Psoriasin is a low molecular weight Ca2+-binding protein with known antimicrobial activity. Since humanairway epithelial cells produce a number of powerful antimicrobial agents as part of their host defence,we investigated whether psoriasin was expressed in human bronchial epithelial cell lines. Expressionwas investigated in 16HBE14o- cells, derived from a normal individual, and compared to CFBE41o- cells,derived from a cystic fibrosis patient. We also examined psoriasin expression following treatment withfactors pertinent to the CF lung-oxidant stress and exposure to pro-inflammatory cytokines. CFBE41o-cells demonstrated much reduced psoriasin levels compared to the 16HBE14o- cells. Increased psoriasin
xidant stressseudomonas aeruginosa
expression was seen following treatment with IL-22 and a cytomix of the pro-inflammatory cytokinesIL-1�, TNF-� and IFN-�; however, the oxidant stressor tert-butyl hydroperoxide had no apparent effect.Over-expression of human psoriasin into both cell lines resulted in increased internalization of Pseu-domonas aeruginosa. In conclusion, expression of psoriasin – which has known anti-microbial activityin other systems – appears to be reduced in CFBE410- compared to 16HBE14o- cells, and its expressionmodified by exposure to pro-inflammatory cytokines.
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. Introduction
Psoriasin (or S100A7) is a low molecular weight, Ca2+-bindingrotein originally described in psoriatic skin lesions (Madsen et al.,991) and involved in keratinocyte differentiation (Broome et al.,003). Psoriasin possesses powerful antimicrobial activity; forxample, it is strongly protective against Escherichia coli in bothormal skin (Glaser et al., 2005) and wounds (Lee and Eckert, 2007),nd though less potent, does demonstrate antimicrobial activitygainst S. aureus and Pseudomonas aeruginosa (Glaser et al., 2005).
Airway epithelial cells produce a number of powerful antimi-robial agents, often in common with the skin, since both barriersncounter significant amounts of microflora (Hiemstra, 2007; Hatand Gallo, 2008). In the airways, these antimicrobial agents areecreted into the airway surface fluid, where they can act as the firstine of defence against inhaled pathogens. Cystic fibrosis (CF) lungisease is characterized by repeated, devastating, cycles of infec-ions with opportunistic pathogens such as P. aeruginosa, many ofhich are now demonstrating resistance against standard antibi-
tics. Strategies which would enhance the innate immune system
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
f the CF airway epithelia would potentially be of benefit as anlternate therapeutic approach. We wished to investigate whetherirway epithelial cells expressed psoriasin, and hypothesized that
rotein extraction, cells were cultured on 100 mm diameter Falconulture dishes (Becton Dickinson, Franklin Lanes, NJ). For cell treat-ents, regular culture media was removed and replaced with one
ontaining no FBS, either in the presence of the agent of interest or vehicle control, for 24 h.
.2. RNA extraction, RT-PCR and quantitative PCR
Total RNA was extracted using TRIzol reagent (Invitrogen) andeverse transcription performed using MMLV reverse transcriptaseInvitrogen) in the presence of 5 mM dNTP (Invitrogen) and 1 �Mligo dT (Amersham Pharmacia, Baie D’Urfe, PQ, Canada). PCR waserformed to investigate psoriasin and IL-22 receptor (IL-22RA1nd IL-10RB) mRNA expression using the following primersforward, reverse): psoriasin: 5′-CCAGCAAGGACAGAAACT-3′
nd 5′-AAGCAAAGATGAGCAACAC-3′; IL-10RB: 5′-TCAGGGCTGAATTTGCA-3′ and 5′-CCCTCGAACTTGAACACA-3′;
L-22RA1: 5′-CTACTATGCCAGGGTCA-3′ and 5′-TCTGTGTCAGGGGTCA-3′. Primers (Invitrogen) were used at00 nM. PCR experiments were performed in the presence of.5 mM MgCl2, 10X Taq buffer with KCl, 2.5 U Taq polymerase (allrom MBI Fermentas, Burlington, ON, Canada) and 5 mM dNTPInvitrogen) in a total reaction volume of 25 �l. After an initialenaturation at 95 ◦C for 1 min, the samples were denatured at4 ◦C for 30 s, allowed to anneal at 52 ◦C (55 ◦C for IL-10RB and
L-22RA1) for 30 s, and extended at 72 ◦C for 30 s for 35 cycles.eactions were completed by a final extension at 72 ◦C for 10 min.ach PCR reaction was performed at least 3 times on differentassages of cells.
Quantitative PCR (qPCR) was used to investigate changesn psoriasin gene expression following exposure to our treat-
ents of interest when compared to that of the housekeepingene hypoxanthine guanine phosphoribosyltransferase (HPRT),sing the Lightcycler thermal cycler system (Roche Appliedcience, Laval, PQ, Canada) as described in Roy et al., 2006.rimers sequences were 5′-GCCAGACTTTGTTGGATTTG-3′ and 5′-TCTCATCTTAGGCTTTGTATTTTG-3′, with conditions as follows:0 min at 95 ◦C, followed by 45 cycles of 95 ◦C for 10 s, 60 ◦C (forPRT) or 52 ◦C (for psoriasin) for 5 s and 72 ◦C for 10 s. The amount ofsoriasin transcript was normalized to the level of HPRT transcriptwhich was unaffected by any of the treatments) and normal-zed data subjected to analysis of variance (ANOVA) followed by
Tukey’s post hoc test as appropriate. All values are reported asean ± S.E.M. with statistical significance reported at p < 0.05.
.3. Immunoblotting
Cells were scraped, spun down and the pellet resuspended in lysis buffer containing 10% SDS and 15 mg/ml DTT in the pres-nce of HaltTM protease inhibitor (Thermo Scientific, Ottawa, ON,anada). Total protein lysate was run on a 12.5% polyacrylamide gelnd transferred to nitrocellulose membrane (Bio-Rad Laboratories,ississauga, ON, Canada). Immunoblotting to confirm psoriasin
rotein expression was performed using a mouse anti-human pso-iasin monoclonal antibody (clone # 47C1068; Imgenex, San Diego,A) at 1:750 dilution. After incubation with the primary antibody,he membrane was incubated with a goat-anti-mouse horse-radisheroxidase (HRP)-conjugated secondary antibody in 5% non-fatilk at 1:7500 dilution (all Jackson ImmunoResearch, West Grove,
A). Proteins were detected using an ECL Plus kit (Amershamharmacia). Unfortunately, peptides corresponding to the epitope
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
equences of the psoriasin antibody were not commercially avail-ble and thus could not be used to confirm specificity. Therefore,ontrol experiments consisted of those in which the primary anti-ody was omitted (results not shown). Additionally, protein lysate
PRESS& Neurobiology xxx (2012) xxx– xxx
from the human mammary epithelial cell line MCF-10A was used
as a positive control (Carlsson et al., 2005).
For protein quantification, a densometric analysis was per-
formed by scanning the film using a UMAX Powerbook III (UMA,
Dallas, TX) and the band images quantified using ImageJ software
(version 1.39, National Institute of Health) using �-actin as the
housekeeping protein. The �-actin primary antibody (rabbit anti-
human polyclonal #4967; Cell Signalling Technology, Danvers, MA)
was used at 1:1000 dilution, while an HRP-conjugated goat anti-
rabbit IgG (Jackson ImmunoResearch) was used at 1:5000 dilution
Fig. 1. Psoriasin expression in human airway epithelial cells. A. cDNA transcripts were detected for psoriasin (246 bp) in both 16HBE14o- and CFBE41o- cells M = 100 bpm s tota1 nd CFe helial
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arker. B. Using qPCR, the relative expression of psoriasin transcript (expressed a6HBE14o- (n = 9). C. Immunoblotting of protein from total cell lysate from HBE axtremely low in CFBE cells. +ve is a sample of lysate from MCF-10A mammary epit
.6. Materials
Human recombinant IL-1�, TNF-�, IFN-� and IL-22 were pur-hased from R&D Systems (Minneapolis, MN) and tert-butylydroperoxide (t-BOOH) from Sigma–Aldrich.
. Results
.1. Expression of psoriasin in model human airway epithelialells
Psoriasin mRNA expression was confirmed via RT-PCR in6HBE14o- and CFBE41o- cells (Fig. 1A). The fragment detectedas of the predicted size (246 bp), and was not detected with-
ut reverse transcription or when water was substituted for theemplate (results not shown). Using qPCR, we next examined theelative expression of psoriasin transcript between the two cellines as total number of copies per �l RNA. Using this approach,
e determined that CFBE41o- cells express significantly less pso-iasin mRNA than 16HBE14o- (Fig. 1B). Protein expression wasonfirmed via immunoblotting on total cell lysate from the twoell lines (Fig. 1C). The protein detected was of the correct size11.4 kD). Although the protein was detected, it was apparenthat the CFBE41o- cells expressed considerably less psoriasin than6HBE14o- cells, since equivalent amounts of total protein were
oaded (30 �g).
.2. Psoriasin expression is unchanged in response to oxidanttress
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
We next investigated whether exposure to oxidant stressffected psoriasin expression in these airway epithelial cell lines,s has previously been reported for mammary epithelia (Carlssont al., 2005). Replicating the protocol of Carlsson et al. (2005),
l number of copies/�l RNA) was considerably less in the CFBE41o- cells than theBE cells demonstrated the presence of psoriasin protein, though expression was
cells, known to express high levels of psoriasin protein.
we used hydrogen peroxide (H2O2, 100 �M) for 1 h as the oxi-
dant stressor, and cells were then allowed to recover for two days.
However, in neither cell line did this protocol result in any signif-
icant changes in psoriasin levels as measured by qPCR (results not
shown).
Therefore we decided next to investigate whether a longer expo-
sure to oxidant stress, which is highly pertinent to the CF airways,
affected psoriasin expression. To do this, we utilized the more sta-
ble oxidant stressor t-BOOH, widely used to induce oxidant stress
(Jung et al., 1998; Matsuno et al., 2008). In order to establish
a non-cytotoxic dose of t-BOOH, we exposed cells to increasing
concentrations of t-BOOH and monitored cell viability using an
MTT assay. After 24 h, no significant cell death was apparent in
the 16HBE14o- cells at t-BOOH concentrations between 50 and
500 �M, while 1000 �M was toxic to nearly all of the cells (Fig. 2A).
In contrast, the CFBE41o- cells succumbed to t-BOOH at much lower
concentrations (Fig. 2B); for example, application of 200 �M t-
BOOH resulted in a cell viability of 98.9 ± 2.1% for the 16HBE14o-
cells versus 4.2 ± 1.3% for the CFBE41o- cells, suggesting the CF cells
are inherently less able to deal with exposure to oxidant stress.
We next examined psoriasin protein expression over a range of
different concentrations of t-BOOH. 16HBE14o- cells were treated
with between 0 and 500 �M t-BOOH for 24 h, protein extracted and
immunoblotting performed. Concentrations greater than 500 �M
t-BOOH induced so much cell death that it was not possible to
extract protein. Following normalization to the house-keeping pro-
tein �-actin, there was no change in psoriasin expression with
oxidant stress (Fig. 2C and D). A similar pattern was apparent
with the CFBE41o- cells, which were treated with between 0 and
100 �M t-BOOH. Again, higher concentrations did not permit pro-
f psoriasin in human airway cystic fibrosis epithelia. Respir. Physiol.
tein extraction due to the high levels of cell death. In this case, the 242
level of protein expression was so low (Fig. 2C) that it was impossi- 243
ble to perform quantification; however, there is no change in signal 244
by eye. This lack of effect on psoriasin expression is in contrast to 245
Fig. 2. Effects of the oxidant stressor tert-butylhydroperoxide (t-BOOH) on cell viability and psoriasin expression. A. Application of increasing doses of t-BOOH to 16HBE14o-c y. In ca ows imd , but
* t.
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ells had little effect on cell viability prior to 1000 �M, as determined by MTT assas the mean ± s.e.m. of 4 individual experiments, with 8 replicates/dose. Panel C shoses of t-BOOH. Densometric analysis was possible only for the 16HBE14o- cellsSignificance at p < 0.05 as determined by ANOVA, followed by Tukey’s post hoc tes
he results observed in mammary cells, where protein expressionas increased (Carlsson et al., 2005) and suggests differential roles
or this protein between the different cell types.
.3. Effects on psoriasin expression following IL-22 treatment
Interleukin (IL)-22 has been proposed as an agent that increaseshe innate immunity of tissues (Whittington et al., 2004; Wolkt al., 2004) and which mainly targets epithelial cells, includinghose of the respiratory and gastrointestinal systems (Wolk et al.,004). It has also been reported to upregulate psoriasin expression
n keratinocytes (Wolk et al., 2006); thus we wished to investigatehether it affected psoriasin expression in airway epithelia. IL-22
chieves its effects through a receptor composed of two subunits –he IL-22RA1 and IL-10RB chains – which co-assemble to produce
functional IL-22R complex (Kotenko et al., 2001). To the best ofur knowledge there have been no previous reports concerninghe expression of the IL-22R complex in these cell lines; there-ore we first examined whether the IL-22RA1 and IL-10RB subunitsere present and thus able to form a functional receptor complex.
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
ndeed, we were able to detect mRNA expression for both subunitsn both cell lines via RT-PCR (Fig. 3A).
Next we investigated psoriasin gene expression in responseo 24 h of IL-22 treatment. 16HBE14o- cells treated for 24 h
ontrast, CFBE41o- cell succumbed at a much lower dose (B). Results are expressedmunoblotting for psoriasin and �-actin protein from cells treated with increasing
revealed no difference in psoriasin expression with increasing oxidant stress (D).
demonstrated a significant increase in psorasin gene expression:
treatment with 40 ng/ml increased expression to 160 ± 3.9% com-
pared to untreated cells (Fig. 3B), while 100 ng/ml resulted in an
increase to 242 ± 14.5% (results not shown). Cell viability after
treatment with IL-22 was measured via the MTT assay, with no
significant cell death apparent at either 40 ng/ml or 100 ng/ml for
either cell line. We then treated CFBE41o- cells with the lower dose
of IL-22 to see whether there was any effect on psoriasin expres-
sion; however, no change was apparent (Fig. 3B).
Finally, we investigated whether IL-22 treatment had any effect
on psoriasin protein expression. Treatment of 16HBE14o- cells
with 40 ng/ml or 100 ng/ml IL-22 resulted in modest, but signifi-
cant, increases in psoriasin protein of 184.8 ± 19.3% and 148.5 ± 6%,
respectively (as normalized to �-actin expression). The same
low, almost non-detectable, level of protein was observed in the
CFBE41o- cells precluding quantification (Fig. 3C and D).
3.4. Effects of exposure to pro-inflammatory cytokines on
psoriasin expression
f psoriasin in human airway cystic fibrosis epithelia. Respir. Physiol.
Although there are no previous reports of psoriasin expression 286
being affected by exposure to pro-inflammatory cytokines, since 287
high levels of pro-inflammatory cytokines have been measured 288
in the CF airway (Bonfield et al., 1999), we wished to investigate 289
Fig. 3. Effects of IL-22 treatment on psoriasin expression. RT-PCR confirmed theexpression of two subunits (IL-22RA1 and IL-10RB chains) which co-assemble toproduce a functional IL-22R complex in both cell types (A). Treatment of cells withIL-22 (40 ng/ml, 24 h) increased psoriasin mRNA expression in 16HBE14o- cells, buthad no effect on CFBE41o- cells. Data are normalized to untreated 16HBEo- values.(pc
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B). This increase was also apparent via western blotting (Panels C and D). Psoriasinrotein was barely detectable in the CFBE41o- cells. Whole cell lysate from MCF-10Aells is shown as a positive control.
hether exposure to IL-1�, TNF-� or IFN-� would have any effectn psoriasin expression. Both gene and protein expression werenvestigated via qPCR and immunoblotting. Exposure to IL-1�2.5 ng/ml) for 24 h had no effect on psoriasin gene expression inhe 16HBE14o- cells; however, a significant increase was seen athe protein level (Fig. 4A and B). A significant increase was alsoeen at the gene level in the CFBE41o- cells (Fig. 4A); however,soriasin protein expression was again too low to permit accurateuantification. Treatment with TNF-� or IFN-� (both at 100 ng/mlor 24 h) resulted in significant increases at the gene level in bothells lines. However, this was not seen at the protein level in6HBE14o- cells (Fig. 4C–F). Finally, treatment with a cytomix ofL-1�, TNF-� and IFN-� together resulted in increases in psoriasinene and protein expression (Fig. 4F and G).
.5. Increased expression of psoriasin enhances P. aeruginosanternalization
Initially, P. aeruginosa strain 8821 was applied to both cell lines
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
or 90 min prior to the application of an antibiotic combination.hen the cells were then lysed, permitting the intracellular bacte-
ia to be plated and quantified, there was a statistically significantncrease in the number of intracellular P. aeruginosa (i.e. bacteria
PRESS& Neurobiology xxx (2012) xxx– xxx 5
that had been internalized during the course of the initial 90 min
exposure) in the CFBE41o- cells when normalized to controls (Fig.
5A). Next, we employed a transient transfection model to increase
psoriasin expression in both cell lines using a commercially avail-
able human psoriasin construct. After 48 h transfection, there was
an increased psoriasin expression of approximately two-fold in
16HBE14o- cells compared to non-transfected and empty-vector
control cells (Fig. 5B), while the fold-increase in the CFBE41o-
cells was somewhat higher (approximately four-fold) over the non-
transfected controls. The protein level in the psoriasin-transfected
CFBE41o- cells was higher than in the non-transfected 16HBE14o-
cells (167.5 ± 6.2% vs 100%; n = 6; Fig. 5B and C).
Cells (16HBE14o- and CFBE41o-) transfected with psoriasin for
48 and 72 h were then exposed to P. aeruginosa for 90 min (as
above). Again, the numbers of intracellular, internalized bacteria
were counted to investigate whether increasing psoriasin proteinexpression affected the numbers of bacteria internalized duringthis short time-frame of infection. In both cell lines the numbersof intracellular P. aeruginosa were significantly reduced following
psoriasin transfection compared to the empty vector control. Dueto high inter-experimental variability, internalized P. aeruginosa
numbers from psoriasin-transfected cells were normalized to the
numbers from vector-alone controls when equivalent bacterial
numbers were applied. In 16HBE14o- cells, intracellular bacterial
counts were 69.7 ± 5.4% and 68.7 ± 8.7% of control after 48 and 72 h,
respectively of psoriasin transfection (both n = 3; Fig. 5D), while for
the CFBE41o- cells, levels fell to 63.1 ± 3.7% (n = 5) and 64.7 ± 4.8%
(n = 4) after 48 and 72 h (Fig. 5E).
4. Discussion
We here demonstrate psoriasin mRNA and protein expression
in a human airway epithelial cell line widely used as a model of nor-
mal bronchial epithelium (16HBE14o-) and in a model CF cell line
(CFBE41o-), in which expression was severely reduced. Psoriasin
expression has previously been described in tissue taken from large
cell lung carcinomas (Zhang et al., 2008); however, we believe this is
the first report of this protein in cell lines derived from normal, non-
cancerous, airway epithelia. The cell lines used in this study were
not derived originally from cancerous sources and are considered
to reflect a normal, and CF, phenotype respectively (Cozens et al.,
1994; Kunzelmann et al., 1993; Ehrhardt et al., 2002). Although
widely used as model cell lines, it is important to note that they are
not a matched pair of CF versus non-CF cell lines; furthermore, our
findings relate only to these particular cell lines, and that caution
must be used when extrapolating these findings to normal and CF
airway phenotypes.
Psoriasin reportedly plays a number of important and diverse
biological roles throughout the body, though its potential role in
the airways is only beginning to be investigated. Our initial find-
ing was that CFBE41o- cells expressed severely reduced levels of
psoriasin mRNA and protein when compared to the 16HBE14o-
cells. This suggests to us that whatever the biological role of psori-
asin in 16HBE14o- airway epithelial cells, this role will be severely
compromised in the CFBE41o- cells. Thus, we sought to investi-
gate whether its expression could be modified under conditions of
physiological stress pertinent to airway infection and inflamma-
tion, hypothesizing that such changes may suggest a potential role
for psoriasin in the airways. To do this, we investigated whether
a number of agents affected psoriasin expression based either on
(a) their known ability to regulate psoriasin in other systems or (b)
f psoriasin in human airway cystic fibrosis epithelia. Respir. Physiol.
their potential relevance to CF, and inflammatory lung disease in 370
general. IL-22, a cytokine reported to increase psoriasin expression 371
in skin cells (Wolk et al., 2006), also had the same effect on the 372
16HBE14o- cells at both the gene and protein levels, while there 373
Fig. 4. Effects of cytokine exposure on psoriasin expression. qPCR and immunoblotting revealed differential changes in expression following treatment of 16HBE14o- andCFBE41o- cells with IL-1� (2.5 ng/ml; A and B), TNF-� (100 ng/ml; C and D), IFN-� (100 ng/ml; E and F) or a cytomix containing all three (G and H), all for 24 h. For the qPCRexperiments, data are expressed in relation to the control 16HBEo- samples, which are set at 100%. Densometric analysis was attempted by also probing membranes for� catioT
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-actin; however, the levels of psoriasin protein were so low that accurate quantifiukey’s post hoc test.
as no apparent effect on the CFBE41o- cells (Fig. 3). This lack ofn effect with the CFBE41o- cells may be a reflection of our findinghat mRNA levels are so low in this cell line, it is simply below theevel of sensitivity of our qPCR approach to detect changes, or it
ay reflect another phenotypic difference between the cell lines.During the course of this study we do clearly identify expres-
ion of IL-22 receptor components in both these airway epithelialell lines, which have not been previously described, while report-ng that IL-22 application has at least one significant potential
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
iological effect on 16HBEo- cells (i.e. up-regulation of psoriasinxpression). Although it has not been studied extensively in theespiratory system, there is an increasing body of evidence that IL-2 plays several important roles in pulmonary epithelial biology.
n was not possible. *Significance at p < 0.05 as determined by ANOVA, followed by
For example, it appears that IL-22 plays a crucial role in mediat-
ing pulmonary host defence against Gram-negative bacteria in a
model of murine pneumonia, partly via increasing production of
antimicrobial peptides (Aujla et al., 2008; Aujla and Kolls, 2009).
It also ameliorates airway inflammation and tissue damage in a
murine-model of allergic lung disease (Taube et al., 2011), while
Hoegl et al. (2011) recently reported that inhaled IL-22 protects
against ventilator-induced lung injury in a rat model. These authors
also demonstrated expression of a functional IL-22 receptor in the
f psoriasin in human airway cystic fibrosis epithelia. Respir. Physiol.
model human alveolar epithelial cell line, A549. We here demon- 396
strate for the first time expression of the components of a functional 397
IL-22 receptor in the model cell lines 16HBE14o- and CFBE41o- 398
. Thus, we believe these cells lines may represent useful in vitro 399
Fig. 5. Transient over-expression of psoriasin increases P. aeruginosa internalization in airway epithelial cells. P. aeruginosa strain 8821 was added to 16HBEo- and CFBE41o-cells at an MOI of 22 for 90 min, after which antibiotics were added to kill external bacteria. Significantly more bacteria were internalized in the CFBE41o- cells compared to16HBEo-. Bacterial colonies were counted in triplicate on 6 separate occasions. Human psoriasin (propagated in pCMV6-XL5) was transfected into 16HBE14o- and CFBE41o-cells for either 48 or 72 h. Panel B shows a representative western blot using antibodies against psoriasin and �-actin for non-transfected, empty vector controls and psoriasin-transfected cells after 48 h transfection. Densometric analysis (Panel C) reveals the relative expression of psoriasin protein compared to 16HBEo- controls (n = 3). Followingp P. aeru4 een 3c
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soriasin transfection, there is a significant decrease in the number of intracellular
8 or 72 h of transfection. Bacterial colonies were counted in quadruplicate on betwontrol as determined by Student’s t-test.
odels to investigate the mechanism of IL-22 action on airwaypithelia, which would appear to be an area of growing interest.
Modest, but significant, increases in psoriasin mRNA and proteinere also detected in response to a cytomix of pro-inflammatory
ytokines (Fig. 4) suggesting a possible role for this product in andaptive host defence response against inflammatory mediators.aution must again be applied in interpreting the results in theFBE41o- cells, since the copies numbers are so low. However,tatistically significant increases in psoriasin mRNA were seen in6HBE14o- cells in response to IL-1�, TNF-� and IFN-� individ-ally as well as together (Fig. 4). However, differences apparent
n the 16HBEo- cells at the gene level were not always seen withrotein, and vice versa, possibly as a result of the low number ofxperimental trials (three) for each data point. However, there was
clear and unequivocal increase in psoriasin message and proteinn response to the cytomix, clearly demonstrating that exposureo pro-inflammatory cytokines can affect expression. However,t is impossible to say which is the key cytokine triggering this
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
ncrease or indeed, whether the combination of all three cytokiness required. These are the first reports of the effects of thesemportant inflammatory mediators on psoriasin expression using
odel airway epithelial cells. Psoriasin has been reported to be
ginosa seen compared to empty vector controls seen for both cell lines after either and 5 separate occasions. *Significance at p < 0.05 vs the appropriate empty vector
down-regulated in response to IFN-� in human breast cancer
epithelial cell lines (Petersson et al., 2007), though the apparentdiscrepancy between our results and those in breast cancer cells
may be related to differences in downstream signaling targets in
the cell types investigated.
We were keen to investigate the role of exposure to oxidant
stress, since this is known to play a central role in the pathogenesis
and progression of a number of inflammatory pulmonary diseases
including CF (van der Vliet et al., 1997). However, we were unable
to detect any changes in psoriasin protein expression in either cell
line over the concentrations of t-BOOH we were able to investigate.
Our findings contrast those of Carlsson et al. (2005), who reported
an increase in psoriasin after a short term exposure to H2O2 and
proposed the protein may be cytoprotective against ROS-induced
cell death in mammary epithelium. However, when we replicated
the oxidant stress protocol used by Carlsson et al. (2005), we were
unable to detect any differences in our model systems. Instead, we
decided to move to a more stable oxidant stressor, t-BOOH, which
f psoriasin in human airway cystic fibrosis epithelia. Respir. Physiol.
should persist in an active form for longer, enabling us to inves- 440
tigate chronic oxidant stress exposure. Interestingly, the CF cells 441
succumbed to oxidant stress at a much lower concentration of t- 442
BOOH than the non-CF, suggesting that their absence of a functional 443
FTR channel may be detrimental to their overall well-being. How-ver, such significant cell death was observed above 500 �M and00 �M t-BOOH in the 16HBEo- and CFBE41o- cells, respectivelyhat it was impossible to extract protein for western blotting. Its possible, of course, that psoriasin expression would be altered athese high concentrations, but the physiological significance is lim-ted. Our finding of a lack of effect on psoriasin expression after 24 hxposure contradicts our initial hypothesis and makes it extremelynlikely, in our opinion, that psoriasin is playing an adaptive roleo combat deleterious oxidant stress, at least in 16HBE14o- cells.
Finally, since psoriasin has reported anti-microbial activity, weished to investigate whether increasing its expression would
ffect how these cell lines treated a bacterial strain of relevanceo CF lung disease. The CF lung is often chronically infected due toolonization by bio-film forming, mucoid, strains of P. aeruginosaHøiby, 2011); therefore we chose to investigate a mucoid strainf P. aeruginosa isolated from a CF patient (strain 8821). Airwaypithelial cells are the first line of defence against inhaled bacte-ia and because we were interested in the innate ability of theseells to cope with an infection burden, we additionally chose tonvestigate only their response to infection in a short time-framei.e. 90 min). Initially, when P. aeruginosa were applied to both cellines at an equivalent amount (MOI of 25), the numbers of bacterianternalized by the CFBE41o- line were significantly higher thanhe 16HBE14o- cells. Furthermore, transfection of a human psori-sin construct into both cell lines, resulting in increased levels ofsoriasin protein, resulted in significantly decreased P. aeruginosa
nternalization when compared to controls. Therefore, we concludehat the more psoriasin protein expressed by a cell, the less P.eruginosa will be internalized.
Previous studies have reported that various strains of P.eruginosa can enter airway epithelial cells; however, the mech-nism of entry and the biological consequences of internalizedacteria are unclear. Plotkowski et al. (1999) determined that P.eruginosa internalization into airway epithelial cells depended notpon the presence of functional CFTR at the cell membrane, butather whether the cells were fully polarized and in possession offfective tight junction complexes. Although normal airway epithe-ial cells are normally highly resistant to apoptosis, infection with. aeruginosa can lead to apoptosis in susceptible cell lines, suchs those that do not develop tight junctions (Rajan et al., 2000).n general it would appear that poorly polarized cells and/or dam-ged epithelial are more susceptible to P. aeruginosa infection. It ismportant to note that cells were not polarized in the present study,hus eliminating the potential effects of tight junction complexes
Cannon et al. (2003) reported there is an inherent defect in thebility of CF cells to undergo apoptosis following P. aeruginosa infec-ion. Their proposed hypothesis was that rapid apoptosis of infectedirway epithelial cells was an essential part of host defence, per-itting infected cells (and thus internalized bacteria) to be cleared
rom the lungs. CF cells displayed a delayed apoptotic response to. aeruginosa infection, which permitted the bacteria to evade hostefence mechanisms and remain in the infected airway cells for
onger. Darling et al. (2004) added to this work by reporting that. aeruginosa are internalized into CFBE41o- cells, which possessF508- CFTR, where they can survive for extended periods with-
ut harming the infected cells, again escaping innate host defenceechanisms. These authors proposed that the presence of a mutant
FTR, such as �F508- CFTR, leads to an increased susceptibility ofnfection by P. aeruginosa, which can remain in the cells for longer.
Although the aims of the present study were not to investigatehe effects of mutant/wild type CFTR on Pseudomonas internaliza-
Please cite this article in press as: Li, T., Cowley, E.A., Reduced expression oNeurobiol. (2012), http://dx.doi.org/10.1016/j.resp.2012.06.028
ion, the results we find do broadly fit into the hypotheses describedbove. Transfection of human psoriasin into either cell line resultedn a significant decrease in the number of colony forming, viable,acteria we were able to recover when compared to controls. This
PRESS& Neurobiology xxx (2012) xxx– xxx
indicates an increased number of bacteria were internalized in the
presence of enhanced psoriasin expression. Psoriasin is predom-
inately a secreted protein, thus one interpretation of our data is
that the higher the level of psoriasin within the cell, the more will
be secreted, which prevents bacteria from becoming internalized.
Conversely, the lower the levels of this protein, the more bacteria
become internalized permitting them to remain within the cells for
longer.
A significant limitation of this approach is that it only mea-
sures bacterial internalization and we have not investigated any
extracellular effects against bacteria. Furthermore, we have not
investigated bacterial killing, which would be a true indication
that this protein is an effective anti-microbial. The initial report
of psoriasin’s anti-bacterial activity reported that it possessed a
potent ability to kill several strains of E. coli, with more modest
anti-microbial effects against S. aereus and P. aeruginosa (Glaseret al., 2005). Finally, we have only investigated one strain of P.aeruginosa in the present study, a strain not examined in theinitial report. However, our data, while modest, do support the
hypothesis that increased levels of psoriasin can result in protec-tion against internalization of mucoid P. aeruginosa and that, while
non-physiological, over-expressing this protein results in a change
in the way airway epithelial cells cope with this pathogen. Psoriasin
is only one of a large number of anti-microbial agents released from
airway epithelial cells (Hiemstra, 2007; Hata and Gallo, 2008) and it
is unclear how this protein would interact with other anti-microbial
proteins in the airway surface liquid. However, we believe that
our data suggests that increased investigation of this protein may
provide another piece of information as to how CF cells are more
susceptible to bacterial infection.
In conclusion, we describe for the first time the expression of
psoriasin in cell culture models of normal and CF airway epithelial
cell lines, as well as its modulation by exposure to oxidant stress,
IL-22 and other inflammatory cytokines. While the present work
does not definitively ascribe a biological role for psoriasin in airway
epithelia, the low levels in CFBE41o- cells hint at the possibility that
the role of this protein may be compromised in the CF airway, and
may be worthy of further investigation in native cells. Furthermore,
we demonstrate that increasing psoriasin expression is effective
in decreasing internalization of a mucoid P. aeruginosa strain, the
first time this has been demonstrated using model human airway
epithelial cells.
Acknowledgments
We wish to thank Christina Jones for excellent technical assis-
tance, Dr Eileen Denovan-Wright for assistance with the qPCR
studies, Dr Andrew Stadnyk for critical reading of the manuscript,
and Drs Tong-Jun Lin and Dr Nikhil Thomas for assistance with
the P. aeruginosa work. This work was supported entirely by Cystic
Fibrosis Canada.
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