Shimpei Furusawa

Interleukin 5 activity in sera from patients with eosinophilia

Summary. Sera from 10 patients with eosinophilia contained eosinophil colony stimulating factor (Eo‐CSF) activity. Using anti murine (m) interleukin‐5 (IL‐5) antibody, we demonstrated that this activity was mainly derived from IL‐5. Administration of prednisolone to patients decreased both Eo‐CSF activity in sera and the number of eosinophils in blood. These results extend our recent study demonstrating that T cells from eosinophilic patients produce IL‐5 with IL‐2 stimulation and may support the speculation that IL‐5 is an important factor which induces eosinophilia.

Effect of human recombinant interleukin 5 and G-CSF on eosinophil colony formation

Human recombinant (r) IL-5 was shown to have the activity to stimulate eosinophil (Eo) colony formation from human non-T, non-adherent bone marrow cells. The majority of these colonies were found to contain a small number of basophils, macrophages or neutrophils. Human rG-CSF, which alone did not stimulate Eo colony formation, showed an enhancing effect on Eo colony formation when added with IL-5. IL-5 seems to stimulate the proliferation and differentiation of CFU-Eo, while G-CSF acts on the early stage of eosinophilopoiesis.

Specific production of eosinophil colony stimulating factor from sensitized T cells from a patient with allergic eosinophilia

Summary. To explore the possibility that an eosinophil colony stimulating factor (EO‐CSF) is elaborated independently of neutrophil CSF (N‐CSF), we compared the effect on the production of EO‐CSF and N‐CSF of adding a specific antigen, an aspergillus extract, to peripheral blood leucocytes of an eosinophilic patient with allergic bronchopulmonary aspergillosis. Conditioned media prepared from the patients mononuclear (MN) and T cells were assayed for EO‐CSF and N‐CSF activities by agar culture technique, using normal human nonphagocytic MN bone marrow cells as target cells. The addition of the specific antigen to the cultures of the patients MN or T cells significantly stimulated the production of EO‐CSF, but not that of N‐CSF, while the patients non‐T cells and normal MN or T cells were not stimulated by the antigen challenge to produce either CSF. These results suggest that EO‐CSF is a factor distinct from N‐CSF, that its production is dependent on the presence of sensitized T cells with antigen‐specific stimulation, and that it might be one of the causes of blood eosinophilia in this patient.

Interleukin 2 stimulates the T‐cells from patients with eosinophilia to produce CFU‐Eo growth stimulating factor

To explore the mechanism of eosinophilopoiesis in patients with reactive eosinophilia, we studied the effect of interleukin 2 (IL‐2) on the production of CSFs, especially CFU‐eosinophil growth stimulating factor (CFU‐Eo GSF) from T‐lymphocytes in patients with reactive eosinophilia. Conditioned media (CM) prepared from patients’E rosette forming cells (ERFC) with or without IL‐2 was assayed for CFU‐Eo, CFU‐monocyte, macrophage (CFU‐M) and CFU‐neutrophil (CFU‐N) GSF. The addition of IL‐2 to the ERFC significantly stimulated the production of CFU‐Eo and CFU‐M GSF while only CFU‐M GSF increased in normals. Serial testing of the CFU‐Eo GSF in ERFC‐CM demonstrated that the ability of ERFC to produce CFU‐Eo GSF with IL‐2 stimulation was retained even when the eosinophilia had disappeared. These results suggest that CFU‐Eo GSF is produced from T‐cells with IL‐2 stimulation and that the T‐cells from patients produce CFU‐Eo GSF with IL‐2 stimulation after the disappearance of eosinophilia.

Interaction of monocytes and T cells in the regulation of normal human megakaryocytopoiesis in vitro: role of IL‐1 and IL‐2

Summary. Autologous or allogeneic peripheral blood T cells can stimulate the human megakaryocyte progenitor cell (CFU‐Meg)‐derived colony formation in a dose‐dependent fashion in agar cultures of nonadherent (NA), T cell‐depleted (NT) bone marrow (BM) cells. Low concentrations of monocytes and T cells can collaborate in the stimulation of CFU‐Meg colony formation or in the production of megakaryocyte colony stimulating factor (Meg‐CSF) by T cells in the presence of mitogens or IL‐2. Monocytes alone can produce only negligible Meg‐CSF under any conditions. When monocyte conditioned medium (CM) was added to T cell‐stimulated NA, NT BM cell cultures, CFU‐Meg colony growth was appreciably increased compared with that stimulated by T cells alone. Dose‐dependent increase in CFU‐Meg colony growth was noted when varying concentrations of IL‐1 were added to T cell‐stimulated NA, NT cell cultures, although IL,‐1 itself could support no CFU‐Meg colony growth in the absence of T cells. These data suggest that a synergistic interaction between T cells and monocytes during the production of Meg‐CSF by T cells could be partly mediated by IL‐1. IL‐2 was found to stimulate Meg‐CSF production by T cells in the presence or absence of mitogens. IL‐2‐stimulated Meg‐CSF production by T cells was augmented by the addition of monocytes. Although IL‐2 itself had no stimulatory effect on CFU‐Meg colony growth, dramatic augmentation in the CFU‐Meg colony number was noted when IL‐2 was added to T cell‐stimulated NA, NT cell cultures. High concentrations of monocytes and prostaglandin E (PGE) inhibited the CFU‐Meg colony formation. These results suggest that IL‐1 and IL‐2 may play a stimulatory role on the normal human in vitro megakaryocytopoiesis and may be involved in the development of reactive thrombocytosis and bone marrow mega‐karyocytic hyperplasia in various inflammatory diseases.

Regulation of interleukin-5 production by interleukin-4, interferon-alpha, transforming growth factor-beta and interleukin-6.

Normal lymphocytes produce IL-5 with PHA and PMA stimulation. In this system, IL-5 production was enhanced by IL-4 and was inhibited by IFN alpha, TGF beta and IL-6.

Heterogeneity of in vitro growth pattern of megakaryocyte progenitors (CFU-M) in myeloproliferative disorders

In groups of 26 patients with myeloproliferative disorders (MPD), 8 with chronic myelogenous leukaemia (CML); 8 with polycythaemia vera (PV); 10 with essential thrombocythaemia (ET); and 6 patients with reactive thrombocytosis (RT), we studied the growth characteristics of bone marrow CFU‐M in agar culture. The bone marrows from all the patients with MPD formed so called endogenous CFU‐M colonies, in the absence of PHA‐LCM, that increased in a dose‐dependent manner with the addition of increasing concentrations of normal human AB‐citrated plasma (NH‐ABCP), while the bone marrows from all the patients with RT and from healthy controls formed few or no endogenous CFU‐M colonies. In MPD, the endogenous CFU‐M growth was enhanced by normal T cells in a dose‐dependent fashion, and was decreased with the depletion of T cells from the marrow cells. These results suggest that the formation of endogenous CFU‐M colonies is caused by hypersensitivity of CFU‐M in MPD to NH‐ABCP, which may contain a small amount of Meg‐CSF, and/or by in vitro T cell stimulation. Among MPD, the endogenous CFU‐M growth in ET was significantly lower than that of other MPD patients; however, the total number of ET CFU‐M grown in the presence of PHA‐LCM was the highest. These data show that the bone marrow CFU‐M in MPD are heterogeneous with respect to in vitro growth pattern or sensitivity to exogenous Meg‐CSF.

Inhibitory effect of PWM-stimulated OKT4+ subsets on erythro-, granulo- and megakaryocytopoiesis in vitro

Summary. Normal human peripheral blood T cells and T‐cell subsets defined by monoclonal antibodies of the OKT series were pretreated with pokeweed mitogen (PWM). Their effects on the haematopoietic precursors, erythroid (BFU‐E, CFU‐E), granulocyte‐macrophage (CFU‐GM) and megakaryocyte (CFU‐M) colony forming cells were evaluated by coculture. While unstimulated T cells and T‐cell subsets enhanced growth of autologous blood BFU‐E, PWM‐stimulated T and OKT4+ cells suppressed it, also inhibiting proliferation of both autologous and allogeneic bone marrow BFU‐E, CFU‐E, CFU‐GM and CFU‐M. PWM‐stimulated OKT8+ cells had little effect on the growth of any of the precursors at the cell concentration at which growth was completely inhibited by PWM‐stimulated OKT4+ cells. Irradiation of T or OKT4+ cells with 3000 rad before PWM stimulation completely abrogated the inhibition. These observations might be related to the mechanism of pancytopenia in some cases of immune‐mediated aplastic anaemia.

Effect of Human Recombinant Interleukin 4 on In Vitro Granulopoiesis of Human Bone Marrow Cells

Utilizing in vitro colony assay, we investigated the effect of human recombinant interleukin 4 (hIL-4) on granulopoiesis of normal human bone marrow cells. Though hIL-4 itself did not possess any colony-stimulating activity, the number of neutrophil (N) colonies, particularly the number of small colonies, supported by human recombinant G-CSF (hG-CSF) was significantly increased when hIL-4 was used as a costimulant. In contrast, the number of eosinophil (Eo) colonies supported by hIL-5 was decreased when hIL-4 was used as a costimulant. Also, the numbers of N and Eo colonies supported by hIL-3 or hGM-CSF were both significantly decreased when hIL-4 was added. These data suggest that hIL-4 has diverse positive and negative regulatory effects on human neutro- and eosinophilopoiesis as a cofactor of various CSFs.

Eosinophil Colony Formation in Acute Nonlymphoblastic Leukemia

Summary Agar culture of bone marrow cells from 52 patients with acute nonlymphoblastic leukemia showed the development of higher than normal incidence of eosinophil colonies in 5 patients. Of these, the growth pattern of one patient was of particular interest since the aggregates formed consisted almost exclusively of eosinophilis which matured by 4 weeks of culture. In this patient, the plating efficiency was extraordinarily high and the growth of eosinophil colonies was significantly delayed. In this single patient karyotypic analysis suggested that the eosinophil colonies may have been derived from a leukemic cell line. Further studies are, however, necessary to determine the nature and origin of eosinophil colonies with acute nonlymphoblastic leukemia. We would like to thank Dr. Yasukazu Tanaka, Department of Experimental Pathology, Metropolitan Institute of Gerontrogy, Tokyo, for his assistance in the preparation of the electron micrographs and for his kind comments. We would also like to thank Professor W. A. Robinson, Department of Medicine, University of Colorado, Denver, for his useful comments and suggestions.