Endothelial injury in transplant glomerulopathy is correlated with transcription factor T-bet expression
2012; Elsevier BV; Volume: 82; Issue: 3 Linguagem: Inglês
10.1038/ki.2012.112
ISSN1523-1755
AutoresQiquan Sun, Mingchao Zhang, Kenan Xie, Xue Li, Caihong Zeng, Minlin Zhou, Zhihong Liu,
Tópico(s)Transplantation: Methods and Outcomes
ResumoTransplant glomerulopathy is an important cause of late graft loss. Inflammatory lesions including glomerulitis and peritubular capillaritis, suggestive of endothelial injury, are prominent in this condition but the mechanism underlying this inflammation remains unclear. Here we measured the expression of T-bet (a member of the T-box family of transcription factors regulating Th1 lineage commitment) and its relationship with inflammation in 70 patients with transplant glomerulopathy. Within this cohort, 32 patients were diagnosed with transplant glomerulopathy, 23 with interstitial fibrosis/tubular atrophy, and 15 with stable grafts. There was a significant increase in T-bet expression in both glomerular and peritubular capillaries of the transplant glomerulopathy group. This expression was strongly correlated with CD4+, CD8+, and CD68+ cell infiltration within glomerular and peritubular capillaries. The expression of GATA3, a Th2 regulator, was rarely found in the transplant glomerulopathy group. Transplant glomerulopathy was associated with diffuse peritubular capillary dilation without reduced capillary density. Moreover, the degree of capillary dilation was significantly correlated with the number of infiltrating CD68+ cells. Since endothelial injury is a typical lesion that follows alloantibody reactivity, our results suggest that T-bet is involved in the pathogenesis of this glomerulopathy. Transplant glomerulopathy is an important cause of late graft loss. Inflammatory lesions including glomerulitis and peritubular capillaritis, suggestive of endothelial injury, are prominent in this condition but the mechanism underlying this inflammation remains unclear. Here we measured the expression of T-bet (a member of the T-box family of transcription factors regulating Th1 lineage commitment) and its relationship with inflammation in 70 patients with transplant glomerulopathy. Within this cohort, 32 patients were diagnosed with transplant glomerulopathy, 23 with interstitial fibrosis/tubular atrophy, and 15 with stable grafts. There was a significant increase in T-bet expression in both glomerular and peritubular capillaries of the transplant glomerulopathy group. This expression was strongly correlated with CD4+, CD8+, and CD68+ cell infiltration within glomerular and peritubular capillaries. The expression of GATA3, a Th2 regulator, was rarely found in the transplant glomerulopathy group. Transplant glomerulopathy was associated with diffuse peritubular capillary dilation without reduced capillary density. Moreover, the degree of capillary dilation was significantly correlated with the number of infiltrating CD68+ cells. Since endothelial injury is a typical lesion that follows alloantibody reactivity, our results suggest that T-bet is involved in the pathogenesis of this glomerulopathy. With the advent of novel immunosuppressive drugs, graft loss due to early acute rejection has been greatly reduced.1.Halloran P.F. Melk A. Barth C. Rethinking chronic allograft nephropathy: the concept of accelerated senescence.J Am Soc Nephrol. 1999; 10: 167-181PubMed Google Scholar However, the incidence of late graft loss remains unacceptably high.2.Meyers C.M. Kirk A.D. Workshop on late renal allograft dysfunction.Am J Transplant. 2005; 5: 1600-1605Crossref PubMed Scopus (20) Google Scholar A recent study suggested that the majority of late graft losses are related to antibody-mediated rejection (AMR).3.Einecke G. Sis B. Reeve J. et al.Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure.Am J Transplant. 2009; 9: 2520-2531Crossref PubMed Scopus (536) Google Scholar Furthermore, earlier reports showed that late graft loss always has a previous emergence of anti-donor antibodies4.Lee P.C. Terasaki P.I. Takemoto S.K. et al.All chronic rejection failures of kidney transplants were preceded by the development of HLA antibodies.Transplantation. 2002; 74: 1192-1194Crossref PubMed Scopus (296) Google Scholar and that late AMR is usually associated with poor graft survival.5.Sun Q. Liu Z.H. Ji S. et al.Late and early C4d-positive acute rejection: different clinico-histopathological subentities in renal transplantation.Kidney Int. 2006; 70: 377-383Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar,6.Pascual J. Samaniego M.D. Torrealba J.R. et al.Antibody-mediated rejection of the kidney after simultaneous pancreas-kidney transplantation.J Am Soc Nephrol. 2008; 19: 812-824Crossref PubMed Scopus (24) Google Scholar Clearly, AMR is one of the most important causes of late graft loss. Transplant glomerulopathy (TG) is a principal form of late AMR.7.Suri D.L. Tomlanovich S.J. Olson J.L. et al.Transplant glomerulopathy as a cause of late graft loss.Am J Kidney Dis. 2000; 35: 674-680Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,8.Solez K. Colvin R.B. Racusen L.C. et al.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 (923) Google Scholar The incidence of TG is ∼20% by 5 years post transplantation.9.Cosio F.G. Gloor J.M. Sethi S. et al.Transplant glomerulopathy.Am J Transplant. 2008; 8: 492-496Crossref PubMed Scopus (195) Google Scholar,10.Fotheringham J. Angel C.A. McKane W. Transplant glomerulopathy: morphology, associations and mechanism.Nephron Clin Pract. 2009; 113 (discussion c7): c1-c7Crossref PubMed Scopus (29) Google Scholar TG is characterized by proteinuria, hypertension, and declining graft function and is often associated with the presence of donor-specific antibodies and positive peritubular capillary (PTC) C4d deposition.9.Cosio F.G. Gloor J.M. Sethi S. et al.Transplant glomerulopathy.Am J Transplant. 2008; 8: 492-496Crossref PubMed Scopus (195) Google Scholar Once TG has been diagnosed, the likelihood of graft survival is extremely poor.7.Suri D.L. Tomlanovich S.J. Olson J.L. et al.Transplant glomerulopathy as a cause of late graft loss.Am J Kidney Dis. 2000; 35: 674-680Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,11.Gloor J.M. Sethi S. Stegall M.D. et al.Transplant glomerulopathy: subclinical incidence and association with alloantibody.Am J Transplant. 2007; 7: 2124-2132Crossref PubMed Scopus (293) Google Scholar Recent studies showed a predominance of glomerulitis and peritubular capillaritis (PTCitis) in TG.11.Gloor J.M. Sethi S. Stegall M.D. et al.Transplant glomerulopathy: subclinical incidence and association with alloantibody.Am J Transplant. 2007; 7: 2124-2132Crossref PubMed Scopus (293) Google Scholar,12.Sis B. Campbell P.M. Mueller T. et al.Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause.Am J Transplant. 2007; 7: 1743-1752Crossref PubMed Scopus (227) Google Scholar Glomerulitis and PTCitis, both of which are forms of microcirculating inflammation, are common features of both acute and chronic AMR.13.Fahim T. Bohmig G.A. Exner M. et al.The cellular lesion of humoral rejection: predominant recruitment of monocytes to peritubular and glomerular capillaries.Am J Transplant. 2007; 7: 385-393Crossref PubMed Scopus (84) Google Scholar Although microcirculating inflammation is generally regarded to be a consequence of antibody reactivity, its mechanism remains poorly understood. T-bet, a member of the T-box family of transcription factors, is a key regulator of Th1 lineage commitment and is required for the optimal production of interferon-γ.14.Szabo S.J. Kim S.T. Costa G.L. et al.A novel transcription factor, T-bet, directs Th1 lineage commitment.Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2725) Google Scholar,15.Miller S.A. Weinmann A.S. Molecular mechanisms by which T-bet regulates T-helper cell commitment.Immunol Rev. 2010; 238: 233-246Crossref PubMed Scopus (60) Google Scholar In addition to its expression in CD4+ T cells, T-bet is also expressed in other immune cells, including CD8+ T cells,16.Sullivan B.M. Juedes A. Szabo S.J. et al.Antigen-driven effector CD8 T cell function regulated by T-bet.Proc Natl Acad Sci USA. 2003; 100: 15818-15823Crossref PubMed Scopus (322) Google Scholar B cells,17.Durali D. de Goer de Herve M.G. Giron-Michel J. et al.In human B cells, IL-12 triggers a cascade of molecular events similar to Th1 commitment.Blood. 2003; 102: 4084-4089Crossref PubMed Scopus (80) Google Scholar dendritic cells,18.Lugo-Villarino G. Maldonado-Lopez R. Possemato R. et al.T-bet is required for optimal production of IFN-gamma and antigen-specific T cell activation by dendritic cells.Proc Natl Acad Sci USA. 2003; 100: 7749-7754Crossref PubMed Scopus (218) Google Scholar and natural killer cells.19.Robbins S.H. Tessmer M.S. Van Kaer L. et al.Direct effects of T-bet and MHC class I expression, but not STAT1, on peripheral NK cell maturation.Eur J Immunol. 2005; 35: 757-765Crossref PubMed Scopus (52) Google Scholar T-bet overexpression was initially reported to be correlated with acute cellular rejection at the level of transcription.20.Hoffmann S.C. Hale D.A. Kleiner D.E. et al.Functionally significant renal allograft rejection is defined by transcriptional criteria.Am J Transplant. 2005; 5: 573-581Crossref PubMed Scopus (117) Google Scholar Recently, increased intragraft T-bet expression was observed in both late AMR21.Ashton-Chess J. Dugast E. Colvin R.B. et al.Regulatory, effector, and cytotoxic T cell profiles in long-term kidney transplant patients.J Am Soc Nephrol. 2009; 20: 1113-1122Crossref PubMed Scopus (58) Google Scholar and TG.22.Homs S. Mansour H. Desvaux D. et al.Predominant Th1 and cytotoxic phenotype in biopsies from renal transplant recipients with transplant glomerulopathy.Am J Transplant. 2009; 9: 1230-1236Crossref PubMed Scopus (46) Google Scholar Our previous study23.Sun Q. Cheng D. Zhang M. et al.Predominance of intraglomerular T-bet or GATA3 may determine mechanism of transplant rejection.J Am Soc Nephrol. 2011; 22: 246-252Crossref PubMed Scopus (18) Google Scholar revealed that T-bet expression was typically located in glomerular capillary loops and PTCs in AMR. Moreover, the predominant intraglomerular expression of the transcription factor T-bet was found to correlate strongly with the infiltration of intraglomerular monocytes,23.Sun Q. Cheng D. Zhang M. et al.Predominance of intraglomerular T-bet or GATA3 may determine mechanism of transplant rejection.J Am Soc Nephrol. 2011; 22: 246-252Crossref PubMed Scopus (18) Google Scholar which is a typical lesion of microcirculating inflammation. The aforementioned data suggest that T-bet is probably the key to clarifying the pathogenesis of microcirculating inflammation. As microcirculating inflammation is common in TG, we postulate that T-bet is involved in the pathogenesis of TG. Our data provide novel insight into the development of antibody-mediated graft injury. From January 2005 through December 2010, 462 renal allograft recipients received renal allograft biopsies in Jinling Hospital, Nanjing University School of Medicine. Among these patients, 70 renal allograft recipients participated in this study, including 32 cases of TG, 23 cases of interstitial fibrosis/tubular atrophy (IF/TA), and 15 cases of stable grafts as controls. The enrollment process of the patients is shown in Supplementary Figure S1 online. The baseline patient characteristics are listed in Table 1. None of the recipients had previously received an organ transplant. There were no significant differences between the three groups with respect to patient age, gender, times of prior transplantation, or incidence of positive panel–reactive antibody; however, the stable graft group had a lower incidence of proteinuria and an earlier biopsy time. The time of biopsy was similar between the IF/TA and TG groups. Each patient received anti-IL-2 receptor monoclonal antibody for the induction of immunosuppressive therapy and was subsequently maintained on a similar immunosuppressive protocol after transplantation (Table 1). Download .jpg (.05 MB) Help with files Supplementary Figure 1Table 1Clinical characteristics of patients participating in this studyCharacteristicsTG (n=32)Stable graft (n=15)IF/TA (n=23)Gender, male (%)24 (75.00)11 (73.33)15 (65.22)Age (years)43.56±11.1642.93±14.4642.48±10.09Donor age (years)37.13±10.2239.00±6.8040.17±7.36Positive pre-transplant PRA (n)000Previous transplant000Previous rejection, n (%)5 (15.63)0 (0.00)1 (4.35)Cold ischemic time (h)8.33±1.808.43±1.407.78±1.69Warm ischemic time (min)5.79±1.525.84±1.336.22±1.35Induction with IL-2R antibody, n (%)32 (100)15 (100)23 (100)Baseline immunosuppressants MMF+Tac+Pred1488 MMF+CsA+Pred15413 Others332Time of biopsy, median (range)4.0 (1–11) years9.07 (0–21) days4.26 (1–11) yearsProteinuria, n (%)29 (90.63)*P<0.001 vs. stable graft group.1 (6.67)16 (69.57)*P<0.001 vs. stable graft group.Abbreviations: CsA, cyclosporine A; IF/TA, interstitial fibrosis/tubular atrophy; MMF, mycophenolate mofetil; Pred, prednisolone; PRA, panel-reactive antibody; Tac, tacrolimus; TG, transplant glomerulopathy.Categorical variables were compared by using Fisher's exact test, continuous variables were compared using one-way analysis of variance. *P 0.4 g/24h.Proteinuria was defined as urine protein >0.4 g/24h.* P<0.001 vs. stable graft group. Open table in a new tab Abbreviations: CsA, cyclosporine A; IF/TA, interstitial fibrosis/tubular atrophy; MMF, mycophenolate mofetil; Pred, prednisolone; PRA, panel-reactive antibody; Tac, tacrolimus; TG, transplant glomerulopathy. Categorical variables were compared by using Fisher's exact test, continuous variables were compared using one-way analysis of variance. *P 0.4 g/24h. Proteinuria was defined as urine protein >0.4 g/24h. On the basis of histological findings, the TG group had a high prevalence of microcirculating inflammation; according to the Banff 05 diagnostic criteria,8.Solez K. Colvin R.B. Racusen L.C. et al.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 (923) Google Scholar 93.8% of the patients in the TG group had glomerulitis and 90.6% had PTC inflammation, both of which were significantly higher than the other two groups (Table 2). Microcirculating inflammation was observed in only a few of the recipients with stable graft function, but was more common in the IF/TA group, with glomerulitis occurring in 20 and 69.6% of the stable graft and IF/TA group, respectively (P=0.021; Table 2). The patients in the TG group also had a higher incidence of tubulitis (68.8% vs. 33.3% and 26.1% for the IF/TA and stable graft groups, respectively, P=0.090, P=0.008), intimal arteritis (59.4% vs. 0.0% and 4.3%, respectively, P<0.001), and C4d deposition (75.0% vs. 6.7% and 0.0%, respectively, P<0.001; Table 2). In the TG and IF/TA groups, the incidences and degrees of tubular atrophy (93.8% vs. 100%, respectively, P=1.000) and interstitial fibrosis (90.6% vs. 100%, respectively, P=1.000) were similar, whereas these lesions were not observed in the stable graft group (Tables 2, 3, 4).Table 2Histological characteristics in the different groupsPost hocTG (n=32)Stable graft (n=15)IF/TA (n=23)P12P13P23PTC inflammation, n (%)29 (90.6)1 (6.7)4 (17.4)<0.001<0.0011.000Glomerulitis, n (%)30 (93.8)3 (20.0)16 (69.6)<0.0010.0780.021Tubulitis, n (%)22 (68.8)5 (33.3)6 (26.1)0.0900.0081.000Intimal arteritis, n (%)19 (59.4)0 (0.0)1 (4.3)<0.001<0.0011.000Interstitial fibrosis, n (%)29 (90.6)0 (0)23 (100)<0.0011.000<0.001Tubular atrophy, n (%)30 (93.8)0 (0)23 (100)<0.0011.000<0.001C4d, n (%)24 (75.0)1 (6.7)0 (0)<0.0011.0001.000Abbreviations: IF/TA, interstitial fibrosis/tubular atrophy; PTC, peritubular capillary; TG, transplant glomerulopathy.Pairwise comparisons were done by Fisher's exact test with Bonferroni correction for P value, where P12 means the adjusted P value for TG group and stable graft group, P13 means the adjusted P value for TG group and IF/TA group, P23 means the adjusted P value for stable graft group and IF/TA group. Open table in a new tab Table 3Banff score of histological characteristics in the different groupsPost hocScoreTG (n=32)Stable graft (n=15)IF/TA (n=23)P12P13P23PTC<0.001<0.0011.000 03 (9.4%)14 (93.3%)19 (82.6%) 110 (31.2%)1 (6.7%)4 (17.4%) 218 (56.3%)00 31 (3.1%)00Glomerulitis<0.001<0.0010.007 02 (6.3%)12 (80.0%)7 (30.4%) 112 (37.5%)3 (20.0%)13 (56.6%) 214 (43.7%)03 (13.0%) 34 (12.5%)00Tubulitis0.0370.0031.000 010 (31.2%)10 (66.7%)17 (73.9%) 116 (50.0%)5 (33.3%)6 (26.1%) 26 (18.8%)00 3000Intimal arteritis<0.001<0.0011.000 013 (40.6%)15 (100%)22 (95.7%) 118 (56.3%)01 (4.3%) 21 (3.1%)00 3000Interstitial fibrosis<0.0010.544<0.001 03 (9.4%)15 (100%)0 112 (37.5%)07 (30.4%) 212 (37.5%)011 (47.9%) 35 (15.6%)05 (21.7%)Tubular atrophy<0.0011.000<0.001 02 (6.3%)15 (100%)0 113 (40.6%)09 (39.1%) 213 (40.6%)011 (47.9%) 34 (12.5%)03 (13.0%)C4d<0.001<0.0010.647 08 (25.0%)14 (93.3%)23 (100%) 11 (3.1%)1 (6.6%)0 210 (31.3%)00 313 (40.6%)00Abbreviations: IF/TA, interstitial fibrosis/tubular atrophy; PTC, peritubular capillary; TG, transplant glomerulopathy.Pairwise comparisons were done by Wilcoxon rank-sum test with Bonferroni correction for P value, where P12 means the adjusted P value for TG group and stable graft group, P13 means the adjusted P value for TG group and IF/TA group, P23 means the adjusted P value for stable graft group and IF/TA group. Open table in a new tab Table 4Immunohistological analysis of microcirculating inflammationPost-hocTG (n=32)Stable graft (n=15)IF/TA (n=23)P12P13P23PTC T-bet0.20 (0.16–0.30)0.00 (0.00–0.04)0.00 (0.00–0.10)<0.001<0.0011.000 GATA30.00 (0.00–0.02)0.00 (0.00–0.00)0.00 (0.00–0.00)0.0220.8191.000 CD40.14 (0.10–0.18)0.00 (0.00–0.04)0.00 (0.00–0.12)<0.001<0.0011.000 CD80.21 (0.16–0.30)0.00 (0.00–0.04)0.00 (0.00–0.014)<0.001<0.0011.000 CD680.36 (0.23–0.51)0.00 (0.00–0.04)0.00 (0.00–0.25)<0.001<0.0011.000GC T-bet1.50 (0.85–2.00)0.00 (0.00–0.00)0.00 (0.00–0.40)<0.001<0.0011.000 GATA30.00 (0.00–0.20)0.00 (0.00–0.00)0.00 (0.00–0.00)0.0750.3921.000 CD41.00 (0.50–1.20)0.00 (0.00–0.00)0.20 (0.00–0.50)<0.001<0.0011.000 CD81.00 (0.73–1.50)0.00 (0.00–0.00)0.20 (0.00–0.60)<0.001<0.0011.000 CD687.18 (3.78–10.50)0.00 (0.00–0.60)0.80 (0.00–1.20)<0.001<0.0011.000Abbreviations: GC, glomerular capillary; IF/TA, tubular atrophy/interstitial fibrosis; PTC, peritubular capillary; TG, transplant glomerulopathy.All continuous variables were not normally distributed and thus were presented as median (25th–75th percentiles), and pairwise comparisons were done by Wilcoxon rank-sum test with Bonferroni correction for P value, where P12 means the adjusted P value for TG group and stable graft group, P13 means the adjusted P value for TG group and IF/TA group, P23 means the adjusted P value for stable graft group and IF/TA group. Open table in a new tab Abbreviations: IF/TA, interstitial fibrosis/tubular atrophy; PTC, peritubular capillary; TG, transplant glomerulopathy. Pairwise comparisons were done by Fisher's exact test with Bonferroni correction for P value, where P12 means the adjusted P value for TG group and stable graft group, P13 means the adjusted P value for TG group and IF/TA group, P23 means the adjusted P value for stable graft group and IF/TA group. Abbreviations: IF/TA, interstitial fibrosis/tubular atrophy; PTC, peritubular capillary; TG, transplant glomerulopathy. Pairwise comparisons were done by Wilcoxon rank-sum test with Bonferroni correction for P value, where P12 means the adjusted P value for TG group and stable graft group, P13 means the adjusted P value for TG group and IF/TA group, P23 means the adjusted P value for stable graft group and IF/TA group. Abbreviations: GC, glomerular capillary; IF/TA, tubular atrophy/interstitial fibrosis; PTC, peritubular capillary; TG, transplant glomerulopathy. All continuous variables were not normally distributed and thus were presented as median (25th–75th percentiles), and pairwise comparisons were done by Wilcoxon rank-sum test with Bonferroni correction for P value, where P12 means the adjusted P value for TG group and stable graft group, P13 means the adjusted P value for TG group and IF/TA group, P23 means the adjusted P value for stable graft group and IF/TA group. In the TG group, we detected a significant increase in T-bet expression in both the PTCs and the capillary loops of the glomeruli, and this result was typically located within the capillary lumen (Figure 1). According to the results of double staining of T-bet and CD4+/CD8+, T-bet expression was detected in a portion of CD4+ and CD8+ (Supplementary Figure S2A and B online) cells and was strongly correlated with intraglomerular and PTC CD4+ cell counts (r=0.943, P<0.001; r=0.906, P<0.001, respectively) and CD8+ cell counts (r=0.936, P<0.001; r=0.895, P<0.001, respectively, Figure 2). Double staining of T-bet and CD68 did not show T-bet expression in CD68+ cells (Supplementary Figure S2C online); however, in situ T-bet expression was strongly correlated with the number of CD68+ cells in both the glomeruli (r=0.579, P=0.001) and the PTCs (r=0.752, P<0.001; Figure 2). We also performed GATA3 staining in this group and detected GATA3 expression in the tubular cells and in some of the T cells; however, both intra-PTC and intraglomerular GATA3 expressions were rare (Supplementary Figure S3 online). We found no correlation between GATA3 expression and microcirculating inflammation. Download .jpg (.03 MB) Help with files Supplementary Figure 2Figure 2The relationship between microcirculating inflammation and in situ T-bet expression. In the transplant glomerulopathy group, T-bet expression was strongly correlated with the number of intraglomerular (a, c and e) and peritubular capillary (PTC; b, d and f), CD4+ (a, b), CD8+ (c, d), and CD68+ (e, f) cells.View Large Image Figure ViewerDownload (PPT) Download .jpg (.05 MB) Help with files Supplementary Figure 3 Intracapillary T-bet expression was also detected in the IF/TA group; however, its expression was much lower than that in the TG group (P<0.001, respectively). Moreover, T-bet expression was observed primarily in the grafts that had microcirculating inflammation. Similarly, T-bet was expressed in a portion of CD4+ and CD8+ cells but not in CD68+ cells, and the counts of CD4+, CD8+, and CD68+ cells were all strongly correlated with the in situ expression of T-bet both in glomeruli and in the PTC area. There were low and nonexistent levels of T-bet and GATA3 expression, respectively, in both the PTCs and glomeruli in the stable graft group. Labeling the endothelial cells for CD31 made it possible to calculate the number and diameters of the capillaries. We compared the PTC density and capillary diameter among the three groups. In the IF/TA group, the density of PTCs was significantly lower compared with the stable graft group (18.14±2.90 vs. 24.40±1.72, respectively P<0.001), whereas the diameter of the existing PTCs was larger (2.43±0.62 vs. 1.07±0.29, respectively, P<0.001), suggesting a rarefaction and dilation of the capillaries (Figure 3). However, in the TG group, we found no such rarefaction of PTCs as observed in the IF/TA group. The capillary density was similar between the TG group and protocol biopsies (23.87±3.92 vs. 24.40±1.72, respectively, P=1.000). However, the capillary diameter in the TG group (6.35±2.15) was significantly higher than both the protocol biopsy group (1.07±0.29, P<0.001) and the IF/TA group (2.43±0.62, P<0.001). In addition, Image-Pro Plus revealed a significantly larger intra-PTC area in the TG group (0.09±0.03) compared with the IF/TA (0.03±0.01, P<0.001) and stable graft (0.02±0.01, P<0.001) groups. In addition to PTCs, capillary dilation was also observed within the glomeruli. The diameter of the intraglomerular capillaries in the TG group was significantly larger than in the IF/TA and stable graft groups (Figure 3). Moreover, the diameter of the glomeruli and intraglomerular area were both significantly larger in the TG group than in the other two groups (Figure 3). Co-staining of CD31 made it possible to explore easily the nature of intracapillary inflammation. Figures 3a–d shows that CD31 staining labeled the capillaries notably well. In the TG group, the infiltrating cells in both the glomeruli and PTCs were primarily constituted of CD68+, CD4+, and CD8+ cells, whereas no CD20+ cells were observed. The dilation of PTCs was strongly correlated with the quantity of infiltrating CD68+ cells (r=0.523, P=0.002) as shown in Figure 3g. In contrast, capillary dilation was rarely observed in areas that lacked PTCitis. We also calculated the ratio of the total intra-PTC area within the cortex to the area of the entire cortex and found that this ratio was also strongly correlated with intra-PTC CD68+ cell counting (r=0.561, P=0.001). However, in the IF/TA group, although there was also a slight enlargement of PTCs, we found no correlation with the degree of microcirculating inflammation. Moreover, co-staining of CD31 and CD68 revealed that the majority of the intraglomerular CD68+ cells were located within the capillary loops. Similarly, the diameter of the capillary loops was strongly correlated with the number of intraglomerular CD68+ cells (r=0.410, P=0.022). In contrast, in the IF/TA group, although glomerulitis with CD68+ cells was common (being present in 69.6% of the cohort), the majority of these cells were located outside of the capillaries (Figure 3c). With respect to the outcome of the allografts, the TG group had a poorer graft outcome than the IF/TA group, with 1-year graft survival rates of 87.5 and 95.7%, respectively, and median survival times of 29 and 36 months, respectively. However, there was no statistical significance between the two groups, and we found no correlation between T-bet expression and graft outcome in the present study. This finding may have been due to insufficient power to detect a significant difference, as the sample size was relatively small. Microcirculating injury is a key lesion in the pathogenesis of antibody-mediated allograft damage. To the best of our knowledge, this study is the first to demonstrate that microcirculating inflammation (including both glomerulitis and PTC inflammation) is strongly correlated with in situ T-bet expression. Our data also revealed that a unique capillary dilation differs between the TG vs. the IF/TA and stable graft groups, and that the degree of dilation is strongly correlated with the degree of microcirculating inflammation. Because microcirculating inflammation typically follows alloantibody reactivity, these results shed new light to the pathogenesis of TG. Microcirculating inflammation is generally regarded to be a lesion that is related to allo-antibody reactivity.3.Einecke G. Sis B. Reeve J. et al.Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure.Am J Transplant. 2009; 9: 2520-2531Crossref PubMed Scopus (536) Google Scholar Recently, a high prevalence of microcirculating inflammation in patients with TG was reported. However, our study revealed an even higher incidence of microcirculating inflammation, as nearly all of the patients in our TG group had microcirculating inflammation. This is partially because all of the biopsies in the TG group were index biopsies, which might reflect active TG. These findings support the hypothesis that TG is mediated by anti-donor antibodies.8.Solez K. Colvin R.B. Racusen L.C. et al.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 (923) Google Scholar Clearly, microcirculating inflammation is a key lesion in antibody-mediated graft injury.3.Einecke G. Sis B. Reeve J. et al.Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure.Am J Transplant. 2009; 9: 2520-2531Crossref PubMed Scopus (536) Google Scholar Thus, it is of great importance to clarify the pathogenesis of microcirculating inflammation. Although glomerulitis was also noted in more than half of the IF/TA patients, the location of their cellular infiltration was notably different from that in the TG patients; specifically, in the TG group, most of the infiltrating cells were located within the capillary lumen, whereas in the IF/TA patients the infiltrating cells were primarily scattered outside of the capillaries. Our data show that the infiltration of intraglomerular and PTC macrophages is strongly correlated with in situ overexpression of the transcription factor T-bet. This correlation is unique to
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