Single Nucleotide Polymorphisms and Long-Term Clinical Outcome in Renal Transplant Patients: A Validation Study.

Genome-wide association studies (GWAS) are designed to investigate single nucleotide polymorphisms (SNPs) and the association with a clinical phenotype. A previous GWAS performed in 300 renal transplant recipients identified two SNPs (rs3811321 and rs6565887) associated with serum creatinine and clinical outcome. We sought to validate these findings. Genotyping of the two SNPs was performed using Taqman assays in 1638 Caucasians participating in the Assessment of LEscol in Renal Transplant (ALERT) study. Primary endpoint was death-censored graft loss, and secondary endpoint was all-cause mortality. Applying Cox regression, no crude association to graft loss was found for rs3811321 on chromosome 14 (hazard ratio [HR] 0.87, 95% CI 0.59-1.29, p = 0.50) or rs6565887 on chromosome 18 (HR 0.88, CI 0.62-1.25, p = 0.48). Multivariable adjustments did not change results, nor did evaluation of the number of risk alleles formed by the two SNPs. No association with mortality was detected. In conclusion, an impact of two SNPs on chromosomes 14 and 18 on death-censored graft survival or all-cause mortality was not confirmed. Our results emphasize the importance of validating findings from high-throughput genetics studies and call for large collaborative research initiatives in the field of transplantation outcomes.

Expression of the T cell-specific adapter protein in human tissues.

T cell-specific adapter protein (TSAd) encoded by the SH2D2A gene is expressed in activated T cells, NK cells and endothelial cells, but its tissue expression has not yet been mapped. Here, we have defined the specificity of two commercially available anti-TSAd monoclonal reagents using peptide arrays. We found them to bind separate epitopes in the C-terminal part of TSAd. We then used immunohistochemistry to examine TSAd expression in various human lymphoid and non-lymphoid tissues. Immunostaining of adjacent tissue sections revealed that a substantial fraction of CD3-positive cells in normal lymphoid and non-lymphoid tissues expressed TSAd. In particular, essentially all intra-epithelial T cells appeared to coexpress TSAd. In addition, TSAd expression was observed in endothelial cells of dermal microvessels, while it was not detected in endothelial cells of the other tested tissues. This work provides insight into the expression pattern of TSAd in various healthy human tissues.

Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells.

Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils. Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We found that cultured human microvascular endothelial cells from the intestine (HIMEC) and from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf ). This observation was corroborated by the immunohistochemical observation of double-positive granules (IL-8(+)vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min, whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8-containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8-containing granules were inducible by treatment with recombinant human IL-1beta for 24 h; additional histamine treatment doubled IL-8 secretion from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.

The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment.

Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEV-derived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (Epstein-Barr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC (plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest.

Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.

Genome-wide transcription profile of endothelial cells after cardiac transplantation in the rat.

Transcriptome analyses of organ transplants have until now usually focused on whole tissue samples containing activation profiles from different cell populations. Here, we enriched endothelial cells from rat cardiac allografts and isografts, establishing their activation profile at baseline and on days 2, 3 and 4 after transplantation. Modulated transcripts were assigned to three categories based on their regulation profile in allografts and isografts. Categories A and B contained the majority of transcripts and showed similar regulation in both graft types, appearing to represent responses to surgical trauma. By contrast, category C contained transcripts that were partly allograft-specific and to a large extent associated with interferon-gamma-responsiveness. Several transcripts were verified by immunohistochemical analysis of graft lesions, among them the matricellular protein periostin, which was one of the most highly upregulated transcripts but has not been associated with transplantation previously. In conclusion, the majority of the differentially expressed genes in graft endothelial cells are affected by the transplantation procedure whereas relatively few are associated with allograft rejection.

Tenascins in fibrotic disorders-from bench to bedside.

Although fibrosis is becoming increasingly recognized as a major cause of morbidity and mortality in chronic inflammatory diseases, available treatment strategies are limited. Tenascins constitute a family of matricellular proteins, primarily modulating interactions of cells with other matrix components and growth factors. Data obtained from tenascin C deficient mice show important roles of this molecule in several models of fibrosis. Moreover there is growing evidence that tenascin C has a strong impact on chronic inflammation, myofibroblast differentiation and recruitment. Tenascin C as well as tenascin X has furthermore been shown to affect TGF-ß activation and signaling. Taken together these data suggest that these proteins might be important factors in fibrosis development and make them attractive both as biological markers and as targets for therapeutical intervention. So far most clinical research in fibrosis has been focused on tenascin C. This review aims at summarizing our up-to-date knowledge on the involvement of tenascin C in the pathogenesis of fibrotic disorders.

Differential interference contrast microscopy combined with immunofluorescence: a new method to phenotype eosinophils in situ.

Several surface receptors are expressed on eosinophils in vitro depending on the state of cellular activation, but immunohistochemical studies of eosinophil-related diseases have mostly focused on the number of infiltrating eosinophils as well as extracellular deposits of eosinophil granule proteins. The present investigation showed that eosinophils display a characteristic granular appearance in cryo-sections and cytospins by differential interference contrast (DIC) imaging. This approach appeared to be more reliable for identification of these cells in situ than immunohistochemical labelling of eosinophil granule proteins. Moreover, combined with immunofluorescence microscopy DIC imaging facilitated three-colour immunofluorescence phenotyping of eosinophils.

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND: Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS: An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS: E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION: Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Expression of functional VCAM-1 by cultured nasal polyp-derived microvascular endothelium.

Induction of endothelial vascular cell adhesion molecule-1 (VCAM-1) by interleukin (IL)-4 is believed to exert a major impact on the extravasation of leukocyte subsets in allergic disease. This notion has recently been challenged because cultured microvascular endothelial cells (ECs) derived from various organs are unable to express VCAM-1 after exposure to IL-4. In this study, we have established a method for isolation and culture of nasal polyp-derived microvascular ECs and report their cytokine-regulated VCAM-1 expression. With a combination of cell enzyme-linked immunosorbent assay, flow cytometry, and reverse transcription polymerase chain reaction, such expression was shown to be induced in a dose- and time-dependent manner not only by IL-1 beta and tumor necrosis factor-alpha but also by IL-4 and IL-13. Therefore, the response of nasal microvascular ECs did not harmonize with that of counterparts from several other tissues. IL-4 or IL-13 combined with submaximal concentrations of IL-1 beta or tumor necrosis factor-alpha increased VCAM-1 expression in a synergistic manner. VCAM-1 was functional as shown by antibody-mediated inhibition of leukocyte adhesion. Taken together, our results supported the notion that selective VCAM-1 induction by IL-4 and IL-13 plays an important role for the preferential recruitment of eosinophils and T lymphocytes seen in human airways affected by allergy.

Regional specialization in the mucosal immune system: primed cells do not always home along the same track.

According to the current paradigm of lymphocyte trafficking, primed B and T cells extravasate in the intestinal lamina propria chiefly by means of the mucosal homing receptor alpha4beta7, which interacts with the vascular addressin MAdCAM-1. However, as discussed here, this mechanism cannot explain the preferential homing of B cells with a high level of J-chain expression to mucosal effector sites outside the gut.

Respiratory burst of intestinal macrophages in inflammatory bowel disease is mainly caused by CD14+L1+ monocyte derived cells.

Macrophages play a crucial role in intestinal mucosal defence, forming dense subepithelial aggregates, particularly in the colon. One of their important bactericidal mechanisms is production of oxygen radicals but this may damage the intestinal epithelium, perhaps as an early step in inflammatory bowel disease (IBD). The potential for release of oxygen radicals from mucosal macrophages in IBD was measured and whether a difference exists between newly arrived (CD14+L1+) monocyte-like cells and resident macrophages (CD14(-)L1-), without or with additional priming in vitro, was investigated. Lamina propria mononuclear cells from six patients with IBD and five with a normal intestine were isolated with an ethylenediaminetetra acetic acid/collagenase/dispase technique and cultured for three days. The cells were tested with or without interferon gamma (200 U/ml) priming in the presence or absence of lipopolysaccharide (1 microgram/ml) for the last 48 hours in cultures. Samples from inflamed IBD mucosa depleted of CD14+ cells by immunomagnetic beads were compared with their undepleted counterparts and with samples from virtually normal mucosa from the same patients. The production of oxygen radicals was measured as the amount of reduced cytochrome C 2.5 hours after triggering with phorbol 12-myristate 13-acetate. The oxygen radical production in macrophages from moderately or severely inflamed mucosa was reduced by median 69% (range 22%-79%, p < 0.027) after depletion of CD14+ cells, reaching a level similar to that found for virtually normal samples from the same IBD patients. Furthermore, this production did not increase significantly in mucosal macrophages from normal reference mucosa and from virtually normal or inflamed IBD mucosa after priming with interferon gamma with or without addition of lipopolysaccharide. Upregulation of a respiratory burst in subepithelial resident macrophages os not a likely pathogenetic step in IBD. The increased oxygen radical production shown by macrophages from IBD lesions can, however, be ascribed to recently extravasated CD14+L1+ monocyte-like cells. Inhibition of extravasation of these reactive cells may form part of a therapeutic approach in the future.

Eosinophil infiltration is related to increased expression of vascular cell adhesion molecule-1 in nasal polyps.

Endothelial adhesion molecules are important in the recruitment of leukocytes to inflammatory sites. Nasal polyps characteristically contain a leukocyte infiltrate in which eosinophils often are remarkably prominent. We have studied whether this feature is related to a particular profile of adhesion molecules on the local microvascular endothelium. Nasal polyps were obtained from 15 patients. Mucosal biopsy specimens of the lower and the middle turbinate from the same patients as well as from three control subjects served as reference tissue. Expression of endothelial adhesion molecules and the relative numbers of eosinophils and neutrophils were examined by two- and three-color immunofluorescence staining. Both the number of eosinophils and the proportion of vessels positive for vascular cell adhesion molecule-1 (VCAM-1) were significantly increased in nasal polyps compared with the turbinate mucosa of the same patients (P = 0.008 and P = 0.001, respectively). By contrast, the number of neutrophils and the relative expression of E-selectin and intercellular adhesion molecule-1 were similar at both tissue sites. Furthermore, the relative number of eosinophils in nasal polyps was well correlated (rs = 0.73, P = 0.006) with the percentage of vessels positive for VCAM-1, but this was not true for neutrophils. Taken together, this direct in situ observation strongly supports the crucial role suggested for VCAM-1 in human eosinophil extravasation at inflammatory sites.

Cytokine-regulated expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human microvascular endothelial cells.

Endothelial cells (EC) recruit circulating leukocytes to sites of inflammation, partly by expression of endothelial-leukocyte adhesion molecules. Whereas the regulation of some adhesion molecules is well characterized in cultured HUVEC, similar data for microvascular human test systems are limited. We studied the cytokine-regulated expression of vascular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in cultured human intestinal microvascular endothelial cells (HIMEC). E-selectin and VCAM-1 were induced, and ICAM-1 was enhanced, in a dose-dependent fashion after stimulation with IL-1beta, TNF-alpha, and LPS. Each adhesion molecule displayed characteristic time-related responses comparable to those obtained with HUVEC, and each molecule supported adhesion of leukocytes. Notable disparities between the two endothelial test systems were that 1) expression of total cellular E-selectin (but not surface membrane expression) was sustained after 72 h of IL-1beta stimulation in HIMEC, contrasting a rapid biphasic response in HUVEC; 2) LPS did not maintain prolonged expression of ICAM-1 and VCAM-1 in HIMEC; and 3) VCAM-1 protein was dose-dependently up-regulated by IL-4 in HUVEC, peaking after 8 h, while IL-4 had only a negligible effect on the expression of this protein in HIMEC. In conclusion, the regulation of these adhesion molecules appears to be somewhat different in HIMEC compared with HUVEC, and the differences from available data on skin-derived microvascular endothelial cell cultures are to some extent substantial. Our findings document the importance of using relevant endothelial cell culture systems for studies of leukocyte-endothelial cell interactions.

Mechanism for enhanced external transfer of dimeric IgA over pentameric IgM: studies of diffusion, binding to the human polymeric Ig receptor, and epithelial transcytosis.

Transport of polymeric Igs (pIgA and pIgM) across secretory epithelia is mediated by the polymeric Ig receptor (pIgR), also known as the transmembrane secretory component. Compared with local production, external transfer of pIgA is favored 6- to 12-fold over that of pIgM on a molar basis. This transfer may be modulated at several levels: diffusion through matrix and basement membranes, ligand affinity for pIgR, and efficiency of epithelial transcytosis. To investigate these possibilities, we compared the ability of Madin-Darby canine kidney epithelial cells transfected with human pIgR to transport pIgA vs pIgM from the basolateral to the apical face, and examined the inhibitory effect of various filter types used for mounting of the monolayer. Binding data showed that pIgR bound pIgA and pIgM with similar affinity. Internalization of both ligands was fast and took place at similar rates; transcytosis was also found to be equally efficient at the molar level. Thus, the overall rate of transport across the epithelial monolayer was comparable for pIgA and pIgM, and was not further enhanced by ligand stimulation over a 20-fold increased concentration level. Conversely, pIgA had a considerable advantage over pIgM in passive diffusion assays performed in vitro. Moreover, in situ immunofluorescence staining showed retention of IgM over IgA and IgG in mucosal basement membrane zones, in contrast to the preferential epithelial uptake of IgA and, less so, IgM. The biologic consequences of the highly efficient epithelial pIg transport, and the diffusion advantage of pIgA over pIgM, are discussed in relation to the evolution and function of secretory Abs.

Cellular and molecular mechanisms for induction of mucosal immunity.

The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal production of Ig polymers are initially stimulated in organized mucosa-associated lympho-epithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive sites they migrate ("home") as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated auto-immune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses in humans. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effector sites of mucosal immunity.

Heterogeneity of endothelial cells: the specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization.

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.