Improved control of childhood asthma with low-dose, short-term vitamin D supplementation: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: In our prior randomized trial on preventing influenza, asthma attacks as a secondary outcome occurred less often in the vitamin D group than in the placebo group. We aimed to clarify whether low-dose, short-term vitamin D supplementation, in addition to standard treatments, improves control of childhood asthma. METHODS: We conducted a randomized, double-blind, placebo-controlled trial comparing vitamin D3 supplements (800 IU/day) with placebo for 2 months in schoolchildren with asthma. The primary outcomes were frequency and severity of asthma judging from changes in asthma control levels defined by the Global Initiative for Asthma (GINA) by collaborating doctors at 2 and 6 months. RESULTS: Japanese schoolchildren with asthma (n = 89) were randomly assigned to receive vitamin D (n = 54) or placebo (n = 35). At 2 months, GINA asthma control was significantly more improved in the vitamin D group compared with the placebo group (P = 0.015). Childhood asthma control test (CACT) scores, a secondary outcome, were also significantly (P = 0.004) improved in the vitamin D group compared with the placebo group at 2 months, and differences remained significant (P = 0.012) at 6 months. The proportion of patients with a peak expiratory flow rate <80% predicted was significantly less in the vitamin D group (8/54: 15%) than in the placebo group (12/35: 34%) at 6 months (P = 0.032). CONCLUSIONS: Low-dose, short-term vitamin D supplementation in addition to standard treatment may improve levels of asthma control in schoolchildren.

RANTES production in a conjunctival epithelial cell line.

PURPOSE: Although corneal tissue damage in allergic ocular diseases is thought to be induced by inflammatory cells that infiltrate from conjunctival tissue, the mechanisms of recruiting these cells remain unclear. The objective of this study was to demonstrate whether conjunctival epithelial cells have the ability to produce "regulated on activation, normal T-cell expressed and secreted" (RANTES). To test this hypothesis, we investigated RANTES expression in the conjunctival tissue and also RANTES production by cytokine stimulation in a human conjunctival epithelial cell line. METHODS: We investigated the expression of the chemokine RANTES in conjunctival epithelium from two patients with atopic keratoconjunctivitis (AKC) and one patient with vernal keratoconjunctivitis (VKC) by using immunohistochemistry. We also investigated the production and suppression of RANTES from a human conjunctival epithelial cell line, Wong-Kilbourne-derived human conjunctiva (WK-hC) by using enzyme-linked immunosorbent assay (ELISA). RESULTS: Conjunctival epithelium from a patient with AKC stained positively for RANTES. We found that tumor necrosis factor-alpha (TNF-alpha) induced de novo production of RANTES, and interferon-gamma (IFN-gamma) synergistically increased the TNF-alpha-dependent production of RANTES from WK-hC cells. Dexamethasone suppressed the RANTES production from the cell line. CONCLUSION: Taken together, human conjunctival epithelial cells were capable of producing RANTES in response to inflammatory stimuli such as TNF-alpha and may play a role in recruiting inflammatory cells such as eosinophils and T lymphocytes toward the ocular surface.

[The role of cytokines and cell adhesion molecules in allergic inflammation].

The mechanisms of late phase allergic reaction (LAR) have been investigated by many researchers in the past decade, since LAR was recognized clinically to be more important than early phase allergic reaction (EAR). The selective eosinophil recruitment in LAR can be explained by the hypothesis of the cascade of cytokines and adhesion molecules. Since cytokines were initially identified as the factors derived from activated T cells, T cells were thought to play an important role in the LAR in the late 1980s. In 1989, murine mast cells were reported to produce many cytokines following activation through Fc epsilon R I. The current topic is to clarify which cell type mainly induce LAR either CD4+ T cells or mast cells.

Acquisition and alteration of adhesion molecules during cultured human mast cell differentiation.

BACKGROUND: Mature human mast cells express several types of adhesion molecules on their surface. Interactions between extracellular matrix (ECM) and adhesion molecules may be important for the migration and localization of mast cells and their precursors in tissues. Little is known about the regulation of adhesion molecules on mast cells during their differentiation. OBJECTIVES: To clarify the evolution of adhesion phenotype and function, we examined the expression of adhesion molecules during cultured human mast cell (CHMC) differentiation and tested adhesion of mature CHMCs to various ECM proteins. METHODS: CHMCs were obtained by culturing human cord blood-derived CD34(+) cells in the presence of stem cell factor and IL-6. Indirect immunofluorescence and flow cytometry was used to study cell surface expression of adhesion molecules and other markers. Mature CHMCs were tested for adhesion molecule function with immobilized matrix proteins. RESULTS: At 1 week of culture, cells expressed CD11a, CD18, CD29, CD49d, and CD49e. At 14 weeks of culture, more mature CHMCs expressed CD11b, CD11c, CD29, CD49b, CD49c, CD49d, CD49e, CD51, CD61, and CD54 and weakly expressed CD18 and CD11a. CD11c, CD51, and CD61 appeared de novo by 4 weeks of culture, whereas CD49b and CD49c appeared by 8 weeks. CD29 decreased at 4 weeks but returned to the identical levels of 1-week-old cells by 8 weeks. Compared with levels at week 1, the levels of CD11a, CD18, CD49d, and CD49e at 4 weeks and beyond decreased during culture. Expression of CD49a, CD49f, and alphad integrin was never detectable during CHMC differentiation. Fourteen-week-old CHMCs significantly adhered to the leucine-aspartic acid-valine-containing connecting segment 1 fragment of fibronectin, the 120-kd argine-glycine-aspartic acid-containing fragment of fibronectin, vitronectin, and laminin through specific integrins. CONCLUSION: Expression of integrins and CD54 is differentially regulated during CHMC differentiation, and mature CHMCs can adhere to many ECM proteins. These changes may facilitate emigration from the bone marrow into the circulation and ultimately contribute to the tissue homing and localization pattern seen with mature mast cells.

Carnoy's fixative reduces the number of chymase-positive cells in immunocytochemical staining of cord-blood-derived human cultured mast cells.

KleinJan et al. (Allergy 1996;51:614-20) reported that Carnoy's fixative reduced the number of chymase-positive mast cells in the nasal mucosa. Therefore, in the present study, we investigated whether Carnoy's fixative reduces the number of chymase-positive cells from cord-blood-derived human cultured mast cells when compared with other types of fixatives. Human mast cells were obtained by culturing cord-blood-derived CD34-positive cells in the presence of stem cell factor and interleukin-6. Staining procedures of the cells in fixation with Carnoy's fixative and with other fixatives gave no differences among the number of tryptase-positive cells, whereas fixation with Carnoy's fixative for 15 min gave a significant decrease in the number of chymase-positive cells compared with acetone for 10 min. The number of chymase-positive cells decreased in a time-dependent manner under fixation with Carnoy's fixative, indicating that Carnoy's fixative had a negative effect on the number of chymase-positive cells from cord-blood-derived human cultured mast cells.

Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-gamma.

BACKGROUND: T(H)1 and T(H)2 cytokines are thought to regulate allergic inflammation. OBJECTIVE: Two key regulatory cytokines, IL-4 and IFN-gamma, were examined for their effects on cytokine production by cultured human mast cells (CHMCs). METHODS: CHMCs were obtained by culturing cord blood-derived CD34(+) cells in the presence of stem cell factor and IL-6 for 14 to 16 weeks. CHMCs were passively sensitized with human myeloma IgE and supplemented with or without IL-4 or IFN-gamma. After the sensitization, CHMCs were stimulated with anti-FcepsilonRIalpha mAb. Concentrations of secreted cytokines were measured by using ELISA, and cytokine messenger RNA was analyzed by using quantitative competitive RT-PCR. RESULTS: IL-4 profoundly enhanced FcepsilonRI-mediated production of macrophage inflammatory protein (MIP) 1alpha, IL-8, and GM-CSF. For example, the enhancement by IL-4 (10 ng/mL) of the production of MIP-1alpha, IL-8, and GM-CSF was 25-, 7-, and 90-fold, respectively, after 6 hours. IL-4 also enhanced levels of FcepsilonRI-induced cytokine messenger RNA but to a lesser degree. In contrast, IFN-gamma inhibited FcepsilonRI-induced production of MIP-1alpha, IL-8, and GM-CSF. For example, the inhibition by IFN-gamma (10 ng/mL) of FcepsilonRI-mediated production of MIP-1alpha, IL-8, and GM-CSF was 80%, 75%, and 95%, respectively. IFN-gamma also suppressed FcepsilonRI-induced messenger RNA expression of these cytokines. Neither IL-4 nor IFN-gamma affected the kinetics of cytokine production by CHMCs. CONCLUSION: These data suggest that IL-4 and IFN-gamma may influence allergic reactions by modulating human mast cell cytokine production.

CCR3-active chemokines promote rapid detachment of eosinophils from VCAM-1 in vitro.

Selective eosinophil recruitment is the result of orchestrated events involving cell adhesion molecules, chemokines, and their receptors. The mechanisms by which chemokines regulate eosinophil adhesion and migration via integrins are not fully understood. In our study, we examined the effect of CCR3-active chemokines on eosinophil adhesion to VCAM-1 and BSA under both static and flow conditions. When eotaxin-2 or other CCR3-active chemokines were added to adherent eosinophils, it induced rapid and sustained eosinophil detachment from VCAM-1 in a concentration-dependent manner. Adhesion was detectably reduced within 3 min and was further reduced at 10-60 min. Simultaneously, eotaxin-2 enhanced eosinophil adhesion to BSA. Preincubation of eosinophils with the CCR3-blocking mAb 7B11 completely prevented chemokine-induced changes in adhesion to VCAM-1 and BSA. Using a different protocol, pretreatment of eosinophils with chemokines for 0-30 min before their use in adhesion assays resulted in inhibition of VCAM-1 adhesion and enhancement of BSA adhesion. By flow cytometry, expression of alpha4 integrins and a beta1 integrin activation epitope on eosinophils was decreased by eotaxin-2. In a flow-based adhesion assay, eotaxin-2 reduced eosinophil accumulation and the strength of attachment to VCAM-1. These results show that eotaxin-2 rapidly reduced alpha4 integrin function while increasing beta2 integrin function. These findings suggest that chemokines facilitate migration of eosinophils by shifting usage away from beta1 integrins toward beta2 integrins.

Cultured basophils but not cultured mast cells induce human IgE synthesis in B cells after immunologic stimulation.

By generating human mast cells and basophils from umbilical cord blood mononuclear cells cultured in the presence of appropriate cytokines, we investigated whether these two cultured cells could provide the cytokine and cell contact signals that are required to induce IgE synthesis in B cells. To activate cultured mast cells and basophils, cross-linking of cell surface high-affinity IgE receptor (Fc epsilonRI) was performed with specific antigen after sensitization with murine IgE. Upon Fc epsilonRI stimulation, basophils, but not mast cells, secreted significant amounts of immunoreactive IL-4 and IL-13 and expressed detectable CD40 ligand (CD40L) and a very low level of Fas ligand (FasL). These observations at the protein level were consistent with the data obtained at the gene transcriptional level, except for the faint expression of only IL-13 mRNA in mast cells. When added to normal human B cells, activated basophils induced IgE and IgG4 synthesis as well as soluble CD23 release. In contrast, neither IgE nor IgG4 synthesis could be induced by the interaction of B cells with activated mast cells, even in the presence of recombinant IL-4. The induction of IgE synthesis by activated basophils was completely abrogated by two neutralizing MoAbs against IL-4 and IL-13 and by a soluble form of CD40. This abrogation was accompanied by abolished mature C epsilon transcription in both cases. Addition of anti-FasL MoAb, however, did not significantly affect IgE induction mediated by activated basophils. These results demonstrate that unlike cultured mast cells, cultured basophils produce biologically active IL-4 and IL-13 and express functional CD40L after Fc epsilonRI stimulation, thereby contributing to IgE production by B cells, and suggest that relatively weak expression of FasL by cultured basophils is not involved in IgE regulation.

Continuous isoproterenol inhalation therapy in children with severe asthmatic attack.

We studied the 1-type isoproterenol inhalation therapy for patients with severe asthmatic attacks who were admitted at the Department of Allergy of National Children's Hospital from 1981 to 1991. One hour after l-type isoproterenol inhalation therapy, statistically significant effects were noted with regard to the asthmatic status. Moreover, no side effect was found amoung the subjects. From these data, 1-type isoproterenol inhalation therapy is thought to be effective for severe asthmatic attacks.

Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells.

To establish the method for generating a large number of mature human mast cells, we cultured cord blood mononuclear cells (CBMC) in several conditions in the presence of Steel factor (SF). Among several cytokines tested, IL-6 enhanced SF-dependent mast cell growth from purified CD34+ cells for more than 8 wk in culture. When CBMC were cultured instead of CD34+ cells, IL-6 enhanced the mast cell development in the presence but not in the absence of PGE2. PGE2 enhanced the SF- and IL-6-dependent development of mast cells from CBMC probably by blocking granulocyte-macrophage CSF (GM-CSF) secretion from accessory cells, because 1) PGE2, or anti-GM-CSF enhanced the mast cell development induced by SF and IL-6 from CBMC, but not from CD34+ cells; 2) GM-CSF inhibited the enhancing effect of IL-6 on the mast cell development from CD34+ cells; and 3) PGE2 inhibited GM-CSF secretion from CBMC. The mast cells cultured in the presence of SF, IL-6, and PGE2 for >10 wk were 99% pure, and seemed to be functionally mature, because 1) they contained 5.62 micrograms of histamine and 3.46 micrograms of tryptase per 10(6) cells; and 2) when sensitized with human IgE and then challenged with anti-human IgE, the cells released a variety of mediators such as histamine, and an increase in intracellular Ca2+ was found in advance of the activation of membrane movement by using a confocal laser-scanning microscope. Electron-microscopic analysis revealed that some of the cultured mast cells are morphologically mature since they filled with scroll granules and contained crystal granules.

Characterization of mast cell-committed progenitors present in human umbilical cord blood.

Human mast cells are derived from CD34(+) hematopoietic cells present in cord blood, bone marrow, and peripheral blood. However, little is known about the properties of the CD34(+) cells. We demonstrated here that mast cell progenitors that have distinct phenotypes from other hematopoietic cell types are present in cord blood by culturing single, sorted CD34(+) cells in 96-well plates or unsorted cells in methylcellulose. The CD34(+) mast cell-committed progenitors often expressed CD38 and often lacked HLA-DR, whereas CD34(+) erythroid progenitors often expressed both CD38 and HLA-DR and CD34(+) granulocyte-macrophage progenitors often had CD33 and sometimes expressed CD38. We then cultured single cord blood-derived CD34(+)CD38(+) cells under conditions optimal for mast cells and three types of myeloid cells, ie, basophils, eosinophils, and macrophages. Of 1,200 CD34(+)CD38(+) cells, we were able to detect 13 pure mast cell colonies and 52 pure colonies consisting of either one of these three myeloid cell types. We found 17 colonies consisting of two of the three myeloid cell types, whereas only one colony consisted of mast cells and another cell type. These results indicate that human mast cells develop from progenitors that have unique phenotypes and that committed mast cell progenitors develop from multipotent hematopoietic cells through a pathway distinct from myeloid lineages including basophils, which have many similarities to mast cells.

Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils.

BACKGROUND: Eosinophils, basophils, and mast cells are believed to be the central tenet cells in allergic conditions including allergic rhinitis, asthma, and eczema. The molecular mechanisms underlying the recruitment of these cells to sites of allergic inflammation are poorly understood. OBJECTIVES: Our aim was to identify a common adhesion molecule that could potentially be responsible for mediating the recruitment of the allergic cell types to the lungs and other sites of allergy. METHODS: We have cloned a sialoadhesin molecule from a human eosinophil library with the use of expressed sequence tag technology and characterized its expression on allergic cells by the use of flow cytometry and specific mAbs. RESULTS: With the use of expressed sequence tag sequencing, we have identified a novel siglec molecule, SAF-2. SAF-2 has homology with other sialoadhesin family members (CD33 and siglec-5) and belongs to a subgroup of the Ig superfamily. SAF-2 is a 431-amino acid protein composed of 3 Ig domains with a 358-amino acid extracellular domain and a 47-amino acid tail. SAF-2 is highly restricted to eosinophils, basophils, and mast cells. Antibodies to SAF-2 do not modulate Ca(++) mobilization or chemotaxis of human eosinophils induced by eotaxin. CONCLUSION: SAF-2 is a highly restricted sialoadhesin molecule, which may be useful in the detection and/or modulation of allergic cells.