ARTICLE IN PRESS

International Journal of Medical Microbiology 296 (2006) 325–329

www.elsevier.de/ijmm

SHORT COMMUNICATION
Streptococcus pneumoniae induces mast cell degranulation
Giovanna Barbutia, Monica Moschionib, Stefano Censinib, Antonello Covaccib,
Cesare Montecuccoc, Pasqualina Montemurroa,
a
Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale, Università di Bari,
P.zza Giulio Cesare 11, I-70124 Bari, Italy
b
Centro di Ricerche IRIS, CHIRON-Vaccines, Siena, Italy
c
Centro CNR Biomembrane e Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Italy

Received 20 July 2005; received in revised form 14 November 2005; accepted 21 November 2005

Abstract

Streptococcus pneumoniae colonizes the nasopharynx of healthy human carriers, but occasionally can spread in the
body causing severe diseases. The mucosa of the respiratory tract is enriched in mast cells, key players of the innate
immune response. Here, we report on the interaction of various strains of S. pneumoniae with the mast cell line RBL-
2H3. Live, but not heat-killed, bacteria were found to induce mast cell degranulation in a dose- and time-dependent
manner, only partially controlled by cytosolic calcium, with no production of TNF-a and IL-6. Non-encapsulated
pneumococcal strains exhibited different potencies in triggering mast cells. We propose here that the induction of mast
cell degranulation by pneumococcal factors not accompanied by the production of pro-inflammatory cytokines may be
a specific strategy elaborated by this bacterium to promote its own spreading from the respiratory mucosa into the
environment.
r 2006 Elsevier GmbH. All rights reserved.

Keywords: Mast cell; Streptococcus pneumoniae; Inflammation

Introduction contribute to inflammation by activating the cytokine,

Streptococcus pneumoniae is a Gram-positive patho-
genic bacterium that colonizes the nasopharynx of
healthy carriers, with a prevalence of more than 40%
of the population. This bacterium is also a common
aetiological agent of upper and lower respiratory tract
diseases. Disseminated infections frequently result in
bacterial pneumonia and otitis media, as well as in
invasive diseases such as meningitis and sepsis (Burman
et al., 1985). Adherence, invasion and death of S.
pneumoniae in the lower respiratory tract can all
Corresponding author. Tel.: +39 080 547 8463;
fax: +39 080 547 8462.
E-mail address: l.montemurro@dimo.uniba.it (P. Montemurro).
complement, and coagulation cascade. The growing
problem of antibiotic-resistant pneumococci has gener-
ated an enhanced interest in the vaccination of high-risk
groups of individuals. However, the limited efficacy of
the current vaccine, particularly for elderly and young
people, has resulted in a greater attention to the
discovery of novel methods to manage and prevent
pneumococcal infections (Di Guilmi and Dessen, 2002).
Mast cells have been most widely studied in the
context of allergic disease, but they have been clearly
shown to have a crucial role in host defence against
several bacteria as elegantly demonstrated with mast
cell-deficient w/wv mice (Malaviya et al., 1996). Mast
cells are strategically located at portals of bacterial entry
and this location is related to their sentinel role in the

1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2005.11.009
 ARTICLE IN PRESS
326 G. Barbuti et al. / International Journal of Medical Microbiology 296 (2006) 325–329
onset of host immune defence. Mast cells respond to a
variety of agonist stimuli and very rapidly release by
exocytosis pre-formed granule-associated mediators of
inflammation such as the vasoactive mediator histamine
which causes the formation of tissue or mucosal
exudates; later on they usually produce cytokines and
chemokines (Marshall, 2004), which trigger a series of
events which include the recruitment of professional
phagocytes.
The relative contribution of mast cells to the
induction of inflammation during S. pneumoniae infec-
tion is ill-known. Therefore, we decided to investigate
the activation of mast cells induced by different strains
of live S. pneumoniae and we report here on the results
obtained which prompted us to propose a pathogenic
mechanism that may be of great relevance.
Materials and methods
Materials
0 0
Ethylene glycol-bis (beta-aminoethylether)-N,N,N ,N -tetra-
acetic acid (EGTA), 1,2 bis (2-aminophenoxy)ethane-
N,N,N0 ,N0 -tetraacetic acid acetoxymethyl ester (BAPTA/
AM), wortmannin, and pertussis toxin (PTX) were from
Sigma (St. Louis, MO, USA).
Cells and bacterial strains
RBL-2H3 cells were maintained in monolayer culture in
DMEM (Euro Clone Ltd., UK) supplemented with 10% foetal
bovine serum, 2% L-glutamine, 100 mg/ml streptomycin, and
100 units/ml penicillin at 37 1C in a humidified 5% CO2
incubator. Cells were harvested with 2.5% trypsin–EDTA–
4Na.
The S. pneumoniae wild-type D39 strain was kindly provided
by S. Falkow (Stanford University), and the R6 strain was a
kind gift of Prof. G. Pozzi, University of Siena. The
S. pneumoniae DCPS2E, capsular isogenic mutant of D39, was
generated by replacing the region comprised between nucleotide
709 and nucleotide 713 of the cps2E gene with a 1.7-kb fragment
from the Escherichia coli aphA-3 gene coding for kanamycin
resistance. The mutant was generated by ‘‘overlap extension’’
essentially as described by Amberg et al. (1995). S. pneumoniae
strains were preserved as stock cultures in 20% glycerol–brain
hearth infusion (BHI; Difco Laboratories, Detroit, USA) at

80 1C. Working cultures for cell stimulation were made by
inoculating 20 ml of this stock into 10 ml of BHI and incubating
at 37 1C until the mid-log phase was reached (0.4–0.5 OD units
at 600 nm). Bacteria were washed twice in phosphate-buffered
saline (PBS) pH 7.4 and were appropriately diluted (see results).
An accurate determination of CFU/ml in the final suspension
was performed by plating onto BHI agar.
b-Hexosaminidase release assay
A total of 1  106 RBL cells/ml in Tyrode’s buffer (135 mM
NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM
glucose, 1 mg/ml bovine serum albumin, and 20 mM Hepes,
pH 7.4) was stimulated with the indicated amount of
S. pneumoniae for different time periods. A23187 was used as
positive control. Degranulation was determined by measuring
the release of a granule marker, b-hexosaminidase, as
previously described (Montemurro et al., 2002). Cellular
viability under every experimental condition was X98% as
assessed by the trypan blue dye exclusion test.
Inhibitory effect of EGTA, wortmannin, pertussis toxin
To evaluate the effect on the pneumoccocal-induced
activation of mast cells, RBL-2H3 (1  106/ml) were loaded
with 40 mM BAPTA/AM at 37 1C for 10 min or treated with
800 ng/ml pertussin toxin (PTX) for 120 min at 37 1C, or
100 nM wortmannin for 10 min at 37 1C. After incubation, the
cells were stimulated with S. pneumoniae (multiplicity of
infection [MOI]: 250) and the release of b-hexosaminidase was
measured. For the EGTA treatment, the cell culture medium
was replaced with calcium-free medium containing 1 mM
EGTA just prior to the addition of S. pneumoniae.
Evaluation of cytokine concentrations
RBL-2H3 cells (1  106/ml) in complete culture medium
were stimulated at 37 1C with the indicated amount of S.
pneumoniae for different time periods (3, 6 and 20 h). As
described previously (Supajatura et al., 2001) these time points
were optimal for production of cytokines from mast cells upon
agonist stimulation. The levels of each cytokine in culture
supernatants were determined by ELISA kits according to the
manufacturer’s instructions (R&D Systems, Minneapolis,
USA).
Results
Effect of S. pneumoniae on mast cell degranulation
Mast cells can be activated by a variety of agonists
and trigger an inflammatory reaction with production of
exudate and recruitment of other inflammatory cells. We
have tested here the effect of the wild-type D39 strain
and of other strains of S. pneumoniae on the well-
established mast cell line RBL-2H3 by measuring the
release of the granule content marker b-hexosaminidase.
Fig. 1A shows that the exocytosis of mast cell incubated
with 250 MOI of live D39 strain for 4 h, is similar to that
induced by the most potent mast cell agonist: the
ionophore A23187. Such an effect is dose-dependent:
with MOI of 10, 100, 200 and 250 the percentage of total
mediator which was released outside the cells was found
to be 7.272.2%, 16.574.3%, 2575.4% and 31.27
12.8%, respectively.
To determine whether degranulation requires live
bacteria, D39 cells were heated at 60 1C for 20 min after
harvesting, a procedure which completely kills the
bacteria, whilst preserving their cellular integrity.

G. Barbuti et al. / International Journal of Medical Microbiology 296 (2006) 325–329
60
β-hexosaminidase release (%)
50
40
30
20
10
0
Control D39 D39
live heat-killed
60
β-hexosaminidase release (%)
50
40
30
20

37 1C. Cell-free supernatants were collected and TNF-a


10



0
0 1
Time (hours)
Fig. 1. (A) b-Hexosaminidase release from RBL-2H3 induced
by S. pneumoniae. RBLcells (1  106/ml) were incubated with
the wild-type encapsulated strain of S. pneumoniae D39, or
with the non-encapsulated strains DCPS2E and R6 (MOI:
250), or with A23187 (1 mM), at 37 1C for 4 h. b-Hexosamini-
dase release was measured as described in Materials and
methods. Each data point is the mean of five independent
experiments7S.D. (B) Time course of RBL-2H3 cell degra-
nulation induced by S. pneumoniae. The release of
b-hexosaminidase into the culture supernatants was measured
at the indicated time points after infection with 2.5  108 CFU/
ml of the D39 strain of S. pneumoniae. Data points are the
average of four different experiments and bars represent S.D.
values.
Fig. 1A shows that such heat-killed S. pneumoniae do
not induce mast cell degranulation. One of the main
virulence factors of pneumococci is the polysaccharide
capsule, and therefore we tested its effect here by using
unencapsulated strains DCPS2E or R6. R6 is a natural
non-encapsulated derivative of serotype 2 D39 strain
and its genome is about 10% smaller in size than that of
the D39 parental strain. DCPS2E is a non-encapsulated
isogenic mutant of D39. The genomic difference
between R6 and D39 strains is not restricted to a partial
deletion of the capsular operon but involves other genes
as well (Hakenbeck et al., 2001). Fig. 1A shows that
DCPS2E is similarly active, whilst R6 is less effective
than the wild-type strain D39 in stimulating b-hexosa-
minidase release from RBL-2H3. This may be taken as
an indication that the R6 strain has lost, in addition to
ARTICLE IN PRESS
327
A the locus encoding for the capsule, other genetic traits
that code for components capable of triggering mast cell
exocytosis. The time course analysis of RBL cell
degranulation induced by a wild-type strain (D39) of
S. pneumoniae is shown in Fig. 1B. At a MOI value of
250, a detectable increase of degranulation is seen after
1–2 h of stimulation; and the maximal effect is recorded
after 4 h of exposure to S. pneumoniae.
S. pneumoniae does not induce the release of TNF-a
∆CPS2E R6 A23187 and IL-6 from RBL-2H3
Activated mast cells have the ability to produce, as a
B late event, various cytokines that play important roles in
the recruitment and activation of inflammatory cells
(Zhang et al., 1992). Therefore, we examined whether
S. pneumoniae stimulates mast cells to secrete proin-




flammatory cytokines. RBL cells were incubated with


live bacteria (MOI from 10 to 250) for 3, 6, or 20 h at


and IL-6 were measured by enzyme-linked immunoab-
sorbent assays. S. pneumoniae did not induce at any time
2 3 4 of incubation the release of preformed TNF-a or de
novo synthesis of TNF-a and IL-6 production by RBL-
2H3, whilst lipopolysaccharide, used as a positive
control, did induce the release of TNF-a and of Il-6
with values of 658 and 2487 pg/ml, respectively.
Intracellular signalling involved in the release of
b-hexosaminidase
To test whether the S. pneumoniae-induced release of
granules requires a rise in cytosolic calcium, experiments
were performed in the presence of either extracellular or
intracellular calcium chelators. Fig. 2 shows that the
inclusion of 1 mM EGTA in the culture medium of
RBL-2H3 cells inhibits by 40% the S. pneumoniae-
induced release of b-hexosaminidase, suggesting that
entry of extracellular calcium is required. Preloading
cells with BAPTA/AM, an intracellular calcium chela-
tor, reduced the release of b-hexosaminidase by 30–40%
in different experiments. These data indicate that a rise
in cytosolic calcium concentration plays a role in the
signalling events triggered upon exposure of cells to
bacteria. Activation of degranulation in mast cells is a
complex phenomenon which depends on the chemical
nature of the stimulus and may implicate G proteins and
activation of protein kinases.
The effect of wortmannin, an inhibitor of the PI-3
kinase (Barker et al., 1995), and of PTX, which inhibits
trimeric G proteins, on pneumococcal-stimulated RBL-
2H3 cells were also tested (Fig. 2). Pretreatment of cells
with 100 nM wortmannin or with 800 ng/ml PTX only
partially reduced granule release induced by S. pneumo-
niae in mast cells.
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328 G. Barbuti et al. / International Journal of Medical Microbiology 296 (2006) 325–329
60
50
Inhibition rate (%)
40
30
20
10
0 for the bacteria residing on the airway mucosal surface
EGTA BAPTA Wortmannin PTX
Fig. 2. Effects of various treatments on the S. pneumoniae-
induced b-hexosaminidase release. Cells (1  106/ml) were
incubated with S. pneumoniae D39 (MOI: 250) at 37 1C for
4 h. For the EGTA treatment, the medium was replaced with
calcium-free medium containing 1 mM EGTA just before
S. pneumoniae was added. RBL cells were loaded with 40 mM
BAPTA/AM at 37 1C for 10 min or 100 nM wortmannin was
added for 15 min prior to bacterial stimulation. In the case of
pertussis toxin (PTX), cells were preincubated with the toxin
(800 ng/ml) for 2 h before the co-cultivation with pneumococci.
Each data point is the mean7S.D. of triplicate experiments.
Discussion
S. pneumoniae colonizes the nasopharynx and is also a
common aetiological agent of upper and lower respira-
tory tract disease. The understanding of the mechanisms
underlying the pathogenesis of pneumococcal infection
is a prerequisite for the development of novel therapeu-
tic strategies. Mast cells are enriched at portals of entry
of many infectious agents and are involved in the
triggering and modulation of the inflammatory re-
sponse.
Here, we have shown that a pathogenic strain of
S. pneumoniae is a powerful inducer of mast cell
degranulation without a concomitant induction of the
production and release of TNF-a and of IL-6. This is at
variance from what we found recently for the neutro-
phil-activating protein of Helicobacter pylori (HP-NAP)
which induces both mast cell degranulation and
production of pro-inflammatory cytokines (Montemur-
ro et al., 2002). HP-NAP is a member of a large family
of mini-ferritin-like proteins, but is so far unique
because of its pro-inflammatory activity (Tonello
et al., 1999). A homology search shows that also
S. pneumoniae encodes a non-heme iron containing
mini-ferritin protein (TIGR SP1572) and it could well be
that this protein is implicated in mast cell activation.
This will be the object of future studies.
The present finding of induction of mast cell
degranulation without release of TNF-a and IL-6 could
account for a reduced neutrophil infiltration and for a
predominance of an inflammatory response character-
ized by an increased secretion of fluids (Nathan, 2002).
S. pneumoniae is not invasive in healthy carriers, and it is
possible that bacterial components, released by secretion
or shedding or upon bacterial lysis at the site of growth,
reach mucosal mast cells. We would like to propose that
the degranulation of mast cells induced by bacterial
products that enter the mucosa, without triggering
the production and release of pro-inflammatory cyto-
kines, is a strategy developed through evolution by
S. pneumoniae in order to gather a source of nutrients
and, perhaps more importantly, to promote their
spreading in the environment via the induction of fluid
secretion with consequent induction of the coughing and
sneezing reflexes. The advantage of an increased fluid
secretion mediated via the induced degranulation of
mucosal mast cells for bacterial spreading is evident.
There is growing evidence that successful pathogens
not only evade or resist the inflammatory response of
the host but can also modulate the activities of some of
the host’s inflammatory cells to favour their growth and
spreading (Feger et al., 2002). The present finding that
S. pneumoniae does not induce mast cells to release pro-
inflammatory cytokines, is intriguing. Clearly it is of
advantage to the bacterium not to promote the
recruitment and activation of neutrophils and macro-
phages. This is in agreement with the recent report that
the susceptibility of CBA/Ca mice to pneumococcal
pneumonia may be due to reduced expression of TNF-a
in lung airways during early infection (Kerr et al., 2002).
Furthermore these authors observed that the number of
mast cell granules increased in lung tissue after infection
with D39.
A number of bacterial molecules may be active in the
process studied here. The S. pneumoniae mutant
DCPS2E, lacking the capsule is as effective as the
encapsulated D39 strain. On the contrary strain R6,
which lacks the capsule and other bacterial components
is less capable of triggering mast cell degranulation,
indicating that the genomic region absent in this strain
encodes bacterial component(s) effective on mast cells.
We have observed that strain R6 increases by 30% the
production of radical intermediates by granulocytes as
compared to the wild-type and DCPS2E strain (our
unpublished results), thus providing further evidence
that this genomic region might play a role in the
modulation of inflammation. The identification of the
proteins responsible for the triggering of mast cell
degranulation and of the signalling pathways involved
will be the subject of future investigations.
Acknowledgements
This work was supported by grants from the
Universities of Bari and of Padova, from MIUR COFIN
 ARTICLE IN PRESS
G. Barbuti et al. / International Journal of Medical Microbiology 296 (2006) 325–329
Project on Inflammation (PRIN 2003062203_006) and the
Network of Excellence on Europathogenomics of the FP6
of the European Community.
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