Department of Microbiology and Immunology
Division of Immunology
Self-defense against invaded pathogenic microorganisms and foreign antigenic
molecules is strictly controlled by the immune system and inflammation.
Our major research interests are to elucidate cells and effector molecules
in innate and acquired immunity and inflammation. In particular, we are
focused on cellular and molecular mechanisms of development and activation
of B cells and IgH class switch recombination under the influence of T
cells, cytokines and adaptor proteins. We are also interested in elucidating
cellular mechanisms of preferential induction of Th1 cells upon immunization
with Mycobacterium-derived, Peptide-25 and its derivatives.
In 2003, Professor Dr. Fritz Melchers, Basel University, who were invited
by the University of Tokyo as the Eminent Scientist of JSPS, has been
joining us for our research projects. Dr. Melchers always encouraged us
and suggested important issues for each project. We would like to appreciate
his enormous contributions.
1. Roles of IL-5 in the B cell differentiation
a. Molecular mechanisms of class switch recombination in CD38-activated
Yumiko Tsukamoto, Atsushi Sato, Kiyoshi Takatsu
Class switch recombination (CSR) is the process that changes physiological
activities of antibodies without changing their specificities to antigens.
Murine B cells differentiate into IgG1- or IgE- secreting cells when activated
by anti-CD40 and IL-4. However, IL-4 cannot induce CSR in anti-CD38-activated
B cells, although IL-5 can induce CSR in both anti-CD40- and anti-CD38-activated
B cells. Anti-CD38 and IL-4-activated B cells differentiate into IgG1-secreting
cells when activated by 8-mercaptugoanosine (8-SGuo). 8-SGuo is a guanosine
analogue, and it induces proliferation and differentiation in B cells.
Activation-induced cytidine deaminase (AID), which is necessary for CSR,
is expressed in anti-CD38, IL-4, and 8-SGuo-activated B cells, and isn't
expressed in_anti-CD38 and IL-4-activated B cells. It suggests that 8-SGuo
induces expression of AID in B cells, and thus induces CSR.
In anti-CD38 and 8-SGuo-activated B cells, AID is expressed; however these
B cells don't differentiate into IgG1-secreting B cells. This suggests
that IL-4 also induces some factor(s) necessary for CSR in anti-CD38,
IL-4, and 8-SGuo-activated B cells. Cell division, transcription of germline
ｳ1, and looping out of ｳ1-ｼ reciprocal DNA are necessary for CSR. In anti-CD38
and 8-SGuo-activated B cells and anti-CD38, IL-4, and 8-SGuo-activated
B cells, cell division and transcription of germline ｳ1 were induced.
However, ｳ1-ｼ reciprocal DNA didn't exist in anti-CD38 and 8-SGuo-activated
B cells. These results show that anti-CD38 and 8-SGuo cannot induce CSR
because of lacking some factor(s) which are involved in CSR other than
cell division, transcription of germline ｳ1, and AID expression. This
factor(s) (Factor X) may be induced by IL-4 in _anti-CD38, IL-4, and 8-SGuo-activated
B cells. We examined the expression of UNG, Bach2, and 53BP1 genes which
are necessary for CSR and found that they were all expressed in anti-CD38
and 8-SGuo-activated B cells. Hence Factor X is not UNG, Bach2, and 53BP1.
Identification of Factor X may reveal some new aspects of CSR.
b. IL-5 induces IgG1 isotype switch recombination in CD38-stimulated murine
Atsushi Sato, Yumiko Tsukamoto, Keisuke Honiara, Kiyoshi Takatsu
As we reported, IL-5 stimulation of anti-CD38-stimulated murine splenic
B (B-2) cells induces ｼ-ｳ1 class switch recombination (CSR) leading to
a high level of lgG1 production. Further addition of IL-4 in the system
enhances IL-5-dependent ｼ-ｳ1 CSR. Stat5a- and Stat5b-deficient B-2 cells
could not show ｼ-ｳ1 CSR and lgG1 production, despite of the intact induction
of ｳ1 germline transcripts. Cell division cycle analyses of Stat5b deficient
B-2 cells using CFSE revealed that Stat5b-deficient B cells were normally
divided to 5 or 6 times, but these they could not express surface IgG1.
These results implied that Stat5 plays pivotal roles in the ｼ-ｳ1 CSR induction.
RT-PCR analysis revealed equivalent levels of AID expression in wild type
and Stat5b-deficient B cells, while expression of the Blimp-1 gene was
impaired in Stat5b deficient B-2 cells.
We examined the involvement of Stat5a and Stat5b in anti-CD38 and 8-SGuo-activated
B cells. Cell division and transcription of germline ｳ1 were induced in
anti-CD38 and 8-SGuo-activated B cells, but those B cells did not undergo
CSR similar to anti-CD38 and IL-5 stimulated Stat5a deficient B cells.
Hence we examined the phosphorylation of Stat5a and Stat5b in anti-CD38
and 8-SGuo-activated B cells. Stat5a and Stat5b were not phosphorylated
in anti-CD38 and 8-SGuo-activated B cells. Downstream molecules of Stat5a
and Stat5b may be involved in the process of CSR induce by anti-CD38,
IL-4, and 8-SGuo.
c. Molecular mechanisms of nuclear factor (NF)-ｺB and the germline ｳ1
transcript expression in CD38-stimulated B cells
Hiroaki Kaku and Kiyoshi Takatsu
CD38 is an ectoenzyme with both ADP ribosyl cyclase and cADP ribosyl hydrolase
activity. Ligation of CD38 on B-2 cells by anti-CD38 mAb induces B cell
proliferation, IL-5Rｱ expression, and the germline ｳ1 mRNA expression.
IL-5 promotes ｼ-ｳ1 CSR and IgM and IgG1 production from anti-CD38-stimulated
B-2 cells in an IL-4 independent manner. We reported that NF-ｺB complexes
play critical roles in the B cell activation in response to CD38 ligation,
and that Btk, PKC, PI3-kinase, BASH/BLNK, PLC-ｳ2 and extracellular Ca2+
influx are involved in NF-ｺB activation induced by anti-CD38 and antibody
production induced by anti-CD38 and IL-5 co-stimulation. By analyzing
the upstream molecules of CD38 signaling pathway, we found that anti-CD38-mediated
NF-ｺB can be induced via activation of Syk and GTP-binding protein. Moreover,
GTP-binding protein but not Syk be co-immunoprecipitated with CD38 in
cell lysates prepared using Brij-58 detergent. These data suggest that
CD38 directly associates with GTP-binding protein or that CD38 constitutively
localizes in GTP-binding protein- or GPCR-associated membrane micro-domain.
Therefore, we speculate that GTP-binding protein may link and modulate
CD38-Syk signaling pathway or that anti-CD38-stimulation induce colocalization
of GTP-binding protein into BCR-associated micro-domain.
2. Role of interleukin-5 (IL-5) and B-1 cells in mucosal immunity and
elicitation of contact sensitivity
a. Origin and differentiation of B-1 cells
Taku Kouro, Masashi Ikutani and Kiyoshi Takatsu
B-1 cells form distinctive subset of B lymphocyte characterized by preferential
distribution to peritoneal cavity and contribution to serum natural antibodies
and intestinal IgA secretion. Although several lines of evidence showed
that constitutive B cell receptor signaling causes expression of B-1 phenotype,
it is still unknown how generation and differentiation of B-1 cells are
controlled. IL-5 is indispensable for normal development of B-1 cells
because IL-5R-/- mice show reduced number and cell size of peritoneal
B-1 cells. To investigate roles of IL-5 in B-1 cell development, we first
checked expression of IL-5R on early lymphoid progenitors in the fetal
liver. IL-5Rｱ is not detected on lineage marker negative (Lin-) fetal
liver cells but weakly detected on B220+ proB cells. Its expression was
also induced when Lin- fetal liver cells were placed in the stroma-free,
serum-free culture with B cell differentiating condition. Then Lin- cells
were stimulated or not stimulated with IL-5 in such cultures and transferred
to lymphocyte-deficient SCID mice. Progenitors from either condition differentiated
to B-1 cells in the SCID mice but more B-1b cells were observed in the
mice received IL-5-stimulated progenitors. Interestingly, feces IgA was
only restored in the mice received IL-5-stimulated progenitors. B-1b cells
differentiated from IL-5-treated progenitors are likely responsible for
intestinal IgA, because SCID mice transferred with B-1b cells secreted
more IgA in the feces than mice received B-1a cells did. From these results,
it is speculated that IL-5 induces B-1b cell differentiation in the early
stage of gestation, which eventually results in production of IgA in the
b. Identification of IL-5 secreting cells in vivo
Masashi Ikutani, Byoung-Gon Moon, Taku Kouro and Kiyoshi Takatsu
B-1 cells differ from B-2 cells in surface markers, anatomical location
and developmental pattern, and are known to be involved in natural immunity
by producing natural antibodies. B-1 cells form distinctive subset of
B lymphocytes characterized by preferential distribution to peritoneal
and pleural cavities as well as unique antigen reactivity. Unlike conventional
B-2 cells, B-1 cells are rather maintained by self-renewal than de novo
differentiation and are activated independent of helper T cells though
details in the proliferation and activation of B-1 cells are unknown.
We are focusing on IL-5 as a candidate of soluble factor that control
B-1 cell development and activation. Indeed we have shown that IL-5 is
indispensable for survival and homeostatic proliferation of peritoneal
B-1 cells and mucosal IgA responses. IL-5 is originally found as a soluble
factor secreted from helper T lymphocytes but recently significant amount
of IL-5 is shown to be secreted from non-lymphoid tissues such as lung.
B-1 cells also depend on IL-5 of non-lymphoid origin because neutralizing
antibody against IL-5 still take effect on peritoneal B-1 cells in the
host mice lacking lymphocytes. However, technical difficulties have been
preventing detection of IL-5 secreting cells that support B-1 cells. To
overcome this problem, we are now generating mouse model in which green
fluorescent protein gene is inserted in the IL-5 gene locus (IL-5/GFP
c. Role of IL-5 and B-1 cells in mucosal immunity
Atsuko Itakura, Yuji kikuchi1, Taku Kouro, Masashi Ikutani, Kiyoshi Takatsu:
1Laboratory of Immunoregulation, Department of Infection Control and Immunology,
Kitasato Institute for Life Sciences, Kitasato University
About a half of IgA is derived from B-1 cells in intestinal and respiratory
mucosa, which plays an important role in primary host defense mechanism
against pathogens that invade through mucosal tissues. B-1 cell numbers
and the size of B-1 cells are decreased in IL-5 receptor ｱ chain-deficient
(IL-5Rｱ-/-) mice, and their serum IgM and intestinal IgA levels were lower
than those of C57BL/6 mice. Therefore, we tried to determine if lack of
IL-5/IL-5R signaling enhances sensitivity to these pathogens.
IL-5Rｱ-/- and C57BL/6 mice were orally or intranasally inoculated with
Salmonella typhimurium or influenza A PR/8 virus, respectively. Results
revealed that IL-5Rｱ-/- mice and C57BL/6 mice died in a similar manner
following inoculation with S. typhimurium, and bacterial loads of Payer's
patches, spleens and mesenteric lymph nodes in IL-5Rｱ-/- mice were comparable
to those in C57BL/6 mice. We found no difference in lung virus titers
between IL-5Rｱ-/- and C57BL/6 mice inoculated with PR/8. ELISA for IgM,
IgG and IgA titers in bronchoalveolar lavage and serum showed that PR/8-specific
antibodies were induced in both IL-5Rｱ-/- and C57BL/6 mice upon inoculation,
and antibody titers in IL-5Rｱ-/- mice were roughly equivalent to those
in C57BL/6 mice. These results indicate that IL-5/IL-5R signaling does
not seem to be involved in host defense against S. typhimurium and influenza
virus, and antibody responses to these pathogens.
d. B-1 cells in elicitation of contact sensitivity
Elicitation of contact sensitivity, a classic example of T cell-mediated
immunity, requires antigen-specific IgM antibodies, which are produced
by B-1 cells within 1 day after skin immunization. Because IL-5 is important
for maintenance of B-1 cells, and promotes antibody production, we examined
if IL-5 is involved in elicitation of contact sensitivity. IL-5Rｱ-/- and
C57BL/6 mice were immunized by painting oxazolone on the chest, abdomen
and feet. On day 4, mice were challenged by topical application of same
antigen on the ears, and ear thickness was measured at 24-hr post-challenge.
We found that ear swelling responses were impaired in IL-5Rｱ-/- mice.
Histological examination of C57BL/6 ears showed edema of the connective
tissue with massive accumulation of inflammatory cells including eosinophils.
In IL-5Rｱ-/- mice, edema and cell infiltration were much milder, and no
eosinophils were observed. These results suggest that IL-5 is required
for full elicitation of contact sensitivity.
3. Role of mast cells, eosinophils, and IL-5 in the development
Yoko Oe-Kikuchi and Kiyoshi Takatsu
Mast cells are though to contribute to the pathogenesis of allergic airway
responses through IgE dependent mechanism. Eosinophilic inflammation is
clearly a hallmark of both allergic and non-allergic asthma. Considerable
evidence suggests that there is association between pulmonary eosinophil
infiltration and AHR in human asthma. An immunopathogenic role for mast
cells is suggested by the role of IL-4, IL-13, IL-5 in IgE synthesis and
eosinophil differentiation and activation, however, the exact mechanism
by which mast cells mediate eosinophilic inflammation and subsequent AHR
are still not entirely clear.
We have studied the role of IL-5 on mast cell and eosinophil activation
and role of activated mast cells in activation and survivability of eosinophils.
First, we have studied IgE-dependent production of histamine and cytokine
(IL-5 and GM-CSF) by mast cells isolated from IL-5R_ KO and IL-5 KO mice
to observe the function of IL-5 on releasability. We observed that mast
cells, expressing detectable IL-5R_, increased their steady state expression
of IL-5 mRNA after cross-linking the IgE receptor. Although we have observed
IL-5, GM-CSF, and histamine release from these mast cells, there were
no significant difference on each releasability, however, we have found
that IL-5 was able to reduce histamine content in mast cells in dose dependent
manner. Second, we have established the system to isolate eosinophils
from a long-term bone marrow culture supplemented with IL-5. Third, to
understand the bioactivity of released product from activated mast cells,
we have further investigated the effect of mast cell supernatants on activation
(such as degranulation and expression of adhesion molecule) and survivability
of eosinophils isolated from a culture of bone marrow cells.