International Immunology, Vol. 23, No. 12, pp. 713–728
doi:10.1093/intimm/dxr083
Advance Access publication 24 October 2011
ª The Japanese Society for Immunology. 2011. All rights reserved.
For permissions, please e-mail: [email protected]
Human eosinophils express RAGE, produce RAGE
ligands, exhibit PKC-delta phosphorylation and
enhanced viability in response to the RAGE ligand,
S100B
Colleen S. Curran and Paul J. Bertics
Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA
Correspondence to: P. J. Bertics; E-mail: [email protected]
Received 12 May 2011, accepted 15 September 2011
Abstract
This study tested the hypothesis that human eosinophils produce ligands for the receptor for
advanced glycation end-products (RAGE), express RAGE and exhibit RAGE-mediated responses. In
examining our microarray data, we identified the presence of RAGE and RAGE ligand (S100A4,
S100A6, S100A8, S100A9, S100A11, S100P, HMGB1) transcripts. Expression of eosinophil RAGE
mRNA was also compared with a known positive control and further assessed via bioinformatics and
sequence analysis of RAGE cDNA. Positive and negative controls were used to identify RAGE,
S100A8 and S100A9 protein in human primary eosinophils. Immunoblot assessment of eosinophils
treated with cytokines (IL-5 or granulocyte macrophage colony-stimulating factor) indicated an upregulation of S100A8 and S100A9 production, whereas co-treatment of eosinophils with a RAGE
ligand and cytokines displayed a down-regulation in the levels of RAGE. Analysis of eosinophilconditioned media revealed that eosinophils are capable of releasing RAGE, S100A8 and S100A9. To
test the eosinophil response to RAGE activation, the most well-characterized RAGE ligand, S100B,
was examined. Treatment of eosinophils with S100B resulted in RAGE-mediated PKC-delta
phosphorylation, a 3-fold dose-dependent increase in cell survival and an increase in the level of
cellular RAGE. Combined, these studies reveal eosinophil expression of RAGE, RAGE ligands and
RAGE-mediated responses. The expression of eosinophil RAGE, soluble RAGE and RAGE ligands
may be pivotal to the functions of eosinophils in various human diseases involving RAGE and S100
ligands.
Keywords: CD11b, GM-CSF, IL-5, S100A8, S100A9
Introduction
Eosinophils are bilobed granulocytic cells predominantly
known to be recruited in response to chronic inflammatory
diseases (1). Of relevance to the present report, the expression, activation and release of the receptor for advanced glycation end-products (RAGE) contribute to disease-associated
inflammation (2, 3). A subset of RAGE ligands, the S100 proteins, is prominently expressed in many human cancers with
indicated functions in tumor progression (4, 5). The S100B
homodimer and S100A8/S100A9 heterodimer are expressed
in tumors known to exhibit eosinophilia such as melanoma
and colon cancer (4, 6). The S100B protein is also a CD8+
T-cell inflammatory cytokine involved in central nervous
system pathologies (7, 8). Eosinophils have been reported
to produce and respond to neuromediators, associate with
peripheral and enteric nerves, and induce neurotoxic activity
(9, 10). The eosinophil is also an established effector cell in
the pathobiology of lung and gastrointestinal inflammation
(10–13). Originally characterized as cystic fibrosis antigens,
the S100A8/S100A9 proteins are increasingly implicated in
chronic airway and intestinal disease (14, 15). Together, this
information suggests that S100 proteins may play a role in
the eosinophil response. However, the intracellular and extracellular mechanisms that enable eosinophils to participate in
various human afflictions are not fully known.
RAGE is a multiligand receptor known to interact with
structures formed via non-enzymatic glycation of proteins
(advanced glycation end-products), amyloid-beta, S100/calgranulins, high-mobility group box-1 (HMGB1) and Mac-1
(CD11b/CD18, aMb2-integrin, complement receptor 3) (16).
In certain cell types, such as monocytes and lung epithelial
714 Eosinophils express RAGE and RAGE ligands
cells, RAGE can exist as a full-length isoform on the cell
membrane and as soluble isoforms as a result of alternative
splicing of pre-mRNA or proteolytic cleavage (17–19). The
development and recruitment of macrophages are indicated
in RAGE-mediated disease progression, resulting in RAGE ligand-induced macrophage activation and the production of
proinflammatory cytokines (16, 20). Studies have also indicated that macrophage RAGE functions as a ligand for
phosphatidylserine in the clearance of apoptotic cells (21,
22). Of interest, increased expression of RAGE ligands is associated with eosinophilia (23), suggesting that, like macrophages, eosinophils may also possess RAGE-mediated
functions. In examining our previously published microarray
of human primary blood eosinophils (24), RAGE and RAGE
ligand mRNA was detected. This preliminary insight suggests that the RAGE system may play a pivotal role in the
eosinophilic response. To date, the study of RAGE or RAGE
ligands in eosinophils has not been extensively explored.
Eosinophils are myeloid cells formed in response to regulatory signals provided by the cytokines IL-3, IL-5 and granulocyte macrophage colony-stimulating factor (GM-CSF)
(25). Eosinophils are also capable of diverse activities involving tissue remodeling/repair, immunological responses
and neurological interactions that are not well understood
(9, 10). Interestingly, RAGE ligands are implicated in these
same functional responses (16). For example, S100B at
nanomolar concentrations has a neurotrophic effect, encouraging repair/regeneration responses. However, at higher
concentrations, S100B functions as a neurotoxin and activates microglia (26). Excessive activation of macrophages,
tissue damage and necrosis are linked to the activities of
RAGE ligands (27) and likewise the recruitment of eosinophils (25, 28). These findings support a potential role of
eosinophils in RAGE-mediated wound repair, inflammation
and neurological function. Therefore, the aim of the present
work was to determine if eosinophils express RAGE and
RAGE ligands and if eosinophils are responsive to a known
inflammation-associated RAGE ligand, namely S100B.
Methods
Isolation and culture of peripheral blood and bronchoalveolar
lavage eosinophils
Peripheral blood and bronchoalveolar lavage (BAL) fluid was
obtained from human allergic rhinitis and allergic asthmatic
patients under informed consent. The study was approved
by the University of Wisconsin–Madison Center for Health
Sciences Human Subjects Committee. Relevant patient information was made available in accordance with institutional
review board protocols. Blood eosinophils were purified from
heparinized blood that was diluted with Hank’s buffered
salt solution (HBSS; Mediatech, Manassas, VA, USA) without
Ca2+ and centrifuged for 20 min at 700 3 g over 1.090 g ml 1
Percoll. A granulocyte fraction was obtained after removal of
the plasma, mononuclear cell band and Percoll. Granulocytes were then subjected to RBC lysis via hypotonic shock,
washed with 4C HBSS supplemented with 2% newborn calf
serum (Life Technologies, Grand Island, NY, USA) and incubated 40 min with magnetic beads coated with anti-CD16,
anti-CD14 and anti-CD3 (Miltenyi Biotechnology, Auburn,
CA, USA) prior to negative selection with an AutoMACS separator (Miltenyi Biotechnology). The recovered mixture
(>97% purity and >98% viability) was evaluated by Giemsa’s-based Diff-Quik stain (Baxter Scientific Products,
McGaw Park, IL, USA) and trypan blue exclusion, respectively. Blood eosinophils were cultured in RPMI 1640 (Mediatech) containing 0.001 or 0.1% human serum albumin
(HSA; Irvine Scientific, Santa Ana, CA, USA). Airway eosinophils were purified by centrifugation of BAL cells through
a Percoll bilayer (1.085/1.100 g ml 1). Eosinophils were recovered at the interface between the Percoll layers and
resuspended in room temperature HBSS supplemented with
2% newborn calf serum. Airway eosinophil purity (>99%)
and viability (>97%) were identified as described in the
blood eosinophil isolation.
Isolation and culture of peripheral blood monocytes
Human blood-derived monocytes were purified as previously
described (29). In brief, heparinized peripheral blood was
obtained from volunteer donors and enriched for peripheral
blood mononuclear cells via centrifugation through a Percoll
monolayer. Platelets were removed by three washes in HBSS
(Mediatech) with 2% heat-inactivated bovine serum. Monocytes were enriched with RosetteSep monocyte enrichment
cocktail and a standard Ficoll centrifugation. The monocytes
were collected at the Ficoll:plasma interface and dispensed
(2 3 106 cells/ml) into 12-well plates (Costar, Corning, NY,
USA) with RPMI 1640 (Mediatech) supplemented with 10%
fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml
penicillin/streptomycin (Mediatech) and 2 mM L-glutamine
(Mediatech) (RPMI 10% complete media). After 2 h in culture at 37C, cells were rinsed with warm HBSS (Mediatech)
to remove non-adherent lymphocytes and resuspended in
RPMI 10% complete media for an additional 24 h at 37C
prior to lysis and processing.
Cell lines
The human HEK-293 cell line was purchased from the
American Type Culture Collection (Manassas, VA, USA).
The human H358 non-small-cell lung cancer (NSCLC) and
DU145 prostate carcinoma cell lines were kindly provided
by Dr Paul Harari and Dr Wade Bushman (University of
Wisconsin, Madison, WI, USA), respectively. The DU145
prostate carcinoma and HEK-293 cells were cultured in
DMEM (Mediatech) supplemented with 10% Cosmic Calf
Serum (Hyclone) and 100 U/ml penicillin/streptomycin
(Mediatech). The H358 NSCLC cells were cultured in RPMI
10% complete media.
Reagents for cell stimulation
Cells, when indicated, were stimulated with GM-CSF (R&D
Systems, Minneapolis, MN, USA), IL-5 (R&D Systems) or
N-formyl-methionyl-leucyl-phenylalanine (fMLF; Sigma Chemical Co., St Louis, MO, USA). The S100B ligand (Calbiochem/
EMD Biochemicals, Inc., Gibbstown, NJ, USA) was used to
activate RAGE given its commercial availability, its known
receptor interactions (30) and its reported functions in certain
cancers (4) and central nervous system pathologies (7) that
have been indicated to recruit eosinophils (6, 9).
Eosinophils express RAGE and RAGE ligands
Analysis of RAGE mRNA expression
Eosinophils (8–10 3 106 cells) were lysed by repeated passage with a 27.5-gauge needle in Qiagen RNeasy Mini Kit
Buffer (Qiagen, Valencia, CA, USA). Lysates were subjected
to a freeze/thaw ( 80C, 15 min or O/N) to ensure adequate
cell lysis and enriched total RNA (0.8–2 lg) was isolated in association with the manufacturer’s protocol (Qiagen RNeasy
Mini Kit). Synthesis of cDNA was performed with the iScript
cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA,
USA) and 0.5–1 lg RNA. PCR was performed in a 10 ll reaction solution (HotStarTaq Master Mix Kit; Qiagen) containing
cDNA corresponding to 50 ng total RNA and 0.5 lM primer
(each for reverse and forward primers). The cycling conditions
were 95C for 5 min, followed by 40 cycles, each consisting of
denaturation at 94C for 30 s, annealing at 65C for 1 min and
extension at 72C for 1.5 min and a final extension at 72C for
10 min. The PCR products were analyzed on 1 or 1.5% agarose gels and visualized by staining with ethidium bromide.
Bands were subsequently excised and purified using QIAquick Gel Extraction Kit (Qiagen). The purified PCR product
sequences were fluorescently labeled with BigDye Terminator
v3.1 Ready Reaction Mix (Applied Biosystems, Foster City,
CA, USA). The cycling conditions were 95C for 5 min, followed by 35 cycles, each consisting of denaturation at 94C
for 30 s, annealing at 52C for 3.5 min and extension at 60C
for 1.5 min and a final extension at 60C for 10 min. Amplification of RAGE cDNA was performed using primer pairs targeting untranslated regions (UTR) and internal sequences of the
gene similar to previous study by Hudson et al. (17). Electrophoresis of PCR product and DNA ladder (Hyladder 10 kb;
Denville Scientific Inc., Metuchen, NJ, USA) were performed
on a 1% agarose gel. Primers utilized are as follows: 5# primer
(RAGE5’UTR1, 5#-AGGAAGCAGGATGGCAGC-3#); 3# primer
(RAGE3’UTR1,
5#-GTCTGAGGCCAGAACAGTTC-3#);
5#
primer (RAGEexon8, 5#-GCCCTGTGCTGATCCTCCCTGAG3#); 5# primer (RAGEexon7, 5#-GCCAGAAGGTGGAGCAGTAGCTC-3#); 3# primer (RAGEexon4, 5#-AGGCAGAATCT
ACAATTTCTGGC-3#).
Bioinformatics
DNA sequence determination was performed with Genescan
software at the University of Wisconsin Biotechnology Center. Sequences corresponding to RAGE were identified in
GenBank using the BLAST Human Sequences tool (http://
blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence data were
viewed and annotated using the BioEdit program (31) and
aligned to RAGE mRNA (NM_001136.3, CDS 25-1239) using
Clustalw (http://www.ebi.ac.uk/clustalw/) and the nucleotide
BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Immunoblotting
Cells were solubilized with lysis buffer (1% SDS, 10 mM
dithiothreitol, 0.5 mM Na3VO4, 1 mM EDTA, 10% glycerol,
10 mM Tris, pH 8.0), sonicated, and boiled (5 min). For analysis of PKC-d phosphorylation/activation, the cell lysates
were additionally subjected to a freeze/thaw cycle ( 80C,
15 min) and a centrifugation (10 min, 15 800 3 g) step prior
to isolating the soluble fraction. Concentrated cell lysis buffer (103) was added to S100A8/S100A9-conditioned media
715
(1:10 dilution) and boiled (5 min). Soluble RAGE-conditioned
media was added to ice-cold acetone (1:3, 20C, 60 min)
and the protein precipitate was pelleted via centrifugation
(4C, 10 min, 15 800 3 g), suspended in lysis buffer (13),
sonicated and boiled (5 min). Protein concentrations were
determined by Micro-BCA protein assay reagents (Thermo
Scientific Pierce, Rockford, IL, USA) and loaded onto a 10,
12.5 or 15% SDS–PAGE gel. Proteins were transferred onto
0.45 lm Immobilion-P polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA), incubated with antibodies raised against human RAGE (R&D Systems goat
polyclonal AF1145, 1:500), actin (BD Biosciences, San Jose,
CA, USA; 612656, 1:5000), phospho-PKC-d (Cell Signaling
Technology, Inc., Danvers, MA, USA; 9374, 1:500), Total
PKC-d (Cell Signaling Technology, Inc.; 2058, 1:500),
S100A8 or S100A9 (Santa Cruz Biotechnology, Santa Cruz,
CA, USA; sc-8112, sc-8114, 1:200). Blots were washed and
subsequently incubated with horseradish peroxide (HRP)conjugated secondary antibodies. The immunoblots that
were re-probed for total PKC-d were stripped with One Minute Western blot Stripping Buffer (GM Biosciences, Inc,
Rockville, MD, USA), incubated with secondary antibodies
and examined for negative chemiluminescence. Bound secondary antibody was visualized following incubation of the
membrane with Super Signal West chemiluminescent HRP
substrate (Thermo Scientific Pierce) and an Epichemi II
darkroom equipped with a 12-bit cooled CCD camera. Luminescence was quantified and evaluated using ImageJ software (National Institutes of Health).
Immunofluorescence
Eosinophils were adhered to coverslips via cytospin, fixed in
4% paraformaldehyde, treated with 0.1% Triton X-100 and
blocked with PBS containing 1% bovine serum albumin
(Sigma) and 1 lg ml 1 normal rabbit IgG (Millipore; 12–
370). Cells were immunostained with polyclonal goat antibodies raised against human RAGE (Santa Cruz sc-8230;
1:50), human S100A8 (Santa Cruz sc-8112; 1:50), S100A9
(Santa Cruz sc-8114; 1:50) or isotype control. The isotype
control and primary antibodies were reacted with rabbit
anti-goat Alexa Fluor 488 antibodies (Invitrogen, Carlsbad,
CA, USA; A-11708, 1:2000). The nucleic acid stain DAPI
was added to identify the bi-lobed nucleus of eosinophils
(Invitrogen; 1:10 000). Coverslips were mounted onto slides
with Fluoro-Gel w/Tris Buffer (Electron Microscopy Sciences,
Hatfield, PA, USA) prior to visualization using a Nikon epifluorescence microscope.
Survival analysis, CD11b expression and activation
Purified blood eosinophils (2 3 105/ml) were cultured atop
150 ll 0.5% w/v agarose-coated 48-well tissue culture plates
(Sarstedt, Newton, NC, USA). Cells were treated with
increasing concentrations of S100B (Calbiochem/EMD Biochemicals, Inc.) and compared with cells treated with buffer
control or GM-CSF (R&D Systems). Eosinophil viability was
determined by assessing the exclusion of propidium iodide
(PI; 3 lg ml 1) staining/fluorescence. The expression and
activation state of CD11b were, respectively, identified by
first incubating with aM/CD11b (MAB1699; R&D Systems) or
716 Eosinophils express RAGE and RAGE ligands
CBRM1/5 (301402; Biolegend, San Diego, CA, USA) antibodies followed by reaction with a secondary 488 Alexa
Fluor-conjugated donkey anti-mouse antibody (A-21202;
Invitrogen) and by comparison with an isotype control. All
analyses were performed using a FACScan flow cytometer
(Becton–Dickinson, Bedford, MA, USA).
Statistical analyses
Measurements of CD11b expression and activation were
assessed among groups using mixed-effects analysis of variance models with a fixed-effect covariate per group and
a random-effect covariate to account for within-patient correlation of measurements. All additional analyses were performed using a paired Student’s t-test. A two-sided P value
of <0.05 was regarded as statistically significant.
Results
Detection of mRNA for RAGE and RAGE ligands in human
primary blood eosinophils
RAGE has multiple reported ligands and has been found to
be expressed in cells of a myeloid lineage (16). In addition,
a previous study has suggested the expression of RAGE in
human primary eosinophils (32). To elaborate upon these
results, we analyzed our previous microarray data of human
primary blood eosinophils stimulated with control buffer, 100
pM IL-5 or 100 pM GM-CSF (4 h) (24). Transcripts identified
as present are summarized in Fig. 1 and compared with
known cytokine responsive transcripts, CD69 and proviral integration site for Moloney murine leukemia virus 1 (PIM-1).
These data revealed the presence of message for RAGE
and RAGE ligands (S100A4, S100A6, S100A8, S100A9,
S100A11, S100P, HMGB1) in human primary blood
eosinophils.
To confirm RAGE mRNA expression, human primary blood
eosinophils were obtained from three different donors and
compared with DU-145 prostate carcinoma cells, an established positive RAGE control (33). Cells were lysed and
mRNA was isolated as detailed under Methods. Primers
specific to the UTR and internal exons were used to amplify
cDNA in the presence or absence of reverse transcriptase.
Electrophoresis of PCR product revealed a migrating band
comparable to RAGE mRNA (coding sequence 25-1239,
NM_001136.3). A representative image of three separate
analyses displays the expression of RAGE in DU-145 prostate carcinoma cells and eosinophils in the presence of reverse transcriptase (Fig. 2), verifying the presence of cDNA
and not genomic DNA.
Sequence and bioinformatics analysis of human primary
blood eosinophil RAGE
Bioinformatic analysis was performed for additional confirmation of eosinophil RAGE expression. Sequences of cDNA
obtained from eosinophils of two separate donors were subjected to a BLAST search (Fig. 2), revealing identity with only
one gene in the human genome, the advanced glycosylation
end-product-specific receptor, Gene ID: 177. Alignment
of the full sequences with RAGE mRNA (coding sequence
25-1239, NM_001136.3) revealed identity of >97%, supporting our data (Fig. 1), indicating eosinophil RAGE transcript
expression.
Identification of RAGE protein expression in human primary
blood eosinophils
To determine if eosinophils express RAGE at the protein
level, the H358 NSCLC cell line and DU-145 prostate carcinoma cell lines were used as controls due to their respective
lack and presence of RAGE protein (33–35). Because each
cell type differs in their cellular concentrations of actin, the
controls are restricted to identifying, and not comparatively
quantifying, RAGE protein expression in eosinophils. In Fig.
3A, a representative immunoblot identifying RAGE in eosinophils is displayed. Repeat experiments involving three different donors were assessed for RAGE levels using
densitometry and normalization to actin controls, and these
data, which support the idea that RAGE is expressed by human eosinophils, are presented in Fig. 3B. In addition, as
shown in Fig. 4, immunofluorescence microscopy with an
Fig. 1. Expression levels of human primary blood eosinophil RAGE and RAGE ligand transcripts. Data are extracted from supplemental results
obtained in our previously published microarray analysis of human primary blood eosinophils stimulated 4 h with control buffer, 100 pM IL-5 or
GM-CSF (24). Data represent 2 independent eosinophil pools from a total of 12 patients. Data displayed represent transcripts identified as
present on the microarray. The abundance of RAGE and RAGE ligand transcripts is represented by model-based expression indexes (MBEI) and
compared with the MBEI of known cytokine (*)-responsive transcripts, CD69 and PIM-1, 6SD.
Eosinophils express RAGE and RAGE ligands
717
teases, such as A Disintegrin And Metalloprotease 10
(ADAM10) and matrix metalloproteinase 9 (MMP-9) (37). In
examining our previous microarray data of human primary
blood eosinophils, transcripts for both ADAM10 and MMP-9
were identified as being present in human eosinophils (24).
To examine if soluble RAGE is produced by human primary
blood eosinophils, these cells (2 3 106 cells/ml) were incubated for 2 or 4 h in 0.001% HSA RPMI and in the presence
of control buffer or 100 pM GM-CSF. Cell lysates and conditioned media were obtained from these cultures and loaded
onto 12.5% SDS–PAGE gels, transblotted and immunostained
for RAGE. In Fig. 6A, a representative immunoblot from four
different eosinophil donors is shown wherein RAGE immunoreactivity is identified in both the cell lysates and in the conditioned media. Densitometry analyses were performed and
these data indicated enhanced soluble RAGE expression
with increased culture time and GM-CSF treatment (Fig. 6B).
Fig. 2. Reverse transcriptase (RT)–PCR detection of human primary
blood eosinophil RAGE by direct observation of ethidium-stained PCR
products. cDNA was generated in the presence or absence of RT and
amplified with primers specific to the UTR of RAGE as detailed in
Methods. Bands displayed correlate in size with RAGE mRNA (coding
sequence 25-1239, NM_001136.3). A representative image is
displayed from three separate experiments involving the DU145 cell
line and three different eosinophil (EOS) donors.
additional RAGE polyclonal antibody (see Methods)
revealed receptor expression throughout the cytoplasm,
thereby supporting our immunoblot assessment (Fig. 3A
and B) of RAGE protein expression in human primary blood
eosinophils. Analysis of BAL eosinophils also revealed the
presence of RAGE in lung eosinophils (Fig. 3C).
RAGE protein expression by human primary blood eosinophils in response to S100B treatment. RAGE ligand–receptor interaction has been reported to amplify RAGE
expression (36). In examining if the RAGE ligand, S100B,
induces eosinophil RAGE expression, 2 3 106 cells/ml human primary blood eosinophils suspended in 0.1% HSA
RPMI were cultured for 1 h with control buffer, 10 lg ml 1
S100B, 100 pM GM-CSF or 100 pM IL-5. In Fig. 5A, a representative immunoblot for RAGE is displayed. These data
suggest that S100B can induce RAGE expression in human
primary blood eosinophils. Repeat experiments involving five
different donors were assessed for RAGE levels using densitometry and normalizing to actin controls. These results reveal a significant increase (P = 0.005) in eosinophil RAGE
expression in response to S100B treatment compared with
the buffer control (Fig. 5B). Treatment with GM-CSF or IL-5
alone or in combination with S100B did not appear to significantly affect cellular RAGE expression.
Soluble RAGE protein production by human primary blood
eosinophils. The most common variants of RAGE include the
full-length form, the C-truncated (soluble RAGE) form and
the N-trunctated isoforms (36). RAGE isoforms have been
proposed to occur via alternative splicing of the pre-mRNA
(17) or in response to proteolytic cleavage by metallopro-
S100A8 and S100A9 production by human primary blood
eosinophils
S100A8 is a common inflammatory protein that has been
reported to function as a dimer with S100A9 in cells of a myeloid lineage (38). As shown in Fig. 1, mRNA expression levels of S100A8 appear to be responsive to GM-CSF and IL-5
treatment in human primary blood eosinophils. To test
whether S100A8 or S100A9 protein production are responsive to these cytokines, human primary blood eosinophils in
0.1% HSA RPMI (2 3 106 cells/ml) were cultured for 1 h with
control buffer, 100 pM GM-CSF or 100 pM IL-5. Samples
were separated on a 15% SDS–PAGE gel and compared
with human blood monocytes, which is a cell type known to
express S100A8 and S100A9 (38), as well as HEK-293 cells,
which serves as a negative control. As shown in Fig. 7A–C,
eosinophils from nine different donors expressed an 11 and
a 14 kDa protein comparable to monocyte control samples.
Addition of GM-CSF or IL-5 to the media significantly enhanced the expression of the bands depicting eosinophilic
S100A8 and S100A9 at 1 h. The presence of these proteins
were further evaluated via immunofluorescence microscopy
and found to be expressed throughout the eosinophil (Fig.
4B and C). In addition, analysis of BAL eosinophils revealed
the presence of S100A8 and S100A9 in lung eosinophils
(Fig. 7D).
S100A8 and S100A9 release by human primary blood and
BAL eosinophils
Increased levels of S100A8 and S100A9 have been indicated to occur in mucosal inflammation (39). To assess if
eosinophils can release these proteins, human primary
blood and BAL eosinophils (5 3 106 cells/ml) were cultured
for 24 h in 0.1% HSA RPMI in the presence of control buffer,
100 pM GM-CSF or 100 pM IL-5. Cell lysates and conditioned media were obtained from these cultures and loaded
onto 15% SDS–PAGE gels, transblotted and immunostained
for S100A8 or S100A9. In Fig. 8A and B, representative
immunoblots from a total of seven different eosinophil donors
are shown wherein S100A8 and S100A9 immunoreactivity
are identified in both the cell lysates and the conditioned
media. Densitometry analyses of immunoblot data were
718 Eosinophils express RAGE and RAGE ligands
Fig. 3. Immunoblot detection of eosinophil RAGE protein. Human primary blood eosinophils (EOS) were assessed for RAGE protein expression
compared to positive (DU145) and negative (H358) controls (A). Cell lysates (50 lg of protein) were loaded onto a 10% SDS–PAGE gel and
examined for the expression of RAGE via immunoblotting as detailed under Methods. Data were also quantified with ImageJ software and
expressed as RAGE mean band densitometry relative to actin controls, 6SEM, N = 3 (B). RAGE expression by BAL eosinophils was assessed
similar to primary eosinophils with the AF1145 polyclonal antibodies. Data displayed represent the expression of RAGE in three BAL donors in
comparison to blood EOS obtained from different donors or the same donor, N = 3 (C).
performed where trends suggest increased S100A9 production in response to treatment with GM-CSF or IL-5 after 24-h
culture. In addition, analysis of BAL eosinophil-conditioned
media also indicated the release of S100A8 and S100A9 by
these cells (Fig. 8E).
S100B induced eosinophil PKC-d phosphorylation
RAGE-mediated activation of PKC-d (as assessed by its
phosphorylation state) has been previously shown to occur
in neurons (40). To test whether eosinophils can also rapidly
(within 5 min) respond to RAGE ligands, eosinophils were
examined for PKC-d phosphorylation in response to fMLF,
a known activator of PKC-d (41), or in response to S100B
treatment. As illustrated in Fig. 9, S100B induced a significant increase (>50%) in the level of eosinophil PKC-d phosphorylation (using either actin or total PKC-d loading
controls), and this observation is consistent with role for
PKC-d in mediating S100B responses in human eosinophils.
Effects of S100B treatment on CD11b expression/activation,
cell size and viability
The S100 proteins have been found to enhance the expression of the adhesion receptor CD11b and to increase cell
survival in monocytes and neurons, respectively (42). In
eosinophils, these responses are regulated by the activation
of PKC-d (43), a serine and threonine kinase we observed
to be activated by S100B (Fig. 9). To examine S100Binduced CD11b expression, eosinophils from six different
donors were cultured in 0.1% HSA RPMI and treated with
buffer control, 1 pM GM-CSF or 10 lg ml 1 S100B for 24 h
and assessed for CD11b expression and activation. As
shown in Fig. 10A, where all donors were analyzed in aggregate, CD11b surface detection was not significantly affected
by S100B treatment, although two donors did exhibit an upregulation in the immunodetection of the active CD11b compared with the vehicle control. In addition, as presented
in Fig. 10B, treatment of eosinophils with GM-CSF led to
the expected and significantly augmented CD11b surface
detection and activation compared with the vehicle control.
Of note, addition of S100B to GM-CSF-treated eosinophil
cultures, as shown in Fig. 10C, appeared to alter the response of several donors when compared with GM-CSF
treatment alone (Fig. 10B). When directly assessing potential
differences in eosinophil CD11b responses between GMCSF and GM-CSF plus S100B treatments (Fig. 10D), we
observed distinct but differential changes in the detection
of CD11b surface expression and activation, indicating
Eosinophils express RAGE and RAGE ligands
719
Fig. 4. Immunofluorescence detection of S100A8, S100A9 and RAGE protein in human eosinophils. Human primary blood eosinophils were
cytospun onto coverslips, fixed, permeabilized and immunostained (as detailed under Methods) using goat polyclonal IgG (A), S100A8 (B),
S100A9 (C) or RAGE (D) antibodies. DAPI nucleic acid stain depicted the eosinophil bi-lobed nuclei (A–D), 1003 images, N > 2.
Fig. 5. Detection of S100B-induced eosinophil RAGE protein via immunoblotting. Human primary blood eosinophils (2 3 106/ml) were cultured
for 1 h in 0.1% HSA RPMI in the presence of buffer control, 100 pM GM-CSF, 100 pM IL-5 or 10 lg ml 1 S100B. Cell lysates (50 lg) of protein
were loaded onto a 10% SDS–PAGE gel and examined for RAGE expression by immunoblotting (A). Data were quantified with ImageJ software
and expressed as RAGE mean band densitometry relative to actin controls, 6SEM, N = 5 (B).
donor-dependent variations in response to GM-CSF versus
GM-CSF plus S100B. Furthermore, in Fig. 10E–G, data are
shown illustrating that cell size and PI staining are reduced
by treatment with GM-CSF or GM-CSF plus S100B compared with the control. These data indicate that eosinophils
cultured above agarose and treated with GM-CSF plus
S100B are more morphologically uniform in cell size and
more viable when compared with other cell treatment
groups.
S100B-induced alterations in eosinophil survival
Cytokines pivotal to eosinophilic function, such as IL-5 and
GM-CSF, are known to enhance eosinophil survival (25). To
further test whether eosinophil survival is affected by S100B
administration, eosinophils from four different donors were
cultured in 0.1% HSA RPMI and treated with buffer control,
100 pM GM-CSF or increasing concentrations of S100B
for 48 h. In Fig. 11, we show that S100B addition was able
to enhance the survival of eosinophils at this time point by
;3-fold; this response was dose dependent and peaked
between 10 and 30 lg ml 1 (480 and 1400 nM) of S100B.
Additional experiments examining Annexin V staining also
suggested that S100B treatment is likely attenuating cell
death that is occurring via apoptosis (Supplemental Figure
1 is available at International Immunology Online). Interestingly, S100B levels (>500 nM) have been associated with
720 Eosinophils express RAGE and RAGE ligands
Fig. 6. Detection of eosinophil soluble RAGE (sRAGE) protein. Human primary blood eosinophils (2 3 106/ml) were cultured in 0.001%
HSA RPMI in the presence of buffer control or 100 pM GM-CSF. Total eosinophil cell lysates from 1 3 106 cells or the total corresponding
conditioned media were loaded onto a 12.5% SDS–PAGE gel and immunoblotted as detailed under Methods. Immunoblots detecting
soluble and cellular RAGE from a representative eosinophil donor are displayed (A). Immunoblot data from multiple patients were
quantified with ImageJ software and expressed as RAGE mean band densitometry relative to actin controls, 6SEM, N = 4 (B).
Fig. 7. Immunoblot detection of cellular levels of eosinophil S100A8 and S100A9. Human primary blood eosinophils (EOS) were cultured for
1 h in 0.1% HSA RPMI (2 3 106/ml) in the presence of buffer control, 100 pM GM-CSF or 100 pM IL-5. Cell lysates (50 lg of protein) were
loaded onto a 15% SDS–PAGE gel and immunoblotted for S100A8 (A and B) and S100A9 (A and C); protein expression was compared with
positive (human peripheral blood monocytes (Mono)) or negative (HEK-293) controls via immnoblot (A) and densitometry analysis with
ImageJ software (B and C). A total of 11 samples of protein were examined from nine different donors where two blots were probed for both
proteins. Data are expressed as S100A8 or S100A9 mean band densitometry relative to actin, 6SEM: treatment versus control; *P < 0.05,
N = 6 (S100A8), N = 5 (S100A9). BAL EOS S100A8 and S100A9 cellular expression were also assessed in comparison to blood EOS from the
same or different donor, N = 3 (D).
Eosinophils express RAGE and RAGE ligands
721
Fig. 8. Immunoblot detection of extracellular eosinophil S100A8 and S100A9. Human primary blood eosinophils (5 3 106/ml) were cultured
in 0.1% HSA RPMI in the presence of buffer control, 100 pM GM-CSF or 100 pM IL-5 for 24 h. Total eosinophil cell lysates from 5 3 105 cells
and the total corresponding conditioned media were loaded onto a 15% SDS–PAGE gel. Representative immunoblots for S100A8 and
S100A9 are, respectively, displayed in (A) and (B). Data were also quantified with ImageJ software and expressed as S100 mean band
densitometry relative to actin controls (C and D), 6SEM, N = 3 (S100A8), N = 4 (S100A9). BAL eosinophil S100A8 and S100A9
extracellular expression were also assessed by immunoblot (E).
Fig. 9. S100B-induced phosphorylation of PKC-d in human eosinophils. Human primary blood eosinophils (2 3 106/ml) were cultured in
0.1% HSA RPMI in the presence of buffer control, fMLF (100 nM) or S100B (10 lg ml 1) for 5 min. Cell lysates (100 lg of protein) were
loaded onto a 10% SDS–PAGE gel and probed for PKC-d phosphorylation. Assessment of PKC-d phosphorylation was ascertained by
immunoblot (A and B) and quantified by ImageJ densitometry analysis (C and D). Data are expressed as PKC-d mean band densitometry
relative to actin, 6SEM: treatment versus control; *P < 0.05, N = 5 (C) or PKC-d mean band densitometry relative to total PKC-d (T-PKC-d),
6SEM: treatment versus control *P < 0.05, N = 3 (D).
722 Eosinophils express RAGE and RAGE ligands
Fig. 10. Continued
neuronal apoptosis and the activation of astrocytes and
microglia (26).
Discussion
Despite the discovery of eosinophils over a century ago, insight into the biochemical functions of these granulocytic,
myeloid cells in various human diseases has remained elusive (44). Of note, the presence of RAGE ligands and the
expression, activation and release of RAGE have been identified in chronic inflammatory disorders associated with eosinophils (1–7, 9, 11, 12, 14). To explore the potential
presence and function of RAGE in eosinophils, we examined
our microarray data (Fig. 1) and confirmed the presence of
Eosinophils express RAGE and RAGE ligands
723
Fig. 10. S100B induced CD11b, size and viability responses after 24 h in culture. Detection of CD11b expression and activation (A–D). Human
primary blood eosinophils (2 3 105/ml) were cultured in 0.1% HSA RPMI atop 0.5% w/v agarose and assessed via flow cytometry (see Methods) for
CD11b expression and activation in response to buffer control, GM-CSF (1 pM) or S100B (10 lg ml 1) after 24 h of treatment. Cells were negatively
selected for PI stain and positively selected for CD11b 488 Alexa Fluor fluorescence. The CD11b data are displayed as the average 488 geometric
mean fluorescence (GMF) of eosinophils from six donors, 6SEM. Analyses were performed comparing control versus S100B treatment (A), control
versus GM-CSF treatment (B), control versus GM-CSF + S100B (C) and GM-CSF versus GM-CSF + S100B (D). Detection of size and viability
changes (E–G). Representative density plot images display eosinophil size (E) and viability (F) in response to buffer control, GM-CSF (1 pM) or
S100B (10 lg ml 1) after 24 h of treatment. Cell gates were established on the GM-CSF + S100B sample and copied to all other samples. Increased
cell size is identified by forward scatter (FSC) and marked by a solid line (E). Increased cell death is identified by PI stain and marked by a dashed
line (F). Percentages marked in Panels E and F were tabulated and charted (G), 6SEM, *P < 0.0001 versuss control, yP = 0.04 versus control, zP =
0.007 versus control. Data displayed (A–G) associate smaller cell size and increased viability with CD11b expression and activation.
eosinophil RAGE transcripts by examining mRNA from cells
obtained from three different donors (Fig. 2). Bioinformatic
analyses on the cDNA sequences obtained from two separate donors also revealed identity with the RAGE gene. We
identified eosinophil RAGE protein in both blood and lung
eosinophils via immunoblot analysis (Fig. 3). In addition,
immunofluorescent detection of RAGE in eosinophils
revealed a distributed pattern (intracellular and plasma
membrane associated; see Fig. 4). Such a pattern may represent multicompartment trafficking and activation of the
receptor and would be consistent with possible RAGE ligation and internalization via one of the many eosinophil RAGE
ligands (Fig. 1) (45). The distributed pattern is also noted in
the immunofluorescent detection of S100A8 and S100A9
(Fig. 4), which are two proteins that have been found to
intracellularly co-localize with RAGE (46). In addition, we
determined that S100B treatment can induce cellular RAGE
production (Fig. 5), whereas cytokines tend to reduce the
detection of cellular RAGE but increase the detection of soluble RAGE (Figs 5 and 6), suggesting that cytokines may
activate cell signals involved proteolytic mechanisms. Interestingly, this form of soluble RAGE has previously been
reported in monocytes (18) and may be increased in the
lung following cytokine exposure, which would be consistent
with our observation, that cellular RAGE levels appear to be
lower in lung versus blood eosinophils (Fig. 3). It is noteworthy that the RAGE gene has been mapped to human chromosome 6P21.3 in the human major histocompatibility
724 Eosinophils express RAGE and RAGE ligands
Fig. 11. S100B induced eosinophil survival after 48 h in culture. Human primary blood eosinophils were cultured in 0.1% HSA RPMI atop 0.5%
w/v agarose in the presence of buffer control, GM-CSF (100 pM) or S100B (0.3, 1, 3, 10, 30 lg ml 1) for 48 h. The percentage of viable cells was
determined by exclusion of PI (3 lg ml 1) via flow cytometric analysis as visualized (A) and charted (B). Cell gates were established on the viable
cells in the GM-CSF-treated sample, which were >70% viable. Cell percentages are averaged, 6SEM: *P = 0.02 versus control, yP = 0.007
versus control, N = 4.
complex (MHC) class III locus near the junction with MHC
class II (47, 48) and that altered expression of the classical
and non-classical MHC genes has been linked to various
diseases, such as diabetes, asthma, rheumatoid arthritis
and cancer (49). Together, these data suggest that the
expression of RAGE, soluble RAGE and RAGE ligands by
eosinophils may play an important role in the progression and persistence of multiple disorders, including chronic
inflammatory diseases.
In our microarray investigations, the S100A8 and S100A9
transcripts were also identified as present (Fig. 1). The
S100A8 and S100A9 proteins are RAGE ligands (50) that
have been reported to be involved in the differentiation of
myeloid cells (50, 51). In examining eosinophil S100A8 and
S100A9 expressions, we found that without stimulation, eosinophils expressed low and comparable levels of both
S100A8 and S100A9 proteins (Fig. 7) throughout the cytoplasm and potentially at the cell surface (Fig. 4). Interestingly, up-regulation of the S100A8 and S100A9 proteins has
been indicated to occur in a RAGE-dependent manner (5).
RAGE and the cytokines, GM-CSF and IL-5, reportedly
induce many of the same intracellular signals [p38 (52, 53),
PKC-d (40, 54), JAK2 (55, 56) STAT5 (56, 57)], suggesting
that GM-CSF and IL-5 may activate RAGE or indirectly
induce cell signals in common with RAGE for S100A8 and
S100A9 production. In response to a 1-h treatment with 100
pM GM-CSF or IL-5, we found an increase in the expression
of both proteins in human primary eosinophils (Fig. 7).
S100A8 and S100A9 are structural proteins known to
promote tubulin polymerization and regulate the migratory
capacity of phagocytic cells (58). These proteins may therefore play a role in the process of eosinophil priming where
enhanced cell signaling in response to chemokines has
been found to occur subsequent to ‘priming’ eosinophils for
1 h with either IL-5 or GM-CSF (59). We found the presence
of S100A8 and S100A9 transcripts in eosinophils after 4 h of
incubation, but only the mRNA levels of S100A8 increased
in response to the addition of GM-CSF or IL-5 (Fig. 1). This
Eosinophils express RAGE and RAGE ligands
result may indicate that S100A9 transcription is comparatively rapid and is sufficient to stabilize S100A8, a mechanistic function of S100A9 that has been previously explored
(58, 60) and/or that control of S100A9 protein levels can
also occur post-transcriptionally. After 24 h of incubation,
S100A8 and S100A9 proteins were identified in eosinophilconditioned media, indicating that these proteins are released by eosinophils (Fig. 8). Elevated human S100A8/
S100A9 serum levels are found in patient studies of type 1
and 2 diabetes mellitus (61, 62), cystic fibrosis (63), cardiac
sarcoidosis (64), acute coronary syndrome (65), Kawasaki
disease (66), inflammatory bowel disease (15), acute pancreatitis (67), psoriasis (68), juvenile idiopathic arthritis (69),
rheumatoid arthritis (70), acute allograft rejection (71, 72),
malaria (73) and cancers of the colon (74) and ovary (75).
Possible eosinophil degranulation associated with S100A8
and S100A9 release, by IL-5- or GM-CSF-activated eosinophils, may aid in our understanding of chronic mucosal
inflammatory diseases, such as cystic fibrosis and inflammatory bowel disease, which are characterized by the presence of these S100 proteins as well as the eosinophilic
granule proteins (14, 15, 76, 77).
Aside from S100A8 and S100A9, other RAGE ligands such
as S100A4, S100A6, S100A10, S100A11 and S100P were
also identified as present on the microarray (Fig. 1), whereas
S100A1, S100A2, S100A3, S100A5, S100A7, S100A12,
S100B and S100G were reported as absent (data not
shown). RAGE is indicated to interact with the S100 proteins
identified as present in the microarray with the exception of
S100A10 (26, 50). This latter protein (S100A10/annexin II
ligand/calpactin I) is predominantly known to co-localize with
annexin A2, leading to the organization of cytoskeletal and
membrane proteins (78). S100A11 (calgazzarin) has similar
interactions via annexin A1 and reported extracellular functions in regulating keratinocyte growth and differentiation
(79, 80). S100A4 (metastasin), S100A6 (calcyclin) and
S100P are produced by various cell types and like most
S100 proteins are found up-regulated in human cancer (4).
These attributes of S100 proteins may offer insight into the
structural changes (shape, size and density) found to occur
in eosinophils upon activation (81, 82) and the function of
eosinophils in the tumor microenvironment (83).
Eosinophil cell survival, changes in cell shape and CD11b
detection have been previously found to be regulated by
PKC-d (43, 84, 85), a cell signal molecule we show to be
phosphorylated in response to micromolar levels of S100B
(Fig. 9). Previous research has indicated that nanomolar
concentrations of S100B can induce neurotrophic effects
and repair/regeneration responses, whereas micromolar levels induce neurotoxicity and microglia activation (26). In our
study, we found that micromolar levels of S100B enhanced
eosinophil viability (Fig. 11), which may, in part, account for
the association of eosinophils in certain types of central nervous system pathologies.
In examining CD11b responses in eosinophils, we found
that S100B can alter CD11b expression and activation in
a subset of individuals, particularly when administered in
conjunction with GM-CSF (Fig. 10D). S100B addition also
resulted in the increased survival and reduced size of
human primary eosinophils. As shown in Fig. 10E–G, these
725
attributes were significantly evident upon GM-CSF treatment
but were further enhanced by the addition of S100B, which
may, in part, be reflective of cell:cell interactions during culture above agarose versus non-specific adhesion and activation known to occur when cells are cultured on
polystyrene (86). In addition, the S100B protein is known to
interact with components of the cytoskeleton and modulate
cell signals associated with cell morphology (87). The uniformity in size as shown in Fig. 10E–G suggests potential
changes in the cytoskeleton and granule compartments that
encourage the retention of granules (low side scatter), viability (lack of PI stain) and a morphologic decrease in size (low
forward scatter). These characteristics are similar to identified BAL hyperdense eosinophils found after allergen challenge in a guinea pig model (88). In the antigen-challenged
microenvironment, release of GM-CSF and S100B by T cells
(8, 89) or certain tumor cells (90–93) may encourage the
accumulation of eosinophils as has been identified in the
asthmatic lung (94) and tumors of the colon (95) and skin
(6). These observations suggest that in the presence of GMCSF and S100B alone, cell signals in eosinophil degranulation are altered, thereby encouraging persistent and chronic
inflammation.
The S100B cytokine is secreted by T cells, astrocytes, oligodendrocytes, found in various isolated human tumor tissue samples and known to bind and activate RAGE (8, 30,
42, 96, 97). Approximately 7 g of S100B is expressed in
a healthy 1.4 kg human brain (98). Inflammation and tumor
formation are indicated to increase tissue-level expression
and the release of S100B (93, 98, 99). Eosinophils have
been indicated to migrate into the brain (100), infiltrate
tumors (83) and associate with neurons (9). While the functions of tissue-associated eosinophilia are not clearly
known, a reparative process is suspected, particularly with
reference to developing subdural hematomas (100). The
interactions of eosinophils with S100B and additional proteins in the microenvironment at these sites may lead to
eosinophil activation, degranulation and the release of fibrinolytic (plasminogen) and cytotoxic (eosinophil cationic
protein, eosinophil-derived neurotoxin) proteins involved in
wound repair and the clearance of damaged cells. Alternatively, eosinophils may release S100A8 and S100A9 and
possibly other additional S100 proteins known to enhance
tumor progression (101) and inflammation (16).
In summary, we have identified the expression of RAGE
and RAGE ligand mRNA and protein in human primary
blood eosinophils. We have also shown that eosinophils
respond to a reported RAGE ligand, S100B. These findings
enhance our understanding of eosinophil function and the
evolution of disease and immunity.
Supplementary data
Supplementary data are available at International Immunology Online.
Funding
This work was supported by National Institutes of Health
(HL088594, HL56396, AI070503 and HL069116) to P.J.B. as
well as by the institutional grant (1UL1RR025011) from the
726 Eosinophils express RAGE and RAGE ligands
Clinical and Translational Science Award program of the
National Center for Research Resources, National Institutes
of Health.
Acknowledgements
The authors wish to thank Lisa Y. Lenertz-Lindemer, PhD, and Lei Shi,
PhD, for their assistance in PCR techniques; Michael D. Evans, MS,
for statistical analysis of the CD11b data and Gregory J. Wiepz, PhD,
for helpful scientific discussion and reviewing this manuscript.
Conflict of Interest. The authors have no financial conflict of interest.
Authorship: C.S.C. designed and performed experiments, analyzed
data and wrote the paper; P.J.B. designed the research and edited
the paper.
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