Mechanisms of chronic renal allograft rejection. II. Progressive allograft glomerulopathy in miniature swine

We have reported that in thymectomized miniature swine treated with a 12-day course of cyclosporin A that major histocompatibility complex class I-mismatched renal allografts either progress to chronic rejection (progression group; n = 4) or tolerance after acute rejection (recovery group; n = 4). Two types of glomerulopathies, termed acute and chronic allograft glomerulopathy, occur in allografts in this model. Morphological and immunohistochemical studies were performed on serial renal biopsies from both groups to examine the pathogenic mechanisms of acute and chronic allograft glomerulopathy. In acute rejection, acute allograft glomerulopathy developed in both groups by Day 18, with antibody deposition and T cell and macrophage infiltration. In situ DNA nick end-labeling (TUNEL)+ injured glomerular endothelial cells appeared from the early phase, followed by destruction of the glomerular capillary network with segmental mesangiolysis. Thereafter, in the progression group, acute allograft glomerulopathy progressed to chronic allograft glomerulopathy during the development of chronic rejection. This process was associated with persistent T cell and macrophage infiltration, antibody deposition, and TUNEL+ glomerular endothelial injury in the glomeruli. Impaired capillary repair, mesangial cell proliferation, and activation were still noted at Day 100, together with accumulation of mesangial matrix and duplication of glomerular basement membrane. In contrast, in the recovery group, acute allograft glomerulopathy recovered by Day 100, associated with the resolution of cellular infiltration and reduction of antibody deposition. We conclude that the acute and persistent cell- and antibody-mediated rejection against glomerular endothelial cells is the key pathogenic determinant of acute allograft glomerulopathy and progression toward chronic allograft glomerulopathy. Impaired capillary repair and phenotypic change of endothelial and mesangial cells also contribute to the development of chronic allograft glomerulopathy. With the development of tolerance, substantial recovery of acute allograft glomerulopathy can occur after the resolution of glomerular inflammation.     

Myeloid cell‐mediated renal injury in rapidly progressive glomerulonephritis depends upon spleen tyrosine kinase

Antibody‐dependent activation of myeloid cells within the glomerulus plays a central role in rapidly progressive forms of glomerulonephritis. The spleen tyrosine kinase (Syk) is expressed by all leukocytes, except mature T cells, and is required for signalling via the B‐cell receptor, Fc receptors, and some integrins. Syk has been proposed as a therapeutic target in glomerulonephritis. However, little is known of Syk activation in human kidney disease, while studies in experimental glomerulonephritis using non‐selective Syk inhibitors require validation via conditional gene deletion. The current study addressed both of these important points. Syk activation (Tyr^525/526 phosphorylation) was examined in a cohort of 96 patients with different glomerulonephritides. Syk activation was evident in infiltrating leukocytes, mainly neutrophils and macrophages, in 36/40 cases of rapidly progressive glomerulonephritis. In contrast, non‐proliferative diseases showed little or no Syk activation. Glomerular and interstitial cells exhibiting Syk activation correlated with renal function and systemic inflammation. Next, we examined mice with conditional Syk gene deletion in myeloid cells (Syk^My) versus Syk^f/f littermate controls in nephrotoxic serum nephritis – a model of rapidly progressive glomerulonephritis. Control Syk^f/f mice featured a transient neutrophil influx at 3 h and severe disease on day 9 of nephrotoxic serum nephritis, with crescent formation, macrophage infiltration, inflammation, kidney fibrosis, and renal dysfunction. In contrast, Syk^My mice had significantly reduced neutrophil and macrophage infiltration despite equivalent glomerular deposition of humoral reactants. Syk^My mice exhibited reduced crescent formation, inflammation, and fibrosis, with improved renal function on day 9 of nephrotoxic serum nephritis. In conclusion, Syk activation is prominent in infiltrating myeloid cells in human rapidly progressive glomerulonephritis, and functional studies demonstrate that Syk deletion in myeloid cells is protective in mouse nephrotoxic serum nephritis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Different Effects of Tacrolimus on Innate and Adaptive Immune Cells in the Allograft Transplantation

While tacrolimus (FK506) is currently used as immunosuppression therapy in transplant recipient, the immunological mechanism remains unknown. Herein, the immunoregulatory effects of FK506 were investigated in the physiological status and allogeneic skin transplantation. FK506 cannot significantly alter the functions of innate immune cells (macrophages and neutrophils) and adaptive immune cells (T cells) in the physiological status. However, it can effectively delay allogeneic skin‐graft rejection through ameliorating the T cell responses, but not myeloid‐derived innate immune cell responses. Importantly, it did not affect the allograft recipient macrophage innate immune defence capacity to bacteria. In clinics, FK506 treatment can significantly control the cytokine production in T cells, but not non‐T cells. This study shows targeting calcineurin signalling, FK506, to be essential in inducing allograft tolerance, but not to damage the innate defence capacity, validating the immune cell phenotypes as a potential marker in transplantation following FK506 treatment.     

Characterization of ectopic lymphoid structures in different types of acute renal allograft rejection

We hypothesize that T cells such as interleukin (IL)‐21⁺B cell lymphoma 6 (BCL6)⁺ T follicular helper cells can regulate B cell‐mediated immunity within the allograft during acute T cell‐mediated rejection; this process may feed chronic allograft rejection in the long term. To investigate this mechanism, we determined the presence and activation status of organized T and B cells in so‐called ectopic lymphoid structures (ELSs) in different types of acute renal allograft rejection. Biopsies showing the following primary diagnosis were included: acute/active antibody‐mediated rejection, C4d⁺ (a/aABMR), acute T cell‐mediated rejection grade I (aTCMRI) and acute T cell‐mediated rejection grade II (aTCMRII). Paraffin sections were stained for T cells (CD3 and CD4), B cells (CD20), follicular dendritic cells (FDCs, CD23), activated B cells (CD79A), immunoglobulin (Ig)D, cell proliferation (Ki67) and double immunofluorescent stainings for IL‐21 and BCL6 were performed. Infiltrates of T cells were detected in all biopsies. In aTCMRI, B cells formed aggregates surrounded by T cells. In these aggregates, FDCs, IgD and Ki67 were detected, suggesting the presence of ELSs. In contrast, a/aABMR and aTCMRII showed diffuse infiltrates of T and B cells but no FDCs and IgD. IL‐21 was present in all biopsies. However, co‐localization with BCL6 was observed mainly in aTCMRI biopsies. In conclusion, ELSs with an activated phenotype are found predominantly in aTCMRI where T cells co‐localize with B cells. These findings suggest a direct pathway of B cell alloactivation at the graft site during T cell mediated rejection.     

CXC and CC chemokines induced in human renal epithelial cells by inflammatory cytokines

Human renal epithelial cells might play an important role during the allograft rejection by producing chemokines in response to proinflammatory cytokines such as tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β produced by endothelial and epithelial cells early after transplantation. The production of chemokines allows inflammatory cells to be drawn into the kidney graft and therefore plays a critical role in the pathophysiologic processes that lead to the rejection of renal transplant. In this process, two chemokine superfamilies, the CC and the CXC chemokines, are the most important. The CC chemokines target mainly monocytes and T lymphocytes, while most of the CXC chemokines attract neutrophils. We showed in our study that in vitro, in unstimulated cells, basal mRNA expression of CXC chemokines (Groα, Groβ, Groγ, ENA‐78 and GCP‐2, IL‐8) that attract neutrophils was detectable and expression of these genes and chemokine release were increased in TNF‐α‐ and IL‐1β‐induced renal epithelial cells. Most of the CC chemokines [monocyte chemotactic protein‐1 (MCP‐1), macrophage Inflammatory protein 1 beta (MIP‐1β), regulated upon activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP‐3α)] showed detectable mRNA expression only after stimulation with proinflammatory cytokines and not in control cells. TNF‐α seems to induce preferably the expression of RANTES, MCP‐1, interferon‐inducible protein (IP‐10) and Interferon‐Inducible T‐cell Alpha Chemoattractant (I‐TAC), while IL‐1β induces mainly IL‐8 and epithelial neutrophil‐activating peptide 78 (ENA‐78).     

Fractalkine expression on human renal tubular epithelial cells: potential role in mononuclear cell adhesion

Fractalkine (CX3CL1) is a transmembrane molecule with a CX3C chemokine domain attached to an extracellular mucin stalk which can induce both adhesion and migration of leucocytes. Mononuclear cell infiltration at renal tubular sites and associated tubular epithelial cell damage are key events during acute renal inflammation following renal allograft transplantation. Using northern and Western blot analysis, we have demonstrated the expression of fractalkine message and protein by renal tubular epithelial cells in vitro. The expression was up-regulated by TNF-alpha, a key proinflammatory cytokine in acute rejection. Investigation of surface expression of fractalkine on cultured proximal tubular epithelial cells revealed only a subpopulation of positively staining cells. Immunohistochemistry revealed that only a proportion of tubules in renal allograft biopsies showed induction of fractalkine expression. Studies using a static model of adhesion demonstrated CX3CR1/fractalkine interactions accounted for 26% of monocytic THP-1 cell and 17% of peripheral blood natural killer cell adhesion to tubular epithelial cells, suggesting that fractalkine may have a functional role in leucocyte adhesion and retention, at selected tubular sites in acute renal inflammation. Thus, fractalkine blockade strategies could reduce mononuclear cell mediated tubular damage and improve graft survival following kidney transplantation.     

Up-regulation of macrophage migration inhibitory factor in acute renal allograft rejection in the rat.

Recent studies have identified a key role for macrophage migration inhibitory factor (MIF) in a number of immune cell-mediated diseases. The current study investigated the potential role of MIF in acute allograft rejection. Lewis rats underwent bilateral nephrectomy and then received an orthotopic DA renal allograft or an orthotopic Lewis renal isograft. Groups of six animals were killed at day 1 or 5 after transplantation. No immunosuppression was used. Animals receiving a renal allograft exhibited severe rejection on day 5, as shown by high levels of serum creatinine, very low rates of creatinine clearance, and severe tubulitis with a dense macrophage and T cell infiltrate. In contrast, isografts had normal renal function on day 5 with no histological evidence of rejection. Northern blotting showed that renal MIF mRNA expression was unchanged at day 1, but was increased 3.5-fold on day 5. In situ hybridization showed a marked increase in MIF mRNA expression by tubular cells and MIF mRNA expression by many infiltrating mononuclear cells in day 5 allografts. Immunostaining confirmed an increase in tubular MIF protein expression, particularly in areas of severe tubular damage with prominent leucocytic infiltration. Double staining showed that many infiltrating macrophages and T cells expressed the MIF protein in day 5 allografts. There was only a minor increase in MIF expression in day 5 isografts, demonstrating that neither surgical injury nor stress cause significant up-regulation of MIF expression in allograft rejection. In conclusion, this study has demonstrated that local MIF production is specifically increased in acute renal allograft rejection. These results suggest that MIF may play an important role in the cellular immune response mediating acute allograft rejection.     

Decreased humoral antibody episodes of acute renal allograft rejection in recipients expressing the HLA-DQβ1*0202 allele

The present investigation was designed to show the effect of human leukocyte antigen (HLA) class II molecular allelic specificities in the recipient on the induction of humoral antibody rejection, identified by C4d peritubular capillary staining, as well as specific antibody identified by Luminex technology. Major histocompatibility complex (MHC) class II molecules are expressed on dendritic cells, macrophages, and B lymphocytes and they present antigenic peptides to CD4 positive T lymphocytes. Human renal peritubular and glomerular capillaries express class II MHC molecules upon activation. Expression of class II molecules on renal microvascular endothelial cells exposes them to possible interaction with specific circulating antibodies. We hypothesize that HLA-DQβ1*0202 expression in recipients decreases the likelihood of antibody-mediated renal allograft rejection. We found that 80% (=25) of DQ2 positive haplotype recipients failed to induce humoral antibody renal allograft rejection and 20% (n=25) of DQ2 positive haplotype recipients induced humoral antibody renal allograft rejection (p=0.008). By contrast, 48% (n=46) of DQ2 negative haplotype recipients failed to induce a humoral antibody component of renal allograft rejection and 52% (n=46) of DQ2 negative haplotype recipients induced humoral antibody-mediated renal allograft rejection. Our results suggest that recipients who express the DQβ1*0202 allele are less likely to induce a humoral antibody component of acute renal allograft rejection than are those expressing DQ1, DQ3, or DQ4 alleles. DQβ1*0202 allele expression in recipients could possibly be protective against acute humoral allograft rejection and might serve as a future criterion in recipient selection and in appropriate therapy for acute renal rejection episodes.     

Cover Picture: Eur. J. Immunol. 2/11

The cover image of this issue consists of the immunohistochemical staining of μ‐calpain in a healthy human kidney and is taken from Letavernier et al. (pp. 473–484). In this article, the authors analyse the role of calpains in acute allograft rejection. The authors demonstrate that μ‐calpain expression is increased in human transplant kidneys undergoing acute or chronic rejection, mainly in infiltrating T cells. The authors further show that calpain inhibition delays allograft rejection. Interestingly, this delay in allograft rejection is associated with a limitation in allograft infiltration by T cells that is due to the slowing down in T‐cell migration rather than proliferation.     

A Preserved Native Kidney Alters Acute Renal Allograft Rejection in the Rat

In this study the effect of a native recipient kidney on acute rat renal allograft rejection is analysed. The authors performed a sequential daily analysis of allograft morphology and infiltration by macrophages and T cells in the presence and the absence of a recipient kidney. Several differences among both experimental groups were observed. Infiltrating macrophages and T cells in the allograft interstitium were more numerous in the presence than in the absence of a recipient kidney. The ratio of macrophages to T cells was 2:1 in the presence and 1:1 in the absence of a recipient kidney. Interstitial allograft infiltration started 1 day earlier in the presence of a contralateral kidney than in its absence. Graft necrosis occurred on day 6 and was complete in the presence of a native kidney. After total nephrectomy a patchy pattern of necrotic and viable tubules was observed from day 5 until the death of the animal. The diameter of monocytes in graft vessels increased only moderately in the presence of a recipient kidney but duplicated in its absence. The authors propose that in experimental renal transplantation contralateral nephrectomy should be performed according to a standardized schedule.     

Spleen tyrosine kinase promotes acute neutrophil-mediated glomerular injury via activation of JNK and p38 MAPK in rat nephrotoxic serum nephritis

Glomerular antibody deposition induces acute neutrophil-mediated glomerular injury via activation of c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). However, the link between antibody deposition and activation of JNK/p38 MAPK signalling is unclear. This study tested the postulate that spleen tyrosine kinase (Syk), which is activated via Fcγ-receptor ligation, is required for activation of JNK and p38 signalling and acute neutrophil-mediated glomerular injury. We used a Syk inhibitor (SYKi) in rat nephrotoxic serum nephritis (NTN) in which neutrophil-mediated glomerular injury is dependent upon JNK and p38 signalling. SYKi or vehicle treatment of Sprague-Dawley rats began 30 min before administration of anti-GBM serum with rats killed 3 or 24 h later. Immunostaining identified de novo glomerular Syk activation (p-Tyr 525/526) in untreated NTN, being most prominent in neutrophils. Vehicle and untreated NTN exhibited heavy proteinuria and glomerular thrombosis at 24 h with P-selectin and fibrin immunostaining within capillaries, glomerular macrophage and T cell infiltration, activation of JNK and p38 MAPK signalling, and upregulation of glomerular mRNA levels of pro-inflammatory molecules (TNF-α, NOS2, MMP-12 and CCL2). In contrast, SYKi treatment provided complete protection from proteinuria, with a profound reduction in glomerular thrombosis and immunostaining for P-selectin and fibrin, and a substantial reduction in glomerular mRNA levels of pro-inflammatory molecules. SYKi treatment also reduced the acute glomerular neutrophil influx and pro-inflammatory response at 3 h in NTN. These protective effects were associated with a significant reduction in glomerular JNK and p38 MAPK activation. In addition, activation of Syk, JNK and p38 was identified in human biopsy samples of acute crescentic glomerulonephritis. In conclusion, this study demonstrates that Syk signalling is required for JNK and p38 MAPK signalling and acute neutrophil-dependent glomerular injury in rat NTN. These findings identify Syk as a potential therapeutic target in antibody-dependent kidney disease.     

Attenuation of folic acid-induced renal inflammatory injury in platelet-activating factor receptor-deficient mice

Platelet-activating factor (PAF), a potent lipid mediator with various biological activities, plays an important role in inflammation by recruiting leukocytes. In this study we used platelet-activating factor receptor (PAFR)-deficient mice to elucidate the role of PAF in inflammatory renal injury induced by folic acid administration. PAFR-deficient mice showed significant amelioration of renal dysfunction and pathological findings such as acute tubular damage with neutrophil infiltration, lipid peroxidation observed with antibody to 4-hydroxy-2-hexenal (day 2), and interstitial fibrosis with macrophage infiltration associated with expression of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in the kidney (day 14). Acute tubular damage was attenuated by neutrophil depletion using a monoclonal antibody (RB6-8C5), demonstrating the contribution of neutrophils to acute phase injury. Macrophage infiltration was also decreased when treatment with a PAF antagonist (WEB2086) was started after acute phase. In vitro chemotaxis assay using a Boyden chamber demonstrated that PAF exhibits a strong chemotactic activity for macrophages. These results indicate that PAF is involved in pathogenesis of folic acid-induced renal injury by activating neutrophils in acute phase and macrophages in chronic interstitial fibrosis. Inhibiting the PAF pathway might be therapeutic to kidney injury from inflammatory cells.     

CCR5 blockade in combination with rapamycin prolongs cardiac allograft survival in mice

Both chemokine receptor 5 (CCR5) blockade and rapamycin (rapa) are effective in modulating transplant immunity and led to prolonged allograft survival, yet a great many grafts were ultimately lost to acute rejection. In this study we examined the inhibition of CCR5 in combination with the treatment with rapa in cardiac transplantation. Fully major histocompatibility complex-mismatched murine cardiac allograft models were randomized to five groups. They were administered with anti-CCR5 antibody or control antibody and rapa or phosphate-buffered saline (PBS), respectively. An additional group was treated with anti-CCR5 antibody, rapa and anti-CD25 antibody. Allograft rejection was investigated by flow cytometric analyses and enzyme-linked immunospot assay. Allografts treated with anti-CCR5 antibody plus rapa showed significantly prolonged survival (83 ± 3 days, P < 0·001) compared with control antibody plus PBS-treated allografts (6 ± 1 days). Treatment with anti-CCR5 monoclonal antibody (mAb) plus rapa inhibited significantly the progression of chronic rejction. Further analysis of donor hearts in the anti-CCR5 antibody plus rapa-treated group demonstrated increased infiltration of CD4⁺CD25⁺forkhead box P3⁺ regulatory T cells, and depletion of CD25⁺ cells resulted in acute rejection of allografts in 18 ± 1 day. CCR5 blockade in combination with rapa is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated by CD25⁺ T cell recruitment and control of T lymphocyte proliferation.     

Expression of nestin after renal transplantation in the rat

Chronic allograft injury (CAI) limits the long‐term success of renal transplantation. Nestin is a marker of progenitor cells, which probably contribute to its pathogenesis. We hypothesize that nestin is induced by ischemia/reperfusion injury and acute rejection, main risk factors for CAI. Syngeneic renal transplantation was performed in Lewis rats and allogeneic transplantation in the Fischer 344 to Lewis strain combination, which results in reversible acute rejection and in CAI in the long‐run. The Dark Agouti to Lewis rat strain combination was used to study fatal acute rejection. In untreated kidneys, nestin immunoreactivity was detected in glomeruli and in very few interstitial or microvascular cells. Syngeneic transplantation induced nestin expression within 4 days, which decreased until day 9 and returned to control levels on day 42. Nestin expression was strong during acute rejection and still detected during the pathogenesis of CAI on day 42. Nestin‐positive cells were identified as endothelial cells and interstitial fibroblast‐like cells co‐expressing alpha‐smooth muscle actin. A sub‐population of them expressed proliferating cell nuclear antigen. In conclusion, nestin is induced in renal grafts by ischemia/reperfusion injury and acute rejection. It is expressed by proliferating myofibroblasts and endothelial cells and probably contributes to the pathogenesis of CAI.     

Neutrophils mediate parenchymal tissue necrosis and accelerate the rejection of complete major histocompatibility complex-disparate cardiac allografts in the absence of interferon-gamma

A major feature of acute rejection of cardiac allografts is an intense mononuclear cell infiltration accompanied by interferon (IFN)-gamma production. In the current study we tested the role of IFN-gamma in acute rejection of allografts by comparing the histopathology of rejection in wild-type versus IFN-gamma-/- recipients of major histocompatibility complex-mismatched cardiac grafts. Wild-type recipients rejected the allografts at days 8 to 9 after transplant but rejection was accelerated 2 to 3 days in IFN-gamma-deficient recipients. During rejection in wild-type recipients, the allografts were heavily infiltrated with CD8+ T cells and other mononuclear cells. In contrast, allografts in IFN-gamma-deficient recipients had few T cells but an intense neutrophil infiltration accompanied by extensive graft parenchymal necrosis. No difference in expression levels of neutrophil chemoattractants including Groalpha/KC, MIP-2, GCP-2, and MIP-1alpha, was observed in allografts retrieved from wild-type and IFN-gamma-/- recipients. Depletion of neutrophils from IFN-gamma-deficient recipients delayed rejection until days 8 to 10 after transplant and restored the histopathology of acute allograft rejection to that observed in allografts rejected by wild-type recipients. These results indicate the potent regulatory properties of IFN-gamma during acute rejection directed at neutrophil infiltration into allografts and mediating graft tissue necrosis.     

Neutrophils sustain pathogenic CD8+ T cell responses in the heart

This study explores the influence of innate immunity on CD8(+) T-cell responses against heart tissue. Adoptive transfer of ovalbumin-specific CD8(+) effector T cells into CMy-mOva mice, which express ovalbumin in cardiac myocytes, results in a lethal acute myocarditis. The inflammatory infiltrate in the heart includes neutrophils as well as T cells. We used anti-Ly6G antibody to transiently deplete neutrophils at the time of onset of disease. By day 7 after receiving 5 x 10(5) CD8(+) effector T cells, 100% of control Ig-treated CMy-mOva mice had died, while 85% of anti-Ly6G-treated mice survived indefinitely. CD8(+) T-cell infiltration and tissue damage were present in both groups, but the disease was limited in the anti-Ly6G-treated mice, with a rapid disappearance of the adoptively transferred CD8(+) T cells within 11 days. Recovery occurred even though blood neutrophil counts began to rise 48 hours after the last anti-Ly6G treatment. Recovery was associated with a chronic CD4(+) cell infiltrate, and a rapid decline in expression of IFN-gamma and IP-10 mRNA in the myocardium. Neutrophil depletion did not effect survival of CMy-mOva mice that received 3 x 10(6) CD8(+) T cells. These data show that granulocytic inflammation sustains CD8(+) T-cell-mediated heart disease, which has important implications for the pathogenesis and treatment of acute myocarditis and allograft rejection.     

Proteomic profiling of renal allograft rejection in serum using magnetic bead–based sample fractionation and MALDI-TOF MS

Proteomics is one of the emerging techniques for biomarker discovery. Biomarkers can be used for early noninvasive diagnosis and prognosis of diseases and treatment efficacy evaluation. In the present study, the well-established research systems of ClinProt Micro solution incorporated unique magnetic bead sample preparation technology, which, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), have become very successful in bioinformatics due to its outstanding performance and reproducibility for discovery disease-related biomarker. We collected fasting blood samples from patients with biopsy-confirmed acute renal allograft rejection (n = 12), chronic rejection (n = 12), stable graft function (n = 12) and also from healthy volunteers (n = 13) to study serum peptidome patterns. Specimens were purified with magnetic bead–based weak cation exchange chromatography and analyzed with a MALDI-TOF mass spectrometer. The results indicated that 18 differential peptide peaks were selected as potential biomarkers of acute renal allograft rejection, and 6 differential peptide peaks were selected as potential biomarkers of chronic rejection. A Quick Classifier Algorithm was used to set up the classification models for acute and chronic renal allograft rejection. The algorithm models recognize 82.64% of acute rejection and 98.96% of chronic rejection episodes, respectively. We were able to identify serum protein fingerprints in small sample sizes of recipients with renal allograft rejection and establish the models for diagnosis of renal allograft rejection. This preliminary study demonstrated that proteomics is an emerging tool for early diagnosis of renal allograft rejection and helps us to better understand the pathogenesis of disease process.     

NK cells contribute to the skin graft rejection promoted by CD4+ T cells activated through the indirect allorecognition pathway

Rejection of solid organ allografts is promoted by T cells. Recipient T cells can directly recognize intact allo-MHC molecules on donor cells and can also indirectly recognize processed donor-derived allo-peptides presented by recipient antigen-presenting cells in the context of self-MHC molecules. Although CD4(+) T cells primed through the indirect allorecognition pathway alone are sufficient to promote acute allograft rejection, it is unknown how they can mediate graft destruction without cognate recognition of donor cells. In this study, we analyzed the indirect effector mechanism of skin allograft rejection using a mouse model in which SCID recipients bearing MHC class II-deficient skin allografts were adoptively transferred with CD4(+) T cells. Histologically, entire graft necrosis was preceded by mononuclear cell infiltration in the graft epithelia with epithelial cell apoptosis, indicating cell-mediated cytotoxicity against donor cells as an effector mechanism. Beside CD4(+) T cells and macrophages, NK cells infiltrated in the rejecting grafts. Depletion of NK cells as well as blocking of the activating NK receptor NKG2D allowed prolonged survival of the grafts. Expression of NKG2D ligands was up-regulated in the rejecting grafts. These results suggest that NK cells activated through NKG2D contribute to the skin allograft rejection promoted by indirectly primed CD4(+) T cells.     

Acute cellular rejection and cyclosporine nephrotoxicity monitored by biopsy in a renal allograft recipient. The differentiation of drug nephrotoxicity from rejection by phenotyping of cellular infiltrates

Serial allograft biopsies were performed on a renal transplant patient who experienced recurrent episodes of acute cellular rejection as well as cyclosporine nephrotoxicity. Five biopsies were performed after acute elevations of the serum creatinine level (15, 46, 155, 244, and 324 days after transplant). Each specimen was evaluated by routine histologic techniques as well as by immunofluorescence analysis and by monoclonal antibody labeling for determination of the cell phenotype of the mononuclear cell infiltrates within each specimen. The first and third specimens disclosed significant T-cell infiltrates with an equal number of T-cytotoxic-suppressor (Leu 2a) and T-helper-inducer (Leu 3a) cells in a diffuse cortical pattern, while the second biopsy showed a slightly milder infiltrate with a marked elevation (7:1) in the Leu 3a:Leu 2a ratio in the cortical-diffuse pattern. Clinically, the patient responded dramatically to cyclosporine dosage reduction following the second biopsy, and bolus steroid antirejection therapy following the first and third biopsies. These findings suggest that phenotypic cell marker analysis within the context of histologic pattern is a useful adjunct to the routine histologic evaluation of renal allograft biopsy specimens and may provide a means of differentiating rejection from cyclosporine nephrotoxicity.