Haplotype analysis on chromosome 6p of tumor necrosis factor alpha, vascular endothelial growth factor A, and interleukin-17F alleles associated with corneal transplant rejection.

OBJECTIVE: The aim of this work was to investigate single-nucleotide polymorphisms (SNPs) in multiple genes on chromosome 6p in corneal transplant recipients known to be at increased risk of failure through immunologic rejection (ie, "high-risk" corneal transplants). Tumor necrosis factor alpha (TNF-α) is a key immunoregulatory cytokine in the ocular environment, interacting with a variety of factors in a synergistic way and playing a crucial role in many stages of the inflammatory response. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors, supporting both hemangiogenesis and lymphangiogenesis, both key in transplant tolerance and rejection. Interleukin-17 (IL-17) is a multifunctional cytokine produced by T-helper 17 cells, exerting specific effector functions during an immune response. Association of SNPs in all 3 genes with corneal transplant outcome was therefore investigated. METHODS: Three hundred five corneal transplant recipients were followed for 3 years, and episodes of allograft rejection were recorded. With the use of patient DNA, 6 SNPs of 3 different genes on chromosome 6p were investigated. The TNF-α promoter SNP -308 G/A (rs1800629) was analyzed with the use of induced heteroduplex generation; 2 VEGF-A functional variants were analyzed, -2578 (rs699947) C/A and -1154 (rs1570360) G/A, with the use of Taqman genotyping assays; and 3 nonsynonymous IL-17F SNPs in exon 3 (negative strand), (rs2397084) A/G, (rs11465553) G/A, and (rs763780) A/G, were investigated with the use of direct sequencing. Haplotypes were inferred with the use of PHASE using positive strand alleles, and exact measures of association were determined with the use of Mid-P exact chi-square. RESULTS: Six common haplotypes were inferred, with the haplotype TNF-α (rs1800629), VEGF-A (rs699947), (rs1570360), IL-17F (rs763780), (rs11465553), and (rs2397084) ACGTCT having a significant association with corneal transplant rejection (odds ratio, 1.78; 95% confidence interval, 1.01-3.11; P = .04). CONCLUSIONS: The results suggest that patients carrying a combination of SNPs for TNF-α, VEGF-A, and IL-17F of ACGTCT haplotype may have an increased risk of corneal allograft rejection compared with patients carrying other haplotypes.

Functional tumor necrosis factor alpha polymorphisms and haplotype analysis in high-risk corneal transplantation.

BACKGROUND: Tumor necrosis factor alpha (TNF-α) plays a critical role in diverse cellular processes including ocular immune tolerance, inflammation, and allograft rejection. The ubiquitous transcription factor nuclear factor kappa B (NF-κB) regulates expression of numerous genes. Induction of the TNF-α pathway is involved in the inflammatory response and loss of transplant tolerance. OBJECTIVES: We investigated functional single nucleotide polymorphisms (SNPs) in the promoter region of TNF-α and an insertion/deletion (indel) polymorphism of NF-κB1 in corneal transplant recipients considered to be at increased risk of immunological rejection (ie, high-risk corneal transplantations) and looked for any associations with corneal transplantation outcome. PATIENTS AND METHODS: Three hundred eighty-four full thickness corneal transplant recipients were followed for 3 years and episodes of reversible and irreversible allograft rejection were recorded. Using DNA obtained from these patients, 5 SNPs located in the promoter region -1031 T/C rs1799964, -863 C/A (rs1800630), -857 C/T (rs1799724), -308 G/A (rs1800629), and -238 G/A (rs361525), and one SNP upstream from the transcription start site (+489) rs1800610 of TNF-α were analyzed using induced heteroduplex generation. A functional NF-κB1 indel (-94) was also investigated. Haplotypes were inferred by PHASE and associations with rejection were determined by chi-square analysis. RESULTS: The TNF-α haplotype TCTGGA was significantly associated with reduced risk of corneal graft rejection (Pc < .005) and TCTAGA was associated with increased risk of rejection (Pc < .005) in high-risk corneal transplants. There was no association with the NF-κB1 indel (Pc > .05). CONCLUSION: According to haplotype frequencies, our results suggest that the TCTGGA haplotype may confer additional protection against risk of immunological rejection whereas TCTAGA may increase risk of corneal allograft rejection in the high-risk setting. However, both haplotypes were relatively rare and thus would not warrant genotyping for individual patient selection for anti-TNF therapy.

The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o-.

Tight junctions (TJs) make a vital contribution to the barrier properties of the airway lining. Opening of TJs, or their frank cleavage, is suspected as a pathophysiological event in the lung, but research into the cellular and molecular mechanisms involved has been impeded by technical limitations of available experimental models. The authors have compared the properties of two epithelial cell lines derived from bronchial epithelium to explore whether these cell lines could constitute appropriate tools for the study of TJ regulation in bronchial epithelium. Investigations of TJs in 16HBE14o- cells and Calu-3 cells were made by fluorescent antibody labelling in conjunction with wide-field, confocal or 2-photon molecular excitation microscopy (2PMEM). The presence of TJ proteins was confirmed by immunoblotting and functional properties of the monolayers were studied by measurements of transepithelial electrical resistance and mannitol permeability. Cells of both lines formed confluent monolayers in which the cells expressed the TJ proteins occludin and ZO-1 in continuous circumferential patterns suggestive of functional TJs. This interpretation was supported by the development of transepithelial electrical resistances and of low paracellular permeability to solutes. Within the limits of resolution offered by 2PMEM, occludin and ZO-1 appeared to colocalize at TJs. These studies suggest that the 16HBE14o- cells and Calu-3 cell lines are potentially useful in vitro models to study how tight junction opening or cleavage changes the functional barrier properties of bronchial epithelium.

Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.

BACKGROUND: House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium. OBJECTIVE: The present study applied novel techniques to compare changes in permeability with quantitative events in tight junctions (TJs) and desmosomes (DMs) of epithelial cells exposed to Der p 1. METHODS: Confluent monolayers of Madin-Darby canine kidney (MDCK) and 16HBE14o-human bronchial epithelial cells were used as experimental models. Permeability was estimated from mannitol clearance. Digital imaging with quantification of TJs and DMs was achieved by fluorescent antibody staining and 2-photon molecular excitation microscopy (2PMEM). Biochemical changes in TJs were studied by immunoblotting, radiolabelling and immunoprecipitation. RESULTS: Der p 1 caused a time-dependent breakage of TJs and reduction in their content of the protein ZO-1. Reduction in ZO-1 immunofluorescence at TJs occurred with a small increase in the amount of diffuse, cytoplasmic immunoreactive ZO-1 staining. Morpho-logical changes in TJs occurred in synchrony with increases in epithelial permeability. DM puncta increased both in size and intensity of staining. Immunoblotting demonstrated that the disruption of TJ morphology was associated with cleavage of ZO-1 and occludin. Cells recovered from allergen exposure by de novo synthesis of occludin. CONCLUSION: Der p 1 could contribute to sensitization and allergic responses by degrading the function of the airway epithelial barrier.

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.

BACKGROUND: Allergenic and non-allergenic proteinases from house dust mites (HDM) cause loss of adhesion between airway epithelial cells that may result in a loss of functional cohesion between the cells and thus assist in allergen presentation. Improved cellular assay systems are needed to ascertain the mechanisms involved. OBJECTIVES: To survey a series of epithelial cell lines (Calu-3, 16HBE14o-, NCI-H292 and A549 from human airways, and MDCK from dog kidney) and establish their utility for studies of the effects of HDM proteinases from D. pteronyssinus on epithelial permeability. To develop an improved method for measuring changes in epithelial permeability induced by HDM proteinases and other provocants. METHODS: The permeability of epithelial monolayer cultures to mannitol was calculated from measurements of clearance using a technique that permits mathematical estimation and reduction of non-cellular diffusional constraints. Permeability was studied under control conditions and after perturbation of monolayers with HDM proteinases (separated into serine- and cysteine-proteinase classes) or chelation of extracellular Ca2+. Fluorescent antibody staining was used to investigate whether the cells expressed tight junctions (staining of ZO-1), desmosomes (staining of desmoplakin) and zonulae adherentes (staining of E-cadherin). RESULTS: The Calu-3 line was identified as an airway cell line that expressed functional tight junctions, desmosomes and zonulae adherentes. Calu-3 monolayers exhibited a low clearance and permeability to mannitol, similar to that seen in the extensively characterized MDCK cell line. Clearance and permeability were significantly increased by treatment with either HDM proteinase fraction or by calcium chelation. 16HBE14o- cells also had a low permeability to mannitol under control conditions and expressed a similar repertoire of functional proteins from major intercellular junctions. In contrast, NCI-H292 and A549 cell lines were functionally deficient in tight junctions, although they did express desmosomes and zonulae adherentes to a greater extent. Epithelial permeability was found to be a more appropriate and sensitive index of epithelial perturbation than was tracer clearance. CONCLUSION: These results suggest that the Calu-3 and 16HBE14o- cell lines are useful tools in studying the mechanism of HDM proteinases on airway epithelial cell function. HDM proteinases of both cysteine and serine mechanistic classes were found to perturb epithelial adhesion and function.

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium.

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.