Immunoreactive corticotropin-releasing factor in rat plasma

Immunoreactive ACTH (I-ACTH) levels in the rat anterior pituitary and plasma, and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence (ME) and plasma were determined after adrenalectomy and in insulin-induced hypoglycemia. I-CRF was detected in plasma from normal rats (mean +/- SD, 5.6 +/- 0.9 pg/ml; n = 6). Gel filtration chromatography of I-CRF from pooled plasma of these rats revealed a single peak which eluted in the position of authentic rat CRF. I-CRF levels in ME and I-ACTH levels in anterior pituitary decreased immediately after adrenalectomy, then gradually increased to high levels 14 days after surgery. Plasma I-CRF and I-ACTH concentrations increased immediately after surgery, slightly decreased to near the control levels at 24 h, and then increased to high concentrations 14 days after surgery. Plasma and ME I-CRF levels 14 days after adrenalectomy, followed by daily dexamethasone replacement, were almost the same as control levels. In insulin-induced hypoglycemia, plasma I-ACTH and I-CRF concentrations increased and ME I-CRF content decreased at 30 and 60 min. These results suggest that plasma I-CRF levels reflect changes in hypothalamic CRF levels.     

A case of psittacosis showing a localized peripheral air-space consolidation]

A 29-year-old man was admitted with fever and anorexia. Radiographic examinations of the chest showed a localized peripheral non-segmental air-space consolidation in the right lower lobe. He had a history of exposure to parakeets, and psittacosis was diagnosed based on the elevated serum complement fixation titer against Chlamydia psittaci. The common radiographic finding of psittacosis is ground-glass attenuation radiating from the hilar areas. We report a rare case of psittacosis presenting a localized consolidation, clearly limited to the subpleural region of the lung.     

Multicentric Castleman's disease associated with renal amyloidosis and pure red cell aplasia

 A 50-year-old man was admitted suffering from severe anemia and renal dysfunction. He had been admitted for the first time at the age of 49, and was diagnosed with multicentric Castleman's disease (MCD) and secondary amyloidosis. At that time, marked erythroid hypoplasia was demonstrated by both aspiration and biopsy of bone marrow. A diagnosis of pure red-cell aplasia (PRCA) was made. Immunosuppressive agents improved his symptoms and laboratory data. We report here a very rare case of PRCA following MCD and amyloidosis, and with reference to the literature, we discuss the relation between MCD and related diseases. Received: February 12, 1998 / Accepted: June 17, 1998     

CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for their maintenance, proliferation, and differentiation. The BM niche is composed of nonhematopoietic and mature hematopoietic cells, including megakaryocytes (Mks). Thrombopoietin (Thpo) is a crucial cytokine produced by BM niche cells. However, the cellular source of Thpo, upon which HSCs primarily depend, is unclear. Moreover, no specific molecular pathway for the regulation of Thpo production in the BM has been identified. Here, we demonstrate that the membrane protein C-type lectin-like receptor-2 (CLEC-2) mediates the production of Thpo and other factors in Mks. Mice conditionally deleted for CLEC-2 in Mks (Clec2^MkΔ/Δ) produced lower levels of Thpo in Mks. CLEC-2–deficient Mks showed down-regulation of CLEC-2–related signaling molecules Syk, Lcp2, and Plcg2. Knockdown of these molecules in cultured Mks decreased expression of Thpo. Clec2^MkΔ/Δ mice exhibited reduced BM HSC quiescence and repopulation potential, along with extramedullary hematopoiesis. The low level of Thpo production may account for the decline in HSC potential in Clec2^MkΔ/Δ mice, as administration of recombinant Thpo to Clec2^MkΔ/Δ mice restored stem cell potential. Our study identifies CLEC-2 signaling as a novel molecular mechanism mediating the production of Thpo and other factors for the maintenance of HSCs.Maintenance of hematopoietic stem cells (HSCs) within the adult BM is crucial for the healthy production of hematopoietic cells (Orkin and Zon, 2008). HSCs reside in a specialized microenvironment in the BM called the niche (Schofield, 1978). Along with cell-intrinsic programs, the niche influences the cell fate of HSCs, which in turn govern the homeostasis of the hematopoietic system (Nakamura-Ishizu et al., 2014a). The HSC niche is chiefly composed of nonhematopoietic cells, including immature osteoblasts (OBLs; Arai and Suda, 2007), endothelial cells (ECs; Butler et al., 2010; Ding et al., 2012), perivascular cells (Sugiyama et al., 2006; Ding et al., 2012), mesenchymal stem cells (MSCs; Méndez-Ferrer et al., 2010), sympathetic nervous cells (Katayama et al., 2006), adipocytes (Naveiras et al., 2009), and nonmyelinating Schwann cells (Yamazaki et al., 2011). Nonetheless, mature hematopoietic cells such as macrophages/monocytes (Chow et al., 2011), osteoclasts (Kollet et al., 2006), and regulatory T cells (Fujisaki et al., 2011) also regulate HSCs, albeit mainly in an indirect manner, through the modulation of nonhematopoietic niche cells. Recently, mature megakaryocytes (Mks) were described as hematopoietic progeny that directly regulate HSC quiescence (Heazlewood et al., 2013; Bruns et al., 2014; Zhao et al., 2014; Nakamura-Ishizu et al., 2014b); one of the mechanisms underlying Mk niche function is the production of the cytokine thrombopoietin (Thpo) by Mks themselves (Nakamura-Ishizu et al., 2014b). However, among the Mk-related niche factors reported to date, no molecular mechanism that is specific to Mks has been identified.Thpo is a crucial cytokine for both the maturation of Mks and the maintenance of quiescent HSCs (Zucker-Franklin and Kaushansky, 1996; Qian et al., 2007; Yoshihara et al., 2007). Thpo is produced in multiple organs, including the liver, kidney, spleen, and muscle (Nomura et al., 1997). Baseline production of serum Thpo is thought to be maintained by the liver and regulated in response to inflammatory stress or changes in glycosylation of aged platelets (Kaser et al., 2001; Stone et al., 2012; Grozovsky et al., 2015). Serum Thpo levels also fluctuate according to circulating platelet number: platelets sequester Thpo via the myeloproliferative leukemia virus oncogene (c-Mpl), the receptor for Thpo (Kuter and Rosenberg, 1995; de Graaf et al., 2010), thereby lowering Thpo levels. Thus, platelet number is not as tightly regulated by Thpo production as erythrocyte number is by erythropoietin production (Fandrey and Bunn, 1993). It is likely that BM HSCs depend on Thpo, which is produced in the BM by niche cells. Depletion of circulating platelets by neuraminidase does not affect HSCs (Bruns et al., 2014), indicating that serum Thpo up-regulation through thrombocytopenia does not affect HSC maintenance. Moreover, HSCs reside near bone-lining OBLs and mature Mks, which both support HSCs by producing Thpo (Yoshihara et al., 2007; Nakamura-Ishizu et al., 2014b). However, the main cellular source of Thpo, upon which BM HSCs depend, and the molecular signaling pathway that mediates BM Thpo production remain elusive.Recent studies showed that signals mediated through C-type lectin-like domain-containing receptors (CLEC-4H1 and CLEC-4H2; also known as Ashwell–Morell receptor) stimulate Thpo production in hepatocytes through recognition of desialylated platelets (Grozovsky et al., 2015). Platelets and Mks express CLEC-2 (Suzuki-Inoue et al., 2006, 2007), which is among the top 25 genes specifically expressed on Mks (Senis et al., 2007). Activation of platelet CLEC-2 through binding to sialylated podoplanin is essential for the segregation of lymphatic and blood vessels during development (Bertozzi et al., 2010; Suzuki-Inoue et al., 2010). CLEC-2–podoplanin signaling also functions in maintenance of lymphocyte- and dendritic cell–related responses in the stroma of lymph nodes (Acton et al., 2012, 2014; Herzog et al., 2013).The significance of CLEC-2 expression on Mks in BM hematopoiesis, and whether it is involved in Thpo production in Mks, has not been previously explored. Here, we demonstrate that Mk-specific deficiency of CLEC-2 disrupts HSC quiescence and alters HSC potential as a result of defective Mk niche function. Moreover, we demonstrate that CLEC-2 signaling is involved in various molecular pathways for production of niche factors, including Thpo in Mks. Through the identification of CLEC-2, a novel Mk-specific factor, our data elucidate the organ-dependent production and function of Thpo and reinforce the idea that Mks contribute to a niche that regulates HSC quiescence.