CC BY
62Assessment of respiratory sensitizers: Cytokine responses in a 3D alveolo-capillary barrier model in vitro
M.I. Hermanns1*, J. Kasper1, R.E. Unger1, G. Carpentier2, E.L. Roggen3, and C.J. Kirkpatrick1
1 Institute of Pathology, Johannes Gutenberg-University Mainz, 55122 Germany 2 Faculte des Sciences et Technologie, Universite Paris Est Creteil Val-de-Marne, 94000 France 3 Novozymes, Bagsvaerd, 2880 Denmark
Inhalation of high- and low-molecular weight chemicals is a leading cause of allergic respiratory diseases. Respiratory epithelium has not only a barrier function, but contributes to pathogenesis via immuno-modulatory effector mechanisms. In this context, the epithelium is the first cell-type which primarily interacts with inhaled allergens. Our aim was to investigate the in vitro effects of respiratory sensitizers (trimellitic anhydride (TMA), diphenylmethane diisocyanate (MDI), ammonium hexachloroplatinate IV (HCPt) and three enzymes) on specific cells of the human distal lung barrier. We examined the influence of these compounds on a human lung epithelial cell line with characteristics of alveolar type II and Clara cells (NCI H441) and on the microvascular endothelial cell line (ISO-HAS-1) in an established co-culture model of the alveolo-capillary barrier. Apical exposure (inhalative exposure) to subtoxic doses of the compounds (maximum of 25% loss of barrier integrity compared to vehicle control) did not markedly affect the barrier function. However, a clear compartmentalized cytokine release was detected. All respiratory sensitizers (HCPt, TMA, MDI and enzymes) induced a specific cytokine release pattern in the basolateral compartment (corresponding to the vascular lumen) after apical exposure, whereas dermal sensitizers (DNCB, CA and the irritant SA) did not show such an effect. This immuno-modulatory effector mechanism could be a possible contribution of the distal lung epithelial and endothelial cells to the pathogenesis of respiratory sensitization. Our data suggest that measuring a basolateral release of cytokines in apically exposed co-cultures may represent a promising in vitro model for the screening of potential chemical respiratory allergens.
Keywords: 3D tissues, in vitro, proinflammatory cytokines, respiratory toxicity, sensitizer
Abbreviations
AEpC—alveolar epithelial cells
AM—alveolar macrophages
CA—cinnamic aldehyde
DC—dendritic cells
DNCB—dinitrochlorobenzene
HCPt—ammonium hexachloroplatinate
HTS—high throughput screening
ISO-HAS-1—human microvascular endothelial cell line
MDI—diphenylmethane-diisocyanate
TER—trans-monolayer or bilayer electrical resistance
TMA—trimellitic anhydride
SA—salicylic acid
SLS—sodium lauryl sulphate
* Corresponding author
Dr. rer. nat. Iris Hermanns, e-mail: [email protected]
1. Introduction
Respiratory allergy is a hypersensitivity reaction in the upper and lower respiratory tract to a protein or chemical xenobiotic. The lungs represent a primary site of entry into the body for innumerable amounts of respired particles and potential irritants, making defence mechanisms in the lung such as cilia, mucous secreting cells, and cytochrome (P450) enzymes involved in the biotransformation of xenobiotics essential [1]. In the general population, respiratory allergy is most frequently induced by environmental proteins, including pollen, dust mite excretions and animal dander. However, in occupational settings, respiratory allergy can be mediated by industrial compounds. These include high molecular weight (HMW) compounds, such as proteins [2], and low molecular weight (LMW) chemicals such as iso-cyanates, reactive dyes, and acid anhydrides, or metals [3, 4]. Due to their small size, LMW chemical allergens first react with proteins to create a complex that is subsequently
Original text © M.I. Hermanns, J. Kasper, R.E. Unger, G. Carpentier, E.L. Roggen, C.J. Kirkpatrick, 2015 © Institute of Strength Physics and Materials Science, Siberian Branch, Russian Academy of Sciences, 2015. All rights reserved.
able to initiate an immune response (haptenation). Development of a respiratory allergy to HMW and LMW compounds can contribute to the formation of asthma, which is the most prevalent occupational lung disease in developed countries. As a result, identification and characterization of compounds which have the potential to act as respiratory allergens represent an important area of research for industrial toxicologists. Respiratory sensitization is also relevant for assessing human health hazards of implantable medical devices. While processing medical devices airborne particles may arise and an inhalation exposure is possible. The respiratory sensitizer diphenylmethane-diisocyanate for example is used as a raw material in manufacture of poly-urethanes. Polyurethane foam and other polyurethane materials are increasingly used in medical applications including wound dressings, breathable adhesive film bandages, catheters, stent coatings, and pacemaker lead insulation. In case of implanted medical devices a leaching of compounds may result in respiratory sensitization. For example ammonium hexachloroplatinate, present in silicone breast implants, is suggested to cause asthma [5]. Additionally, elec-trodeposition from ammonium hexachloroplatinate solution has been proposed for fabrication of Pt-coated implantable electrodes for cardiac electrotherapy [6] and for implantable glucose fuel cells [7].
The evolution of hypersensitivity resulting in respiratory allergy consists of two distinct stages. The first is sen-sitization, which involves the development of an immune status, while the second is elicitation, which results in the clinical manifestation of allergy [8]. In the early events following allergen exposure the respiratory sensitizer reacts with the epithelial layer of the lung tissue. With an area of approximately 100-140 m2 [9] the alveoli of the human lung represent the largest exposure area of the respiratory tract, compared to an area of 12 m2 for the upper respiratory tract. Besides the lung epithelium, the basement membrane [10], the connective tissue [11], and the capillary endothelium [12, 13] also serve as structural barriers in the deep lung. Upon contact with a sensitizing compound the alveolar epithelial cells (AEpC) as well as the directly neighbouring endothelial cells (EC) cross-talk, resulting in a selective pattern of cytokines which attract immune cells, such as alveolar macrophages (AM) and dendritic cells (DCs).
The aim of the present study is to assess the ability of an in vitro alveolo-capillary barrier model to discriminate between skin contact and respiratory sensitizers by analyzing the pattern of cytokines secreted after contact with the different compounds. The model consists of a 3D co-culture consisting of an adenocarcinoma epithelial cell line (NCI H441) and a capillary endothelial cell line (ISO-HAS-1) on opposite sides of 24-Transwell® filter membranes which results in a functional alveolo-capillary barrier in vitro. This 3D model was shown to mimic the layered structure of the lower respiratory tract barrier, allowing cross-talk between
both cell types and resulting in the generation of a distinct alveolar and capillary compartment. The final goal is the development of a high throughput screening (HTS) approach to facilitate a simple, accurate and cost-effective assessment of the respiratory allergy potential of a compound by detecting cytokine release into the alveolar (upper well) or capillary (lower well) compartment.
2. Materials and methods
2.1. Cell culture
Monocultures of ISO-HAS-1 and NCI H441: Prior to seeding cells the 96-well plates (TPP Switzerland) were coated with 50 ¡xl fibronectin for 1 h at 37°C (5 ¡xg/ml, Roche Diagnostics, Mannheim). The human microvascular endothelial cell line, ISO-HAS-1 [14, 15], and the human lung adenocarcinoma cell line, NCI H441 (purchased from ATCC, ATCC-HTB-174, Promochem, Wesel, Germany), were used in these experiments. The cells were seeded into 96-well plates at 1.6 x104 and 3.2 x 104 cells/well, ISO-HAS-1 and NCI H441, respectively. Cells were cultured in RPMI 1640 medium (Gibco) with L-glutamine supplemented with 10% FCS and Pen/Strep (100 U/100 ¡g/ml) and cultivated at 37°C, 5% CO2 for 24 h prior to compound exposure.
Co-cultures of ISO-HAS-1 with NCI H441: The co-culture was performed as described previously [16] with some minor alterations. Briefly, HTS 24-Transwell® filters (polycarbonate, 0.4 ¡xm pore size; Costar, Wiesbaden, Germany) were coated with rat tail collagen type I (12.12 ¡xg/cm2, BD Biosciences, Heidelberg, Germany). ISO-HAS-1 (6.9 x 104/cm2) were seeded on the lower surface of the inverted filter membrane. After 2 h of adhesion at 37°C and 5% CO2, NCI H441 (2.6 x104/cm2) were placed on the upper surface of the membrane. The cells were cultured for about 10 days in co-culture. Beginning at day 3 of cultivation the NCI H441 were treated with dexamethasone (1 ¡m, Sigma, Taufkirchen, Germany). After 7 days the barriers generally achieve trans-bilayer electrical resistance (TEER) values with an average of (560 ± 6) Q x cm2. Confluent barrier-forming NCI H441 cells on the upper well surface showed a circumferential staining of the tight junction protein ZO-1 (see Fig. 1). The integrity of the ISO-HAS-1 cell layer on the opposite side of the membrane was proven by an uninterrupted distribution of VE-Cadherin at the cell-cell junctions (Fig. 1). Establishment of TEER values that average (560 ± 6) Q x cm2 was chosen for exposure of the co-culture to the learning set of compounds from the epithelial side (apical exposure).
2.2. Sensitizing and irritating compounds
A learning set of compounds was investigated, including six respiratory sensitizers (trimellitic anhydride (TMA), diphenylmethane-diisocyanate (MDI), ammonium-hexa-chloroplatinate (HCPt), three enzymes (amylase, lipase, protease)), three dermal sensitizers (dinitrochlorobenzene (DNCB), cinnamic aldehyde (CA), eugenol (E)), and two
w
VE-Cadherin
Fig. 1. Assembly of the in vitro co-culture model (upper row). TEM micrograph and immunofluorescent staining on day 10 of co-cultivation (lower row). Apical-basolateral differentiated (polarized) NCI H441 (ZO-1, red) on the upper surface and ISO-HAS-1 (VE-Cadherin, green) on the opposite side of the filter membrane; nuclear counter stain blue (scale bar = 10 |im).
non-sensitizers or irritants (salicylic acid (SA) and sodium lauryl sulphate (SLS)). The following compounds were insoluble in water and were therefore dissolved in DMSO: TMA, MDI, DNCB. The final in-well concentration of DMSO was set at 0.1% as maximum. The same level of DMSO present in the respective concentration of a particular test compound was applied to parallel cells as a control (vehicle control).
2.3. Effect of compounds on cell viability
Following exposure for 24 h cytotoxicity of the compounds was investigated in mono-culture by detecting mi-tochondrial enzymatic perturbation (MTS assay, Cell titer Glo Promega). Dose-response curves were created and cytotoxic effect expressed as IC50 value (IC50 = chemical concentration required to reduce cellular metabolic activity— corresponding to cell viability—to 50% of the maximum value compared to vehicle exposed cultures) was determined. To calculate the IC50 a four parameter logistic equation: "log(inhibitor) versus response-Variable slope" in GraphPad Prism 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com) was used.
2.4. Bioelectrical measurements
The trans-bilayer electrical resistance was expressed in Q x cm2 and measured using an EVOM voltohmmeter (World Precision Instruments, Berlin, Germany) equipped
with a pair of STX-2 chopstick electrodes. Briefly, HTS 24-Transwell® filter membranes without cells, filter membranes with cells in the culture medium, and filter membranes with cell lines in medium supplemented with 1 ^M dexame-thasone on day three of co-cultivation were examined over a cultivation period of 10 to 14 days. Electrodes were placed in the upper and lower chamber and resistance measured with the voltohmmeter. Resistance was recorded as the mean resistance x area of three independent experiments. Calculations for Q x cm2 were made by subtracting the resistance measurement of the blank filter coated with collagen type I (approximately 110 Q) and multiplying by the area of the monolayer (0.33 cm2).
2.5. Effect of compounds and vehicles on tissue viability
The alveolo-capillary barrier model was exposed to sub-toxic concentrations of the sensitizers (evaluated before by MTS assay in monoculture). The co-cultures were treated with increasing concentrations of the sensitzers and irritants to determine the concentration at which tissue viability became impaired. Dosing solutions of compound or vehicle control were prepared in RPMI 1640 with increasing concentrations of the test vehicle (0.005-0.1% DMSO). These solutions were added to the tissues in triplicate according to the dosing protocol described below. After 24 h incubation, at 37°C with 5% CO2 and humidity, dose-determining experiments for the co-cultures were performed using reduction in TEER as read-out.
2.6. Exposure
NCI H441 cells were grown to confluency in co-culture with ISO-HAS-1 cells, following which an appropriate concentration of respiratory sensitizers (TMA, MDI, HCPt, amylase, lipase, protease), dermal sensitizers (DNCB, CA, Eugenol), and irritants (SA, SLS), causing a maximum of 25% loss of barrier integrity, were added to the NCI H441 cells (upper well) prior to incubation for 4 and 24 hours. In brief, co-cultures were exposed to the respective sensitizer on the apical surface (luminal side) on day 7 of co-culture, when a stable barrier with TEER-values of (560 ± 6) Q x cm2 was established. A volume of 210^l of the appropriate concentration of sensitizer in RPMI 5% FCS w/o Dex was added to the apical surface (upper well, NCI H441). The corresponding opposite side of the co-culture was exposed to culture medium without test substance. Tissues were exposed for 4 and 24 hours and TEER was measured at each time point. After 24 hours of exposure the culture supernatant was taken from the upper well (corresponding to the alveolar lumen side) and the lower well (corresponding to the intravascular side) for further cytokine analysis.
2.7. Detection of cytokine expression
For the parallel determination of the relative levels of selected human cytokines and chemokines a Human Cyto-kine Array (Proteome Profiler™ Array R&D Systems, Wiesbaden, Germany) was used following the instructions of the manufacturer. In brief, cell culture supernates were diluted and mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture was then incubated with the multiplex array. Any cytokine/detection antibody complex present was bound by its cognate immobilized capture antibody on the membrane. Following a wash step to remove unbound material, streptavidin-horseradish pero-xidase and chemiluminescent detection reagents were added sequentially. Western blotting images were taken after ten minute exposures under the Molecular Imager Chemidoc XRS+ (Bio-Rad, Munich, Germany) and density was analyzed in ImageJ [17]. Image panels and densitometric analysis were accomplished using the previously described ImageJ tool (ImageJ protein array analyzer macro by G. Carpentier [18]). "IntDen" (the product of Area and Mean Gray Value) was used as parameter of relative level of the selected human cytokine. The relative levels of cytokine release into the upper and lower well are expressed as ratio [%] between the compound and the vehicle-treated co-culture ("IntDen" of compound / "IntDen" of vehicle control x100). The cytokines and chemokines detected by the multiplex array are listed in Table 1.
2.8. Statistical analysis
From several independent measurements, means and standard deviations were calculated. Data are shown as mean ± S.E. from at least three separate experiments. Testing for significant differences between means was carried out
Table 1. List of cytokines monitored by cytokine array
Abbreviation Cytokine name
C5a Complement component 5a
CD154 CD40 Ligand
G-CSF Granulocyte-colony stimulating factor
GM-CSF Granulocyte macrophage-colony stimulating factor
CXCL1 Neutrophil-activating protein 3
CCL1 Chemokine (C-C motif) ligand 1
sICAM-1 Soluble intracellular adhesion molecule 1
IFN-y Interferon y
IL-1a Interleukin 1 a
IL-1 ß Interleukin 1 ß
IL-1ra Interleukin receptor agonist
IL-2 Interleukin 2
IL-4 Interleukin 4
IL-5 Interleukin 5
IL-6 Interleukin 6
CXCL8 Interleukin 8
IL-10 Interleukin 10
IL-12 Interleukin 12
IL-13 Interleukin 13
IL-16 Interleukin 16
IL-17 Interleukin 17
IL-17E Interleukin 17E
IL-23 Interleukin 23
IL-27 Interleukin 27
IL-32a Interleukin 32a
CXCL10 Interferon y-inducible protein 10 (IP10)
CXCL11 Interferon y-inducible protein 9 (IP9)
CCL2 Monocyte chemotactic protein 1 (MCP1)
MIF Macrophage migration inhibitory factor
CCL3 Macrophage inflammatory protein 1a (MIP1a)
CCL4 Macrophage inflammatory protein 1ß (MIP-1ß)
PAI-1 Plasminogen activator inhibitor 1
CCL5 Regulated on activation, normal T cell expressed and secreted (RANTES)
CXCL12 Stromal cell-derived factor 1
TNF-a Tumor necrosis factor a
sTREM-1 Soluble triggering receptor expressed on myeloid cells 1
using one- and two-way ANOVA followed by Dunnett's Multiple Comparison or Bonnferoni's post test at a probability of error of 5% (*), 1% (**) and 0.1% (***).
Fig. 2. MTS assay of NCI H441 monocultures with dose-response curves. If applicable, the IC50 was calculated for the respective compound [n.d. = not detectable].
3. Results
3.1. Assessment of IC50 values for each compound in monocultures
In dose finding experiments monocultures of NCI H441 and ISO-HAS-1 were exposed to six respiratory sensitizers (TMA, MDI, HCPt, enzymes (amylase, lipase, protease)), three dermal sensitizers (DNCB, CA, eugenol), and two non-sensitizers or irritants (SA and SLS). Figure 2 depicts the dose-response curves of all compounds tested for the epithelial cell line NCI H441 compared to the dose-response curve of the vehicle exposed cultures (DMSO or culture medium). In general, the ISO-HAS-1 cells showed similar but more sensitive responses to each compound tested (data not shown). IC50 values were determined for HCPt, Protease, DNCB, CA, SA and SLS (Fig. 2). Exposure with MDI, TMA, amylase, lipase and eugenol did not measurably effect cell vitality at the concentrations tested.
3.2. Assessment of TEER values for each compound in co-culture
Dose-response experiments for subtoxic concentrations of the sensitizers were performed using reduction in TEER (maximum of 25% loss of barrier integrity compared to vehicle control) as read-out in co-culture. The IC50 values evaluated for the monocultures of NCI H441 were comparable to a subtoxic effect on TEER values in co-culture for HCPt, protease, DNCB, SA and SLS. The effects of TMA, MDI, amylase, lipase, CA and eugenol on TEER were not attributable to their cytotoxicity on the epithelial cells. TMA, MDI, amylase and lipase did not show obvious changes in cell vitality at any of the concentrations tested in monoculture (see Fig. 2). In co-culture concentrations >0.5 mM TMA clearly reduced TEER after 4 and 24 h of exposure (Fig. 3). MDI at concentrations >0.05 mM caused an increase in TEER compared to the vehicle treated control.
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Fig. 3. Transepithelial electrical resistance (TEER) of the co-culture with time of exposure. Co-culture of H441/ISO-HAS-1 was exposed to subtoxic concentrations of the compounds and respective vehicle controls for 4 and 24 h. Corresponding concentrations of the respective solvent (medium, DMSO).
For amylase, lipase and eugenol a subtoxic dose could also be defined by TEER decreases at concentrations of 433, 80 and 433 |Ag/ml, respectively. The subtoxic concentration of CA (IC50 = 0.652 mM in monoculture) had to be modified after TEER reading to 1 mM CA in co-culture.
3.3. Cytokine expression after exposure to compounds (cytokine multiplex array)
The alveolo-capillary barrier model was challenged from the apical side of the epithelium with subtoxic levels of chemicals and the effects evaluated by TEER measurement. The impact of the test compounds on the cells was investigated using a multiplex array for measuring cytokine release into both upper and lower compartments of the 24-transwell unit. In Fig. 4 the changes in cytokine concen-
trations are depicted as % of the respective vehicle-control treated co-culture. A red colour describes an increase and a blue colour a decrease in cytokine concentration in the respective compartment compared to vehicle-control.
For the respiratory sensitizers, even if the pattern of the cytokines secreted after 24 hours exposure was not the same for MDI, TMA and HCPt, an obvious increase in basolateral release of different cytokines was observed. The same was true for the selected lipase and protease. The cytokine response to a subtoxic dose of metal HCPt and the selected amylase was less intense but in common are increases in basolateral CCL5 or PAI-1, respectively.
In contrast, two of the three dermal sensitizers, DNCB and CA, did not show significant effects on cytokine secretion, even though at certain concentrations the levels were
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Fig. 4. Cytokine multiplex array. The array was performed using the medium in the upper well (representing the luminal side of the lung epithelium) as well as on the medium in the lower well (representing the luminal side of the lung endothelium or the baso-lateral side of the epithelium, resp.) after topical exposure (upper well) to subtoxic concentrations of the selected compounds. Red—elevated concentration; blue—reduced concentration compared to control treated with corresponding concentrations of the respective solvent (medium, DMSO).
slightly lower than values of the vehicle controls. In general, the cytokine response of the dermal sensitizer and irritant exposed co-cultures was less than with the respiratory sensitizers. In addition, the two irritants tested, SA, and SLS, induced a more marked increase in cytokine release into the upper well. 2.5 mM SA showed small increases in upper well CD154, IFN-gamma and PAI-1 concentrations, whereas 0.25 mM SLS strongly stimulated IL-8, and MIF release into the upper well compared to the vehicle control. Furthermore, a basolateral increase of sICAM-1 and IL-8 appeared to be more related to irritation than to respiratory sensitization. On the other hand, basolateral CCL-5 (RANTES) secretion may provide a useful marker to discriminate respiratory sensitizers from irritants. CCL-5 released into the lower well was stimulated by all three respiratory sensitizers and by two of the three enzymes tested.
A more in-depth analysis of the results allowed the identification of cytokine patterns capable of discriminating the different classes of compounds. Thus, skin sensitizers ap-
peared to be characterized by a reduced release of most of the cytokines and chemokines on the apical and the baso-lateral side of the epithelium. If increases in cytokine release were detectable, for example, results observed when cells were exposed to the skin sensitizer eugenol, cytokine release was observed on the apical side. Interestingly, the specific pattern for respiratory sensitizers appeared to occur in the opposite way, with an increased release of most of the cytokines occurring into the basolateral compartment. All respiratory sensitizers induced a preferential cytokine release into the basolateral compartment (corresponding to the vascular lumen) after apical exposure, thus also discriminating them from the two irritants tested.
4. Discussion
To date, the risk assessment for potential lung sensitiz-ers depends completely on the results obtained from animal testing. Although animal models have offered significant insight into the identification of respiratory sensitizers
and allergens, significant differences in both the respiratory anatomy and function in rats and mice make extrapolation to humans very difficult. Therefore, a continued challenge has been the development of human in vitro test systems for assessing and identifying the relevance and expression patterns of in vitro markers, pathways and networks relevant for sensitization of humans. The Precision Cut Lung Slices (PCLS) technology represents a promising test system for the assessment of sensitizing agents and clearly resembles the in vivo situation [19-21]. There have also been attempts to develop models with the less complex monocultures using human primary bronchial cells [22]. In order to gain more insight and enhanced understanding of the relevant interactions of inflammatory processes involving the interplay of several different cell types and mechanisms, in vitro co-culture systems may be valuable tools for allowing a better prediction of effects to be expected in vivo. By using a co-culture system, at least two cell types directly interact and the system has been shown to mimic both the barrier and gene expression patterns observed in the in vivo situation. Although co-culture systems are still lacking many details and characteristics of a differentiated tissue, such systems have been shown to allow cell-cell communication (reviewed by [23]).
We have developed a co-culture system using human distal lung epithelial and microvascular endothelial cells in which stable and confluent monolayers were co-cultivated for 7 to 10 days on the upper and lower surface of collagen-coated HTS 24-Transwell® filter membranes, establishing a functional barrier in a 24-multiwell-test-system. This model mimics the layered interfaces of the alveolo-capil-lary barrier and allows cell-to-cell communication to occur that influences the cellular morphology, differentiation and barrier properties [16]. The system is shown to have in vivo-like functionality in terms of morphology and responses to well-known physiological stimuli, e.g. cytokines [24], and chemical compounds, such as sulfur mustard [25] and nano-particles [26, 27]. Apical exposure to subtoxic concentrations of foreign materials elicits cytokine release to the lower compartment, even if barrier properties remain unaffected. On account of the barrier properties a clear compartmen-talization is generated. Exposure of chemicals to this functional alveolo-capillary barrier in vitro enables studies on their effects in two distinct compartments, the alveolar lumen (apical) and the vascular lumen (basolateral). Thus, this assay can either detect the penetration of two barriers (epithelial and endothelial) by a certain substance (sensitizing, irritating, toxic) or the effects on cross-talk between both cell types following epithelial activation, which simulates inhalation of a foreign substance
In general, test systems based on lung tissue or lung cells have been shown to be more responsive to respiratory sensitizers as compared to skin sensitizers and irritants [19, 22]. In the present study, after exposure of the co-culture
system to respiratory sensitizers, the amount and level of secreted cytokines was generally higher than after exposure to skin sensitizers. Since induction of CCL5 occurs by respiratory sensitizers, this marker may have the potential to discriminate between both respiratory and skin sensiti-zers. The learning set of compounds used in this study clearly demonstrated that increases in the basolateral CCL5 release were solely caused by the respiratory sensitizers. In addition, cytokine patterns were identified that permit discrimination between the different classes of compounds. The metal HCPt is the only compound that induced an apical release of IL-1ra. IL-8 release was induced by respiratory sensitizers and irritants but not by the three skin sensitizers examined. Instead, skin sensitizers appear to be characterized by a decrease of most cytokines on the baso-lateral side compared to vehicle-treated control. Interestingly, the respiratory sensitizers examined all revealed an increased release of distinct, specific cytokine patterns into the baso-lateral compartment.
However, there are limitations to using the alveolo-capil-lary model for potential lung-sensitizer risk assessment since this model is missing the immune cell component, such as AM or DCs that are also present in the lung in the in vivo situation. In addition to the barrier epithelial cells, both macrophages and airway DCs make up one of the first lines of defence against inhaled agents such as allergens and endotoxins [28]. Furthermore, in allergic airway inflammation it was recently shown that the ability of AM to prevent DC activation is impaired [29]. To date, researchers have focused on the early events occurring in the lung (e.g. bioavai-lability, chemical reactivity, EpC and DC responses) in their efforts to develop in vitro tools for assessing the sensitizing potential ofvarious compounds (reviewed by [30, 31]). The pattern of cytokines released after exposure to sensitizers may be essential to induce chemotaxis of immune cells. Based on this, the addition of AM to the existing model described in these studies could give further insight into the early events of sensitization that occur in a healthy alveolo-capillary unit. Due to the intense interactions between the various cell types in vivo, a physiologically relevant model should consist of an AM-AEpC-EC 3D triple-culture cell system or optimally with a more complex AM-AEpC-EC-DC quadruple-culture system.
Nevertheless, the studies described here have shown that the alveolo-capillary co-culture system appears to be a rapid and reproducible model to investigate the response of distal lung epithelial and endothelial cells to respiratory sensi-tizers. Further studies are needed to develop a predictive respiratory allergy model and determine how the cytokines released by these cells might further influence AM, DCs and DC-like cells (e.g. THP-1, MUTZ-3) in the initiation of a functional immune response. A recently developed triple-culture using the described alveolo-capillary model with different phenotypes of AM (J. Kasper, J Tissue Eng Regen Med. 2015; Jun 15. doi 10.1002/term.2032. [Epub
ahead of print]) could be helpful in addressing these questions in future.
In conclusion, the learning set of compounds revealed promising differences in cytokine regulation, especially with respect to basolateral release of CD5a, CD154, IFN-gamma, MIF and PAI-1, depending on which respiratory sensitizers and irritants were applied. Basolaterally released CCL5 discriminated between sensitizers and irritants, as well as respiratory and skin sensitizers. Our understanding of the key events in respiratory sensitization is growing and more complex human test systems are emerging.
Acknowledgments
The authors wish to thank Mrs M. Moisch, E. Hübsch and A. Sartoris for their excellent technical assistance with the cell culture, the cytokine assays and immunocytoche-mical studies, respectively. This study was supported by the FP6 EU-project Sens-it-iv.
Conflict of interest
The authors declare to have no conflict of interests.
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