Renal tubulitis is a pathological phenomenon defined by kidney-infiltrating mononuclear cells that have traversed the tubular basement membrane to lie beneath and between the tubular epithelial cells (TEC). Along with vasculitis, the extent of tubulitis is used in the quantitative “Banff” scoring protocol to define the severity of the lesions associated with renal allograft rejection ( 1 , 2 ). In addition to this role in rejection diagnosis, it is clear that injury to the basement membrane and to the tubular epithelium itself can contribute to the organ dysfunction observed during rejection ( 3 ). The Banff score is now used widely to diagnose rejection and to monitor the efficacy of antirejection therapy.
Our group has shown previously that TEC express CC chemokines after renal transplantation ( 4 ); these may attract recipient CD3+ lymphocytes, which are capable of intratubular proliferation during episodes of acute rejection ( 5 ). There is a positive correlation between such T cell proliferation and the proliferation of adjacent TEC, which is indicative of ongoing epithelial damage and repair ( 5 ). This is supported by demonstration that many intratubular T cells contain cytotoxic effector molecules, including perforin ( 6 , 7 ).
Study of mucosal epithelia, including those of the gut and lung, has identified the presence of a characteristic population of intraepithelial lymphocytes (IEL). It was shown recently that the majority (>95%) of these cells express a specific adhesion molecule, the αEβ7-integrin and that these cells are typically CD4− CD8+ T cells; αEβ7 is present on fewer than 2% of peripheral lymphocytes. This integrin is identified by antibodies specific for the CD103 antigen, which is present on the αE subunit of the heterodimeric protein. There have been sporadic reports of weak expression of CD103 on some activated mononuclear phagocytes and mast cells. However, in these cases the antigen is often identified by application of the HML-1 antibody, which has recently been shown to be cross-reactive with a nonintegrin activation marker on monocytes ( 8 ). Significantly, the only known molecule to which the αEβ7-integrin binds with high affinity is E-cadherin, a molecule constitutively expressed by most epithelial cells (9, 10, 11).
E-cadherin is well known for its ability to form homophilic adhesive bonds between adjacent epithelial cells. These bonds are essential to enable tight-junction formation to occur so that epithelial tissues can perform transport functions. However, the consequences for epithelial function and viability of additional heterophilic bond formation with IEL expressing the αEβ7-integrin are poorly understood ( 10 ). It is clear that adhesive bonding to stabilize interaction between TEC and activated, cytotoxic intratubular T cells has the potential to be particularly damaging during acute renal allograft rejection.
Previous study has shown that CD103+ T cells can be isolated from disaggregated renal tissue during “acute on chronic” rejection ( 12 ). Although these cells express a reduced level of the LFA-1 integrin, generally associated with adhesion before immune-mediated target cell lysis, it has been demonstrated that they do have the capacity to lyse cultured TEC ( 13 ). This has led to speculation that expression of the αEβ7-integrin by allospecific T cells within a renal allograft confers the capacity to kill E-cadherin expressing target cells, such as TEC. In this system, the low level of LFA-1 effectively prevents the lysis of non-E-cadherin expressing cells within the graft ( 13 ). This model is clearly consistent with the importance of tubulitis as a defining feature of renal allograft rejection. However, there have been no reports describing the physical location of CD103+ cells within renal tissue during acute allograft rejection.
In this study, the distribution and number of CD103+ T cells has been determined in renal transplant biopsy sections, graded for rejection in accordance with the Banff 97 criteria. In addition to immunolocalization of CD103, sections have also been stained to detect other relevant antigens including CD8, E-cadherin, and TGFβ1.
MATERIALS AND METHODS
The study was carried out using cryostat sections from biopsies taken routinely from renal allografts during episodes of clinical dysfunction. In all cases, the patients were immunosuppressed with a Cyclosporin-A based regimen; episodes of acute rejection were treated with high-dose methylprednisolone. Cryostat sections from 34 transplant biopsies were available. These were cut from small pieces of tissue removed from diagnostic cores at time of sampling and snap frozen, in accordance with agreed ethical guidelines. The larger core, on which diagnosis was made, was sent for routine processing. In addition, normal control biopsies were available from 7 kidneys before grafting (day 0) and tissue from a small number of transplant nephrectomies which demonstrated chronic rejection, some with significant inflammatory infiltrate. Formalin-fixed, paraffin-embedded transplant biopsy and nephrectomy blocks were available for use in the detection of TGFβ1.
Antibodies and other immunological reagents for tissue staining.
Mouse monoclonal antibodies to CD103 (BerAct 8) and CD8 (CD8/144B), mouse IgG1 isotype control, biotinylated rabbit anti-mouse immunoglobulins and a streptavidin biotin peroxidase kit were all obtained from Dako Ltd (Ely, UK). Chicken polyclonal anti-TGFβ1 (IgG fraction) and normal chicken IgG were obtained from R&D Systems Europe Ltd (Abingdon, UK), mouse monoclonal anti-E-cadherin from Immunotech (Coulter Electronics, High Wycombe, UK), FITC-conjugated rabbit anti-chicken IgG from Sigma-Aldrich Co (Poole, UK), biotinylated goat anti-chicken and streptavidin biotin alkaline phosphatase kit from Vector Laboratories (Peterborough, UK). Normal rabbit serum (NRS) and normal lamb serum (NLS) were obtained from Gibco-BRL (Paisley, UK).
Acetone-fixed cryostat sections were treated to minimize endogenous peroxidase activity and to block endogenous biotin (Biotin Blocking System; Dako). Adjacent sections were incubated simultaneously with primary antibodies to detect CD8 and CD103 (both at 1/50 dilution).
Subsequently, sections were incubated with biotinylated rabbit anti-mouse immunoglobulins (at 1/250 dilution) followed by streptavidin biotin peroxidase complex (in TBS). The immune complex was detected with nickel-enhanced diaminobenzidine (pH 6.0).
Nephrectomy sections, demonstrating chronic rejection and labeled for CD103, were stained subsequently to detect E-cadherin. After incubation with anti-human E-cadherin or mouse isotype control (IgG1), both at 20 μg/ml, the secondary antibody was biotinylated rabbit anti-mouse immunoglobulins and the detection system was streptavidin biotin alkaline phosphatase complex. The substrate/chromagen was Vector Red, with levamisole to inhibit endogenous alkaline phosphatase activity.
In all cases nonspecific binding of antibodies was minimized by prior incubation of sections with “blocking serum” which contained 20% NRS and 5% normal human serum in Tris-buffered saline (TBS; pH 7.6). Antibodies were diluted in blocking serum and negative control sections were included for each biopsy, incubated with blocking serum only, in place of primary antibody. All primary antibody incubations were overnight at 4°C. Washing between stages was carried out using TBS. Sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted in DPX.
Immunostaining of paraffin sections for quantitative assessment of TGFβ1.
Serial sections were dewaxed, blocked, and incubated with polyclonal chicken anti-human TGFβ1 or nonimmune chicken IgG (control), at the same concentration. The primary antibody was detected with rabbit anti-chicken IgG, conjugated to FITC with which sections were incubated for 2 hr at 37°C before being mounted in immunofluorescence mounting medium (Dako).
Enumeration of stained T cells and statistical methods.
CD8 and CD103-positive IEL were counted across the total area of the biopsy section at 400× magnification. The total number of positive IEL per section was divided by number of tubular cross-sections (or 10 TEC) per stained tissue section to give the mean CD8 or CD103+ cells per tubular cross-section (TCS) in the area counted. The ratio of mean CD103+ to CD8+ cells per TCS was also calculated for each biopsy section. Biopsies were graded in accordance with Banff 97 criteria for acute rejection ( 2 ) and then grouped as 0, 1, and 2, where 0 was no rejection and “suspicious for rejection,” 1 was predominantly acute cellular rejection with interstitial and tubular scores of i2 and t1, and 2 was predominantly acute cellular rejection with scores of at least i2 and t2/t3. A Mann-Whitney U test (Minitab software, v11) was applied to compare median CD8+ and CD103+ IEL/TCS values and median CD103+ to CD8+ IEL/TCS ratios between the different groups.
Scanning laser confocal microscopy.
The indirect immunofluorescence procedure and analysis by scanning laser confocal microscopy was performed as described by Robertson et al. ( 14 ), with minor modifications. Fluorescence images were collected using an MRC-600 confocal imaging system with krypton/argon laser (Bio-Rad, Hemel Hempstead, UK) implemented on a Nikon Optiphot microscope. The single channel mode was based on the laser line at 488 nm with the confocal aperture, gain and black-level settings kept constant. Optical sectioning was performed by scanning across the XY axis at 3-μm increments in the Z-axis and a “Z-series” image was constructed from digitally stored serial images. Quantitative information was obtained using COMOS software applying histogram analysis. “Color banding” of the selected area permitted specific inclusion of tubular epithelium in the analysis and excluded unstained features such as tubular lumens. The expression of TGFβ1 was calculated as the ratio of mean fluorescence intensity over the selected area of experimentally stained tissue to the corresponding value in control sections and termed the TGFβ1 index: (mean fluorescence intensity × % selected tubular area TEST stained)/(mean fluorescence intensity × % selected tubular area CONTROL stained).
Repeated staining and examination of a series of sections taken from each of three biopsy specimens determined the reproducibility of this method. In each case, it was found that the CV was less than 12%.
Quantification of CD103+ T cells after allospecific activation in vitro.
Peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation (Lymphoprep; Nycomed-Pharma, Little Chalfont, UK) of heparinised peripheral blood from normal volunteers. The cells were adjusted to 1×107 cells/ml in RPMI1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Paisley, UK). An allogeneic EBV-transformed B cell line ( 15 ) was then irradiated (50Gy; 137Cs source) and added to the PBMC at a 1:2 ratio and the mixture cultured at 37°C in humid air containing 5% CO2. After 0, 3, or 5 days, separate aliquots of the cell mixture were supplemented with an optimal concentration of TGFβ1 (10ng/ml; R&D Systems, Abingdon, UK) and culture was continued.
Cells were recovered periodically from each culture for quantification of CD8 and CD103 expression. These antigens were detected using anti-CD8-PE (UCHT-1; Sigma, Poole, UK) and anti-CD103-FITC (BerACT-8, Becton Dickinson, Cowley, UK) antibody for 30 min at 4°C. After staining, the cells were washed three times before analysis by dual-color immunofluorescence flow cytometry (FACScan; Becton Dickinson). Data analysis was performed using WinMDI software, v2.8 (http://facs.scripps.edu).
Eight of the 34 biopsies were grouped in category 0; of these, 5 showed no rejection and 3 were considered “suspicious for rejection.” The former subgroup contained the following diagnoses: pretransplant ischemic damage with possible pyelonephritis ( 1 ), old ischemic damage ( 1 ), acute ischemic damage ( 1 ), possible cyclosporin A toxicity accompanied by acute tubular necrosis ( 1 ), and previous rejection with no reason for continued clinical dysfunction ( 1 ). A small number of CD103+ cells was observed in only two of the eight biopsies in this group; one of these cases showed cyclosporin A toxicity and the other evidence of previous rejection. No evidence of infection or recurrence of original disease was found in any of the biopsies that were diagnosed with acute cellular rejection. The most commonly observed minor diagnostic features were glomerular and vascular sclerosis (sometimes predating transplantation), mild interstitial fibrosis and cyclosporin A toxicity.
Vascular inflammation was observed in eight of the biopsies but this was never greater than mild arteritis (v1) except in one case in which, interestingly, no CD103+ IEL were identified in the tubules. Insufficient frozen tissue was available to extend the study to include involvement of CD103+ cells in intimal arteritis, but this was not an obvious phenomenon observed during analysis.
Detection of CD8+ and CD103+ cells.
The biopsy sections from normal and transplanted kidneys all contained CD8+ cells within the tubular interstitium (Table 1;Fig. 1a). These cells were not enumerated precisely but the interstitial CD8 infiltrate was assessed using a scale ranging from + (the slight infiltrate seen in normal sections) to ++++ (the dense infiltrate observed during acute rejection). Levels of interstitial CD8 positivity reflect the overall interstitial infiltrate observed in diagnostic sections except in one case similar to normal kidney levels and one borderline case with a CD8 score greater than normal. In addition, the sections contained a variable number of CD8+ intratubular lymphocytes (Fig. 1a); these were enumerated within each tubular cross-section (Table 1).
Twenty-two of 34 posttransplant biopsies contained CD103+ lymphocytes. During acute rejection, these cells were located almost exclusively in close juxtaposition to TEC (Fig. 1,b and c); the tubules containing CD103+ IEL were often distributed focally. On rare occasions, CD103+ cells were found within the interstitium but this was always restricted to areas containing a dense inflammatory infiltrate. No intratubular CD8+ or CD103+ cells were observed in sections from normal control biopsies.
Mean CD8+ and CD103+ lymphocytes per tubular cross-section, ranked by rejection grade.
Comprehensive morphometric data relating to intratubular CD8+ and CD103+ cells per TCS within each of the 34 posttransplant biopsies are presented in Table 1; a graphical representation of the grouped data is shown in Figure 2. Statistical analysis showed that median CD8+ and CD103+ cells per TCS was significantly greater in biopsy sections in group 2 (n=12) than in group 1 (n=14:P =0.005 for CD8;P =0.009 for CD103). The difference between median CD8+ and CD103+ cells per TCS in group 0 (n=8) and group 1 rejection sections was also significant (P =0.023 for CD8;P =0.037 for CD103). In addition, the median ratio of CD103+ to CD8+ cells per TCS was significantly greater in sections in group 2 (median=0.4) than in group 1 (median=0.2) sections (P =0.02).
TGFβ1 expression related to rejection grade.
Serial control and test sections were stained for TGFβ1 and corresponding areas of tubular cortex were selected for analysis. In all sections showing acute cellular rejection, the expression of TGFβ1 was concentrated mainly in the tubular epithelium where it was often distributed diffusely; however, concentration was occasionally observed at the baso-lateral surface of a tubule or within individual epithelial cells (Fig. 1 d). Focal distribution was sometimes seen in isolated groups of tubules across the section. In some sections, the highest concentration of TGFβ1 was detected in isolated cells in the inflammatory infiltrate and frequently included intratubular lymphocytes. These immune cells were excluded from quantita-tive analysis of the expression of TGFβ1 by the tubular epithelium.
Examination of selected areas of tubular epithelium showed that the TGFβ1 index ranged from 1 to 2.12 in group 1 rejection sections (n=16) and from 1 to 3.48 in group 2 sections (n=13). Only 2 group 0 biopsies were included in the TGFβ1 analysis; both of these had an index of 1. The expression of TGFβ1 was significantly greater in the TEC of group 2 rejection sections than in group 1 sections (P =0.034).
It was found that paraffin sections from renal transplant tissue showing chronic rejection, with pronounced tubular atrophy, demonstrated widespread interstitial expression of TGFβ1. This was especially the case within dense inflammatory cell aggregates (Fig. 1e).
CD103+ cells in relation to E-cadherin expression in chronic rejection.
Cryostat sections from renal transplant nephrectomy tissue showing chronic rejection were stained using a dual label technique; these sections showed interstitial fibrosis and tubular atrophy in addition to an inflammatory cell infiltrate. The expression of CD103+ cells was much higher in the tubules than in acute rejection but, significantly, was not restricted to the tubules. Such extratubular location of CD103+ cells in these cases was demonstrated by simultaneous identification of E-cadherin and CD103 (Fig. 1 f); this shows clearly the presence of CD103+ cells both within the tubules and in areas remote from tubular epithelium. Irrelevant mouse IgG isotype control antibodies did not produce any positive staining of the tubular epithelium. Unfortunately, insufficient frozen tissue from nephrectomies demonstrating chronic rejection was available, at this stage, to perform a quantitative analysis of CD103+ IEL for comparison with acute rejection.
Expression of CD103 by alloreactive T cells.
Dual color immunofluorescence flow cytometry allowed simple and reproducible analysis of the proportion of cells expressing both CD8 and CD103 at various times after the stimulation of peripheral lymphocytes by culture with allogeneic antigen presenting cells (Fig. 3a). Using this technique it was demonstrated that the proportion of CD103+ cells within the CD8+ population did not rise above 10% in a basic allogeneic coculture system. However, it was found that the proportion of cells expressing CD103 increased markedly to reach levels in excess of 60% of the CD8+ population after supplementation of the culture medium with TGFβ1 (Fig. 3 b). Resting lymphocytes did not express CD103 after the addition of TGFβ1 (data not shown). The effect of TGFβ1 addition on the proportion of CD103+ cells was independent of the day on which the factor was added, with a similar proportional increase being produced by addition on day 0 or after culture for 3 or 5 days (Fig. 3 b).
Despite the presence of a small number of interstitial CD8+ T cells, this study has demonstrated clearly that normal renal tissue contains no intratubular lymphocytes or CD103+ cells. Biopsies taken during episodes of acute renal allograft rejection showed interstitial inflammation and tubulitis in which there was a high proportion of CD8+ lymphocytes. In addition foci of CD103+ cells were identified but these were restricted exclusively to the tubular epithelium. Such intraepithelial cells are typically CD8+ lymphocytes that may be retained in the epithelial site by binding to E-cadherin ( 9 , 10 ). Furthermore, the ratio of CD103+ cells to CD8+ cells within the tubular infiltrate was shown to increase with severity of tubulitis. Although tubulitis is a feature used to diagnose and monitor the progression of acute rejection ( 1 , 2 ), the presence of CD103+ cells within the tubular infiltrate has not been reported previously.
It has been suggested previously that the αEβ7-integrin is not a homing receptor ( 16 ) but is induced locally on activated T cells by the action of TGFβ1( 10 ). Indeed, it is most likely that lymphocytes are recruited to sites such as the gut after interaction between the α4β7-integrin, which is expressed commonly by peripheral lymphocytes, and the mucosal addressin, MAdCAM-1. These cells may then undergo α-chain switching after local stimulation by TGFβ, and the resultant αEβ7-integrin is able to interact with and bind E-cadherin.
During allograft rejection, it is understood that recipient peripheral T cells are activated during the presentation of alloantigens by donor antigen-presenting cells derived from the graft “passenger” leukocyte population ( 17 ). In our study, this process was modeled by coculture of peripheral lymphocytes with allogeneic antigen-presenting cells. Significantly, CD103 expression was only observed at a high level in these cultures after the addition of exogenous TGFβ1; this result is consistent with previous reports ( 12 ). In these cultures, the rate of increase of the proportion of CD103+ cells within the population was similar in cultures stimulated with TGFβ1 on days 0, 3, or 5. This suggests that TGFβ1 acts by stimulation of a gradual differentiation of the allospecific T cell population toward expression of the αEβ7-integrin.
In this study, it was shown that the distribution of TGFβ1 within renal allograft tissue is concentrated in the tubules during acute rejection. On this basis, the intratubular location of CD103+ T cells is consistent with local differentiation of activated allospecific cells within the TGFβ1-rich tubular microenvironment. Indeed, the increased proportion of CD103+ cells observed within the tubules in biopsy sections with high tubulitis scores may be a direct consequence of the elevated expression of TGFβ1 also observed in higher rejection grades. The role of TGFβ1 in local induction of CD103 is further supported by the presence of CD103+ cells in the tubular interstitium of grafts showing evidence of chronic rejection. In sections showing this pathology, it was found that TGFβ1 was widespread within the interstitial tissue. Here, a major source of the growth factor would appear to be aggregates of inflammatory cells. The presence of substantial numbers of CD103+ cells in some cases of chronic rejection is in agreement with a previous report showing that up to 61% of the CD8+ cells isolated from cases defined as “acute on chronic” rejection coexpressed CD103 ( 12 ).
It has been shown previously that addition of TEC to activated T cells is sufficient to induce up-regulation of CD103 expression in vitro; furthermore, this process can be inhibited by the addition of TGFβ-blocking antibodies ( 18 ). These data provide evidence that TEC can support the differentiation of IEL and are consistent with the observed proximity of CD103+ T cells and TGFβ1 within the tubular epithelium of renal allograft tissue.
The precise functional significance of interaction between E-cadherin and T cells expressing the αEβ7-integrin remains unclear. However, it has been shown that cross-linking the αEβ7-integrin can augment both the proliferative and cytotoxic function of IEL from the gut ( 19 , 20 ), whilst stimulation of the T cell antigen receptor can increase the affinity of this integrin for E-cadherin ( 11 ). It is known that cytotoxic lymphocytes can reversibly modulate the physiological function of intra-TEC tight-junctions ( 21 ), and that blockade of the αEβ7-integrin can prevent cell-mediated lysis of E-cadherin expressing target cells in vitro ( 19 ). Recent in vivo evidence of a direct role for the αEβ7-integrin in human pathology comes from a study of Sjogren’s syndrome, in which a close association is demonstrated between CD103+ T cells and apoptotic acinar epithelial cells ( 22 ). Apoptotic TEC are a commonly observed feature of acute renal allograft rejection ( 23 ).
The conventional immunosuppressive agent, Cyclosporin A, is unable to control the proliferation of activated T cells and is known to augment the production of TGFβ by human TEC ( 24 ). It is paradoxical that this widely used drug might actually potentiate the differentiation of cytotoxic T cells expressing the αEβ7-integrin within the tubular epithelium during acute renal allograft rejection. Recently, the Banff group has proposed that the identification of cytotoxic T lymphocytes could enhance the diagnosis of rejection and/or predict later graft dysfunction ( 2 ). It is possible that this identification should be refined to include immunolocalisation of CD103.
Several studies have addressed the possibility that adhesion molecule blockade might provide a useful therapeutic strategy for the control of acute renal allograft rejection; in most cases this has focused on control of the interaction between LFA-1 and ICAM-1. Although such strategies have achieved some success in experimental cardiac transplantation ( 25 , 26 ), a recent clinical trial in renal transplantation has been disappointing ( 27 ). In our study, it is suggested that specific T cell adhesion to E-cadherin might play an important role in tubulitis and renal allograft rejection. It follows that adhesion between the αEβ7-integrin and E-cadherin could provide a target for therapeutic intervention during acute rejection after renal transplantation
The authors are grateful to Dr. Trevor Booth for help with the confocal microscopy and to Drs. Anne Cunningham, Simi Ali, and David Jones for their help during preparation of this manuscript.
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