Akimasa Someya

A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis

The mast cell is one of the major effector cells in inflammatory reactions and can be found in most tissues throughout the body. During inflammation, an increase in the number of mast cells in the local milieu occurs, and such accumulation requires directed migration of this cell population. As it has previously been reported that the human cathelicidin‐derived antibacterial peptide, LL‐37, stimulates the degranulation of mast cells, we hypothesized that LL‐37 could be a mast cell chemotaxin. The present study shows that LL‐37 is a potent chemotactic factor for mast cells. The chemotactic response was dose‐dependent and bell‐shaped, reaching an optimal concentration of 5 µg/ml. In addition, checkerboard analysis showed that cell migration towards this peptide was chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125I‐labelled LL‐37‐derived peptide revealed that LL‐37 has at least two classes of receptors, namely high‐ and low‐affinity receptors, on mast cells. Furthermore, the competitive binding assay suggested that LL‐37 is unlikely to utilize formyl peptide receptor‐like 1 (FPRL1), a functional LL‐37 receptor for neutrophil and monocyte migration, on mast cells. In addition, the treatment of cells with pertussis toxin and phospholipase C inhibitor, U‐73122, inhibited LL‐37‐mediated migration, indicating that LL‐37 induces mast cell chemotaxis through a Gi protein‐phospholipase C signalling pathway. These results show that besides its antibacterial activities, LL‐37 may have the potential to recruit mast cells to inflammation foci.

ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage

To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide‐producing activity using HL‐60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91phox, p67phox, and p47phox were expressed after myelocyte stages, whereas p22phox, p40phox, and rac‐2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO‐induced HL‐60 cells indicated that gp91phox, p67phox, and p47phox were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22phox, p40phox, and rac‐2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO‐induced HL‐60 cells were obtained using human bone‐marrow cells by immunocytochemical staining and NBT assay, except that p22phox was detected by immunocytochemical staining after myelocyte stages in bone‐marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide‐producing activity is obtained after myelocyte stages during neutrophil maturation.

Purification of the 260 kDa cytosolic complex involved in the Superoxide production of guinea pig neutrophils

A 260 kDa cytosolic complex (SP‐1) was purified from guinea pig neutrophils. SP‐1 was composed of 63 kDa, 47 kDa and 39 kDa proteins. The 63 kDa and 47 kDa proteins proved to correspond to human p67phox and p47phox by Western blot analysis, whereas Western blot and amino acid sequence analyses revealed that the 39 kDa protein was a novel protein. The 47 kDa protein was separated from the 63 kDa and 39 kDa proteins by dithiothreitol (DTT)‐treatment. On the other hand, the 63 kDa and 39 kDa proteins were not separated with DTT, detergent and ethanol treatment. These results suggest that the 39 kDa protein tightly associates with the 63 kDa protein and may regulate the function of the 63 kDa protein.

Response of superoxide anion production by guinea pig eosinophils to various soluble stimuli: Comparison to neutrophils

The effect of various soluble stimuli on the superoxide production by guinea pig eosinophils was studied in comparison to neutrophils. Phorbol myristate acetate, A23187, digitonin, NaF, concanavalin A (Con A), and cytochalasin E stimulated eosinophils and neutrophils to release O2-. The O2- production by these active agents, excluding Con A and cytochalasin E, was much greater in eosinophils than in neutrophils. Formyl-Met-Leu-Phe stimulated the O2- production in neutrophils but not in eosinophils. Neither histamine nor Val/Ala-Gly-Ser-Glu stimulated the O2- production in both types of leukocytes. A23187- or Con A-stimulated O2- production was greatly enhanced by cytochalasin B pretreatment in neutrophils but not in eosinophils. Lineweaver-Burk analysis of NADPH oxidase in particulate fractions showed that eosinophils possessed the same Km values as neutrophils and greater Vmax values than neutrophils, suggesting that eosinophils have a similar, but more active, O2- -generating enzyme system than neutrophils.

Evaluation of the expression of human CAP18 gene during neutrophil maturation in the bone marrow

To understand the gene expression of CAP18 (18‐kDa cationic antibacterial protein), a member of cathelicidins, we evaluated mRNA and protein expression of CAP18 using human bone marrow cells and mature neutrophils. Northern blot analysis revealed that CAP18 mRNA was expressed more abundantly in bone marrow cells than mature neutrophils, whereas Western blot analysis indicated that CAP18 protein was more abundant in mature neutrophils than bone marrow cells. Consistent with this, in situ hybridization using bone marrow cells demonstrated that the expression of CAP18 mRNA was neutrophil lineage‐specific and was observed primarily in myelocytes (>95%) with limited expression in more immature cells (promyelocytes) and mature cells (metamyelocytes, band cells, and segmented neutrophils). Furthermore, immunohistochemical study indicated that, coincident with the increase of CAP18 mRNA levels, CAP18‐positive cells increased markedly at myelocyte stage, and the increased levels remained almost constant (>95%) in metamyelocytes, band cells, and segmented neutrophils, although the mRNA levels were remarkably reduced in these cells. Together these observations indicate that CAP18 gene transcription likely occurs lineage‐ and stage‐specifically at the myelocyte stage of neutrophil maturation in the bone marrow and results in the synthesis and cytoplasmic accumulation of CAP18, which is present in the subsequent stages of neutrophil maturation. J. Leukoc. Biol. 64:845–852; 1998.

Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase.

NADPH oxidase, a superoxide‐producing enzyme in phagocytic cells, consists of membrane‐associated cytochrome b558 and cytosolic components (p47‐phox, p67‐phox, p40‐phox, rac 1/2). Activation of NADPH oxidase is accompanied by the phosphorylation of cytosolic components (p47‐phox and p67‐phox). In this study, we have examined whether p40‐phox, a newly identified cytosolic component, is phosphorylated during neutrophil activation, and the relationship between p40‐phox phosphorylation and NADPH oxidase activation. When 32P‐labeled guinea pig neutrophils were stimulated by phorbol 12‐myristate 13‐acetate, p40‐phox was phosphorylated as p47‐phox. It is interesting that phosphorylation of p40‐phox was markedly inhibited by protein kinase C inhibitor, H‐7, but not by casein kinase II inhibitor, A‐3, and H‐7 inhibited translocation of p40‐phox and activation of NADPH oxidase. Furthermore, purified protein kinase C but not casein kinase II directly phosphorylated p40‐phox of p40‐phox/p47‐phox/p67‐phox complex. Together these observations suggest that p40‐phox is phosphorylated by protein kinase C during neutrophil activation, and phosphorylation of p40‐phox may be important for the activation of NADPH oxidase. J. Leukoc. Biol. 66: 851–857; 1999.

Characterization of cDNA clones encoding guinea pig neutrophil cationic peptides

cDNA clones encoding antimicrobial guinea pig neutrophil cationic peptides GNCP‐1 and GNCP‐2 were isolated from a bone marrow cell cDNA library. Analysis of these clones indicated that both GNCPs were produced as precursor proteins comprising 93 amino acid residues, which were composed of signal sequences (N‐terminal 19 residues), pro‐peptide sequences (43 residues) and mature GNCP sequences (31 residues). The deduced amino acid sequence showed that there were only two amino acid differences between GNCP‐1 and GNCP‐2, one in the pro‐peptide region and one in the mature peptide region. Interestingly, Northern blot analysis and transcription run‐off assay revealed that the expression of GNCP mRNA and the transcription of GNCP gene was observed in bone marrow cells but not in mature neutrophils. These observations suggest that mature neutropils, despite their abundant content of GNCPs, lose the capacity to synthesize GNCPs.

GEP100/Arf6 Is Required for Epidermal Growth Factor-Induced ERK/Rac1 Signaling and Cell Migration in Human Hepatoma HepG2 Cells

Background Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. Methodology/Principal Findings We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration. Conclusions/Significance Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

Evaluation of the expression of the cationic peptide gene in various types of leukocytes

To understand the regulation of the production or antimicrobial cationic peptide (CP) in leukocytes, expression of the CP gene was evaluated in various types of leukocytes using guinea pig neutrophils, monocytes, macrophages, cosinophils, lymphocytes and bone marrow cells. Acid‐urea PAGE and SDS‐PAGE/immunoblot analyses showed that CP was present in neutrophils and bone marrow cells, but not in other leukocytes. Northern blot and transcription run‐off analyses revealed that only bone marrow cells expressed CP mRNA and transcribed the CP gene. Interestingly, in situ hybridization analysis using bone marrow cells demonstrated that CP mRNA was expressed in the neutrophil precursor cells, such as promyclocytes and myclocytes, but was not detected in the mature neutrophils and other bone marrow cells. Moreover, immunocytochemical study indicated flat CP present in the neutrophil precursor cells and the mature neutrophils in the bone marrow. Thus, the CP gene appears to be expressed during a limited period of neutrophil maturation, and CP is likely synthesized by the neutrophil precursor cells in the bone marrow.