Muneo Inaba

Differential Regulation of Human Blood Dendritic Cell Subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a+/CD11c+ population (CD11c+ DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a−/CD11c− population (CD11c− DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-αβ)-producing cells. Here, we investigate the effects of IFN-αβ and IFN-γ as well as other cytokines on CD11c+ and CD11c− DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-γ and IFN-α, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c+ DCs. Incubation of CD11c+ DCs with IFN-γ also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-α did not induce IL-12 but, rather, augmented IL-10 production. IFN-α-primed matured CD11c+ DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-α by itself neither matured CD11c− DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c− DCs. Unlike IFN-α, IL-3 was a potent survival factor and induced the maturation of CD11c− DCs. The IL-3-primed CD11c− DCs activated T cells to produce IL-10, IFN-γ, and IL-4. Thus, CD11c+ and CD11c− DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.

Plasmacytoid Dendritic Cells Regulate Th Cell Responses through OX40 Ligand and Type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-α in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-γ-producing Th cells depending on their capacity to residually produce IFN-α. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-α-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.

Cutting Edge: Essential Role of Phospholipase C-γ2 in B Cell Development and Function

Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-γ2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-γ2 in murine lymphopoiesis, the PLC-γ2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-γ2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-γ2 is a critical component of BCR signaling pathways and is required to promote B cell development.

Prevention of senile osteoporosis in SAMP6 mice by intrabone marrow injection of allogeneic bone marrow cells

The SAMP6 mouse (a substrain of senescence‐accelerated mice) spontaneously develops osteoporosis early in life and is, therefore, a useful model for examining the mechanisms underlying osteoporosis. We have recently established a new bone marrow transplantation (BMT) method: the bone marrow cells (BMCs) of normal allogeneic mice are directly injected into the bone marrow (BM) cavity of irradiated (5.5 Gy × 2) recipients (IBM‐BMT). Using IBM‐BMT, we attempted to prevent osteoporosis in SAMP6 mice. The hematolymphoid system was completely reconstituted with donor‐type cells after IBM‐BMT. Thus‐treated SAMP6 mice showed marked increases in trabecular bones even at 12 months of age, and the bone mineral density remained similar to that of normal B6 mice. In concordance with these findings, urinary deoxypyridinoline also remained continuously low until 10 months of age, indicating that IBM‐BMT was effective in the prevention of bone absorption.

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells). Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction. These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.

Apoptosis of colorectal adenocarcinoma (COLO 201) by tumour necrosis factor-alpha (TNF-α) and/or interferon-gamma (IFN-γ), resulting from down-modulation of Bcl-2 expression

Fas antigen is constitutively expressed in the normal colon epithelium, but considerably diminished in most colorectal carcinomas. In the present study, we examine the relationship between Fas antigen expression and apoptosis using the colorectal carcinoma cell line COLO 201, on which a low grade of Fas antigen is expressed. Anti‐Fas antibody had no effect on the induction of apoptosis of COLO 201. However, TNF‐α and/or IFN‐γ, independently and additively, up‐regulated Fas antigen expression on COLO 201 and induced apoptosis in a dose‐dependent manner. Both cytokines also increased the COLO 201 sensitivity to anti‐Fas antibody, resulting from the down‐modulation of Bcl‐2 and the up‐regulation of Bax. These findings indicate that cytokine(s) plus anti‐Fas antibody (which mimics natural Fas ligand) are more effective in inducing apoptosis of COLO 201 than cytokine(s) alone. These findings suggest that immunotherapy in combination with cytokine(s) and lymphokine‐activated killer (LAK) cells will become a more effective therapy for cancer than cytokine(s) or LAK cells alone, since the Fas ligand is expressed on activated T cells, natural killer cells and macrophages.

Comparative analyses of megakaryocytes derived from cord blood and bone marrow

Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34+ cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)‐3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c‐mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL‐3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.

A New Method for Bone Marrow Cell Harvesting

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new “perfusion method” for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate‐buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4+ and CD8+ T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this “perfusion method” are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the “perfusion method” is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft‐versus‐host‐disease in allogeneic BM transplantation.

Experimental autoimmune thyroiditis induced by thyroglobulin-pulsed dendritic cells

Dendritic cells (DCs), which are the most effective professional antigen-presenting cells (APCs), initiate and regulate immune responses. In this report, we examine the role of DCs in the induction of autoimmune thyroiditis. Experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin (Tg) plus adjuvant is considered to be an animal model of autoimmune thyroiditis, and is categorized as a T cell-mediated autoimmune disease. To examine the contribution of DCs to EAT, naive DCs were purified from high responder B10BR mice and pulsed with various concentrations of porcine Tg (pTg). These pTg-pulsed DCs were transferred without adjuvant to syngenic BIOBR mice to induce EAT. Mice that had received pTg-pulsed DCs showed thyroiditis, and the degree of thyroiditis induced was positively correlated to the amounts of pTg used for the incubation (pulsing) of DCs. The severity of thyroiditis was also correlated to the amounts of anti-pTg IgG2a antibodies and IFN-gamma in the recipient sera, but not to IL-4 or IL-10, indicating that Th1 cells are mainly activated by pTg-pulsed DCs and attributable to the pathogenesis of EAT.

EFFECT OF BONE MARROW TRANSPLANTATION ON ANTI-PHOSPHOLIPID ANTIBODY SYNDROME IN MURINE LUPUS MICE

The (NZW x BXSB)F1 (W/BF1) mouse is known to be an animal model of systemic lupus erythematosus (SLE) and immune thrombocytopenic purpura (ITP). These mice produce not only anti-DNA antibodies but also anti-platelet antibodies, resulting in decreased platelet counts. They show a high level of proteinuria, increased white blood cell (WBC) counts, hypertension, and myocardial infarction due to the high levels of anti-cardiolipin antibodies. When W/BF1 mice (4-5 months) were lethally irradiated and then reconstituted with T cell-depleted bone marrow cells of normal BALB/c mice (8 weeks), 60% of the mice survived more than one year. The WBC and platelet counts in the mice were normalized, and the levels of anti-DNA and anti-platelet antibodies decreased. The renal dysfunction was also ameliorated as indicated by a lower level of proteinuria, lower levels of serum creatinine (S-CRTN) and blood urea nitrogen (BUN), and by improved histology. The blood pressure (BP) of the treated W/BF1 mice decreased due to the improved renal functions. In contrast to the non-treated W/BF1 mice which died of myocardial infarction or renal failure by the age of 7 months, the treated W/BF1 mice showed no evidence of myocardial infarction even one year after BMT. This was due to the lower cardiolipin levels.