Annual Report 2004

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 B cells.
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 B cells

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 gut.

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 knock-in mice).

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 of asthma
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.