Tetsuya Ohtsuki

Immunohistochemical identification of proteoglycan form of macrophage colony-stimulating factor on bone surface

Several studies using op/op mice have shown that macrophage colony-stimulating factor (M-CSF) was necessary for osteoclast formation in vivo. Previously we reported that osteoblastic cells produced two molecular forms of M-CSF; one is an 85-kDa M-CSF, and the other is a proteoglycan form of M-CSF (PG-M-CSF) which has a binding affinity for bone-derived collagens and is extractable from human bone. In this study, we performed immunostaining of human bone using a newly established anti-PG-M-CSF antibody, and showed positive staining PG-M-CSF, probably produced by bone lining cells, on the bone surface. This observation suggests that the bone surface is suitable for osteoclast formation because of the presence of PG-M-CSF.

A human osteoblastic cell line, MG-63, produces two molecular types of macrophage-colony-stimulating factor

A human osteoblastic cell line, MG-63, mouse primary osteoblasts, and a mouse osteoblastic cell line, MC3T3-E1, were shown to produce macrophage-colony-stimulating factor (M-CSF) by bone-marrow-cell colony assay, using a specific neutralizing antibody for M-CSF. Immunoblot analysis of M-CSF, produced by MG-63 cells, revealed the presence of a higher-molecular-weight species of M-CSF, in addition to the 85-kDa M-CSF. The higher-molecular-weight species had a high affinity to the DEAE-Sephacel column and was sensitive to chondroitinase ABC and AC. These physico-chemical profiles were wholly compatible with those of the proteoglycan form of M-CSF (PG-M-CSF), which was recently identified by our group in the conditioned medium of Chinese hamster ovary cells transfected with the 4.0-kb cDNA of the M-CSF gene. Conditioned medium of MG-63 cells was fractionated by DEAE-Sephacel column chromatography, and the M-CSF of each fraction was measured by both enzyme-linked immunosorbent assay and bone-marrow-cell colony assay. The fractions eluted by 0.3-0.6 M NaCl, which were shown to contain only PG-M-CSF on immunoblot analysis, also have macrophage-colony-stimulating activity.

Quantitative analysis of the two macrophage colony-stimulating factor mRNA expressed in a human stromal cell line by reverse transcription-polymerase chain reaction (RT-PCR).

We established a quantitative analysis system for 4.0 kb and 1.6 kb macrophage colony-stimulating factor (M-CSF) mRNA, using reverse transcription-polymerase chain reaction. Using this system, we performed quantitative analysis of the two mRNAs expressed in the human stromal cell line, KM102, in the resting condition and when stimulated by various concentrations of interferon-gamma (IFN-gamma). The expression of 1.6 kb M-CSF mRNA was more efficiently stimulated by IFN-gamma than that of 4.0 kb M-CSF mRNA. The alternative splicing of a single M-CSF gene has been shown to generate several M-CSF proteins with different localization; we believe that molecular analysis of the transcription products by this system is important to better understand the physiological significance of the different species of M-CSF derived from each mRNA.

Proteoglycan form of macrophage colony-stimulating factor binds low density lipoprotein.

We recently isolated a proteoglycan form of macrophage colony-stimulating factor (PG-M-CSF) that carries a chondroitin sulfate glycosaminoglycan chain. Here, we examined the interaction of PG-M-CSF with low density lipoprotein (LDL). When LDL preincubated with PG-M-CSF was fractionated by molecular size sieving chromatography, it was eluted earlier than untreated LDL. When LDL was preincubated with chondroitin sulfate-free 85-kD M-CSF instead of PG-M-CSF, the elution profile of LDL remained unchanged, indicating specific interaction between PG-M-CSF and LDL. The level of PG-M-CSF binding in the wells of a plastic microtitration plate precoated with LDL was significant, this binding being completely abolished by pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate. The addition of exogenous chondroitin sulfate or apolipoprotein B inhibited the binding of PG-M-CSF to LDL in a dose-dependent manner, indicating that the interaction between PG-M-CSF and LDL was mediated by the binding of the chondroitin sulfate chain of PG-M-CSF to LDL apolipoprotein B. PG-M-CSF was also demonstrated in the arterial wall, and there were increased amounts of PG-M-CSF in atherosclerotic lesions. The in vitro interaction between PG-M-CSF and LDL thus appears to have physiological significance.

Two Cases of Mature B-Cell Acute Lymphocytic Leukemia with Normal Karyotype in Adults

Acute lymphocytic leukemia with mature B-cell phenotype (B-ALL) is a rare type of ALL. Although B-ALL cells commonly have a characteristic chromosomal translocation that includes 8q24 (the location of the c-myc protooncogene), the leukemic cells of the two patients reported in this paper showed normal karyotypes. This finding was confirmed by Southern blot analysis of bone marrow cells in case 1. A c-myc probe was used, and no rearrangement or amplification of c-myc expression was found; B-ALL with the translocation including 8q24 is reported to show high levels of expression of rearranged c-myc. Investigation of anti-Epstein-Barr virus (EBV) antibody revealed that both cases 1 and 2 had a past history of EBV infection. However, at least in case 1, integration of the EBV genomic DNA into the leukemic cells was not detected by the polymerase chain reaction in which specific primers for EBV were employed. The difference in pathophysiology between B-ALL with and without the 8q24 translocation is unclear, however, the prognosis of the two patients with B-ALL with the normal karyotype was as poor as that of B-ALL with the chromosomal translocation.

Granulocyte Colony-Stimulating Factor-Producing Malignant Lymphoma

A 71-year-old woman with leukocytosis was admitted for treatment of malignant lymphoma. During the clinical course, neutrophilia of unknown origin occurred in parallel with the progression of the malignant lymphoma. The supernatant of lymphoma tissue culture contained a high titer of granulocyte colony-stimulating factor (G-CSF), and lymphoma cells were positive when immunohistochemically stained by anti-G-CSF antibody. Western blot analysis and mouse colony assay of the supernatant also confirmed that the lymphoma produced G-CSF.

Tretinoin induces bone marrow collagenous fibrosis in acute promyelocytic leukaemia: new adverse, but reversible effect

In 11/13 acute promyelocytic leukaemia (APL) cases treated with tretinoin (RA) we observed RA‐induced inaspirable collagenous fibrosis of the bone marrow. To study the mechanism of RA on collagen production, we cultured a human bone marrow derived stromal cell line and an osteoblastic cell line with RA in vitro. 10−7 and 10−6 M of RA stimulated collagen production. Clinical and experimental observation may be important to understand this adverse effect of RA as it is useful in cancer chemoprevention as well as treatment for APL. This adverse effect is spontaneously reversible after stopping RA or following chemotherapy.

Human Herpesvirus-6-associated Generalized Vesiculobullous Eruptions After Allogeneic Bone Marrow Transplantation

Human herpesvirus-6 (HHV-6) is a member of the beta herpesvirus subfamily causing exanthema subitum in children. In patients having undergone allogeneic bone marrow transplantation (BMT), HHV-6 is detected in blood samples by polymerase chain reaction (PCR) at a prevalence ranging from 38 to 60% [1]. The virus causes fever, skin rash, marrow suppression, interstitial pneumonitis and encephalitis in patients receiving a bone marrow transplant [1,2]. We report a patient with HHV-6associated generalized vesiculobullous eruptions during the early phase after allogeneic BMT, successfully treated with ganciclovir. A 22-year-old man noticed ulceration of the parietal skin in October 1997. Since the skin biopsy showed infiltration of immature abnormal lymphoid cells, he received total 40 Gy of irradiation to the lesion. In April 1998, the bilateral post auricular lymph nodes became enlarged and lymph node biopsy demonstrated lymphoblastic lymphoma of precursor B-cell type, i.e. CD19, CD10, CD34, and TdT. He received an autologous peripheral blood stem cell transplant after entering complete remission. After 6 months, massive infiltration of lymphoblastic lymphoma cells in the bone marrow appeared. He entered complete remission again with the Hyper C-VAD regimen [3]. In December 1999, he was admitted to Jichi Medical School Hospital to receive an allogeneic bone marrow transplant. The conditioning regimen consisted of total body irradiation at 12 Gy in six fractions from days 29 to 27, etoposide 150 mg/m twice a day from days 26 to 24, cyclophosphamide 60 mg/kg on days 23 and 22. Graft-versus-host disease (GVHD) prophylaxis was 1.5 mg/kg of cyclosporin twice a day and short-term methotrexate on days 1, 3 and 6. Granulocyte colony-stimulating factor was administered from day +1. He received 1,000 mg/day of acyclovir from day 27 to prevent herpes virus infection and cytomegalovirus (CMV) hyperimmunoglobulin at a dose of 12.5 g every two weeks to prevent CMV infection from day +7. In January 2000, he received an allogeneic bone marrow transplant from an HLA-identical sister. The serum antibody to HHV-6 was positive both in the donor and the recipient. A total of 4:77 £ 10 mononuclear cells/kg was infused. On day +19, a macropapular-like skin rash appeared on the cheeks and the backs of the hands along with low-grade fever. On day +24, skin rash extended on the face, hands and frontal chest. Acute GVHD was suspected, although skin biopsy was not performed. He was administered prednisolone (PSL) at a dose at 1.0 mg/kg initially and subsequently at an increasing dose of 1.5 mg/kg without improvement of skin rash. On day +26, the white blood cell count was 0:9 £ 10=l and he continued to depend on platelet and red cell transfusion. Bone marrow aspiration showed engraftment of donor cells by chromosomal analysis and maturation arrest of myeloid cells. The rash extended over his whole body with blisters, in particular around the mouth (Fig. 1). Conjunctivitis and high fever were noted. We suspected that the rash was not caused by GvHD but by herpes virus infection, since peri-oral blisters and conjunctivitis are not typical in GvHD and PSL worsened