Human umbilical cord blood monocyte-derived eosinophils produce superoxide but not nitric oxide.

OBJECTIVE: To investigate whether in vitro derived eosinophils release nitric oxide (NO), whose role in the pathogenesis of asthma is under intense debate. MATERIALS AND METHODS: Human umbilical cord mononuclear cells were isolated from umbilical cord blood cells and cultured in vitro in presence of interleukin-3 and interleukin-5. Superoxide generation was monitored with dihydrorhodamine-123, NO release was estimated by measuring the accumulation of nitrite. Expression of NO synthases proteins was detected by immunoblotting. RESULTS: Both N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine, and 1-O-Alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine induced superoxide release in umbilical cord eosinophils, while no response was observed with lipopolysaccharide, interleukin-4 and/or interferon-gamma. Furthermore, upon activation with different inflammatory stimuli, neither induction of nitric oxide synthesis nor expression of the constitutive and/or inducible nitric oxide synthase were observed in these eosinophils derived in vitro. CONCLUSION: Human umbilical cord derived eosinophils are able to produce superoxide as peripheral blood eosinophils. Whether human peripheral eosinophils are capable of NO synthesis is still the subject of considerable debate, nevertheless, our results suggest that these in vitro derived eosinophils are not capable of nitric oxide synthesis.

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium.

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.

[Selective differentiation and proliferation of human eosinophils from umbilical cord blood: calcium fluxes and superoxide ion secretion]. / Différenciation sélective et prolifération d'éosinophiles humains issus de cellules souches de sang ombilical: flux calciques et sécrétion d'ions superoxydes.

Investigation of the physiologic mechanisms involved in the activation of eosinophils is crucial to comprehend their role in the pathogenesis of allergic reactions. To overcome the difficulty of obtaining large numbers of eosinophils, we differentiated in vitro eosinophils from human umbilical cord blood mononuclear cells. These cells responded to fMLP or PAF with an increase in [Ca2+]i, associated with O2 production. Deprivation or chelation of extracellular calcium induced a reduction of fMLP or PAF-induced [Ca2+]i rise and O2- production. Similar results were obtained with extracellular Ni2+ addition. Chelation of intracellular calcium induced an inhibition of fMLP- or PAF-induced [Ca2+]i rise and a decrease in O2- production. Our results indicate that fMLP- and PAF-dependent O2- production in eosinophils requires intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.

Physiological characteristics of identified motor units in the mouse extensor digitorum longus muscle: an in vitro approach.

Physiological, histochemical, and morphometric properties of fast-twitch single motor units were studied in mouse extensor digitorum longus muscles in an in vitro ventral root-nerve-muscle preparation. Single motor units were functionally isolated by microdissection of the ventral root, and the glycogen depletion technique was used to demonstrate the component muscle fibers. Monoclonal antibodies were used to identify their myosin heavy chain composition. The technique allows one to correlate physiological characteristics of single motor units with fiber type but is less useful for morphological assessment of motor unit size as a result of failure to deplete glycogen from all fibers of motor units containing fibers with high oxidative capacity.

Human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5 respond to fMLP with [Ca2+]i variation and O2- production.

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing normal morphology and cyanide-resistant peroxidase. O2- production and [Ca2+]i rise were measured in these in vitro differentiated eosinophils after fMLP stimulation; with dihydrorhodamine-123 and fura-2, respectively. Umbilical cord blood-derived eosinophils responded to fMLP (0.01 nM to 3 microM) with a concentration-dependent production of O2- (EC50 = 63.1 +/- 17.2 nM; Emax = 71.0 +/- 6.2 pmol/min/10(6) cells). O2- production was correlated with an fMLP concentration-dependent increase in [Ca2+]i (EC50 = 32.5 +/- 14.9 nM; Emax = 200.0 +/- 23.9 nM). These results indicate that human umbilical cord blood-derived eosinophils demonstrate functional characteristics similar to adult human peripheral blood eosinophils after activation by fMLP. Therefore, the large numbers of eosinophils (2-3 x 10(6)/ml cord blood) which can be obtained by culture of human cord blood mononuclear cells may serve as a useful model for future studies which will provide insight into the pathogenesis of diseases associated with eosinophils.

Identification, distribution, and myosin subunit composition of type IIX fibers in mouse muscles.

The purpose of the present investigation was to study the distribution and subunit composition of type IIX fibers in mouse muscles. The existence of a population of type IIX fibers in fast-twitch muscles of the mouse was shown by mean of immunohistochemistry and gel electrophoresis. In the hindlimb muscles, tibialis anterior (TA) and extensor digitorum longus (EDL), type IIX fibers account for approximately one third of the total fiber number, with the superficial portion of the TA (TAS) being composed exclusively of type IIB and IIX fibers. A similar proportion of IIX fibers was found in diaphragm (DIA) while in tongue muscles approximately 40% of the fibers were IIX. Single fiber gel electrophoresis revealed a significant number of fibers in TAS that contain both IIB and IIX myosin heavy chain (MyHC). This was confirmed with immunohistochemistry, which revealed the presence of fibers with various degrees of staining intensity. This suggests that there may exist a degree of plasticity which results in the conversion of IIX fibers to IIB fibers and vice versa. Analysis of myosin light chain (MyLC) composition of type IIX fibers revealed that the ratio of MyLC3f to MyLC1f was significantly lower than in type IIB fibers.