Tsunefumi Shibuya

Involvement of interferon-γ and macrophage colony-stimulating factor in pathogenesis of haemophagocytic lymphohistiocytosis in adults

Summary We investigated the role of monocyte/macrophage‐activating cytokines in pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in 21 adult patients. Sera from patients with active HLH contained extremely high levels of macrophage colony‐stimulating factor (M‐CSF) and of interferon‐γ (IFN‐γ). These levels returned to almost normal during remission. Neither interleukin‐4 nor granulocyte/macrophage colony‐stimulating factor could be detected. Active HLH sera also contained high concentrations of inflammatory monokines, such as interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Serum concentrations of soluble CD8 and soluble interleukin‐2 receptor were extremely high during active HLH, and returned to virtually normal levels during remission. Circulating CD2+ T‐cells obtained from patients with active HLH spontaneously secreted M‐CSF and IFN‐γin vitro, whereas circulating monocytes did not produce detectable levels of both M‐CSF and IFN‐γ, but produced high levels of IL‐6 and TNF‐α. These findings suggest that IFN‐γ and M‐CSF at least partly from T‐cells, such as CD8+ T‐cells, might contribute to activation of monocytes or histiocytes, resulting in the up‐regulated monokine production and haemophago‐cytosis in HLH.

Cytomegalovirus (CMV) antigenaemia for rapid diagnosis and monitoring of CMV‐associated disease after bone marrow transplantation

A technique for the rapid detection of cytomegalovirus (CMV) antigen‐positive blood leucocytes (CMV antigenaemia) was evaluated in 15 marrow transplant patients as a means of diagnosis and for monitoring CMV‐associated disease. CMV antigenaemia was determined by direct immunoperoxidase staining of leucocytes with a peroxidase‐labelled monoclonal antibody, HRP‐C7, which binds an immediate‐early antigen of human CMV.

Central nervous system involvement in adult T-cell leukemia/lymphoma

Central nervous system (CNS) involvement was reviewed in 99 patients with adult T‐cell leukemia/lymphoma (ATLL). Fifteen episodes of CNS involvement developed in ten of 99 patients (10.1%); nine had leptomeningeal involvement, whereas two developed intracerebral invasion, one developed cord involvement, and one developed both. CNS involvement was more frequent in the lymphoma type than in the other types of ATLL. Nuchal rigidity was not common (33%) and a syndrome of inappropriate secretion of antidiuretic hormone (ADH) occurred in association with CNS involvement (40%). Three episodes of marked hypoglycorrhachia also were noticed. The systemic progression of ATLL was the most common setting of CNS involvement (80%) and the major cause of death (80%). As for the acute and lymphoma types of ATLL, no significant difference was observed in survival between patients with and those without CNS involvement. These results indicate that CNS involvement is not an essential prognostic factor of ATLL and that it should be treated with systemic chemotherapy coupled with intrathecal chemotherapy. The control of systemic ATLL is important for the prophylaxis of CNS involvement.

Increased incidence of cytomegalovirus (CMV) infection and CMV-associated disease after allogeneic bone marrow transplantation from unrelated donors

Cytomegalovirus (CMV) infection and CMV-associated disease were monitored using the CMV antigenemia assay in 72 patients who received allogeneic bone marrow transplantation (BMT), and their incidences were compared between related and unrelated donor transplant patients. The incidence of CMV infection after BMT was significantly higher in patients who received transplants from HLA-matched unrelated donors than from HLA-matched sibling donors (87% vs 53%, P < 0.05). cmv-associated disease developed in 73% of unrelated and in 14% of sibling donor transplant patients (P < 0.01). the peak levels of cmv antigenemia were significantly higher in unrelated donors than in sibling donor transplant patients (16 vs 1 CMV antigen-positive cells per 50 000 WBCs, P < 0.01). the median number of cmv antigen-positive cells on first detection was also significantly higher in unrelated donor transplant patients (15 vs 1, P < 0.01). the detection of cmv antigen-positive cells preceded the development of cmv-associated disease in 18% of unrelated donor transplant patients, suggesting a lower predictive value of cmv antigenemia for subsequent cmv- associated disease in unrelated donor bmt. careful monitoring and further studies are needed for the early diagnosis and prevention of cmv-associated disease in unrelated donor bmt.

Interleukin 4 suppresses the spontaneous growth of chronic myelomonocytic leukemia cells.

We studied the effects of IL-4 on the spontaneous proliferation of chronic myelomonocytic leukemia (CMMoL) cells in vitro. IL-4 (100 U/ml) suppressed the spontaneous DNA synthesis by approximately 50% in 5 of 8 cases examined. IL-4 (100 U/ml) also inhibited the spontaneous colony formation by CMMoL cells in a methylcellulose culture by 50-97% in all of the 10 cases in which spontaneous colonies were formed. This IL-4-mediated suppression of the growth of CMMoL cells was completely abolished by the addition of anti-IL-4 neutralizing antibodies. The spontaneous CMMoL colonies were substantially suppressed by the addition of either anti-IL-6 or anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibodies to the colony assay system: the addition of both anti-IL-6 and anti-GM-CSF antibodies resulted in greater than 80% inhibition of the colony formation by CMMoL cells. On the other hand, none of anti-IL-1-beta, anti-granulocyte-CSF, anti-macrophage-CSF, or anti-tumor necrosis factor-alpha antibodies affected the CMMoL colony formation. In the supernatants from 24-h cultures of CMMoL cells, high levels of IL-6 and GM-CSF were demonstrated in 9 of 9 and 2 of 9 cases examined, respectively. IL-4 (100 U/ml) almost completely inhibited the secretion of IL-6 and GM-CSF by CMMoL cells. These observations suggest that IL-4 suppresses the spontaneous proliferation of CMMoL cells by inhibiting their production of IL-6 and/or GM-CSF, both of which could act in vitro as an autocrine growth factor for CMMoL cells.

Cytokine production by peripheral blood monocytes and T cells during haemopoietic recovery after intensive chemotherapy.

Summary We studied the production of cytokines by peripheral blood monocytes and T cells during the period of haematological recovery following intensive chemotherapy. Twelve adults with haematological malignancies received consolidation chemotherapy of complete remission. Monocytes and T cells were collected during the phase of recovery from intensive chemotherapy, and were incubated for 24 h in a culture medium with 10% FCS. Concentrations of cytokines in the culture supernatant were measured with an enzymelinked immunosorbent assay. During the recovery phase, concentrations of IL‐6, G‐CSF and IL‐1β in the culture supernatant of the collected monocytes significantly exceeded those of the monocytes obtained from normal healthy subjects. Similarly, the concentrations of GM‐CSF and IFN‐γ in the supernatant of recovery phase T cells significantly exceeded those of normal T cells. Plasma levels of these cytokines were also elevated. These data suggest that the monocytes and T cells may be activated in vivo to produce haemopoietic cytolines during haematological recovery, and that, during haematological recovery, the monocytes and T cells may be actively involved in the induction of haematopoiesis following the myelosuppression induced by chemotherapy.

Influence of transplanted dose of CD56+ cells on development of graft-versus-host disease in patients receiving G-CSF-mobilized peripheral blood progenitor cells from HLA-identical sibling donors.

Summary:We investigated effects of variations in the cellular composition of G-CSF-mobilized peripheral blood progenitor cell (G-PBPC) allografts on clinical outcomes of allogeneic PBPC transplantation. We retrospectively analyzed transplanted doses of various immunocompetent cells from 27 HLA-identical sibling donors in relation to engraftment, incidence of graft-versus-host disease (GVHD), and survival. Significant variability was documented in both absolute numbers and relative proportions of CD34+, CD2+, CD3+, CD4high+, CD4+25+, CD8high+, CD19+, CD56+, and CD56+16+ cells contained in these allografts. Stepwise Cox regression analysis revealed that the CD56+ cell dose was significantly inversely correlated with the incidence of GVHD. Thus, there was a significantly higher incidence of grade II acute GVHD in patients receiving a lower CD56+16+ cell dose (hazard ratio (HR) 0.0090; 95% confidence interval (CI), <0.00001–3.38; P=0.031), a higher incidence of chronic GVHD in those receiving allografts with a lower CD56+16+ to CD34+ ratio (HR <0.00001; 95% CI <0.00001–0.0007; P=0.0035), and a higher incidence of extensive chronic GVHD in those receiving allografts with a lower CD56+ to CD34+ ratio (HR <0.00001; 95% CI <0.00001–0.053; P=0.0083). These results suggest that CD56+ cells in G-PBPC allografts from HLA-identical sibling donors may play an important role in preventing the development of GVHD.

Fas antigen (CD95) and hematopoietic progenitor cells.

We investigated the expression of an apoptosis associated antigen (Fas) (CD95) on hematopoietic progenitor cells. Freshly isolated CD34+ cells from bone marrow did not express Fas. However, interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha) induced dose-dependent expression of both Fas mRNA and Fas protein on the surface of CD34+ cells after 48 hours of serum-free culture. TNF-alpha-induced Fas expression was mediated by p55-TNF-alpha receptor. Induced Fas was functional as it could transduce apoptotic signals in response to anti-Fas monoclonal antibody (MoAb). The Fas-defective 1pr mice are reported to have abnormally radio-resistant hematopoietic stem cells. Consequently, we also investigated the relation between Fas and ionizing radiation. Human CD34+ cells expressed Fas following low-dose ionizing radiation in a dose-dependent fashion. Fas induced on CD34+ cells mediated apoptosis in response to anti-Fas MoAb. We evaluated the expression of Fas and Bel-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured with hematopoietic growth factors for 7 days. Approximately half of the freshly isolated CD34+ cells expressed Bel-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas and rapidly lost Bel-2 expression. Furthermore apoptosis was induced in the cultured CD34+ population in response to anti-Fas MoAb. Thus, functional Fas can be induced on hematopoietic progenitor cells in vitro by negative hematopoietic regulators, ionizing radiation, as well as positive hematopoietic regulators. The Fas system is thought to play an important role at the level of hematopoietic progenitor cells in both physiologic and pathologic conditions.

Multiple autoimmune haemopoietic disorders and insidious clonal proliferation of large granular lymphocytes.

We report a patient with clonal proliferation of CD3+8+TCRαβ+ large granular lymphocytes (LGL) presenting multiple episodes of autoimmune cytopenia, including autoimmune neutropenia, idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, and pure red cell aplasia. Each disorder appeared separately or as a combination during an 11‐year clinical course. The increase of blood CD3+8+TCRαβ+ LGL was detected 6 years after the initial diagnosis of cytopenia, but the absolute number of LGL cells was always < 1.0 × 109/l. LGL cells were of monoclonal origin and had a chromosomal abnormality. LGL cells transiently responded to cyclosporine A therapy, which was also effective on all of these autoimmune cytopenias. Accordingly, an undetectable level of proliferation of a clonal LGL population could cause various autoimmune haemopoietic disorders.

Haematopoiesis in the aged as studied by in vitro colony assay

Changes occurring in human haematopoiesis with advancing age were studied using an in vitro haematopoietic colony assay in 22 elderly subjects with unexplained anaemia, and in 15 elderly and 15 young subjects without anaemia. Both elderly groups were found to have significantly lower numbers of bone marrow early erythroid‐committed progenitors (BFU‐E) than the young controls. The elderly anaemic group also showed significantly lower numbers of granulocyte/macrophage progenitors (CFU‐GM) than the young controls. However, the responses of the erythroid‐committed progenitors in the elderly groups to erythropoietin and burst‐promoting activity were similar to those observed in the young controls. Therefore, it is probable that anaemia tends to occur easily in elderly individuals as a result of reduction of reserves of haematopoietic progenitors with advancing age.