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.

Characterization of a receptor for interleukin 5 on human eosinophils: variable expression and induction by granulocyte/macrophage colony-stimulating factor.

Interleukin 5 (IL-5) acts on eosinophil differentiation and activation, suggesting the existence of a membrane receptor for IL-5 on eosinophils. Here, we report that 125I-labeled recombinant human IL-5 bound, at 4 degrees C, to high affinity receptors on human eosinophils. The association constant was higher for hypodense eosinophils (1.93 x 10(9) M-1) than for normodense cells (0.39 x 10(9) M-1), with a closely related number of receptor sites per cell. No specific binding occurred on neutrophils. The specific binding of IL-5 was induced by overnight incubation at 37 degrees C of human eosinophils with granulocyte/macrophage (GM)-CSF. The levels of increase were significantly higher for normodense than for hypodense eosinophils, suggesting a previous in vivo activation of the later subpopulation by GM-CSF. IL-3 was ineffective by itself but synergistically enhanced the effect of GM-CSF. Specificity studies showed that the binding of 125I-labeled IL-5 was inhibited by IL-5, but not by other cytokines, on human eosinophils. These results show the existence of a specific binding site for IL-5 on human eosinophils with a variable affinity on eosinophil hypodense or normodense subpopulations, as previously reported for other membrane receptors.

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.

Association of vitamin D receptor gene polymorphism with prostate cancer and benign prostatic hyperplasia in a Japanese population.

Recent studies have suggested that vitamin D is an important determinant of prostate cancer risk and inherited polymorphisms in the 3'-untranslated region (3'UTR) of the vitamin D receptor (VDR) gene are associated with the risk and progression of prostate cancer. This study was conducted to explore the association of VDR gene polymorphisms with prostate cancer risk in Japanese men who are considered to be much less influenced by environmental risk factors for prostate cancer. We studied 222 prostate cancer patients, 209 benign prostatic hyperplasia (BPH) patients, 128 male controls who were over 60 years old and without any evidence of prostate cancer or BPH, and 198 female controls. A PCR-RFLP method was used to determine three VDR gene polymorphisms in the 3'UTR characterized by restriction enzymes BsmI, ApaI and TaqI. In the BsmI polymorphism, heterozygosity or homozygosity for the absence of the BsmI restriction site was associated with one-third the risk of prostate cancer (P < 0.0001; odds ratio, 3.31; 95% confidence interval, 2.05-5.32) and with one-half the risk of BPH (P < 0.005; odds ratio, 2.07; 95% confidence interval, 1.33-3.22) compared with the male controls. The TaqI and ApaI polymorphisms did not show any significant association with either prostate cancer or BPH. The results indicate that the BsmI polymorphism in the VDR gene plays a significant role in protection against prostate cancer and BPH. Because of the racial difference in the strength of the linkage disequilibrium between the three polymorphisms, additional studies are required to apply the present results to other racial-ethnic groups.

Induction of beta 2 integrin expression on an eosinophilic cell line (EoL-1) by the supernatant of mononuclear cells stimulated with specific allergen from asthmatic patients.

Adhesion molecules recently have been considered to play an important role in inflammatory processes in bronchial asthma. Our previous study revealed high expression of beta 2-integrin family (CR3, LFA-1 alpha, CD18) on hypodense eosinophils. Thus, from the point of view of cell-to-cell interaction between mononuclear cells and eosinophils, we examined whether the supernatant of mononuclear cells obtained from mite-allergic asthmatic patients cultured with specific allergen mite-allergen is involved in adhesion molecule expression using an eosinophilic cell line (EoL-1). These characteristics of beta 2-integrin family expression (high expression of beta 2 integrin) were induced by the supernatant of mononuclear cells from mite-allergic asthmatic patients stimulated with mite-allergen as well as with a combination of the recombinant eosinophilopoietic growth cytokines (IL-3, GM-CSF and IL-5). Thus, we could conclude that some cytokines produced by specific allergen stimulated mononuclear cells in asthmatics might be involved in allergic inflammation through the induction of adhesion molecule expression on eosinophils in asthma or allergic disorders.

Possible involvement of mononuclear cells stimulated with specific allergen from asthmatic patients in ICAM-1 expression on the eosinophilic cell line.

Adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), recently have been considered to play a key role in inflammatory processes in asthma. Thus, from the point of view of cell interactions between mononuclear cells and eosinophils, we examined whether the supernatant of mononuclear cells (MNC) obtained from mite-allergic asthmatic patients cultured with specific allergen is involved in ICAM-1 expression using an eosinophilic cell line (EoL). ICAM-1 expression was induced by the supernatant of MNC from mite-allergic asthmatic patients stimulated with mite allergen as well as by a combination of IL-3, GM-CSF, and IL-5. Thus, we could conclude that some cytokines produced by specific allergen-stimulated MNC in asthmatics might be involved in allergic inflammation through the induction of adhesion molecule expression such as ICAM-1 on eosinophils in asthma or allergic disorders.

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.

Coincident induction of K rev-1/rap 1A, rap 1B and H-ras mRNAs in the rat spinal cord by noxious stimulation.

Two cDNA fragments, K rev-1/rap 1A and rap 1B, were amplified from total cellular RNA of the rat spinal cord by reverse transcription-polymerase chain reaction with a set of oligonucleotide primers specific for the human rap 1A cDNA. We report here using Northern blot analysis with these cDNA probes that noxious stimulation causes a marked and coincident increase in rap 1A, rap 1B and H-ras mRNAs in the rat spinal cord. This suggests that Rap 1 participates in sensory processing in spinal neurons in parallel with Ras.

Mast cells in the cutaneous lesions of sarcoidosis: their subtypes and the relationship to systemic manifestations.

Possible involvement of mast cells in pulmonary sarcoidosis has been suggested, however whether mast cells are involved in cutaneous sarcoidosis remains unknown. We undertook a morphological study of mast cells in the lesional skin from 17 patients with cutaneous sarcoidosis using immunohistochemical methods. Mast cells were present in non-parenchymal fibrous areas, but not in granulomatous areas, in the biopsy specimens from the cutaneous lesions. However, there were no significant differences between the number of mast cells in the lesional skin and that in non-lesional skin from the patients. Mast cells containing substantial quantities of both tryptase and chymase (MC(TC) cells) were present in 41% of the patients, and cells containing tryptase but not chymase (MC(T) cells) were present in 59% of patients. All patients of the former group showed systemic manifestations of the disease concomitantly. Serum angiotensin I-converting enzyme levels were elevated in 71.4% of the former group, and in 30% of the latter group. This study for the first time demonstrated that mast cells were present in non-parenchymal fibrous areas of the cutaneous lesions of sarcoidosis, and the mast cell subtypes may be related to systemic manifestations.

Cultured human mast cells derived from umbilical cord blood cells in the presence of stem cell factor and interleukin-6 cannot be a model of human skin mast cells: fluorescence microscopic analysis of intracellular calcium ion mobilization.

To know whether cultured human mast cells raised from umbilical cord blood cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6) can be a model of human skin mast cells, the cells were stimulated, and intracellular calcium ion ([Ca(2+)](i)) mobilization was analyzed by fluorescence microscopic techniques in parallel with a measurement of histamine released from the cells. When IgE-sensitized mast cells were activated by anti-IgE, [Ca(2+)](i) elevation began at the periphery and subsequently proceeded toward the center of the cells. The increase in [Ca(2+)](i) in calcium ionophore A23187-stimulated mast cells began at the center and spread to the periphery of the cells. Significant histamine release was observed by each stimulation. However, either compound 48/80 or substance P failed to increase [Ca(2+)](i) with no appreciable histamine release. This study shows that there is heterogeneity of [Ca(2+)](i) mobilization in the activated human mast cells, and that cultured human mast cells derived from umbilical cord blood cells in the presence of SCF and IL-6 can not be a model of human skin mast cells.

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 saiboku-to (TJ-96) on bronchial asthma. Induction of glucocorticoid receptor, beta-adrenaline receptor, IgE-Fc epsilon receptor expression and its effect on experimental immediate and late asthmatic reaction.

A remarkable "steroid sparing" effect of Saiboku-to was noted within 6 to 12 months of treatment in steroid-dependent asthmatic patients. Saiboku-to spared the downregulation of glucocorticoid receptor of human lymphocytes, plasma ACTH, and cortisol levels. It also spared downregulation of beta 2 receptor by beta 2 agonists and suppressed mACh receptor at the same time. Saiboku-to increased tyrosine aminotransferase (TAT) production, which was inhibited by actinomycin-D, thus having steroid-like activity. In mite-allergic asthma, Saiboku-to inhibited the induction of expression of IgE-Fc epsilon R/CD23 in the lymphocytes by mite allergen. It also inhibited IgE production by mite allergens. In experimental asthma in guinea pigs the use of Saiboku-to resulted in a decrease in the number of eosinophils in the bronchoalveolar lavage fluid during late asthmatic response. These findings suggest that Saiboku-to may be effective in inhibiting both the expression of IgE-Fc epsilon R2 and the induction of expression of IgE-Fc epsilon R1. Saiboku-to also has a steroid-like action and polyhedral anti-asthmatic activities.

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.

Evidence for a link between glycoxidation and lipoperoxidation in patients with chronic renal failure.

AIM: The purpose of this study is to examine whether or not there is a relationship between glycoxidation and lipid peroxidation in patients with chronic renal failure. SUBJECTS AND METHODS: Dermal samples from 26 living or autopsied subjects were sequentially extracted with NaCl, pepsin, collagenase, and NaOH to obtain four fractions (salt-soluble fraction: SSF; pepsin-soluble fraction: PSF; collagenase-soluble fraction: CSF; and insoluble fraction: ISF). The glycoxidation product was measured by pentosidine-linked fluorescence (ex: 335/em: 385) and the levels of lipid peroxide, malondialdehyde (MDA), were assessed by determining the MDA-linked fluorescence (ex: 390/em: 460) which was further confirmed by HPLC. RESULTS: In patients undergoing hemodialysis, MDA-linked fluorescence markedly increased in collagen-rich fractions, PSF, CSF, and ISF, while pentosidine-linked fluorescence increased in PSF and CSF, in comparison to the controls and the pre-dialysis patients with CRF. Interestingly, the increase in the lipid peroxides strongly correlated with the level of glycoxidation product in PSF, CSF, and ISF (p < 0.0001 in PSF, CSF; p < 0.01 in ISF). The HPLC data of MDA in the PSF was in good correlation with logistic levels of both MDA- (n = 9, r = 0.738, p = 0.023) and pentosidine-linked fluorescence (n = 9, r = 0.721, p = 0.028). In contrast, in SSF, the collagen-poor fraction (collagen content: less than 3% of the total extracted collagen), the data showed a significant increase in the MDA-linked fluorescence only in the pre-dialysis patients with CRF, but not in the HD patients with no correlation with the glycoxidation products. CONCLUSION: These findings suggest that both the lipid peroxidation and glycoxidation increased in close relation to each other in the matrix collagen and thus demonstrate a synergetic contribution to the tissue damage observed in patients with CRF

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.

[The roles of adhesion molecules, cytokines and chemokines in relation to eosinophil activation in allergic inflammation].

Several characteristics of human eosinophil heterogeneity, as well as the existence of eosinophil subpopulations, "normodense" and "hypodense", have been reported in diseases associated with hypereosinophilia. Hypodense eosinophils can be distinguished by the increased expression of various membrane receptors including IL-5 receptor (J Exp Med 172:1347) and various protein expression (J Immunol 142:4416). Recently, adhesion molecules, as well as eosinophils, have been found to play an important role in the inflammatory processes in allergic disease. We demonstrated here as below: 1) Characteristics of adhesion molecules expression on eosinophils in asthma, namely, high-intensity expression of adhesion molecules. 2) Induction of adhesion molecule expression by PAF and RANTES and in addition induction by the supernatant of mononuclear cells from mite-allergic asthmatic patients stimulated with mite-allergen as well as with a combination of the recombinant IL-3, GM-CSF, and IL-5 (Immunol Lett 42:25, 1994 & 46:241, 1995). 3) Elevated soluble ICAM-1 in bronchial asthma (Lancet 343:1108, 1994).

[The roles of adhesion molecules, cytokines and chemokines in allergic inflammation].

Recently, adhesion molecules, as well as eosinophils, have been found to play an important role in the inflammatory processes in allergic disease. We demonstrated here as below. Characteristics of adhesion molecules expression on eosinophils in asthma, namely, high-intensity expression of adhesion molecules. Induction of adhesion molecule expression by PAF and RANTES and in addition induction by the supernatant of mononuclear cells from mite-allergic asthmatic patients stimulated with mite-allergen as well as with a combination of the recombinant IL-3, GM-CSF and IL-5. Elevated soluble ICAM-1 in bronchial asthma. Moreover, the presence of a large variety of membrane receptors and the identification of cytotoxic molecules (mainly granule basic proteins) have indicated that eosinophils should be considered as effector cells. We therefore investigated the possible release of granule proteins in response to signaling from ICAM-1 and its ligands. The concentrations of eosinophil cationic protein and eosinophil-derived neurotoxin in supernatants of eosinophils were significantly greater (p < 0.05) in the presence of recombinant soluble ICAM-1 than without it. These results suggest that signaling from ICAM-1 and its ligands might induce eosinophil activation and might be involved in degranulation of eosinophil granule proteins. In addition, reactive oxygen species generated by eosinophils have also been considered capable of causing airway injury at the inflamed focus. We examined the effect of recombinant soluble ICAM-1 and its ligands on eosinophil-induced radical oxygen products. Recombinant soluble ICAM-1 augmented eosinophil oxidative metabolism. It was concluded that signaling via adhesion molecules might play an important role in the pathogenesis of allergic inflammation through activation of eosinophils, such as through an increase in oxidative metabolism or degranulation of eosinophil granule proteins.