Fumihiko Hato

Selective Activation of p38 Mitogen-Activated Protein Kinase Cascade in Human Neutrophils Stimulated by IL-1β

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1β. IL-1β induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1β for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1β. IL-1β primed neutrophils for enhanced release of superoxide (O2−) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1β also induced O2− release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1β-induced O2− release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1β and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1β induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1β and activation of this cascade mediates IL-1β-induced O2− release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.

Activation of Human Neutrophil by Cytokine-Activated Endothelial Cells

Abstract— Cytokine activation of vascular endothelial cells renders the hyperadhesiveness for neutrophils. During the processes of inflammation and atherosclerosis, the production of reactive oxygen species by neutrophils contributes to endothelial cell (EC) damage and injury. However, the precise mechanisms for neutrophil activation by ECs remain unknown. Thus, we investigated what kinds of pathophysiological factors synthesized by inflammatory cytokine-activated ECs potentiated the activity of neutrophil functions. The magnitude of O2− release from neutrophils, which is one of pivotal neutrophil functions, was measured as an indicator potentiated by activated ECs. Neutrophils release massive amounts of O2− on coculture with activated ECs. Anti–granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (Ab) or specific platelet-activating factor (PAF)-receptor antagonist suppressed the O2− release from neutrophils on coculture with the activated ECs by 50% to 70%. The supernatants from activated ECs also induced O2− release by neutrophils. This stimulatory effect of activated EC supernatants on O2− release by neutrophils was abolished by anti–GM-CSF Ab or by PAF-receptor antagonist. As we previously reported, we demonstrated the expression of GM-CSF mRNA by Northern blotting and protein synthesis of GM-CSF by ELISA on tumor necrosis factor as well as interleukin-1–activated ECs. Although phosphorylation of mitogen-activated protein kinases was observed in ECs stimulated by tumor necrosis factor and interleukin-1, treatment of ECs with PD98059 (MEK1 inhibitor) and SB203580 (p38 mitogen–activated protein kinase inhibitor) in the presence of the cytokine failed to attenuate the stimulatory effect of activated ECs on neutrophil activation. We found that activated ECs regulated neutrophil function on coculture. We show here for the first time, to our knowledge, that the collaboration between GM-CSF and PAF synthesized by activated ECs markedly potentiated neutrophil activation.

Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2

We have recently demonstrated that granulocyte‐colony stimulating factor (G‐CSF) delays human neutrophil apoptosis via up‐regulation of cellular inhibitor of apoptosis 2 (cIAP2), which is dependent on activation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Here, we show that type I and type II interferons (IFNs), which bind to the distinct receptors, exert the antiapoptotic effect on human neutrophils through the similar mechanism. IFN‐α (type I IFN) and IFN‐γ (type II IFN), like G‐CSF, delayed human neutrophil apoptosis through the protein synthesis‐dependent mechanism. Stimulation of neutrophils with IFN‐α or IFN‐γ resulted in tyrosine phosphorylation of STAT1 and STAT3 but not phosphorylation of STAT5, Akt, extracellular signal‐regulated kinase, and p38 mitogen‐activated protein kinase. IFN‐α and IFN‐γ induced the expression of transcripts of cIAP2 and suppressor of cytokine signaling 1 and 3, but not cIAP1, Mcl‐1, and A1. IFN‐α‐ and IFN‐γ‐induced up‐regulation of cIAP2 mRNA and protein, phosphorylation of STAT3, and antiapoptotic effect were inhibited significantly by pretreatment of cells with AG490, a specific inhibitor of JAK2. These findings suggest that cIAP2 expression is up‐regulated by IFN‐α and IFN‐γ through, at least in part, activation of the JAK2‐STAT3 pathway, and increased expression of the cIAP2 protein may contribute to an IFN‐α‐ and IFN‐γ‐mediated antiapoptotic effect on human neutrophils.

Antiapoptotic effect of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and cyclic AMP on human neutrophils: protein synthesis-dependent and protein synthesis-independent mechanisms and the role of the Janus kinase-STAT pathway.

Spontaneous neutrophil apoptosis during culture was delayed by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or dibutyryl-cyclic adenosine monophosphate (cAMP), whereas apoptosis was accelerated by cycloheximide or actinomycin D. G-CSF-mediated antiapoptosis was completely abolished by cycloheximide or actinomycin D, whereas GM-CSF-mediated antiapoptosis was not completely abolished by these inhibitors. Antiapoptosis induced by dibutyryl-cAMP was highly resistant to cycloheximide, and that induced by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone was unaffected by cycloheximide. G-CSF- and GM-CSF-mediated antiapoptosis and phosphorylation of signal transducer and activator of transcription 3 (STAT3) and STAT5 were inhibited by AG490, an inhibitor of Janus kinase.The level of Mcl-1 protein was not associated with neutrophil apoptosis. The results suggest that (a) neutrophil survival in the resting state is primarily regulated by the constitutive synthesis of antiapoptotic proteins; (b) the prevention of spontaneous apoptosis is mediated through the protein synthesis-dependent and/or protein synthesis-independent mechanisms according to the stimuli used; and (c) the Janus kinase-STAT pathway is involved in G-CSF- and GM-CSF-mediated antiapoptosis.

Cleavage of Mitogen-Activated Protein Kinases in Human Neutrophils Undergoing Apoptosis: Role in Decreased Responsiveness to Inflammatory Cytokines

Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) are major signaling molecules activated in human neutrophils stimulated by cytokines. Both molecules were cleaved at the N-terminal portion in neutrophils undergoing apoptosis induced by in vitro culture alone or treatment with TNF and/or cycloheximide. The cleavage of both molecules was inhibited by G-CSF and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a caspase inhibitor, both of which can inhibit neutrophil apoptosis. In a cell-free system, ERK and p38 MAPK were not cleaved by recombinant caspase-3 or caspase-8 while gelsolin was cleaved by caspase-3 under the same condition. The cleavage of both molecules appears to be specific to mature neutrophils, since it was not detected in immature cells (HL-60 and Jurkat) undergoing apoptosis, indicating that proteases responsible for the cleavage of both molecules may develop during differentiation into mature neutrophils. Concomitant with the cleavage of ERK and p38 MAPK, GM-CSF- and TNF-induced superoxide release, adherence, and phosphorylation of ERK and p38 MAPK were decreased in neutrophils undergoing apoptosis. In addition, GM-CSF- and TNF-induced superoxide release and adherence were inhibited by PD98059 MAPK/ERK kinase inhibitor) as well as SB203580 (p38 MAPK inhibitor), suggesting possible involvement of ERK and p38 MAPK in superoxide release and adherence induced by these cytokines. These findings indicate that ERK and p38 MAPK are cleaved and degraded in neutrophils undergoing apoptosis in a caspase-dependent manner and the cleavage of both molecules may be partly responsible for decreased functional responsiveness to inflammatory cytokines.

Toll-like receptor agonists stimulate human neutrophil migration via activation of mitogen-activated protein kinases

Human neutrophil migratory responses to Toll‐like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)(3)‐lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10u2003min after stimulation with LPS or P3CSK4, and was sustained for more than 80u2003min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK), which preceded neutrophil migration. TLR‐mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or platelet‐activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP‐ or PAF‐induced neutrophil chemotaxis is not affected by the pre‐exposure of cells to TLR agonists.

Peroxisome proliferator-activated receptor gamma activation induces cell cycle arrest via the p53-independent pathway in human anaplastic thyroid cancer cells.

Anaplastic thyroid carcinoma is one of the most aggressive human malignancies. Outcomes of intensive multimodal therapy have been far from satisfactory. Furthermore, p53 gene dysfunction, often found in this type of cancer, is known to impair the efficacy of the therapeutic agents. Specific ligands for peroxisome proliferator activated receptor gamma (PPAR‐γ) induce growth suppression in some tumor cells. In this study, we investigated the role of PPAR‐γ in anaplastic thyroid cancer cell lines (OCUT‐1, ACT‐1). PPAR‐γ was expressed and functional in both cell lines. Activation of PPAR‐γ with its specific ligands, troglitazone and 15‐deoxy‐Δ12, 14‐prostaglandin J2, inhibited cell growth in a dose‐dependent manner through inducing G1 cell cycle arrest. P53 protein expression differed in OCUT‐1 and in ACT‐1, though the levels stayed constant irrespective of ligand exposure in both cell lines. In contrast, p21 and p27 proteins were induced in a dose‐dependent manner in both situations. This study showed that PPAR‐γ ligands were able to induce growth suppression in anaplastic thyroid cancer cells via a p53‐independent, but p21‐ and p27‐dependent cytostatic pathway. These tumor‐suppressive effects of PPAR‐γ may provide a novel approach to the treatment of anaplastic thyroid cancer.

Ex vivo expansion of mature human neutrophils with normal functions from purified peripheral blood CD34+ haematopoietic progenitor cells.

Purified CD34+ haematopoietic progenitor cells were cultivated with stem cell factor, interleukin 3 (IL‐3), granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and granulocyte CSF (G‐CSF) for 7u2003d, and thereafter non‐adherent cells were divided into two groups. Cells in one group (group A) were further cultivated for 7u2003d with four cytokines, and cells in the other group (group B) were further cultivated for 7u2003d with G‐CSF alone. On day 14, 220‐fold and 130‐fold increases in the numbers of non‐adherent cells were achieved for groups A and B respectively. These cell preparations contained 65% granulocytes for group A and 95% granulocytes for group B. These cells gained the ability to respond effectively with chemotaxis, phagocytosis and superoxide (O2−) release. Cells in group B were appropriately primed by G‐CSF, GM‐CSF, tumour necrosis factor α and IL‐1β for enhanced release of . The responsiveness of these cells was identical to that of peripheral blood neutrophils, indicating that cells in group B may be in the resting state. In contrast, cells in group A were not primed by these cytokines for enhanced release of O2− and released a large amount of O2− spontaneously, indicating that cells in group A may be in the activated state. These findings indicate that mature neutrophils with normal functions were expanded ex vivo in group B and suggest that these cells could be used for possible autologous neutrophil transfusion to prevent bacterial infections during severe neutropenia after cytotoxic chemotherapy.

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 3–5u2003min after G‐CSF stimulation, reached a maximum at 10u2003min, and was sustained for approximately 35u2003min. The maximum migration rate was 84·4u2003±u20032·9u2003μm/5u2003min. 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.

Decreased anti-Saccharomyces cerevisiae antibody titer by mesalazine in patients with Crohn's disease.

Saccharomyces cerevisiae may contribute to the pathophysiology of Crohn’s disease. We determined serum anti‐Saccharomyces cerevisiae antibody (ASCA) levels in patients with inflammatory bowel disease.