Immunophenotypic analysis of leukocyte infiltration in the renal transplant

Immunohistological analyses of transplant biopsy material can provide valuable insight into the intragraft events following transplantation. The magnitude, composition and phenotype of the leukocyte infiltration and up-regulation of parenchymal antigen expression within the renal allograft have been major areas of interest. Immunohistological analyses have demonstrated the presence of leukocyte infiltration not only during clinical rejection episodes, but also during periods of stable graft function. Nevertheless the infiltration is significantly increased during clinical rejection. Indeed, quantitation of the leukocyte infiltration using a morphometric point counting technique or image analysis can be used prospectively to diagnose rejection in the renal transplant recipient. Macrophages and T lymphocytes (both of the CD4 and CD8 phenotype) are the major infiltrating cells, B lymphocytes and NK cells being present, but in the minority. Activated and proliferating cells have been demonstrated during allograft rejection and may provide a means of identifying a functionally active infiltrate.     

Increased expression of peripheral blood leukocyte genes implicate CD14+ tissue macrophages in cellular intestine allograft rejection

Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.     

Chemokines: directing leukocyte infiltration into allografts

Chemokines have been shown to play a critical role in the recruitment of leukocytes to transplanted organs. Animal models and clinical studies have demonstrated predictable temporal and spatial correlations between chemokine production and leukocyte infiltration into allografts. Antagonism of chemokines or chemokine receptors has been shown to delay leukocyte infiltration and prolong graft function, demonstrating an important role for chemokines in allograft rejection.     

Presence of intragraft B cells during acute renal allograft rejection is accompanied by changes in peripheral blood B cell subsets

B cells have various functions, besides being plasma cell precursors. We determined the presence of intragraft B cells at time of acute rejection (AR) and looked for correlates of B cell involvement in peripheral blood. Renal biopsies at time of AR or stable graft function were analysed for the presence of B cells and B cell-related gene expression, as well as C4d staining. Peripheral blood B cell subset distribution was analysed at various time-points in patients with AR and controls, alongside serum human leucocyte antigen (HLA) antibodies. AR was accompanied by intragraft CD20⁺ B cells, as well as elevated CD20 (MS4A1) and CD19 gene expression compared to controls. B cell infiltrates were proportional to T cells, and accompanied by the chemokine pair C-X-C motif chemokine ligand 13 (CXCL13)–C-X-C motif chemokine receptor 5 (CXCR5) and B cell activating factor (BAFF). Peripheral blood memory B cells were decreased and naive B cells increased at AR, in contrast to controls. While 22% of patients with AR and 5% of controls showed de-novo donor-specific antibodies (DSA), all biopsies were C4d-negative. These results suggest a role for B cells in AR by infiltrating the graft alongside T cells. We hypothesize that the shift in peripheral blood B cell composition is related to the graft infiltration at time of AR.     

Chemokine receptor-dependent alloresponses

Summary:  Immunologists have typically viewed alloreactivity schematically as a function of antigen presentation, expansion of alloreactive T and B cells within regional lymphoid tissues, and cellular infiltration and destruction of an allograft. Actual details of the steps between immune activation and accumulation of effector cells within a graft typically have not received much attention. However, just how cells 'know' to move to and migrate within a graft or not is proving to be of increasing interest, as the chemokine-dependent mechanisms underlying leukocyte recruitment to a transplant are dissected. Experimentally, chemokine receptor targeting can prolong or induce permanent allograft survival, despite preservation of alloresponses within secondary lymphoid tissues, whereas current immunosuppressive protocols have only modest effects on chemokine production and leukocyte homing. Recent knowledge of the chemokine-dependent nature of allograft rejection, acceptance, and tolerance induction are presented as a basis for understanding the rationale for preclinical trials of chemokine receptor-targeted therapies currently underway in primate recipients of solid organ allografts.     

Renal allograft rejection: the contribution of chemokines to the adhesion and retention of alphaE(CD103)beta7 integrin-expressing intratubular T cells

Recruitment of activated T cells to the tubules is a defining feature of cell-mediated renal allograft rejection. Many of these intratubular T cells express the alphaE(CD103)beta7 integrin, potentially allowing adhesion to epithelial cells which express the only defined counter-receptor, E-cadherin. However, the potential of rejection-associated intratubular chemokines to modulate the adhesive function of this integrin has not been investigated. This study demonstrated that CCL7 is expressed within the tubules during renal allograft rejection. Modelling with CD103-expressing MOLT-16 T cells demonstrated chemotactic responses to the chemokines CXCL10, CXCL12, CCL5 and, most significantly, CCL7 (p<0.001); these responses were consistent with the expression of CXCR3, CXCR4 and CCR1 by these cells. A solid-phase adhesion assay showed little background binding of MOLT-16 cells to immobilised human E-cadherin.Fc fusion protein but alphaEbeta7 integrin-specific adhesion was greatly increased by the addition of either Mn(2+) or 10nM CCL7 (p<0.01 or <0.001, respectively). Treatment of activated human peripheral T cells with TGFbeta(1) for 3 days induced the expression of CD103 on a mean 53% of these cells; a similar proportion of CD103+ and CD103- T cells within these cultures expressed receptors for the chemokine CCL7. CD103+ T cell fractions were sorted from mitogen- or alloantigen-activated, TGFbeta(1)-treated T cell cultures and also showed specific enhancement of adhesion to E-cadherin.Fc fusion protein following stimulation with Mn(2+) or 10nM CCL7 (p<0.01 in all cases); CD103- T cells were not adherent under any conditions. Together these data suggest that although the alphaEbeta7 integrin is induced on activated intratubular T cells by the presence of TGFbeta, the adhesive function of this integrin is promoted by the presence of chemokines such as CCL7, which are also expressed within tubules during renal allograft rejection.     

Expression of CXCL16 in human rectal cancer

Local immunoregulation mediated by infiltration of inflammatory cells into colorectal adenocarcinomas is important for tumour progression. Tumour-associated macrophages and T cells are predominant components of chemokine-guided infiltrate of most colorectal tumours. CXCL16 is a newly discovered CXC chemokine expressed by antigen presenting cells attracting Th1, Tc and NK T cells. In this study, which is the first report on expression of the chemokine CXCL16 in human rectal cancer, CXCL16 gene and protein expression were analysed in cancer and normal adjacent tissue. Immunohistochemistry revealed CXCL16 expression in macrophages in normal tissue. The CXCL16 was found to a very limited extent in tumour-associated macrophages. Western blot analysis showed a suppression of CXCL16 protein in rectal cancer compared to non-cancer tissue in 83% of the patients (n=23, P=0.003). However, with real-time PCR mRNA was not down-regulated in the cancer compared to normal tissue, which may depend on regulated factor(s) at the level of translation and/or post-translation. The results may reflect one of the immunological mechanisms underlying carcinogenesis.     

On the edge: the physiological and pathophysiological role of chemokines during inflammatory and immunological responses

Most, if not all, chemokines bind to seven transmembrane spanning G protein-coupled receptors and activate cellular migration. Stimulated chemokine expression is essential for directing leukocyte emigration from the circulation into sites of inflammation and tissue damage. In contrast, constitutive chemokine expression plays a role in the development of lymphoid cells, organs, and tissues. The present review examines rheumatoid arthritis and transplantation rejection as two examples of pathological conditions where chemokine directed leukocyte infiltration aids in the pathogenesis of the disease. We further discuss insights into leukocyte trafficking gained by chemokine and chemokine receptor transgenic and null mutant mice.     

Reduced intragraft mRNA expression of matrix metalloproteinases Mmp3, Mmp12, Mmp13 and Adam8, and diminished transplant arteriosclerosis in Ccr5-deficient mice

Experimental and human organ transplant studies suggest an important role for chemokine (C-C-motif) receptor-5 (CCR5) in the development of acute and chronic allograft rejection. Because early transplant damage can predispose allografts to chronic dysfunction, we sought to identify potential pathophysiologic mechanisms leading to allograft damage by using wild-type and Ccr5-deficient mice as recipients of fully MHC-mismatched heart and carotid-artery allografts. Gene expression in rejecting heart allografts was analyzed 2 and 6 days after transplantation using Affymetrix GeneChips. Microarray analysis led to identification of four metalloproteinase genes [matrix metalloproteinase (Mmp)3, Mmp12, Mmp13 and a disintegrin and metalloprotease domain (Adam)8] with significantly diminished intragraft mRNA expression in Ccr5-deficient mice at day 6. Accordingly, allografts from Ccr5-deficient mice showed less tissue remodeling and hence better preservation of the myocardial architecture compared with allografts from wild-type recipients. Moreover, survival of cardiac allografts was significantly increased in Ccr5-deficient mice. Carotid artery allografts from Ccr5-deficient recipients showed better tissue preservation, and significant reduction of neointima formation and CD3+ T cell infiltration. Ccr5 appears to play an important role in transplant-associated arteriosclerosis that may involve metalloproteinase-mediated vessel wall remodeling. We conclude that early tissue remodeling may be a critical feature in the predisposition of allografts to the development of chronic dysfunction.     

CXCL10 and CXCL13 Expression were Highly Up-regulated in Peripheral Blood Mononuclear Cells in Acute Rejection and Poor Response to Anti-Rejection Therapy

Background  

Acute rejection is still one of the main complications which enhances the cost and the risk to renal graft failure. Chemokines, interacting with respective receptors, can recruit leukocytes into grafts and mediate allograft rejection. In this study, we aimed to analyze gene expression of chemokines including CCL5/RANTES, CXCL10/IP-10, CXCL13/BCA-1, and receptors of CCR5, CXCR3, CXCR5 in peripheral blood mononuclear cells (PBMCs) during acute renal allograft rejection     

DNA Microarray-Based Gene Expression Profiling in Porcine Keratocytes and Corneal Endothelial Cells and Comparative Analysis Associated with Xeno-related Rejection

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.     

Anti-CCL25 Antibody Prolongs Skin Allograft Survival by Blocking CCR9 Expression and Impairing Splenic T-Cell Function

Chemokines, by virtue of their ability to recruit immune cells into allografts, play critical roles in acute transplantation rejection. CCR9 and its ligand, CCL25, is one of the key regulators of thymocyte migration and maturation in normal and inflammatory conditions. Moreover, several studies have revealed that high expression of CCR9 and CCL25 participated in many kinds of diseases. However, the role of CCR9 in allograft rejection is still unclear. In this study, we established a murine skin transplantation model of acute rejection. Our findings showed that the proportion of CCR9-expressing T cells was significantly increased in the spleen of allotransplanted mice compared with syngeneic transplantation. Furthermore, expression of CCL25 in allograft was similarly increased. Neutralization of CCL25 by intravenous injection of anti-CCL25 monoclonal antibody significantly prolonged skin allograft survival, decreased the number of infiltrating cells, and simultaneously suppressed the chemotactic ability and the proliferation of the splenic T cells in response to allogeneic antigens. Finally, blockade of CCL25 also diminished the secretion of IFN-γ by splenic T cells. These studies indicated that CCR9/CCL25 was involved in acute transplantation rejection and anti-CCL25 strategies might be useful in preventing acute rejection.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1^lv1) rats received orthotopic corneal isografts or Wistar-Furth (RT1^u) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8⁺ than CD4⁺ cells was found at days 1–3 following rejection, whereas there was a higher proportion of CD4⁺ cells at days 5–8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1–2, 4 and 7–10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4⁺ > CD8⁺ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1–2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes—from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4⁺ cells was found in both aqueous and graft at later times.     

Anti-interleukin-12/23p40 antibody attenuates chronic rejection of cardiac allografts partly via inhibition γδT cells

In our previous study, we showed that treatment with an anti‐interleukin (IL)‐12/23p40 antibody inhibits acute cardiac allograft rejection via inhibiting production of interferon (IFN)‐γ and IL‐17a. However, the impact of this antagonistic anti‐p40 antibody on chronic cardiac rejection was unclear. Hearts of B6.C‐H2bm12/KhEg mice were transplanted into major histocompatibility complex (MHC) class II‐mismatched C57Bl/6J mice (wild‐type, γδTCR ^–/– and IL‐17^–/–), which is an established murine model of chronic allograft rejection without immunosuppression. The mice were treated with control immunoglobulin (Ig)G or 200 µg anti‐p40 monoclonal antibody on post‐operative days, respectively. Abdominal palpation and echocardiography were used to monitor graft survival. The mice administered with anti‐p40 antibody showed a significant promotion in graft survival (median survival time >100 days), and histological analyses revealed that cardiac allograft rejection was attenuated. Quantitative real‐time polymerase chain reaction (qRT–PCR) and immunofluorescence analyses demonstrated that anti‐p40 antibody down‐regulated the level of ingraft cytokine and chemokine expression (IL‐6, IFN‐γ, IL‐17a, CCL2 and CCL20). Flow cytometry analyses showed that γδ T cells are an important ingraft source of IFN‐γ and IL‐17a and inhibit the production of inflammation cytokine by anti‐p40 antibody. Compared with the wild‐type group, the graft survival time in the γδ T cell receptor^–/– and IL‐17^–/– mice was prolonged significantly. Therefore we propose that, in the chronic allograft rejection model, treatment with anti‐p40 antibody prolongs graft survival possibly by reducing the amount of reactive inflammatory cells, especially γδ T cells.