Histaminase release from human eosinophils.

Phagocytosis of opsonized zymosan by human eosinophils results in a dose-dependent noncytotoxic release of histaminase as well as arylsulfatase and beta-glucuronidase. The calcium ionophore A23187 also stimulates release of eosinophil histaminase at concentrations of ionophore which barely release arylsulfatase and beta-glucuronidase. Zymosan-induced histaminase release from eosinophils but not from neutrophils was abolished or markedly reduced in the presence of cytochalasin B, suggesting a difference in the mechanisms of histaminase release from the two granulocyte cell types.     

Stimulation of degranulation from human eosinophils by platelet-activating factor

Platelet-activating factor (PAF) is a highly active mediator which has been implicated in allergic inflammation and bronchial asthma, possibly by interacting with eosinophils. We have examined the effect of PAF on activation of purified human eosinophils as measured by degranulation (eosinophil peroxidase, eosinophil cationic protein, arylsulfatase B, beta-glucuronidase, and alkaline phosphatase) and oxidative metabolism (superoxide anion production). PAF induced enzyme release at concentrations ranging from 1 pM to 10 microM in a rapid (t1/2 5 to 8 min), Ca2+-dependent and noncytotoxic manner from both the specific and small granules, whereas its biologic precursor and metabolite, lyso-PAF, had no effect. For all enzymes, maximal enzyme release occurred at 100 nM PAF with a mean ED50 value of 1.47 +/- 0.4 nM. At this concentration the mean percentage of total enzyme release by PAF from specific granules was 20.3 +/- 1.6% (17.9% for eosinophil peroxidase, 20.6% for beta-glucuronidase, 22.4% for alkaline phosphatase) and 28.8 +/- 2.2% from small granules (arylsulfatase B). Calcium ionophore A23187, PMA, and opsonized zymosan also induced eosinophil degranulation but their peak effect after 10-min incubation with maximal release 14.7%, 12.9%, or 14.1%, respectively, was lower when compared with PAF. Incubation of eosinophils with the PAF-antagonist WEB 2086 led to a parallel shift of the dose-response curve to the right, indicating a competitive antagonism. PAF also caused generation of superoxide anions by human eosinophils but this occurred at higher concentrations of PAF (1 microM to 30 microM) with an ED50 of 8.4 +/- 0.9 microM. Again, this effect was competitively inhibited by WEB 2086. These studies demonstrate that PAF activates human eosinophils to release granule constituents and generate superoxide anions. Since both PAF and eosinophil products are associated with pathogenesis of bronchial asthma our findings may be of particular pathophysiologic relevance.     

Formation and proliferative effects of lipoxins in human bone marrow

Lipoxins A4 and B4 together with the all-trans lipoxin (LX) isomers were produced by normal human bone marrow cell suspensions after incubation with ionophore A23187. Both LXA4 and LXB4 enhanced the growth of myeloid progenitor cells in semisolid agar in the presence of suboptimal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lipoxin A4 at 10(-10) M stimulated the colony formation in 13 out of 15 tested human bone marrows with a mean (+/- SEM) increase of 47 +/- 11% (p = 0.001). A similar stimulatory effect was observed after addition of LXB4 (10(-10) M). The monohydroxyeicosatetraenoic acids 5-, 12- and 15-HETE did not affect colony growth. In addition, LXA4 (10(-8) M) efficiently counteracted the increased colony formation induced by leukotriene C4 (10(-10) M), suggesting an antagonistic relationship between these lipoxygenase products. The results support a role for lipoxins in the regulation of human myelopoiesis.     

Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils

Background

Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.

Objective

In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.

Methods

Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.

Results

The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.

Conclusions

Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.     

Design, synthesis and bioactions of novel stable mimetics of lipoxins and aspirin-triggered lipoxins

The lipoxins (LX) are a class of potent endogenous oxygenated products that are enzymatically generated from arachidonic acid and have novel anti-inflammatory properties and promote resolution. Elucidation of the biochemical pathways involved in the metabolic inactivation of LX and the discovery of the aspirin-triggered lipoxins (ATL) provided the basis for the design and synthesis of stable analogs of LX and ATL. This special issue review describes the efforts that led to the design and synthesis of stable LX/ATL mimetics, which permitted the detailed elucidation of their novel biological roles, leading to the development of new anti-inflammatory agents that mimic their actions. These synthetic molecules provided the means to uncover the physiologic roles of both the LX and the ATL biosynthetic pathways which led to several unexpected discoveries. Among these findings is the involvement of polyisoprenyl phosphates (PIPP) in intracellular signaling mediated by presqualene diphosphate (PSDP), and the recognition of the novel roles of these lipid mediators in regulating cell trafficking during inflammation as well as in promoting resolution of inflammatory processes. These efforts also provided the basis for examining the potential therapeutic role of LX/ATL stable mimetics and led to the development of new analogs with improved pharmacokinetics that opened the way to potentially new approaches to treating human diseases.     

Morphometry of eosinophils in human blood

Ultrastructural characteristics of human blood eosinophils have been analysed by morphometric methods in 18 healthy adults. Data were obtained relating to whole cell, nucleus, mitochondria and specific granules. No significant differences were found between the eosinophils of the 9 male and 9 female subjects. The results provide normal values against which changes in eosinophils in disease states may be assessed.     

SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils

The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.     

Activated human eosinophils synthesize new proteins

In order to study the biochemical consequences of prolonged in vitro activation of human blood eosinophils, aqueous whole cell lysates, cell-free supernatants from resting eosinophils, and cells activated with opsonized zymosan, calcium ionophore (A23187), N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and phorbol 12-myristate 13-acetate (PMA) were analyzed by polyacrylamide gel electrophoresis (PAGE). In comparison to resting eosinophils, opsonized zymosan-activated eosinophil extracts demonstrated altered protein composition on both the native PAGE and sodium dodecyl sulfate (SDS) -PAGE. Three new polypeptides of apparent molecular mass 24 kDa, 43 kDa and 60 kDa appeared on SDS-PAGE gels when opsonized zymosan-activated eosinophil extracts were electrophoresed. In contrast, extracts from fMet-Leu-Phe, A23187, and PMA-activated eosinophils demonstrated neither altered polypeptide composition nor new polypeptides. Opsonized zymosan also induced the incorporation of L-[35S]methionine into eosinophil proteins and this was completely blocked by pretreating the cells with cycloheximide. This finding suggests that eosinophils activated by certain stimuli synthesize new proteins. These newly synthesized proteins, which are freely secreted into the medium during cell activation, may possess important immunological functions.     

Singlet oxygen production by human eosinophils

Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.     

Comparison of different methods for the purification of eosinophils from human peripheral blood

Eosinophils are often purified in discontinuous gradients. Since continuous gradients usually provide a greater recovery of more highly purified cells, the present investigation was undertaken to compare the purification of eosinophils from normal whole blood in continuous and discontinuous gradients of Percoll. Contrary to our expectations, recovery and purity of eosinophils obtained from the discontinuous gradients were comparable to or higher than those from the continuous gradients of Percoll that were tested with whole blood. The purity of the modal fractions of eosinophils from the discontinuous gradients was between 88 and greater than 99% of the nucleated cells and from the continuous gradients, 80 to 93% of the nucleated cells. We have compared continuous and discontinuous gradients with many different kinds of cells. This is the first time we have found continuous and discontinuous gradients equally effective. We speculate this finding is related to the fact that the band capacities are vastly overloaded in these gradients. In addition, we tested the rate of superoxide production by eosinophils from the same donors after their purification by two different methods in discontinuous gradients. Eosinophils purified from normal whole blood in gradients of Percoll by a modification of the method of Roberts and Gallin [1985) Blood 65, 433-440) had a higher rate of superoxide production after stimulation with phorbol myristate acetate than those purified from leukocyte-rich plasma in gradients of Metrizamide by the method of Vadas et al. [1979) J. Immunol. 122, 1228-1236).     

Effects of dexamethasone on TNF-alpha-induced release of cytokines from purified human blood eosinophils

A 25-year-old, with type I Diabetes Mellitus with a previous diagnosis of Protamine Allergy but not to human Insulin, started to notice anaphylactic reactions inmmediatly after bolus with Insulin. Skin prick and intradermal test were positive to all insulins. Skin tests to other potential allergens resulted negative. Examination after bolus of Human Insulin revealed urticaria. Daily insulin requirement were around 2-2,4 U/Kg/day. Slow desensitisation with Aspart insulin, the insulin with lowest size of skin test, was performed using subcutaneous insulin pump. Six months after the end of desensitisation his daily insulin requirement decreased to 0.8 U/Kg/day and oral corticosteroids are being reduced with no symptoms.     

Transcellular conversion of endogenous arachidonic acid to lipoxins in mixed human platelet-granulocyte suspensions

Incubation of mixed human platelet/granulocyte suspensions with ionophore A23187 led to a platelet dependent formation of several lipoxin isomers from endogenous substrate. The major metabolite coeluted with authentic lipoxin A4 (5(S), 6(R), 15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) in several HPLC-systems and showed an identical UV-spectrum. Furthermore, a similar profile of lipoxins was formed in pure platelet suspensions incubated with exogenous leukotriene A4 (5(S) -5, 6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid). The conversion of exogenous leukotriene A4 to lipoxin A4 was markedly increased in the presence of ionophore A23187.     

Effects of dexamethasone on TNF-alpha-induced release of cytokines from purified human blood eosinophils

SUMMARY: BACKGROUND: TNF-alpha is an important mediator in allergy also for its effects on eosinophils. METHODS: The effect of dexamethasone on TNF-alpha induced eosinophils survival, degranulation (ECP), cytokines release (IL-8, GM-CSF) and adhesion to VCAM-1, ICAM-1 and IgG coated wells (EPO release) were evaluated. RESULTS: The drug inhibited IL-8 and GM-CSF production, but not viability, degranulation or adhesion in human peripheral blood eosinophils. CONCLUSION: These results indicate that part of the activity of glucocorticosteroids on eosinophils may be mediated by their ability to inhibit cytokine secretion that in turn is important for the perpetuation of the allergic inflammation.     

Interactions between electron and proton currents in excised patches from human eosinophils

The NADPH-oxidase is a plasma membrane enzyme complex that enables phagocytes to generate superoxide in order to kill invading pathogens, a critical step in the host defense against infections. The oxidase transfers electrons from cytosolic NADPH to extracellular oxygen, a process that requires concomitant H+ extrusion through depolarization-activated H+ channels. Whether H+ fluxes are mediated by the oxidase itself is controversial, but there is a general agreement that the oxidase and H+ channel are intimately connected. Oxidase activation evokes profound changes in whole-cell H+ current (IH), causing an approximately -40-mV shift in the activation threshold that leads to the appearance of inward IH. To further explore the relationship between the oxidase and proton channel, we performed voltage-clamp experiments on inside-out patches from both resting and phorbol-12-myristate-13-acetate (PMA)-activated human eosinophils. Proton currents from resting cells displayed slow voltage-dependent activation, long-term stability, and were blocked by micromolar internal [Zn2+]. IH from PMA-treated cells activated faster and at lower voltages, enabling sustained H+ influx, but ran down within minutes, regaining the current properties of nonactivated cells. Bath application of NADPH to patches excised from PMA-treated cells evoked electron currents (Ie), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both IH and Ie was delayed, and sometimes prevented, by cytosolic ATP and GTP-gamma-S. A good correlation was observed between the amplitude of Ie and both inward and outward IH when a stable driving force for e- was imposed. Combined application of NADPH and DPI reduced the inward IH amplitude, even in the absence of concomitant oxidase activity. The strict correlation between Ie and IH amplitudes and the sensitivity of IH to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.     

New antihypertensive peptides isolated from rapeseed

Four potent angiotensin converting enzyme (ACE) inhibitory peptides, IY, RIY, VW and VWIS, were isolated from subtilisin digest of rapeseed protein. Among them RIY and VWIS are new peptides with IC(50) 28 and 30 microM, respectively. All isolated peptides lowered blood pressure of spontaneously hypertensive rats (SHR) following oral administration. The maximum effect in the case of RIY was observed 4h after administration, while maximum effect of other peptides on blood pressure occurred 2h after administration. Furthermore, the antihypertensive effect of RIY was observed even in old rats, in which ACE inhibitors become less effective, suggesting that a different mechanism other than ACE inhibition is also involved in lowering blood pressure by this peptide. Subtilisin digest of rapeseed protein also significantly lowered blood pressure of SHR after oral administration of a single dosage 0.15 g/kg, exerting maximum antihypertensive effect 4h after administration. This digest appears promising as a functional food, which may be useful in the prevention and treatment of hypertension.     

Pasteurella strains isolated from human sputum

Summary In four years ten strains ofPasteurella were isolated from the sputum of eight patients. Six strains proved to bePasteurella multocida (with two lactose-positive variants); the other strains were classified asPasteurella haemolytica var.ureae. (Henriksen andJyssum, 1960) orPasteurella ureae (Jones, 1962).