Takayuki Nakazawa

Theophylline accelerates human granulocyte apoptosis not via phosphodiesterase inhibition.

Theophylline, in addition to its bronchodilator effect, is reported to have an antiinflammatory action that may account for its clinical effectiveness in the reduction of inflammatory cells in the airway. In bronchial asthma, such inflammatory cytokines as GM-CSF and IL-5 are upregulated and have been proposed to cause granulocyte infiltration (neutrophils and eosinophils) in the airway by inhibition of granulocyte apoptosis. We examined the abilities of theophylline to counteract the prolongation of human granulocyte survival caused by cytokines. Theophylline was shown to shorten granulocyte survival in a dose-dependent manner. Upon incubation with a therapeutical concentration of theophylline (0.1 mM; 18 microg/ml), percentages of GM-CSF (10 ng/ml)-induced delayed apoptosis increased from 18+/-2% to 38+/-3% (p < 0.02) in neutrophils and from 21+/-2% to 35+/-2% (p < 0.02; 24-h incubation) in eosinophils. The percentage of IL-5 (5 ng/ml)-induced delayed eosinophil apoptosis also increased from 22+/-4% to 33+/-2% (P < 0. 05). In contrast, cyclic AMP (cAMP)-increasing agents (3-isobutylmethylxanthine, dibutyryl cAMP, and rolipram) inhibited granulocyte apoptosis in the control and anti-Fas antibody-treated cells. In eosinophils, the expression of bcl-2 protein decreased after incubation with theophylline. These findings suggest that theophylline accelerates granulocyte apoptosis, which may play an essential role in inflammation, and controls granulocyte longevity regardless of the elevation of intracellular cAMP levels.

Theophylline induces neutrophil apoptosis through adenosine A2A receptor antagonism.

This study was designed to determine whether theophylline would augment granulocyte apoptosis via a mechanism of adenosine A2A receptor antagonism. A selective adenosine A2 receptor agonist (CGS‐21680, 1 μM) exhibited the most efficient potency for decreasing neutrophil apoptosis for 16 h from 63 ± 5 to 19 ± 4% (P < 0.001); it exerted poor and adverse effects on eosinophil survival. A selective protein kinase A inhibitor KT‐5720 (10 μM) reversed the capacity of dibu‐tyryl cAMP but not CGS‐21680 to induce an inhibitory effect on neutrophil apoptosis, suggesting that occupancy of adenosine A2 receptors inhibit neutrophil apoptosis by a cAMP‐independent mechanism. Theophylline derivatives show the following pattern of potency for inducing neutrophil apoptosis competing with CGS‐21680: 8‐phenyltheophylline = 8‐p‐sulfophenyltheophylline > theophylline ≫ enprofylline. This pattern is consistent with the affinity established for A2A receptors. Theophylline demonstrated an additive effect to that of anti‐Fas antibody (CH11, 1 μg/mL) in inducing neutrophil apoptosis, but not to that of adenosine deaminase or KF‐17837 (a selective A2 receptor antagonist; 1 μM), suggesting conflicting effects on the receptor antagonism. These findings suggest that theophylline has an immunomodulatory action on neutrophil apoptosis via a mechanism of A2A antagonism. J. Leukoc. Biol. 67:529–535; 2000.

CD27/CD70 interaction directly induces natural killer cell killing activity.

The CD27 molecule is expressed on a portion of natural killer (NK) cells as well as T and B cells. To provide the functional capacity of CD27 molecule on NK cells, we here highly purified CD3− CD56+ NK cells by flow cytometry (purity >98%), and investigated their NK cell activity via CD27/CD70 interaction using a CD70‐transfectant by a 4h 51Cr‐release assay. The enhancement of NK activity by purified NK cells in the presence of interleukin‐2 (IL‐2) or interleukin‐12 (IL‐12) against CD70/Nalm‐6 was not recognized as compared to against mock/Nalm‐6. However, after a coculture with the fixed CD70/300‐19, the purified NK cells increased the NK cell activity against K562, the value being 10 to 20% higher than coculture with the mock/300‐19 in the presence of IL‐2 or IL‐12. The enhancement of NK activity was blocked by the addition of anti‐CD70 monoclonal antibody (mAb). In addition, conjugation of NK cells to the target was increased by coculture with the CD70/300‐19 without increased expression of adhesion molecules. In the parallel experiment, there was no increase in the killing capacity of NK cells. These results strongly show that CD27/CD70 interaction directly enhances NK activity in the presence of IL‐2 or IL‐12 by increasing the effector and target conjugate formation, indicating that CD27/CD70 interaction plays an important role in the cytotoxic function of NK cells.

Later development of Fas ligand-mediated cytotoxicity as compared with granule-mediated cytotoxicity during the maturation of natural killer cells.

We classified CD56+ CD3− natural killer (NK) cells into CD2− CD56dim (CD2− NK), CD2+ CD56dim (CD2+ NK) and CD2+ CD56bright populations, and investigated mainly functional differences between the former two populations. CD2− and CD2+ NK cells were the same in their morphology and several surface molecules except for CD2. The percentages of CD2− NK cells in total NK cells were higher in the cord blood and bone marrow than in the peripheral blood of adults or children. Freshly isolated CD2− NK cells had CD2 in the cytoplasm, and gradually expressed it on the surface upon incubation with interleukin‐2 (IL‐2). These results demonstrated that CD2 is an antigen which appears on the surface during the maturation of NK cells. The granule‐mediated cytotoxicities, which are mainly performed by the perforin molecule, of CD2+ NK cells against K562 and Daudi cells were higher than those of CD2− NK cells, and they were inhibited to the levels of CD2− NK cells by the addition of a blocking anti‐CD2 monoclonal antibody (mAb). Fas ligand (FasL) mRNA was expressed in freshly isolated CD2+ NK cells but not in the CD2− NK cells. Neither freshly isolated NK populations showed FasL‐mediated cytotoxicity, and only CD2+ NK cells lysed Fas‐transfected targets after the 24‐hr incubation with IL‐2. Based on these results, CD2− NK cells have already developed granule‐mediated cytotoxicity equal to that of CD2+ NK cells except for the CD2‐associated activity, but they, unlike CD2+ NK cells, totally lack FasL‐mediated cytotoxicity. These findings suggest that FasL‐mediated cytotoxicity may be acquired at more mature stages of NK‐cell maturation than granule‐mediated cytotoxicity.

Presenility of granulocytes in Down syndrome individuals

Neutrophil function defects occur in individuals with Down syndrome (DS). We examined apoptosis of granulocytes (neutrophils and eosinophils) in DS individuals and control healthy subjects. Granulocyte survival was shortened in DS individuals, and the percentage of apoptotic granulocytes from DS during incubation was significantly higher than that from healthy subjects. The difference was time-dependent, and that between DS and healthy subjects was nearly 30% after longer periods of incubation. In control granulocytes, both granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interleukin-5 (5 ng/ml) counteracted the programmed cell death and delayed the apoptosis caused by anti-Fas antibodies, whereas those inflammatory cytokines were not able to completely prevent cellular apoptosis in DS patients. Apoptosis and functional impairment of granulocytes may contribute to the risk of infections underlying pathological conditions of DS, and accelerated apoptosis of granulocytes may be a factor to prevent chronic airway inflammation and bronchial asthma in DS individuals.

Abnormal proliferation of CD4− CD8+ γδ+ T cells with chromosome 6 anomaly: Role of fas ligand expression in spontaneous regression of the cells

We report a case of granular lymphocyte proliferative disorder accompanied with hemolytic anemia and neutropenia. Phenotypes of the cells were T cell receptor γδ+ CD3+ CD4− CD8+ CD16+ CD56− CD57−. Southern blot analysis of T cell receptor β and γ chains demonstrated rearranged bands in both. Chromosomal analysis after IL‐2 stimulation showed deletion of chromosome 6. Sorted γδ+ T cells showed an increase in Fas ligand expression compared with the levels in sorted αβ+ T cells. The expression of Fas ligand on these γδ+ T cells increased after IL‐2 stimulation. The patients anemia improved along with a decrease in granular lymphocyte count and disappearance of the abnormal karyotype without treatment. The expression of Fas ligand may be involved in spontaneous regression of granular lymphocyte proliferation with hemolytic anemia. Am. J. Hematol. 60:305–308, 1999.

Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells

It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.

Cytolytic mechanisms involved in non‐MHC‐restricted cytotoxicity in Chediak‐Higashi syndrome

To determine the mechanisms responsible for the impaired lymphocyte‐mediated cytotoxicity in Chediak‐Higashi syndrome (CHS), we investigated the killing ability of peripheral blood lymphocytes (PBL) from three patients with CHS using several kinds of target cells that were sensitive to perforin, Fas ligand (FasL), and/or tumour necrosis factor‐alpha (TNF‐α). Freshly isolated CHS PBL did not kill K562 target cells, killing of which by normal PBL was perforin‐dependent, as demonstrated by complete inhibition by concanamycin A (CMA), an inhibitor of perforin‐based cytotoxicity. In contrast, the CHS PBL exhibited substantial cytotoxicity against Jurkat cells, which was only partially inhibited by CMA treatment but not by the addition of neutralizing anti‐FasL or anti‐TNF‐α antibodies. IL‐2‐activated CHS PBL exhibited substantial levels of cytotoxicity against K562 and Jurkat cells, the levels being 74% and 83% of the respective normal control values, respectively. CMA treatment showed that while the cytotoxicity of IL‐2‐activated CHS PBL against K562 was largely dependent on perforin, that against Jurkat was largely not. IL‐2‐activated CHS PBL expressed FasL mRNA, and killed Fas transfectants. These findings indicate that CHS PBL have an ability to kill some target cells via a perforin‐mediated pathway, especially when they are activated by IL‐2. It was also demonstrated that CHS PBL can exert cytotoxicity against certain target cells by utilizing FasL and an undefined effector molecule other than perforin, FasL, or TNF‐α.

Beneficial effect of granulocyte colony-stimulating factor in an infant with Pasteurella multocida brain abscess.

Sir: A 2-month-old boy was hospitalized with fever and irritability on 9 October 1990. Six weeks earlier, he had been scratched on the anterior fontanelle by a domestic cat. Computed tomography (CT) scans revealed a large multilocular abscess in the left anterior part of the brain which communicated with the left lateral ventricle. Cultures of CSF grew Paswurella multocida, which was also isolated f rom the oral cavity of the family cat. Intravenous injection of large doses of antibiotics and continuous irrigation of the abscess cavity with antibiotics resulted in a temporary improvement in the CSF findings, fol lowed by a further exacerbat ion (cell count in CSF, 7300/gl) and by absence of increase in the neutrophit count (1600/111), As presented in Fig. 1, random migration and chemotaxis of the patient s neutrophils showed lower levels as compared with those of age-matched controls, but their 0 2 production did not [1, 2]. Accordingly, on 27 October , we injected