Retinoic acids up-regulate functional eosinophil-driving receptor CCR3.

Eotaxins and their receptor CCR3 have a definitive role for tissue accumulation of eosinophils both under homeostatic and pathologic conditions. However, physiological stimuli that can up-regulate CCR3 in blood-derived human eosinophils have not been recognized. As a prior gene microarray study revealed up-regulation of CCR3 in eosinophils stimulated with retinoic acids (RAs), the expression of functional CCR3 was examined. We found that 9-cis RA and all-trans RA (ATRA) significantly induced surface CCR3 expression regardless of the presence of IL-3 or IL-5. Pharmacological manipulations with receptor-specific agonists and antagonists indicated that retinoic acid receptor-α activation is critical for CCR3 up-regulation. RA-induced CCR3 was associated with its functional capacity, in terms of the calcium mobilization and chemotactic response to eotaxin-1 (CCL11). Our study suggests an important role of vitamin A derivatives in the tissue accumulation of eosinophils.

Clinical evaluation after a notification policy of linezolid use: a case series of 22 patients.

Linezolid exhibits a broad spectrum of activity against Gram-positive cocci, including Methicillin-resistant Staphylococcus aureus (mRSA) and vancomycin-resistant enterococci (VRe). However, recent studies have already reported the emergence of linezolid-resistant mRSA or VRe. the purpose of this study is to evaluate not only the efficacy of linezolid for the treatment of nosocomial mRSA infections but also the effect of a notification policy of linezolid use. the charts of inpatients who had been treated with linezolid were reviewed for clinical outcome. After introduction of the notification policy of linezolid use, the clinical success rate was 73.3%, and the rate of appropriate linezolid use was 80%, whereas the success rate was 14.2% and the appropriate use rate was 14.3% before the policy. in conclusion, appropriate use controlled by a notification policy of antibiotics use is essential for prevention of the emergence and spread of linezolid-resistant bacteria, and for proper demonstration of its antibacterial ability.

Thioredoxin reduces C-C chemokine-induced chemotaxis of human eosinophils.

BACKGROUND: Human thioredoxin (TRX) is one of redox-active proteins that regulate reactive oxidative metabolisms. In recent study, we found that serum levels of TRX were elevated in asthmatic patients with exacerbation; however, few details are known about the physiological role of TRX in allergic inflammation, involving eosinophil infiltration. OBJECTIVE: In the present study, we examined whether TRX modulated C-C chemokine-induced chemotaxis of human eosinophils. METHODS: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16 negative selection. After incubation with or without recombinant TRX, chemotaxis of human eosinophils was measured using Boyden chamber. RESULTS: Preincubation with TRX suppressed eotaxin- and regulated on activation, normal T-cell expressed and secreted (RANTES)-induced chemotaxis of eosinophils. Although, TRX had no effect on the expression of C-C chemokine receptor 3, which is a receptor of eotaxin and RANTES, we demonstrated that the activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases, which play an important role in eosinophil migration, was attenuated by the treatment with TRX. CONCLUSION: Our results suggest that the elicited TRX is beneficial to reduce allergic inflammation through negative regulation of eosinophil functions and has potential in the treatment of allergic diseases, such as asthma.

Soluble vascular cell adhesion molecule-1 induces human eosinophil migration.

BACKGROUND: Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses including asthma. Adhesion molecules are known to play an important role in the accumulation of eosinophils in allergic inflammatory foci, and they contribute to eosinophil activation. Elevated levels of the soluble forms of adhesion molecules in the body fluid of asthmatic patients have been observed, although their pathophysiological significance remains to be fully elucidated. METHODS: Peripheral blood eosinophils were purified, and the effect of soluble vascular cell adhesion molecule-1 (sVCAM-1) on eosinophil migration was investigated using in vitro systems. RESULTS: We found that sVCAM-1 (1 to 10 mug/ml) induced eosinophil chemotaxis, rather than chemokinesis, in a concentration-dependent fashion. In addition, sVCAM-1 induced cell shape change and actin polymerization, which are necessary for cell movement. Manipulations with very late antigen (VLA)-4-neutralizing antibody and signal inhibitors indicated that the sVCAM-1-induced chemotaxis was mediated through ligand-dependent activation of tyrosine kinase Src, p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK) MAPK. Rapid phosphorylation of these signaling molecules was observed using a bead-based multiplex assay. CONCLUSION: Our results raise the possibility of sVCAM-1 in the fluid phase as a significant contributor to the heightened eosinophilic inflammatory response.

Sex-related splenocyte function in a murine model of allergic asthma.

BACKGROUND: The prevalence and severity of asthma are higher among boys than girls, but the ratios are reversed after puberty. These observations strongly suggest that sex hormones have a role in the pathogenesis of the disease. However, the mechanisms underlying the gender differences in asthma are not fully understood. OBJECTIVE: The aim of this study was to investigate sex differences in allergic inflammation in terms of immune function. METHODS: Male and female C57BL/6 mice were sensitized and challenged with ovalbumin (OVA). OVA-specific IgE in serum and airway inflammation were compared between sexes. Splenocytes from OVA-sensitized male or female donor mice were transferred to male or female naïve recipient mice. Subsequently, the recipient mice were challenged, followed by the evaluation of OVA-specific IgE and airway inflammation. Cytokines secreted from splenocytes of the sensitized mice were measured. RESULTS: The levels of OVA-specific IgE and the allergen-induced airway inflammation were higher in female than in the male mice. The contents of T-helper type 2 (Th2) cytokines, IL-4, IL-5 and IL-13, in the bronchoalveolar lavage fluid from female mice were higher than those from male mice. The airway inflammation in female recipients transferred with splenocytes from female donors was more severe than that in any other combination of recipients and donors. Splenocytes from the sensitized female mice produced more of the Th2 cytokine, IL-5, than those from the sensitized male mice upon stimulation with OVA. CONCLUSION: Our findings suggest that the sex difference in allergic airway inflammation may be attributable to the sex difference in not only the hormonal environment but also in the immune cells themselves.

Hepatocyte growth factor attenuates eotaxin and PGD2-induced chemotaxis of human eosinophils.

BACKGROUND: Hepatocyte growth factor (HGF) is known to influence a number of cell types, and regulate various biologic activities including cell migration, proliferation, and survival. In a recent study, we found that, in vivo, HGF suppresses allergic airway inflammation, i.e. the infiltration of inflammatory cells including eosinophils into the airway, and further, that HGF reduces Th2 cytokine levels; however, the directly physiologic role of HGF with eosinophils remains unclear. In this study, we investigate the potential of recombinant HGF to regulate the factor-induced chemotaxis of human eosinophils. METHODS: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16-negative selection. After culture with or without recombinant HGF, esoinophil chemotaxis was measured by Boyden chamber and KK chamber. RESULTS: Treatment with HGF prevented eotaxin or prostaglandin D(2) (PGD(2))-induced chemotaxis of eosinophils. Moreover, we demonstrated that extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinases as well as the enhancement of Ca(2+) influx, which are indispensable for eosinophil chemotaxis, were attenuated by HGF treatment. CONCLUSION: Taken together, these data suggest that in allergic diseases, HGF not only mediates eosinophils through the inhibition of Th2 cytokines, but also regulates the function of eosinophils directly, provides further insight into the cellular and molecular pathogenesis of allergic reactions.

Red blood cells regulate eosinophil chemotaxis by scavenging RANTES secreted from endothelial cells.

BACKGROUND: Eosinophils play a critical role in the pathogenesis of allergic diseases. CC chemokines, such as regulated on activation, normal, T cell expressed, and secreted (RANTES), are key regulators of eosinophil locomotion. Although eosinophils migrate from the bloodstream into tissues, mechanisms that generate a chemogradient across the endothelium remain to be fully elucidated. OBJECTIVE: We first examined the polar secretion of RANTES by endothelial cells. We also studied the functional scavenging effect of red blood cells (RBCs) on RANTES secreted into the intravascular side. METHODS AND RESULTS: Endothelial cells were cultured in a transwell chamber with a membrane pore size of 0.45, 3.0, and 8.0 microm and stimulated with TNF-alpha, IL-1beta, or IFN-gamma from the apical or basolateral side for 16 h. The measurement of RANTES in the supernatant was performed by ELISA. We did not see any difference in the amount of RANTES secreted from the cytokine-stimulated endothelium between inner (intravascular side) and outer (extravascular side) wells separated by the 8.0-microm membrane, although apical polarization was observed with the 0.45-microm membrane. The addition of RBCs (hemoglobin (Hb): 0.5-15 g/dL) to the apical supernatant of TNF-alpha-stimulated endothelial cells reduced the RANTES level in a concentration-dependent manner. The treatment of supernatant on the intravascular side with RBCs significantly enhanced the migration of eosinophils. CONCLUSION: RBCs possess a scavenging effect on intravascular RANTES, and thereby regulate transendothelial migration of eosinophils. Our findings suggest a new role of RBCs in allergic inflammation.

CD28 and secretory immunoglobulin A-dependent activation of eosinophils: inhibition of mediator release by the anti-allergic drug, suplatast tosilate.

BACKGROUND: Eosinophils are major effector cells in allergic diseases. After their recruitment to sites of inflammation, they contribute to the pathophysiology of the disease by releasing granule proteins and cytokines. Suplatast tosilate (IPD-1151T), a new anti-allergic agent, has shown beneficial effect in the treatment of asthma, associated with reduced bronchoalveolar lavage eosinophil infiltration and eosinophilic cationic protein (ECP) release in serum and sputum. OBJECTIVE: We investigated whether suplatast tosilate could exert direct effects on human eosinophil activation. METHODS: Eosinophils from hypereosinophilic patients or normal donors were purified by Percoll gradient and the magnetic cell separation system. Chemotaxis was studied using the Boyden chamber technique using three chemoattractants, formyl-methionine-leucine-phenylalanine (fMLP), IL-5 and eotaxin. Oxidative metabolism was determined by a luminol-dependent chemiluminescence assay after activation with eotaxin or secretory IgA (sIgA). The release of ECP and eosinophil derived neurotoxin (EDN) was measured by radioimmunoassay and cytokine production was determined by ELISA following activation with sIgA or anti-CD28. RESULTS: The chemotactic response to fMLP, IL-5 and eotaxin was significantly inhibited by IPD-1151T. Suplatast tosilate was partially inhibiting the release of reactive oxygen species (ROS) induced by eotaxin and sIgA. Activation by sIgA and CD28 ligation resulted in the release of ECP and EDN, which was inhibited by IPD-1151T. Upon activation by anti-CD28, only IL-13 production was inhibited by IPD-1151T, whereas release of IL-2 and IFN-gamma was not affected. IL-10 release induced by sIgA was also inhibited by IPD-1151T. Additionally, the pro-inflammatory cytokine IL-6, which was secreted following anti-CD28 and sIgA stimulation, was strongly inhibited by IPD-1151T. CONCLUSION: Through inhibition of chemotaxis, IPD-1151T might limit the number of eosinophils at the inflammation site. Furthermore, it could reduce the pathological potential of eosinophils by inhibiting the release of ROS and cationic proteins, main inflammatory mediators produced by eosinophils. Moreover, the inhibition of immunoregulatory cytokines released by eosinophils could locally modify the immune response.

Elemental diet modulates the growth of Clostridium difficile in the gut flora.

BACKGROUND AND AIMS: Tube feeding is regarded as a risk factor for Clostridium difficile-associated diarrhoea. Recently, we reported that C. difficile toxin was frequently found in patients receiving an elemental diet. The present study was conducted to clarify whether elemental diets are associated with the growth of C. difficile in the gut flora. METHODS: C. difficile was cultured for 72 h in various concentrations of elemental diet containing 3% thioglycollate, and the growth rate or activity of C. difficile was evaluated by Gram stain or by measuring optical density at 560 nm. Faecal samples from 10 healthy adults were cultured in elemental diet + 3% thioglycollate. RNA was extracted from faeces with glass powder, which can eliminate PCR inhibitors, and mRNA of C. difficile toxin B was measured by reverse transcription PCR. RESULTS: Maximum OD560 value during culture in thioglycollate-containing elemental diet was 2.4 times higher than that in thioglycollate alone (P = 0.0163). Viability of C. difficile was decreased in thioglycollate but not in thioglycollate-containing elemental diet. Toxin B mRNA was detected in five faecal samples (50%) before culture and in all samples after culture. CONCLUSIONS: Our results suggest that an elemental diet can modulate the growth of C. difficile in the gut flora.

Upregulated response to chemokines in oxidative metabolism of eosinophils in asthma and allergic rhinitis.

Reactive oxygen species (ROS) from eosinophils are known to cause tissue damage in allergic inflammation. CC chemokines, especially eotaxin and regulated on activation, normal T-cell expressed and secreted (RANTES), are involved not only in chemotaxis but also in eosinophil activation, such as ROS production. It has been shown that eosinophils from allergic patients are not functionally equivalent to those from normal subjects. In the present study, the characteristics of chemokine-primed ROS production in eosinophils from allergic patients and normal controls were compared. After pretreatment with chemokines, eosinophils were stimulated with calcium ionophore A23187. ROS production by eosinophils was measured using luminol-dependent chemiluminescence. Both RANTES and eotaxin exhibited a priming effect on calcium ionophore-induced ROS production from eosinophils. Despite there being no difference in expression of CC chemokine receptor 3, the priming effect of RANTES and eotaxin was significantly enhanced in eosinophils from the patients. Interleukin-5 further enhanced the priming effect of chemokines in eosinophils from normal subjects, but not those from allergic subjects. The present results suggest an upregulated response to chemokines in eosinophils from allergic patients, and that interleukin-5 can induce a similar phenotype to that found in vivo in allergic patients.

Possible involvement of C-C chemokines in functional augmentation of adhesion molecules in asthmatic patients.

Adhesion molecules and C-C chemokines play an important role in the accumulation of eosinophils in allergic inflammation. In the present study, the expression and function of adhesion molecules on eosinophils from asthmatic patients and involvement of RANTES and eotaxin were examined. Eosinophils isolated by the CD16 negative selection method were stimulated with or without RANTES or eotaxin. Expression of b integrins on eosinophils and the functional adherence to recombinant soluble intercellular adhesion molecule-1 (r-sICAM-1)-coated plates were examined. Compared with normal subjects, eosinophils from asthmatic patients showed increased expression of b2 integrins and functional adherence to r-sICAM-1-coated plates. RANTES and eotaxin augmented the functional adherence of eosinophils without a significant upregulation of b2 integrins. Anti-b2 integrin antibody inhibited the augmentative effect on eosinophil adherence of RANTES and eotaxin. Pertussis toxin, wortmannin, and genistein inhibited chemokine-induced adherence. RANTES and eotaxin are closely related to eosinophil accumulation not only as chemotactic agents but also as augmentative agents for eosinophil adherence through involvement in functional eosinophil adherence to ICAM-1 by a possible qualitative change of b2 integrins. Pertussis toxin-sensitive G proteins, PI3 kinase, and tyrosine kinase are involved in signal transduction leading to activation of b2 integrins on eosinophil following stimulation with RANTES and eotaxin.

[Chemokine (RANTES, eotaxin, etc.)].

Chemokine is the general term for the cytokines that possess chemotactic activity for leukocytes. The chemokines, secreted 8-10 kD proteins, active and attract leukocytes to inflammatory foci through binding to specific G protein-coupled seven transmembrane-spanning cell surface receptors. Two subfamilies, CXC and CC chemokines, are distinguished according to the position of the first two cysteines, which may be either separated by one amino acid or adjacent. The CXC chemokine family mainly have chemotactic activity for neutrophils. Since RANTES and eotaxin, which are CC chemokines, have chemotactic activity on eosinophils, these substance are considered to play a key role in allergic inflammation. Moreover, RANTES and eotaxin are considered to be closely related to not only chemotaxis but also activation of eosinophils such as degranulation, oxidative metabolism and adherence.

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils.

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.

Expression of adhesion molecules on cord-blood-derived, cultured human mast cells and effect of dexamethasone on intercellular adhesion molecule-1 expression on the mast cells treated by phorbol myristate acetate.

BACKGROUND: Increase in mast-cell number at sites of allergic inflammation has been observed, and glucocorticoids applied to the sites have been shown to result in a significant reduction in mast cells. However, the expression of adhesion molecules on cultured human mast cells and their regulation by glucocorticoids is poorly understood. METHODS: Cultured human mast cells were raised from human umbilical cord-blood cells, and the expression of adhesion molecules on the mast cells was analyzed by flow cytometry. The cells were also incubated with 10 ng/ml phorbol myristate acetate (PMA) for the indicated time, and the effect of dexamethasone on adhesion molecule expression on PMA-treated, cultured human mast cells was examined. RESULTS: Cord-blood-derived, cultured human mast cells constitutively expressed intercellular adhesion molecule-1 (ICAM-1), very late antigen-4 (VLA-4), and macrophage-1 antigen (Mac-1). Weak expression of lymphocyte function-associated antigen-1 (LFA-1) was observed on the cells, whereas they failed to express vascular cell adhesion molecule-1 (VCAM-1). Kinetic studies showed that after a transient downregulation reaching a minimum at 8 h, the expression of ICAM-1 was markedly upregulated on PMA-treated mast cells after a 24-h incubation. In contrast, the expression of VLA-4 and Mac-1 was decreased after the incubation with PMA for 24 h. The PMA-induced upregulation of ICAM-1 was inhibited by dexamethasone in a concentration-dependent manner. CONCLUSION: Our results indicate that cord-blood-derived, cultured human mast cells constitutively express integrins and ICAM-1, but not VCAM-1, and demonstrate for the first time that dexamethasone inhibits the upregulation of ICAM-1 on PMA-treated, cultured human mast cells.

[Eosinophils and related chemokines].

Chemokines such as RANTES, eotaxin, MIP-1 and MCP-4 are considered to be involved in the pathophysiology of allergic inflammation because of their ability to drive eosinophils through their binding sites, chemokine receptors, expressed on eosinophils. Among those chemokines, RANTES and eotaxin are considered to play important roles in the process of the maturation, migration and activation of eosinophils. An overview of the effect of chemokines on eosinophils throughout their migration from bone marrow to the inflammatory focus is described in this paper. Furthermore, our observations on the effects of chemokines on eosinophils such as adherence through beta-2 integrin, the production of reactive oxygen species, intracellular EG2 content and production of RANTES by eosinophils are reported.

Expression of VLA-4 on eosinophils decreases in patients with eosinophilia.

BACKGROUND: It is assumed that the very late antigen-4 (VLA-4) plays a key role in selective migration and accumulation of eosinophils to the allergic inflammatory focus. The regulatory mechanism for VLA-4 expression is poorly understood, as is its relationship between other adhesion molecules. OBJECTIVE: The aim of the study was to elucidate the relationship between VLA-4 expression and the activation of eosinophils. METHODS: The surface expression of VLA-4, Mac-1, ICAM-1, CD4, CD25, CD69, CD89, IL-5 receptor and GM-CSF receptor on eosinophils isolated from the peripheral blood of 15 patients with eosinophilia and 16 healthy volunteers was measured. RESULTS: The surface expression of VLA-4 presented in mean fluorescent intensity by flow-cytometric analysis showed a significant decrease in the patients with eosinophilia (>700 eosinophils/microl) compared to that of the subjects without eosinophilia. On the other hand, the surface expression of Mac-1 was significantly increased in the patients with eosinophilia. There was an inverse correlation between the expression of VLA-4 and that of Mac-1 (r = -0.81) on the eosinophils obtained from the patients with eosinophilia. CONCLUSION: The changes on the surface expressions of Mac-1 and VLA-4 may be indicating the activation of eosinophils in the patients with eosinophilia and may contribute to their migration to the allergic inflammatory focus.

Effect of roxithromycin on eotaxin-primed reactive oxygen species from eosinophils.

BACKGROUND: The CC chemokine eotaxin not only attracts eosinophils to inflamed sites but also promotes adhesion, degranulation and reactive oxygen species production of eosinophils. Reactive oxygen species released from eosinophils are believed to injure epithelial cells at inflamed sites, resulting in airway hyperresponsiveness. Roxithromycin has been reported to have antiasthmatic effects, although its mechanism of action is not thoroughly understood. Therefore, the effect of roxithromycin on eotaxin-primed reactive oxygen species production from eosinophils was studied. METHODS: Reactive oxygen species production by eosinophils cultured with or without roxithromycin was evaluated using luminol-dependent chemiluminescence. RESULTS: Roxithromycin inhibited the release of reactive oxygen species from eosinophils evoked with the calcium ionophore A23187, regardless of pretreatment with or without eotaxin. CONCLUSION: Roxithromycin may protect epithelial cells at inflamed sites, at least partly by inhibiting the release of reactive oxygen species from eosinophils.

Serum eosinophil derived neurotoxin may reflect more strongly disease severity in childhood atopic dermatitis than eosinophil cationic protein.

Serum levels of eosinophil cationic protein (ECP) have been shown to be a good parameter of the disease severity of patients with atopic dermatitis (AD). However, the relationship between the disease severity and the eosinophil derived neurotoxin (EDN) has not been established in AD patients. The purpose of this study is to examine serum ECP and EDN levels in relation to the disease severity in AD children. Serum ECP and EDN levels were assessed in relation to the skin scores in 34 AD children (18 boys and 16 girls; age 0.6 to 7years: mean+/-S.D. 2.2+/-1.9) and six non-atopic control children (three boys and three girls; age 1 to 3years: mean+/-S.D. 1.7+/-0.9). Serum ECP and EDN levels of the patients with AD were significantly increased compared with the non-atopic controls. Serum EDN levels of the patients were also related to the disease severity. The skin scores were more significantly correlated with serum EDN levels than ECP levels. We concluded that serum EDN may reflect more strongly disease severity as eosinophilic activation in AD children than serum ECP.

Effect of reactive oxygen species on fetal lung maturation.

OBJECTIVES: Fetal lung maturation in preterm infants with chorioamnionitis is known to be accelerated. However, the molecular basis of this pathological acceleration has not been elucidated. We investigated whether reactive oxygen species play a role in the acceleration of fetal lung maturation. STUDY DESIGN: On the 16th day of gestation, xanthine (1mM) and xanthine oxidase solution (0.1-100mU/ml) were injected into the intrauterine cavity of pregnant rats. On the 19th day of gestation, we examined the expression of the mRNA of surfactant associated proteins A, B and C (sp-A, sp-B and sp-C) by reverse transcription-polymerase chain reaction. RESULTS: sp-A, sp-B and sp-C mRNAs were observed in lung tissue from fetal rats stimulated by xanthine-xanthine oxidase in contrast to the control. CONCLUSION: Reactive oxygen species in amniotic fluid might be an important factor in accelerated fetal lung maturation associated with chorioamnionitis in the rat experimental model.