis) according to Ludwig classification [16]. Controls consisted of 236 patients with other liver diseases, 249 patients with nonhepatic diseases, and 75 healthy subjects. Patients with other liver diseases included 14 primary sclerosing cholangitis, 67 autoimmune hepatitis (AIH) type I, 43 AIH type II, 60 chronic viral hepatitis C, 30 chronic viral hepatitis B, and 22 alcoholic liver disease. Serum samples from 92 patients with systemic lupus erythematosus (nonhepatic autoimmune disease) and 40 patients with inflammatory bowel diseases (Crohn’s disease, 20; ulcerative colitis, 20) were also evaluated. In order to assess the nonspecific binding in the ELISA test, we also studied serum samples from 22 patients without liver disease who had elevated serum gamma-globulin concentrations (15 with elevated IgG, 4 with elevated IgM, and 3 with both elevated IgG and IgM levels) and serum samples from 20 patients with rheumatoid arthritis and high rhumatoid factor levels. Because several bacterial membrane components (lipid A and cardiolipin) are candidates for antiphospholipid antibodies targeting, we also tested serum samples from 35 patients with primary antiphospholipid syndrome.
Furthermore, we included serum samples from 40 patients with gram-negative septicemia and high levels of endotoxins (including E. coli, 19; Pseudomonas aeruginosa, 11; Proteus mirabilis, 5) as well as serum samples from 75 healthy blood donors. All serum samples were stored at 80(C until assayed. 2.2. Enzyme-linked immunosorbent assay (ELISA) Monophosphoryl lipid A extracted from Salmonella minnesota R595 LPS (List Biological Laboratories, Campbell, CA, USA), containing less than 0.2% ketodeoxyoctonate, was reconstituted at 1 mg/ml in 0.5% triethylamine in sterile pyrogen-free water, and 250 µl aliquots were stored at 80(C until used. ELISA assays were carried out as follows: wells of flat-bottom polystyrene microtiter plates (Microwell, Nunc, Denmark) were coated with 100 µl of a 20 µg/ml solution of lipid A dissolved in ethanol. The dried plates (4 (C overnight) were washed 3 times with phosphatebuffered saline (PBS) at pH 7.4 containing 0.5 ml/l of Tween -20 (PBS-Tween). One hundred microliters of 1:100 PBS diluted serum sample was added to each well and incubated for 1 h at room temperature. Unbound antibodies were removed by washing three times in PBS–Tween. Bound antibodies were detected by the addition to each well of 100 µl of an alkaline phosphatase-conjugated goat anti-human IgM (Biosys, Compie`gne, France) diluted 1:1000 in PBS. After 1 h incubation, wells were washed twice in PBS–Tween and once in 0.9% NaCl. One hundred microliters of a 1 mg/ml p-nitropenylphosphate substrate (Sigma, St Louis, MO, USA) in glycine buffer at pH 10.4 were added to each well and incubated for 20 min at 37 (C. Optical density (OD) at 405 nm was read with a Dynatech MR 5000 automatic plate reader (Dynatech Laboratories, Chantilly, VA, USA). Serum samples with an OD value above 0.9 were considered to be positive for IgM to lipid A (cut-off: mean for healthy individuals+two standard deviations). 2.3. Quantitation of serum immunoglobulins Serum concentrations of IgM were determined by nephelemetry (BNA, Behring). 2.4. Liver biopsies Baseline liver biopsies and repeated liver biopsies were staged according to Ludwig classification [16]. The lesions of cholangitis and the ductopenia were also noted. Cholangitis was defined by the presence of a mononuclear inflammatory infiltrate surrounding and penetrating the bile duct epithelium with associated epithelial damage and/or destruction of the basement
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Table 1 Prevalence of IgM anti-lipid A antibody in 36 PBC patients before UDCA administration and in 560 controls
The prevalence of anti-lipid A antibody was very low in the other controls.
Diseases
3.2. Baseline characteristics of PBC patients with or without anti-lipid A antibody
N
IgM anti-lipid A N
%
Primary biliary cirrhosis
36
23
64
Liver diseases Primary sclerosing cholangitis Autoimmune hepatitis type I Autoimmune hepatitis type II Chronic viral hepatitis C Chronic viral hepatitis B Alcoholic liver disease
14 67 43 60 30 22
0 15 9 1 0 0
0 22 21 2 0 0
Non-hepatic diseases Systemic lupus erythematosus Inflammatory bowel diseases Elevated serum globulin Rheumatoid arthritis Primary antiphospholipid syndrome Septicemia (gram negative bacteria)
92 40 22 20 35 40
5 0 0 1 3 0
6 0 0 5 8 0
Healthy individuals
75
3
4
membrane. Ductopenia was defined by the absence of bile ducts in at least 50% of the portal tracts. 2.5. Quantitative measurement of serum soluble human CD-14 The quantitative measurement of serum soluble human CD-14 was done using an immunoenzymometric assay (sCD-14 EASIA, Biosource, Europe, SA; normal range: 1.4–4.5 µg/ml). 2.6. Statistical analysis The means of the two independent groups were compared using Student’s t-test or the Mann–Whitney test. The Wilcoxon signed rank test was used to compare nonparametric variables in paired groups. Fisher’s exact test was used to compare frequencies of dichotomous variables. The minimum level of statistical significance was set at P<0.05. Data are presented as meanstandard error (SE).
3. Results
The biochemical and histological data did not differ significantly between these two groups except for total serum IgM levels (Table 2) and the presence of cholangitis lesions and/or ductopenia. In fact, patients with anti-lipid A had significantly higher IgM levels than those without this antibody (8.71.1 g/l vs 4.40.8 g/l, P<0.02). Total serum IgM levels correlated with IgM anti-lipid A antibody levels estimated by OD of the ELISA test (r=0.65, P<0.02) (Fig. 1). Similarly, antilipid A-positive patients had significantly more lesions of cholangitis and/or ductopenia than anti-lipid A-negative patients (100% vs 50%, Fisher exact test, P<0.02). There is no correlation between the titer of antimitochondrial antibody and IgM anti-lipid A antibody (Fig. 2). 3.3. Biological data of PBC patients after UDCA administration After UDCA therapy, a significant decrease in prevalence of anti-lipid A antibody was observed [22% (8/36) after UDCA vs 64% (23/36) before (P<0.001)]. Serum total IgM levels decreased significantly after drug administration in patients with anti-lipid A antibody before UDCA (Wilcoxon test, P<0.03). Mean IgM levels were found to be significantly higher in the group anti-lipid A-positive both before and after UDCA than in the group anti-lipid A-positive before and anti-lipid A-negative after UDCA (8.11 g/l vs 4.80.6 g/l, Mann–Whitney test, P<0.02). In patients anti-lipid A-negative prior to treatment, IgM levels did not differ significantly after UDCA therapy (Wilcoxon test). Under UDCA treatment, normalization of serum alanine and serum aspartate aminotransferases and alkaline phosphatase levels was obtained in 27% antilipid A-positive patients compared to 45% anti-lipid A-negative patients (NS). 3.4. Detection of soluble CD-14 Soluble CD-14 were assayed in serum samples from 21 healthy subjects, 27 PBC patients, and 64 patients with various liver diseases. PBC patients had significantly higher soluble CD-14 levels than healthy individuals (6.80.3 vs 4.60.3 µg/ml, Mann–Whitney test, P<0.001). There was no significant difference between CD-14 levels in PBC patients and those with others liver diseases.
3.1. Detection of IgM anti-lipid A antibody As shown in Table 1, the prevalence of anti-lipid A antibody was 64% (23/36) in PBC patients before UDCA therapy and 22% (24/110) in patients with AIH.
4. Discussion As with other autoimmune diseases, bacterial infection has been incriminated in the pathogenesis of PBC,
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Table 2 Baseline characteristics of PBC patients with or without anti-lipid A antibody
Anti-lipid A positive (n=23) Anti-lipid A negative (n=13)
AP N<100 IU/l
-GT* N<33 IU/l Bilirubin* N<17 µmol/l
Total IgM** N 0.8–2.2 g/l
Liver biopsy* Stage 1–2
41360 452108
46963 36767
8.71.1 4.40.8
67% 60%
25.44.3 28.710.3
AP=serum alkaline phosphatase; -GT=serum -glutamyl transpeptidase. * Not significantly different. ** Significantly different (P<0.02).
Fig. 1. Correlation between serum total IgM and anti-lipid A antibody in 36 patients with PBC before UDCA therapy.
,J0 DQWLOLSLG $ E\ RSWLFDO GHQVLW\
U S 16
0
$QWLPLWRFKRQGULDO DQWLERG\ WLWHUV
Fig. 2. Relation between IgM anti-lipid A antibody and antimitochondrial antibody titer in patients with PBC before UDCA therapy.
the main argument being the existence of a crossreactivity between anti-mitochondrial antibodies and antigens of E. coli or other mycobacteria [12,17]. No definite proof of a bacterial aetiology has been identified to date [18]. In our study, we focused on lipid A, a substance that is the carrier of the main biological activities of LPS (a component of gram-negative bacteria). We confirmed the high prevalence (64%) of IgM anti-lipid A in PBC patients and the significant correlation between the level
of this antibody and total IgM (P<0.02). This data could explain the hyper IgM-emia observed in PBC patients. This anti-lipid A hyper IgM-emia appears specific of this disease because no anti-lipid A antibody was found in patients with hyper IgM-emia without liver disease. These results are in agreement with a previous report by Ide et al. [15]. A valid comparison between our study and the latter report [15] is difficult, however, because the authors did not use the prevalence of anti-lipid A antibody, but rather a ratio of OD in patients serum samples to normal controls serum samples. Futhermore, the number of healthy subjects that served as controls was small (n=19) and did not allow determination of a threshold. In our study, which involved a large population of controls (n=560), anti-lipid A antibody prevalence was very low, except in the group of AIH (24/110, 22%). This prevalence is not easily interpretable because we have little information on histological activity and treatment in this group. Further studies are required to determine the prevalence of anti-lipid A in patients with AIH according to treatment. The role of LPS, a potent immunostimulant which is a polyclonal B cell activator, has been suggested, although it has not been possible to design an experimental animal model for PBC. Masanaga et al. [19], however, induced autoimmune cholangitis by immunizing neonatally thymectomized mice with both recombinant protein containing the major mitochondrial autoantigens and LPS. Their results suggest that endotoxins may increase the immunopathogenicity of this disease. Some authors have shown that NK cells and NK1.Ag+T cells in mice are stimulated by superantigens like LPS through IL-12 production from Kupffer cells [20,21]. At the interface between innate and adaptive immunity, the NK1.Ag+T cells have cytotoxic activities in the absence of prior antigenic stimulation and are able to regulate the secondary adaptive immune response through cytokine production [22]. Interestingly, these cells are able to recognize the nonclassical antigen-presenting molecule CD1d and to respond to lipid or glycolipid components of the bacterial cell wall [23]. In PBC liver, Tsuneyama et al. have reported aberrant expression of CD1d on bile duct epithelium
E. Ballot et al. / Journal of Autoimmunity 22 (2004) 153–158
and epithelioid granuloma around lipid A [24]. Sakisaka et al. also found a significant retention of endotoxin and an increase in apoptosis in the biliary epithelial cells of PBC patients [10]. In our study, the lesions of cholangitis were more frequent and more severe in the group of PBC patients with antibodies to lipid A. This result supports the hypothesis that LPS may contribute to the pathogenesis of cholangitis. Furthermore, the inactivation by dephosphorylation of lipid A under the influence of alkaline phosphatase is pH-dependent [25]. It has been shown that the secretion and alkalinization of the bile are deficient in PBC because of the markedly reduced activity of the AE2 exchanger in the biliary tract [26]. Thus, the LPS present in the biliary tract may be partly responsible for the inflammation and destruction of lesser biliary ducts. The liver plays a central role in eliminating the endotoxins from the portal blood. The cell surface receptors responsible for binding LPS include the CD14/ Toll-like receptor 4, but also the Fc receptor for the complexes LPS-anti-LPS [27]. Thus, accelerated internalization and detoxification of endotoxins by anti-LPS appear to be a Fc receptor mediated process. Indeed, profound disturbances of Fc receptor mediated immune clearance by anti-LPS have been reported in PBC [28]. CD14 is present as both a membrane-bound and a soluble form [29,30]. Soluble CD14 is found in human serum under normal conditions and mediates LPS activation of non-CD14-bearing cells [31]. A recent report has shown that human hepatocytes produce CD14. This local production may have important biological consequences for the liver’s response to LPS [32]. In our study, we have shown a significant increase of the soluble CD14 level in PBC patients but also in other various liver diseases. UDCA is currently the only drug approved specifically for the treatment of PBC [33,34]. The mechanisms underlying the beneficial effects of UDCA in PBC are still ill-defined. It has been reported that UDCA can decrease the translocation of endotoxin from the gut [35] and restore the activity of the AE2 exchanger [26]. Therefore, UDCA may participate in inactivating LPS present in the lesser biliary ducts. Recently, Sasatomi et al. demonstrated the abnormal accumulation of endotoxin in biliary epithelial cells and the possible beneficial effects of UDCA on the intra-hepatic metabolism of endotoxin [11]. Ide et al. have shown that the serum level of IgM anti-lipid A antibody decreased with UDCA therapy [15]. In the present study, according to Ide et al. [15], we have demonstrated a significant decrease of the prevalence of anti-lipid A antibody after UDCA therapy, associated to a decrease of serum total IgM level. In conclusion, our data confirm that a large part of IgM antibodies in PBC patients’ serum samples are directed against lipid A and suggest that disturbance to
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innate immunity in response to an infection may be involved in some cases of PBC.
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