Apical Supernatants Research Articles

Basolateral activation with TLR agonists induces polarized cytokine release and reduces barrier function in RPE in vitro

PurposeSystemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1β in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability.MethodsRPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1β. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining.ResultsNone of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells.ConclusionsDanger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.

Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia.

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

Does the circadian clock make RPE-mediated ion transport “tick” via SLC12A2 (NKCC1)?

ABSTRACTThe presence of a circadian clock in the retinal pigment epithelium (RPE) was discovered recently. However, little is known about mechanisms or processes regulated by the RPE clock. We cultured ARPE-19 monolayers in a transwell culture system, and we found rhythmic mRNA expression of the sodium-potassium-chloride co-transporter SLC12A2. We localized the corresponding protein product, NKCC1, on the apical membrane of ARPE-19 cells. We found that concentrations of sodium, potassium, and chloride oscillated in apical supernatants. The ion concentration gradients between supernatants strongly correlated with SLC12A2 mRNA expression. Our results suggest that the circadian clock regulates ion transport by the RPE via NKCC1 expression.

The Diacetyl-Exposed Human Airway Epithelial Secretome: New Insights into Flavoring-Induced Airways Disease.

Bronchiolitis obliterans (BO) is an increasingly important lung disease characterized by fibroproliferative airway lesions and decrements in lung function. Occupational exposure to the artificial food flavoring ingredient diacetyl, commonly used to impart a buttery flavor to microwave popcorn, has been associated with BO development. In the occupational setting, diacetyl vapor is first encountered by the airway epithelium. To better understand the effects of diacetyl vapor on the airway epithelium, we used an unbiased proteomic approach to characterize both the apical and basolateral secretomes of air-liquid interface cultures of primary human airway epithelial cells from four unique donors after exposure to an occupationally relevant concentration (∼1,100 ppm) of diacetyl vapor or phosphate-buffered saline as a control on alternating days. Basolateral and apical supernatants collected 48 h after the third exposure were analyzed using one-dimensional liquid chromatography tandem mass spectrometry. Paired t tests adjusted for multiple comparisons were used to assess differential expression between diacetyl and phosphate-buffered saline exposure. Of the significantly differentially expressed proteins identified, 61 were unique to the apical secretome, 81 were unique to the basolateral secretome, and 11 were present in both. Pathway enrichment analysis using publicly available databases revealed that proteins associated with matrix remodeling, including degradation, assembly, and new matrix organization, were overrepresented in the data sets. Similarly, protein modifiers of epidermal growth factor receptor signaling were significantly altered. The ordered changes in protein expression suggest that the airway epithelial response to diacetyl may contribute to BO pathogenesis.

COMPARISON OF EFFECTS INDUCED BY FORMALDEHYDE EXPOSURE ON ALVEOLAR AND BRONCHIAL EPITHELIAL CELLS IN VITRO

Background and Aims Exposure to formaldehyde (FA) at environmental concentrations is suspected to be associated to asthma and respiratory disorders. Nevertheless, the physiopathology of this relation is still unclear and experimental data are needed. In vitro experimentation is complementary to epidemiological studies. However experimental procedure parameters need to be adapted, such as the choice of target cells of respiratory tract. The aim of this study was to compare the inflammatory response of FA exposure at environmental level on alveolar and bronchial epithelial cells. Methods Human alveolar (A549) and bronchial (BEAS-2B) epithelial cells were exposed at the air-liquid interface in a Vitrocell exposure module to FA (50 μg/m3) (or air for control) during 30 min. After 24h post-incubation, cellular viability was assessed using LDH release. Two chemokines (IL-8, MCP-1) were assayed in the apical supernatant by ELISA. In addition, to modulate inflammatory response, pre-sensitization was performed using LPS (5 μg/mL) (to mimic a bacterian activation), TNFα (1 ng/mL) or macrophages conditioned media (to mimic the macrophages/epithelial cells cooperation) during 16h before exposure. Results Whatever the cell type, cell viability was unaffected by air or FA exposure compared to cells not exposed at the air-liquid interface. Without presensitization or after LPS presensitization, FA had not effect on A549 or BEAS-2B, compared to air-exposed cells. FA exposure, after TNFα presensitization, resulted in an inflammatory response, an increase of IL-8 production by A549 cells; after macrophage conditioned media presensitization, an increase of IL-8 production by A549 cells, and decrease of MCP-1 production by BEAS-2B cells was observed. Conclusions After presensitization FA exposure at environmental levels provokes an inflammatory response in epithelial cells of the respiratory tract. Moreover, this response is differently modulated depending on the level of the cells in the respiratory tract.

TIMP-3: A novel target for glucocorticoid signaling at the blood–brain barrier

Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood–brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.

T Cell Chemotaxis and Chemokine Release afterStaphylococcus aureusInteraction with Polarized Airway Epithelium

In response to bacterial infection, airway epithelium releases inflammatory mediators including cytokines and chemokines that lead to immune cell efflux and could stimulate the adaptive T cell immune response. The aim of our study was to analyze, in a double chamber culture, the chemokine changes in response to Staphylococcus aureus and their consequences for T cells. Our data show that S. aureus stimulates basolateral and apical release of IL-8 and eotaxin by airway epithelial cells. We also observed increased chemokine receptor expression on CD8+ and CD4+ T cells and enhanced chemotaxis of CD4+ T cells toward apical supernatant. Our data strongly suggest that S. aureus interaction with airway epithelium contributes to specific migration of T cells to inflamed sites.

037 Comparative chemokine secretory profiles in normal and CF polarized epithelial cells following S. aureus infection

Introduction Staphylococcm aureus (S. aureus) is one of the most common causes of infections in human and one the first pathogens infltrating the lung in patients with cystic fibrosis. Branchial epithelial cells are actively involved in the pathogenesis of airway inflammation through the production of multiple factors such as cytokines and chemokines. Cytokines release can lead to the efflux of immune cells, involved in the innate immune response, and stimulate the adaptative immune response. The aim of our study was to analyze 1) the apical and basolateral chemokine profiles obtained by polarized normal human airway epithelial cells (MM39) and CF human airway epithelial cells KM4, 2) the effect of S. aureus interaction with polarized MM39 and KM4 on these chemokine profiles. Methods Polarized monolayer of transformed human tracheal gland cell lines were prepared by seeding 5.10 5 cells/cm 2 on the top of microporous polyester supports. The cells were maintained in a submerged System. For each experiment, confluent airway epithelial cells were apically infected with alive S. aureus . The apical and basolateral supernatants were coUected at different time points, and the chemokine profiles for each condition were analyzed. Results A number of chemokines are secreted by the MM39 and KM4 airway among them IL-8, NAP-2, MIP-1β, Eotaxin-2, RANTES. Following S. aureus contact, the secretion of these chemokines by MM39 cells is increased, while their secretion by KM4 cells is maintained in the basal level. Differences in the levels of the chemokines present in the apical or basolateral compartment of each cell line culture are observed. Looking at the time-dependent and bacteria-dependent IL-8 secretion by MM39 and KM4, we observed an increase of the apical IL-8 secretion by MM39 while the IL-8 secretion level was constant for the KM4. Conclusion Following stimulation of the cells by S. aureus , the chemokine secretion alteration by the MM39 polarized epithelium is more pronounced than for the KM4 polarized airway epithelium. Furthermore, for most of the chemokines studied, the variation is more important in the apical supernatant. This could represent an important factor to be taken into consideration in the development of the lung immune response.

Intraepithelial Cell Neutralization of HIV-1 Replication by IgA

HIV is transmitted sexually through mucosal surfaces where IgA Abs are the first line of immune defense. In this study, we used paired IgA and IgG mAbs against HIV gp160 to study intraepithelial cell neutralization and inhibition of HIV replication. African green monkey kidney cells, Vero C1008, polarizable epithelial cells transfected to express the polymeric Ig receptor (pIgR), were transfected with HIV proviral DNA, and intracellular neutralization mediated by the mAbs was assessed. D47A and D19A IgA, which neutralized HIV in a conventional assay, potently inhibited intracellular HIV replication as assessed by infecting HeLa-CD4-long terminal repeat/beta-galactosidase cells (human cervical carcinoma cell line) and CEMx174 cells (human T cell line) with apical supernatant, basolateral medium, and cell lysate from transfected cells. D47A also inhibited the production of virus as assessed by direct assay of p24. In contrast, D47 and D19 IgG, sharing the same V regions, but which were not transcytosed by the pIgR, did not inhibit intracellular HIV replication, nor did D47A and D19A IgA in pIgR- cells, incapable of transcytosing IgA. Confocal immunofluorescence microscopy showed prominent colocalization of HIV protein and D47A, in agreement with the intracellular neutralization data. D10A, which did not neutralize HIV in the conventional assay, and irrelevant IgA did not show intracellular neutralization or colocalization. Control studies with two kinds of conditioned medium confirmed that HIV neutralization had indeed occurred inside the cells. Thus, during its transcytosis through epithelial cells, HIV-specific IgA can neutralize HIV replication.

Red blood cells regulate eosinophil chemotaxis by scavenging RANTES secreted from endothelial cells.

Eosinophils play a critical role in the pathogenesis of allergic diseases. CC chemokines, such as regulated on activation, normal, T cell expressed, and secreted (RANTES), are key regulators of eosinophil locomotion. Although eosinophils migrate from the bloodstream into tissues, mechanisms that generate a chemogradient across the endothelium remain to be fully elucidated. We first examined the polar secretion of RANTES by endothelial cells. We also studied the functional scavenging effect of red blood cells (RBCs) on RANTES secreted into the intravascular side. Endothelial cells were cultured in a transwell chamber with a membrane pore size of 0.45, 3.0, and 8.0 microm and stimulated with TNF-alpha, IL-1beta, or IFN-gamma from the apical or basolateral side for 16 h. The measurement of RANTES in the supernatant was performed by ELISA. We did not see any difference in the amount of RANTES secreted from the cytokine-stimulated endothelium between inner (intravascular side) and outer (extravascular side) wells separated by the 8.0-microm membrane, although apical polarization was observed with the 0.45-microm membrane. The addition of RBCs (hemoglobin (Hb): 0.5-15 g/dL) to the apical supernatant of TNF-alpha-stimulated endothelial cells reduced the RANTES level in a concentration-dependent manner. The treatment of supernatant on the intravascular side with RBCs significantly enhanced the migration of eosinophils. RBCs possess a scavenging effect on intravascular RANTES, and thereby regulate transendothelial migration of eosinophils. Our findings suggest a new role of RBCs in allergic inflammation.

Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model.

Three defense functions of immunoglobulin A (IgA), immune exclusion, intracellular neutralization, and virus excretion, were assessed in a measles virus model using polarized epithelial cells expressing the polymeric immunoglobulin receptor and monoclonal antibodies against the viral H and F envelope proteins and the internal N protein. Anti-H IgA was the most effective antibody at preventing infection via the apical surface, i.e., immune exclusion. This IgA was also the most effective at intraepithelial cell neutralization after infection at the apical surface and endocytosis of IgA at the basolateral surface, although an antibody against the internal N protein was also effective. In the intracellular neutralization experiments, confocal immunofluorescence microscopy showed prominent colocalization of anti-H IgA and H protein inside virus-infected cells, whereas colocalization of anti-F and F protein and of anti-N and N protein was much less, in agreement with the neutralization results. Combinations of IgA anti-H, anti-F, and anti-N showed no synergistic effects in intracellular neutralization. In the immune excretion experiments, virus immune complexes with either anti-H or anti-F IgA placed beneath polarized epithelial cells could be transported to the apical supernatant. Anti-F IgA, which was relatively poor at immune exclusion and intracellular neutralization, was the most robust at virus excretion. Thus, the studies collectively demonstrated three different antiviral functions of IgA in relation to epithelium and also suggested that the particular viral component with which a given IgA antibody reacts is an important determinant of the magnitude of the antiviral effect.

Neutrophil-mediated epithelial injury during transmigration: role of elastase.

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, beta-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.

Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes.

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% +/- 12.5% of the total virus yield compared with 0.23% +/- 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.

Intracellular neutralization of virus by immunoglobulin A antibodies.

IgA is thought to neutralize viruses at the epithelial surface of mucous membranes by preventing their attachment. Since IgA, a polymeric immunoglobulin, is transported through the lining of epithelial cells by the polymeric-immunoglobulin receptor and since viruses are obligate intracellular parasites, we hypothesized that IgA antibodies may also interfere with viral replication by binding to newly synthesized viral proteins within infected cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the polymeric-immunoglobulin receptor were infected on the apical surface with Sendai virus. Anti-Sendai virus IgA monoclonal antibody delivered from the basolateral surface colocalized with viral protein within the cell, as documented by immunofluorescence. More importantly, anti-viral IgA reduced virus titers greater than 1000-fold (P less than 0.0001) in apical supernatants and greater than 10-fold (P less than 0.0001) in cell lysates from monolayers treated with anti-viral IgA compared with those treated with either anti-viral IgG or an irrelevant IgA monoclonal antibody. We believe that the differences in viral titers between cell layers treated with specific IgA, which enters the epithelial cell by binding to the polymeric-immunoglobulin receptor, and those treated with specific IgG, which does not enter the cells, or irrelevant IgA indicate that specific intracellular IgA antibodies can inhibit viral replication. Thus, in addition to the classical role of humoral antibodies in extracellular defense, IgA antibody may be able to neutralize microbial pathogens intracellularly, giving IgA a role in host defense that has traditionally been reserved for cell-mediated immunity.