INFECTION AND IMMUNITY, Oct. 2008, p. 4489–4497
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 10
Human Mast Cell Activation by Staphylococcus aureus: Interleukin-8
and Tumor Necrosis Factor Alpha Release and the Role of
Toll-Like Receptor 2 and CD48 Molecules?
Claudio M. Rocha-de-Souza,1Beata Berent-Maoz,1David Mankuta,2
Allon E. Moses,3and Francesca Levi-Schaffer1*
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of
Jerusalem, Jerusalem, Israel1; Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel2; and
Department of Clinical Microbiology and Infectious Diseases, Hadassah University Hospital, Jerusalem, Israel3
Received 27 February 2008/Returned for modification 17 April 2008/Accepted 10 July 2008
The ability of Staphylococcus aureus to invade and survive within host cells is believed to contribute to its
propensity to cause persistent and metastatic infections. In addition, S. aureus infections often are associated
with atopic diseases such as dermatitis, rhinitis, and asthma. Mast cells, the key cells of allergic diseases, have
a pivotal role in innate immunity and have the capacity of phagocytosis, and they can destroy some pathogenic
bacteria. However, little is known about the ability of some other bacteria to survive and overcome mast cell
phagocytosis. Therefore, we were interested in evaluating the interplay between mast cells and S. aureus. In this
study, we show that human cord blood-derived mast cells (CBMC) can be infected by pathogenic S. aureus. S.
aureus displayed a high adherence to mast cells as well as invasive and survival abilities within them. However,
when infections were performed in the presence of cytochalasin D or when CBMC were preincubated with
anti-Toll-like receptor 2 (TLR2) or anti-CD48 antibodies, the invasiveness and the inflammatory response were
abrogated, respectively. Furthermore, we observed an increase of TLR2 and CD48 molecules on CBMC after
S. aureus infection. The infection of CBMC with S. aureus also caused the release of tumor necrosis factor alpha
(TNF-?) and interleukin-8 (IL-8). Both live and killed S. aureus organisms were found to trigger TNF-? and
IL-8 release by CBMC in a time-dependent manner. Cumulatively, these findings suggest that S. aureus
internalizes and survives in mast cells. This may play an important role in infections and in atopic diseases
associated with S. aureus.
Staphylococcus aureus, a gram-positive bacterium colonizing
the human skin and nasopharynx, is one of the most important
human pathogens, and it causes various superficial, systemic,
and nosocomial infections (13). S. aureus infections often are
followed by the bacterial invasion of the vascular system, lead-
ing to bacteremia and sepsis. The ability of S. aureus to be
internalized by and survive within some host cells, such as
endothelial cells, keratinocytes, and epithelial cells, may con-
tribute to the development of persistent or chronic infections
and may eventually lead to deeper tissue infections or dissem-
ination (14, 15, 23). In particular, the invasion of vascular
endothelial cells is thought to be a critical step in the devel-
opment of metastatic infections in patients with S. aureus bac-
teremia, since it results in the upregulation of procoagulant
activity, the expression of surface adhesion molecules, and the
release of proinflammatory cytokines (36, 43). The coloniza-
tion of human skin by S. aureus is also a characteristic feature
of several inflammatory skin diseases, which often are followed
by tissue invasion and severe cell damage in a fibronectin-
binding protein (FnBP)-dependent manner (15, 27). S. aureus
also can survive engulfment by professional phagocytes such as
neutrophils and monocytes. In both of these cell types, S.
aureus promptly escapes from the endosomes/phagosomes and
proliferates within the cytoplasm (44). Furthermore, recent in
vitro studies showed that S. aureus survived inside macro-
phages in a metabolically active form that did not affect the
viability of these cells until the intracellular environment be-
came suitable for escape. This finding suggests that the ability
of S. aureus to survive phagocytosis in human macrophages
contributes to the dissemination of the infection and may be
detrimental to the host (16).
An interesting link has been shown between S. aureus and
atopic diseases, such as dermatitis, rhinitis, and asthma (5), in
which it has been hypothesized that S. aureus can exacerbate
the immunoglobulin E (IgE)-mediated reactions. For example,
studies have shown greater S. aureus colonization in the skin of
patients with atopic eczema/dermatitis syndrome than in the
skin of normal healthy subjects (7, 38). Moreover, in chronic
rhinosinusitis, S. aureus enterotoxin B shifts the cytokine pat-
tern toward Th2. S. aureus enterotoxin B also stimulates the
production of interleukin-5 (IL-5) and induces polyclonal IgE
production, which might contribute to severe inflammation via
the activation of the mast cells (4, 12). IgE antibodies specific
to the S. aureus superantigen are present in nasal polyp tissue,
and their levels correlate with markers of eosinophil activation
and recruitment (25).
Mast cells are known key effector cells of IgE-mediated
hypersensitivity reactions. In addition, more recently it has
been recognized that mast cells, being strategically stationed at
* Corresponding author. Mailing address: Department of Pharma-
cology and Experimental Therapeutics, School of Pharmacy, Faculty of
Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusa-
lem 91120, Israel. Phone: 972-2-6757512. Fax: 972-2-6758144. E-mail:
?Published ahead of print on 21 July 2008.
sites exposed to the external environment, such as lung, skin,
gastrointestinal, and urinogenital tracts, play a critical role in
host defense as cardinal cells of innate immune response
against infectious pathogens, including S. aureus (17, 28). In-
deed, mast cells have been found to bind and internalize gram-
positive and gram-negative bacteria (2, 18). Mast cells can
recognize and attach to a wide variety of opsonized bacteria.
For example, Salmonella enterica serovar Typhimurium coated
with the iC3b fragment of complement is recognized through
complement receptor 3 (CR3) on the mast cell membrane (9).
In addition, mast cells express several Fc? receptors that are
involved in the binding of IgG-coated bacteria (9). Recently,
various strains of Escherichia coli, Enterobacter cloacae, and
Klebsiella pneumoniae were found to bind avidly to mouse bone
marrow-derived mast cells in opsonin-dependent conditions,
followed by their internalization within vacuoles (19). In vitro
and in vivo studies have shown that mast cells release proin-
flammatory and chemotactic mediators upon contact with
pathogens (2, 19, 21). For example, mast cell-derived tumor
necrosis factor alpha (TNF-?) modulates neutrophil influx and
bacterial clearance in Klebsiella pneumoniae infection (21).
Immune cells, including mast cells, express pattern recogni-
tion receptors that recognize pathogen-associated molecular
patterns and Toll-like receptors (TLRs), a family of proteins
that resemble the antimicrobial Toll proteins of Drosophila
(26, 35). The involvement of TLRs has been implicated in the
host response to several staphylococcal infection models (8,
41). TLR2, expressed on mast cells, has been found to recog-
nize and respond to several pathogen-associated molecular
patterns, including peptidoglycan (PGN), lipoproteins, and
lipoteichoic acid (37). PGN from S. aureus stimulates mast cells
in a TLR2-dependent manner to produce TNF-?, IL-4, IL-5,
IL-6, and IL-13 (40). The intradermal injection of PGN also
led to increased vasodilatation and inflammation through the
TLR2-dependent activation of mast cells (40). Furthermore, in
response to PGN or other TLR2 activators, mast cell degran-
ulation has been attributed a critical role in exacerbating al-
lergic diseases, especially atopic dermatitis (5).
Another important molecule in bacterium-mast cell interac-
tion is CD48, a glycosylphosphatidylinositol-anchored protein
(39). The cleavage of CD48 from the mast cell surface with
phospholipase C or its neutralization with CD48-specific anti-
bodies prevented subsequent bacterial adherence (22). The
engagement of CD48 by FimH-expressing type 1 fimbriated
Escherichia coli also was found to trigger TNF-? release by
mast cells (22).
In spite of all the aforementioned evidence of an interplay
between S. aureus and mast cells and of its importance in the
dynamics of an allergic disease concomitant to infection, the
direct cross-talk between human mast cells and S. aureus has
not been studied in depth. Therefore, we decided in the
present study to analyze the interactions between human cord
blood-derived mast cells (CBMC) and S. aureus.
We report here that, in vitro, S. aureus adheres to, invades,
survives in, and stimulates human mast cells with the involve-
ment of CD48 and TLR2.
MATERIALS AND METHODS
Bacterial culture conditions. Staphylococcus aureus ATCC 25923 and S. aureus
HUH1, a methicillin-susceptible strain isolated from a patient with a blood-
stream infection in the Department of Clinical Microbiology and Infectious
Diseases at the Hadassah University Hospital, were used. Bacteria maintained at
?70°C in skim milk-glycerol (Difco) were subcultured onto blood agar and
incubated at 37°C overnight. A sweep of colonies was inoculated into tryptic soy
broth (TSB; Difco) and incubated at 37°C for 24 h. The culture density was
estimated by the measurement of the optical density at 650 nm. Bacteria were
diluted to achieve a multiplicity of infection (MOI) of 10 before use. In some
experiments, S. aureus was heat killed (for 30 min at 60°C). After treatment,
bacteria were washed three times with phosphate-buffered saline (PBS) prior
Labeling of the bacteria. Bacteria (109ml?1) were washed twice with PBS,
suspended in fluorescein isothiocyanate (FITC; 1 ?g ml?1; Sigma, Israel) dis-
solved in PBS, and incubated for 30 min under constant shaking at 37°C. FITC-
labeled bacteria were washed three times with PBS prior to use. In some exper-
iments, sulfo-N-hydroxysuccinimide–hexanoate linker chain–biotin (Perbio
Science, Germany) was dissolved at a final concentration of 1 ?g ml?1in PBS.
Identical volumes of bacteria in FITC-PBS and biotin were combined and further
incubated under the same conditions as those described above (1).
CBMC. CBMC were obtained by culturing umbilical cord blood mononuclear
cells as previously described (3). Briefly, fresh cord blood was diluted with
Hank’s balanced salt solution, loaded onto Ficoll-Paque, and centrifuged (350 ?
g for 25 min). Mononuclear cells were washed twice with Hank’s balanced salt
solution and resuspended in 100 ml minimal essential medium alpha (MEM-?)
containing 10% (vol/vol) fetal calf serum (FCS), penicillin (100 U ml?1), strep-
tomycin (100 ?g ml?1), ribonucleosides/deoxyribonucleosides, and stem cell
factor (100 ng ml?1) (a gift from Amgen). Culture medium was replaced weekly.
CBMC were used after 8 to 12 weeks of culture, when ?97% were positive for
tryptase as assessed by intracellular flow cytometry. Cord blood was obtained
according to the Institutional Helsinki Committee guidelines of Hadassah Hos-
pital, and its use was approved by the committee.
Adherence assay. For quantitative adhesion assays, bacterial suspensions were
incubated with CBMC (2.5 ? 105ml?1) in 48-well tissue culture plates (Nunc,
Denmark). After incubation periods of 30, 60, 120, and 180 min at 37°C, S.
aureus-associated CBMC were washed three times with PBS, lysed with 0.1%
Triton X-100 in PBS, diluted, and cultured onto blood agar at 37°C for 24 h for
bacterial viable counting (in CFU per milliliter). Bacterial inocula corresponded
to the number of viable bacterial cells in the supernatant at each time point plus
the number of viable bacteria associated with CBMC (i.e., intracellular plus
extracellular bacteria) (34). In order to differentiate between extracellular and
intracellular bacteria, CBMC were grown for 24 h on circular glass coverslips
coated with 0.01% poly-L-lysine solution (Sigma, Israel) on 48-well tissue culture
plates (Nunc, Denmark). Biotinylated and FITC-labeled S. aureus (MOI of 10)
cells were incubated with CBMC (2.5 ? 105ml?1) for 180 min at 37°C. After
incubation, infected cells were fixed with 4% paraformaldehyde in PBS, washed
three times with PBS, and stained with streptavidin-allophycocyanin (BD Bio-
sciences Pharmingen) diluted 1:200 in PBS. Samples were evaluated using an
LSM 510 confocal laser-scanning microscope (Zeiss, Germany) (1).
Invasion and intracellular viability assays. The ability of S. aureus to invade
CBMC was assessed by the gentamicin protection assay (34). Briefly, after 180
min of incubation, S. aureus-associated cells were washed three times with PBS
and subsequently incubated with MEM-? containing gentamicin (300 ?g ml?1).
The wells then were incubated for 30 min at 37°C and washed three times with
PBS. Cell lysis was carried out as described for the quantitative adhesion assay,
and the lysates (100 ?l) from each well were diluted and plated onto blood agar
for the determination of viable intracellular bacteria (IC). In preliminary exper-
iments, S. aureus strains were tested for gentamicin sensitivity; no colonies were
present in blood agar after 180 min of incubation with 300 ?g ml?1of gentamicin
in MEM-? medium. The internalization assay also was carried out by flow
cytometric analysis (i.e., fluorescence-activated cell sorting [FACS]) (32), and
CBMC were incubated with killed or live FITC-labeled S. aureus. After 180 min,
the S. aureus-associated CBMC were washed twice with ice-cold flow buffer (PBS
containing 1% FCS) and resuspended in flow buffer. The samples were kept in
the dark on ice until the analysis. To eliminate signals from extracellular bacteria,
trypan blue solution (0.4%; Sigma, Israel) was added to a final concentration of
0.2% directly before analysis. Samples were analyzed using a Becton-Dickinson
FACSCalibur and Cell Quest software. To assess the amount of internalized
bacteria, the percentage of FITC-positive cells was multiplied by the mean
fluorescence intensity of these cells to obtain the uptake index (u.i.). No toxic
effect was detected toward S. aureus strains during FITC or biotin labeling.
Furthermore, to check the validity of the quenching approach, FITC-labeled S.
aureus alone with or without trypan blue treatment was analyzed by flow cytom-
etry. In previous experiments, the FITC-labeled S. aureus signal was abrogated
after trypan blue treatment. To observe intracellular killing or bacterial survival,
4490 ROCHA-DE-SOUZA ET AL.INFECT. IMMUN.
the experimental design of the protection antibiotic assay was changed. CBMC
were infected with S. aureus for 180 min, and at this point the medium was
replaced with MEM-? containing 300 ?g ml?1gentamicin. Instead of analyzing
all samples after 30 min of gentamicin treatment, samples were taken out after
30, 60, 120, and 180 min. The number of viable bacteria at 30 min was considered
Cytochalasin D treatment. To study the role of the cytoskeleton in S. aureus
uptake, CBMC were incubated with cytochalasin D (2.5 to 5 ?g ml?1; Sigma,
Israel) for 60 min at 37°C before S. aureus interaction and also during the entire
gentamicin protection assay, as described above. In preliminary experiments,
cytochalasin D was assessed for toxicity for both S. aureus strains and CBMC and
was found either to have no adverse effects or to change adherence and invasion
properties at concentrations of up to 5 ?g ml?1.
CD48- and TLR2-mediated inhibition invasion assay. The involvement of
CD48 and TLR2 molecules in CBMC during exposure to S. aureus also was
investigated. CBMC were incubated with anti-human CD48 (MEM-102; 10 ?g
ml?1; Santa Cruz Biotechnology), anti-human TLR2 (clone TL2.1; 10 ?g ml?1;
Ebioscience), or isotype-matched IgG1 (MP Biomedicals, Germany) at room
temperature for 30 min before the adherence and internalization assays were
TLR2 and CD48 expression. To analyze the expression of TLR2 and CD48 on
CBMC surfaces, killed or live S. aureus-associated CBMC were incubated with
anti-human TLR2 (clone TL2.1; 10 ?g ml?1; Ebioscience), anti-human CD48
(MEM-102; 10 ?g ml?1; Santa Cruz Biotechnology), or isotype-matched IgG1
(MP Biomedicals, Germany) and were labeled with FITC-goat anti-mouse IgG
(1:100; Santa Cruz Biotechnology). Cells were incubated with the antibodies at
room temperature for 30 min. Analysis was performed by FACS.
Evaluation of cytokine concentration. CBMC suspensions were analyzed for S.
aureus-specific IL-8 and TNF-? cytokine production. CBMC were incubated with
killed or live S. aureus at different time points at 37°C. The levels of each cytokine
in culture supernatants were determined by using specific enzyme-linked immu-
nosorbent assay kits according to the manufacturer’s instructions (R&D Sys-
tems). The results are expressed as the concentration of cytokine per 106CBMC,
as extrapolated from a standard curve with recombinant cytokine. To define the
possible role of S. aureus invasion in IL-8 and TNF-? release, CBMC were
treated with cytochalasin D for 60 min before infection. To rule out the possi-
bility that cytochalasin D treatment affected the capacity of CBMC to release
IL-8 and TNF-?, cytochalasin D-treated cells (5 ?g ml?1) and nontreated cells
were incubated with PGN (10 ?g m?1; Sigma, Israel), a well-known TLR2
agonist and cell activator. To study the possible role of TLR2 and CD48 mole-
cules on S. aureus-induced IL-8 and TNF-? release, CBMC were treated with
anti-human CD48 (10 ?g ml?1), anti-human TLR2 (10 ?g ml?1), both antibod-
ies, or isotype-matched IgG1 for 60 min before infection.
Statistical analysis. All assays were performed in triplicate and repeated at
least three times. Results are presented as the means ? standard deviations. The
Student’s t test or analysis of variance followed by the Tukey-Kramer multiple
comparison test were used to compare means, with a P ? 0.05 considered
S. aureus invades and survives in CBMC. It has been rec-
ognized that adherence to and invasion in host cells are im-
portant steps in the pathogenesis of many bacteria, including
that of S. aureus (15). As shown in Table 1, S. aureus strains
quickly adhered to CBMC (within 30 min). The number of
adherent bacteria increased steadily with time similarly for
both strains. In fact, at 30 min, 9.86 (strain 25923) and 10.66%
(HUH1) of the bacteria adhered to CBMC, whereas at 180
min we found 41.14 and 34.29% adherent bacteria, respec-
tively. To check whether bacterial internalization takes place,
CBMC were incubated with biotinylated and FITC-labeled S.
aureus. Confocal microscopy clearly differentiated between
several small clusters of extracellular bacteria adhering to the
CBMC surface and small clusters of intracellular bacteria (Fig.
1). In addition, the antibiotic protection assay detected viable
internalized bacteria at 30 min postinfection. The percentage
of internalized bacteria increased with time, with a mean of
0.09 and 1.49% for the HUH1 strain and 0.06 and 1.36% for
strain 25923 after 30 and 180 min, respectively (Table 1). Fur-
thermore, we compared the internalization of live bacteria to
that of heat-killed bacteria by CBMC at 180 min. FACS anal-
ysis (Fig. 2) revealed that approximately 60% more S. aureus
cells were internalized by CBMC when the bacteria were live
than when they were heat killed (u.i., 4.05 ? 106? 1.3 ? 106
for live and 1.69 ? 106? 0.3 ? 106for heat killed [P ? 0.001]).
Furthermore, FACS analysis detected approximately 65 and
41% of the cell population with a strong fluorescent signal
TABLE 1. Staphylococcus aureus viable bacteria associated with and internalized by CBMCa
No. (%) of associated cells No. (%) of internalized cellsNo. (%) of associated cellsNo. (%) of internalized cells
3.38 ? 105? 1.08 ? 105(9.87)
1.07 ? 106? 4.93 ? 106(14.53)
2.47 ? 106? 7.02 ? 106(22.12)
7.69 ? 106? 1.29 ? 106(41.14)
2.87 ? 103? 4.78 ? 103(0.06)
1.43 ? 104? 3.69 ? 104(0.23)
4.31 ? 104? 1.31 ? 104(0.49)
1.52 ? 105? 3.65 ? 105(1.36)
4.18 ? 105? 4.76 ? 105(10.66)
1.78 ? 106? 4.98 ? 106(19.87)
4.89 ? 106? 1.19 ? 106(28.96)
1.20 ? 107? 4.08 ? 107(34.29)
3.25 ? 103? 2.88 ? 103(0.09)
1.91 ? 104? 5.45 ? 104(0.26)
8.17 ? 104? 4.84 ? 104(0.66)
3.48 ? 106? 1.96 ? 106(1.49)
aS. aureus strains (MOI of 10) were incubated for up to 180 min with CBMC (2.5 ? 105ml?1) at 37oC and analyzed by quantitative adherence and antibiotic
protection assays. The numbers are given as absolute values, and numbers in parentheses are the percentages of the recovered bacteria compared to the total number
of bacteria in the inocula (i.e., the number of viable bacterial cells in the supernatant at each time point plus the number of viable bacteria associated with CBMC
?intracellular plus extracellular? or plus the number of intracellular bacteria only). Data are means ? standard deviations from three experiments performed in triplicate.
FIG. 1. S. aureus invades CBMC. CBMC were incubated with
biotinylated and FITC-labeled S. aureus (MOI of 10) for 180 min at
37°C. After infection, samples were fixed with 4% paraformaldehyde in
PBS and stained with streptavidin-allophycocyanin. Samples were eval-
uated by confocal microscopy. (A) CBMC and small clusters of extra-
cellular bacteria (arrow) plus intracellular bacteria (thin arrow).
(B) CBMC and small clusters of extracellular bacteria.
VOL. 76, 2008S. AUREUS-INDUCED MAST CELL ACTIVATION4491
when CBMC were incubated with live or heat-killed S. aureus,
respectively. Since fluorescence emitted by extracellular bacte-
ria and CBMC-attached bacteria was quenched by trypan blue,
this strong fluorescent signal from CBMC suggests the pres-
ence of intracellular bacteria.
To assess the role of the cytoskeleton on S. aureus internaliza-
tion by CBMC, the infections were carried out in the presence of
cytochalasin D (2.5 and 5 ?g ml?1). The data presented in Fig.
3 show that cytochalasin D inhibited S. aureus internalization
by up to 80% (P ? 0.001).
To evaluate whether intracellular S. aureus is killed by
CBMC, after 180 min of bacterium-cell interaction, the CBMC
were incubated with gentamicin at different time points. Sur-
prisingly, an increase of bacterial survival was found (?57%
increase) for both strains after 180 min compared to the per-
centage of recovered bacteria after 30 min (100% of viable
bacteria recovery) (Table 2). This is consistent with an in-
creased survival of bacteria after being internalized by CBMC.
S. aureus stimulates TLR2 and CD48 expression on CBMC
surface. Uninfected CBMC expressed low levels of TLR2 and
CD48 on the cell surface (Fig. 4A, B). However, after infection
with S. aureus, an increase of TLR2 and CD48 molecule ex-
pression on the CBMC surface was detected. The increase of
these molecules was evident at 30 min and was augmented at
180 min after S. aureus invasion and survival (Fig. 4). Infected
CBMC incubated with isotype-matched IgG1 expressed low
levels of TLR2 and CD48 that were similar to those expressed
by uninfected CMBC. These results indicate that S. aureus is
capable of upregulating TLR2 and CD48 on CBMC in a time-
dependent fashion. To determine whether TLR2 expression
induced in response to S. aureus is dependent on bacterial
viability, CBMC were incubated with killed S. aureus. Killed S.
aureus did not result in the stimulation of TLR2 expression on
CBMC (data not shown).
CD48 and TLR2 molecules are involved in S. aureus inter-
nalization by CBMC. It has been recognized that CD48 and
TLR2 are involved in bacterium-mediated cell activation (22,
37). Therefore, CBMC were preincubated with TLR2- and
CD48-neutralizing antibodies. As detected by FACS and by
the antibiotic protection assay (Fig. 5A, B), anti-TLR2 and
anti-CD48 neutralizing antibodies decreased S. aureus inter-
nalization by CBMC by approximately 40% compared to S.
FIG. 2. FACS analysis of S. aureus internalization. The FACS of CBMC in the presence of trypan blue discriminates cell-associated extracel-
lular bacteria from intracellular bacteria. CBMC were incubated with killed FITC-labeled S. aureus (A) or live FITC-labeled S. aureus (B) for 180
min at 37°C. After the addition of trypan blue, samples were analyzed by FACS. Data are from a representative experiment (n ? 3) that depicts
the percentage of CBMC intracellular S. aureus as well as the percentage of the u.i. M1 is the marker set at approximately 95% of the total events
using nonlabeled S. aureus.
FIG. 3. Cytochalasin D inhibits S. aureus invasion of CBMC.
CBMC treated with cytochalasin D for 60 min before infection were
incubated with S. aureus strains (MOI of 10) for 180 min at 37°C, and
the percentage of internalized bacteria was evaluated. Results are the
means (? standard deviations) from three independent experiments
(? and ??, P ? 0.001 compared to results for untreated cells).
TABLE 2. S. aureus intracellular survival after internalization
No. (%) of viable bacteria recovered
ATCC 25923 HUH1
1.32 ? 105? 1.03 ? 105(100)
1.44 ? 105? 3.21 ? 105(109)
1.63 ? 105? 2.37 ? 105(124)
1.94 ? 105? 0.45 ? 105(147)
2.63 ? 105? 0.32 ? 105(100)
2.97 ? 105? 1.25 ? 105(113)
3.44 ? 105? 0.05 ? 105(131)
4.29 ? 105? 0.62 ? 105(157)
aCBMC (2.5 ? 105ml?1) were incubated with S. aureus strains (MOI of 10)
for 180 min at 37°C. After infection, CBMC were incubated with gentamicin (300
?g ml?1) for the indicated periods of time, and the number of CFU protected
from gentamicin killing was counted (calculated as described in footnote a of
Table 1). Data are means ? standard deviations from three experiments per-
formed in triplicate.
4492 ROCHA-DE-SOUZA ET AL.INFECT. IMMUN.
aureus internalization by nontreated CBMC (for anti-TLR2,
4.38 ? 106? 1.4 ? 106IC for isotype-treated CBMC and
1.53 ? 106? 1.7 ? 106IC for pretreated CBMC; for anti-
CD48, 3.72 ? 106? 0.3 ? 106IC for isotype-treated CBMC
and 1.62 ? 106? 1.8 ? 106IC for pretreated CBMC [P ?
0.001]). In contrast, S. aureus adherence was not affected by the
neutralizing antibody treatment (4.25 ? 107? 0.2 ? 107ad-
herent bacteria for non-pretreated CBMC and 4.43 ? 107?
1.6 ? 107adherent bacteria for pretreated CBMC; P ? 0.05%)
(data not shown).
S. aureus induces IL-8 and TNF-? release by CBMC. The
infection of CBMC with S. aureus induced IL-8 and TNF-?
release. Time course experiments showed that there was a
positive correlation between IL-8 and TNF-? and the duration
FIG. 4. Invasive S. aureus upregulates TLR2 and CD48 molecules on CBMC. CBMC were incubated in medium or infected with S. aureus
(MOI of 10) for 180 min at 37°C. Cells then were stained with anti-TLR2 (A) or anti-CD48 antibody (B) and analyzed by FACS. Noninfected cells
are indicated by the shaded areas, and the infected ones are indicated by the white areas. These results are representative of three independent
FIG. 5. TLR2 and CD48 receptors are involved in S. aureus uptake by CBMC. CBMC were preincubated with anti-human TLR2 (A) or
anti-human CD48 (B) neutralizing antibody before incubation with S. aureus (MOI of 10) for 180 min at 37°C. After infection, cells were incubated
with gentamicin for 60 min to kill extracellular bacteria, and the samples were analyzed by FACS. Pretreated CBMC are indicated by the shaded
areas in the histograms. These results are representative of three independent experiments.
VOL. 76, 2008S. AUREUS-INDUCED MAST CELL ACTIVATION4493
of S. aureus-CBMC interaction (for IL-8, 266.67 ? 11.11 pg at
30 min and 600.96 ? 12.40 pg at 360 min; for TNF-?, 420 ?
14.14 pg at 30 min and 1,145.5 ? 265.06 pg at 360 min [P ?
0.001]) (Fig. 6). Furthermore, noninfected CBMC released
200.5 ? 33.23 pg of TNF-? (Fig. 6A) and 150.54 ? 14.53 pg of
IL-8 (Fig. 6B) after 360 min of incubation.
To determine whether proinflammatory cytokine release
was dependent on bacterial viability, CBMC were incubated
with killed S. aureus. Killed S. aureus preserved the ability to
adhere to and trigger IL-8 and TNF-? release from CBMC
in a time-dependent manner (for IL-8, 164.51 ? 9.49 pg at
30 min and 401.40 ? 11.38 pg at 360 min; for TNF-?,
450.5 ? 42.42 pg at 30 min and 1,060 ? 155.56 pg at 360 min
[P ? 0.001]) (Fig. 6).
We next evaluated whether proinflammatory cytokine re-
lease was dependent on S. aureus uptake. Therefore, the S.
aureus-CBMC interaction was performed in the presence of
cytochalasin D. The results in Fig. 7 show that in this case, IL-8
(Fig. 7A) and TNF-? (Fig. 7B) release was drastically de-
creased compared to IL-8 and TNF-? release by infected
CBMC without cytochalasin D treatment (P ? 0.01 and P ?
0.001, respectively). The cytochalasin D treatment did not in-
fluence the PGN-induced IL-8 and TNF-? release by CBMC,
and no synergistic effect was observed when PGN was incu-
bated with infected CBMC (Fig. 7).
We also investigated the role of TLR2 and CD48 molecules
on S. aureus-induced cytokine release. CBMC were incubated
with anti-human TLR2 and anti-human CD48 antibodies or
both antibodies before infection (Fig. 8). Anti-TLR2 signifi-
cantly reduced the IL-8 (Fig. 8A) and TNF-? (Fig. 8B) release
by CBMC (P ? 0.001). Anti-CD48 also decreased the cytokine
release (P ? 0.01). Furthermore, the combination of these
antibodies had a significant synergistic effect (P ? 0.001). The
amount of IL-8 and TNF-? released by CBMC incubated with
isotype-matched IgG was similar to that of infected cells (for
IL-8, 602 ? 32.72 pg and 624.94 ? 45.94 pg; for TNF-?, 787.51 ?
56 pg and 808.61 ? 11.95 pg).
This study provides, for the first time, evidence of a direct in
vitro interaction between bacterial pathogens such as S. aureus
and human mast cells. In fact, we have demonstrated that
human CBMC recognize S. aureus and respond to its infection
FIG. 6. S. aureus induces TNF-? and IL-8 release by CBMC. The kinetics of TNF-? (A) and IL-8 (B) release by uninfected CBMC ( ) and
by CBMC following exposure to live (?) and killed (f) S. aureus strains (MOI of 10) at 37°C for the indicated periods is shown. The values are
the means ? standard deviations from three independent experiments (? and ??, P ? 0.001).
FIG. 7. IL-8 (A) and TNF-? (B) release by CBMC is dependent on S. aureus internalization. CBMC were left uninfected, infected with S.
aureus at 37°C for 180 min in the presence or absence of cytochalasin D, or incubated with PGN (10 ?g m?1) at 37°C for 180 min in the presence
or absence of cytochalasin D. Supernatants were analyzed for IL-8 and TNF-?. The values are the means ? standard deviations from three
independent experiments (?, P ? 0.01; ??, P ? 0.001).
4494 ROCHA-DE-SOUZA ET AL.INFECT. IMMUN.
by releasing proinflammatory cytokines. S aureus displayed a
high adherence to, as well as invasive and survival abilities in,
the mast cells. FACS analysis showed the percentage of inter-
nalized live S. aureus to be higher than that of the antibiotic
protection assay at the same time point (30%; P ? 0.01) (data
not shown). This can be attributable to differences in bioassays.
FACS analysis detects both live and killed internalized bacte-
ria, while the antibiotic protection assay detects only live bac-
teria (32). FACS analysis also detected the uptake of heat-
killed S. aureus, suggesting that the mast cells have phagocytic
ability. Although there are several previous reports indicating
that mast cells have the capacity to recognize, phagocytize, and
destroy different bacterial species and, thus, play a potentially
crucial role in innate immunity (18, 19, 28), in some cases mast
cells can be both infected and eventually destroyed by the
pathogenic bacteria. Because the percentage of bacterial re-
covery after 180 min of gentamicin treatment was higher than
that after 30 min, it is suggested that internalized S. aureus not
only survived but also multiplied within the mast cells. To the
best of our knowledge, this is the first report showing in vitro
S. aureus survival and proliferation within human mast cells. In
fact, Arock et al. (2) reported an appreciable and time-depen-
dent decrease in the viability of S. aureus (CI127 strain) asso-
ciated with mast cells. This discrepancy may be due to genetic
and virulence differences among S. aureus strains.
The mechanisms underlying bacterial entry, phagosome
maturation, and dissemination include strategies, as well as
unique tactics, evolved by individual species to establish infec-
tion. Invasive bacteria actively induce their own uptake by
phagocytosis in normally nonprofessional phagocytic cells and
then either establish a protected niche within which they sur-
vive and replicate or disseminate from cell to cell by means of
an actin-based motility process. Therefore, the uptake of bac-
teria by mast cells may account for the virulence of S. aureus in
diseases, especially those associated with atopy in which there
is an increase in mast cell numbers. This organism also may use
mast cells to establish inflammatory responses and metastatic
foci of infection.
Our data also demonstrate that S. aureus-infected mast cells
evoke significant IL-8 and TNF-? release. Mast cell-derived
TNF-? and IL-8 have been shown to be responsible for neu-
trophil recruitment. TNF-? is of particular interest, because
mast cells are the only known cells to store this cytokine and,
thus, are able to release this mediator immediately upon acti-
vation (21). Furthermore, the mast cell enhancement of early
neutrophil recruitment is considered a potential mechanism of
host defense during bacterial infection (20, 21). Therefore,
mast cells also may contribute to immunity against S. aureus
infection through this mechanism. Killed S. aureus also was
able to trigger proinflammatory cytokine release from the mast
cells. There are several reports showing the ability of commer-
cial purified PGN and lipoteichoic acid to induce proinflam-
matory cytokine release by different types of cells (8, 11, 37,
40). Since heat-killing treatment preserves the integrity of bac-
teria, it is not surprising that killed S. aureus also preserves this
ability. These data also suggest that cytokine release by the
mast cells does not require an active bacterial infection. This is
particularly interesting because during an infection, bacteria
can be killed and release several surface molecules, like PGN
and lipoteichoic acid, that can activate mast cells and trigger an
inflammatory response (8, 10). We can hypothesize that these
data are particularly relevant to atopic dermatitis, in which the
interactions of mast cells with products from S. aureus could be
critical in the local enhancement of chronic inflammation at
specific skin sites (24). In previous works it was reported that
S. aureus components may lead to an increase in the release of
Th2-related cytokines (4, 42, 48). Specifically, it was shown that
PGN may increase the release of IL-5, IL-10, and IL-13 from
human mast cells (48). However, the influence of other S.
aureus components on human mast cell degranulation and
Th2-related cytokine synthesis and release requires further
Several glycosylphosphatidylinositol-anchored proteins are
located in the lipid rafts of the mast cell plasma membrane.
CD48 is one protein that has been implicated specifically in
bacterial adherence, invasion, and/or bacterium-mediated cell
activation (9, 20, 22). Moreover, we have recently shown a
selective increase of the expression of CD48 both in human
and in murine allergic asthma. Its selective downregulation by
the use of CD48-neutralizing antibodies in murine asthma
resulted in an amelioration of the disease (29, 30). Taken
together, these data suggest an important link between CD48
and bacterial infection and allergy. TLR2 is another important
receptor on the mast cells, and it is involved in the recognition
of lipoproteins, lipoteicoic acid, and PGN from gram-positive
bacteria (40). Our results show that CD48- and TLR2-neutral-
izing antibodies highly decrease the S. aureus internalization by
the mast cells, indicating the involvement of these two mole-
cules in S. aureus internalization. Furthermore, we found that
the S. aureus infection upregulated the expression of TLR2 and
CD48 on the mast cell surface. Therefore, it is possible to
speculate that, in vivo, invasive S. aureus could prime mast cells
to be more responsive to CD48- and TLR2-specific ligands.
CD48 also has been implicated in Mycobacterium tuberculosis
internalization by rat peritoneal mast cells and the adherence
of FimH-expressing type 1 fimbriated E. coli on the mast cell
surface (22, 31). In addition, our data are in accordance with
recent reports that have demonstrated that Porphyromonas
gingivalis, Mycobacterium avium, and Klebsiella pneumoniae in-
FIG. 8. Role of TLR2 and CD48 in IL-8 (A) and TNF-? (B) re-
lease by CBMC. CBMC were left uninfected or were infected with S.
aureus at 37°C for 180 min in the presence or absence of anti-TLR2
and CD48 antibodies. Supernatants were analyzed for IL-8 and
TNF-?. The values are the means ? standard deviations from three
independent experiments. ?, P ? 0.01; ??, P ? 0.001.
VOL. 76, 2008S. AUREUS-INDUCED MAST CELL ACTIVATION4495
vade endothelial cells, macrophages, and airway epithelial cells
as well as upregulate TLR expression and elicit an inflamma-
tory response (33, 45, 47). Interestingly, TLR2 could be in-
volved in S. aureus survival in the mast cells. The reason for this
speculation comes from two recent reports showing that S.
aureus and P. gingivalis were cleared in TLR2-deficient mac-
rophages more rapidly than in the wild type (6, 46). However,
it remains to be clarified whether the TLR2-mediated survival
of internalized bacteria is accomplished as a part of the host
immune response or as a bacterial strategy to evade, survive,
and spread in the host organism and whether this mechanism
also is observed in mast cells. Although our data suggest that
TLR2 and CD48 are involved in S. aureus internalization,
neutralizing antibodies to these two receptors did not com-
pletely abolish the bacterial internalization. This finding rein-
forces the complex multifactorial nature of S. aureus interac-
tion with host cells.
In summary, we have demonstrated in vitro that S. aureus
has invasive and survival abilities in human mast cells, and
together these abilities upregulate its proinflammatory poten-
tial. Three observations support this statement: (i) internalized
bacteria induced IL-8 and TNF-? release; (ii) the cytochalasin
D treatment decreased the bacterial internalization and, con-
sequently, the inflammatory response; and (iii) anti-human
TLR2 and anti-human CD48 antibodies inhibited the inflam-
matory response and S. aureus internalization by the mast cells.
Our data strengthen the previous observations of the wors-
ening of allergic diseases concomitantly with S. aureus infec-
tions and indicate novel therapeutical approaches.
This project was supported by a grant from the Israel Science Foun-
dation (no. 213105) and of the Aimwell Charitable Trust (United
Kingdom). C. M. Rocha-de-Souza’s work on this project was sup-
ported by the Valazzi-Pikovsky Fellowship Fund (The Hebrew Uni-
versity of Jerusalem). F. Levi-Schaffer is affiliated with the David R.
Bloom Center of Pharmacy and the Adolph and Klara Brettler Center
for Research in Molecular Pharmacology and Therapeutics at The
Hebrew University of Jerusalem.
The authors have no financial conflicts of interest.
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Editor: B. A. McCormick
VOL. 76, 2008 S. AUREUS-INDUCED MAST CELL ACTIVATION4497