Intragraft Expression of the IL-10 Gene Is Up-Regulated in Renal Protocol Biopsies with Early Interstitial Fibrosis, Tubular Atrophy, and Subclinical Rejection
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090411
ISSN1525-2191
AutoresMiguel Hueso, Estanis Navarro, Francesc Moreso, Francisco O’Valle, Mercè Pérez‐Riba, Raimundo García del Moral, Josep M. Grinyó, Daniel Serón,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoGrafts with subclinical rejection associated with interstitial fibrosis and tubular atrophy (SCR+IF/TA) show poorer survival than grafts with subclinical rejection without IF/TA (SCR). Aiming to detect differences among SCR+IF/TA and SCR, we immunophenotyped the inflammatory infiltrate (CD45, CD3, CD20, CD68) and used a low-density array to determine levels of TH1 (interleukin IL-2, IL-3, γ-interferon, tumor necrosis factor-α, lymphotoxin-α, lymphotoxin-β, granulocyte-macrophage colony-stimulating factor) and TH2 (IL-4, IL-5, IL-6, IL-10, and IL-13) transcripts as well as of IL-2R (as marker for T-cell activation) in 31 protocol biopsies of renal allografts. Here we show that grafts with early IF/TA and SCR can be distinguished from grafts with SCR on the basis of the activation of IL-10 gene expression and of an increased infiltration by B-lymphocytes in a cellular context in which the degree of T-cell activation is similar in both groups of biopsies, as demonstrated by equivalent levels of IL-2R mRNA. These results suggest that the up-regulation of the IL-10 gene expression, as well as an increased proportion of B-lymphocytes in the inflammatory infiltrates, might be useful as markers of early chronic lesions in grafts with SCR. Grafts with subclinical rejection associated with interstitial fibrosis and tubular atrophy (SCR+IF/TA) show poorer survival than grafts with subclinical rejection without IF/TA (SCR). Aiming to detect differences among SCR+IF/TA and SCR, we immunophenotyped the inflammatory infiltrate (CD45, CD3, CD20, CD68) and used a low-density array to determine levels of TH1 (interleukin IL-2, IL-3, γ-interferon, tumor necrosis factor-α, lymphotoxin-α, lymphotoxin-β, granulocyte-macrophage colony-stimulating factor) and TH2 (IL-4, IL-5, IL-6, IL-10, and IL-13) transcripts as well as of IL-2R (as marker for T-cell activation) in 31 protocol biopsies of renal allografts. Here we show that grafts with early IF/TA and SCR can be distinguished from grafts with SCR on the basis of the activation of IL-10 gene expression and of an increased infiltration by B-lymphocytes in a cellular context in which the degree of T-cell activation is similar in both groups of biopsies, as demonstrated by equivalent levels of IL-2R mRNA. These results suggest that the up-regulation of the IL-10 gene expression, as well as an increased proportion of B-lymphocytes in the inflammatory infiltrates, might be useful as markers of early chronic lesions in grafts with SCR. Subclinical rejection (SCR), ie, the presence of histological lesions typical of acute allograft rejection (according to Banff criteria) in patients with stable renal function, has been associated with silent progression of interstitial fibrosis and tubular atrophy (IF/TA).1Nankivell BJ Borrows RJ Fung CL O'Connell PJ Allen RD Chapman JR The natural history of chronic allograft nephropathy.N Engl J Med. 2003; 349: 2326-2333Crossref PubMed Scopus (1653) Google Scholar, 2Nickerson P Jeffery J Gough J McKenna R Grimm P Cheang M Rush D Identification of clinical and histopathologic risk factors for diminished renal function 2 years posttransplant.J Am Soc Nephrol. 1998; 9: 482-487PubMed Google Scholar Furthermore, there is evidence that the simultaneous presence of subclinical rejection, interstitial fibrosis and tubular atrophy (SCR+IF/TA) could be associated with a poorer graft survival when compared with grafts with subclinical rejection without IF/TA (SCR), or with grafts with IF/TA but without subclinical rejection (IF/TA), suggesting that different mechanisms of immune response could be involved in the process of graft degeneration.3Moreso F Ibernon M Goma M Carrera M Fulladosa X Hueso M Gil-Vernet S Cruzado JM Torras J Grinyo JM Seron D Subclinical rejection associated with chronic allograft nephropathy in protocol biopsies as a risk factor for late graft loss.Am J Transplant. 2006; 6: 747-752Crossref PubMed Scopus (266) Google Scholar CD4+ T helper (TH) lymphocytes are essential regulators of the immune response. After activation by antigen-presenting cells, TH lymphocytes differentiate into effector cells, specialized in cytokine secretion, that can be further classified as type 1 (TH1) or type 2 (TH2) based on their profiles of cytokine gene expression and immune regulatory function. The process of acute rejection has been related to the activation of the TH1 response, with the subsequent production of interferon (IFN)-γ, interleukin IL-2, and other cytokines and chemokines, whereas the TH2 response, which mediates humoral immunity, has been associated with chronic allograft rejection.4D'Elios MM Josien R Manghetti M Amedei A de Carli M Cuturi MC Blancho G Buzelin F del Prete G Soulillou JP Predominant Th1 cell infiltration in acute rejection episodes of human kidney grafts.Kidney Int. 1997; 51: 1876-1884Crossref PubMed Scopus (95) Google Scholar, 5Kirk AD Jacobson LM Heisey DM Radke NF Pirsch JD Sollinger HW Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function.Transplantation. 1999; 68: 1578-1582Crossref PubMed Scopus (58) Google Scholar, 6Shirwan H Chronic allograft rejection. Do the Th2 cells preferentially induced by indirect alloantigen recognition play a dominant role?.Transplantation. 1999; 68: 715-726Crossref PubMed Scopus (124) Google Scholar However, cytokines of both TH cell subtypes have been detected in patients during episodes of acute rejection, in which the levels of IL-7, IL-8, IL-10, IL-15, γ-IFN, perforin, granzyme-B, and Fas ligand transcripts, but not of IL-2, were found to be up-regulated.7Strehlau J Pavlakis M Lipman M Shapiro M Vasconcellos L Harmon W Strom TB Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation.Proc Natl Acad Sci USA. 1997; 94: 695-700Crossref PubMed Scopus (388) Google Scholar Studies of renal biopsies from clinically-stable transplanted patients, performed two to three years after transplantation, evidenced a low degree of immune activation in the allografts.5Kirk AD Jacobson LM Heisey DM Radke NF Pirsch JD Sollinger HW Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function.Transplantation. 1999; 68: 1578-1582Crossref PubMed Scopus (58) Google Scholar Furthermore, in these biopsies, the severity of the inflammatory lesions associated with interstitial fibrosis and tubular atrophy could be correlated with an increase in IL-6 gene expression, a TH2 cytokine.5Kirk AD Jacobson LM Heisey DM Radke NF Pirsch JD Sollinger HW Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function.Transplantation. 1999; 68: 1578-1582Crossref PubMed Scopus (58) Google Scholar On the other hand, expression of TNF-α and IL-8 genes was only correlated with tubular atrophy, and no correlation was detected among changes in the expression of the IFN-γ, IL-1β, IL-2, or IL-4 genes with any histological injury.5Kirk AD Jacobson LM Heisey DM Radke NF Pirsch JD Sollinger HW Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function.Transplantation. 1999; 68: 1578-1582Crossref PubMed Scopus (58) Google Scholar Finally, in this complex context of pathophysiological alterations and changes in the expression of immune-related genes, no morphological trait has been found that could be used as a marker of the degree of immune activation to specifically detect those infiltrates associated with chronic lesions. Low-density arrays (LDA), based on the use of real-time RT-PCR, have been introduced as a novel method for gene expression profiling and offer higher throughput than usual single gene analysis.8Goulter AB Harmer DW Clark KL Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue.BMC Genomics. 2006; 7: 34Crossref PubMed Scopus (38) Google Scholar We underwent the analysis of the expression of TH1 (IL-2, IL-3, γ-IFN, tumor necrosis factor [TNF]-α, lymphotoxin-α, lymphotoxin-β, granulocyte-macrophage colony-stimulating factor [GM-CSF]) and TH2 (IL-4, IL-5, IL-6, IL-10, and IL-13) cytokine genes in protocol biopsies of kidney allograft using LDA, with the aim to characterize the population of cells that infiltrated renal allografts, as well as to identify differences in gene expression that could facilitate the early detection of chronic lesions. In this pilot study we used protocol biopsies from renal allografts performed during the first year of follow-up, obtained between 1995 and 1998, from which total RNA was available for analysis. Protocol biopsies were performed as previously described9Seron D Moreso F Bover J Condom E Gil-Vernet S Canas C Fulladosa X Torras J Carrera M Grinyo JM Alsina J Early protocol renal allograft biopsies and graft outcome.Kidney Int. 1997; 51: 310-316Crossref PubMed Scopus (215) Google Scholar in patients who gave their informed consent and had a serum creatinine level below 250 μmol/L, proteinuria below 1 g/day, and stable renal function defined as a variability of serum creatinine of less than 15% during the two weeks before and after of the biopsy. For all biopsies, two cores of tissue were obtained, one of them was processed for conventional histology and the second was immediately snap frozen in liquid nitrogen and stored at −80°C for RNA extraction. Renal lesions were blindly evaluated according to the 2005 Banff working classification criteria.10Solez K Colvin RB Racusen LC Sis B Halloran PF Birk PE Campbell PM Cascalho M Collins AB Demetris AJ Drachenberg CB Gibson IW Grimm PC Haas M Lerut E Liapis H Mannon RB Marcus PB Mengel M Mihatsch MJ Nankivell BJ Nickeleit V Papadimitriou JC Platt JL Randhawa P Roberts I Salinas-Madriga L Salomon DR Seron D Sheaff M Weening JJ Banff '05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN′).Am J Transplant. 2007; 7: 518-526Crossref PubMed Scopus (920) Google Scholar We identified nine patients with the simultaneous presence of SCR and IF/TA and 6 patients with SCR without IF/TA, and selected 16 additional normal protocol biopsies matched by the time of biopsy, to conduct a case–control study. Jurkat T-leukemia cells, activated with 20 nmol/L phorbol 12-myristate 13-acetate (PMA) and 1 μmol/L ionomycin, were used as positive control for T-lymphocyte activation in the cytokine mRNA detection by real-time PCR. The Hospital Ethics Committee approved this study, and a written informed consent was obtained from each patient. For immunohistochemical studies, paraffin-embedded specimens were sliced at 4 μm, incubated with monoclonal antibodies to CD45 (pan-leukocytes), CD3 (T-lymphocytes), CD68 (monocytes/macrophages), CD20 (B-lymphocytes; all from Master Diagnóstica SL, Granada, Spain), or IL-10 (R&D Systems, Minneapolis, MN) and subsequently stained with a goat–anti-mouse secondary antibody labeled with horseradish peroxidase (Master Diagnóstica SL) in an automated immunohistochemical stainer (Lab Vision, Fremont, CA).11Moreso F Seron D O'Valle F Ibernon M Goma M Hueso M Cruzado JM Bestard O Duarte V del Moral RG Grinyo JM Immunephenotype of glomerular and interstitial infiltrating cells in protocol renal allograft biopsies and histological diagnosis.Am J Transplant. 2007; 7: 2739-2747Crossref PubMed Scopus (31) Google Scholar In all cases, sections were counterstained with hematoxylin to make nuclei evident. Tonsil sections and biopsies from human kidney with acute rejection were used as positive controls in the identification of IL-10–expressing cells. A positive result was defined, by the pathologist, as the detection of IL-10–stained cells in the infiltrates. Positively-stained cells in the whole renal cortex were counted at ×20 to ×40 magnification and acquired with a digital camera. For the leukocyte markers, results were expressed as the number of positive cells/mm2, or as the percentage of each population regarding the CD45 cells. With the aim to identify antibody-related rejection, C4d deposits were evaluated using a polyclonal antibody (Biomedica Medizinprodukte GmbH&co KG, Vienna, Austria). The negative control replaced the primary antibody by rabbit normal serum.11Moreso F Seron D O'Valle F Ibernon M Goma M Hueso M Cruzado JM Bestard O Duarte V del Moral RG Grinyo JM Immunephenotype of glomerular and interstitial infiltrating cells in protocol renal allograft biopsies and histological diagnosis.Am J Transplant. 2007; 7: 2739-2747Crossref PubMed Scopus (31) Google Scholar Positive C4d staining was defined as the presence of C4d deposition in more than 50% of peritubular capillary sections. Total RNA isolation and purification, reverse transcription, and quality analysis of the synthesized cDNA were done as previously described.12Hueso M Beltran V Moreso F Ciriero E Fulladosa X Grinyo JM Seron D Navarro E Splicing alterations in human renal allografts: detection of a new splice variant of protein kinase Par1/Emk1 whose expression is associated with an increase of inflammation in protocol biopsies of transplanted patients.Biochim Biophys Acta. 2004; 1689: 58-65Crossref PubMed Scopus (7) Google Scholar A sample of approximately 500 ng of cDNA was run (by duplicate) on the ABI PRISM7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) using the LDA technology (microfluidic cards) with commercial probes and primers, and according to the manufacturer's protocol. The LDA contained predesigned primer probe sets for the following human cytokine and cytokine receptor genes (Applied Biosystems): IL-2 (ID number: Hs00174114_m1); IL-2 receptor (ID number: Hs00166229_m1); IL-3 (ID number: Hs00174117_m1); IL-4 (ID number: Hs00174122_m1); IL-5 (ID number: Hs00174200_m1); IL-6 (ID number: Hs00174131_m1); IL-10 (ID number: Hs00174086_m1); IL-13 (ID number: Hs00174379_m1); lymphotoxin α (ID number: Hs00236874_m1); lymphotoxin β (ID number: Hs00242739_m1); γ- IFN (ID number: Hs00174143_m1); TNF-α (ID number: Hs00174128_m1); and GM-CSF (ID number: Hs00171266_m1); as well as the following endogenous controls glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) (ID number: s99999905_m1); and 18S rRNA, (ID number: 4342379-18S). Transcript levels were calculated using the delta threshold cycle (Ct) method based in the relative quantification of cytokine expression normalized to the housekeeping gene after determining the first cycle of fluorescence detection (Ct), and calculating the difference of these threshold cycles. In a second step we normalized the results observed in grafts with SCR to the expression in normal protocol biopsies by applying the 2−ΔΔCT method, as described by the manufacturer (Applied Biosystems; User Bulletin 2). The normalization to GAPDH or 18S rRNA as internal controls gave comparable results. In the case of the data presented in this manuscript, normalization was referred to the 18S rRNA internal control. When no detectable level of transcript was found, a value equal to half the minimum observed 18S-normalized level was assigned.13Muthukumar T Dadhania D Ding R Snopkowski C Naqvi R Lee JB Hartono C Li B Sharma VK Seshan SV Kapur S Hancock WW Schwartz JE Suthanthiran M Messenger RNA for FOXP3 in the urine of renal-allograft recipients.N Engl J Med. 2005; 353: 2342-2351Crossref PubMed Scopus (486) Google Scholar To analyze the normal distribution of variables, the Kolmogorov-Smirnov test was applied. Student t test and the analysis of variance were used in normal quantitative variables. Mann–Whitney U test and Kruskal–Wallis test were applied to those not following a normal distribution and to ordinal variables. Differences between categorical data were assessed using a 2 × 2 contingency table analysis and the Fisher exact tests. Correlation between transcript levels and lymphocyte infiltration was analyzed by the Spearman rank correlation coefficient. Results were expressed as mean ± SD. P values were corrected for the number of variables compared according to the Bonferroni method. The outlier values (values outside 1.5 times the interquartile range above the third quartile or below the first quartile) in box-plot graphs were not considered for the quantification of cytokine transcript levels. All P values were two-tailed, and a P value 20%4 (25%)1 (17%)2 (22%)nsHLA mismatches2.69 ± 1.252.50 ± 1.043.11 ± 1.45nsCIT, hours21 ± 422 ± 323 ± 6nsATN, no/yes14/26/08/1nsAcute rejection, no/yes14/24/24/50.07At protocol biopsy Time after transplantation, days189 ± 106138 ± 39242 ± 140ns Serum Creatinine level, μmol/L129 ± 28141 ± 41184 ± 49*P ≤ 0.005 vs Normal (Bonferroni test);0.006 Creatinine clearance, ml/min58 ± 1155 ± 1350 ± 20ns Proteinuria, g/day0.22 ± 0.100.18 ± 0.060.50 ± 0.29*P ≤ 0.005 vs Normal (Bonferroni test);†P = 0.006 vs SCR (Bonferroni test).0.001PRA indicates panel reactive antibodies; HLA, human leukocyte antigen; CIT, cold ischemia time; ATN, acute tubular necrosis.* P ≤ 0.005 vs Normal (Bonferroni test);† P = 0.006 vs SCR (Bonferroni test). Open table in a new tab PRA indicates panel reactive antibodies; HLA, human leukocyte antigen; CIT, cold ischemia time; ATN, acute tubular necrosis. All renal biopsies contained at least one arterial section, and the mean number of glomeruli in the biopsy core was 10 ± 6. Sample size distribution according to 1997 Banff criteria were as follows: 16 (51.6%) were adequate (≥10 glomeruli, more than 2 arteries), 10 (32.2%) represented minimum sample adequacy (between seven and nine glomeruli, and at least one arterial section), and 5 (16.1%) biopsies were inadequate (between one and six glomeruli and at least one arterial section). The severity of IF/TA, according to the Banff score, in the group of patients of SCR+IF/TA was mild in eight cases and moderate in one case. Table 2 scores acute and chronic lesions in all patients of the study according to Banff criteria. The histological diagnosis of marginal biopsies was normal in 4 cases, and the fifth case displayed subclinical rejection with interstitial fibrosis and tubular atrophy (SCR+IF/TA).Table 2Severity of Acute and Chronic Lesions According to Banff CriteriaNormalSCRSCR+IF/TAPn1669g0.00 ± 0.000.17 ± 0.400.33 ± 0.500.055i0.19 ± 0.401.00 ± 0.001.00 ± 0.00<0.0001t0.00 ± 0.001.00 ± 0.000.78 ± 0.44<0.0001v0.00 ± 0.000.00 ± 0.000.00 ± 0.00ah0.06 ± 0.250.33 ± 0.510.13 ± 0.350.27cg0.00 ± 0.000.17 ± 0.400.44 ± 0.520.012ci0.00 ± 0.000.00 ± 0.001.00 ± 0.50<0.0001ct0.06 ± 0.250.00 ± 0.001.00 ± 0.50<0.0001cv0.00 ± 0.000.00 ± 0.000.13 ± 0.350.26Shown are the mean ± SD of the Banff scores. The abbreviations used in the above Table correspond to the following pathological categories: g, glomerulitis; i, interstitial infiltrate; t, tubulitis; v, vasculitis; ah, arteriolar hyalinosis; cg, chronic glomerular lesions; ci, chronic interstitial lesions; ct, chronic tubular lesion; cv, chronic vascular lesions. Open table in a new tab Shown are the mean ± SD of the Banff scores. The abbreviations used in the above Table correspond to the following pathological categories: g, glomerulitis; i, interstitial infiltrate; t, tubulitis; v, vasculitis; ah, arteriolar hyalinosis; cg, chronic glomerular lesions; ci, chronic interstitial lesions; ct, chronic tubular lesion; cv, chronic vascular lesions. Grafts with SCR+IFTA were clinically stable, and graft function was followed for 118 ± 27 months. Figure 1 demonstrates that grafts diagnosed of SCR did not display a non-overt clinical rejection, because creatinine levels were mostly stable in the different groups. In this period of time, one patient from the group of normal protocol biopsies lost their graft due to chronic allograft dysfunction associated with nephrotic proteinuria 93 months after transplantation (no diagnostic biopsy was performed), and another three died with a functioning graft (one due to a sepsis, and other two due to coronary syndrome). In the group with SCR only, one patient lost their graft due to chronic allograft dysfunction 43 months after transplantation (no diagnostic biopsy was available for further study), but this patient died later of myeloma. Finally, three patients in the group of SCR+IF/TA lost their grafts, two due to biopsy-proven IF/TA at 98 and 120 months after transplantation, and one due to a "de novo" glomerulonephritis. In addition, one patient died with a functioning graft due to acute myocardial infarction in the group of SCR+IF/TA. We studied the cellularity of interstitial infiltrates by immunohistochemistry (IHC) using antibodies specific for B-lymphocytes (anti-CD20), T-lymphocytes (anti-CD3), macrophages (anti-CD68), and total leukocytes (anti-CD45). Table 3 shows that grafts with interstitial fibrosis and tubular atrophy (SCR+IF/TA) displayed a more severe infiltration of total (T and B) lymphocytes, but not of macrophages, expressed as the absolute number of infiltrating cells per mm2, when compared with renal tissue from normal protocol biopsies (598 ± 268 CD3+ cells/mm2 in SCR+IFTA grafts versus 253 ± 149 CD3+ cells/mm2 in normal protocol biopsies, P = 0.005; 353 ± 264 CD20+ cells/mm2 in SCR+IFTA grafts versus 79 ± 108 CD20+ cells/mm2 in normal protocol biopsies, P = 0.015; 261 ± 99 CD68+ cells/mm2 in SCR+IFTA grafts versus 193 ± 133 CD68+ cells/mm2 in normal protocol biopsies, P = 0.112). When grafts with SCR+IFTA and grafts with SCR were compared, no significant differences in the absolute number of infiltrating cells were found.Table 3Density of Infiltrating Cells in Protocol Biopsies According to Their Immunophenotype (in Number of Cells per mm2)mAbNormal n = 11SCR n = 6SCR+IF/TA n = 7PCD45460 ± 334696 ± 2931034 ± 500*P = 0.01 vs Normal (Bonferroni test);0.019CD3253 ± 149435 ± 176598 ± 268†P = 0.005 vs Normal (Bonferroni test).0.005CD2079 ± 108175 ± 154353 ± 264*P = 0.01 vs Normal (Bonferroni test);0.015CD68193 ± 133326 ± 116261 ± 990.112Cellularity of interstitial infiltrates was studied by immunohistochemistry using antibodies specific for B-lymphocytes (anti-CD20), T-lymphocytes (anti-CD3), macrophages (anti-CD68), and total leukocytes (anti-CD45). Positively stained cells in the whole renal cortex were counted at ×40 magnification, and results were expressed as the mean ± SD of the number of positive cells per mm2. The number of patients studied is stated below each clinical condition.* P = 0.01 vs Normal (Bonferroni test);† P = 0.005 vs Normal (Bonferroni test). Open table in a new tab Cellularity of interstitial infiltrates was studied by immunohistochemistry using antibodies specific for B-lymphocytes (anti-CD20), T-lymphocytes (anti-CD3), macrophages (anti-CD68), and total leukocytes (anti-CD45). Positively stained cells in the whole renal cortex were counted at ×40 magnification, and results were expressed as the mean ± SD of the number of positive cells per mm2. The number of patients studied is stated below each clinical condition. We also determined the relative weight of each cell population in the composition of the inflammatory infiltrate by normalizing the number of infiltrating cells to the total number of CD45 cells, expressing the result as percentage. Table 4 shows that the value for B lymphocytes was significantly higher in SCR+IF/TA grafts than in SCR grafts or in normal tissue (35 ± 23% in SCR+IF/TA biopsies versus 12 ± 10% in normal protocol biopsies, and 23 ± 18% in SCR biopsies, P = 0.04), whereas no significant differences were found for T lymphocytes (62 ± 21% in SCR+IF/TA biopsies versus 61 ± 18% in normal protocol biopsies and 64 ± 10% in SCR biopsies P = ns). Regarding macrophages, a higher proportion was detected in normal biopsies and SCR grafts than in SCR+IF/TA grafts (44 ± 19% in normal protocol biopsies, 51 ± 16% in SCR biopsies versus 28 ± 9% in SCR+IF/TA grafts, P = 0.04).Table 4Relative Proportions of CD3+, CD20+, and CD68+ Cells Regarding CD45+ Cells in Protocol BiopsiesmAbNormal n = 11SCR n = 6SCR+IF/TA n = 7PCD361 ± 18%64 ± 10%62 ± 21%nsCD2012 ± 10%23 ± 18%35 ± 23%*P = 0.038 vs Normal (Bonferroni test);0.04CD6844 ± 19%51 ± 16%28 ± 9%†P = 0.056 vs Normal (Bonferroni test).0.04For each case, we calculated the number of CD3+, CD20+, and CD68+ infiltrating cells as percentage of total CD45+ cells, and expressed the result as the mean ± SD.* P = 0.038 vs Normal (Bonferroni test);† P = 0.056 vs Normal (Bonferroni test). Open table in a new tab For each case, we calculated the number of CD3+, CD20+, and CD68+ infiltrating cells as percentage of total CD45+ cells, and expressed the result as the mean ± SD. With the aim to study a potential pathogenic role of the humoral immunity in the development of SCR and SCR+IF/TA, we analyzed the diffuse deposits of C4d along the peritubular capillaries from 26 biopsies, but no differences were evident among the groups (with 2 of 12 normal protocol biopsies, 2 of 6 SCR biopsies, and 3 of 8 SCR+IF/TA grafts presenting C4d deposits, P = ns). In addition, the number of infiltrating B lymphocytes detected did not correlate with the diffuse C4d deposition found in peritubular capillaries (161 ± 195 B cells/mm2 in grafts without C4d staining versus 196 ± 159 in grafts with diffuse C4d staining, P = ns). We studied the expression of 13 cytokine genes (TH1 and TH2 responses) in our group of protocol biopsies by using the LDA technology, and the results obtained are shown in Table 5. The most significant finding was the frequent detection of IL-10 transcripts in SCR+IF/TA grafts (detected in 77.8% of SCR+IF/TA grafts versus 33.3% of SCR grafts and 37% of normal protocol biopsies; P = 0.06). On the contrary, we could not detect IL-4 or IL-13 transcripts in any of our samples, and IL-2 (6.5%), IL-3 (9.7%), IL-6 (19.4%), and GM-CSF (3.2%) mRNAs were detected only in a small number of the entire group of protocol biopsies.Table 5Number of Protocol Biopsies in which the Stated Cytokine Transcripts Were DetectedCytokinesNormal n = 16SCR n = 6SCR+IF/TA n = 9PIL-22 (12%)01 (11.1%)nsIL-2 receptor9 (56%)5 (83.3%)6 (66.7%)nsIL-33 (19%)00nsIL-4000nsIL-5001 (11.1%)nsIL-63 (19%)2 (33.3%)1 (11.1%)nsIL-106 (37%)2 (33.3%)7 (77.8%)0.06IL-13000nsγ-IFN7 (43%)3 (50%)6 (66.7%)nsTNF-α12 (75%)5 (83.3%)6 (66.7%)nsLymphotoxin α (LTA)3 (19%)2 (33.3%)3 (33.3%)nsLymphotoxin β (LTB)10 (62%)5 (83.3%)8 (88.9%)nsGM-CSF001 (11.1%)nsTotal RNA was extracted from the protocol biopsies, reverse transcribed to cDNA, and used for cytokine transcript quantification by real-time RT-PCR using low-density arrays (Applied Biosystems). Shown are the number of protocol biopsies in which expression of the stated cytokine genes was detected in the groups studied.IL indicates interleukin; γ-IFN, gamma interferon; TNF-α, tumor necrosis factor-α; GM-CSF, granulocyte-macrophage colony-stimulating factor. Open table in a new tab Total RNA was extracted from the protocol biopsies, reverse transcribed to cDNA, and used for cytokine transcript quantification by real-time RT-PCR using low-density arrays (Applied Biosystems). Shown are the number of protocol biopsies in which expression of the stated cytokine genes was detected in the groups studied. IL indicates interleukin; γ-IFN, gamma interferon; TNF-α, tumor necrosis factor-α; GM-CSF, granulocyte-macrophage colony-stimulating factor. Expression data were also quantitatively analyzed. Regarding the expression of TH1-associated cytokines that were prevalently detected in protocol biopsies (γ-IFN, TNF-α, lymphotoxin-α, lymphotoxin-β), no differences were found among normal, SCR, and SCR+IF/TA grafts (data not shown). On the other hand, expression analysis of TH2-associated cytokines showed a significant increase in the expression of IL-10 transcripts in biopsies with SCR+IF/TA when compared with normal or SCR biopsies (−19 ± 3 cycles in biopsies with SCR+IF/TA, −24 ± 3 cycles in biopsies with SCR, and −23 ± 3 cycles in normal biopsies, P = 0.017) as shown in Figure 2A. To determine the relative increase of IL-10 gene expression in SCR+IFTA ov
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