Akihiro Konno

Cellular expression of murine Ym1 and Ym2, chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.

Targeted disruption of MAIL, a nuclear IκB protein, leads to severe atopic dermatitis-like disease

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IκB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor κB (IκB) ζ. In this study, we generated Mail–/– mice to investigate the roles of MAIL in whole organisms. Mail–/– mice grew normally until 4–8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail–/– mice than in normal. Histopathological analysis indicated that the Mail–/– skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail–/– skin lesions, similar to that observed in the skin of patients with AD. In Mail–/– mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail–/– mouse is a valuable new animal model for research on AD.

Altered balance of inhibitory and active Fc gamma receptors in murine autoimmune glomerulonephritis

Mag is an MRL-derived glomerulonephritis susceptibility locus that includes the Fcgr2b and Fcgr3 genes encoding the inhibitory Fc gamma receptor IIB (FcgammaRIIB) and active FcgammaRIII, respectively. We measured changes in gene balance in three B6.MRLc1 congenic mouse strains containing the 82-86, 92-100 and 100 cM regions of the MRL chromosome 1. We found that only the strain that has 92-100 (which includes Fcgr loci) developed glomerulonephritis. These congenic mice had splenomegaly, elevated blood urea nitrogen, anti-dsDNA antibodies and higher urinary albumin excretion compared to the parental strain C57BL/6(B6). Prior to the development of glomerulonephritis, large CD3- (T cell) and B220- (B cell) positive areas were identified in the spleens of B6.MRLc1(92-100) mice. Both Fc receptors were found in mesangial and dendritic cells; important sites of immune-complex clearance and antigen presentation. The FcgammaRIII-positive areas were more prominent in the congenic strain. Fcgr2b mRNA was lower in the B6.MRLc1(92-100) kidney and spleen than in those organs of the B6 mice while Fcgr3 expression and the Fcgr3 to Fcgr2b mRNA ratio was higher in the congenic strain kidneys, spleen and thymus than in those of the B6 prior to and at an early stage of glomerulonephritis. We conclude that the imbalance of inhibitory and active Fc gamma receptors influences the pathogenesis of glomerulonephritis.

Presence of B7-2+ Dendritic Cells and Expression of Th1 Cytokines in the Early Development of Sialodacryoadenitis in the IQI/Jic Mouse Model of Primary Sjögren's Syndrome

Subpopulations of infiltrating lymphocytes, professional antigen-presenting cells (APCs), and Th1/Th2 cytokines that could initiate an autoimmune sialodacryoadenitis were studied in the IQI/Jic mouse model of primary Sjögrens syndrome. Although lymphocytic infiltrations were first seen in submandibular glands (SMGs) of females and in lacrimal glands (LGs) of males at 8 weeks of age, clusters of MHC class II+, CD11c+, B7-2+ dendritic cells (DCs) were already localized in these tissues at 4 weeks. At 8 weeks, the infiltrating lymphocytes consisted of almost equal numbers of B cells and CD4+ T cells. In the inflammatory foci, MHC class II+, CD11c+, B7-2+ DCs formed network-like structures. Duct cells in the lesions showed immunoreactivities for MHC class II and ALCAM (a costimulatory adhesion molecule). IL-12 and IFN-γ transcripts were detected by RT-PCR in SMGs of females and in LGs of males at 8–12 weeks. These results suggest that the clustered DCs might play an important role in the initiation of the adenitis, and further suggest that the DCs and epithelial cells may participate in the activation of CD4+ T cells. It is also likely that Th1 cytokines mediate the functional interactions between the APCs and CD4+ T cells in the early lesions.

Expression of γδ T cell receptor on caprine globule leukocytes

Abstract Histochemical characteristics and immunological surface phenotypes of globule leukocytes (GLs) of normal goats were investigated in the intestine. In the small intestine, GLs were concentrated in the base of the villus and around the crypt, whereas in the cecum and colon they were randomly distributed. Their cytoplasmic granules exclusively stained with phosphotungstic acid hematoxylin, and were negative for peroxidase and histamine in contrast to those of subepithelial mast cells. The existence of chondroitin sulfate in some granules of GLs and heparin in most granules of mast cells were revealed by alcian blue staining and digestion with chondroitinase ABC. Isolated intestinal GLs were positive for T cell receptor (TcR) 1-N24 (γδ) and CD8α, and negative for WC1-N3 and WC1-N4. Cryostat sections of ileum revealed preferential intraepithelial distribution of both TcR1-N24 + cells and CD8 + cells. WC1-N3 + and WC1-N4 + cells were rarely seen in the epithelium and lamina propria. These results indicate that caprine GLs are a γδ T cell subset, which is a different cell population from WC1 positive γδ T cells.

Oocytes in Newborn MRL Mouse Testes

Abstract Although mammals produce either sperm or eggs depending on their sex, we found oocytes in the testes of newborn MRL/MpJ male mice. In the present study, we report the morphological characteristics of testicular oocytes, the postnatal change of oocyte number per testis, and the expression of a few oocyte-specific genes in the testes of MRL/MpJ mice. The testicular oocytes had a diameter of 50–70 μm and were surrounded by zonae pellucidae, which were observed between oocytes and follicular epithelial cells. Ultrastructurally, the testicular oocytes contained numerous microvilli and cortical granules, receiving cytoplasmic projections from follicular epithelial cells. The testicular oocytes appeared as early as at birth, and the largest number was found on Day 14. The testicular oocytes were detected in only MRL strains and B6MRLF1, but not in C57BL/6, C3H/He, BALB/c, DBA/2, A/J, and MRLB6F1. The expression of the oocyte-specific genes Zp1, Zp2, Zp3, and Omt2a was detected in testes from MRL/MpJ mice. These results suggest that newborn male MRL/MpJ mice with XY chromosomes can produce oocytes in their testes and that one of the genes causing this exists on the Y chromosome.

Tissue distribution of CD6 and CD6 ligand in cattle: expression of the CD6 ligand (CD166) in the autonomic nervous system of cattle and the human.

We studied the tissue distribution of CD6+ lymphocytes and cells expressing the CD6 ligand (also known as activated leukocyte cell adhesion molecule CD166) in calves by immunohistochemistry using an anti‐bovine CD6 monoclonal antibody (mAb), a human CD6 (huCD6)‐immunoglobulin G1 fusion protein (huCD6‐Ig), and an anti‐human CD166 (anti‐huCD166) mAb. The huCD6‐Ig and anti‐huCD166 mAb bound to the sympathetic and parasympathetic nerve fibers but not to myelinated nerve fibers in the spinal nerve. Studies with human tissue using the anti‐huCD166 mAb yielded identical patterns of labeling. Dense accumulations of CD6+ lymphocytes were present in areas of the thymuses and spleens of calves, in areas innervated by huCD6‐Ig+ nerves. The cDNAs encoding the bovine CD166 and CD6 were isolated from the sympathetic ganglion and spleen, respectively. Predicted amino acid residues that are important for human and mouse CD6‐CD166 binding were also conserved in bovine CD6 and CD166. Bovine CD166 transcripts were detected by reverse transcriptase‐PCR in all the tissues that bound huCD6‐Ig. These results show that the bovine orthologue of CD166 was constitutively expressed in the autonomic nervous systems of cattle and suggest that CD6+ lymphocytes adhere to CD166+ autonomic nerve terminals via CD6.

Hepatocyte Nuclear Factor 4 Alpha Is Related to Survival of the Condensed Mesenchyme in the Developing Mouse Kidney

Hepatocyte nuclear factor 4 alpha (Hnf4α) is a transcription factor required for embryogenesis and organogenesis. In the adult kidney, Hnf4α is expressed at a high level in proximal tubules. Although its expression begins from the embryonic period, its function in the developing kidney has remained almost unknown. In this study, we investigated the role of Hnf4α in the cultured embryonic mouse kidney by gene silencing using the RNA interference method. Additionally, we identified the dynamics of Hnf4α in the microenvironment of the developing kidney. As a result of gene silencing, the cellular organization in the condensed mesenchyme (CM) fell into disorder and many apoptotic cells appeared. In addition, laser microdissection‐reverse transcriptase‐polymerase chain reaction provided evidence that Hnf4α gene expression began first in the CM. These results suggest the possibility that Hnf4α plays an important role in the regulation of the cell survival at the CM stage in nephrogenesis. Developmental Dynamics 239:1145–1154, 2010.

Ovarian cysts in MRL/MpJ mice originate from rete ovarii.

In MRL mice aged more than 1 year, but not in C57BL/6 mice, ovaries had grossly visible cysts presenting unilaterally or bilaterally. Postnatally, all MRL mice developed ovarian cysts by 8 months of age. Observations by light microscopy, including lectin histochemistry, indicated that the cysts sometimes included papillomatous tissues located at the hilar region and were similar to the rete ovarii system, but not to follicles. Two types of epithelial cells, ciliated and non‐ciliated, were arranged on the cysts, in which both cell types had many microvilli projecting in various directions and random ramifications in the cystic lumen. These characteristics suggest that ovarian cysts developing in MRL mice originate mostly from the rete ovarii. Cysts derived from the rete ovarii at 8 months of age were histologically detected in all C3H mice as well as MRL mice, with variable incidence in ICR, AKR, CBA/N and ddY, and none in C57L/6, DBA/2, BALB and A/J mice. However, measurement of the maximum diameters of the ovarian cysts indicated that MRL mice regularly possessed the largest cysts visible to the naked eye. This is the first report of ovarian cysts in this inbred strain, suggesting that ovarian cysts in MRL mice appear with stable incidence and development.

Expression of Hepatocyte Nuclear Factor 4α in Developing Mice

Hepatocyte nuclear factor (HNF) 4α, a transcription factor of the nuclear hormone receptor family, is generally expressed in some endoderm‐derived epithelial tissues such as hepatocytes. In mice, an alternative promoter referred to as the P2 promoter is located upstream from the P1 promoter, resulting in the transcription of at least nine isoforms. In this study, we investigated the expression of Hnf4α in adult and embryonic mouse tissues, paying special attention to the developing metanephros by using immunohistochemistry and reverse transcriptase‐polymerase chain reaction for the detection of P1 and/or P2 promoter‐derived products. In adult mouse tissues, the kidney was the only organ expressing Hnf4α controlled only by the P1 promoter, and HNF4α was detected in the nuclei of epithelial cells in the proximal tubules, but not in other components of the nephron. In the metanephros, HNF4α was detected first at the epithelial cell nuclei in part of the comma‐shaped body, distributed widely throughout the developing nephron and finally restricted to the proximal tubules. Interestingly, it was noted that Hnf4α mRNAs from stomach, pancreas and kidney tissues in embryonic periods were transcribed by both promoters. Immunohistochemistry for HNF4α and HNF1α revealed that both factors involved the same network of transcription factors, giving the impression that HNF4α was upstream of HNF1α.