Cell-specic Interleukin-15 and Interleukin-15 receptor

subunit expression and regulation in pneumococcal

pneumoniaComparison to chlamydial lung infection
Andreas C. Hocke
a
, Matthias P. Lampe
a
, Martin Witzenrath
a
, Hans Mollenkopf
b
,
Jens Zerrahn
b
, Bernd Schmeck
a
, Ulrich Kessler
a
, Matthias Kru ll
a
, Sven Hammerschmidt
c
,
Stefan Hippenstiel
a
, Hartwig Schu tte
a
, Norbert Suttorp
a
, Simone Rosseau
a,
*
a
Charite Universita tsmedizin Berlin, Department of Internal Medicine, Infectious and Respiratory Diseases, Chariteplatz 1, 10117 Berlin, Germany
b
Department of Immunology, Max Planck Institute for Infection Biology, Chariteplatz 1, 10117 Berlin, Germany
c
Research Center for Infectious Diseases, University of Wu rzburg, Ro ntgenring 11, 97070 Wu rzburg, Germany
Received 11 October 2006; received in revised form 21 March 2007; accepted 3 May 2007
Abstract
Interleukin (IL)-15 has critical impact on the homeostasis and activation of natural killer cells, natural killer T cells, cdT cells, and
CD8
+
T cells, and contributes to antimicrobial defenses particularly at mucosal sites. The respiratory tract comprises a large mucosal
surface and harbors signicant amounts of lymphocytes, however the expression pattern of IL-15 in the lung and its role in local immune
responses are largely unknown. We therefore analyzed the dierential expression of IL-15 and the IL-15 receptor (IL-15R) complex in
the lungs of mice and demonstrated substantial constitutive expression in bronchial and alveolar epithelial cells, alveolar macrophages,
and vascular smooth muscle cells, implicating contribution to pulmonary immune cell homeostasis already under normal conditions. The
induction of pneumococcal pneumonia but not the infection with Chlamydophila pneumoniae evoked a signicant up-regulation of IL-15
on alveolar macrophages and bronchial epithelial cells, with the latter presenting de-novo expression of IL-15 on their basolateral surface
and additional up-regulation of IL-15Ra. Moreover, transcriptome analysis as well as semi-quantitative PCR indicated at least partial
transcriptional regulation in mice lungs. In conclusion IL-15 is suggested being of functional importance in the pulmonary immune
response against pneumococcal pneumonia.
2007 Elsevier Ltd. All rights reserved.
Keywords: Chlamydophila pneumoniae; IL-15; Mice; Pneumococci; Pneumonia; Interleukin 15; IL-15 receptor mice; Streptococcus pneumoniae
1. Introduction
Interleukin (IL)-15 is a pleiotropic pro-inammatory
cytokine with particular impact on the activation of innate
and tissue-associated immune responses. It plays a pivotal
role in the generation and maintenance of natural killer
(NK)-cells, NKT cells, cdT, and memory CD8
+
T cells
[1], contributes to the functional maturation of dendritic
cells and macrophages [2], and activates phagocytosis and
cytokine production of neutrophil granulocytes [3]. In
addition, IL-15 initiates chemokinesis and functional
polarization of T cells [4], and induces proliferation and
dierentiation of B cells [5]. Furthermore, IL-15 is a potent
inhibitor of several apoptosis pathways via the induction of
antiapoptotic molecules like Bcl-2 [6,7], or blocking tumor
necrosis factor (TNF) receptor-1-mediated cell death [8].
Although IL-15 mRNA is found in variable tissues and
cell types, concurrent protein expression is known only for
a limited number of cells including monocytes, macro-
phages, and epithelial cells. IL-15 protein consists of two
isoforms; the longer signaling peptide is targeted to the
secretory pathway, whereas the shorter peptide appears
to be restricted to the cytoplasm and nucleus [9]. Both
1043-4666/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cyto.2007.05.009
*
Corresponding author. Fax: +49 30 450 553979.
E-mail address: simone.rosseau@charite.de (S. Rosseau).
www.elsevier.com/locate/issn/10434666
Cytokine 38 (2007) 6173
isoforms are subjected to tight post-transcriptional regula-
tion. Signaling is mediated via interaction with the widely
distributed heterotrimeric IL-15 receptor (IL-15R), which
consists of a b-chain (shared with IL-2), the common c-
chain, and the unique a-chain [10]. The latter is capable
of binding IL-15 with high anity in the absence of
IL-15Rb and IL-15Rc, and cells bearing IL-15Ra can bind
and transpresent IL-15 to immune cells lacking IL-15Ra
[11]. IL-15 has low secretion potential, and soluble IL-15 is
hardly detected in biological systems. Therefore, extracellu-
lar signaling is supposed to be mediated predominantly by
IL-15Ra- or membrane-bound IL-15 [12,13].
Previous studies have implicated a role for IL-15 in host
resistance to intracellular pathogens and viruses, including
Salmonella choleraesuis [14], Listeria monocytogenes [7],
Mycobacteria spp. [15], Toxoplasma gondii [16], and Herpes
simplex virus [17]. In particular, up-regulation of IL-15 at
mucosal sites appeared to account for its protective host
defense mechanisms [18,19]. In conjunction with the nding
of abnormal high IL-15 expression in the intestinal mucosa
in inammatory bowel disease [20], these data implicate a
critical role for IL-15 in mucosal immune responses.
The respiratory tract represents one of the largest muco-
sal surfaces of the body, and it harbors large amounts of
immune cells, including macrophages, dendritic cells, gran-
ulocytes, and various lymphocyte populations. In addition,
previous studies revealed strong pro- and anti-inamma-
tory cytokine expression in lung epithelial, endothelial as
well as vascular smooth muscle cells under inammatory
conditions suggesting an important role of these cell types
for the pulmonary immune-homeostasis. Therefore, the
localization of the cellular expression pattern is important
for the understanding of the interaction between anti-
and pro-inammatory mediators within the complex net-
work in the lungs.
The role of IL-15 in pulmonary immunity is currently
unknown, and the denite prole of IL-15 and IL-15R
complex expression in the lung has not been established.
In the present study, we thus detailed the expression pat-
tern of IL-15 and the IL-15R subunits a, b, and c in the
lungs of mice, and immunohistochemistry suggested a role
for IL-15 in pulmonary immune cell homeostasis already
under normal conditions. Analyses of transcriptional regu-
lation, immunolocalization, and quantitative protein
expression further implicated the contribution of IL-15
and IL-15Ra to pulmonary host defense mechanisms in
the course of pneumonia. Of note, increases of IL-15
expression in the lung depended on the type of microbial
challenge, since up-regulation of IL-15 was detected in
Streptococcus pneumoniae induced pneumonia, but was
not observed after infection with Chlamydophila pneumo-
niae (C.p.).
1
2. Materials and methods
2.1. Experimental protocol
The expression of IL-15 and the IL-15R subunits a, b,
and c was analyzed in female, 8- to 10-week-old C57BL/
6 mice (Charles River, Sulzfeld, Germany). Mice were
housed in the central animal facility at the Charite, main-
tained on a 12-h light/12-h dark cycle under pathogen-free
conditions, and fed autoclaved food and water ad libitum.
All experiments were approved by local authorities (LAGe-
tSi, Berlin).
For the preparation of lungs, mice were deeply anesthe-
tized by intraperitoneal injection of ketamine and xylazine,
and subsequently tracheotomized and ventilated (Mini
Vent type 845; Hugo Sachs Elektronik, March-Hugstetten,
Germany). After sternotomy, animals were sacriced by
cardiotomy, and lungs were perfused with sterile saline at
4 C for 3 min via a pulmonary artery catheter. Thereafter,
lungs were dissected and processed for microscopic analy-
sis, immunohistochemistry, Western blotting, microarray
transcriptome analysis, RT-PCR, or ow cytometry,
respectively. A total of 10 completely untreated mice served
as baseline controls.
2.2. Bacterial strains
Single-colony isolates of S. pneumoniae (serotype 3;
NCTC 7978) were maintained at 37 C and 5% CO
2
on
Columbia agar supplemented with 5% sheep blood (Bec-
tonDickinson, Heidelberg, Germany). Single colonies
were expanded by resuspension in ToddHewitt broth
(BectonDickinson) supplemented with 10% heat inacti-
vated fetal calf serum (Invitrogen, Karlsruhe, Germany)
and 0.5% yeast extract, and incubation at 37 C for 34 h
to the mid-log phase (A
600
, 0.35). Pneumococci were har-
vested by centrifugation, and resuspended in phosphate
buered saline (PBS).
Chlamydophila pneumoniae (TW-183; VR2282, ATCC,
Rockville, Maryland) was cultured and puried as
described previously [21]. Aliquots were diluted in
sucrosephosphateglutamate buer supplemented with
10% fetal calf serum, and stored at 75 C until use.
HEp-2 cells and the C.p. stocks were determined to be free
of Mycoplasma contamination by PCR.
2.3. Induction of pneumonia
For the induction of pneumococcal pneumonia, C57Bl/
6 mice were lightly anaesthetized by intraperitoneal injec-
tion of ketamine and xylazine, and subsequently infected
with 5 10
6
colony forming units of S. pneumoniae in
20 ll PBS by transnasal application. Preparation of mouse
lungs was performed at 6, 12, 24, 48 or 60 h after infection
(n = 5 each) as described above. Control mice received
20 ll of sterile PBS and were sacriced at 6 or 60 h
(n = 3, each), respectively.
1
Abbreviations used: AEC, alveolar epithelial cells; AM, alveolar
macrophages; BEC, bronchial epithelial cells; C.p., Chlamydophila
pneumoniae; IL, Interleukin; NK, natural killer; PMN, polymorphonu-
clear neutrophils.
62 A.C. Hocke et al. / Cytokine 38 (2007) 6173
For the induction of chlamydial lung infection, anesthe-
tized C57Bl/6 mice received 5 10
6
inclusion forming units
of C.p. in a total volume of 25 ll via intratracheal injection
by means of a microsprayer (Penn-Century, Philadelphia,
PA). Animals receiving 25 ll of normal saline or HEp2-cell
suspension served as control groups. Infected mice were
sacriced at 6, 24, 72, 144, or 216 h (n = 5, each), and con-
trol mice (n = 5 each) at 72 or 216 h after treatment.
2.4. Histology
Dissected lungs were instilled with TissueTek OCT com-
pound via the tracheal cannula (Plano, Wetzlar, Germany),
and frozen in liquid nitrogen. Ten micrometers sections
were cut from separated right and left lung tissue blocks
using a cryostat HM560 (Microm International, Walldorf,
Germany). Histopathological assessment was performed
on slides stained with hematoxylin eosin.
2.5. Immunohistochemistry
Immunohistochemistry was performed as previously
described [22]. In brief, lung tissue sections were xed with
paraformaldehyde and rinsed in PBS. The xed sections
were blocked with corresponding serum in PBS and subse-
quently incubation with polyclonal primary antibodies
(Ab) was performed. The specicity of the primary Ab
was veried by preincubation of Ab with corresponding
blocking peptides. Polyclonal Ab against IL-15 (goat
anti-mouse; sc-1296; dilution 1:100), IL-15Ra (goat anti-
mouse; sc-5526; dilution 1:100), IL-15Rb (rabbit anti-
mouse; sc-672; dilution 1:20), and IL-15Rc (rabbit anti
mouse; sc-670; dilution 1:100), as well as the corresponding
blocking peptides were obtained from Santa Cruz (Heidel-
berg, Germany). Replacement of primary Ab with rabbit
or goat serum, respectively, served as negative control.
After overnight incubation sections were washed in PBS
and incubated with alkaline phosphatase-conjugated goat
anti-rabbit F(ab)
2
or rabbit anti-goat F(ab)
2
Ab (Rock-
land, Gilbertsvill, PA, USA; dilution 1:2000) likewise.
Immunostaining was developed at constant conditions
using a Vector Red Substrate Kit (Vector Laboratories,
Peterborough, UK). Levamisole (2.5 mM; Vector Labora-
tories) was added to inhibit endogenous alkaline phospha-
tase activity.
In baseline controls, indirect immunostaining of IL-15
was reassessed by direct immunostaining applying a
cross-reacting monoclonal mouse anti-human IL-15 Ab
(dilution 1:25; R&D Systems, Minneapolis, MN) which
has been previously labeled by the use of the Zenon
Alexa Flour 680 Mouse IgG Labeling Kit (Molecular
Probes, Leiden, Netherlands).
Intracellular staining of IL-15 was achieved by treatment
of lung sections with 1% Triton X-100 (SigmaAldrich) for
15 min. Prior to the microscopic evaluation, lung sections
were counterstained with methyl green. The analysis of each
slide was blinded to two-independent investigators applying
an arbitrary visual scale with grading scores of 0, 1, 2, 3, and
4 representing no, weak, moderate, strong, and intense
staining, respectively. Microscopic examination was per-
formed at a magnication of 200 (PlanNeoFluar; NA
0.5) and included two slides from both right and left lung,
respectively. Each analysis comprised the examination of
all positively stained structures.
2.6. Quantitative analysis of immunostaining by digital
image analysis
Image analysis was performed as described previously
[23]. Briey, measurement was performed with a custom-
designed lter for Vector Red in optical quality (central
wavelength 525 nm; half bandwidth 10 2 nm; manufac-
tured by Chroma Technology Corp., Brattleboro, USA).
A stabilized 12-V/100-W tungstenhalogen lamp was used
for illumination. 12-bit gray-scale images (objective: 40/
numerical aperture 1.3 oil; Zeiss Plan-Neouar) were
acquired using a cooled charge-coupled device camera
(AxioCam MRm, Zeiss, Jena, Germany) mounted on an
Axioskop 2 mot (Zeiss), and processed with ImagePro-
Plus

4.5 (Media Cybernetics, Silver Springs, USA). Back-

ground measurement was performed to evaluate the
inuence of nonspecic antibody binding, and a shade of
gray of 995 (12-bit range: 04095) was set as threshold
for positive staining. For constant microscope settings,
each measurement was preceded by system calibration
using a reference slide. In controls, 10 complete cross-sec-
tions from right or left lungs were measured. Five cross-
sections were analyzed in infected groups. From each
section, 510 images per structure were digitized, and the
region of interest was manually dened. The mean gray
values were automatically measured (ImageProPlus) and
analyzed with GraphPadPrism

4.0 (GraphPad, CA). In

each mouse lung, the data of the dierent structures were
averaged as mean, and considered as one-independent
experiment. The signal intensities in control lungs were
set 100%, and the changes in infected groups were
expressed as percentage of the corresponding control. For
direct visualization of staining intensity a pseudocolor scale
was applied, and bright-eld images were taken with a
cooled high-range-color AxioCam HRc (Zeiss) using the
same magnication.
2.7. Western blotting
Dissected lungs were snap-frozen in liquid nitrogen,
minced, lysed, forwarded to sodium dodecyl sulfate10%
polyacrylamide gel electrophoresis, and blotted onto
Hybond-ECL membranes (Amersham Biosciences, Drei-
eich, Germany). The membranes were blocked, washed,
and hybridized with polyclonal Ab against IL-15 and
ERK2 (Santa Cruz). Protein detection was performed by
visualization of IRDye800- or Cy5.5-labeled secondary
Ab (Odyssey infrared imaging system; LI-COR Inc.,
Lincoln, Nebraska) [24].
A.C. Hocke et al. / Cytokine 38 (2007) 6173 63
2.8. Semi-quantitative RT-PCR
Total RNA was extracted from frozen mouse lungs
using the RNEasy Mini Isolation Kit (Quiagen, Hilden,
Germany), and reverse-transcribed using the Pro Stare
First-strand RT-PCR Kit (Stratagene Europe, Amsterdam,
Netherlands). cDNA was amplied by PCR using primers
for murine IL-15 (337 bp; 5
0
-GCC ATA GCC AGC TCA
TCT TC-3
0
and 5
0
-GCA ATT CCA GGA GAA AGC
AG-3
0
) and murine G3PDH (496 bp; 5
0
-TGA TGG GTG
TGA ACC ACG AG-3
0
and 5
0
-TCA GTG TAG CCC
AAG ATG CC-3
0
). After 40 cycles, PCR products were
analyzed on 1.2% agarose gels by ethidium bromide stain-
ing, using a ChemiGenius bio-imager (Syngene,
Cambridge, UK).
2.9. Microarray transcriptome analysis
Microarray experiments were done as two-color hybrid-
izations. RNA labeling was performed with a Fluorescent
Linear Amplication Kit (Agilent Technologies). In brief,
cDNA was reverse transcribed from 4 lg of total RNA
(derived from ve lungs in each group and time point,
respectively) with an oligo(dT)-T7 promoter primer and
MMLV-RT. Second-strand synthesis was carried out with
random hexamers. Fluorescent antisense cRNA was syn-
thesized with either cyanine 3-CTP (Cy3-CTP) or cyanine
5-CTP (Cy5-CTP) and T7 polymerase. The uorescent-
labeled antisense cRNA was precipitated overnight with
LiCl, ethanol washed and resuspended in water. The puri-
ed products were quantied at A
552nm
for Cy3-CTP and
A
650nm
for Cy5-CTP. Before hybridization, 1.25 lg labeled
cRNA of each product were fragmented and mixed with
control targets and hybridization buer according to the
suppliers protocol (Agilent Technologies). Hybridizations
were done overnight for approximately 17 h at 60 C.
The slides were washed according to the manufacturers
manual and scanning of microarrays was performed with
5 lm resolution using a DNA microarray laser scanner
(Agilent Technologies). In order to compensate dye specic
eects, and to ensure statistically relevant data, a color
swap was performed. The RNA samples were labeled vice
versa with the two uorescent dyes (uorescence reversal).
Features were extracted with the image analysis tool Ver-
sion A6.1.1.1 from Agilent Technologies. Subsequent data
analysis was carried out on the Rosetta Inpharmatics plat-
form Resolver Built 3.2.2. The data discussed in this publi-
cation have been deposited in NCBIs Gene Expression
Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and
are accessible through GEO Series Accession Number
GSE5289 [25].
2.10. Statistical analysis
Data are expressed as means SEM. KruskalWallis
test for non-parametric data was used to determine median
signicance between all groups by ranks. Two-tailed
MannWhitney test was used to determine statistical dier-
ences between stimulated time points vs. control. p Values
<0.05 were considered signicant.
3. Results
Immunostaining was absent in control sections where
the primary antibodies for IL-15 (Fig. 1D), IL-15Ra,
IL-2Rb, and IL-2Rc (Fig. 7C, F, and I) were blocked with
their corresponding blocking peptides or nonspecic
immune serum was applied. Connective tissue or collagen
bers remained negative in all experiments. For IL-15,
identical results were obtained when direct immunostaining
was performed using a primarily uorochrome-labeled
monoclonal anti-human IL-15 Ab (data not shown).
3.1. Histopathology and clinical course of pneumococcal
pneumonia
Histological analysis of infected mice revealed inam-
matory leukocyte inux into the lung emerging 24 h after
transnasal infection. Leukocyte inltrates were predomi-
nantly composed of polymorphonuclear neutrophils
(PMNs) and could be found in the alveolar compartment
as well as in perivascular and peribronchial regions (data
not shown). The perivascular and peribronchial inltrates
additionally contained signicant amounts of lymphocytes.
Pulmonary leukocyte recruitment was absent in baseline
controls and mock-infected control mice, and it was absent
in infected mice 6 and 12 h after transnasal infection.
Forty-eight hours after infection, parts of the lung paren-
chyma appeared severely destroyed. Without exception,
infected mice became moribund after 60 h and all animals
had to be sacriced (data not shown).
3.2. Immunolocalization of IL-15 in normal mice lungs
Indirect immunostaining revealed considerable constitu-
tive expression of intracellular IL-15 in bronchial epithelial
cells (BEC) of proximal and distal airways, as well as in
alveolar epithelial cells (AEC) and alveolar macrophages
(AM) (Fig. 1AC). A weaker staining was noted in vascu-
lar smooth muscle cells of pulmonary veins and bronchial
arteries (Fig. 1A). Extracellular expression of IL-15 was
limited to AM and BEC, with the latter presenting IL-15
solely on their apical surface (Fig. 5A and C).
3.3. Up-regulation of IL-15 protein expression in
pneumococcal pneumonia
Western blot analysis revealed a strong increase of pul-
monary IL-15 expression in the course of pneumococcal
pneumonia emerging 24 h after transnasal infection
(Fig. 2A and B). Immunohistochemistry localized
enhanced IL-15 expression to BEC (Fig. 3A and B) of
proximal and distal airways and to AM (Fig. 3E and F),
whereas IL-15 expression in vascular smooth muscle cells
64 A.C. Hocke et al. / Cytokine 38 (2007) 6173
remained unchanged (Fig. 3B). AEC only moderately
increased their IL-15 levels solely in inltrated areas
(Fig. 3C and D) whereas IL-15 levels of AEC in unaected
regions remained unchanged. A weak staining for IL-15
was noted in inltrating PMNs, which remained constant
in the course of pneumonia (Fig. 3B).
Quantitative digital image analysis conrmed signicant
time-dependent up-regulation of IL-15 in BEC (Fig. 4A
and B) and AM (Fig. 4E and F), only minor changes were
noted in AEC (Fig. 4C and D). Up-regulation of IL-15
protein expression in BEC and AM occurred very rapidly
within 6 h after infection, and persisted or further increased
up to the endpoint 60 h after the application of pneumo-
cocci. Interestingly, BEC and AM of both inltrated areas
and non-aected regions showed signicant up-regulation
of IL-15 levels. Beside the increase of intracellular IL-15,
AM, and BEC also presented strong up-regulation of
extracellular IL-15 (Fig. 5AD). It is worth mentioning
that BEC not only increased IL-15 expression on their api-
cal side, but also displayed de-novo expression of IL-15 on
their basolateral surface (Fig. 5B). In contrast to infected
mice, there was no dierence between pulmonary IL-15
expression in baseline controls compared to mock-infected
control mice 6 or 60 h after transnasal application of PBS,
respectively, as assessed by immunohistochemistry.
3.4. Increase of pulmonary IL-15 mRNA in pneumococcal
pneumonia
Semi-quantitative RT-PCR provided evidence for tran-
scriptional up-regulation of IL-15 in the course of pneumo-
coccal pneumonia emerging 12 h after transnasal infection
(Fig. 6A and B). The results of microarray transcriptome
analysis supported these ndings, and further revealed
Fig. 2. Up-regulation of pulmonary IL-15 protein expression in pneumo-
coccal pneumonia. Mice were transnasally infected with 5 10
6
cfu S.
pneumoniae and sacriced at 6, 12, 24, 48, and 60 h after infection. Lungs
were harvested, the protein fraction was isolated, and immunodetection of
IL-15 was performed by Western blotting (A). Densitometry of IL-15
blots was performed in relation to the loading control ERK2 (B). Blots
representative of three dierent experiments with similar results are
presented.
Fig. 1. Immunolocalization of IL-15 in lung sections of normal C57BL/6 mice assessed by Vector Red/methyl green staining. IL-15 staining was abundant
in BEC (arrows; A), smooth muscle cells of bronchial arteries (arrowhead; A), alveolar epithelial cells (arrows; B); and alveolar macrophages (arrowhead;
C). The specicity of immunostaining was conrmed by prior incubation of the anti-IL-15 Ab with the corresponding blocking peptide (arrows; D).
A representative of ve dierent experiments with similar results is presented. Original magnications 400, (C) 630.
A.C. Hocke et al. / Cytokine 38 (2007) 6173 65
transcriptional up-regulation of IL-15Ra 12 h after infec-
tion (Fig. 6C). Taken together, these ndings implicated
at least partial transcriptional regulation of pulmonary
IL-15 and IL-15Ra expression in pneumococcal
pneumonia.
3.5. Immunolocalization of the IL-15R subunits a, b, and c in
the lung
In untreated mice, IL-15Ra, b, and c were constitutively
expressed on BEC, vascular smooth muscle cells and AM.
Like IL-15, IL-15Ra was mainly expressed on the apical
surface of BEC under baseline conditions (Fig. 7A). IL-
15Rb was constitutively expressed on BEC, vascular
smooth muscle cells, AEC, and AM (Fig. 7D). IL-15Rc
showed constitutive expression on BEC and vascular
smooth muscle cells (Fig. 7G).
Pneumococcal pneumonia induced strong up-regulation
of IL-15Ra exclusively on BEC, which also displayed
de-novo expression of IL-15Ra on their basolateral surface
(Fig. 7B). AEC presented a moderate increase of their IL-
15Ra levels, whereas up-regulation of IL-15Ra was neither
observed on vascular smooth muscle cells nor on AM.
Inltrating leukocytes showed surface expression of IL-
15Ra, which remained unchanged in the course of pneu-
monia. In contrast to IL-15Ra, pneumococcal pneumonia
did not aect pulmonary levels of IL-15Rb which remained
unchanged in all cell types (Fig. 7E).
Although macrophages, neutrophil granulocytes, and
lymphocytes have been described to express IL-15Rc, leu-
kocyte c chain expression could neither be seen in normal
mice lungs nor in infected lungs. However, additional ow
cytometric analysis (with the same polyclonal primary Ab,
a biotinylated secondary Ab, and streptavidine-coupled
allophycocyanine; data not shown) proved IL-15Rc
expression on lung leukocytes. Therefore, lack of detection
in immunohistochemistry may be due to expression levels
below the microscopic detection limit.
Fig. 3. Digital image analysis with pseudocolor depiction of intracellular IL-15 immunostaining in lung sections from control mice (A, C, and E) and mice
with pneumococcal pneumonia 48 h after transnasal infection (B, D, and F). (A and B) IL-15 expression was strongly increased in bronchial epithelial cells
(arrows), whereas expression in smooth muscle cells of bronchial arteries remained unchanged (arrowheads). Inltrating leukocytes (PMNs) expressed low
levels of IL-15 (asterisk). (C and D) Alveolar epithelial cells showed moderate up-regulation of IL-15 expression (arrows). (E and F) Enhanced IL-15
expression was noted in alveolar macrophages (arrows). Representative gures of ve dierent experiments with similar results are given. Original
magnications 400.
66 A.C. Hocke et al. / Cytokine 38 (2007) 6173
Fig. 4. Time course of intracellular IL-15 expression in bronchial and alveolar epithelial cells and alveolar macrophages in murine pneumococcal
pneumonia quantied by digital image analysis. Quantitative IL-15 expression is depicted as percent of gray scale values normalized to baseline controls
(set as 100%). Mice were transnasally infected with 5 10
6
cfu S. pneumoniae and sacriced at 6, 12, 24, 48, and 60 h after infection. Control mice received
a transnasal challenge with 20 ll PBS, and were sacriced after 60 h. (A) Bronchial epithelial cells of proximal airways; and (B) of distal airways.
(C) Alveolar epithelial cells in areas without leukocyte inltrates and (D) in inltrated areas. (E) Alveolar macrophages in unaected regions as well as
(F) in inltrated areas. Mean SEM of ve-independent experiments are given.
*
p < 0.05,
**
p < 0.01,
***
p < 0.001 vs. control.
Fig. 5. Up-regulation of extracellular IL-15 expression on BEC and AM in pneumococcal pneumonia. Images show extracellular IL-15 Vector Red
immunostaining in lung sections from control mice (A and C) and mice 48 h after transnasal infection (B and D). (A and B) In normal mice IL-15 was
expressed solely on the apical side of bronchial epithelial cells (arrows). Infected mice showed signicant up-regulation of apical IL-15 (arrows) as well as a
marked increase of IL-15 expression on the basal surface of bronchial epithelial cells after 48 h (arrowhead). (C and D) Alveolar macrophages from
infected mice also showed a marked rise of surface IL-15 expression compared to control mice (arrowheads). Representative gures of ve dierent
experiments with similar results are shown. Original magnications of (A) and (B) 400, (C) and (D) 630.
A.C. Hocke et al. / Cytokine 38 (2007) 6173 67
3.6. Histopathology and clinical course of chlamydial
pneumonia
Pulmonary chlamydial infection induced only moderate
leukocyte inltration within peribronchial and perivascular
regions, and alveolar inltrates were not observed. Inam-
matory inltrates exclusively contained mononuclear cells,
and they did not emerge until 6 days after infection. They
reached a maximum from 9 to 12 days (data not shown).
Infected mice presented only minor clinical symptoms
14 days after infection, and survival rate was 100%. Lungs
of control mice receiving intratracheal saline or HEp-2 cell
suspension showed no lymphocyte inltrations and were
not dierent from baseline controls.
3.7. Infection with C. pneumoniae neither aected pulmonary
IL-15 expression nor lung lymphocyte subset composition
and activation
In contrast to pneumococcal pneumonia, chlamydial
infection failed to increase pulmonary IL-15 levels 6, 12,
24, or 72 h after bacterial challenge. IL-15 expression also
remained constant when lymphocyte inltrates became
apparent 9 or 12 days after infection. In particular, BEC
of inltrated bronchial regions did not up-regulate their
IL-15 levels neither on the apical nor on the basolateral
surface (Fig. 8A and B). Additionally, IL-15 expression
of AEC and AM remained unchanged 12 days after infec-
tion (Fig. 8C and D). Control mice receiving saline or
HEp-2 cells also showed no dierence in pulmonary IL-
15 expression compared to baseline controls.
Flow cytometric analysis of lymphocyte subset composi-
tion 12 days after intratracheal application of C.p. showed
no dierence between baseline controls and infected mice.
Furthermore, CD69 expression on pulmonary NK and
NKT cells was not increased compared to baseline controls
(data not presented).
4. Discussion
IL-15 is thought to be a crucial factor for the activation
of innate immune responses, especially at mucosal surfaces
of the gastrointestinal and urogenital tract [1820,2631].
We now present evidence for a role of IL-15 and its related
receptor complex in pulmonary immunity, both under
physiological conditions and in the course of pneumococ-
cal pneumonia. IL-15 protein levels were already constitu-
tively detected in the lungs of healthy mice, and
microscopic immunolocalization revealed cell-type specic
expression of IL-15 most notably in BEC and AM, but also
in AEC and vascular smooth muscle cells. The induction of
pneumococcal pneumonia evoked a signicant rise of pul-
monary IL-15 expression predominantly originating from
BEC, AM, and the huge amount of PMNs expressing
low levels of IL-15. Additionally, increases of intracellular
IL-15 were accompanied by extracellular IL-15 as well as
IL-15Ra expression. In contrast, enhancement of IL-15
expression was not detected in chlamydial lung infection,
suggesting pathogen-dependent regulation of IL-15 synthe-
sis in pulmonary cell types.
IL-15 mRNA is widely distributed and expressed in mul-
tiple cell types and tissues including the lung, which mainly
comprises the long secretory isoform of IL-15 [32]. IL-15
protein synthesis is assumed being more limited and subject
to a complex regulatory process aecting translation, intra-
cellular tracking, and secretion [33,34]. Dening the func-
tional role of IL-15 thus required the dierential analysis of
IL-15 protein production on a single cell level. In this
Fig. 6. Transcriptional up-regulation of pulmonary IL-15 expression in
pneumococcal pneumonia. Mice were transnasally infected with
5 10
6
cfu S. pneumoniae and sacriced at 12, 24, and 48 h after infection.
(A) Lungs were harvested, RNA was isolated, and IL-15 mRNA was
detected by RT-PCR. (B) Semi-quantitative PCR densitometry was
performed in relation to G3PDH. A representative of three dierent
experiments is presented. (C) Microarray transcriptome analysis also
revealed signicant up-regulation of IL-15 mRNA and additionally
showed elevated levels of IL-15Ra mRNA in infected lungs compared
to controls (set as 1). Pooled data of three identical experiments are given.
68 A.C. Hocke et al. / Cytokine 38 (2007) 6173
regard, IL-15 protein has been detected in monocytes, mac-
rophages and dendritic cells [35,36], in bone marrow stro-
mal cells [32,37], and in several epithelial cells including
kidney epithelial cells [38], keratinocytes [39], retinal pig-
ment epithelium [40], as well as pulmonary and intestinal
epithelial cells in vitro and ex vivo [26,41].
IL-15 produced by the intestinal epithelium was shown
to account for the survival and expansion of intraepithelial
lymphocytes in the gut [26,42], which is further substanti-
ated by the nding of markedly reduced intestinal intraep-
ithelial lymphocytes in IL-15- and IL-15Ra-decient mice
being deprived of NK and NKT cells [43,44]. The complex
and tight post-transcriptional regulation of IL-15 might
also indicate that overproduction of IL-15 is detrimental.
This hypothesis is supported by the ndings of increased
IL-15 synthesis in chronic inammatory diseases including
rheumatoid arthritis, psoriasis, or inammatory bowel dis-
ease [20,45].
In the lung, IL-15 protein expression has been previ-
ously detected in human AM [4648], predominantly under
inammatory conditions including sarcoidosis, cryptogenic
brosing alveolitis, chronic obstructive pulmonary disease,
tuberculosis, and HIV infection [49,50]. Beside, the lung
also harbors large amounts of immune cells including sig-
nicant numbers of IL-15 responsive leukocyte popula-
tions. The considerable constitutive expression of IL-15
on the surface of AM thus may contribute to the homeo-
stasis of intrapulmonary leukocytes in the alveolar com-
partment. Additionally, upon pneumococcal activation,
the substantial intracellular amounts of IL-15 may be
transferred to the cellular surface where membrane-bound
IL-15 can induce bidirectional signaling [13], by autocrine
signaling or transpresenting IL-15 to other immune cells
bearing the IL-15R complex. Furthermore, AM expressed
the signaling heterodimer IL-15Rb and c
c
, enabling them
to respond to extrinsic IL-15, however, the determination
if secreted IL-15 in the alveolar uid exerts relevant func-
tions was shown to be quite dicult [47]. In addition, our
data conrm previous reports demonstrating abundant
IL-15 synthesis by AM [47,48], again proving the fact that
Fig. 7. Immunohistochemical analysis of IL-15 receptor complex expression in lung sections from control mice (A, D, and G), and mice with
pneumococcal pneumonia 48 h after transnasal infection (B, E, and H). (A and B) Bronchial epithelial cells constitutively expressed IL-15Ra on their
apical surface. Strong up-regulation of IL-15Ra on the apical surface was noted in pneumococcal pneumonia. Concomitantly, de-novo expression of IL-
15Ra on the basal surface of bronchial epithelium was observed. Likewise, vascular smooth muscle cells and alveolar macrophages exhibited signicant
constitutive expression of IL-15Ra, which showed no increase in infected mice. IL-15Ra was also expressed on inltrating leukocytes. (C) Preincubation of
the primary Ab with the corresponding blocking peptide inhibited IL-15Ra immunostaining. (D and E) IL-15Rb was constitutively expressed on bronchial
epithelial cells, vascular smooth muscle cells, and alveolar macrophages. IL-15Rb expression was not aected by pneumococcal infection, but inltrating
leukocytes showed IL-15Rb immunostaining. (F) Preincubation of the primary Ab with the corresponding blocking peptide inhibited IL-15Rb
immunostaining. (G and H) IL-15Rc was constitutively expressed on bronchial epithelium and vascular smooth muscle cells. Expression was not aected
by pneumococcal infection. (I) Preincubation of the primary Ab with the corresponding blocking peptide inhibited IL-15Rc immunostaining. Original
magnications 400, (B) and (C) 200.
A.C. Hocke et al. / Cytokine 38 (2007) 6173 69
cells of the monocyte/macrophage lineage are among the
functionally relevant IL-15 sources in the body [51]. These
ndings underline that IL-15 expression of AM might con-
tribute signicantly to the pulmonary immune response,
where continuous inspiration of foreign particles and
microorganisms requires a rapid and eective activation
of resident pulmonary leukocytes.
Interestingly, epithelial cells also signicantly contrib-
uted to the constitutive expression of IL-15 protein in the
lung, supporting the concept of considering BEC and
AEC as important constituents of pulmonary immunity
[52]. The nding of IL-15 protein synthesis in respiratory
epithelial cells conrms previous reports showing IL-15
expression in epithelial cells of dierent compartments
[26,39,40,53]. In the human lung, IL-15 gene and protein
expression have been previously detected in bronchial biop-
sies from healthy controls as well as from patients with
inammatory lung diseases [48]. However, analyzing iso-
lated human BEC demonstrated IL-15 gene expression,
but failed to detect constitutive protein synthesis, which
was induced only after the stimulation with IFNc [41].
These observations indicate that IL-15 translation in
BEC depends on additional factors produced in the bron-
chial microenvironment. However, constitutive and
induced IL-15 might also contribute to pulmonary leuko-
cyte activation. In particular, IL-15 and IL-15Ra were
shown to exert function on the phagocytic capacity of
PMNs [54] and IL-15 exposed AEC up-regulated adhesion
molecules leading to adherence of PMNs in vitro [55].
These results are well in line with the AEC expression of
IL-15 and IL-15Ra in this study suggesting a role of
IL-15/Ra complex for attracting PMNs to the alveolar
compartment during pneumococcal pneumonia but not in
chlamydial lung infection.
Beside, in the salivary glands, epithelial IL-15 is a critical
factor for the dierentiation of B-1 cells into soluble IgA
producing cells [31], and polyvalent IgA is also one of the
most important humoral factors of innate defenses in the
lower respiratory tract. With regard to the abundant levels
of IL-15 expression in BEC contribution of these cells to
the synthesis of soluble IgA in the bronchial mucosa has
to be further investigated.
Beyond acting on local immune cells, epithelial IL-15
might also serve as an autocrine survival factor [53], or
contributes to epithelial proliferation [26]. Similar to other
epithelial cells [53], BEC and AEC express the signaling
subunits IL-15Rb and c
c
, enabling them to respond to
extrinsic IL-15.
We noticed constitutive expression of IL-15 and
IL-15Ra, b, and c
c
in vascular smooth muscle cells of bron-
chial arteries. In skeletal muscle, IL-15 exerts anabolic
eects and improves muscle function [56]. IL-15 has been
previously localized in aortic smooth muscle cells of
C57Bl/6 mice [57], but the functional impact of IL-15
expression in smooth muscle cells has still to be dened.
The fast and substantial up-regulation of pulmonary
IL-15 protein expression in pneumococcal pneumonia
underlines a possible role of this cytokine in innate defenses
of the lung. In this respect, the dierential analysis of IL-15
expression on a single cell level emphasizes the impact of
Fig. 8. Immunohistochemical analysis of IL-15 expression in lung sections from HEp-2 challenged control mice (A and C) and mice with chlamydial
pneumonia (B and D) assessed by Vector Red/methyl green staining (arrows). In contrast to pneumococcal pneumonia, IL-15 expression was not up-
regulated in lungs of Chlamydophila pneumoniae infected mice, as depicted by representative sections (of ve-independent experiments with similar results).
Neither BEC (A and B) nor AEC (arrows) or AM (arrowheads) (C and D) showed dierences in the expression level of IL-15 within inltrated areas 9 days
after intratracheal challenge. Original magnication 400.
70 A.C. Hocke et al. / Cytokine 38 (2007) 6173
BEC, AM, and most probably the huge amount of invad-
ing PMNs. The rst two cell types increased their expres-
sion levels of IL-15 as well as IL-15Ra, enabling them to
recruit and activate, as well as to respond to local or invad-
ing immune cells bearing IL-15R subunits. The fraction of
invading PMNs exhibited low levels of IL-15 and IL-15Ra,
which might indicate an autocrine activation thereby
increasing their host defense competence and delaying their
apoptosis [54,58]. Additionally, IL-15 has been suggested
to contribute to LPS-induced PMN recruitment into the
lung in an IL-17-dependent manner [59], which has also
been shown to induce PMN inltration during Klebsiella
pneumoniae pneumonia [60]. This might implicate an
IL-15IL-17 relevant axis for PMN recruitment during
Gram-positive pneumococcal pneumonia. However, in
the course of pneumococcal pneumonia, pulmonary
CD4
+
T cells markedly decreased (data not shown), which
were demonstrated as the main source of IL-17 [59]. Inter-
estingly, in pneumococcal pneumonia two recent studies
have shown that TNF and IL-1 receptor signaling as well
as IL-12 induced IFNc expression in the pulmonary com-
partment is crucial for PMN recruitment to infected lungs
[61,62].
Additionally, in certain circumstances IL-15 is also che-
moattractant for lymphocyte subsets. In the course of
pneumococcal pneumonia, beside pulmonary CD4
+
T cells
also B cells markedly decreased, but the numbers of
CD8
+
T, cdTCR
+
T, and NK cells remained constant, and
NKT cells even increased (data not shown). These features
may be of great importance in pneumococcal pneumonia
calling for further studies to discover if these events neces-
sitate IL-15 expression and function.
Since chlamydial infection did not evoke a rise in pul-
monary IL-15 expression, up-regulation of IL-15 in the
lung does not seem to be a general feature of bacterial
pneumonia. There may be several reasons for the dierent
IL-15 responses in both infection models: First, microor-
ganisms themselves may induce IL-15 expression via the
interaction with specic pathogen-recognition receptors
(PRRs), as has been described for macrophages and Myco-
bacterium leprae [63]. Although both pathogens have been
shown to activate host cells via overlapping subsets of
PRRs (e.g. TLRs, NODs) [64,65] optimal IL-15 induction
additionally requires appropriate priming and triggering
stimuli [35], therefore implicating a role for indirect activa-
tors including cytokines as well. Second, chlamydial lung
infection in the used model resulted in mild pneumonia
emerging after several days, whereas pneumococci induced
acute and severe symptoms. Inhibition of host cell apopto-
sis and suitable PMN recruitment are typical features of
the acute model suggesting the necessity for increased IL-
15. In addition, C. pneumoniae, as obligate intracellular
bacteria, are dependent on particular environmental condi-
tions in the host cells. For example, Chlamydia actively
inuence host cell apoptosis, and they promote the forma-
tion of specic replication vacuoles of infected cells depen-
dent on their life cycle [66]. It could be speculated that the
control of host cell apoptosis would be inaccessible for
C. pneumoniae if high intracellular IL-15 levels predomi-
nate, which may prompt Chlamydia to an IL-15 suppres-
sion. Interestingly, a previous study demonstrated that
IL-15 mRNA was up-regulated by Chlamydia trachomatis
but not by C. pneumoniae in infected synovial tissue sam-
ples [67]. Furthermore, although both Chlamydia species
replicated in human endothelial cells, only C. pneumoniae
induced pro-inammatory cell activation in these cells
[68]. Thus, species dierences (C. pneumoniae vs. C. tracho-
matis) may also be important for cell activation, including
IL-15 induction. Since pneumococci belong to the group of
Gram-positive bacteria and Chlamydia to Gram-negative
pathogens, this might also contribute to dierent IL-15
expression levels in pneumonia. However, IL-15 has been
shown to protect mice against a variety of infections
including S. choleraesuis [14], L. monocytogenes [7], Myco-
bacteria spp. [15], Escherichia coli [69], T. gondii [16],
Malaria [70], and HSV [17] among which Gram-negative
pathogens, too. Taken together, a multitude of factors
are suggested to account for dierent IL-15 expression
and function in both models having to be addressed in
future studies in more detail.
Based on the data presented herein, the functional rele-
vance of this cytokine in pneumococcal pneumonia is thus
currently under investigation. In this respect, it is notewor-
thy that patients with acquired immunodeciency syn-
drome showed severely impaired IL-15 protein synthesis
and increased susceptibility against S. pneumoniae infec-
tions [71].
In essence, our data revealed a cell-type specic IL-15
regulation during bacterial pneumonia which implicated
an important role in the homeostatic regulation of pulmon-
ary immune and non-immune cells both in the bronchial
and in the alveolar compartment. The up-regulation of
IL-15 during pneumococcal pneumonia further indicates
critical impact on pathogen-specic immune responses at
the mucosal surface of the respiratory tract.
Acknowledgments
We thank S. Wagner for excellent technical assistance.
Parts of this work will be included in the MD thesis of
Matthias P. Lampe. This work was supported by the Bun-
desministerium fu r Bildung und Forschung (Kompetenz-
netzwerk ambulant erworbene Pneumonie; CAPNETZ)
to N.S., J.Z., B.S., S.H., and S.R., and by the Deutsche
Forschungsgemeinschaft, Schwerpunktprogramm (Kr2197/
1-2) to M.K. and N.S.
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