Báo cáo khoa học: Tec family kinases: regulation of FceRI-mediated mast-cell activation ppt

MINIREVIEW
Tec family kinases: regulation of FceRI-mediated mast-cell
activation
Wilfried Ellmeier, Anastasia Abramova and Alexandra Schebesta
Division of Immunobiology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna,
Austria
Mast cells are crucial regulators of
type I hypersensitivity reactions
Mast cells are key players in type I hypersensitivity
reactions and are critically involved in the development
of allergic rhinitis, allergic asthma and systemic ana-
phylaxis. Mast cells (as well as basophils) express the
high-affinity Fc receptor type I for IgE (FceRI), and
thus one of their most characteristic features is their
ability to bind IgE. Cross-linking of the IgE ⁄ FceRI
complex with antigen (e.g. an allergen) causes the acti-
vation of mast cells and induces a variety of effector
functions. As a part of the early-phase response that
occurs within minutes, mast cells secrete preformed
mediators like histamine, proteolytic enzymes and pro-
teoglycans. In addition, rapidly synthesized lipid medi-
ators such as leukotrienes (LTs) and prostaglandins
Keywords
cytokine production; degranulation; early-
phase and late-phase effector functions; Itk;
mast cell signaling; Tec; TLR activation; type I
hypersensitivity reactions; tyrosine kinases
Correspondence
W. Ellmeier, Division of Immunobiology,
Institute of Immunology, Center for
Pathophysiology, Infectiology and

Immunology, Medical University Vienna,
Lazarettgasse 19, 1090 Vienna, Austria
Fax: +43 1 40160 965003
Tel: +43 1 40160 65003
E-mail:
(Received 31 August 2010, revised 3
November 2010, accepted 25 February
2011)
doi:10.1111/j.1742-4658.2011.08073.x
Mast cells express the high-affinity receptor for IgE (Fc eRI) and are key
players in type I hypersensitivity reactions. They are critically involved in
the development of allergic rhinitis, allergic asthma and systemic anaphy-
laxis, however, they also regulate normal physiological processes that link
innate and adaptive immune responses. Thus, their activation has to be
tightly controlled. One group of signaling molecules that are activated
upon FceRI stimulation is formed by Tec family kinases, and three mem-
bers of this kinase family (Btk, Itk and Tec) are expressed in mast cells.
Many studies have revealed important functions of Tec kinases in signaling
pathways downstream of the antigen receptors in lymphocytes. This review
summarizes the current knowledge about the function of Tec family kinases
in FceRI-mediated signaling pathways in mast cell.
Abbreviations
BMMC, bone marrow-derived mast cells; Btk, Bruton’s tyrosine kinase; Bmx, bone marrow kinase on the X chromosome; Erk, extracellular
signal-regulated kinase; FceRI, high-affinity Fc receptor type I for IgE; Grb2, growth factor receptor bound 2; IL, interleukin; Itk, IL-2-inducible
T-cell kinase; JNK, c-jun N-terminal kinase; LAT, linker for the activation of T cells; LT, leukotriene; MAPK, mitogen-activated protein kinase;
NFAT, nuclear factor for activated T cells; NFjB, nuclear factor kappa-light chain enhancer of activated B cells; PDK1, 3-phosphoinositide-
dependent protein kinase 1; PH, pleckstrin homology; PK, protein kinase; PLC, phospholipase C; PtdIns3K, phosphatidylinositol 3-kinase;
PtdIns(4,5)P
3
, phosphatidylinositol-4,5-bisphosphate; Rlk, resting lymphocyte kinase; Src, sarcoma; Syk, spleen tyrosine kinase; Tec, tyrosine

kinase expressed in hepatocellular carcinoma; TFK, Tec family kinase; TNF, tumor necrosis factor; TLR, Toll-like receptor; xid, X-linked
immunodeficiency; ZAP-70, zeta-chain-associated kinase 70.
1990 FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS
are released within a short time after activation. More-
over, mast-cell activation leads to the production of
various cytokines and chemokines. This occurs within
hours after activation and is a part of the late-phase
response. The plethora of mediators and factors pro-
duced in the early- and late-phase activation by mast
cells cause many physiological and pathophysiological
changes associated with type I hypersensitivity reac-
tions. These include vasodilation and increased vascu-
lar permeability, and functional changes that are
dependent on the affected tissues such as enhanced
mucus production, bronchoconstriction, diarrhea and
vomitting, as well as the infiltration of many other
types of immune cells, such as eosinophils, basophils,
monocytes, neutrophils and lymphocytes [1,2].
In addition to their crucial role in type I hypersensi-
tivity reactions, mast cells can also be activated by
IgE-independent triggers such as FccR or complement
receptors [3]. Moreover, mast cells express a variety of
Toll-like receptors (TLRs) and it has been shown that
mast cells can contribute to the host defense against
bacterial infections [4,5]. Furthermore, mast cells have
been implicated in the defense against toxins, but also
in the pathology of autoimmune diseases such as rheu-
matoid arthritis and inflammatory bowel disease, in
cardiovascular diseases and in cancer [6]. Thus, mast
cells are not only critically involved in type I hypersen-

sitivity reactions, but they also have to be considered
an important positive as well as negative regulator of
normal physiological processes that link innate and
adaptive immune responses [7].
It has been shown that mast cells originate from
CD13
+
CD34
+
CD117
+
hematopoietic bone marrow
progenitor cells [8–10]. Mast cells are most abundant
in the gut, skin and airways, and mature in tissues
from their committed progenitors [11]. Dependent on
the microenvironment of the surrounding tissue, or on
the conditions under which they have been generated
in vitro, mast cells acquire distinct phenotypic profiles
and thus produce different types of early- and late-
phase mediators. Moreover, there are differences
between murine and human mast cells [12]. These
issues have to be taken into account when comparing
data obtained from different studies.
Mast-cell activation pathways
The signaling pathways that regulate the activation of
mast cells upon FceRI cross-linking have been investi-
gated in great detail. Many of the signaling mole-
cules that have important functions in FceRI
signaling pathways are also expressed in lympho-
cytes, thus there are similarities in the basic signaling

machinery of the FceRI in mast cells and antigen
receptors in lymphocytes. Several recent reviews have
comprehensively summarized FceRI-mediated signal-
ing in mast cells [13–15], therefore the key steps in
the activation of mast cells are only briefly described
in this review.
The FceRI is a multimeric cell surface receptor con-
sisting of an a, b and two c chains. The a chain binds
IgE, whereas the b and c chains are involved in the
signal transduction [16]. One of the earliest steps after
FceRI cross-linking is the tyrosine phosphorylation of
several cellular proteins, including immunoreceptor
tyrosine-based activation motifs in the FceRI b
and c chains (Fig. 1). This leads to the recruitment and
activation of the sarcoma (Src) family kinase Lyn, and
subsequently to the activation of spleen tyrosine kinase
(Syk), a member of the Syk ⁄ zeta-chain-associated kinase
70 (ZAP-70) kinase family. This results in the phosphor-
ylation of linker for the activation of T cells (LAT) and
the formation of a large signaling complex that also con-
tains Bruton’s tyrosine kinase (Btk). Subsequently, Syk
and Btk activate phospholipase C (PLC)c1 and PLCc2,
which convert phosphatidylinositol-4,5-bisphosphate
[PtdIns(4,5)P
3
] into inositol-1,4,5-trisphosphate and
diacylglycerol, leading to Ca
2+
mobilization and the
activation of protein kinase (PK)C isoforms. Formation

of the LAT signaling complex leads also to activation of
the mitogen-activated protein kinase (MAPK) path-
ways. Ca
2+
mobilization and MAPK activation lead to
the activation of downstream transcription factors
required for cytokine expression such as members of the
nuclear factor for activated T cells (NFAT) family and
of the activator protein-1 complex, respectively.
A complementary signaling pathway that is also
required for the activation of mast cells is mediated by
Fyn, another member of the Src kinase family [17]. Fyn
associates with the FceRI b chain, and activates the
adaptor protein Grb2-associated binding protein 2,
which is a positive effector molecule for the activation
of the phosphatidylinositol 3-kinase (PtdIns3K) path-
way. This leads to membrane recruitment of pleckstrin
homology (PH) domain containing signaling molecules
such as 3-phosphoinositide-dependent protein kinase 1
(PDK1) and PKB ⁄ Akt. Activation of PDK1 is impor-
tant for degranulation, and activated PKB ⁄ Akt leads to
the activation of the nuclear factor kappa-light chain
enhancer of activated B cells (NFjB) pathway, essential
for the induction of cytokine expression. Thus, both
Lyn- and Fyn-mediated pathways are required for
efficient mast-cell activation. Together, this complex
signaling network downstream of the FceRI induces
the various mast-cell responses, ranging from early-
phase effector function such as degranulation and the
W. Ellmeier et al. Tec kinases in mast cells

FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS 1991
production of lipid mediators to late-phase effector
function such as the production of various cytokines.
A brief overview of Tec family kinases
As described above, FceRI signaling pathways have
been investigated in great detail. Studies using mast cells
generated from mice deficient in the Src family kinase
members Lyn [18–20] or Fyn [17], or from mice which
lack the Syk ⁄ ZAP-70 kinase family member Syk [21]
have convincingly demonstrated a critical role of these
two kinase families during mast-cell activation. Tec fam-
ily kinases (TFKs) represent another class of protein
tyrosine kinases that are implicated in FceRI-mediated
mast-cell activation [22]. The Tec kinase family consti-
tutes the second largest family of nonreceptor protein
Fig. 1. A simplified scheme of FceRI signaling. IgE ⁄ FceRI cross-linking by antigen induces the phosphorylation of immunoreceptor tyro-
sine-based activation motifs in the FceRI b and c chains. This leads to the recruitment and activation of the Src family kinase Lyn,
which activates Syk, a member of the Syk ⁄ ZAP-70 kinase family. This results in the phosphorylation of several tyrosine residues in LAT
and the formation of a signaling complex containing the guanine nucleotide exchange factor Vav and adaptor molecules such as Src
homology 2-domain containing leukocyte protein of 76 kDa, growth factor receptor bound 2 (Grb2), Grb2-related adaptor protein 2
(Gab2), kinases such as Btk and PLCc (PLCc1 and PLCc2). Btk recruitment to the membrane is regulated via PH domain-mediated bind-
ing to PtdIns(3,4,5)P
3
binding, generated by activated PtdIns3K. Syk and Btk activate PLCc1 and PLCc2, which convert PtdIns(4,5)P
2
into inositol-1,4,5-trisphosphate and diacylglycerol, leading to Ca
2+
mobilization and the activation of PKC isoforms. FceRI cross-linking
leads also to the activation, via Grb2 ⁄ son of sevenless (SOS) of the MAPK Erk1 ⁄ 2, p38 and JNK1 ⁄ 2. Together, the signals lead to the
activation of transcription factors such as of the NFAT and activator protein-1 gene families. The association of Fyn, another member of

the Src kinase family, with the FceRI b chain induces a complementary signaling pathway required for efficient mast-cell activation. Fyn
activates the adaptor protein Grb2-associated binding protein 2, which activates PtdIns3K, leading to the generation of PtdIns(3,4,5)P
3
from PtdIns(4,5)P
2
and subsequently to PH domain-mediated recruitment of PDK1 and PKB ⁄ Akt. PDK1 activation leads to degranulation
(via the activation of PKCd). PKB ⁄ Akt signaling leads to activation of the NFjB pathway. Together, this complex signaling network
downstream of the FceRI leads to the activation of various mast-cell responses, ranging from early-phase effector function, such as
degranulation and the production of lipid mediators, to late-phase effector function, such as cytokine production. Btk is required for effi-
cient PLCc activation, and loss of Btk leads to impaired Ca
2+
mobilization and impaired early- and late-phase effector functions. Tec pos-
itively regulates cytokine production and lipid mediator production, although it negatively regulates Erk 1 ⁄ 2 activation. Itk is a negative
regulator of cytokine production. The role of Rlk in mast cells is not known. For simplicity, not all cross-talks between the various sig-
naling molecules and pathways are indicated. DAG, diacylglycerol; IP
3
, inositol-1,4,5-triphosphate.
Tec kinases in mast cells W. Ellmeier et al.
1992 FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS
tyrosine kinases and consists of five members [23]: bone
marrow kinase on the X chromosome (Bmx; also
known as Etk) [24–26], Btk [27–29], interleukin (IL)-2-
inducible T-cell kinase (Itk; also known as Emt)
[24,30,31], resting lymphocyte kinase (Rlk; also known
as Txk) [32,33] and tyrosine kinase expressed in hepato-
cellular carcinoma (Tec) [34]. They are preferentially
expressed in cells of the immune system, although
expression of some members outside the hematopoietic
system has been described [25,34]. Btk, Itk and Tec are
expressed in mast cells and are activated upon cross-

linking of FceRI [35–37], suggesting that they are part
of the signaling machinery in mast cells. In addition,
Rlk expression has been described in mast cell lines [38].
Ever since their discovery, TFKs have received a lot
of attention in studies investigating signal transduction
pathways in immune cells. Mutations in Btk are the
molecular cause of X-linked agammaglobulinemia, a
rare human genetic disorder characterized by a reduc-
tion in serum Ig levels due to defects in B lymphocyte
development [27,28,39]. In contrast to the large num-
ber of different mutations described for the human
BTK gene, a single point mutation (R28C) in the PH
domain in the murine Btk gene leads to a similar, but
less severe, syndrome in mice, X-linked immunodefi-
ciency (xid) [29,40]. More recently, a homozygous
mutation in the Src homology 2 domain of Itk has
been linked with an Epstein Barr virus-associated lym-
phoproliferative disease [41].
The key regulatory steps that lead to the activation
of TFK have been defined by using a variety of bio-
chemical and functional studies, as well as genetic
approaches. The majority of studies dissecting the acti-
vation steps have been performed in lymphocytes,
which revealed that Tec kinases are regulated at vari-
ous levels. They are recruited to the membrane via the
interaction of their PH domain with PtdIns(3,4,5)P
3
phosphate, which is generated from PtdIns(4,5)P
2
by

PtdIns3K. Upon membrane localization, they are
phosphorylated by Src family kinases such as Lck or
Lyn, and subsequent autophosphorylation leads to
their full activation. TFK form intra- and intermolecu-
lar interactions, which can regulate their activation,
and Tec kinases are also part of larger signaling com-
plexes and membrane-associated adaptor proteins.
Moreover, the detailed analysis of B cells isolated from
X-linked agammaglobulinemia patients and xid mice,
and the generation of mice deficient for Tec family
members have led to a comprehensive insight into
TFK-dependent biological processes induced by anti-
gen receptor stimulation in lymphocytes. These studies
clearly demonstrated that TFK are important regula-
tors of lymphocyte function. Several recent reviews
comprehensively summarize the current knowledge
about the evolution, structure, activation pathways
and function of TFK in lymphocytes [39,42–47] and
are therefore not described further.
The role of TFK in the myeloid lineage has not been
studied as extensively as in the lymphoid lineage. Nev-
ertheless, studies performed in human and murine
monocytes ⁄ macrophages [48–52], osteoclasts [53], den-
dritic cells [54], neutrophils [55,56], platelets [57] and
erythroid cells [58] indicate crucial functions for TFK
also in the nonlymphoid lineages of the hematopoietic
system [59,60]. This minireview summarizes studies
describing the role of Btk, Itk and Tec in mast cells.
Mast-cell development in the absence
of TFK

Mast-cell development appears to be normal in the
absence of Btk (both in Btk-null or xid mice), Tec or
Itk. Analysis of various types of mast cells in Btk
) ⁄ )
(skin, tracheobronchial airways), Tec
) ⁄ )
(skin) and
Itk
) ⁄ )
(skin, peritoneum, tracheobronchial airways,
lung) mice showed no differences compared with wild-
type mice with respect to tissue distribution, cell quan-
tities or morphological features [37,61–63]. However,
ex vivo analyzed peritoneal and splenic Itk
) ⁄ )
mast
cells had higher levels of FceRI on their surface, most
likely due to the elevated IgE serum levels in the
absence of Itk [63].
In vitro IL-3 or IL-3 ⁄ stem cell factor-generated
Tec
) ⁄ )
, Btk
) ⁄ )
or Itk
) ⁄ )
bone marrow-derived mast
cells (BMMC) showed similar histological staining
characteristics and had similar expression levels of
FceRI that were upregulated to comparable levels

upon overnight incubation with IgE [37,61–63]. How-
ever, Btk- or Tec-deficient BMMC generated in the
presence of either IL-3 or IL-3 ⁄ stem cell factor yielded
higher numbers of mast cells compared with wild-type
cultures [37,64,65]. Btk-null mast cells showed reduced
cell death during BMMC culture and this might be the
cause of increased cell numbers, because Btk-deficient
mast cells die more slowly upon IL-3 deprivation than
wild-type cells [64,65]. Btk-deficient mast cells showed
reduced c-Jun N-terminal kinase (JNK)1 ⁄ 2 activation
upon IL-3 withdrawal, and the impaired JNK1 ⁄ 2 acti-
vation could be reversed by enforced retroviral expres-
sion of a wild-type, but not a kinase-dead, version of
Btk in Btk-deficient mast cells, indicating an essential
role for Btk kinase activity in the activation of
JNK1 ⁄ 2 [64]. A side-by-side comparison of Tec- and
Btk-deficient cultures (as well as Tec
) ⁄ )
Btk
) ⁄ )
cul-
tures) revealed that the mast cell numbers were signifi-
cantly higher in Tec
) ⁄ )
cultures than in Btk
) ⁄ )
W. Ellmeier et al. Tec kinases in mast cells
FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS 1993
cultures, but did not increase further in Tec
) ⁄ )

Btk
) ⁄ )
cultures [37]. The reason for the negative role of Tec in
these in vitro cultures is not known. Because in vivo
numbers of mast cells are similar between wild-type,
Tec
) ⁄ )
and Btk
) ⁄ )
mice, it is likely that the cytokine
milieu and ⁄ or the different microenvironment in vitro
leads to increased numbers of mast cells in the absence
of Tec kinases.
Btk regulates early- and late-phase
effector functions upon FceRI-mediated
mast-cell activation
Soon after the discovery that Btk is mutated in
X-linked agammaglobulinemia ⁄ xid, studies performed
by Kawakami and colleagues [61] with either xid or
with gene-targeted Btk-deficient mice revealed that Btk
is a crucial regulator of FceRI-mediated mast cell
functions in vivo and in vitro. Btk-deficient mice were
subjected to various passive cutaneous anaphylaxis
experiments to test early- and late-phase mast-cell
effector functions. In one type of passive cutaneous
anaphylaxis experiment, mice were ‘sensitized’ with
intradermal injection of anti-dinitrophenol IgE fol-
lowed by intravenous injection of antigen (dinitrophe-
nol–BSA) and Evans blue dye. Btk-mutant mice
showed a dose-dependent decrease in blood vessel per-

meability as measured by the extravasations of Evans
blue dye shortly after antigen exposure. In another
passive cutaneous anaphylaxis model system, mice
were intravenously injected with anti-dinitrophenol IgE
followed by applying dinitrofluorobenzene to the ear
of sensitized mice. Btk-mutant mice displayed reduced
edema, as indicated by a reduced increase in ear thick-
ness 24 h after dinitrofluorobenzene exposure [61].
Thus, both early and late effector phases are impaired
in Btk-deficient mice.
In addition to the in vivo experiments, many studies
performed with in-vitro-generated BMMCs extended
the observation that Btk plays an important role in the
regulation of FceRI signaling and helped to character-
ize in detail the biochemical and molecular function of
Btk. Degranulation as measured by histamine secretion
is delayed ⁄ impaired in a dose-dependent manner in
Btk-null mast cells [37,61,65], and the release of LTs
(without distinguishing between C
4
⁄ D
4
⁄ E
4
) was found
to be similar in control and Btk-deficient mast cells
[61,65]. However, more recent studies indicated that
LTC
4
production is reduced in BMMC in the absence

of Btk [37,66] in addition to prostaglandin D
2
and
reactive oxygen species production [66]. Biochemically,
the impairment of early effector functions can be
attributed, despite a normal total tyrosine phosphory-
lation pattern, to impaired PLCc1 and PLCc2
phosphorylation and hence impaired inositol-1,4,5-
triphosphate generation and Ca
2+
mobilization
[37,61,65,66], which is important for mast cell degranu-
lation [67,68]. Other FceRI-signaling defects detected
in the absence of Btk were reduced activation of
JNK1 ⁄ 2 and p38 [65,66], whereas PKCa and PKCbII
phosphorylation appeared to be slightly enhanced [66].
No differences in the phosphorylation ⁄ activation kinet-
ics of Syk, FceRIb, PKB ⁄ AKT, and extracellular sig-
nal-regulated kinase (Erk) 1 ⁄ 2 have been reported
[37,61,65,66]. The signaling defects in the absence of
Btk resulted in impaired activation of the transcription
factors NFjB and NFAT [69], providing an explana-
tion for the impaired but not fully abrogated secretion
of inflammatory cytokines [such as IL-2, tumor necro-
sis factor (TNFa), IL-6, IL-13 and GM-CSF) in xid
and Btk-deficient mast cells [37,61,66,69]. In agreement
with the observed defects in cytokine production, the
promoter regions of Il2 and Tnfa show reduced activ-
ity in Btk-null compared with wild-type mast cells in
transient luciferase reporter assays performed in mast

cells [61]. Furthermore, it has been demonstrated that
Btk regulates FceRI-induced IL-2 transcription
through a pathway including JNK1 ⁄ 2, activator
protein-1 and NFAT, whereas Btk also positively
regulates IL-2 and TNFa expression via PKB ⁄ Akt
dependent pathways [61,64,70].
One notable exception from the list of cytokines
that were reduced in Btk-deficient mast cells is IL-4,
which is enhanced in the absence of Btk [37]. It is
clear that Btk, dependent on the cellular context
and ⁄ or type of receptor stimulation, can function as
a positive and negative regulator of signaling path-
ways. It has been demonstrated that Btk can act as
either a pro- or anti-apoptotic signaling intermediate
in FasL- or radiation-induced apoptosis, respectively
[71,72]. Moreover, it was shown that Btk-deficient
murine bone marrow-derived dendritic cells were
more mature and displayed an increased in vitro and
in vivo T-cell stimulatory capacity compared with
wild-type bone marrow-derived dendritic cells [54].
Although a molecular mechanism to explain the
enhanced IL-4 levels remains to be determined, the
finding that Btk-deficient mast cells showed enhanced
IL-4 secretion further supports the general concept
that Btk can also have negative regulatory roles.
Another interesting Btk function was revealed by
the observation that Btk plays a role in regulating
mast-cell adhesion upon monomeric IgE binding [73].
During recent years, it has been shown that mono-
meric IgE molecules not only lead to the upregulation

of FceRI expression on mast cells [74,75], but that
Tec kinases in mast cells W. Ellmeier et al.
1994 FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS
certain IgE molecules (termed highly cytokinergic
IgEs) also promote the adhesion to fibronectin and the
survival of mast cells [76,77]. This effect is mediated
via b1 and b7 integrins, and monomeric IgE–Fce RI-
mediated inside-out signaling activates Lyn, Syk and
Btk. Adhesion to fibronectin of Btk-deficient BMMC
was partially impaired, indicating a regulatory role of
Btk in this process. This was specific to FceRI-medi-
ated adhesion pathways, because stem cell factor-stim-
ulated wild-type and Btk-deficient mast cells showed a
similar adhesion [73].
Itk and FceRI-mediated mast-cell
activation
A role for Itk in mast cells has long been suspected
ever since it was shown that Itk is activated upon
FceRI signaling [36]. Experimental evidence for a func-
tion of Itk during mast-cell activation was initially
provided by Forssell et al. [62] using an ovalbumin-
induced acute phase plasma extravasation model that
is dependent on IgE-mediated mast-cell degranulation.
Mice were immunized twice with ovalbumin, subse-
quently injected with Evans blue dye and rechallenged
with aerosolized ovalbumin. Under these conditions,
Itk-deficient mice showed a dramatically reduced
Evans blue dye extravasation compared with wild-type
mice. In line with this finding, it was observed that
Itk

) ⁄ )
airway mast cells showed a decreased degranu-
lation response compared with wild-type airway mast
cells [62]. However, another study from August and
colleagues [63] showed that transfer of Itk
) ⁄ )
mast
cells into mast cell-deficient W ⁄ W
v
mice fully rescued
the histamine defect of W ⁄ W
v
mice in a systemic ana-
phylactic reaction model, in which mice were injected
with anti-dinitrophenol IgE followed by systemic
administration of dinitrophenol–human serum albu-
min. Because IgE levels are elevated in Itk
) ⁄ )
mice
[62,78,79], it is conceivable that the reduced mast-cell
responses of Itk
) ⁄ )
mice in in vivo model systems were
most likely due to IgE-loaded or occupied FceRI on
mast cells which may impair binding of newly synthe-
sized or administered IgE [63]. A detailed phenotypic
in vitro analysis of Itk
) ⁄ )
BMMC showed that they
have a normal degranulation response (as revealed by

assaying hexosaminidase release) and Ca
2+
mobiliza-
tion, however, Itk-deficient BMMC produced increased
levels of IL-13 and TNFa compared with wild-type
BMMCs. The increase in cytokine production corre-
lated with enhanced expression of NFATc1 and
NFATc2, and enhanced nuclear localization of
NFATc2 already in nonstimulated Itk
) ⁄ )
mast cells
[63]. These data indicate that Itk is dispensable for
degranulation, but they suggest that Itk is a crucial
negative regulator of cytokine production in mast cells.
Tec is essential for efficient cytokine
production upon FceRI-mediated
mast-cell activation
Tec is the third member of the Tec kinase family for
which a function in mast cells could be identified.
Studies from our laboratory showed that Tec is acti-
vated upon FceRI stimulation, clearly indicating that
Tec is a part of the signaling machinery downstream
of the FceRI signalosome [37]. An examination of
early-phase mast cell effector function revealed that
histamine release upon in vitro FceRI activation was
normal in Tec
) ⁄ )
BMMC. Moreover, Tec-deficient
mice displayed normal serum histamine levels in an
anaphylactic reaction model, in which mice were

primed with anti-trinitrophenol IgE followed by sys-
temic injection of trinitrophenol. Thus, unlike Btk, Tec
is not controlling the release of histamine upon mast-
cell activation. However, Tec has an essential function
in pathways leading to the generation of LTC
4
,
because LTC
4
levels were severely reduced in Tec
) ⁄ )
BMMC. LTs are generated from arachidonic acid by
the activity of 5-lipoxygenase. This generates LTA
4
,
which is converted into LTB
4
or LTC
4
by LTA
4
hydrolase or LTC
4
synthase, respectively [80]. Because
LTB
4
levels (and thus LTA
4
hydrolase activity) were
comparable between Tec

+ ⁄ +
and Tec
) ⁄ )
BMMC, Tec
might specifically contribute to the regulation of LTC
4
synthase activity [37].
Tec is also required in BMMC for efficient cytokine
production. In the absence of Tec, TNFa, IL-13 and
GM-CSF levels were reduced, although IL-5 and IL-6
were not affected by the loss of Tec. In contrast, IL-4
production was almost completely abolished, indicat-
ing an essential role of Tec in the regulation of IL-4.
Biochemically, the defects in the generation of LTC
4
and in cytokine production were accompanied by mild
defects in Ca
2+
mobilization, although PLCc1 (Y783)
and PLCc2 phosphorylation (Y1217) as well as LAT
and PKB ⁄ Akt phosphorylation were similar between
Tec
+ ⁄ +
and Tec
) ⁄ )
BMMC. However, activation of
the MAPK pathways was altered in the absence of
Tec, because JNK1 ⁄ 2 phosphorylation was reduced,
whereas p38 activation appeared to be normal. Inter-
estingly, Tec

) ⁄ )
BMMC displayed enhanced Erk1 ⁄ 2
activation upon FceRI stimulation. Although a link
between enhanced Erk1 ⁄ 2 phosphorylation and a bio-
logical phenotype of Tec
) ⁄ )
BMMC could not be
established in this study, the results clearly demon-
strate that Tec is a negative regulator of signaling
W. Ellmeier et al. Tec kinases in mast cells
FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS 1995
pathways that lead to the activation of Erk1 ⁄ 2 in mast
cells [37].
Compensatory pathways among TFK in
mast cells
It is well documented that there are compensatory
functions among members of the Tec kinase family.
Hence, combined deletions result in more severe phe-
notypes than individual deletions. This has been dem-
onstrated for Itk and Rlk in T cells [81], and for Tec
and Btk in B cells [82], macrophages [51,52], osteo-
clasts [53] and platelets [57]. Because mast cells express
multiple members of Tec kinases, it was not surprising
that compensatory functions have been observed for
Tec and Btk in mast cells. Although early-phase effec-
tor functions such as histamine release were not fur-
ther reduced in Tec
) ⁄ )
Btk
) ⁄ )

BMMC compared with
Btk-deficient mice, the production of TNFa, GM-CSF,
IL-5 and IL-6 was severely affected in the combined
absence of Tec and Btk [37]. This indicates that the
activity of either Btk or Tec is required for efficient
cytokine production. One notable exception was IL-4,
which was enhanced in Btk-deficient mast cells but
severely reduced in Tec
) ⁄ )
and in Tec
) ⁄ )
Btk
) ⁄ )
mast
cells compared with wild-type cells. This indicates dif-
ferential utilization of Tec and Btk in the regulation of
IL-4 production. The reason for the differential
requirement of Tec and Btk is not known and it
remains to be determined whether this is due to tran-
scriptional alterations or post-transcriptional effects,
because it has been shown that Btk and Bmx control
mRNA stability in human monocytes [48–50].
The role of Tec kinases in other
mast-cell activation pathways
In addition to FceRI stimulation, mast cells can also
be activated via other cell-surface receptors such as
triggering of TLRs [4,5]. TFK have been implicated in
TLR signaling in other cell lineages such as mono-
cytes ⁄ macrophages [60], and Btk or Bmx-deficient
human monocytes ⁄ macrophages showed, in part,

impaired responses upon TLR stimulation [48–50].
Because Btk has been shown to interact with compo-
nents of the TLR2 and TLR4 signaling machinery
[83,84], it has been investigated whether the loss of Btk
affects TLR signaling in mast cells. Activation of Btk-
deficient mast cells with various TLR ligands did not
impair cytokine production. In fact, Btk-deficient cells
showed slightly elevated levels of IL-6 and TNFa upon
lipopolysaccharide stimulation, indicating a potential
negative role in TLR signaling [85].
Another important cell-surface receptor that contrib-
utes to the activation of mast cells is c-Kit. It has been
shown that c-Kit is essential for development, growth
and survival of mast cells [86]. In addition, c-Kit syner-
gizes with FceRI and c-Kit activation enhances FceRI-
mediated degranulation and cytokine production
[87–89]. Interestingly, Btk seems to also play an impor-
tant role in c-Kit signaling. Gilfillan and colleagues [69]
have observed that the enhancement of mast cell effec-
tor functions after c-Kit stimulation was impaired
shown in Btk
) ⁄ )
BMMCs, indicating that Btk is essen-
tial in c-Kit-mediated amplification pathways that
enhance FceRI-mediated mast-cell activation.
Future studies
The studies described above clearly indicate that TFK
play an important role in the regulation of mast-cell
activation. The phenotypic analysis of the various Tec
kinase-deficient mast cells revealed distinct functions

for different members of the Tec kinase family.
Whereas Btk positively regulates almost any aspect of
FceRI-mediated mast cell function (with the exception
of IL-4 production which is enhanced in the absence
of Btk), the role of Itk and Tec is more specialized and
in part also distinct to the role of Btk. The observation
that Itk-deficient mast cells display enhanced cytokine
production indicates a complex utilization of TFK in
mast cells. Thus, future studies on the role of Tec kin-
ases in mast cells have to address how they are bio-
chemically and molecularly integrated into the various
FceRI-induced signaling pathways. To the best of our
knowledge, there is no study published that investi-
gates Rlk-deficient mast cells. Thus, the potential role
of Rlk in mast cells should be analyzed. Moreover, the
comparative analysis of mast cells with combinatorial
deletion in Tec kinases (double or triple knockouts)
helps us gain a more detailed picture of biochemical
and biological functions of TFK in mast cells. It will
also be important to reconstitute Tec kinase-deficient
mast cells with wild-type or kinase-dead version of Tec
kinases to determine kinase-independent (i.e. scaffold-
ing) functions of Tec family members, because small
molecule inhibitors for TFK have been developed [90–
92]. Most of the studies on TFK in mast cells have
been performed with murine mast cells. Because there
are species-specific differences between murine and
human mast cells [12], it is essential to extend the anal-
ysis of TFK to human mast cells. Insight from the
analysis of human mast cells will indicate whether

pharmacological inhibition ⁄ targeting of TFK provides
a promising therapeutic strategy for the inhibition of
mast-cell activation.
Tec kinases in mast cells W. Ellmeier et al.
1996 FEBS Journal 278 (2011) 1990–2000 ª 2011 The Authors Journal compilation ª 2011 FEBS
Acknowledgements
The authors thank Dr Shinya Sakaguchi for critical
reading of the manuscript. The work on Tec family
kinases in mast cells was supported by the START
program (Grant Y-163) of the Austrian Science Fund
and the Austrian Ministry of Science and Research,
and in part by the FP6 EU Marie Curie RTN ‘Chro-
matin Plasticity’.
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