Effects of human lung fibroblasts on eosinophil degranulation.

BACKGROUND: Although eosinophils and fibroblasts are thought to contribute to the mechanisms of chronic asthmatic inflammation, the interaction between eosinophils and fibroblasts has not been thoroughly clarified. We examined eosinophil cationic protein (ECP) release from human eosinophils cultured in the presence of human lung fibroblast HFL-1. METHODS: Eosinophils from healthy donors were cultured with or without C5a for 16 h in the presence of human fetal lung fibroblasts which had previously been incubated with or without TNF for 4 h. ECP in supernatants was measured by RIA. RESULTS: ECP release was potentiated only when both eosinophils and fibroblasts were activated by C5a and TNF, respectively, while it was not significantly potentiated when either eosinophils or fibroblasts were activated. Paraformaldehyde fixation of fibroblasts had some suppressive effect on ECP enhancement, and mAb against GM-CSF partly inhibited ECP enhancement. Coculture of eosinophils and fibroblasts with stimulus treatment resulted in the enhancement of both eosinophil adhesion and ECP release. The potentiation of ECP release was partially inhibited by anti-ICAM-1 mAb, anti-CD18 mAb, and anti-CD29 mAb, which caused partial and comparable inhibition of the enhancement of eosinophil adhesion. CONCLUSIONS: This study suggests that the activation of fibroblasts may have some role in the potentiation of ECP release from cocultured eosinophils, and that adhesion of eosinophils to fibroblasts may partly be involved in the mechanism of ECP potentiation.

Air pollution and allergy.

Epidemiological studies have found a recent increase in the prevalence of allergic diseases, especially in industrialized countries. A change in environmental factors may be considered as one of the causes of this increase. It has been reported that the prevalence of allergic diseases is higher in polluted areas than in unpolluted ones. Therefore, we focused on the effect of one air pollutant, suspended particulate matter (SPM), on allergic responses. We showed that SPM had an enhancing effect on the IgE antibody production in mice. In Japan, the number of cars with diesel engines has increased rapidly, and it has been calculated that 35-80% of SPM in large cities consists of diesel exhaust particulates (DEP). We demonstrated that DEP had an adjuvant effect on the IgE antibody production in mice when administered intraperitoneally or intranasally. In humans, it has been shown that nasal challenge with DEP enhanced total IgE and specific IgE production in nasal lavages. Furthermore, it has been demonstrated that DEP had an enhancing effect on Th2-type cytokine synthesis in both mice and humans. It cannot be excluded that DEP may be related to the increase in prevalence of allergic diseases through the effect on the IgE antibody production and Th2-cytokine synthesis.

Release of granule proteins from human eosinophils stimulated with mast-cell mediators.

BACKGROUND: It has been suggested that mast cells and eosinophils are major effector cells in the pathogenesis of allergic diseases. However, the interaction of these cells has not been thoroughly elucidated. We examined eosinophil cationic protein (ECP) release and cytosolic free calcium concentration ([Ca2+]i) in human eosinophils induced by the major mast-cell mediators including cytokines. METHODS: Eosinophils from healthy donors were stimulated with the major mast-cell mediators for 20 min after preincubation with cytochalasin B for 10 min. ECP in supernatants was measured by radioimmunoassay. Moreover, to examine changes of [Ca2+]i in eosinophils, Fura-2-loaded eosinophils were monitored for fluorescence changes after stimulus addition. RESULTS: Of the tested mediators (prostaglandin [PG]D2, leukotriene (LT)B4, platelet-activating factor (PAF), histamine, LTC4, and eosinophil chemotactic factor of anaphylaxis [ECF-A]), LTB4 and PAF induced ECP release from eosinophils. Any cytokines produced by human mast cells, i.e., interleukin (IL)-4, IL-5, IL-8, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), did not induce ECP release in our system. ECP release triggered with LTB4 and PAF occurred at concentrations of 10(-8)-10(-6) M concentration-dependently. LTB4 and PAF also elicited a rise in [Ca2+]i in eosinophils. Neither PGD2, histamine, nor LTC4 induced ECP release, although they increased cytosolic calcium in eosinophils. CONCLUSIONS: Of mast-cell mediators, LTB4 and PAF induced eosinophil degranulation. The contribution of LTB4 and PAF from mast cells to eosinophil degranulation may be important in the pathogenesis of allergic inflammatory diseases.

Eosinophil degranulation in the presence of lung fibroblasts.

BACKGROUND: Although eosinophils (Eos) and fibroblasts (Fb) are closely approximated in the bronchial submucosae of asthmatics, and are believed to play important roles in the pathogenesis of bronchial asthma, the interaction between Eos and Fb has not been thoroughly elucidated. In this study, we have examined eosinophil cationic protein (ECP) release from human Eos cultured in the presence of human lung Fb. METHODS: Eos from healthy donors were cultured with or without C5a for 16 h in the presence of human fetal lung Fb which had previously been incubated with or without some cytokines for 4 h. ECP in supernatants was measured by RIA. RESULTS: ECP release was potentiated only when both Eos and Fb were activated by C5a and TNF, respectively, while it was not significantly potentiated when either Eos or Fb were activated. ECP release from Eos activated by C5a was also potentiated when Fb were stimulated by IL-1beta. The enhancement of ECP release in cocultured Eos and Fb with stimulation was partly inhibited by monoclonal antibodies against GM-CSF and was accompanied by the enhancement of adhesion of Eos to Fb. CONCLUSIONS: This study shows that the stimulation of both Eos and Fb increases ECP release. It is suggested that Fb may influence Eos degranulation and play a role in the pathogenesis of bronchial asthma.

Eosinophil adhesion to human bronchial epithelial cells: regulation by cytokines.

BACKGROUND: Eosinophil infiltration into the airways and interaction with bronchial epithelial cells are important in the pathogenesis of asthma. The purpose of the present study was to elucidate the regulatory mechanisms of eosinophil adhesion to human bronchial epithelial cells. METHODS: We cultured a human bronchial epithelial cell line, BEAS-2B, on a collagen-coated glass slide. Highly purified human eosinophils were added to each well to allow attachment to epithelium for 30 min. The number of attached eosinophils was counted. RESULTS: Eosinophil adhesion to epithelial cells was increased when the BEAS-2B cells were pretreated with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Although IFN-gamma upregulated ICAM-1 expression as shown by flow cytometry, specific neutralizing antibody to ICAM-1 failed to block eosinophil adhesion. Dexamethasone significantly suppressed eosinophil adhesion to bronchial epithelial cells. CONCLUSION: Eosinophil adhesion to bronchial epithelium was dynamically regulated by cytokines, and this process might be a target for therapeutic intervention in the treatment of asthma.

Effects of interleukin (IL)-3 and IL-5 on human eosinophil degranulation induced by complement components C3a and C5a.

It is suggested that eosinophils (Eos) play an important role in the pathogenesis of bronchial asthma by releasing cytotoxic cationic eosinophil granule proteins and damaging bronchial epithelial cells. However, the exact nature of the actual inducer of eosinophil degranulation in vivo is unclear. We examined eosinophil cationic protein (ECP) release from human Eos in response to soluble agonists such as C5a, C3a, platelet-activating factor, and FMLP with or without interleukin (IL)-3 or IL-5 priming. Eosinophil degranulation induced by these soluble agonists required the pretreatment of Eos by cytochalasin B even in IL-3 priming. Among four agonists, C5a was the most effective stimulus of ECP release either with or without IL-5 priming. IL-3 and IL-5 remarkably enhanced ECP release in Eos triggered by C3a and C5a. The enhancement of ECP release by IL-3 and IL-5 occurred at 0.1-0.3 ng/ml and became maximal at 10-30 ng/ml, concentration-dependently. The enhancement of ECP release by cytokines became optimal at an interval of 10 min. Our data support the importance of complement components and cytokines in eosinophil degranulation in vivo.

Eosinophil degranulation in the presence of bronchial epithelial cells. Effect of cytokines and role of adhesion.

It has been suggested that eosinophils (Eos) are responsible for damage to bronchial epithelial cells by releasing toxic eosinophil granule proteins in bronchial asthma. We examined eosinophil cationic protein (ECP) release from human Eos cultured in the presence of human bronchial epithelial cell line BEAS-2B (Ep). ECP release was potentiated only when both Eos and Ep were activated by IL-5 and TNF, respectively, while it was not potentiated when either Eos or Ep were activated. ECP release from Eos activated by IL-5 was also enhanced when Ep was stimulated by IFN-gamma. Paraformaldehyde fixation of Ep had no effect on ECP enhancement, excluding the possibility that soluble factors from Ep contribute to ECP potentiation. Coculture of Eos and Ep with cytokine treatment resulted in the enhancement of eosinophil adhesion and ECP release, and eosinophil adhesion preceded ECP release in the kinetic study. The enhancement of ECP release was partially inhibited by anti-CD18 mAb, which caused partial and comparable inhibition on the potentiation of eosinophil adhesion. These results suggest that the activation of Ep may profoundly affect the ability of cocultured Eos to release ECP and that CD18-dependent adhesion of Eos to Ep may be considered as one of the mechanisms of ECP enhancement.

Effects of physiologic soluble agonists on leukotriene C4 production and degranulation by human eosinophils.

The capacities of physiologic soluble agonists to induce leukotriene C4 (LTC4) formation and eosinophil cationic protein release from normal human eosinophils were studied. The most effective stimulus for LTC4 production by eosinophils was N-formyl-methionyl-leucyl-phenylalanine, while that for eosinophil cationic protein release was complement factor 5a. Interleukin-3 (IL-3) and IL-5 modulated both LTC4 formation and eosinophil cationic protein release induced by soluble agonists in a similar fashion, whereas tumor necrosis factor and nerve growth factor affected only LTC4 production. The optimal preincubation period for priming of LTC4 production by IL-3 or IL-5 was 90 min, while that for eosinophil cationic protein release was 10 min. These results indicate that degranulation and the generation of lipid mediators are separately regulated cellular responses, and priming by cytokines may qualitatively change the pathophysiologic consequences of eosinophil activation by soluble agonists.

Cefotiam-induced IgE-mediated occupational contact anaphylaxis of nurses; case reports, RAST analysis, and a review of the literature.

Cefotiam (CTM) is one of the most popular cephem antibiotics in Japan. Recently we experienced two cases of nurses with CTM-induced contact anaphylaxis. When they were preparing drip infusions of antibiotics or working around other nurses doing so, they suddenly fell into shock with other symptoms such as flushing, urticaria, abdominal distress, vomiting, dyspnoea and/or loss of consciousness. The symptoms never occurred after they avoided exposure to CTM. Passive cutaneous or open patch tests were positive for CTM. Histamine release was induced by CTM from washed leucocytes. RAST analysis using CTM-human serum albumin-coupled discs showed high % RAST count, suggesting that these reactions were mediated by IgE antibodies. A RAST inhibition test suggested that the methyl-thiotetrazole side-chain was the main antigenic determinant. Both patients had hand dermatitis that had appeared preceding the episodes of anaphylaxis. Although the dermatitis had been resistant to treatments, it also disappeared after they avoided exposure to CTM. It seemed likely that it was also induced or exacerbated by CTM and facilitated the penetration of CTM to cause anaphylaxis. The literature is also reviewed.

Degranulation from human eosinophils stimulated with C3a and C5a.

It is unclear what actually induces eosinophil degranulation in vivo. We examined eosinophil cationic protein (ECP) release from normal human eosinophils (Eos) in response to C3a and C5a. C3a and C5a induced remarkable ECP release when Eos were preincubated with cytochalasin B. ECP release induced by C3a or C5a was greater than that induced by PAF at a concentration of 10(-7) M. The ED50 for ECP release was 3 x 10(-8) M and 3 x 10(-9) M for C3a and C5a, respectively. C3a or C5a elicited a rapid and transient rise in [Ca2+]i. These results suggest that C3a and C5a may contribute to hypersensitivity diseases by inducing eosinophil degranulation.

The effect of terfenadine on the immediate and late-phase reactions mediated by immunoglobulin E.

Basophils in mononuclear cell populations were challenged with allergens, anti-immunoglobulin E (anti-IgE), C5a or formyl-methyl-leucyl-phenylalanine (FMLP), with or without a short pre-incubation with interleukin-3 (IL-3), in the presence of increasing concentrations of terfenadine. At doses of 0.1-1 micrograms/ml, terfenadine inhibits histamine release and generation of the sulfidoleukotrienes, leukotriene C4, D4 and E4 in basophils challenged with an IgE-dependent trigger. At concentrations above 10 micrograms/ml, however, terfenadine induces the histamine release but abolishes the formation of leukotrienes, and this may be due to a cytotoxic effect. In eosinophils, by contrast, terfenadine appears to inhibit the production of leukotrienes by eosinophils, triggered by FMLP only at concentrations above 10 micrograms/ml (which are toxic to basophils at least). In a double-blind, placebo-controlled study, 15 allergic patients were given skin challenges with specific allergen and with histamine, before and at 3 days, 2 and 4 weeks after treatment with terfenadine (120 mg/day for 3 days). The skin reactions were evaluated visually and followed kinetically by thermography. Terfenadine caused a significant decrease in both the immediate and late-phase reactions. Late-phase reactions to histamine were shown with thermography in some of the patients tested.

Opposing effects of tumor necrosis factor-alpha and nerve growth factor upon leukotriene C4 production by human eosinophils triggered with N-formyl-methionyl-leucyl-phenylalanine.

Tumor necrosis factor (TNF) as well as the hematopoietic growth factors interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor affect several eosinophil functions. We previously reported (J. Exp. Med. 1989. 170: 467; 1990. 172: 1577) that the hematopoietic growth factors also potentiate leukotriene C4 (LTC4) formation by eosinophils as well as basophils stimulated with soluble chemotactic peptides such as N-formyl-methionyl-leucyl-phenylalanine (FMLP), but whether TNF also modulates lipid mediator generation in normodense eosinophils triggered with FMLP is unknown. Here we show that a short preincubation (10 min) of human eosinophils purified from healthy donors with low concentrations of TNF (5-150 pM) strongly enhances LTC4 formation in response to FMLP. However, basophil mediator release is not affected by TNF preincubation. Nerve growth factor (NGF), the receptor of which is structurally related to the TNF receptors, tended to suppress lipid mediator synthesis in eosinophils, in contrast to its profound potentiating capacity on basophil mediator release. Thus, the present study demonstrates a first difference in susceptibility of eosinophils and basophils towards cytokines, indicating that TNF and NGF may regulate the relative importance of effector functions of these otherwise closely related cell types.

IL-3 and IL-5 prime normal human eosinophils to produce leukotriene C4 in response to soluble agonists.

Eosinophils (Eos) produce large amounts of leukotriene C4 (LTC4) and platelet-activating factor (PAF) in response to calcium ionophore. However, the capacity of naturally occurring soluble agonists to promote lipid mediator formation by Eos is largely unknown. Our previous studies on neutrophils and basophils showed that certain hematopoietic growth factors are important regulators of lipid mediator formation. We examined LTC4 production by normal human Eos from healthy donors in response to soluble agonists with or without preincubation with the cytokines IL-3 and IL-5. Among three agonists (FMLP, C5a, PAF) tested over a wide concentration range, only FMLP induced some LTC4 formation by itself in normal Eos. However, after preincubation with IL-3 or IL-5, Eos produced detectable amounts of LTC4 in response to all three agonists. Eos primed by IL-3 or IL-5 generated at least 1 order of magnitude more LTC4 in response to FMLP as compared to C5a or PAF. FMLP-induced LTC4 production was enhanced by 26 to 635% (n = 16) and 67 to 611% (n = 12) after preincubation with IL-3 or IL-5, respectively. Priming for LTC4 production was concentration dependent occurring at IL-3 or IL-5 concentrations of 3 to 30 ng/ml and required an optimal preincubation period of 90 min. Thus, IL-3 and IL-5 profoundly modulate the production of lipid mediators by Eos in response to the soluble agonists FMLP, C5a, and PAF. Our data further support the importance of these cytokines in inflammatory reactions involving Eos.

Topical thrombin-induced IgE-mediated anaphylaxis: RAST analysis and skin test studies.

Bovine topical thrombin (BTT) is a heterologous plasma thrombin concentrate that has been frequently used for the hemostasis since the 1940s. Recently, three patients in Japan went into shock after the topical application of BTT at lesion sites, and two of these patients had received BTT repeatedly. The clinical symptoms and the increased anti-BTT percent RAST counts suggest that these reactions were shock mediated by anti-BTT IgE antibodies. The RAST-inhibition analysis suggested that the antigenic substance(s) were bovine-specific moiety(ies) mainly involved in the contaminant rather than bovine thrombin itself. The skin tests were studied to predict such allergic reactions. The intracutaneous test provoked nonspecific reactions even at the low concentrations of BTT. The prospective study on the predictive value of the prick test with 1000 U/ml (1 mg/ml) of BTT in 192 patients suggested that it is useful to detect highly sensitive patients. In addition, the increased levels of anti-BTT IgE antibodies in patients 1 month after the single administration of BTT suggested the immunogenicity of the topical application of BTT.

Regulation of mediator release by human basophils: importance of the sequence and time of addition in the combined action of different agonists.

Biologically active molecules affecting basophil function can be divided into 4 groups according to their capacity to induce basophil degranulation and/or leukotriene generation: (1) full agonists such as anti-IgE or fMLP, which induce both histamine and leukotriene release; (2) partial agonists such as C5a, which induces degranulation only; (3) incomplete agonists such as neutrophil-activating peptide-1, platelet-activating factor or C3a, which induce mediator release only after cytokine preincubation, and (4) basophil response modifiers, such as interleukin-3, interleukin-5 and granulocyte/macrophage- colony-stimulating factor, which (a) enhance the releasability to all basophil agonists, (b) change the mediator profile, (c) enhance the rate of mediator release, (d) render basophils responsive to lower agonist concentrations and (e) render basophils responsive to incomplete agonists. We demonstrated that histamine release and de novo synthesis of lipid mediators are clearly separately regulated, and that combined actions of different molecules are of importance. In particular, the type(s), the time interval and the sequence of action of basophil-activating molecules are crucial for the final outcome of the basophil release reaction.

Influence of environmental factors on IgE production.

The prevalence of atopic diseases appears to have increased rapidly, especially in industrialized countries. The increase may be explained by a change in certain environmental factors. This article focuses on the influence of environmental factors on IgE production. Epidemiological or experimental reports have shown that tobacco smoke, virus infection and mercuric chloride may enhance IgE production. We demonstrated the enhancing effect of diesel-exhaust particulates (DEP), which seem to have increased in urban environments, on IgE antibody production. The IgE antibody responses in mice immunized by intraperitoneal injection of antigens mixed with DEP were higher than those in animals immunized with the antigens alone. DEP also had an adjuvant activity for IgE antibody production in mice after entry via the respiratory tract (the natural mode of entry). The enhancing effect of DEP on IgE antibody responses was demonstrated even when a small dose such as 1 micrograms of DEP was given intranasally at three-week intervals. Our further study has indicated that suspended particulate matter including materials other than DEP has an adjuvant activity for IgE antibody production.

Enhancing effect of suspended particulate matter on the IgE antibody production in mice.

Suspended particulate matter (SPM), suspended in the polluted environmental atmosphere, are perpetually inhaled into the human body and are considered to have profound effects on human health. This study investigated the enhancing effect of SPM on the IgE antibody production in mice. The IgE antibody responses in mice immunized with intranasal administration of ovalbumin (OA) plus SPM at 3-week intervals were higher than responses in the animals immunized with OA alone. When the dose of OA administered as an antigen was 0.25 microgram, the time course and magnitude of enhancement by SPM was comparable to those by killed Bordetella pertussis, a common adjuvant. SPM had an enhancing effect on IgE antibody production even in a small dose such as 0.25 microgram administered at 3-week intervals. The possibility cannot be excluded that the natural exposure of humans to SPM in the environmental atmosphere may explain the high prevalence rate of allergic rhinitis caused by pollens in polluted districts in Japan.

Diesel-exhaust particulates inoculated by the intranasal route have an adjuvant activity for IgE production in mice.

Our previous study indicated that the IgE antibody responses in mice immunized with intraperitoneal injection of the antigens mixed with diesel-exhaust particulates (DEP) were higher than those in the animals immunized with the antigens alone. We examined the adjuvant activity of DEP inoculated by the intranasal route, i.e., the natural entrance of DEP. In 3-week interval immunization, the IgE antibody responses in mice immunized with intranasal inoculation of ovalbumin (OA) mixed with DEP were higher than responses in the animals immunized with OA alone. DEP had an adjuvant activity for anti-OA IgE antibody production, even in a small dose such as 1 micrograms administered with a 3-week interval. Also in 1-week interval immunization, the enhancing effect of DEP on anti-OA IgE antibody production was demonstrated when mice were immunized with intranasal inoculation of OA and DEP. The possibility cannot be excluded that DEP, which are kept buoyant in the environmental atmosphere of urban districts, may exert an adjuvant activity for IgE antibody production after being inhaled into the human body and have some relation to the mechanism of the outbreak of allergic rhinitis caused by pollens in Japan.