Hiroshi Tachimoto

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 (FcεRI) was performed with specific antigen after sensitization with murine IgE. Upon FcεRI 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 Cε 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 FcεRI 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.

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 α4 integrins and a β1 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 α4 integrin function while increasing β2 integrin function. These findings suggest that chemokines facilitate migration of eosinophils by shifting usage away from β1 integrins toward β2 integrins.

Transendothelial migration of human basophils

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1β significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that β2 integrins play the primary role in basophil TEM, but β1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.

Usefulness of Wheat and Soybean Specific IgE Antibody Titers for the Diagnosis of Food Allergy

BACKGROUND Since the first suggestion of threshold values for food specific IgE antibody levels in relation to clinical reactivity, several authors have proposed different threshold values for different allergens. We investigated the relationship between wheat/soybean specific IgE antibody levels and the outcome of wheat/soybean allergy diagnosis in children of different ages. METHODS A retrospective study was conducted in 536 children admitted consecutively to our clinic with the suspicion of wheat and/or soybean allergy. The children underwent an oral food challenge and blood samples for specific IgE measurement were obtained. RESULTS The children who reacted to the oral food challenge had higher specific IgE titers to the specific allergen compared to the non-reacting group. The risk for reaction increased 2.33-fold (95% CI 1.90-2.87) for wheat and 2.08-fold (95% CI 1.65-2.61) for soybean, with increasing levels of specific IgE. A significant difference between the ages of subjects pertained only to wheat. CONCLUSIONS We found a relationship between the probability of failed challenge and the concentration of IgE antibodies to both wheat and soybean. Age influences the relationship of allergen specific IgE levels to wheat and oral food challenge outcome. Younger children are more likely to react to low levels of specific IgE antibody concentration to wheat than older children.

RANTES production in a conjunctival epithelial cell line

Purpose Although corneul 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-α (TNF-α) induced de novo production of RANTES. and interferon-γ (IFN-γ) synergistically increased the TNF-a-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-a and may play a role in recruiting inflammatory cells such as eosinophils and T lymphocytes toward the ocular surface.

Utility of the Peripheral Blood Basophil Histamine Release Test in the Diagnosis of Hen’s Egg, Cow’s Milk, and Wheat Allergy in Children

Background: The diagnosis of food allergy (FA) is made by oral food challenge tests (OFCs) that occasionally produce serious symptoms in patients; therefore, whether to perform OFCs should be carefully considered. The utility of the histamine release test (HRT) in the diagnosis of childhood FA has not been fully examined. Methods: Sixty-four subjects with suspected hen’s egg allergy, cow’s milk allergy (CMA), and wheat allergy (WA) were enrolled. The diagnosis of FA was made based on the outcomes of OFCs or a convincing history of symptoms after food ingestion within 6 months before or after sample collection. HRT was performed using an HRT Shionogi kit. The threshold of histamine release (HRT threshold), which was defined as the minimum concentration of food antigen to induce a 10% net histamine release, was analyzed in association with FA diagnosis. Results: Receiver operating characteristic analysis showed that the HRT threshold was useful in the diagnosis of heated egg allergy (HEA), raw egg allergy (REA), CMA, and WA. We were able to determine the cutoff value for the HRT threshold in relation to outcomes of OFCs. The cutoff value was 6 ng/ml of egg white antigen in HEA and REA (p < 0.01), 40 ng/ml of milk antigen in CMA (p < 0.01), and 500 ng/ml of wheat antigen in WA (p < 0.05). The efficiency was 70.3% for HEA, 78.0% for REA, 77.6% for CMA, and 70.7% for WA. Conclusions: We conclude that the HRT threshold measurement for egg white, milk, and wheat antigen is related to outcomes of OFCs and is useful in determining when OFCs should be performed.

Cross-Talk between Integrins and Chemokines That Influences Eosinophil Adhesion and Migration

Eosinophils accumulate selectively at allergic inflammation sites. Numerous studies have been done to clarify the mechanisms of selective eosinophil accumulation during allergic inflammation. Adhesion molecules and chemokines play important roles in selective eosinophil accumulation [1]. For example, eosinophils express ·4ß1 integrins, one of the ligands for VCAM-1, but neutrophils do not. Among chemokine receptors, eosinophils express CCR3. Like ·4ß1 integrins, CCR3 is not expressed on neutrophils, so its activation effectively and selectively induces eosinophil chemotaxis. In this review, we discuss the regulation of eosinophil integrin function by CCR3active chemokine. Expression of Integrins on Eosinophils

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 Carnoys fixative reduced the number of chymase‐positive mast cells in the nasal mucosa. Therefore, in the present study, we investigated whether Carnoys 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 Carnoys fixative and with other fixatives gave no differences among the number of tryptase‐positive cells, whereas fixation with Carnoys fixative for 15 min gave a significant decrease i n 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 Carnoys fixative, indicating that Carnoys fixative had a negative effect on the number of chymase‐positive cells from cord‐blood derived human cultured mast cells.

Phorbol esters alter α4 and αd integrin usage during eosinophil adhesion to VCAM-1

We examined the effect of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA) on the human eosinophil adhesion molecule phenotype and attachment to VCAM-1 via α4 and αd integrins under static and flow conditions. PMA increased surface expression of αd integrins and decreased α4 integrin expression. Under static conditions, eosinophils bound well to VCAM-1, primarily via α4β1 integrins, with a minor αdβ2 integrin component. Unexpectedly, PMA-stimulated eosinophils bound equally well to VCAM-1 and albumin in a temperature- and divalent cation-dependent manner, yet adhesion was independent of β1 and β2 integrins. Under flow conditions, eosinophils readily attached to VCAM-1, and adhesion was inhibited by both α4 and αd mAbs (95 and 50% inhibition, respectively). Many fewer PMA-stimulated eosinophils bound to VCAM-1 under flow conditions, but both α4 and αd mAbs inhibited adhesion equally. Thus, PMA alters eosinophil integrin expression and the relative contributions of α4 and αd integrins during attachment to VCAM-1.

Chemokine Production in Conjunctival Epithelial Cells

The inflammatory cells that accumulate in inflammatory sites differ depending upon the cause of the inflammation. Bacterial infection or the precipitation of IgG immunocomplexes leads to the accumulation of neutrophils; viral infection or delayed hypersensitivitiy, to the accumulation of monocytes and lymphocytes; and parasite infection and the late-phase reaction of allergic inflammation, to the accumulation of eosinophils and basophils.