A Monocyte Chemoattractant Protein-1 (MCP-1) Polymorphism And Outcome After Renal Transplantation
Bernd Krüger*,
Bernd Schröppel,
Rami Ashkan,
Brad Marder,
Carl Zülke,
Barbara Murphy,
Bernhard K. Krämer* and
Michael Fischereder*
*Klinik für Innere Medizin II, University of Regensburg, Germany; Division of Nephrology, Mount Sinai School of Medicine, New York, New York; and Klinik für Chirurgie II, University of Regensburg, Germany.
Correspondence to Dr. Bernd Schröppel, Division of Nephrology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1243, New York, NY 10029. Phone: 212-241-8004; Fax: 212-987-0389;
ABSTRACT. Among the factors modulating transplant rejectionand cardiovascular disease, chemokines and their respectivereceptors deserve special attention. In this respect, increasedexpression of MCP-1 and the corresponding receptor CCR2 havebeen demonstrated in renal transplant rejection and coronaryartery disease. The impact of the MCP-1–2518G and CCR2–64Igenotypes on renal allograft function was investigated in 232patients who underwent transplantation over an 11-yr period.Genomic DNA was genotyped using PCR with sequence-specific primersfollowed by restriction fragment length polymorphism analysis.Eighteen (7.8%) patients were homozygous for the MCP-1–2518Gmutation. The G/G allele of MCP-1 -2518 behaved as a determinantfor long-term allograft survival and resulted in reduction ofthe mean graft survival, as compared with the heterozygous (A/G)or wild-type (A/A) allele (67 ± 14 versus 95 ±4 mo; Log rank P = 0.0052). The 64I mutation of CCR2 had noeffect on kidney graft failure (93 ± 6 and 91 ±5 mo, respectively; P = 0.81). None of the investigated polymorphismsshowed a significant shift in gene frequency in acute rejectionand rejection-free groups. In conjunction with these findings,peripheral blood mononuclear cells from kidney transplant recipientscarrying the G-allele were characterized by a 2.5-fold higherMCP-1 secretion (P < 0.05). In conclusion, recipients ofrenal transplants homozygous for the –2518 G mutationof the MCP-1 gene are at risk for premature kidney graft failure.This variant of MCP-1 may be a future predictor for long-termkidney graft failure. E-mail: Bernd.Schroppel@mssm.edu
During acute allograft rejection, monocytes and T effector cellsare directed into the transplant and produce a characteristictubular or vascular infiltrate (1). The complex process of extravasationand influx of leukocyte subsets into the site of tissue injuryappears to be mediated to a significant extent by the expressionof specific chemokines and chemokine receptors (2). Specifically,the expression of the CC-chemokine MCP-1 together with the correspondingchemokine receptor CCR2 could be detected on the mononuclearcells infiltrating the kidney graft (3–8). Furthermore,elevated levels of MCP-1 were also detected in the serum andurine of patients at the time of acute and chronic rejection(9,10).
Currently, in experimental transplantation targeted deletionof CCR1, CCR5, CX3CR1, or CXCR3 or specific blockade of thesereceptors, have resulted in improved allograft survival (1,11).Furthermore, we have recently shown that human transplant recipientswithout a functional CCR5 due to a 32-bp deletion in the CCR5gene (32 mutation) have significantly better allograft survival(12). The interaction of MCP-1 and CCR2 has not been blockedexperimentally in a transplant model to date. Preliminary data,however, suggests a prolonged vascularized cardiac allograftsurvival in CCR2 knockout mice compared with wild-type (11).
A substantial number of allografts are lost due to death ofthe recipient from cardiovascular disease (13,14). Detailedstudies of atherosclerotic vascular disease in a rodent modelwere able to demonstrate the involvement of MCP-1/CCR2 in thecomplex process of atherogenesis (15,16,17). Interestingly,the MCP-1–2518G variant in homozygous patients appearsto be a genetic risk factor for CAD (18). Thus, chemokines andchemokine receptors may affect allograft function not only throughacute and chronic allograft rejection but perhaps indirectlythrough the accelerated cardiovascular comorbidity of the recipients.
Common mutations are described for CCR2 and MCP-1. Althoughthere is to date no evidence that the CCR2–64I polymorphismalters CCR2 expression or function on leukocytes, this variantis associated with delayed progression of HIV infection anda lower risk for the development of sarcoidosis (19,20,21).The MCP-1 polymorphism at position -2518 (G or A) relative tothe major transcriptional start site was shown to influencethe level of MCP-1 production in response to interleukin1 (IL-1)in vitro (22). In this study, we analyzed whether MCP-1–2518and CCR2–64I variants are associated with altered kidneyallograft outcome.
Patients Demographics
A total of 232 first renal allograft recipients transplantedat two centers from 1990 through 2001 were analyzed. Demographicdata for donor and recipient age and gender, HLA-mismatch, panelreactive antibodies, cold ischemia time, immunosuppressive therapy,presence of rejection episodes, and graft survival were extractedfrom the hospital record. Acute rejection was determined byallograft biopsy in 84% of renal transplant recipients. In theremaining, rejection was defined by an increase in creatininelevel by 30% from baseline that was not attributable to othercauses with subsequent return to baseline after treatment withpulse steroids or antilymphocytic antibodies. Graft survivalwas defined as recipient survival with a functioning renal transplant.The Internal Review Board approved the study, and written consentwas obtained at the time of enrollment.
Identification of Genotypes
Genotype analysis for CCR2-64I and MCP-1-2518 was performedon genomic DNA isolated from peripheral blood with PCR withsequence-specific primers followed by restriction fragment lengthpolymorphism analysis as described previously (18,20). Briefly,after PCR amplification, PCR products were digested with PvuII(recognizes the MCP-1-2518 A/G transition) or BsaBI (CCR2-64Imutation) (New England Biolabs, Waltham, MA). MCP-1-2518 G/Aand CCR2–64I variants were detected by electrophoresison 4% NuSieve (3:1) gel.
MCP-1 Production
MCP-1 released from unstimulated and stimulated peripheral bloodmononuclear cells (PBMC) was determined by enzyme-lined immunosorbentassay (ELISA) as described (22). Briefly, PBMC from kidney transplantrecipients were isolated with density gradient centrifugation.PBMC were cultured for 24 h with and without addition of 10ng/ml IL-1 (Endogen, Wolburn, MA). MCP-1 concentrations wereassayed in cell-free supernatants by specific ELISA accordingto the manufacturer’s instructions (BioSource International,Inc., Camarillo, CA).
Statistical Analyses
Patient and graft survival was analyzed with Kaplan-Meier estimates.Groups were compared with log rank test. Multivariate analysisfor the respective risk factors was performed with Cox regression.P values were adjusted for multiple testing according to themethod described by Bonferroni. P < 0.05 was considered significant.
The overall gene frequencies of MCP-1-2518 G (28.3%) and CCR2–64I(11.8%) were similar to those previously reported (18,20,22).Eighteen patients (7.8%) were homozygous for the MCP-1-2518Gmutation, and 94 patients (40.5%) were heterozygous. Three patients(1.3%) were homozygous for the CCR2-64I mutation; 45 patients(19.4%) were heterozygous. Individuals with or without the MCP-1-2518G/G genotype did not differ with respect to other risk factorsfor allograft failure such as donor age, cold ischemia time,ethnic background, living donor, HLA-mismatch, panel of reactiveantibodies, immunosuppressive therapy, recipient age and gender,and occurrence of acute rejections (Table 1).
Table 1. Demographic and clinical data for 232 recipients of first kidney transplants according to the MCP-1 -2518 genotype
Association of the Polymorphism with Acute Rejection
The overall incidence of acute rejection within the first yearwas 36.4%. Neither of the investigated polymorphisms showeda significant shift of gene frequency in acute rejectors versusnonrejectors. The gene frequency for the MCP-1-2518 G allelewas 28.4% for rejectors and 28.2% for nonrejectors (P = 0.67).CCR2-64I gene frequency was 11.4% in the rejection group versus12.0% in the rejection free group (P = 0.37). Similarly, nodifference was found by analyzing by genotypic distribution(A/A&A/G versus G/G) of MCP-1-2518 (Table 1).
Effect of Graft Survival on Genotypes
A total of 39 grafts (17%) lost function, and mean graft survivalwas 91 mo. Patient and graft survival with respect to the MCP-1genotype is shown in Figure 1. In accordance with a previousstudy, the G/G genotype was compared with the combined A/A andA/G genotypes (18). The G/G allele of MCP-1-2518 behaves asa determinant for long-term allograft survival and results insignificant reduction of the mean graft survival, as comparedwith the heterozygous (A/G) or wild-type (A/A) allele (67 ±14 versus 95 ± 4 mo; log rank P = 0.0052; Table 2 andFigure 1).
Figure 1. Kaplan-Meier estimate of graft survival in recipients of renal transplants and numbers at risk with respect to MCP-1 genotype. Triangles represent the GG patients who have been censored for observation time. Homozygosity for the mutated -2518G allele is associated with significantly higher rates of graft failure (log rank P = 0.0052).
Table 2. Survival time of the first kidney graft according to the MCP-1 -2518 and CCR2-64I genotype
After multivariate correction for the risk factors listed inTable 1 the Cox proportional hazard analysis revealed that onlythe presence of the G/G genotype was associated with graft loss.The G/G genotype represented a 4.5-fold risk for graft loss(95% CI, 2.3 to 8.8; P = 0.027).
The reason for three graft losses within 12 mo after transplantationfor the G/G MCP-1 genotype were transplant vein thrombosis inone case, and two patients lost their graft because of severerejection.
The 64I mutation of CCR2 had no effect on kidney graft survival(Table 2). Death with a functioning graft occurred in eightpatients (3.4%). Censoring these patients’ graft survivaldata did not change the significantly poorer outcome for patientswith the MCP-1 G/G genotype (data not shown).
MCP-1 Production
The functional effect of this MCP-1 mutation in immunosuppressedtransplant recipients is not known. We therefore examined theeffect of the -2518 G-allele on MCP-1 production after stimulationwith the proinflammatory cytokine IL-1. Baseline MCP-1 productionwas comparable in PBMC with or without the G-allele (543 ±88 versus 305 ± 131; P = 0.10). However, after stimulationthe G-allele was associated with a significant 2.5-fold higherMCP-1 production compared with PBMC without the G-allele (Figure 2).At the time of blood collection, the two groups studiedfor MCP-1 production did not differ in the immunosuppressivetherapy (P = 1.0 by Fisher’s exact test).
Figure 2. MCP-1 production in the presence or absence of the G-allele in kidney transplant recipients. Peripheral blood mononuclear cells (PBMC) from kidney transplant recipients were cultured and stimulated with recombinant interleukin-1 (IL-1). MCP-1 concentrations were assayed in cell-free supernatants by specific ELISA according to the manufacturer’s instructions (BioSource International, Inc.). PBMC from individuals with -2518 G-allele produced significantly more MCP-1 than PBMC without the G-allele (*P < 0.05). Experiments were performed in duplicate. Data are presented as mean ± SEM.
Despite advances in immunosuppression over recent years, long-termgraft function remains adversely affected by a disproportionatelyhigh rate of first-year graft failure and accelerated declinedue to allograft nephropathy or recipient mortality. Multipleassociations have been reported for gene polymorphisms on ofHLA, cytokine, and costimulatory molecules on clinical outcomesof kidney transplantation (23). Few studies however have exploredconsequences of genetically determined alterations of receptor-ligandinteractions in the chemokine system.
Our group was the first to report a significant associationbetween an allelic variation of a chemokine gene with kidneygraft survival (12). Whereas the CCR532 mutation demonstrateda beneficial effect on kidney graft survival, our present studydemonstrates a significant association of allograft failurewith G allele of MCP-1-2518 polymorphism. Renal allograft recipientswith the homozygous for the G allele experience premature graftloss with a significantly shortened mean graft survival of 28mo. Our functional data has also shown that PBMC isolated fromkidney transplant recipients who are carriers of this alleleshow an increased production of MCP-1.
The presence or absence of heterozygous CCR2–64I variantdid not influence the kidney graft outcome. This is in accordancewith our previous observation that CCR2-64I was not associatedwith acute rejection or graft survival in 207 liver transplantrecipients (24). Our data contrast however with a recent studywhere the heterozygous CCR2-64I variant was associated withdecreased incidence of acute rejection (P = 0.014) in 163 kidneytransplant recipients. Long-term graft survival was not addressedin this study (25).
The increased levels of MCP-1 expressed by carrier of the Gallele of the -2518 polymorphism has two potentially negativeeffects on kidney graft outcome. An increased mononuclear cellinfiltration attracted by increased levels of MCP-1 can enhanceboth rejection and the development of transplant-associatedatherosclerosis (26). Although we cannot exclude subclinicalrejections, we found no influence of the MCP-1 variant on acuterejection in our population. This is in accordance with dataobtained from a fully MHC-disparate mouse cardiac rejectionmodel where the MCP-1/CCR2 had a primary role in the developmentof transplant vasculopathy while playing only a secondary rolein acute rejection (WW Hancock, personal communication).
The role of MCP-1 and CCR2 in the pathogenesis of atherosclerosismay be through increasing mononuclear cell recruitment intothe intima, thereby contributing to both antigen specific andnonspecific effects. This role is supported by the fact thatCCR2 as well as MCP-1 knockout mice, when backcrossed with apoE-deficient or LDL receptor—deficient mice, have an inhibitoryeffect on the formation and development of atherosclerotic lesions(15,16). The findings in animal models are consistent with humancardiovascular studies. The MCP-1-2518 (G/G) so-called highMCP-1 producer genotype was at increased risk for CAD. The factthat this effect was not seen in heterozygotes -2518 (A/G) suggestsa recessive mode of action of this chemokine on CAD (18). Thesame study found that individuals homozygous for CCR2–64Iwere at reduced risk for severe CAD, tested by coronary angiography(18). Due to the low number of CCR2-64I homozygotes, we wereunable to assess whether this variant is associated with reducedrisk of premature kidney graft loss. Our data indicate howeverthat the heterozygous genotypes for MCP-1-2518 as well as CCR2-64Ido not have an apparent effect on graft survival.
Identification of MCP-1-2518 (G/G) genotype as a risk factorfor premature allograft failure expands our knowledge of thecomplex genetic factors contributing to successful transplantation(23). Beyond an improvement in understanding the pathophysiologyof allograft failure, this interaction also offers an interestingpotential for future specific therapy. Just as with CCR1, CCR5,and CXCR3, specific blockade of MCP-1/CCR2 interaction appearspossible. In experimental atherosclerosis, another disease modelthat is modulated by MCP-1/CCR2, overexpression of inactiveMCP-1 resulted in significant inhibition of atheroscleroticlesion formation (27,28). Similar interventions might improveallograft survival. Future studies with experimental transplantationin animals with pharmacologic blockade of MCP-1 will possiblydetermine the effects of interventions specifically directedagainst this chemokine.
Acknowledgments
Barbara Murphy is supported by NIH research grant RO1 AI 49289-01.
Footnotes
Bernd Krüger and Bernd Schröppel contributed equallyto the work presented in this paper.
Nelson PJ, Krensky AM: Chemokines, chemokine receptors, and allograft rejection. Immunity 14: 377–386, 2001[CrossRef][Medline]
Nadeau KC, Azuma H, Tilney NL: Sequential cytokine dynamics in chronic rejection of rat renal allografts: Roles for cytokines RANTES and MCP-1. Proc Natl Acad Sci USA 92: 8729–8733, 1995[Abstract/Free Full Text]
Pavlakis M, Strehlau J, Lipman M, Strom TB: Use of intragraft gene expression in the diagnosis of kidney allograft rejection. Transplant Proc 28: 2019–21, 1996[Medline]
Nagano H, Nadeau KC, Takada M, Kusaka M, Tilney NL: Sequential cellular and molecular kinetics in acutely rejecting renal allografts in rats. Transplantation 63: 1101–1108, 1997[CrossRef][Medline]
Grandaliano G, Gesualdo L, Ranieri E, Monno R, Stallone G, Schena FP: Monocyte chemotactic peptide-1 expression and monocyte infiltration in acute renal transplant rejection. Transplantation 63: 414–420, 1997[CrossRef][Medline]
Robertson H, Morley AR, Talbot D, Callanan K, Kirby JA: Renal allograft rejection: Beta-chemokine involvement in the development of tubulitis. Transplantation 69: 684–687, 2000[CrossRef][Medline]
Segerer S, Cui Y, Eitner F, Goodpaster T, Hudkins KL, Mack M, Cartron JP, Colin Y, Schlöndorff D, Alpers CE: Expression of chemokines and chemokine receptors during human renal transplant rejection. Am J Kidney Dis 37: 518–531, 2001[Medline]
Corsi MM, Leone G, Fulgenzi A, Wasserman K, Leone F, Ferrero ME: RANTES and MCP-1 chemokine plasma levels in chronic renal transplant dysfunction and chronic renal failure. Clin Biochem 32: 455–460, 1999[CrossRef][Medline]
Prodjosudjadi W, Daha MR, Gerritsma JS, Florijn KW, Barendregt JN, Bruijn JA, van der Woude FJ, van Es LA: Increased urinary excretion of monocyte chemoattractant protein-1 during acute renal allograft rejection. Nephrol Dial Transplant 11: 1096–1103, 1996[Abstract/Free Full Text]
Hancock WW, Gao W, Faia KL, Csizmadia V: Chemokines and their receptors in allograft rejection. Curr Opin Immunol 12: 511–516, 2000[CrossRef][Medline]
Fischereder M, Luckow B, Hocher B, Wüthrich RP, Rothenpieler U, Schneeberger H, Panzer U, Stahl RA, Hauser IA, Budde K, Neumayer H, Krämer BK, Land W, Schlöndorff D: CC chemokine receptor 5 and renal-transplant survival. Lancet 357: 1758–1761, 2001[CrossRef][Medline]
Ojo AO, Hanson JA, Wolfe RA, Leichtman AB, Agodoa LY, Port FK: Long-term survival in renal transplant recipients with graft function. Kidney Int 57: 307–313, 2000[CrossRef][Medline]
Kasiske BL: Epidemiology of cardiovascular disease after renal transplantation. Transplantation 72 [Suppl 6]: S5–S8, 2001
Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ: Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell 2: 275–281, 1998[CrossRef][Medline]
Dawson TC, Kuziel WA, Osahar TA, Maeda N: Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice. Atherosclerosis 143: 205–211, 1999[CrossRef][Medline]
Boring L, Gosling J, Cleary M, Charo IF: Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 394: 894–897, 1998[CrossRef][Medline]
Szalai C, Duba J, Prohaszka Z, Kalina A, Szabo T, Nagy B, Horvath L, Csaszar A: Involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (CAD). Coincidence of elevated Lp(a) and MCP- 1: 2518 G/G genotype in CAD patients. Atherosclerosis 158: 233–239, 2001[CrossRef][Medline]
Lee B, Doranz BJ, Rana S, Yi Y, Mellado M, Frade JM, Martinez-A C, O’Brien SJ, Dean M, Collman RG, Doms RW: Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR 5, and CXCR4. J Virol 72: 7450–7458, 1998[Abstract/Free Full Text]
Smith MW, Dean M, Carrington M, Winkler C, Huttley GA, Lomb DA, Goedert JJ, O’Brien TR, Jacobson LP, Kaslow R, Buchbinder S, Vittinghoff E, Vlahov D, Hoots K, Hilgartner MW, O’Brien SJ: Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC). ALIVE Study. Science 277: 959–965, 1997[Abstract/Free Full Text]
Hizawa N, Yamaguchi E, Furuya K, Jinushi E, Ito A, Kawakami Y: The role of the C-C chemokine receptor 2 gene polymorphism V64I (CCR2–64I) in sarcoidosis in a Japanese population. Am J Respir Crit Care Med 159: 2021–2023, 1999[Abstract/Free Full Text]
Rovin BH, Lu L, Saxena R: A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun 7: 259–344, 1999
Akalin E, Murphy B: Gene polymorphisms and transplantation. Curr Opin Immunol 13: 572–576, 2001[CrossRef][Medline]
Schröppel B, Fischereder M, Ashkar R, Lin M, Krämer BK, Marder B, Schiano T, Murphy B: The impact of polymorphisms in chemokine and chemokine receptors on outcomes in liver transplantation. Am J Transplant 2: 640–645, 2002[CrossRef][Medline]
Abdi R, Tran TB, Sahagun-Ruiz A Murphy PM, Brenner BM, Milford EL, McDermott DH: Chemokine receptor polymorphism and risk of acute rejection in human renal transplantation. J Am Soc Nephrol 13: 754–758, 2002[Abstract/Free Full Text]
Stark VK, Hoch JR, Warner TF, Hullett DA: Monocyte chemotactic protein-1 expression is associated with the development of vein graft intimal hyperplasia. Arterioscler Thromb Vasc Biol 17: 1614–1621, 1997[Abstract/Free Full Text]
Egashira K, Koyanagi M, Kitamoto S, Ni W, Kataoka C, Morishita R, Kaneda Y, Akiyama C, Nishida KI, Sueishi K, Takeshita A: Anti-monocyte chemoattractant protein-1 gene therapy inhibits vascular remodeling in rats: Blockade of MCP-1 activity after intramuscular transfer of a mutant gene inhibits vascular remodeling induced by chronic blockade of NO synthesis. FASEB J 14: 1974–1978, 2000[Abstract/Free Full Text]
Ni W, Egashira K, Kitamoto S, Kataoka C, Koyanagi M, Inoue S, Imaizumi K, Akiyama C, Nishida Ki K, Takeshita A: New anti-monocyte chemoattractant protein-1 Gene Therapy attenuates atherosclerosis in apolipoprotein E-knockout mice. Circulation 103: 2096–2101, 2001[Abstract/Free Full Text]
Received for publication May 31, 2002.
Accepted for publication July 3, 2002.
This article has been cited by other articles:
H.-J. Anders, V. Vielhauer, and D. Schlondorff Current paradigms about chemokines as therapeutic targets
Nephrol. Dial. Transplant.,
December 1, 2004;
19(12):
2948 - 2951.
[Full Text][PDF]
B. Schroppel, N. Zhang, P. Chen, W. Zang, D. Chen, K. L. Hudkins, W. A. Kuziel, R. Sung, J. S. Bromberg, and B. Murphy Differential Expression of Chemokines and Chemokine Receptors in Murine Islet Allografts: The Role of CCR2 and CCR5 Signaling Pathways
J. Am. Soc. Nephrol.,
July 1, 2004;
15(7):
1853 - 1861.
[Abstract][Full Text][PDF]
B. Schroppel and B. Murphy Gene Variants Affecting Bioavailability of Drugs: Towards Individualized Immunosuppressive Therapy?
J. Am. Soc. Nephrol.,
July 1, 2003;
14(7):
1955 - 1957.
[Full Text][PDF]
C. Viedt and S. R. Orth Monocyte chemoattractant protein-1 (MCP-1) in the kidney: does it more than simply attract monocytes?
Nephrol. Dial. Transplant.,
December 1, 2002;
17(12):
2043 - 2047.
[Full Text][PDF]
Top
Abstract
Introduction
32 mutation) have significantly better allograft survival (12). The interaction of MCP-1 and CCR2 has not been blocked experimentally in a transplant model to date. Preliminary data, however, suggests a prolonged vascularized cardiac allograft survival in CCR2 knockout mice compared with wild-type (11). 
(IL-1
