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Defective Interleukin-1 Receptor Antagonist Production Is Associated with Resistance of Acute Liver Graft Rejection to Steroid Therapy

2000; Elsevier BV; Volume: 157; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)64805-5

1525-2191

Filoména Conti, Sylvie Breton, Frédéric Batteux, Valérie Furlan, D Houssin, Bernard Weill, Yvon Calmus,

Organ Transplantation Techniques and Outcomes

Thirty percent of acute liver graft rejection episodes are resistant to steroids. As interleukin-1 (IL-1) is an important target of steroid therapy, we examined the possible involvement of reduced sensitivity of IL-1 production to steroids or defective production of its antagonist, IL-1Ra. Patients were assessed during steroid-sensitive or -resistant rejection and 2 years later. In situ IL-1β and IL-1Ra expression was evaluated by immunohistochemistry; their production was assayed by enzyme-linked immunosorbent assay and the gene polymorphisms by reverse transcriptase-polymerase chain reaction on blood cells. Hepatic IL-1β and IL-1Ra expression were enhanced during rejection. IL-1 production and its inhibition by steroids were similar in steroid-responsive and steroid-resistant rejection. However, IL-1Ra production was lower in steroid-resistant than in steroid-responsive rejection, and this difference was still observed 2 years after rejection. IL-1β and IL-1Ra gene polymorphisms did not differ between patients with and without steroid resistance. Low IL-1Ra production is associated with steroid resistance of acute rejection and is due to a constitutional defect. The early identification of such patients might qualify them for stronger anti-rejection therapy, including IL-1Ra. Thirty percent of acute liver graft rejection episodes are resistant to steroids. As interleukin-1 (IL-1) is an important target of steroid therapy, we examined the possible involvement of reduced sensitivity of IL-1 production to steroids or defective production of its antagonist, IL-1Ra. Patients were assessed during steroid-sensitive or -resistant rejection and 2 years later. In situ IL-1β and IL-1Ra expression was evaluated by immunohistochemistry; their production was assayed by enzyme-linked immunosorbent assay and the gene polymorphisms by reverse transcriptase-polymerase chain reaction on blood cells. Hepatic IL-1β and IL-1Ra expression were enhanced during rejection. IL-1 production and its inhibition by steroids were similar in steroid-responsive and steroid-resistant rejection. However, IL-1Ra production was lower in steroid-resistant than in steroid-responsive rejection, and this difference was still observed 2 years after rejection. IL-1β and IL-1Ra gene polymorphisms did not differ between patients with and without steroid resistance. Low IL-1Ra production is associated with steroid resistance of acute rejection and is due to a constitutional defect. The early identification of such patients might qualify them for stronger anti-rejection therapy, including IL-1Ra. After liver transplantation, ∼50% of patients experience at least one episode of acute rejection.1Adams DH Neuberger JM Patterns of graft rejection following liver transplantation.J Hepatol. 1990; 10: 113-119Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 2Klintmalm G Nery J Husberg B Gonwa T Tillery G Rejection in liver transplantation.Hepatology. 1989; 10: 978-985Crossref PubMed Scopus (141) Google Scholar This can be reversed in many cases by high doses of steroids, but 20 to 30% of patients do not respond and need additional therapy.2Klintmalm G Nery J Husberg B Gonwa T Tillery G Rejection in liver transplantation.Hepatology. 1989; 10: 978-985Crossref PubMed Scopus (141) Google Scholar, 3Wiesner R Wahlstrom H Gores G Kamath P Krom R Early cellular rejection treated with high-dose intravenous corticosteroid therapy is associated with a decrease in the incidence of steroid-resistant rejection and graft failure from rejection.Transplant Proceed. 1993; 25: 1791-1793PubMed Google Scholar, 4Adams D Neuberger J Treatment of acute rejection.Semin Liver Dis. 1992; 12: 80-87Crossref PubMed Scopus (41) Google Scholar, 5Wagner F Reichenspurner H Uberfuhr P Kur F Kaulbach H Meuser B Ziegler U Reichart B How successful is OKT3 rescue therapy for steroid-resistant acute rejection episodes after heart transplantation?.J Heart Lung Transplant. 1994; 13: 438-442PubMed Google Scholar, 6Portela D Patel R Larson-Keller J Ilstrup D Wiesner R Steers J Krom R Paya C OKT3 treatment for allograft rejection is a risk factor for cytomegalovirus disease in liver transplantation.J Infect Dis. 1995; 171: 1014-1018Crossref PubMed Scopus (123) Google Scholar, 7Hosenpud J Norman D Pantely G Cobanoglu A Starr A OKT3-induced hypotension in heart allograft recipients treated for steroid-resistant rejection.J Heart Transplant. 1989; 8: 159-165PubMed Google Scholar, 8Freese DK Snover DC Sharp HL Gross CR Kay Savick S Payne WD Chronic rejection after liver transplantation: a study of clinical, histopathological and immunological features.Hepatology. 1991; 13: 882-891Crossref PubMed Scopus (121) Google Scholar Interleukin-1 (IL-1) plays a key role in inflammatory and immune-mediated diseases.9Dinarello C Biologic basis for interleukin-1 in disease.Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar During allograft rejection, IL-1 production precedes allograft dysfunction and injury.10Salom R Maguire J Hancock W Endothelial activation and cytokine expression in human acute cardiac allograft rejection.Pathology. 1998; 30: 24-29Crossref PubMed Scopus (53) Google Scholar, 11Mannon R Sundar S Sanfilippo F Coffman T Alterations in renal interleukin-1 production during kidney transplant rejection in the rat. The effects of high-dose methylprednisolone.Transplantation. 1993; 56: 1157-1162Crossref PubMed Scopus (17) Google Scholar, 12Hoffmann MW Wonigeit K Steinhoff G Herzbeck H Flad HD Pichlmayr R Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection.Transplantation. 1993; 55: 329-335Crossref PubMed Scopus (105) Google Scholar Inhibition of IL-1 production is an important mechanism by which corticosteroids suppress immune response.13Barnes P Adcock I Anti-inflammatory actions of steroids: molecular mechanisms.Trends Pharmacol Sci. 1993; 14: 436-441Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 14Kern J Lamb R Reed J Daniele R Nowell P Dexamethasone inhibition of interleukin 1 beta production by human monocytes. 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Enhanced interleukin-1 receptor antagonist in rejection-free kidney transplant patients.Transplantation. 1997; 63: 1751-1756Crossref PubMed Scopus (28) Google Scholar The mechanisms of steroid resistance after organ transplantation are unknown. We explored the possible roles of a reduced sensitivity of IL-1 production to corticosteroid therapy, or defective IL-1Ra production. Our results indicate that both IL-1 production and its inhibition by corticosteroids are normal in patients whose acute rejection is steroid-resistant whereas, in contrast, IL-1Ra secretion is consistently reduced, pointing to a constitutional defect of IL-1Ra production in such patients. The study population consisted of adult liver transplant recipients. After liver transplantation, patients were treated initially with corticosteroids, azathioprine, and cyclosporine. The dose of cyclosporine was adjusted to maintain whole-blood levels between 150 and 250 μg/L. Azathioprine was discontinued after 3 months, and steroids were tapered to 5 to 10 mg/day. Acute rejection was defined by both biochemical and histological criteria, including at least two of the following: portal inflammatory infiltrate, endothelial inflammation, and biliary damage. Rejection was considered resistant when liver function tests improved by <50% of the highest values after three steroid boluses, in addition to persistent histological features of acute rejection on control liver biopsy. Episodes of acute rejection were initially treated for 3 days with a 1-g bolus of intravenous methylprednisolone daily. Cases of steroid-resistant acute rejection were treated with the monoclonal antibody OKT3 (Ortho Pharmaceutical Corp., Raritan, NJ). Blood and liver biopsy specimens were obtained, with their informed consent, from 29 patients with suspected rejection (Table 1). Histological confirmation of acute rejection was obtained in 21 of these cases. Nine episodes responded to steroids whereas 12 resisted. The remaining eight patients had no histological evidence of acute rejection, either initially or during follow-up. Transplant recipients without acute rejection were assessed at approximately day 15, so as to be as close as possible to the median time of acute rejection in the two other groups. A portion of each biopsy specimen was immediately fixed and embedded in paraffin. Sections were stained with hematoxylin-eosin-safran for histological diagnosis. The remaining portion was snap-frozen in liquid nitrogen and stored at −80°C for immunohistochemistry. Blood and liver samples were obtained on days 14.5 ± 2.5, 15.1 ± 3.1, and 14.7 ± 2.8, respectively, from patients without rejection and patients with steroid-responsive and steroid-resistant rejection. In the latter two subgroups, samples were obtained before anti-rejection therapy. Blood and liver biopsy specimens were also obtained from the same patients, with their informed consent, during a routine work-up, a mean of 2 years posttransplantation. At that time, the patients were clinically stable and receiving minimal doses of steroids. Moreover, the total doses of steroids received since liver transplantation, as well as the cyclosporine dose and blood concentrations, were similar in the three groups of patients (Table 1). Blood samples were also obtained, with their informed consent, from 44 healthy volunteers.Table 1Patient CharacteristicsSteroid-responsive acute rejection (n = 9)Steroid-resistant acute rejection (n = 12)No rejection (n = 8)Age55.245.152.5(48–66)(32–62)(36–59)Sex4M, 5F5M, 7F3M, 5FInitial pathologyPosthepatitic cirrhosis553Alcoholic cirrhosis232Others243Long-term assessmentTime after transplantation (years)2.752.52.75(2–4)(2–4)(2–4)Dose of steroids (mg/d)5.55.755.6(5–10)(3–10)(3–10)Total dose of steroids (mg)5,585 ± 9555,175 ± 8505,170 ± 835Cyclosporine dose (mg/d)166 ± 45171 ± 51144 ± 52Cyclosporine trough level (μg/l)124 ± 35112 ± 41131 ± 44 Open table in a new tab Five-micrometer cryostat sections of each biopsy specimen were mounted on polysine-coated slides (CML-CBE, Nemours, France). Serial slides were air-dried, fixed in acetone, and stored at −20°C until staining. Cytokine expression was tested using an indirect immunoenzymatic method.30Cordell JL Falini B Erber WN Ghosh AK Abdulaziz A MacDonald S Pulfpord KAF Stein H Mason DY Immunoenzymatic labelling of monoclonal antibodies using immune complex of alkaline phosphatase and monoclonal anti-alkaline phosphatase.J Histochem Cytochem. 1984; 32: 219-229Crossref PubMed Scopus (2923) Google Scholar Anti-IL-1β monoclonal antibody (Genzyme, Cambridge, MA) and anti-IL-1Ra polyclonal antibody (Genzyme. were used at a dilution of 1:20. In positive controls, some blood mononuclear cells activated by lipopolysaccharide (LPS) (1 μg/ml) (Sigma Chemical Company, St Louis, MO) expressed IL-1β and IL-1Ra, whereas no inactivated cells expressed IL-1β and IL-1Ra (data not shown). Cryostat sections were refixed in acetone then incubated with Tris-buffered saline, 20% human AB serum (Sanofi Diagnostics Pasteur, Marnes La Coquette, France), then with the primary antibody. Slides treated with the polyclonal primary antibody (anti-IL-1Ra) were incubated with 1:20 mouse anti-rabbit immunoglobulin (DAKO, Dakopatts A/S, Glostrup, Denmark). All slides were then incubated with 1:20 rabbit anti-mouse immunoglobulin (DAKO) and subsequently with alkaline phosphatase-anti-alkaline phosphatase complexes (DAKO). Alkaline phosphatase activity was revealed after incubation in fast-red TR and napthol-phosphate solution containing levamisole.31Gaulard P Kanavaros P Farcet J Rocha F Haioun C Divine M Reyes F Zafrani E Bone marrow histologic and immunohistochemical findings in peripheral T-cell lymphoma: a study of 38 cases.Hum Pathol. 1991; 22: 331-338Abstract Full Text PDF PubMed Scopus (64) Google Scholar The slides were counterstained with hematoxylin. In negative controls, the primary antibody was replaced by an irrelevant antibody at the same dilution. Anti-CD3 (DAKO) was used as a positive control. The slides were examined by two of the authors (FC and SB), who were blinded to the pathological diagnoses. Positive cells were counted on each slide in a portal and a lobular field at a magnification of ×200. The number of positive cells recorded was a consensus between the two observers. Five milliliters of heparinized blood were obtained from each patient, diluted 1:5 in RPMI 1640 medium containing l-glutamine and penicillin/streptomycin (Gibco-BRL, Cergy-Pontoise, France), then cultured for 24 hours with or without LPS (1 μg/ml),21Dinarello C Interleukin-1 and interleukin-1 antagonism.Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar with various concentrations of dexamethasone (0 to 10−5 mol/L). IL-1β and IL-1Ra were measured in duplicate in 24-hour supernatants of blood cells, using an ELISA kit (R&D System Europe Ltd., Abingdon, UK). The detection limits were 4 pg/ml IL-1β and 31 pg/ml IL-1Ra, and the within-run variation was <5% in each case. Twenty-four patients (nine with steroid-responsive, nine with steroid-resistant acute rejection, and six with no rejection) were investigated for IL-1Ra and IL-1β gene polymorphisms. Genomic DNA was isolated from blood by the salting-out method.32Miller K Anderson J Human monocyte/macrophage activation and interleukin 1 generation by biomedical polymers.J Biomed Mater Res. 1988; 22: 713-731Crossref PubMed Scopus (134) Google Scholar IL-1Ra exon 2 polymorphism was analyzed as previously described.33Tarlow J Blakemore A Lennard A Solari R Hughes H Strenkasserer A Duff G Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat.Hum Genet. 1993; 91: 403-404Crossref PubMed Scopus (648) Google Scholar Oligonucleotides 5′-CTCAGCAACACTCCTAT-3′ and 5′-TCCTGGTCTGCAGGTAA-3′ were used as primers.34Hurme M Santtila S IL-1 receptor antagonist (IL-1Ra) plasma levels are co-ordinately regulated by both IL-1Ra and IL-1beta genes.Eur J Immunol. 1998; 28: 2598-2602Crossref PubMed Scopus (387) Google Scholar The polymerase chain reaction products were separated by electrophoresis on 2% agarose gel and stained with ethidium bromide. Allele 1 (four repeats) was 410 bp long and allele 2 (two repeats) was 240 bp long. The region containing the Ava I polymorphic site at position −511 of the IL-1β gene was amplified by polymerase chain reaction. The oligonucleotides 5′-TGGCAATTGATCTGGTTCATC-3′ and 5′-GTTTAGGAATCTTCCCACTT-3′ flanking this region were used as primers.35Di Giovine F Takhsh E Blakemore A Duff G Single base polymorphism at -511 in the human interleukin-1 beta gene (IL1 beta).Hum Mol Genet. 1992; 1992: 450-451Crossref Scopus (348) Google Scholar Polymerase chain reaction products were digested with 6 U of Ava I at 37°C for 3 hours. Fragments were analyzed on 2% agarose gel. This revealed products of 190 bp + 114 bp (allele 1) and 304 bp (allele 2). Position +3953 within exon 5 of the IL-1β gene exhibited single bp polymorphism.36Pociot F Molvig J Wogesen L Worsaae H Nerup J A TaqI polymorphism in the human interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro.Eur J Clin Invest. 1992; 22: 396-402Crossref PubMed Scopus (866) Google Scholar The polymorphic region containing the Taq I restriction site was amplified using the following primers: 5′-GTTGTCATCAGACTTTGACC-3′ and 5′-TTCAGTTCATATGGACCAGA-3′. Taq I digestion of the 249-bp fragments resulted in products that either remained intact (allele 2. or were cut into two fragments of 135 bp and 114 bp (allele 1). The immunohistochemical and ELISA results are expressed as means ± SE. Statview IV software (Abacus Concepts, Berkeley, CA. running on a Macintosh computer was used for statistical analyses (analysis of variance followed by Fisher's exact test). Positive cells were rare in liver grafts without rejection, whereas they were abundant during acute rejection (P < 0.03 in the portal tract and P < 0.01 in the lobule), but there was no difference between patients with steroid-responsive and steroid-resistant acute rejection (Figure 1a). Stained cells were mostly endothelial, Kupffer, and inflammatory cells, infiltrating the sinusoids and portal tracts. Positive cells were rare in liver transplants without acute rejection (Figure 2A) and abundant in both steroid-responsive (Figure 2B) and steroid-resistant acute rejection (Figure 2C). The number of cells expressing IL-1Ra was higher in patients with acute rejection than without acute rejection (P < 0.05 in the portal tract and P < 0.02 in the lobule), but there was no difference in this respect between steroid-responsive and -resistant acute rejection (Figure 1b). Stained cells were Kupffer and inflammatory cells infiltrating the sinusoids and portal tracts. Few hepatic cells expressed IL-1β and IL-1Ra, with no significant difference between the three groups of patients (data not shown). IL-1β concentrations were low in the supernatants of unstimulated blood cells from patients without acute rejection, with steroid-responsive and with steroid-resistant acute rejection (46 ± 29 pg/ml, 46 ± 29 pg/ml, and 64 ± 46 pg/ml, respectively). After LPS stimulation, the corresponding IL-1β concentrations rose to 1,634 ± 724 pg/ml, 1,027 ± 612 pg/ml, and 1,959 ± 264 pg/ml (NS). IL-1β production was inhibited by dexamethasone in a concentration-dependent manner and to a similar extent in each group (Figure 3a). Supernatants of unstimulated cells contained 1,824 ± 394 pg/ml, 1,002 ± 583 pg/ml, and 1,422 ± 417 pg/ml IL-1β, respectively, in patients without rejection and those with steroid-responsive and -resistant acute rejection (NS). After LPS stimulation, the corresponding IL-1β concentrations were 6,728 ± 1,497 pg/ml, 4,155 ± 1,231 pg/ml, and 5,149 ± 1,098 pg/ml (NS). IL-1β production was inhibited by dexamethasone to a similar extent in each group (Figure 3b). IL-1Ra concentrations were 565 ± 275 pg/ml, 2,012 ± 1,486 pg/ml, and 1,034 ± 496 pg/ml, respectively, in the supernatants of unstimulated cells from patients without rejection and those with steroid-responsive or steroid-resistant rejection. After stimulation, the corresponding IL-1Ra concentrations rose to 6,461 ± 1,386 pg/ml, 9,010 ± 2,119 pg/ml, and 3,129 ± 349 pg/ml (P < 0.01 between the steroid-responsive and steroid-resistant acute rejection groups). Dexamethasone inhibited LPS-stimulated IL-1Ra production to a similar extent in the three groups (Figure 3c). IL-1Ra concentrations were 3,573 ± 573 pg/ml, 3,156 ± 798 pg/ml, and 1,934 ± 250 pg/ml in the supernatants of unstimulated cells from patients without rejection and those with steroid-responsive and -resistant acute rejection, respectively. After stimulation, the corresponding IL-1Ra concentrations rose to 6,331 ± 1,210 pg/ml, 7,269 ± 1,301 pg/ml, and 3,661 ± 366 pg/ml (Figure 3d). IL-1Ra secretion was inhibited by corticosteroids to a similar extent in the three groups of patients. IL-1Ra secretion was lower in patients with a history of steroid-resistant rejection than in the other two groups, both in the absence and presence of dexamethasone (P < 0.01). In contrast, IL-1Ra secretion did not significantly differ between patients with a past history of acute rejection (5,208 ± 701 pg/ml) and those with no such history (6,331 ± 121 pg/ml). Taking a cut-off value of 5,750 pg/ml, eight of the nine patients with steroid-responsive acute rejection exhibited IL-1Ra levels above this value, whereas all 12 patients with resistant acute rejection had values below the cut-off (Figure 4). The distribution of IL-1β and IL-1Ra genotypes and corresponding allelic frequencies were similar to those previously reported (Table 2). None of the alleles examined was associated with the resistance of acute rejection to steroid therapy in the patients tested. In addition, the association of IL-1Ra allele 2 with IL-1β −511 allele 2 or IL-1β +3,953 allele 2 had no effect on steroid resistance (Table 3).Table 2IL-1Ra and IL-1β Genotypes (Base Exchange Polymorphisms at Positions −511 and +3,953) and the Corresponding Allele FrequenciesGenotypeAllele1.11.22.212IL-1RaNo rejection (n = 6)5100.910.09Steroid-responsive acute rejection (n = 9)7110.83*NS versus patients without rejection.0.17Steroid-resistant acute rejection (n = 9)5400.77*NS versus patients without rejection.†NS versus patients with steroid-responsive acute rejection.0.23IL-1β (−511)No rejection (n = 6)5010.830.17Steroid-responsive acute rejection (n = 9)6210.78*NS versus patients without rejection.0.22Steroid-resistant acute rejection (n = 9)4410.67*NS versus patients without rejection.†NS versus patients with steroid-responsive acute rejection.0.33IL-1β (+3,953)No rejection (n = 6)2220.500.50Steroid-responsive acute rejection (n = 9)5220.67*NS versus patients without rejection.0.33Steroid-resistant acute rejection (n = 9)4320.61*NS versus patients without rejection.†NS versus patients with steroid-responsive acute rejection.0.39* NS versus patients without rejection.† NS versus patients with steroid-responsive acute rejection. Open table in a new tab Table 3The Effect of IL-1Ra and IL-1β Genotypes on Steroid ResponseGenotypeSteroid-responsive acute rejection (n = 9)Steroid-resistant acute rejection (n = 9)IL-1Ra-2+24IL-1Ra-2−75IL-1Ra-2+/IL-1β-511-2+12IL-1Ra-2−/IL-1β-511-2+23IL-1Ra-2+/IL-1β-511-2−12IL-1Ra-2−/IL-1β-511-2−52IL-1Ra-2+/IL-1β+3,953-2+02IL-1Ra-2−/IL-1β+3,953-2+43IL-1Ra-2+/IL-1β+3,953-2−22IL-1Ra-2−/IL-1β+3,953-2−32 Open table in a new tab To determine whether the low IL-1Ra levels observed in transplant patients with steroid resistance were also found in the general population, IL-1Ra production was measured in blood samples from 44 healthy volunteers. Large interindividual differences were found in IL-1Ra secretion (Figure 4). Low IL-1Ra production (<5,750 pg/ml) by LPS-stimulated mononuclear cells, similar to that found in the steroid-resistant acute rejection group, was observed in 34% of healthy volunteers. This is the first report suggesting that the steroid resistance of acute rejection is associated with low levels of IL-1Ra production by blood cells. In all of the patients with acute rejection, whatever their response to steroid therapy, a high level of IL-1β production was found by immunohistochemistry on liver biopsies and by ELISA in the supernatants of stimulated blood cells. These data are in agreement with those previously obtained by reverse transcriptase-polymerase chain reaction in the same clinical setting.11Mannon R Sundar S Sanfilippo F Coffman T Alterations in renal interleukin-1 production during kidney transplant rejection in the rat. The effects of high-dose methylprednisolone.Transplantation. 1993; 56: 1157-1162Crossref PubMed Scopus (17) Google Scholar, 12Hoffmann MW Wonigeit K Steinhoff G Herzbeck H Flad HD Pichlmayr R Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection.Transplantation. 1993; 55: 329-335Crossref PubMed Scopus (105) Google Scholar Because steroid-resistant and steroid-responsive acute rejection did not differ in terms of IL-1β production by blood mononuclear cells, a defect in IL-1β sensitivity to steroid therapy is probably not involved in steroid resistance, although a lower sensitivity of hepatocytes and sinusoidal endothelial cells to steroids could not be excluded. IL-1Ra expression in the liver grafts was also increased in patients with acute rejection relative to patients without rejection. To our knowledge, this is the first clear evidence that hepatic IL-1Ra production is enhanced during acute liver graft rejection. This IL-1Ra overexpression may result from an increased stimulation through the local production of cytokines, and may restrict the rejection process by inhibiting the effects of IL-1.37Gabay C Smith M Eidlen D Arend W IL-1 receptor antagonist (IL-1Ra) is an acute-phase protein.J Clin Invest. 1994; 99: 2930-2940Crossref Scopus (260) Google Scholar, 38Arend W Dayer J Inhibition of the production and effects of interleukin 1 and tumor necrosis factor alpha in rheumatoid arthritis.Arthritis Rheum. 1995; 38: 151-160Crossref PubMed Scopus (927) Google Scholar In those patients, IL-1Ra was only expressed by the inflammatory cells infiltrating the sinusoids and portal tracts. In our study, hepatocytes, which may secrete IL-1Ra, especially when stimulated by IL-1 and IL-6,37Gabay C Smith M Eidlen D Arend W IL-1 receptor antagonist (IL-1Ra) is an acute-phase protein.J Clin Invest. 1994; 99: 2930-2940Crossref Scopus (260) Google Scholar were consistently found to be negative for IL-1Ra expression by immunohistochemistry. It is noteworthy that the number of the infiltrating cells expressing IL-1Ra was similar during steroid-responsive and -resistant episodes of acute rejection. In contrast, IL-1Ra production by blood cells was lower in steroid-resistant than in steroid-sensitive patients, both at the time of acute rejection and throughout the long term. To determine whether this difference reflected the existence of high and low IL-1Ra producers within the general population, we compared LPS-stimulated IL-1Ra production by blood cells from the transplant patients with that of healthy volunteers, and found the same broad range of responses in both groups. All steroid-resistant patients exhibited IL-1Ra levels <5,750 pg/ml, as did ∼30% of the healthy volunteers, who may therefore be at risk of steroid resistance. It remains to be determined if this distinction between weak and strong IL-1Ra producers could be used to predict an increased risk of steroid resistance in other transplant settings, as well as in other inflammatory or autoimmune diseases. We then investigated whether the variability in IL-1Ra production could be explained by the recently described IL-1β and IL-1Ra gene polymorphisms. Indeed, certain IL-1β or IL-1Ra alleles are associated with severe forms of inflammatory or autoimmune diseases.35Di Giovine F Takhsh E Blakemore A Duff G Single base polymorphism at -511 in the human interleukin-1 beta gene (IL1 beta).Hum Mol Genet. 1992; 1992: 450-451Crossref Scopus (348) Google Scholar, 36Pociot F Molvig J Wogesen L Worsaae H Nerup J A TaqI polymorphism in the human interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro.Eur J Clin Invest. 1992; 22: 396-402Crossref PubMed Scopus (866) Google Scholar, 39Santtila S Savinainen K Hurme M Presence of the IL-1Ra allele 2 (IL1RN*2) is associated with enhanced IL-1 beta production in vitro.Scand J Immunol. 1998; 47: 195-198Crossref PubMed Scopus (500) Google Scholar, 40Chang D Interleukin-1 and interleukin-1 receptor antagonist in systemic lupus erythematosus.Immunol Invest. 1997; 26: 649-659Crossref PubMed Scopus (19) Google Scholar, 41Andus T Daig R Volg D Aschenbrenner E Lock G Hollerbach S Kollinger M Scholmerich J Gross V Imbalance of the interleukin 1 system in colonic mucosa-association with intestinal inflammation and interleukin 1 receptor antagonist genotype 2.Gut. 1997; 41: 651-657Crossref PubMed Scopus (134) Google Scholar, 42Heresbach D Alizadeh M Dabadie A Le Berre N Colombel J Yaouanq J Bretagne J Semana G Significance of interleukin-1beta and interleukin-1 receptor antagonist genetic polymorphism in inflammatory bowel diseases.Am J Gastroenterol. 1997; 92: 1164-1169PubMed Google Scholar, 43Roussomoustakaki M Satsangi J Welsh K Louis E Fanning G Targan S Landers C Jewell D Genetic markers may predict disease behavior in patients with ulcerative colitis.Gastroenterology. 1997; 112: 1845-1853Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar In addition, allele 2 of the IL-1Ra gene is associated with higher concentrations of the molecule in plasma.34Hurme M Santtila S IL-1 receptor antagonist (IL-1Ra) plasma levels are co-ordinately regulated by both IL-1Ra and IL-1beta genes.Eur J Immunol. 1998; 28: 2598-2602Crossref PubMed Scopus (387) Google Scholar We found no link between the known IL-1β and IL-1Ra polymorphisms and steroid resistance. The number of patients tested was however small, and this result needs to be confirmed. Other polymorphisms of IL-1Ra gene should also be investigated. The mechanisms of steroid resistance are still poorly understood. 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Clinical resistance to steroids is probably potentiated by the inhibitory effect of steroids on IL-1Ra production. This study defines a new concept in the pathophysiology of steroid resistance and suggests IL-1Ra as a potential treatment. 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We thank Mrs. Voltoury and the Blood Transfusion Center at Hôpital Saint-Vincent de Paul (Paris) (Etablissement de Transfusion Sanguine de l'AP-HP), who collected the blood samples from normal volunteers; François Durand and Christophe Duvoux, who collected samples from liver transplant recipients; and Pierre Thomopoulos, Françoise Russo-Marie, and David Young for their help in preparing the manuscript.