Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

Expression and tyrosine phosphorylation of Crk-associated substrate lymphocyte type (Cas-L) protein in human neutrophils.

Crk-associated substrate lymphocyte type (Cas-L) protein, also known as human enhancer of filamentation 1 (Hef1) or neural precursor cell-expressed, developmentally down-regulated gene 9 (Nedd9), belongs to the Cas family of adapter proteins, which are involved in integrin signaling. Previous reports showed that Cas-L is expressed preferentially in lymphocytes and epithelial cells. Cas-L mediates signals from integrins, T-cell receptors, B-cells receptors, and transforming growth factor beta, leading to cell movement and cell division. Here, we report the expression of Cas-L in neutrophils. Cas-L was tyrosine-phosphorylated when human neutrophils were stimulated by fMLP, tumor necrosis factor-alpha (TNF), or lipopolysaccharide. The tyrosine phosphorylation of Cas-L in fMLP- or TNF- stimulated neutrophils was further enhanced by adhesion of the cells to their substrates. Cas-L was found to be localized at focal adhesions in stimulated neutrophils based on immunofluorescence microscopy. These findings suggest that Cas-L is one of the targets of inflammatory cytokines and is also modulated by cell adhesion process in neutrophils.

Effect of normal human erythrocytes on blood rheology in microcirculation.

BACKGROUND: Effects of RBCs on blood rheology were studied using a microchannel array flow analyzer (MC-FAN). METHODS: Fluidity of four types of samples prepared from the venous blood of healthy volunteers was examined in terms of passage time through the microchannel array of MC-FAN (model KH-3): (1) physiological saline-RBC suspensions and plasma-RBC suspensions, each adjusted to a predetermined hematocrit value; (2) suspensions of glutaraldehyde-treated hardened RBCs; (3) fibrinogen-RBC and albumin-RBC suspensions; and (4) dextran-RBC suspensions. RESULTS: Hematocrit positively correlated with passage time. Both plasma and fibrinogen prolonged passage time significantly. Hardened RBCs completely obstructed the microchannel. The passage time of dextran-RBC suspensions was prolonged in a dextran molecular weight- and concentration-dependent manner and was dependent on the passage time of the solution alone. CONCLUSIONS: Blood rheology, as determined by MC-FAN, is affected not by RBC aggregation but hematocrit, RBC deformability, and the passage time of the solution.

Cyclic AMP delays neutrophil apoptosis via stabilization of Mcl-1.

Human neutrophils underwent spontaneous apoptosis, which was accompanied by degradation of Mcl-1, but not other anti-apoptotic molecules (cIAP1, cIAP2, A1, survivin and Bcl-2). Spontaneous neutrophil apoptosis and Mcl-1 degradation were prevented by cyclic AMP (cAMP) agonists (dibutyryl cAMP and prostaglandin E(1)), and the effects of cAMP agonists on neutrophils were highly resistant to cycloheximide, a protein synthesis inhibitor, although slight increase in Mcl-1 mRNA expression was induced by cAMP agonists. Proteasome inhibitors (epoxomicin and lactacystin) also prevented spontaneous neutrophil apoptosis and Mcl-1 degradation to the same extent as cAMP agonists, and no additive effect was obtained by combination of cAMP agonists and proteasome inhibitors. These findings suggest that cAMP agonists, like proteasome inhibitors, delay neutrophil apoptosis primarily via stabilization of Mcl-1.

Serine protease inhibitors inhibit superoxide release and adherence in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

Stimulation of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor-alpha (TNF) results in increased superoxide (O2-) release and adherence. O2- release and adherence are dependent on activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Possible participation of serine proteases in GM-CSF- or TNF-induced activation of human neutrophils was explored with various serine protease inhibitors, including phenylmethylsulfonyl fluoride, L-1-tosylamido-2-phenylethyl-chloromethyl ketone and N-alpha-p-tosyl-L-lysine-chloromethyl ketone. GM-CSF- or TNF-induced O2- release and adherence were inhibited in parallel by pretreatment of neutrophils with these inhibitors. On the other hand, GM-CSF- or TNF-induced phosphorylation of ERK and p38 MAPK was unaffected by these inhibitors at the concentrations effective for the inhibition of O2- release and adherence. These findings suggest that serine proteases are involved in GM-CSF- and TNF-induced O2- release and adherence in human neutrophils and that serine proteases function downstream or independently of the activation of ERK and p38 MAPK.

Activation of human neutrophils by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha: role of phosphatidylinositol 3-kinase.

Stimulation of human neutrophils with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or tumor necrosis factor alpha (TNF) resulted in phosphorylation of Akt, the potency being GM-CSF > G-CSF = TNF, which was inhibited by wortmannin. The findings indicated that phosphatidylinositol 3-kinase (PI3K) is activated by these cytokines. The possible participation of PI3K in activation of neutrophil functions induced by these cytokines was explored with PI3K inhibitors (wortmannin and LY294002). Superoxide release and adherence induced by GM-CSF or TNF were inhibited by PI3K inhibitors. Actin reorganization and morphological changes induced by G-CSF or GM-CSF were also inhibited by wortmannin, whereas these responses induced by TNF were unaffected by wortmannin. These findings suggested that PI3K is differentially involved in cytokine-mediated activation of neutrophil functions depending on the cytokines used. The results also showed that activation of extracellular signal-regulated kinase, but not p38 mitogen-activated protein kinase, induced by these cytokines is partly mediated by PI3K activation.

Enhanced neutrophil motility by granulocyte colony-stimulating factor: the role of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

The effect of granulocyte colony-stimulating factor (G-CSF) on human neutrophil motility was studied using videomicroscopy. Stimulation of neutrophils with G-CSF resulted in enhanced motility with morphological change and increased adherence. Enhanced neutrophil motility was detected within 3-5 min after G-CSF stimulation, reached a maximum at 10 min, and was sustained for approximately 35 min. The maximum migration rate was 84.4 +/- 2.9 microm/5 min. A study using the Boyden chamber method revealed that G-CSF-stimulated neutrophils exhibited random migration but not chemotaxis. Enhanced neutrophil motility and morphological change were inhibited by MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] inhibitors (PD98059 and U0126), and a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), but not by a p38 MAPK inhibitor (SB203580). These findings are consistent with the fact that G-CSF selectively activates MEK/ERK and PI3K, but not p38, in neutrophils. MEK/ERK activation was associated with G-CSF-induced redistribution of F-actin and phosphorylated myosin light chain. Enhanced neutrophil motility was observed even in the presence of neutralizing anti-CD18 antibody, which prevented cell adherence. These findings indicate that G-CSF induces human neutrophil migration via activation of MEK/ERK and PI3K.

An Alternative Approach to Atopic Dermatitis: Part II-Summary of Cases and Discussion.

In the first part of this Review, we presented case-series where Kampo treatment was introduced for those atopic dermatitis (AD) patients who had failed with conventional therapy, in an attempt to prove that there exists a definite subgroup of AD patients for whom Kampo treatment is effective. In this second part, we will first provide the summary of the results for 140 AD patients we treated in 2000. The results suggest that Kampo treatment is effective for more than half of AD patients who fail with conventional therapy. In the Discussion, we will examine the evidential basis for conventional AD therapy and discuss how Kampo treatment should be integrated into the guidelines for AD therapy. We contend that Kampo treatment should be tried before systematic immunosuppressive agents are considered. As each Kampo treatment is highly individualized, it should be regarded more as 'art' than technology, and special care should be taken to assess its efficacy in clinical trial.

Proteolytic conversion of STAT3alpha to STAT3gamma in human neutrophils: role of granule-derived serine proteases.

Four isoforms (alpha, beta, gamma, and delta) have been identified for signal transducer and activator of transcription 3 (STAT3). It has been reported that STAT3gamma, which is derived from STAT3alpha by limited proteolysis during granulocytic differentiation, is a major STAT3 isoform expressed in human neutrophils. We confirmed that STAT3gamma was a major STAT3 isoform detected in human neutrophil lysates prepared with the conventional lysis buffer. The enzymes capable of converting STAT3alpha to STAT3gamma in vitro were localized in neutrophil granule fraction and were released into the medium upon ionomycin stimulation. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, CuSO(4), and ONO-5046 (a specific inhibitor of neutrophil elastase), but not by aprotinin, leupeptin, benzamidine, and EDTA. STAT3gamma was effectively generated in vitro from STAT3alpha by limited proteolysis with human neutrophil elastase or proteinase 3 but not cathepsin G. The converting activity in neutrophil lysates was reduced by immunodepletion of elastase but not proteinase 3. Unexpectedly, STAT3gamma was undetected in the lysates of neutrophil-derived cytoplasts, which lack granules, and the cytosol fraction prepared by nitrogen cavitation. The STAT3 isoform detected in these preparations was primarily STAT3alpha. STAT3gamma was also undetected in the lysates of PMSF-pretreated neutrophils and was markedly decreased in the lysates of ionomycin-pretreated neutrophils. These findings indicate that, in contrast to the previous reports, STAT3alpha, but not STAT3gamma, is primarily expressed in human neutrophils, and STAT3gamma is rapidly generated from STAT3alpha by limited proteolysis with granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer.

An Alternative Approach to Atopic Dermatitis: Part I-Case-Series Presentation.

Atopic dermatitis (AD) is a complex disease of obscure pathogenesis. A substantial portion of AD patients treated with conventional therapy become intractable after several cycles of recurrence. Over the last 20 years we have developed an alternative approach to treat many of these patients by diet and Kampo herbal medicine. However, as our approach is highly individualized and the Kampo formulae sometimes complicated, it is not easy to provide evidence to establish usefulness of this approach. In this Review, to demonstrate the effectiveness of the method of individualized Kampo therapy, results are presented for a series of patients who had failed with conventional therapy but were treated afterwards in our institution. Based on these data, we contend that there exist a definite subgroup of AD patients in whom conventional therapy fails, but the 'Diet and Kampo' approach succeeds, to heal. Therefore, this approach should be considered seriously as a second-line treatment for AD patients. In the Discussion, we review the evidential status of the current conventional strategies for AD treatment in general, and then specifically discuss the possibility of integrating Kampo regimens into it, taking our case-series presented here as evidential basis. We emphasize that Kampo therapy for AD is more 'art' than technology, for which expertise is an essential pre-requisite.

Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases.

Stimulation of human neutrophils with tumor necrosis factor-alpha (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF. Cytokine-induced actin depolymerization was sustained for at least 30 min after stimulation. In contrast, N-formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly (within 45 s) induced an increase in the fluorescence intensity of FITC-phalloidin (actin polymerization) and morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK, and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished by PD-98059 and were unaffected by SB-203580. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils. Phosphorylated ERK and p38 MAPK were not colocalized with F-actin in neutrophils stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF induce actin depolymerization and morphological changes through activation of ERK and/or p38 MAPK and that cytokine-induced actin reorganization may be partly responsible for the inhibitory effect of these cytokines on neutrophil chemotaxis.

Hematopoietic, angiogenic and eye defects in Meis1 mutant animals.

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.