Immunology of Aging: the Birth of Inflammaging.

The inflammaging concept was introduced in 2000 by Prof. Franceschi. This was an evolutionary or rather a revolutionary conceptualization of the immune changes in response to a lifelong stress. This conceptualization permitted to consider the lifelong proinflammatory process as an adaptation which could eventually lead to either beneficial or detrimental consequences. This dichotomy is influenced by both the genetics and the environment. Depending on which way prevails in an individual, the outcome may be healthy longevity or pathological aging burdened with aging-related diseases. The concept of inflammaging has also revealed the complex, systemic nature of aging. Thus, this conceptualization opens the way to consider age-related processes in their complexity, meaning that not only the process but also all counter-processes should be considered. It has also opened the way to add new concepts to the original one, leading to better understanding of the nature of inflammaging and of aging itself. Finally, it showed the way towards potential multimodal interventions involving a holistic approach to optimize the aging process towards a healthy longevity.

Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis.

Synovial tissue affected by rheumatoid arthritis is characterized by proliferation, which leads to irreversible cartilage and bone destruction. Current and experimental treatments have been aimed mainly at correcting the underlying immune abnormalities, but these treatments often prove ineffective in preventing the invasive destruction. We studied the expression of cyclin-dependent kinase inhibitors in rheumatoid synovial cells as a means of suppressing synovial cell proliferation. Synovial cells derived from hypertrophic synovial tissue readily expressed p16INK4a when they were growth-inhibited. This was not seen in other fibroblasts, including those derived from normal and osteoarthritis-affected synovial tissues. In vivo adenoviral gene therapy with the p16INK4a gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis. Thus, the induction of p16INK4a may provide a new approach to the effective treatment of rheumatoid arthritis.

Medullary but not cortical thymic epithelial cells present soluble antigens to helper T cells.

Thymic epithelial cell lines (TECs) were established from newborn C57BL/6 mice. They were classified into two types (medullary and cortical TECs) by using the monoclonal antibody (Th-3) that recognizes the meshwork structure of thymic cortical epithelial cells. Antigen-presenting activity of each TEC was determined by using ovalbumin-specific, I-Ab-restricted helper T cell lines. It was demonstrated that the medullary but not the cortical TECs functioned as antigen-presenting cells. This is the first evidence for the functional difference between the cortical and the medullary TEC.

A novel type of cell death of lymphocytes induced by a monoclonal antibody without participation of complement.

A monoclonal antibody, RE2, raised by immunizing a rat with cell lysate of a mouse T cell clone, was found to directly kill interleukin 2-dependent T cell clones without participation of serum complement. Fab fragments of RE2 had no cytolytic activity, while the cross-linking of Fab fragments with anti-rat immunoglobulin reconstituted the cytotoxicity. The cytotoxicity was temperature dependent: the antibody could kill target cells at 37 degrees C but not at 0 degrees C. Sodium azide, ethylenediaminetetraacetic acid, and forskolin did not affect the cytolytic activity of RE2, while the treatment of target cells with cytochalasin B and D completely blocked the activity. This suggested that the cell death involves a cytoskeleton-dependent active process. Giant holes on the cell membrane were formed within 5 minutes after the treatment with RE2, as observed by scanning electron microscopy. There was no indication of DNA fragmentation nor swelling of mitochondria during the cytolysis, suggesting that the cell death is neither apoptosis nor typical necrosis. The antibody also killed T cell lymphomas and T and B cell hybridomas only when these cells were preactivated with concanavalin A, lipopolysaccharide, or phorbol myristate acetate. Preactivated peripheral T and B cells were sensitive to the cytotoxicity of RE2, while resting T and B cells were insensitive. These results provide evidence for a novel pathway of cell death of activated lymphocytes by membrane excitation.

Immunosenescence is both functional/adaptive and dysfunctional/maladaptive.

Alterations in the immune system with aging are considered to underlie many age-related diseases. However, many elderly individuals remain healthy until even a very advanced age. There is also an increase in numbers of centenarians and their apparent fitness. We should therefore change our unilaterally detrimental consideration of age-related immune changes. Recent data taking into consideration the immunobiography concept may allow for meaningful distinctions among various aging trajectories. This implies that the aging immune system has a homeodynamic characteristic balanced between adaptive and maladaptive aspects. The survival and health of an individual depends from the equilibrium of this balance. In this article, we highlight which parts of the aging of the immune system may be considered adaptive in contrast to those that may be maladaptive.

Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta.

Osteopontin is a phosphorylated, sialic acid-rich, noncollagenous bone matrix protein containing the Arg-Gly-Asp-Ser amino acid sequence responsible for cell adhesion. The protein strongly binds to hydroxyapatite and play an important role in calcification. Expression of osteopontin mRNA was analyzed in human aortic atherosclerotic lesion by Northern blot hybridization, as well as by in situ hybridization. The expression of osteopontin mRNA was detected in 24 out of 25 samples of aorta obtained from 17 autopsy cases, but not in one normal aortic sample. The magnitude of expression was proportional to the stage of atherosclerosis. In situ hybridization revealed that the cells expressing osteopontin mRNA were detected in the wall surrounding atheroma and closely associated with calcification. They were morphologically identified as foam cells and immunohistologically positive with HHF35, appearing to be derived from smooth muscle cells. These findings have suggested that smooth muscle cell-derived foam cells express osteopontin mRNA and play an important role in calcification of the atherosclerotic lesions.

Characterization of cytokine LD78 gene promoters: positive and negative transcriptional factors bind to a negative regulatory element common to LD78, interleukin-3, and granulocyte-macrophage colony-stimulating factor gene promoters.

Cytokine LD78 is a human counterpart of the mouse macrophage inflammatory protein 1 alpha/hematopoietic stem cell inhibitor. Promoters of the LD78 alpha and LD78 beta genes showed similar inducible activities in two leukemic cell lines, K562 and Jurkat, but the induction mechanisms differed between the two cell lines. Further characterization of the LD78 alpha promoter indicated that multiple positive and negative regulatory elements are present, some of which are differentially required for induction and repression of the promoter activity in different cells. One of the negative regulatory elements, ICK-1, functioned in both cell lines in the absence and presence of stimulation and was shown to be a recognition site for positive and negative transcriptional factors. This ICK-1 element contained a direct repeat, and similar repeats were also found in the negative regulatory elements of hematopoietic growth factor interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoters. Nuclear extracts from K562 and Jurkat cells formed several protein-DNA complexes with the LD78 alpha ICK-1 element, one of which was also observed with the IL-3 and GM-CSF ICK-1 elements. Results from in vivo and in vitro analyses suggested that the protein forming this complex functions as a negative factor. The binding affinity of this protein, ICK-1A, to the LD78 alpha ICK-1 element was low and was significantly affected by the incubation temperature and the salt concentration in the binding buffer. ICK-1B, another protein bound specifically by the LD78 alpha ICK-1 element, was shown to be a positive factor important for induction of the promoter. These results suggested that ICK-1A plays an important role in balanced expression of LD78, IL-3, and GM-CSF during hematopoietic cell growth and differentiation.

Induction of immunological tolerance by oral, but not intravenous and intraportal, administration of ovalbumin and the difference between young and old mice.

BACKGROUND: Induction of immunological tolerance is dependent on the route of antigenic administration, the dose of an antigen and the age of animals. OBJECTIVES: We investigated the effect of age on the tolerance induction in mice by administration of antigen through different routes and at different doses. DESIGN: Young and old BDF1 mice were orally, intraportally or intravenously administrated with a low or a high dose of ovalbumin (OVA). Then, delayed-type hypersensitivity (DTH) responses and serum anti-OVA antibody levels were assessed after systemic immunization of OVA with alum after appropriate intervals. RESULTS: In the young mice, oral administration of OVA suppressed DTH response and anti-OVA IgG1, IgG2b, IgM and IgE level in a dose-dependent manner. In the old mice, however, the suppression of IgG1 and IgE levels was induced by oral administration of a low dose of OVA, but no suppression by a high dose. On the other hand, intraportal or intravenous injection of OVA did not suppress DTH response and enhanced anti-OVA antibody levels in a dose-dependent manner in both young and old mice. Production of anti-OVA IgG2a antibody after systemic injection of OVA was detected in the mice, which had been treated with intraportal or intravenous injection of OVA, but not detected in the mice, which had been treated with oral administration of OVA. On the contrary, suppression of anti-OVA IgE antibody was observed only in the mice, which had been treated with oral administration of OVA. CONCLUSION: The oral administration of OVA, neither intravenous nor intraportal, induced immunological tolerance to OVA. An adequate dose of OVA for the tolerance induction and the suppression of antibody production are different between young and old mice. The suppression of IgE antibody was observed only by oral administration of OVA, much obviously in young mice than in the old. The results also indicated that the antigen processing in the liver did not play a major role in the induction of oral tolerance to OVA.

The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneurons in the spinal cord during development.

Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. We performed reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons by panning to identify RTKs involved in the early development of motoneuron. In situ hybridization revealed that Cek8, a member of the eph family, was specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles, and disappeared after the naturally occurring cell death period (E6-E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons.

Signal pathway of mitogen-induced Ca2+-activated K+ currents in young and aged T-cell clones of C57BL/6 mice.

Signal transduction pathways of mitogenic plant lectin, concanavalin A (Con A)- and ionomycin (INM)-induced (Ca2+-dependent K+ currents (I(Con A) and I(INM)) have been compared in young and aged T-cell clones by using the nystatin perforated patch-clamp whole-cell recording technique. In young T-cell clones, Con A evoked a long-lasting outward current which is mediated by the activation of the Ca2+-dependent K+ channels. The Ca2+ ionophore, INM, evoked a short-lasting Ca2+-dependent outward K+ current (I(INM)). The protein tyrosine kinase (PTK) inhibitor, herbimycin A (3 x 10(-6) M), but not the G protein blocker, pertussis toxin (PTX, 500 ng ml(-1)), completely prevented the I(Con A), but did not affect the I(INM). In aged T-cell clones, Con A fails to evoke any current response, while INM evokes an outward current which is comparable to that in a young T-cell clone. It is concluded that PTK, but not PTX-sensitive G proteins, plays a critical role in mediation of the signal transduction from Con A stimulation to activation of the Ca2+-dependent K+ channels, and that an impairment of the early signal pathway, perhaps the PTK, might be involved in the mechanism of the age-related decline of the proliferative response of T-lymphocytes to mitogenic stimulation.

Abnormality in the early signal transduction pathway is responsible for the impaired proliferative response and low K+ current in a T-cell clone by stimulation with anti-CD3 antibody.

Two T-cell clones were established from young and old C57BL/6 mice, respectively. The proliferative response to anti-CD3 stimulation was significantly greater in the young (YT5) than in the old (OT13) T cell clone. However, a similar high response was observed in both T-cell clones upon stimulation with phorbol myristate acetate (PMA) and ionomycin (INM). The calcium-dependent K+ current (IK(Ca)) was recorded with the patch-clamp method in these T-cell clones. With anti-CD3 stimulation, the amplitude of the outward K+ current was significantly lower in OT13 than in YT5 cells. With stimulation with PMA and INM, however, no significant difference in IK(Ca) was obtained between the two types of cells. The level of proliferative response of T-cells to mitogens was well reflected by the amplitude of IK(Ca). Some abnormality in the early pathway of signal transduction, which led to the intracellular Ca2+ influx, appears to be responsible for the impaired proliferation of the old T-cell clone.

Associations of menstrual pain with intakes of soy, fat and dietary fiber in Japanese women.

OBJECTIVE: Intakes of soy, fat, and dietary fiber may be associated with the symptoms of dysmenorrhea through their biological effects on estrogens or prostaglandin production. The present study was to examine the relationships between intakes of soy, fat, and dietary fiber and the severity of menstrual pain. DESIGN: Cross-sectional study. SETTING: Three colleges and two nursing schools. SUBJECTS: A total of 276 Japanese women aged 19-24 y. METHODS: Intakes of nutrients and foods including soy products, isoflavones, fats and dietary fiber were estimated by a validated semiquantitative food frequency questionnaire. Severity of menstrual pain was assessed by the multidimensional scoring system reported by Andersch and Milson. RESULTS: Intake of dietary fiber was significantly inversely correlated with the menstrual pain scale (r=-0.12, P=0.04) after controlling for age, smoking status, age at menarche and total energy intake. Neither soy nor fat intake was significantly correlated with menstrual pain after controlling for the covariates. CONCLUSIONS: The cross-sectional difference in dietary fiber intake across the level of menstrual pain was small in magnitude but warrants further studies.

Cell-free transmission of Fv-4 resistance gene product controlling Friend leukemia virus-induced leukemogenesis in mice.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). The Fv-4' env antigen that binds to the cell surface of Fv-4'-bearing C3H cells was found in sera from normal Fv-4'-bearing C4W mice. The serum Fv-4' env antigen binds to ecotropic MuLV receptors, shown by specific binding to transfectant cells expressing ecotropic MuLV receptors but not to parental mink cells. To determine whether the binding of Fv-4' env antigen to the putative MuLV receptors would block FLV infection, C3H thymocytes or spleen cells that had been preincubated with C4W serum were mixed with FLV and the subsequent production of MuLV specific antigens was examined. C3H thymocytes or spleen cells treated with C4W serum became refractory to binding by FLV. These results provide evidence that the Fv-4' env antigen is released from C4W-derived cells in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with FLV. The implication of these findings for gene therapy of retrovirus-induced disease such as acquired immune deficiency syndrome (AIDS) is discussed.

A gene therapy model for retrovirus-induced disease with a viral env gene: expression-dependent resistance in immunosuppressed hosts.

At the initial stage of retroviral infection, virion envelope glycoprotein (env product) binds to cell surface receptors. Cells infected with retrovirus or into which the env gene was introduced, become resistant to superinfection by other retroviruses with the same receptor specificity, a phenomenon known as receptor interference. We have demonstrated previously that the introduction of an env gene from a truncated endogenous ecotropic murine leukemia virus (MuLV), the Fv-4 resistance (Fv-4r) gene, into the bone marrow hematopoietic cells of Fv-4 sensitive (Fv-4s) mice protected mice from ecotropic retrovirus-induced disease. Using the gene transfer system under the control of the retroviral vector and bone marrow transplantation (BMT), here we could show that the expression of an introduced Fv-4r gene in hematopoietic cells continued for more than 1 year after BMT. To determine the inhibitory mechanism of Fv-4r env gene expression against FLV-infection in this model system, peripheral blood mononuclear cells (PBMCs), or spleen cells from chimeras with various degrees of env-expression, were mixed with green fluorescence protein (GFP)-conjugated Friend MuLV envglycoprotein (GFP-Fr-ENV). The amount of GFP-Fr-ENV bound to these cells inversely correlated with the expression intensity of the transduced env gene indicating the receptor interference effect. Next, to see whether transduction of the Fv-4r gene would protect an immunosuppressed host from FLV-induced leukemogenesis, we generated immunocompromised chimeras by transplanting env-transduced bone marrow cells into a thymectomized host. These chimeras also resisted FLV-induced leukemogenesis, indicating that receptor interference-based gene therapy could become a therapeutic basis for immunodeficiency virus-induced diseases in vivo.

Expression of inducible nitric oxide synthase (NOS) in bone marrow cells of myelodysplastic syndromes.

Nitric oxide (NO) is a biological mediator which is synthesized from L-arginine by a family of nitric oxide synthases (NOS). We have studied the expression of the inducible NOS (iNOS) by bone marrow cells from the patients with myelodysplastic syndromes (MDS) at the mRNA level by RT-PCR assay and at the protein level by immunohistochemical staining using a specific anti-iNOS monoclonal antibody. The iNOS message was present in 92% of bone marrow tissues from MDS patients (11 out of 12) by an examination using RT-PCR. Basically, iNOS message was negative or very weak in control (1/9) and AML (0/7) cases. This was supported by immunohistochemical findings that the iNOS was positive in most of the bone marrow samples from MDS patients (9 out of 12), while bone marrow cells of control (O out of 12) and AML (O out of 5) cases were basically negative. Double immunostaining for CD68 antigen, which is a marker for macrophage lineage cells, and iNOS was performed on MDS bone marrow sections. iNOS was dominantly localized to bone marrow macrophages, although a part of myeloid cells were also positively stained with anti-iNOS antibody in a part of cases. These results indicated that there is some in vivo induction of iNOS expression for local NO production that might be involved in the dysregulation of hematopoiesis in bone marrow of MDS.

Localization of Fas and Fas ligand in bone marrow cells demonstrating myelodysplasia.

Frequent apoptosis in the bone marrow of patients with myelodysplastic syndromes (MDS) was demonstrated on frozen sections using the terminal deoxytransferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. The overall mean percentage of TUNEL-positive cells was about 17% in the bone marrow of MDS, while bone marrow from control cases exhibited a mean of 3.4% (P < 0.001). To elucidate the mechanism of apoptosis in bone marrow cells of MDS, the expression of Fas antigen and Fas ligand (FasL) was examined by RT-PCR and immunohistochemistry. All MDS cases showed expression of Fas mRNA (12/12) and most exhibited an expression of FasL mRNA (10/12) by RT-PCR. Basically, control cases did not show positive signals for Fas and FasL mRNA, however, a very weak band was detected in three cases (3/10) for Fas and in one case (1/10) for FasL mRNA by RT-PCR. Immunohistochemical examination revealed positive staining for Fas (11/12) and FasL (12/12) in the bone marrow of MDS, while all the bone marrow samples from control cases were negative for anti-Fas (0/15) and for anti-FasL (0/15) antibody. Double staining clarified that TUNEL-positive apoptotic cells expressed Fas antigen on the cell surface, although not all Fas-positive cells were TUNEL positive. The Fas-positive cells of MDS bone marrow included hematopoietic cells expressing CD34 antigen, neutrophil elastase, a marker for myeloid series of cells, or glycophorin A, a marker for erythroid cells. However, CD68-positive cells which were macrophage lineage cells, did not express Fas antigen strongly. In contrast, positive staining for FasL was detected in hematopoietic cells and CD68-positive cells in the bone marrow of MDS. These results suggest that the Fas-FasL system plays an important role in inducing apoptosis in the bone marrow of MDS and works in an autocrine (hematopoietic cell-hematopoietic cell interaction) and/or paracrine (hematopoietic cell-stromal cell interaction) manner.

Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes.

To clarify whether regulatory cytokines inhibit hematopoiesis in patients with myelodysplastic syndromes (MDS), malignancies characterized by the formation of cytopenias despite the presence of cellular bone marrow, expression of TNF-alpha and IFN-gamma by bone marrow cells was investigated using specific reverse transcriptase-polymerase chain reaction assays. An enhanced expression of the mRNA for TNF-alpha was observed in most of the samples from MDS patients (11/14, 79%), whereas no enhancement was observed in bone marrow samples from AML (0/6), CML (0/2) or control cases (0/8). The expression of IFN-gamma was also enhanced in some of MDS cases (5/12, 42%) while AML (0/5), CML (0/2) and control cases (0/6) showed very low levels of IFN-gamma mRNA expression. Immunohistochemical examination confirmed the scattered presence of TNF-alpha or IFN-gamma producing cells in the bone marrow of MDS patients. The majority of these cells were CD68-positive macrophage lineage cells. These results suggested that disruption of hematopoiesis in MDS might be caused by enhanced production of inhibitory regulatory cytokines especially TNF-alpha and occasionally IFN-gamma by bone marrow macrophages.

Protection of retrovirus-induced disease by transplantation of bone marrow cells transduced with MuLV env gene via retrovirus vector.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We have demonstrated previously that bone marrow chimeras in which hematopoietic cells were replaced with cells expressing Fv-4 resistant (Fv-4r) gene product became refractory to Friend leukemia virus (FLV)-induced leukemogenesis. To induce in vivo resistance against retrovirus-induced diseases by retroviral vector-mediated gene transduction, we introduced Fv-4 env gene into bone marrow cells of FLV-susceptible C3H/He (C3H) mice with retroviral vector (pLSF) derived from murine Friend spleen focus forming virus (SFFV) and the cells were transplanted into lethally irradiated C3H mice. After the bone marrow transplantation, Fv-4r gene product was successfully expressed on erythroid and myeloid cells, while lymphoid cells were only weakly expressing Fv-4r gene product. The C3H mice expressing relatively higher amounts of Fv-4r gene product were rendered resistant to FLV-induced erythroleukemia, while mice expressing lower amounts of the Fv-4r gene product were still susceptible. Effective protection of FLV-induced leukemia in these mice suggested that the Fv-4r gene expression by erythroid cells that were the major target of FLV infection might be critical for resisting FLV-induced leukemia. Thus, gene therapy model by transducing Fv-4r env gene using bone marrow transplantation would provide a useful protection model system of retrovirus-induced diseases.

Distribution of Fv-4 resistant gene product in Friend leukemia virus-resistant Fv-4r mouse strain.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We demonstrated previously that the Fv-4 resistant (Fv-4r) gene product, Fv-4r env antigen, is released from Fv-4r-bearing BALB/c-Fv-4Wr (C4W) mouse-derived cells into serum in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with Friend leukemia virus (FLV) by receptor interference. This unique resistance mechanism against retroviral infection might provide a possible therapeutic model system of human retroviral infection such as AIDS. To further investigate the Fv-4r gene action in vivo, we examined the distribution and character of Fv-4r env antigen in serum and systemic organs from C4W mice. The Fv-4r env antigen was immunohistochemically localized to the lympho-hematopoietic cells and exocrine glandular cells, such as those of the salivary gland and pancreas. Using immunoprecipitation followed by Western blotting, we determined two types of gp70-related Fv-4r env antigen in the serum of C4W mice, showing molecular weights of either 70-75 kDa and 80-85 kDa. When thymocytes from Fv-4 susceptible gene (Fv-4r)-bearing C3H mouse were mixed with C4W mouse serum, the 70-75k Da molecule of the C4W serum dominantly bound to C3H thymocytes and thus contributed to receptor interference function. Using immunoelectron microscopy, Fv-4r env antigen was mainly localized to the cell surface membrane of thymic lymphoid cells, while acinar cells of the salivary gland possessed Fv-4r env antigen in the endoplasmic reticulum (ER) as well as on the cell surface membrane. These data indicate that several glandular organs, as well as lymphohematopoietic organs of C4W mice, may contribute to the production of cell-free Fv-4r env antigen, resulting in protection of cells from infection with FLV by receptor interference.

Inhibition of myeloid differentiation by Hoxa9, Hoxb8, and Meis homeobox genes.

OBJECTIVE: The homeobox gene Hoxb8 is activated in the murine myelomonocytic cell line WEHI-3B as a result of intracisternal A particle integration. Cooperative activation between Hoxa9 and Meis1 is induced by retroviral integration in BXH2 murine myeloid leukemias and the myeloid leukemia cell line M1. The present study was conducted to examine possible Meis gene activation and cooperative DNA binding of homeobox proteins in WEHI-3B and to reveal the specific role of Hox and Meis genes in myeloid differentiation. MATERIALS AND METHODS: Northern blot analysis and reverse transcriptase polymerase chain reaction were performed to examine homeobox genes expression. Electrophoretic mobility shift assay was performed to evaluate DNA binding of homeobox proteins. Myeloid differentiation of 32Dcl3 was induced by granulocyte colony-stimulating factor. RESULTS: Meis2 was coactivated with Hoxb8 in WEHI-3B cells. DNA-protein complexes including Hox, Meis, and Pbx were observed in WEHI-3B and 32Dcl3. Expression and the DNA-binding complex of Hoxa9, Hoxb8, Meis1, and Meis2 were down-regulated during myeloid differentiation of 32Dcl3 cells. Enforced expression of Hox or Meis genes inhibited myeloid differentiation of 32Dcl3. CONCLUSION: The results indicate that Meis2 is an important Meis gene for myeloid leukemogenesis and that Hox and Meis are important genes for myeloid leukemogenesis through differentiation block.