Generation and Metabolism of Leukotrienes and Release of Histamine from Human Dispersed Tonsillar Cells

We studied the generation and metabolism of leukotrienes (LT) and the release of histamine by human tonsillar cell suspensions. Human tonsils were dissected and mechanically dispersed. This procedure yielded a single cell suspension with 1.6 +/- 0.5 X 10(8) cells/g tissue consisting of 97.3 +/- 0.4% lymphocytes, 1.4 +/- 0.3% granulocytes, 1.3 +/- 0.3% macrophages/monocytes, and 0.03 +/- 0.02% mast cells/basophils. The cells were stimulated either with Ca-ionophore A 23187, melittin, or anti-human IgE. Determination of the 5-lipoxygenase products LTB4 and LTC4 was performed with specific radioimmunoassays (RIA), and histamine release was measured by the fluorophotometric technique. A time- and dose-dependent release of the mediators was monitored. LTB4 exceeded the amount of LTC4 in the supernatants. The concentration of leukotrienes ranged between 0.8 and 5.4 ng LTB4/1 X 10(8) cells or 0.5 and 1.5 ng LTC4/1 X 10(8) cells, depending on the stimulus. Histamine release after stimulation ranged between 25 and 35% of the total histamine content, whereas buffer controls amounted to 17%. The incubation of the cells (1 X 10(8) with exogenously added LTB4 resulted in the formation of omega-oxidated products (20-OH and 20-COOH-LTB4) and a novel unpolar metabolite, as identified by thin layer chromatography. This metabolite was not immunoreactive in the LTB4-RIA used. LTC4 and LTD4 were converted into LTE4 when added either to sonicated cells or to the cell-free supernatants of prestimulated tonsillar cells, indicating the release of gamma-glutamyltranspeptidase and dipeptidase, respectively. Our data clearly demonstrate the generation and metabolism of the 5-lipoxygenase products LTB4 and LTC4 as well as the release of histamine from human dispersed tonsillar cells, suggesting that they have a modulatory function with respect to the inflammatory potential at local sites.     

Differential modulation of mediator release from human basophils and mast cells by mizolastine

BACKGROUND: Basophils and mast cells play a major role in the pathogenesis of allergic disorders by releasing several proinflammatory mediators. Some histamine H1 receptor antagonists exert anti-inflammatory activities by modulating mediator release from basophils and mast cells. OBJECTIVE: To study the in vitro effects of mizolastine, an H1 receptor antagonist, on the release of eicosanoids, histamine and IL-4 from human basophils and lung mast cells. METHODS AND RESULTS: Mizolastine (10(-7)-10(-5) M) concentration-dependently inhibited the release of cysteinyl leukotriene C4 from anti-IgE-stimulated basophils (IC(50): 3.85+/-0.28 microM) and mast cells (IC(50): 3.92+/-0.41 microM). The same concentrations of mizolastine did not affect anti-IgE-induced prostaglandin D2 release from lung mast cells. In contrast, mizolastine enhanced up to 80% IgE-mediated histamine release (EC(50): 4.63+/-0.14 microM) from basophils, but not from mast cells and it significantly potentiated IL-4 release from basophils induced by anti-IgE. Mizolastine did not affect histamine release from basophils induced by formyl peptide, whereas it inhibited cysteinyl leukotriene C4 release (IC(50): 1.86+/-0.24 microM). Blockade of cytosolic phospholipase A2 and arachidonic acid mobilization by pyrrolidine-1 did not alter the effect of mizolastine on histamine release from basophils, thereby excluding accumulation of arachidonic acid metabolic intermediates as the cause of this effect. Mizolastine did not influence anti-IgE-induced activation of extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) in human basophils. CONCLUSIONS: Mizolastine efficiently inhibits LTC4 synthesis in human basophils and mast cells presumably by interfering with 5-lipoxygenase. In contrast, it enhances histamine and IL-4 release only from anti-IgE-stimulated basophils. Therefore, mizolastine differentially regulates the production of mediators from basophils and mast cells in a cell- and stimulus-specific fashion.     

Effects of the new 5-lipoxygenase inhibitor E6080 on leukotriene release in vitro.

Fundamental studies were conducted to examine the release of histamine and leukotriene (LT) C4 from lung fragments of guinea pigs and the effects of E6080 on the release of LTB4 and LTC4 from lung fragments or inflammatory cells. The release of histamine and LTs showed large interindividual variations and a marked dependence on experimental conditions. Addition of 10 mM L-cysteine significantly increased LTC4 release compared with that in its absence (about 1.7 times, in terms of mean value). E6080 inhibited antigen-stimulated LTB4 and LTC4 release from passively sensitized human (IC50: LTB4 0.08 microM, LTC4 0.2 microM) and guinea-pig lung fragments (IC50: LTC4 1.1 microM). The LTB4 and LTC4 releases from healthy human polymorphonuclear leukocytes (calcium ionophore A23187) and from allergic patients' leukocytes (basophils, antigen) were inhibited by E6080 with IC50 values of below 1.0 microM. Furthermore, the LTC4 release from rat alveolar macrophages (silica particles) was inhibited by E6080 with an IC50 of 0.2 microM. The potent inhibition by E6080 might be a result of the inhibition of 5-lipoxygenase, since 5-lipoxygenase in rat basophilic leukemia cell was inhibited by E6080 with an IC50 of 0.2 microM. The results confirm the potent inhibitory effects of E6080 on the release of LTs.     

Modulation of leukotriene release from human polymorphonuclear leucocytes by PMA and arachidonic acid

Stimulation of human neutrophils (PMN) with Ca ionophore A23187, opsonized zymosan and formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) led to a time- and dose-dependent release of LTB4, 20-OH-LTB4, 20-COOH-LTB4, 6-trans-LTB4, 12-epi-6-trans LTB4 and LTC4, as detected by reverse-phase HPLC. Preincubation of the PMN suspension in the presence of Ca2+ and Mg2+ with phorbol-12-myristate-13-acetate (PMA) did not release leukotrienes by itself, but modulated the subsequent Ca ionophore-induced leukotriene release. The release of LTC4, 20-OH-LTB4 and 20-COOH-LTB4 was significantly decreased. Lesser effects were observed for the release of LTB4 and the non-enzymatic LTB4 isomers. In contrast, opsonized zymosan and FMLP enhanced the release of LTB4 and LTB4-omega-oxidation products from cells pretreated with PMA. With arachidonic acid as prestimulus, the amounts of the LTB4 isomers (6-trans-LTB4 and 12-epi-6-trans-LTB4) were enhanced significantly on subsequent stimulation with Ca ionophore. Prestimulation of lymphocytes, monocytes and basophilic granulocytes (LMB) with PMA had no significant effects on the ionophore-induced release of LTC4 and LTB4. PMN, but not LMB, suspensions prestimulated with PMA convert exogenously added LTC4 to LTB4 isomers and LTC4 sulphoxide. Our data suggest that preincubation of human granulocytes with PMA modified leukotriene release by activation or inhibition of different metabolic pathways for LTC4 and LTB4.     

Signal transduction for interleukin-3-dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase.

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin-3 (IL-3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL-3 in these differentiated, non-dividing blood leukocytes. IL-3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL-3 pretreatment, indicating that the priming effect of IL-3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a-induced histamine release nor IL-3-dependent LTC4 formation in contrast to the IgE receptor-dependent basophil response. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) induced histamine release without leukotriene formation. PMA-treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL-3 effect on C5a-induced LTC4 synthesis. Only the C5a signal but not the IL-3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG-50864 and herbimycin A, were both very efficient blockers of IL-3-dependent lipid mediator formation whereas C5a-induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine-sensitive serine/threonine kinase. To the contrary, IL-3-dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.     

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils

BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS : Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.     

Enhanced granulocyte cytotoxicity by mediators derived from anti-IgE-stimulated human leucocytes.

Basophil-containing leucocyte fractions stimulated with an anti-human IgE, F(ab')2, generated histamine and the leukotrienes LTB4 and LTC4 with significant correlations between LTB4 and histamine (P less than 0.01; n = 26) and LTC4 and histamine release (P less than 0.001; n = 29) in the cell-free supernatants (SN). SN from these anti-IgE-treated cells enhanced the cytotoxicity of eosinophils and neutrophils (against complement-coated schistosomula of Schistosoma mansoni) in vitro. When SN were fractionated by reverse phase-high performance liquid chromatography (RP-HPLC), the enhancing activity for neutrophils was almost totally confined to fractions having LTB4 immunoreactivity (co-eluting as a single peak with the synthetic LTB4 marker). In contrast, the LTB4-containing fraction had minimal effects on eosinophil cytotoxicity, whereas synthetic histamine gave comparable enhancement to the unfractionated SN. The generation of LTs (but not histamine), as well as enhanced neutrophil cytotoxicity from basophil-containing leucocytes by anti-IgE treatment, was maximally inhibited by the 5-lipoxygenase inhibitors U-60, 257 and BW755C. Conversely, the cyclooxygenase inhibitor indomethacin did not significantly affect LT release, nor did it affect the subsequent cytotoxicity enhancing activity of SN from such cells. These results indicate that LTB4 and LTC4 are released from basophils, together with histamine, by IgE-dependent mechanisms, LTB4 enhances the cytotoxicity of bystander neutrophils, and histamine (and to a lesser extent, LTB4) augments eosinophil cytotoxicity.     

IL-3 in the development and function of basophils

The β common chain (βc) cytokine family includes granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5, all of which use βc as key signaling receptor subunit. GM-CSF, IL-3 and IL-5 have specific roles as hematopoietic growth factors. IL-3 binds with high affinity to the IL-3 receptor α (IL-3Rα/CD123) and then associates with the βc subunit. IL-3 is mainly synthesized by different subsets of T cells, but is also produced by several other immune [basophils, dendritic cells (DCs), mast cells, etc.] and non-immune cells (microglia and astrocytes). The IL-3Rα is also expressed by immune (basophils, eosinophils, mast cells, DCs, monocytes, and megacaryocytes) and non-immune cells (endothelial cells and neuronal cells). IL-3 is the most important growth and activating factor for human and mouse basophils, primary effector cells of allergic disorders. IL-3-activated basophils and mast cells are also involved in different chronic inflammatory disorders, infections, and several types of cancer. IL-3 induces the release of cytokines (i.e., IL-4, IL-13, CXCL8) from human basophils and preincubation of basophils with IL-3 potentiates the release of proinflammatory mediators and cytokines from IgE- and C5a-activated basophils. IL-3 synergistically potentiates IL-33-induced mediator release from human basophils. IL-3 plays a pathogenic role in several hematologic cancers and may contribute to autoimmune and cardiac disorders. Several IL-3Rα/CD123 targeting molecules have shown some efficacy in the treatment of hematologic malignancies.     

The degradation product of the C5a anaphylatoxin C5adesarg retains basophil-activating properties

The complement cleavage product C5a is a potent agonist of different leukocyte types and also has anaphylatoxic properties through the release of mediators by basophils and tissue mast cells. C5a is very rapidly degraded by serum carboxypeptidase N which cleaves the functionally important carboxy-terminal arginine, generating C5a_desarg, a chemotactic agonist with little mast cell-activating ability. Here we show that natural human C5a_desarg is still a trigger for basophil mediator release superior to other endogenous IgE-independent agonists such as monocyte chemotactic protein (MCP)-1, interleukin (IL)-8, C3a and platelet-activating factor. On a molar basis C5a_desarg is only one order of magnitude less potent and about half as efficacious as C5a at inducing basophil degranulation. Priming of basophils with either IL-3, IL-5, granulocyte-macrophage-colony-stimulating factor (GM-CSF) or nerve growth factor (NGF) (with comparable efficacies, but different potencies: IL-3>NGF>IL-5>GM-CSF) enhanced histamine release and conditioned the cells to produce large amounts of leukotriene C4 (LTC4), which is not generated by basophils exposed to C5a_desarg alone. The efficacy of C5a and C5a_desarg at inducing histamine and LTC4 release by primed basophils was similar. Thus, C5a_desarg is a stable inducer of release of inflammatory mediators by human basophils, particularly in primed cells, and complement may, therefore, play a role in immediate-type hypersensitivity diseases in allergic late-phase reactions.     

Effect of cytokines on mediator release from human dispersed lung mast cells

To evaluate the contribution of human lung mast cells (HLMC) to allergic inflammation, we investigated whether or not cytokines, including stem-cell factor (SCF), monocyte chemotactic and activating factor (MCAF), and RANTES, activate HLMC. SCF induced histamine release from dispersed HLMC in a dose-dependent fashion (P<0.01). The release was 7.8 ± 1.0% at 500 ng/ml SCF (n= 9). This response was also observed in chopped lung tissue. HLMC from which surface IgE molecules had been removed by treatment with lactic acid responded to SCF, while these cells lost their response to anti-IgE. The process was relatively rapid and reached a maximum in 5 min. This response required extracellular calcium, and it was observed at 37°C, but not at 4°C or 20°. A brief preincubation (10 min) with lower concentrations of SCF, which were ineffective in releasing histamine, enhanced anti-IgE-induced histamine release (P<0.05), while its enhancing effect was lost by the longer preincubation (30 min). SCF did not prime basophils to enhance stimulated-histamine release. Interleukin (IL)-1α, IL-1β, IL-3, IL-4, IL-5, granulocyte/macrophage-colony stimulating factor (GM-CSF), MCAF, and RANTES neither induced histamine release nor enhanced the release stimulated by anti-IgE after a 10- or 30-min preincubation. The combination of IL-3 and IL-4 showed no effect on histamine release from HLMC. Leukotriene (LT)C₄/D₄/E₄ production by SCF was negligible, as compared with anti-IgE-induced LT production. SCF at 1.5 ng/ml augmented anti-IgE-induced LT generation significantly (536+ 117 pg/10⁵ mast cells and 1569 ± 258 pg/10⁵ mast cells; P<0.01). These results provide further evidence that numerous aspects of the phenotype of mast cells and basophils are heterogeneous, including structure, relevant secretagogues, and pharmacologic control.     

Effect of deuterium oxide on leukotriene C4 generation in immunologically stimulated human leukocytes

Addition of deuterium oxide (D2O), 6-36%, resulted in a dose-dependent increase in allergen- or anti-IgE-induced leukotriene C4 (LTC4) generation from human basophils. In the presence of 36% D2O, the enhancement was 260 +/- 135% for allergen stimulation and 480 +/- 152% for anti-IgE stimulation as compared with the control incubated in normal buffer. The increasing effect of D2O on LTC4 generation from basophils was completely reversed by washing the cells before incubation with allergen. Vinblastine as well as colchicine, at a concentration of 100 microM, counteracted the effect of D2O. The enhanced release of histamine and LTC4 from basophils challenged with allergen was suppressed by Dimaprit, a histamine H2 receptor agonist, at a concentration required to inhibit the release by 50% of 5 X 10(-5) M for histamine and 10(-5) M for LTC4. These observations suggest that microtubules may be involved in LTC4 generation from immunologically stimulated basophils.     

Role of tyrosine kinases in IgE-mediated signal transduction in human lung mast cells and basophils.

Recent evidence suggests that tyrosine kinases play an important role in signal transduction mechanisms utilized by a range of different agonists in many cell types. We have investigated the effects of four different inhibitors of tyrosine kinases on IgE-dependent histamine release from human lung mast cells and basophils. Genistein inhibited the anti-IgE-induced histamine release from human basophils (at 10 microM genistein, inhibition = 55 +/- 5%, n = 17, P < 0.005) with an IC50 of 8 microM, but was much less effective in the human lung mast cell (at 10 microM, inhibition = 18 +/- 6%, n = 11, P < 0.05). Two inactive analogs of genistein, genistin and diadzein, failed to affect anti-IgE-induced histamine release significantly in either mast cells or basophils. A second inhibitor of tyrosine kinases, tyrphostin 25, inhibited IgE-dependent release from basophils (at 10 microM, inhibition = 25 +/- 7%, n = 6, P < 0.05) though it was less effective than genistein and failed to affect IgE-induced histamine release from human lung mast cells (at 10 microM, inhibition = 22 +/- 16%, n = 5, P = NS). In contrast, methyl 2,5 dihydroxycinnamate (MDC) failed to inhibit anti-IgE-dependent histamine release in human basophils (at 10 microM, inhibition = 3 +/- 3%, n = 5, P = NS) but proved to be an effective inhibitor of anti-IgE-induced degranulation in human lung mast cells (at 10 microM, inhibition = 53 +/- 16%, n = 5, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of four hemolysin-producing organisms (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) for release of inflammatory mediators from various cells.

We investigated the role of various hemolysin-producing strains (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) in induction of inflammatory mediators, e.g., histamine release from rat mast cells as well as the chemiluminescence response and the release of lipoxygenase transformation products from human polymorphonuclear neutrophils. Our data show that the hemolysin-positive bacteria as well as the hemolysin-positive culture supernatants were active in inducing the chemiluminescence response, leukotriene (LTB4 and LTC4) release from human granulocytes, and histamine release from rat mast cells. The degree of leukotriene release was dependent on the hemolysin type and on the expression of hemolysin activity. The E. coli alpha-hemolysin and the aerolysin-producing A. hydrophila were the most potent stimuli whether washed bacteria or bacterial supernatant was used. Bacteria expressing the S. marcescens hemolysin and the listeriolysin were only poor inducers of leukotriene generation. In contrast to leukotriene generation, all hemolysin-positive strains induced nearly the same histamine release in a dose-dependent manner. Our data suggest a potent role for various hemolysins as virulence factors in inducing the release of inflammatory mediators.     

The release of leukotriene B4 from human skin in response to substance P: evidence for the functional heterogeneity of human skin mast cells among individuals

Substance P is located in cutaneous nerve fibres and induces wheal and flare responses, accompanied by granulocyte infiltration, upon intradermal injection. Studies with animal skin and rat peritoneal mast cells have suggested that substance P induces the release of histamine and leukotriene B4 (LTB4), a potent chemoattractant for granulocytes, from skin mast cells. However, the release of LTB4 has not been detected from mast cells enzymatically isolated from human skin. In order to investigate the mechanism of granulocyte infiltration induced by substance P in human skin, we studied the release of LTB4 and histamine in response to substance P, and the effect of dexamethasone using human skin obtained from 22 nonallergic individuals. Histamine was released from all skin tissue samples in a dose-dependent manner. However, the amount of LTB4 release, both constitutive and inducible, was variable among skin preparations. Substance P induced a large release of LTB4 from the skin of eight donors (twice to six times that of the spontaneous release), but no or only negligible release from the skin of 14 donors. The amount of constitutive release of LTB4 correlated with the amount of tissue histamine. Dexamethasone selectively abolished the inducible release of LTB4, without an effect on histamine release and the constitutive release of LTB4. These results suggest that substance P induces the release of LTB4 in a certain population of human individuals by a glucocorticosteroid-dependent mechanism, and plays an important role in neurogenic inflammation with granulocyte infiltration.     

Oestradiol enhances in vitro the histamine release induced by embryonic histamine-releasing factor (EHRF) from uterine mast cells.

The relationship between maternal hormones and factors secreted by the implanting embryo is still controversial. We have analysed the in-vitro effect of oestradiol and human embryo-derived histamine-releasing factor (EHRF) on histamine release from rat uterine mast cells. Rat uterine mast cells which were preincubated with oestradiol and then challenged with human EHRF gave histamine release values two- to threefold higher than those without preincubation. The enhancement observed was time- and temperature-dependent. A similar enhancement was obtained with human sensitized basophils but not with rat peritoneal mast cells. Oestradiol, used as a direct challenge, did not induce any histamine release from either rat uterine or peritoneal mast cells, or from human sensitized basophils. Oestradiol preincubation also enhanced the histamine release induced by anti-IgE but did not enhance the histamine release induced by substance P or compound 48/80, two secretagogues that are not mediated by IgE. Moreover, uterine fragments derived from rats at various oestrus phases, with different amounts of endogenous oestrogen, were challenged in vitro with EHRF. The release of histamine by mast cells was higher at the proestrus and preimplantation phases than at dioestrus. All these findings suggest that the interaction of oestradiol with rat uterine mast cells was capable of enhancing in vitro the histamine releasing effect of EHRF.     

The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) is a potent inhibitor of mediator release from human dermal mast cells and peripheral blood basophils

BACKGROUND: The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) has recently been developed as a novel and cell-selective inhibitor of inflammatory cytokine secretion; it has fewer adverse effects than currently available drugs. OBJECTIVE: In this study, we investigated the capacity of pimecrolimus to directly inhibit in vitro mediator release from human skin mast cells and basophils. METHODS: Purified cutaneous mast cells or basophil-containing peripheral blood leukocytes were obtained from healthy human donors and preincubated with pimecrolimus (0.1 nmol/L to 1 micromol/L) in the absence or presence of its specific antagonist (rapamycin), cyclosporin A (100 nmol/L to 1 micromol/L), or dexamethasone (1 micromol/L) and then stimulated with anti-IgE or with calcium ionophore A23187 plus phorbol myristate acetate. Cell supernatants were kept for analysis of histamine, tryptase, LTC4, and TNF-alpha. RESULTS: Pimecrolimus caused a strong and dose-dependent inhibition of anti-IgE--induced release of histamine from mast cells and basophils (maximally 73% and 82%, respectively, at 500 nmol/L pimecrolimus) and of mast cell tryptase (maximally 75%) and a less pronounced inhibition of LTC4 (maximally 32%) and of calcium ionophore plus phorbol myristate acetate--induced mast cell TNF-alpha release (90% maximum at 100 nmol/L pimecrolimus). In contrast, inhibition achieved during mast cell histamine release was maximally 60% with cyclosporin A and only 28% with dexamethasone. CONCLUSION: These data demonstrate a marked inhibitory capacity of pimecrolimus on mediator release from human mast cells and basophils with a potency exceeding that of cyclosporin A and dexamethasone. Pimecrolimus might thus be expected to be effective in the treatment of mast cell-- and basophil-dependent diseases.     

FK506 inhibition of histamine release and cytokine production by mast cells and basophils

Histamine release and cytokine production by mast cells and basophils are thought to be closely involved in the pathogenesis of allergic diseases. Some reports show that FK506 (tacrolimus hydrate) inhibited histamine release and cytokine production by mast cells and basophils. However, as the effects of FK506 has not been compared with those of clinically used drugs in those reports, the clinical relevancy of FK506 inhibition remained unclear. In this paper, we compared the actions of FK506 with those of steroids or disodium cromoglycate (DSCG) which has been clinically used. FK506 inhibited histamine release by Brown-Norway rat peritoneal mast cells more potently than steroids and especially DSCG. FK506 also inhibited histamine release by a mast rat basophilic leukemia (RBL)-1 cell line and human peripheral blood basophils, whereas steroids failed to inhibit histamine release by human basophils. FK506 as well as steroids inhibited TNF-alpha and IL-4 production by RBL-1 cells. FK506 was therefore more effective than steroids and DSCG in inhibiting histamine release, and it also had the ability of inhibiting cytokine production by mast cells as steroids do. We concluded that FK506 might regulate allergic diseases via these actions, judging from the viewpoint of clinical relevancy.     

Effects of mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients on inflammatory mediator release from human polymorphonuclear granulocytes and rat mast cells.

Mucoid Pseudomonas aeruginosa causing chronic bronchopulmonary infection in cystic fibrosis (CF) patients may interfere with host defence mechanisms. We investigated 13 P. aeruginosa strains isolated from sputa of CF patients with regard to the induction or modulation of inflammatory mediator release from human neutrophils (PMN) and rat mast cells. The effects of mucoid as compared to non-mucoid bacteria were studied using a mucoid strain and its non-mucoid revertant. The release of leukotrienes (LT) and histamine in response to the majority of the CF strains was insignificant. However, preincubation of PMN with P. aeruginosa caused a dose-dependent decrease (50-95%) of LTB4 and LTC4 generation and LTB4 metabolism induced by the Ca(2+)-ionophore A23187 or opsonized zymosan (ZX) (P less than 0.001). The mucoid strains caused a three- to 10-fold higher impairment of LTB4 release (P less than 0.05) and a concomitant down-regulation of LTB4 receptors on neutrophils. Inhibitory effects were also obtained for mucoid and non-mucoid bacteria when the phorbol-ester or the Ca(2+)-ionophore induced luminol enhanced chemiluminescence response (P less than 0.001) or the histamine release from rat peritoneal mast cells (P less than 0.01) was studied. The bacteria-cell contact with non-mucoid strains was associated with an increased Ca2+ influx into PMN, whereas mucoid bacteria had no effect. In addition, a protein kinase C-dependent decrease of the C3bi receptor was suppressed by the mucoid--and less effectively--by the non-mucoid strain. The results suggest that the impairment of the phagocytic and inflammatory system may contribute to the pathogenesis and persistence of mucoid P. aeruginosa infection in CF.     

Calcineurin antagonists differentially affect mediator secretion, p38 mitogen-activated protein kinase and extracellular signal-regulated kinases from immunologically activated human basophils

BACKGROUND: Basophils participate in allergic diseases by invading affected tissues and secreting histamine, leukotriene (LT)C4, IL-4 and IL-13 following FcepsilonRI cross-linking. A reduction of basophil mediator production is therefore of considerable therapeutical interest. Macrolactam derivatives, which inhibit calcineurin activation, may be candidates for antiallergic therapy as they reduce both symptoms of inflammatory skin disease in animal models and mast cell degranulation. OBJECTIVE: To investigate the effects of the calcineurin antagonists ascomycin and cyclosporin A on IgE-dependent mediator release from human basophils. METHODS: Basophils were purified by Ficoll density centrifugation, elutriation and negative selection. Histamine release was measured spectrofluorometrically; LTC4, IL-4 and IL-13 secretions were assayed by enzyme-linked immunosorbent assay (ELISA). Lysed cells were subjected to Western blotting using specific antibodies to phospho-p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)-1 and -2. RESULTS: Ascomycin (0.01 nm to 1 micro m) and cyclosporin A (0.1 nm to 10 micro m) strikingly inhibited (maximally 100%) anti-IgE-induced histamine and cytokine release from basophils, and these actions were unaffected by IL-3 priming. Ascomycin, however, was less potent at blocking LTC4 secretion, whereas cyclosporin A was unable to block production of this mediator. In immunoblotting studies, ascomycin and cyclosporin A reduced IgE-dependent p38 MAPK activation but were less potent at reducing ERK phosphorylation in basophils. CONCLUSION: Calcineurin antagonists like ascomycin and cyclosporin A block IgE-dependent basophil degranulation and cytokine synthesis. Calcineurin may target p38 MAPK activation, but seems to have less activity on ERK phosphorylation. This is paralleled by a reduced or even absent effect of calcineurin antagonists on eicosanoid production.