Comparative enzymology of native and recombinant house dust mite allergen Der p 1.

BACKGROUND: The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. METHODS: Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. RESULTS: Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. CONCLUSION: The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens.

Interactions between mature Der p 1 and its free prodomain indicate membership of a new family of C1 peptidases.

BACKGROUND: Studies in vivo have shown that the cysteine peptidase activity of group 1 house dust mite allergens contributes to their allergenicity. These allergens are synthesized initially as proenzymes and removal of the propiece is necessary to unmask their proteolytic activity. In related C1 family cysteine peptidases of enzyme clan CA, liberated propieces continue to inhibit the mature peptidase as tight binding inhibitors. As it is not known whether mite peptidase allergens behave similarly, our objective was to investigate the effect of the Der p 1 propiece on the catalytic activity of Der p 1 and Der f 1. METHODS: Enzymatic activity of natural Der p 1 and Der f 1 was assessed using a specific substrate and the effect of the recombinant propiece on its enzyme kinetics defined. The integrity of the propiece during these interactions was studied functionally and by analysis of the reaction mixtures. RESULTS: Der p 1 propiece was a potent competitive inhibitor of Der p 1 and Der f 1. In contrast to other cysteine peptidase prodomains, which are cognate tight binding inhibitors, the Der p 1 propiece behaves as a substrate and is fully degraded during this interaction. CONCLUSION: Mature Der p 1-prodomain interactions differ from other C1 family cysteine peptidases, suggesting that group 1 mite allergens are a new subgroup among C1 family cysteine peptidases. The rapid inactivation of Der p 1 prodomain is a newly identified mechanism that may contribute to the potency of this allergen.

Protein kinase C alpha expression in normal breast, ductal carcinoma in situ and invasive ductal carcinoma.

The purpose of this study was to determine if Protein Kinase C alpha (PKC alpha) is altered in expression or localisation in normal breast, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). We obtained 14 mixed cases of invasive ductal carcinoma (IDC) and DCIS, 36 pure DCIS cases and 25 cases of normal breast. The sections were stained immunohistochemically for PKC alpha expression. Staining was cytoplasmic. The results showed a progressive reduction in staining intensity from normal breast to invasive ductal carcinoma. The staining pattern was heterogeneous in the cytoplasm of DCIS and IDC, but homogeneous in the cytoplasm of normal breast ductal epithelium. Interestingly, mitotic cells and cells with aberrant nuclear morphology showed increased cytoplasmic staining in DCIS and IDC. PKC alpha activity is altered in dividing or abnormal cells, but overall expression is reduced in IDC. This raises the possibility of an alteration in the subcellular localisation of PKC alpha which may relate to changes in desmosomal adhesive state.

The detection of IgG and IgA autoantibodies to desmocollins 1-3 by enzyme-linked immunosorbent assays using baculovirus-expressed proteins, in atypical pemphigus but not in typical pemphigus.

BACKGROUND: We have shown previously that human desmocollin (Dsc) 1 is recognized by IgA autoantibodies of subcorneal pustular dermatosis (SPD) type IgA pemphigus. However, the presence of IgG anti-Dsc autoantibodies is still controversial, and antibodies to Dsc2 and Dsc3 have not been clearly identified. OBJECTIVES: To investigate this by producing recombinant proteins consisting of the entire extracellular domains of human Dsc1, 2 and 3 in baculovirus, and to use them to establish an enzyme-linked immunosorbent assay (ELISA). METHODS: By this ELISA, we examined in total 165 cases of various types of autoimmune bullous diseases, as well as 23 normal controls. RESULTS: None of 45 sera of classical pemphigus showed either IgG or IgA antibodies to any Dsc. In contrast, one atypical pemphigus serum showed both IgG and IgA antibodies to Dsc1, which were adsorbed by incubation with Dsc1 baculoprotein. Furthermore, this ELISA detected both IgA and IgG anti-Dsc3 antibodies in one atypical case, and IgA antibodies to both Dsc2 and Dsc3 in another. This reactivity was confirmed by positive IgA immunofluorescence with Dsc2 and Dsc3 expressed on COS-7 cells. These results show that both IgG and IgA autoantibodies against all of Dsc1-3 are present in the sera of particular cases of nonclassical pemphigus, except for IgG antibodies to Dsc2, but that they are not detected in classical pemphigus. Unexpectedly, although IgA antibodies of all of eight SPD type IgA pemphigus sera reacted with Dsc1 expressed on COS-7 cells, only one serum was positive in Dsc1 ELISA for IgA. CONCLUSIONS: This result indicates either that Dscs expressed by baculovirus may not adopt the correct conformation or that Dscs may need association with other molecules to express all the epitopes for autoantibodies.

Altered expression of desmosomal components in high-grade squamous intraepithelial lesions of the cervix.

We have used immunohistochemistry to test the hypothesis that components of the desmosome are disrupted during neoplastic progression of squamous epithelial cells in the uterine cervix. Sections of normal cervix and squamous intraepithelial lesions (SILs) were immunostained for desmosomal proteins and glycoproteins, and results were assessed using a semi-quantitative grading system. No difference between normal cervix and low-grade SIL (LSIL) was found. A significant reduction in expression of desmogleins was seen between high-grade SIL (HSIL) and LSIL (P<0.01) and normal cervix (P<0.001). Desmocollin expression was not reduced significantly, although scores showed significantly greater variation in HSIL compared with LSIL (P<0.05) and normal cervix (P<0.05). There was no significant difference in desmoplakin expression among the three groups. The results suggest that there may be sequential disruption of desmosomal function during neoplastic progression of cervical squamous intraepithelial cells, with downregulation of desmogleins during the progression from LSIL to HSIL and loss of desmocollin expression occurring in some cases of established HSIL.

Desmosomal adhesion regulates epithelial morphogenesis and cell positioning.

Desmosomes are intercellular junctions of epithelia and are of widespread importance in the maintenance of tissue architecture. We provide evidence that desmosomal adhesion has a function in epithelial morphogenesis and cell-type-specific positioning. Blocking peptides corresponding to the cell adhesion recognition (CAR) sites of desmosomal cadherins block alveolar morphogenesis by epithelial cells from mammary lumen. Desmosomal CAR-site peptides also disrupt positional sorting of luminal and myoepithelial cells in aggregates formed by the reassociation of isolated cells. We demonstrate that desmosomal cadherins and E-cadherin are comparably involved in epithelial morphoregulation. The results indicate a wider role for desmosomal adhesion in morphogenesis than has previously been considered.

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.

Desmosomes are reduced in the mouse uterine luminal epithelium during the preimplantation period of pregnancy: a mechanism for facilitation of implantation.

Dynamic regulation of intercellular junctions is an essential aspect of many developmental, reproductive, and physiological processes. We have shown that expression of the desmosomal protein desmoplakin decreases in the luminal uterine epithelium during the preimplantation period of pregnancy in mice. By the time of implantation (between Days 4.5 and 5 of pregnancy), desmoplakin protein can barely be detected by SDS-PAGE and Western blotting, and by immunocytochemistry, it is restricted to well-spaced, punctate dots at the apicolateral junction. Using confocal XZ series and electron microscope quantitation, both the density and distribution of desmosomes along the lateral cell surfaces of luminal epithelial cells were observed to change during early pregnancy. On Day 1 of pregnancy, desmosomes were found at high density in the apicolateral junctional complex, being present here in 79% of ultrathin sections examined, whereas on Day 5, the density was much reduced (present in only 18% of ultrathin sections examined). Desmosomes were found along the lateral surfaces, at or below the level of the nucleus, in 15% of ultrathin sections examined on Day 1 of pregnancy but in only 1% on Day 5. Desmoplakin mRNA declined during the first 4-5 days of pregnancy, along with the protein, suggesting that these changes are controlled at the level of mRNA. This study shows that desmosomes are regulated during early pregnancy, and we propose that a reduction in desmosome adhesion facilitates penetration of the luminal epithelium by trophoblast cells at implantation.

Parenteral administration of an activating monoclonal antibody to the alpha1beta1 integrin in dogs.

In mice, monoclonal antibody (mAb) to the alpha1 integrin abrogate gastro-intestinal damage during graft-versus-host-disease (GVHD), suggesting anti alpha1 mAb as candidates for treatment in humans as well. Our current data show that one such reagent, mAb 1B3.1, when immobilized to plastic wells via rabbit- anti murine (ram) immunoglobulin (Ig) induces a protein kinase-dependent spreading of activated human T cells. Furthermore, it significantly increases the proliferative response, and expression of interleukin-2 (IL-2) receptors (R) and CD69, of resting T cells, expressing minimal integrin on the cell surface, to sub-optimal stimulation by anti-CD3 mAb. We found, in addition, that mAb 1B3.1 a) immuno-precipitates alpha1beta1 integrins from cell-surface iodinated canine epithelial cells b) is highly reactive with canine T cells after their activation and c) inhibits adhesion of canine T cells to collagen IV. Despite the potential ability of the mAb to co-activate T cells in vitro, two dogs that received 4 injections of 0.5-0.3 mg/Kg of mAb 1B3.1 remained healthy, developing only marginal transient lymphopenia. Injection of 0.75mg/Kg in a third dog induced a more marked lymphopenia, and an additional dose of 1.0 mg/Kg 2 weeks later was followed by gastrointestinal hemorrhage. importantly, the lymphopenia was associated with a greater and more persistent decrease of CD8+ than of CD4+ T cells, leading to an increase in the CD4/CD8 ratio 24 hours after the injection. Thus, despite it's co-activating effects in vitro, administration of this mAb in vivo is feasible when appropriately dosed, and may have immuno-modulatory effects.

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.

Expression of a single pair of desmosomal glycoproteins renders the corneal epithelium unique amongst stratified epithelia.

PURPOSE: To determine desmosomal glycoprotein isoform expression in bovine corneal, limbal, and conjunctival epithelium and desmosomal profile and distribution during corneal re-epithelialization. METHODS: Immunofluorescence (IF) for desmosomal components on cryostat sections of fresh epithelia was supported by immunoblot analysis of tissue lysates. Wounded corneas maintained in organ culture were examined by IF at times up to full re-epithelialization (96 hours). RESULT: Immunofluorescence for desmoplakin confirmed desmosome presence throughout all three epithelia. Plakoglobin was also ubiquitous. Of the desmosomal glycoproteins, desmocollin 2 (Dsc2) and desmoglein 2 (Dsg2) were expressed throughout, but Dsc3 and Dsg3 were confined to the limbus and conjunctiva, and Dscl and Dsgl were absent. Dsc2 and Dsg2 IFs were stronger in superficial layers, but Dsc3 and Dsg3 were stronger basally, fading suprabasally. Glycoprotein expression in cornea and conjunctiva was confirmed by immunoblot analysis. No change in glycoprotein expression occurred during re-epithelialization. CONCLUSIONS: Uniquely among stratified epithelia, cornea expresses only a single pair of desmosomal glycoproteins, Dsc2 and Dsg2. Expression of Dsc3 and Dsg3 in limbus and conjunctiva coincides with their association with cell proliferation in other epithelia, but corneal epithelial cells did not express Dsc3 or Dsg3 during re-epithelialization. Absence of Dscl and Dsgl correlates with lack of keratinization in ocular epithelia. These expression patterns may have significance for the specific properties and differentiation patterns of the epithelia. Presence of desmosomes throughout re-epithelialization raises the question of how migrating cells mutually re-position.

Molecular map of the desmosomal plaque.

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the 'a' form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter 'b' form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

Complementary peptides against the major epitope in the NC16A domain of BP180 show no specificity as vaccines to bullous pemphigoid.

A stretch of 14 amino acids (542-555) (MCW-1) in the NC16A domain of BP180 has been shown to be an immunogenic and pathogenic epitope for bullous pemphigoid (BP). Therefore, it provides an excellent target for treatment through a complementary peptide approach, which has been established in other autoimmune diseases, including experimental autoimmune myasthenia gravis. We examined two synthetic complementary peptides BP3CP5 and BP5CP3 against this region. These peptides were derived, respectively, by reading the antisense RNA of this region of BP180 in 3'-5' and 5'-3' directions. We found evident complementarities in hydropathic scores between MCW-1 and both complementary peptides. However, by enzyme-linked immunosorbent assay (ELISA), the complementary peptides BP3CP5 and BP5CP3 did not bind to either synthetic peptide BPNP or glutathione-S-transferase (GST) fusion proteins BP180NC16a and GST-BP-1050. BPNP, BP180NC16a and GST-BP-1050 cover the MCW-1 region of BP180 and were used as the natural peptides in this study. In addition, neither BP3CP5 nor BP5CP3 blocked the reaction between BPNP and anti-BPNP antibody, nor did they block immunofluorescent staining of the basement membrane zone by BP sera. Pre-incubation with BP3CP5 and BP5CP3 did not block the binding of BP sera to the BP18NC16a fusion protein in immunoblotting. Furthermore, rabbit antisera raised against BP3CP5 and BP5CP3 did not bind BP sera in ELISA. Pre-incubation with these rabbit antisera did not inhibit or reduce the binding of BP sera to the autoanltigen in either imnmunoblotting or immunofluorescence. Thus, we concluded that complementary peptides against this particular epitope in BP180 NC16A domain showed no specificity as vaccines to BP, although this approach should be tried for other epitopes in various autoimmune bullous diseases.

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.

Activation of protein kinase C modulates cell-cell and cell-substratum adhesion of a human colorectal carcinoma cell line and restores 'normal' epithelial morphology.

Abnormal cell adhesion is an important contributing factor in invasion and metastasis. Here, we show that morphologically 'normal' cell-cell and cell-substratum adhesion can be restored to a poorly differentiated carcinoma cell line by activation of protein kinase C (PKC). This cell line, VACO 10MS, grows as multicellular aggregates loosely attached to the substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 7.5 nM) induces rapid adhesive changes with 2 components. First, within 15 min of TPA the cells become closely apposed, an event resembling the 'compaction' seen in the mouse early embryo. Next, over 2 hr, the cells spread, forming a monolayer. We show that compaction depends on extracellular calcium, E-cadherin-mediated adhesion and F-actin but not on protein synthesis, microtubules or substratum adhesion. By contrast, cell spreading is independent of cadherin and extracellular Ca2+ but involves the formation of focal contacts containing alpkha(v) integrin. TPA treatment causes rapid translocation of PKC-alpha to the insoluble fraction. During compaction, actin- and PKC-alpha-containing lamellae form over the entire aggregate surface, those adjacent to the substratum appearing to initiate spreading. Compaction does not involve increased phosphorylation of the cadherin/catenin complex. We conclude that activation of PKC-alpha restores 'normal' morphology to these poorly differentiated cells. Our results are of general interest in relation to the regulation of cell adhesion and, through further investigation, may lead to identification of novel targets for therapeutic suppression of invasion and metastasis.

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.

Keratin filament deployment and cytoskeletal networking in a sensory epithelium that vibrates during hearing.

The intricate and spatially precise ways in which keratin intermediate filaments are deployed in certain cochlear epithelial cells, called supporting cells, suggests that these filaments make a micromechanically important contribution to the functional design of the guinea pig organ of Corti. Filament arrays that include keratins 8, 18, and 19 are confined mainly to regions close to the ends of large transcellular microtubule bundles in supporting cells. These cells and their microtubule bundles link sensory hair cells to a specialized basement membrane that vibrates during hearing. The keratin filament arrays apparently help anchor the ends of the microtubule bundles to cell surfaces. Filaments are concentrated at the apices and bases of most cells that contact hair cells. Substantial arrays of adherens junctions link the apices of these cells. Hence, keratin filaments may contribute to a cytoskeletal network that distributes mechanical forces from cell to cell and that coordinates the displacement of neighboring hair cells. However, high concentrations of keratin filaments have not been detected at the apices of one of the supporting cell types, which apparently has a mechanical role that is different from that of the others. Transmission electron microscopy has revealed previously undescribed filament networks at all the locations where the binding of antibodies to keratins is most marked. There is evidence that intercellular linkage of the keratin networks via their association with actin-containing meshworks and adherens junctions is more extensive than linkage provided by desmosomes.