Innate immunity - Signal Transduction シグナル伝達

SYK deficient mice show defective in phagocytosis of macrophages and in generaction of reactive oxygen intermediates of neutrophils.

1: J Exp Med. 1997 Oct 6;186(7):1027-39.

A critical role for Syk in signal transduction and phagocytosis mediated by
Fcgamma receptors on macrophages.

Crowley MT, Costello PS, Fitzer-Attas CJ, Turner M, Meng F, Lowell C, Tybulewicz
VL, DeFranco AL.

G.W. Hooper Foundation, University of California, San Francisco, California
94143-0552, USA. [email protected]

Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of
responses important for host immunity. Signaling events necessary for these
responses are likely initiated by the activation of Src-family and Syk-family
tyrosine kinases after FcgammaR cross-linking. Macrophages derived from
Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by
FcgammaRs, as well as in many FcgammaR-induced signaling events, including
tyrosine phosphorylation of a number of cellular substrates and activation of
MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another
potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and
Escherichia coli bacteria was also not affected. Syk- macrophages exhibited
formation of polymerized actin structures opposing particles bound to the cells
by FcgammaRs (actin cups), but failed to proceed to internalization.
Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked
FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate
in a Syk-dependent signaling pathway critical for FcgammaR-mediated
phagocytosis. Macrophages derived from mice deficient for the three members of
the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited
poor Syk activation upon FcgammaR engagement, accompanied by a delay in
FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is
critical for FcgammaR-mediated phagocytosis, as well as for signal transduction
in macrophages. Additionally, our findings provide evidence to support a model
of sequential tyrosine kinase activation by FcgammaR's analogous to models of
signaling by the B and T cell antigen receptors.

Publication Types:
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 9314552 [PubMed - indexed for MEDLINE]
1: Mol Cell Biol. 1998 Jul;18(7):4209-20.

The Syk protein tyrosine kinase is essential for Fcgamma receptor signaling in
macrophages and neutrophils.

Kiefer F, Brumell J, Al-Alawi N, Latour S, Cheng A, Veillette A, Grinstein S,
Pawson T.

Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute,
Mount Sinai Hospital, Toronto, Ontario M5G 1X5.

The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains
that engage phosphorylated immunoreceptor tyrosine-based activation motifs in
the signaling subunits of immunoreceptors. Syk, in conjunction with Src family
kinases, has been implicated in immunoreceptor signaling in both lymphoid and
myeloid cells. We have investigated the role of Syk in Fcgamma receptor
(FcgammaR)-dependent and -independent responses in bone marrow-derived
macrophages and neutrophils by using mouse radiation chimeras reconstituted with
fetal liver cells from Syk-/- embryos. Chimeric mice developed an abdominal
hemorrhage starting 2 to 3 months after transplantation that was ultimately
lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of
generating reactive oxygen intermediates in response to FcgammaR engagement but
responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient
macrophages were defective in phagocytosis induced by FcgammaR but showed normal
phagocytosis in response to complement. The tyrosine phosphorylation of multiple
cellular polypeptides, including the FcgammaR gamma chain, as well as Erk2
activation, was compromised in Syk-/- macrophages after FcgammaR stimulation. In
contrast, the induction of nitric oxide synthase in macrophages stimulated with
lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly,
Syk-deficient macrophages were impaired in the ability to survive or proliferate
on plastic petri dishes. Taken together, these results suggest that Syk has
specific physiological roles in signaling from FcgammaRs in neutrophils and
macrophages and raise the possibility that in vivo, Syk is involved in signaling
events other than those mediated by immunoreceptors.

Publication Types:
Research Support, Non-U.S. Gov't

PMID: 9632805 [PubMed - indexed for MEDLINE]

ZAP-70 deficient mice impair the developmnt of NK1.1+ alpha beta T cells.

1: Immunol Lett. 2000 Jul 3;73(1):65-9.

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in
ZAP-70 deficient mice.

Tone S, Iwabuchi K, Iwabuchi C, Negishi I, Onoe K.

Division of Immunobiology, Section of Pathophysiology, Institute for Genetic
Medicine, Hokkaido University, Kita-15 Nishi-7, Kita-Ku, Sapporo 060-0815,
Japan.

The mechanism of development of a unique subset of T cells, thymic NK1.1(+)
alpha beta T cells, has been poorly understood. We found that the development of
thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70.
Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected
in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present
report, we examined whether biochemical treatments that replace TCR-mediated
positive selection signals could restore the generation of thymic NK1.1(+) alpha
beta T cells in ZAP -/- mice using the thymus organ culture. We found that a
higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell
generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells.
Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population
suggested that these cells expressed CD8 but not CD4 molecules, which is a
different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These
results suggest that differential signaling is required for the generation of
mainstream T cells and thymic NK1.1(+) alpha beta T cells.

PMID: 10963813 [PubMed - indexed for MEDLINE]

ITK-/- mice reduce mast cell degranulation and acute allergic responses.

1: Am J Respir Cell Mol Biol. 2005 Jun;32(6):511-20. Epub 2005 Mar 18.

Interleukin-2-inducible T cell kinase regulates mast cell degranulation and
acute allergic responses.

Forssell J, Sideras P, Eriksson C, Malm-Erjefalt M, Rydell-Tormanen K, Ericsson
PO, Erjefalt JS.

Transplantation Center, Foundation for Biomedical Research, Academy of Athens,
Athens, Greece.

Bruton's tyrosine kinase (Btk) is thought to positively regulate mast cell
activation, implying a role in allergic responses. We have compared acute and
late phase allergic airway reactions in mice lacking either Btk or
interleukin-2-inducible T cell kinase (Itk), another Tec kinase expressed in
mast cells. Btk(-/-) mice showed minor protection against allergic symptoms when
challenged with allergen via the airways. In sharp contrast, both acute and late
phase inflammatory allergic responses were markedly reduced in Itk(-/-) mice.
Notably, airway mast cell degranulation in Itk(-/-) mice was severely impaired,
despite wild-type levels of allergen-specific IgE and IgG1. The degranulation
defect was confirmed in DNP-conjugated human serum albumin-challenged mice
passively sensitized with anti-DNP IgE antibodies, and was also observed after
direct G-protein stimulation with the mast cell secretagogue c48/80. Moreover,
late phase inflammatory changes, including eosinophilia, lymphocyte
infiltration, and Th2 cytokine production in the lungs, was eliminated in
Itk(-/-) mice. Collectively, our data suggest a critical role of Itk in airway
mast cell degranulation in vivo that together with an impaired T cell response
prevents the development of both acute and late phase inflammatory allergic
reactions.

Publication Types:
Research Support, Non-U.S. Gov't

PMID: 15778496 [PubMed - indexed for MEDLINE]

Vav-2,3(-/-) neurons exhibit impaired axon guidance.

1: Neuron. 2005 Apr 21;46(2):205-17.

Comment in:

Vav family GEFs link activated Ephs to endocytosis and axon guidance.

Cowan CW, Shao YR, Sahin M, Shamah SM, Lin MZ, Greer PL, Gao S, Griffith EC,
Brugge JS, Greenberg ME.

Neurobiology Program, Children's Hospital, Boston, Massachusetts 02115, USA.

Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or
repulsion of axonal growth cones during development. However, the mechanisms
that determine whether Eph signaling promotes attraction or repulsion are not
known. We show here that the Rho family GEF Vav2 plays a key role in this
process. We find that, during axon guidance, ephrin binding to Ephs triggers
Vav-dependent endocytosis of the ligand-receptor complex, thus converting an
initially adhesive interaction into a repulsive event. In the absence of Vav
proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone
collapse in vitro and significant defects in the ipsilateral retinogeniculate
projections in vivo. These findings suggest an important role for Vav family
GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive
signaling during axon guidance.

Publication Types:

PMID: 15848800 [PubMed - indexed for MEDLINE]

Vav-1(-/-) NK cells are dramactically diminished

1: Eur J Immunol. 2001 Aug;31(8):2403-10.

Vav-1 regulates NK T cell development and NK cell cytotoxicity.

Chan G, Hanke T, Fischer KD.

Abteilung Physiologische Chemie, Universitat Ulm, Ulm, Germany.

The hematopoietic-specific Rho-family GTP exchange factor Vav-1 is a regulator
of lymphocyte antigen receptor signaling and mediates normal maturation and
activation of B and T cells.Recent findings suggest that Vav-1 also forms part
of signaling pathways required for natural and antibody dependent cellular
cytotoxicity (ADCC) of human NK cells. In this study, we show that Vav-1 is also
expressed in murine NK cells. Vav-1(-/-) mice had normal numbers of splenic NK
cells, and these displayed a similar expression profile of NK cell receptors as
wild-type mice. Unexpectedly, IL-2-activated Vav-1(-/-) NK cells retained normal
ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In
contrast, Vav-1(-/-) NK cells exhibited reduced natural cytotoxicity against
EL4, C4.4.25, RMA and RMA/S. Together, the results demonstrate that Vav-1 is
dispensable for mainstream NK cell development, but is required for NK natural
cytotoxicity. Unlike the findings for NK cells, NK T cells were dramatically
diminished in Vav-1(-/-) mice and splenocytes from Vav-1 mutant mice failed to
produce IL-4 in response to in vivo CD3 stimulation. These data highlight the
important role of Vav-1 in NK T cell development and NK cell function.

Publication Types:
Research Support, Non-U.S. Gov't

PMID: 11500824 [PubMed - indexed for MEDLINE]

2B4

2B4 is a cell-surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T-lymphocyte function. It appears that the primary function of 2B4 is to modulate other receptor-ligand interactions to enhance leukocyte activation.

PLCG2-deficient mice exhibit a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function.

1: Immunity. 2000 Jul;13(1):25-35.

Phospholipase Cgamma2 is essential in the functions of B cell and several Fc
receptors.

Wang D, Feng J, Wen R, Marine JC, Sangster MY, Parganas E, Hoffmeyer A, Jackson
CW, Cleveland JL, Murray PJ, Ihle JN.

Department of Biochemistry, St. Jude Children's Research Hospital, Memphis,
Tennessee 38105, USA.

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of
PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have
decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell
deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens
are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent
antibody production is absent. The similarity to Btk- or Blnk-deficient mice
demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma
signaling is also defective, resulting in a loss of collagen-induced platelet
aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4
function. The results define a signal transduction pathway broadly utilized by
immunoglobulin superfamily receptors.

Publication Types:

PMID: 10933392 [PubMed - indexed for MEDLINE]

GAB3 has no effect for phenotype.

1: Mol Cell Biol. 2003 Apr;23(7):2415-24.

Gab3-deficient mice exhibit normal development and hematopoiesis and are
immunocompetent.

Seiffert M, Custodio JM, Wolf I, Harkey M, Liu Y, Blattman JN, Greenberg PD,
Rohrschneider LR.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109-1024, USA.

Gab proteins are intracellular scaffolding and docking molecules involved in
signaling pathways mediated by various growth factor, cytokine, or antigen
receptors. Gab3 has been shown to act downstream of the macrophage
colony-stimulating factor receptor, c-Fms, and to be important for macrophage
differentiation. To analyze the physiological role of Gab3, we used homologous
recombination to generate mice deficient in Gab3. Gab3(-/-) mice develop
normally, are visually indistinguishable from their wild-type littermates, and
are healthy and fertile. To obtain a detailed expression pattern of Gab3, we
generated Gab3-specific monoclonal antibodies. Immunoblotting revealed a
predominant expression of Gab3 in lymphocytes and bone marrow-derived
macrophages. However, detailed analysis demonstrated that hematopoiesis in mice
lacking Gab3 is not impaired and that macrophages develop in normal numbers and
exhibit normal function. The lack of Gab3 expression during macrophage
differentiation is not compensated for by increased levels of Gab1 or Gab2 mRNA.
Furthermore, Gab3-deficient mice have no major immune deficiency in T- and
B-lymphocyte responses to protein antigens or during viral infection. In
addition, allergic responses in Gab3-deficient mice appeared to be normal.
Together, these data demonstrate that loss of Gab3 does not result in detectable
defects in normal mouse development, hematopoiesis, or immune system function.

PMID: 12640125 [PubMed - indexed for MEDLINE]

GAB2 deficient mast cells in mice result in impaired response of Fc epsilon RI.

1: Nature. 2001 Jul 12;412(6843):186-90.

Essential role for Gab2 in the allergic response.

Gu H, Saito K, Klaman LD, Shen J, Fleming T, Wang Y, Pratt JC, Lin G, Lim B,
Kinet JP, Neel BG.

Cancer Biology Program, Division of Hematology and Oncology, Beth Israel
Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
02215, USA [email protected].

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay
molecules, including the protein-tyrosine phosphatase Shp-2 and
phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth
factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die
during embryogenesis and show defective responses to several stimuli. Here we
report that Gab2-/- mice are viable and generally healthy; however, the response
(for example, degranulation and cytokine gene expression) of Gab2-/- mast cells
to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor
Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive
cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice.
Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a
critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast
cells. Our data identify Gab2 as the principal activator of PI(3)K in response
to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab
family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated
signalling molecules may be new targets for developing drugs to treat allergy.

PMID: 11449275 [PubMed - indexed for MEDLINE]

GAB2 deficient macrophages in mice result impaired phagocytosis.

1: J Cell Biol. 2003 Jun 23;161(6):1151-61.

Critical role for scaffolding adapter Gab2 in Fc gamma R-mediated phagocytosis.

Gu H, Botelho RJ, Yu M, Grinstein S, Neel BG.

Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, HIM 1047 Boston, MA
02115, USA. [email protected]

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding
molecules, plays important roles in regulating the growth, differentiation, and
function of many hematopoietic cell types. In this paper, we reveal a novel
function of Gab2 in Fcgamma receptor (FcgammaR)-initiated phagocytosis in
macrophages. Upon FcgammaR activation, Gab2 becomes tyrosyl phosphorylated and
associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K),
and the protein-tyrosine phosphatidylinositol Shp-2. FcgammaR-mediated
phagocytosis is severely impaired in bone marrow-derived macrophages from
Gab2-/- mice. The defect in phagocytosis correlates with decreased
FcgammaR-evoked activation of Akt, a downstream target of PI3K. Using confocal
fluorescence microscopy, we find that Gab2 is recruited to the nascent
phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires
the pleckstrin homology domain of Gab2 and is sensitive to treatment with the
PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary
role in recruitment to the phagosome. Because PI3K activity is required for
FcgammaR-mediated phagocytosis, our results indicate that Gab2 acts as a key
component of FcgammaR-mediated phagocytosis, most likely by amplifying PI3K
signaling in the nascent phagosome.

PMID: 12821647 [PubMed - indexed for MEDLINE]