Hideo Enokihara

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.

Eosinophilia in myelodysplastic syndrome with a (12;21)(q23;q22) translocation

Cytogenetic analysis of bone marrow (BM) cells from a patient with myelodysplastic syndrome (MDS) associated with eosinophilia showed a 45,XY,t(12;21)(q23;q22), -17 karyotype. We performed clonal and suspension cultures using the patients BM mononuclear cells to clarify the mechanism of eosinophilia. Eosinophil colonies formed in the presence of interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF), but not in the presence of IL-3. When BM cells were cultured in suspension in the presence of IL-5, they differentiated to mature eosinophils, and chromosome analysis identified the 45,XY,t(12;21)(q23;q22), -17 karyotype in all metaphases. The patients serum did not stimulate eosinophil proliferation or differentiation in comparison with normal serum, however, these data suggest that the abnormal clone with 45,XY,t(12;21)(q23;q22), -17 karyotype may have an increased responsiveness to IL-5 and GM-CSF, resulting in 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.

Biallelic DNA rearrangements and deletions within the BCL-6 gene in B-cell non-Hodgkin's lymphoma

Summary. Chromosomal translocations involving band 3q27 are recently described common specific cytogenetic abnormalities in B‐cell neoplasms, and the BCL‐6 gene, identified on 3q27, was shown to be disrupted and over‐expressed in lymphoma cells having these chromosomal translocations. In the present study we found rearrangements within the BCL‐6 gene in seven out of 3 5 cases with B‐cell non‐Hodgkins lymphoma (NHL). Further analysis revealed that three of these patients with BCL‐6 abnormality had multiple rearranged bands hybridized with probes from a single restriction fragment within the major translocation cluster (MTC). suggesting that independent DNA rearrangements would occur on both alleles. Additionally, Southern blot analysis indicated that three patients carry deletions encompassing the area containing the first exon of the BCL‐6 gene. Our results suggest that biallelic DNA rearrangements and deletions would occasionally occur in NHL patients with BCL‐6 abnormality.

Serum levels of major basic protein in patients with or without eosinophilia : measurement by enzyme-linked immunosorbent assay

Summary. A bone marrow proteoglycan (BMPG) has been purified which consists of the same amino acid sequence as that of pro‐MBP, and produced two anti‐BMPG mAbs. Serum levels of major basic protein (MBP), a cationic protein rich in the eosinophil granule, were measured in patients with eosinophilia or allergic diseases by an enzyme‐linked immunosorbent assay (ELISA) using these mAbs. The serum levels of MBP in patients with eosinophilia (n= 64) and in those with allergic diseases without eosinophilia (n= 32) were elevated significantly (P < 0·001 and P= 0·038, respectively). There was a weak positive correlation between the serum levels of MBP and the eosinophil counts in the patients with eosinophilia (r= 0·38). Among these patients, extremely high serum levels of MBP were found in those with hypereosinophilic syndrome (HES) and Kimuras disease. Serum levels of MBP decreased more slowly than the eosinophil counts in patients with eosinophilia when treated by glucocorticoids. We conclude that measurement of serum levels of MBP is useful in evaluating the total‐body proliferation and infiltration of eosinophils more accurately than following‐up the eosinophil counts alone.

Clonal involvement of eosinophils in therapy‐related myelodysplastic syndrome with eosinophilia, translocation t(1;7) and lung cancer

We report a therapy‐related MDS (RAEB) patient with eosinophilia, unbalanced translocation der(7)t(1;7) (q12;q22) and lung cancer. We observed no increase in cytokine levels in serum or in the conditioned medium (CM) of peripheral T cells cultured with or without IL‐2. When bone marrow (BM) cells were cultured with GM‐CSF, IL‐3 and SCF in a semisolid system, the colonies were exclusively eosinophilic. Cytogenetic analysis of the colony cells identified the same chromosome abnormality in all metaphases to that of BM cells. Suspension and clonogenic colony assay of BM cells cultured with various cytokines showed predominant eosinophilic growth and differentiation with GM‐CSF, but not with the other cytokines examined. These findings, together with mild morphological abnormalities of eosinophils, indicate clonal involvement of eosinophils in the myelodysplastic syndrome (MDS) clone, and that the eosinophilia was derived from the neoplastic clone with the translocation and was not associated with the patient’s lung cancer.

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.