Epithelial Cells LID-PEG Skills Course

Becky Vonakis, Ph.D. Assistant Professor of Medicine,

Johns Hopkins Asthma & Allergy Center

July 24, 2012

Outline

• Physiological function of epithelial cells in the immune system

• Pathological changes in asthma and Chronic Obstructive Pulmonary Disease (COPD).

• Human airway epithelial cell culture models

• Functional assays for Air-Liquid Interface (ALI) cultures

Immune Functions of Epithelial Cells

• Define the border between ourselves and the environment – Respiratory, intestinal, urogenital mucosa

• Transport – Ions, macromolecules – Dynamic endocytic network

• Host Defense – Against environmental toxins, bacteria, viruses – innate immune system – adaptive immune system – Specialized cellular functions: cilia

Immune System Functions

• Barrier function – Structural and functional polarization

• Apical surface exposed to the environment • Basolateral surface communicates with mucosal immune

system • Paracellular barrier via intercellular (tight) junctions

• Secreted factors • Mucus • Immunoglobulins

• Associated cells • direct contact : dendritic cells • secreted mediators: chemokines, cytokines

Molecular Structure of Tight Junctions

Multiple Levels of Interaction Between the Epithelium and Immune Cells

Epithelium in Innate Immune Recognition: Homeostatic Response

Epithelium in Innate Immune Recognition: Protective Response

Epithelium in Innate Immune Recognition: Pathogenic Response

Mechanisms in Atopic Asthma

Asthma Classifications

• Phenotypes – Trigger

• Allergic (atopic) • Non-allergic (cold-air)

– Severity • Medication use • Airflow obstruction / reversibility

– Cellular components • Neutophils (Fatal asthma) • Granulocytes (eosinophils, mast cells, basophils) • B cells (immunoglobulins) • Monocytes, Dendritic cells (antigen presentation) • T lymphocytes (subsets polarized by cytokine exposure)

Epithelium in Asthma Progression

Chronic Inflammation and Airway Remodeling

Tight Junction Disruption In Asthma

Coordination of Mucous Metaplasia and Chronic Airway Inflammation

Respiratory Viruses Target the Epithelium Leading to Asthma Exacerbations

COPD

• Emphysema and chronic bronchitis

• Limited airflow in the airway that is not fully reversible

• Associated with chronic inflammation and alveolar destruction

• Primary risk factor is tobacco smoke

Epithelium in Cigarette Smoke Inflammation

Airway Mucus Hypersecretion: a Common Feature in Asthma and COPD

Culture Models of Human Airway Epithelial Cells

• Cell lines – Simple, inexpensive to grow

– Transformed phenotype

• Submerged Primary Culture – Moderate expense

– Can be passaged to expand

• Air-Liquid Interface Culture – Most physiological system

– Expensive!!

Cell Lines

• BEAS2B, Calu-3, A549

• Phenotype often passage sensitive, submerged culture for growth

• Useful for studies of respiratory viruses with epithelium and cytokine/chemokine secretion

• Calu-3 can be grown on inserts to form tight junctions and apical media removed for few hours but grow very slowly

Submerged Culture

• Cell Source

– Cadaveric human lungs or animals

– Commercial (Lonza)

• Stimuli to apical surface through liquid not air

– Cigarette smoke extract

– Diesel particle extract

ALI Culture

• Most closely mimics human airway epithelium in terms of cell composition and molecular structure

• Permits physiological stimulation with airborne substances (smoke, diesel exhaust)

Establishment of Air-Liquid Interface (ALI) Cultures

• Cells are harvested from cadaveric human lungs /lung&nose brushing through IRB-approved protocols.

• After dissection, airway mucosa is digested with proteases and cells are plated in normal submerged culture for expansion

• For experiments, cells are plated on transwells consisting of a filter support that allows manipulation of separate apical and basolateral compartments.

• After growth to confluency, media is removed from the apical surface and the culture differentiates into an pseudostratified columnar epithelial layer with ciliated cells and goblet cells readily apparent.

• These cultures are then used to study synthesis and secretion of mucus components, transepithelial barrier and transport, ciliary beat frequency, gene expression and other features specific to airway epithelia.

conner.med.miami.edu/web/conner.html

Electron Microscopy of Mature ALI: Multiple Cell Types Evident

Transmission electron micrograph of PFC/OsO4-fixed human airway epithelia grown on a semipermeable membrane filter.

Labeled structures include air, filter, ASL, cilia (arrowheads), goblet cells (G), basal cells (B), and ciliated cells (C). Scale bar equals 5 μm.

Electron Microscopy of Mature ALI: Cross-section

Scanning electron micrograph of PFC/OsO4-fixed differentiated human airway epithelia. View is from edge of filter where epithelium has separated from membrane filter

Labeled structures are filter (F), filter pores (arrowheads), basal cells (B), cilia (C), epithelia (E), and mucus layer (M). Scale bar equals 5 μm.

Differentiation of ALI Cultures Times after seeding: 3 d (A,B), 6 d (C,D), 10 d (E,F), and 14 d (G,H). In panels A, C, E, and G, the scale bar indicates 37.5 μm, and in panels B, D, F, and H, the scale bar indicates 5 μm.

Stimuli Employed

• Cytokines/ Chemokines – TH2 cytokines for asthma studies (IL-13, TSLP) – TH1 cytokines for COPD studies (IL-17, IL-1beta)

• Innate Receptor Ligands – Products of bacteria, viruses

• Bacteria, Viruses – Frequent cause of disease exacerbations

• Environmental toxins – Cigarette smoke – DEP

Functional Assays: Barrier Function

• Transepithelial electrical resistance – Measured with “Chopstick” ohmmeter

– Ionic permeability

• Macromolecular permeability – FITC-dextran (4 KD)

– AlexaFluor-488-BSA (60 KD)

– Add labeled molecule to apical surface in buffer, incubate for 45 min., 37deg.C, measure fluorescence in basolateral supernatant

Functional Assays: Viability

• WST-1 metabolic assay

– Measures cell health through quantification of cellular NADPH levels

– Kit from Roche

– Add reagent to apical layer, incubate at 37deg.C for 1 hr., read absorbance in ELISA plate reader.

– Compare untreated, media, stimulated cells.

– Higher the value, more viable cells recovered

Functional Assays: Cellular Signaling

• Proteomics and Phosphoproteomics – Scrape cells or detach with non-trypsin

dissociation buffer

– Lyse in detergent-containing buffer with inhibitors (protease, phosphatase, kinase cocktail)

– Proceed with Western blot

• Receptor Binding Assays – Harvest cells, incubate with fluorescently-labeled

ligand, filter to separate cell-associated ligand from free ligand, measure fluorescence on filters

Functional Assays: Cytokine/Chemokine Arrays

• Proteome Profiler arrays from R & D Systems

• 21 cytokines and chemokines measured from 1 supernatant

• Mix supernatant with cocktail of biotinylated antibodies. Membrane contains capture antibodies to cytokines/chemokines, incubate membrane with Bt-antibody- supernatant, detect with streptavidin-HRP and chemiluminesence on film.

Functional Assays: Gene Expression

• Real-Time PCR

– Scrape cells, extract total RNA with Trizol, PCR to detect cytokine gene expression

• Gene expression arrays

– Messenger RNA

– Micro RNA

– Core facility

References

• Holgate, ST, The sentinel role of the airway epithelium in asthma pathogenesis. Immunol. Reviews, 242: 205-219, 2011

• Karp, PH, et al An In Vitro Model of Differentiated Human Airway Epithelia: Methods for Establishing Primary Cultures, Methods in Molecular Biology, vol. 188: Epithelial Cell Culture Protocols, Edited by: C. Wise, Humana Press Inc., Totowa, NJ