The FcγRIIa polymorphism in patients with chronic kidney graft rejection

The Fc␥RIIa Polymorphism in Patients With Chronic Kidney Graft Rejection A. Pawlik, M. Florczak, L. Bak, E. Da˛browska-Zamojcin, J. Rozanski, L. Domanski, and B. Gawronska-Szklarz ABSTRACT The Fc␥RIIa receptors, which provide a crucial link between cellular and humoral components of the immune response, display allelic polymorphism. Individuals are homozygous for either arginine 131 (RR131) or histidine 131 (HH131) or are heterozygous for these two alleles (RH131). The HH131 genotype binds human IgG2 with high RR131 with low, and RH131 with intermediate affinity. The aim of the study was to evaluate the Fc␥RIIa polymorphism in patients with chronic kidney graft rejection. The study included 121 renal transplant recipients: 53 patients with long-term stable graft function and 68 with chronic allograft rejection. The distribution of Fc␥RIIa genotypes in patients with chronic kidney graft rejection did not differ significantly from that in patients with stable graft function. The results suggest that the Fc␥RIIa polymorphism is not an important genetic risk factor for chronic rejection of kidney allografts.

T

HE pathophysiology of chronic allograft rejection is much less well understood than that of acute rejection. It is clear that alloantigen-dependent mechanisms initiate and are important in the progression of chronic rejection. However, alloantigen-independent mechanisms such as graft ischemia and viral infections undoubtedly contribute to chronic allograft dysfunction. Both the cellular and the humoral immune response play important roles in this process. Antibodies can be produced in response to the allograft. Depending on their isotype, they may activate the complement cascade or bind to Fc receptors on inflammatory cells, initiating the antibody-dependent cell-mediated cytotoxicity (ADCC) via hematopoietic cells, which express Fc receptors.1 The Fc receptors are glycoproteins found on the surface of hematopoietic cells that bind the Fc portion of immunoglobulin to provide a link between the humoral and cellular immune systems.2 In addition to host defense, Fc receptors play a role in autoimmune diseases and immunohematologic disorders. The Fc␥RIIa, which binds IgG in its oligomeric form or when it is bound to cells, is found on the surface of monocytes, macrophages, neutrophils, platelets, basophils, eosinophils, and other cells.3 The Fc␥RIIa gene is polymorphically expressed, resulting in an amino acid change (arginine: or histidine). This polymorphism alters ligand-binding properties. Histidine/ histidine (H/H131) cells have markedly higher binding

affinity for human IgG2 than do arginine/arginine (R/R131) cells, whereas heterozygotes (H/R131) have intermediate affinity. Also, major differences exist in binding of murine IgG1 (R/R131 ⬎ H/H131) and minor differences with human IgG3 (H/H131 ⬎ R/R131).4 Differences in the binding affinity have been demonstrated in a variety of assays measuring phagocytosis, monocyte-dependent T-cell proliferation, rosetting of opsonized erythrocytes, antibody-mediated platelet activation, and anti-Fc␥RIIa monoclonal antibody (MAb) reactivity. The variation in ligand specificity of Fc␥RIIa polymorphic forms has clinical relevance. Human IgG2 targets are bacteria with a polysaccharide capsule; Fc␥RIIa is the only Fc receptor with binding affinity for human IgG2. Only the H/H131 genotype can effectively bind human IgG2. The Fc␥RIIa genotype may influence the ability to clear encapsulated organisms.5 The Fc␥RIIa polymorphism is also related to risk of other immune-mediated diseases, including heparin-associated thrombocytopenia6 and lupus neFrom the Department of Pharmacokinetics and Therapeutic Drug Monitoring, and Department of Transplantology, Nephrology, and Internal Diseases, Pomeranian Medical University, Szczecin, Poland. Address reprint requests to Andrzej Pawlik, MD, Department of Pharmacokinetics and Therapeutic Drug Monitoring, Pomeranian Medical University, 70-111 Szczecin, Powst. Wielkopolskich 72.

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0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.05.076

Transplantation Proceedings, 36, 1311–1313 (2004)

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phritis.7 Previous studies have demonstrated that T-cell activation and proliferation are dependent on the presence of cells bearing Fc␥RIIa receptors.8 Genetic polymorphism of Fc␥RIIa influenced the in vitro release of Il-2, Il-6, INF␥, TNF␣, and in vivo release during rejection treatment after renal transplantation.9,10 Moreover, immune complexes of IgG bound to antigen can interact through binding to the immunoglobulin Fc position to cell-surface Fc␥ receptors, resulting in modulation of the immune response and induction of graft rejection. We have shown a correlation between Fc␥RIIa polymorphism and renal allograft survival.11 The association of Fc␥RIIa polymorphism with the acute and chronic rejection of allografts is not known. The aim of the present study was to evaluate the correlation between the Fc␥RIIa polymorphism and chronic renal allografts rejection. MATERIALS AND METHODS One hundred twenty-one recipients of first renal transplants were selected for the study. The study included 53 patients with longterm stable graft function (32 males, 31 females; aged 24 to 64 years; mean, 45.7 years; duration of allograft, 2 to 12 years; mean, 5.9 years; serum creatinine in range of normal values; absence of hypertension and proteinuria; absence of abnormalities in biopsy as well as in ultrasound and nuclear scans) and 68 patients with chronic allograft rejection (37 males, 31 females; aged 20 to 67 years; mean, 48.5 years; duration of allograft 2–11 years; mean, 6.2 years). Chronic rejection (chronic allograft nephropathy) was diagnosed by eliminating other causes of chronic renal dysfunction (infections, urinary obstruction, allograft artery stenosis, or cyclosporine [CyA] toxicity) and by changes in biopsy samples. This process was diagnosed clinically in patients having a slow and persistent rise in serum creatinine at least 30% above baseline, usually accompanied by new or worsening hypertension and proteinuria (above 500 mg/24 h). Anatomical problems were excluded by ultrasound and nuclear scans. Biopsy criteria included the presence of interstitial fibrosis, tubular athrophy, and particularly the characteristic vascular changes such as hypertrophy of the arterial intima and smooth muscle (intimal thickening) and glomerular sclerosis.12–14 Twelve patients were classified as Banff IB, 37 as Banff IIB, and 19 as Banff IIIB.15 Immunosuppressive therapy consisted of CyA (2–2.5 mg/kg/d depending on levels), azathioprine (1–2 mg/kg/d), and prednisone (5–20 mg/d).

Fc␥RIIa Genotyping Genomic DNA was extracted manually (precipitation with trimethylammonium bromide salts) from leukocytes contained in 450 ␮L of venous blood with ethylendiaminetetraacetic acid (EDTA) as an anticoagulant. We performed 25-␮L (PCRs) containing 2.5 ␮L of genomic DNA (approx. 100 ng), 2.5 ␮L of 10 ⫻ PCR buffer (containing 15 mmol/L MgCl2, Gibco BRL), 200 ␮mol/L of each dNTP (Gibco BRL), and 0.5 U of Taq polymerase (Gibco BRL). We used 0.5 ␮mol/L H131-specific-sense primer (5⬘-ATCCCAGAAATTCTCCCA-3⬘) from the second extracellular domain or 0.5 ␮mol/L R131-specific-sense primers (5⬘-ATCCCAGAAATTCTCCCG-3⬘ (all primers were synthesized by Gibco BRL) and 0.5 ␮mol/L common antisense primer from an area of the downstream intron where the sequences for Fc␥RIIa, Fc␥RIIb, and Fc␥RIIc diverge (5⬘-CAATTTTGCTGCTATGGGC-3⬘). The resulting fragment was 253 bp (base pair) in length. As internal

PAWLIK, FLORCZAK, BAK ET AL control, we used 0.125 ␮mol/L human growth hormone (HGH)-I primer (5⬘-CAGTGCCTTCCCAACCATTCCCTTA-3⬘) and 0.125 ␮mol/L HGH-II primer (5⬘-ATCCACTCACGGATTTCTGTTGTGTTTC-3⬘), which resulted in a 439-bp fragment. We used a thermal cycler (Mastercycler Eppendorf) to perform a hot-start PCR following by 5 minutes at 95°C, 10 cycles of 1 minute at 95°C, 2 minutes at 57°C, and 1 minute at 72°C; thereafter, to enhance the sensitivity, we used 22 cycles of 1 minute at 95°C, 2 minutes at 54°C, and 1 minute at 72°C, and a final extension step for 5 minutes at 72°C. Each PCR analysis was performed with control samples for R/R, H/R, and H/H genotypes. The PCR amplification products were separated on 1.5% agarose and visualized by using ethidium bromide.16

Statistical Analysis The distribution of genotypes in patients with chronic kidney graft rejection was compared with subjects showing stable graft function and statistically evaluated using the chi-square test with Yates’ correction for small groups (Epi Info 6 program, version 6.2, World Health Organization, Geneva, Switzerland).

RESULTS

Among patients with stable graft function (Table 1) the RR131 genotype was detected in 11 (20.7%), RH131 in 26 (49.1%), and HH131 in 16 (30.2%) patients. The distribution of alleles was as follows: H131 allele 54.7%; R131 allele 45.3%. In patients with chronic allograft rejection, the RR131, RH131, and HH131 genotypes were found in 14 (20.6%), 35 (51.5%), and 19 (27.9%) cases, respectively. The distribution of alleles was as follows: H131 allele, 53.7%; R131 allele, 46.3%. There were no significant differences in distribution of Fc␥RIIA genotypes and alleles between patients with chronic kidney allograft rejection and stable graft function (Table 1). DISCUSSION

Chronic graft rejection, which may occur as early as a few months after transplantation, is characterized by gradual and progressive deterioration of graft function. The formation of plaques and intimal smooth muscle cell proliferation in the arterioles and arteries of allografts results in arterial occlusion or other forms of allograft arteriosclerosis.17 Arteriosclerosis and fibrosis are the common denominators of chronic rejection, with persistent perivascular and interstitial inflammation. In renal transplantation, the histological features of chronic rejection include interstititial fibrosis, tubular atrophy; intimal thickening, and glomelular sclerosis.13,14,18 The etiology of chronic rejection is considered to be multifactorial, including both alloantigen-independent and alloantigen-dependent factors, but much still needs to be learned about the mechanisms causing and supporting graft deterioration. The alloantigen-independent factors that can lead to an inflammatory response and endothelial cell injury include ischemia, reperfusion injury, hyperlipidemia, cytomegalovirus (CMV) infection, and also hypertension. In some instances (e.g., ischemia and reperfusion injury) the inflam-

POLYMORPHISM IN KIDNEY GRAFT REJECTION

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Table 1. Distribution of Fc␥RIIa Genotypes in Patients With Stable Graft Function and Chronic Kidney Graft Rejection Fc␥RIIa R/R131

Patients with stable graft function (n ⫽ 53) Patients with chronic kidney graft rejection (n ⫽ 68) P value

Fc␥RIIa R/H131

Fc␥RIIa H/H131

(n)

(%)

(n)

(%)

(n)

(%)

11

(20.7)

26

(49.1)

16

(30.2)

14

(20.6)

35

(51.5)

19

(27.9)

NS

NS

NS

matory response is triggered by direct trauma to the vascular endothelium. Hyperlipidemia results in endothelial cell injury attibutable to lipid deposition in the vascular wall, whereas CMV infection may also cause inflammation of the vascular wall.19 The immunological response involves Tcells, B-cells, macrophages, and upregulation of class II major histocompatibility complex (MHC) and adhesion molecules. Both T-cell-derived IFN-␥ and TNF-␣ activate macrophages and macrophage-derived cytokines. In the present study we did not observe an association between the Fc␥RIIa polymorphism and chronic kidney allografts rejection. Some workers believe that the cellular immune response is the main factor involved in chronic kidney allograft rejection.20 Although previous studies have shown associations of Fc␥-receptors, Fc␥R-blocking factors, as well as the Fc␥RIIa polymorphism with renal allograft survival,11,21–23 it seems that these receptors may not play an important role in chronic kidney allograft rejection.

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