Kishiko Nakamura

Nicotine induces human neutrophils to produce IL‐8 through the generation of peroxynitrite and subsequent activation of NF‐κB

Leukocytosis in tobacco smokers has been well recognized; however, the exact cause has not been elucidated. To test the hypothesis that tobacco nicotine stimulates neutrophils in the respiratory tract to produce IL‐8, which causes neutrophilia in vivo, we examined whether nicotine induces neutrophil‐IL‐8 production in vitro; the causative role of NF‐κB in its production, in association with the possible production of reactive oxygen intermediates that activate NF‐κB; and the nicotinic acetylcholine receptors (nAChRs) involved in IL‐8 production. Nicotine stimulated neutrophils to produce IL‐8 in both time‐ and concentration‐dependent manners with a 50% effective concentration of 1.89 mM. A degradation of IκB‐α/β proteins and an activity of NF‐κB p65 and p50 were enhanced following nicotine treatment. The synthesis of superoxide and the oxidation of dihydrorhodamine 123 (DHR) were also enhanced. The NOS inhibitor, nω‐Nitro‐l‐arginine methyl ester, prevented nicotine‐induced IL‐8 production, with an entire abrogation of DHR oxidation, IκB degradation, and NF‐κB activity. Neutrophils spontaneously produced NO whose production was not increased, but rather decreased by nicotine stimulation, suggesting that superoxide, produced by nicotine, generates peroxynitrite by reacting with preformed NO, which enhances the NF‐κB activity, thereby producing IL‐8. The nAChRs seemed to be involved in IL‐8 production. In smokers, blood IL‐8 levels were significantly higher than those in nonsmokers. In conclusion, nicotine stimulates neutrophil‐IL‐8 production via nAChR by generating peroxynitrite and subsequent NF‐κB activation, and the IL‐8 appears to contribute to leukocytosis in tobacco smokers.

Liver damage in patients with colony-stimulating factor-producing tumors

PURPOSE We have demonstrated that colony-stimulating factor (CSF)-producing tumor cell lines produce not only CSF but also interleukin-1 (IL-1) and interleukin-6 (IL-6). Clinically, we have observed that patients bearing such tumors present with liver dysfunction and fever in addition to marked leukocytosis. The purpose of this study was to determine whether or not the liver damage was specifically related to CSF-producing tumors. PATIENTS AND METHODS Clinicopathologic examinations were performed in six autopsied patients with CSF-producing tumors. We also transplanted two tumor cell lines (KHC287 and CHU-2), which produce granulocyte (G)-CSF, IL-1, and IL-6, to nude mice. RESULTS Of the six patients, five had G-CSF- and one had granulocyte/macrophage (GM)-CSF-producing tumors. IL-1 and IL-6 concentrations in plasma or culture supernatant were elevated in these patients. Biochemical examinations revealed high serum enzyme levels of the biliary system in contrast to normal or slight increases in transaminase levels in all patients studied. Serum direct bilirubin was elevated in five of the six patients. Three common pathologic changes of the liver were found: (1) focal necrosis associated with neutrophil infiltration in the centrilobular zones, (2) fibrous change and enlargement of the portal area associated with neutrophil infiltration, and (3) intrahepatic cholestasis. The same pathologic changes, except for cholestasis, were reproduced in the liver of mice transplanted with KHC287 or CHU-2. CONCLUSION These results indicate that patients with CSF-producing tumors have characteristic liver damage, and suggest a new paraneoplastic syndrome of leukocytosis and liver damage.

Expression of the erythropoietin receptor on a human myeloma cell line.

We demonstrated the expression of the erythropoietin (EPO) receptor by a human myeloma cell line (MM-S1) which was established in our laboratory. EPO dose-dependently stimulated the proliferation of MM-S1 cells. Binding of radioiodinated EPO (125I-Epo) to MM-S1 cells was competitively inhibited by unlabeled EPO, but not by other recombinant cytokines. Specific binding of 125I-Epo to MM-S1 cells was saturable, and the Scatchard analysis revealed 330 EPO binding sites per cell with a Kd of 0.56 nmol/L. Bound EPO was internalized by MM-S1 cells during incubation at 37 degrees C. This is the first report describing the expression of the EPO receptor by human cells other than those of the erythroid lineage.

IL-1 production as a regulator of G-CSF and IL-6 production in CSF-producing cell lines.

We previously demonstrated that colony stimulating factor (CSF)-producing cell lines co-produce interleukin-1 (IL-1) and IL-6 in addition to CSFs. In the present study, we examined the role of IL-1 production in three human tumour cell lines producing granulocyte (G)-CSF, IL-1 and IL-6. Addition of anti-human IL-1 alpha antiserum to the culture caused a 90-62% reduction of G-CSF and a 85-44% reduction of IL-6 production, respectively, as evaluated by enzyme immunoassay in all three cell lines. The decrease of G-CSF and IL-6 production by the anti-IL-1 alpha antiserum was also confirmed at the level of mRNA expression. The anti-IL-1 alpha antiserum did not affect the growth of these cell lines. Excess recombinant IL-1 alpha exogenously added to the culture enhanced G-CSF and IL-6 production in all three cell lines. However, IL-1 alpha had little effect on the growth of these three cell lines. Neither anti-IL-6 nor anti-G-CSF antibodies affected the production of the other cytokines. These results indicate that IL-1 alpha regulates G-CSF and IL-6 production in these tumour cell lines, and suggest that the IL-1 production plays an important role in CSF-producing tumours.

Colony-stimulating factor-producing tumours: production of granulocyte colony-stimulating factor and interleukin-6 is secondary to interleukin-1 production.

We have reported that tumours producing colony-stimulating factor (CSF) also secrete interleukin-1 (IL-1) and IL-6. In the present study, we characterised the role played by IL-1 in CSF production. IL-1 receptor antagonist inhibited the production of granulocyte (G)-CSF and IL-6 by approximately 90% in CSF-producing human lung carcinoma cell lines. Similar results were obtained with hydrocortisone, which suppresses IL-1 gene expression. In contrast, 15 non-CSF-producing human lung carcinoma cell lines did not show detectable IL-1 production, although seven of them were induced to produce G-CSF and IL-6 by exogenous IL-1 alpha. Cell lines that responded to IL-1 alpha, including the CSF-producing lines, expressed receptors for IL-1 alpha. These results indicate that CSF-producing tumours can be characterised by their constitutive IL-1 production, IL-1 receptor expression and IL-1-dependent excess production of G-CSF and IL-6. Furthermore, transcription factor(s) may be involved in the abnormal IL-1 alpha production.

Megakaryocyte potentiating activity of IL‐1, IL‐6 and GM‐CSF as evaluated by their action on in vitro human megakaryocytic colonies

Summary We examined whether recombinant cytokines enhance the in vitro platelet production of interleukin‐3 (IL‐3)‐induced human megakaryocytic colonies (Meg‐colony). We classified Meg‐colonies into four categories based on platelet production during in situ observation on day 14: type 0, absence of cytoplasmic processes in a colony; type 1, one to three processes in at least one megakaryocyte in a colony; type 2, four to eight processes; type 3, more than nine processes or division of cytoplasm. Type 3 colonies were considered to be platelet‐producing. In control cultures, type 1 Meg‐colonies were dominant, followed by type 2, type 3 and type 0. Of the cytokines added at the initiation of culture, interleukin‐1 alpha (IL‐1α), interleukin‐6 (IL‐6), and granulocyte/macrophage colony stimulating factor (GM‐CSF) significantly increased the number of colonies. Furthermore, these three cytokines significantly elevated the proportion of type 3 colonies. Interleukin‐4 (IL‐4), granulocyte‐CSF, macrophage‐CSF and erythropoietin did not affect the colony count or distribution of colony type. IL‐1α. IL‐6 and GM‐CSF also significantly elevated the proportion of type 3 colonies, even when added to the culture on days 8 or 11. These results indicate that IL‐1α, IL‐6 and GM‐CSF promote platelet production of in vitro Meg‐colonies.

Establishment of an erythroid cell line (JK‐1) that spontaneously differentiates to red cells

The authors established a new hemopoietic cell line (JK‐1) from a patient with chronic myelogenous leukemia in erythroid crisis. This JK‐1 line predominantly consists of immature cells, but a small number of mature erythroblasts and red cells can be consistently seen without any specific differentiation inducer. The JK‐1 cells grow in suspension culture supplemented with human plasma and carry double Philadelphia chromosomes. Hemoglobin staining with benzidine was positive for about 20% of cells and the type of the hemoglobin was for the most part HbF. Surface‐marker analysis revealed JK‐1 cells positive for glycophorin A, EP‐1, and HAE9. The proportion of mature cells was elevated by the addition of δ‐aminolevulinic acid. Erythropoietin (EPO) enhanced the growth of JK‐1 cells either in the suspension or in methylcellulose semisolid culture. The total number of EPO receptors was 940 per cell, of which 220 sites had an affinity higher than the other 720 sites. This is the first report of an established human erythroid cell line which spontaneously undergoes terminal differentiation.

Differentiation of Myeloid Cells and 1, 25-Dihydroxyvitamin D3

It is well established that 1,25(OH)2D3 induces monocyte/macrophage (Mo/Mphi) colonies when added to culture of granulocyte/macrophage progenitors. Recently, we demonstrated that one of the target cells of 1,25(OH)2D3 in Mo/Mphi differentiation is the neutrophilic promyelocyte that is believed to belong to the neutrophilic lineage. This fact overthrows the established theory that normal hematopoietic precursors are committed to respective cell lineages and do not deviate from their own lineage. The lineage switching from the promyelocyte to Mo/Mphi was suggested to be operating in vivo because 1,25(OH)2D3 is a physiological substance produced by Mphi. More recently, we have shown that transient exposure (24 h) of promyelocytes to 1,25(OH)2D3 causes Mo/Mphi differentiation. This strategy could be useful for examining the effects of 1,25(OH)2D3 on the growth and differentiation of normal myeloblasts and myeloid progenitor cells. Recent advances in molecular biology have enabled investigators to identify a number of genes involved in Mo/Mphi differentiation induced by 1,25(OH)2D3. Some of these may be the determinant genes for Mo/Mphi differentiation; however, further studies are required to determine the underlying mechanisms of Mo/Mphi differentiation.

Multiple Recurrence of Acute Idiopathic Thrombocytopenic Purpura

Takayuki Takahashi, MD, Second Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606 (Japan) Acute idiopathic thrombocytopenic purpura (acute ITP) is characterized by the sudden onset of severe throm-bocytopenia following an infection which is usually of viral origin, and spontaneous resolution of the thrombocytope-nia is the general rule. The recovery of the disease is usually permanent, but multiple recurrences have been reported in some exceptional patients [1-3]. A 41-year-old man presented to our hospital with generalized petechial hemorrhages on March 28, 1984. Two days earlier, he had developed a common cold with symptoms of sore throat, dry cough, and a temperature of 38.2 °C. He had not taken any medications prior to the onset of hemorrhage. Physical examination showed a typical feature of common cold. Laboratory examinations revealed a platelet count of 20 × 109/1 and a white cell count of 10 × 109/1 (61% neutrophils, 7% monocytes, 4% eosino-phils, 26% lymphocytes, and 2% atypical lymphocytes). The hemoglobin concentration was 16.3 g/dl. Examination of the coagulation system and serum biochemistry gave nonspecific results. The C-reactive protein level was slightly elevated to 1.4 mg/dl (normal: < 0.3 mg). Serologi-cal tests for autoimmune disorders and viral infections were nonspecific. The patient was not given any medications, and 1 week later hematological examination showed complete recovery of the platelet count to 453 × 109/1. The subsequent clinical course of this patient is displayed in figures 1 and 2. Bone marrow aspirates obtained at the second and third attacks of thrombocytopenia showed a moderate reduction of megakaryocyte number without any other abnormalities, whereas that obtained during sustained remission of the disease revealed normal megaka-ryocytopoiesis. At the third attack of thrombocytopenia, oral prednisolone therapy was started because of severe bleeding tendency, and was continued until 1989, when the platelet count remained within normal limits. At the last attack of thrombocytopenia in 1986, the platelet-associated IgG level was 210 ng/l07 platelets (normal range: 9-25 ng) as measured by ELISA [4], whereas the level was 12.0 ng/l07 platelets in November 1989 when the platelet count was normal.