IL-3 up-regulates and activates human eosinophil CD32 and αMß2 integrin causing degranulation.

BACKGROUND: Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. OBJECTIVE: We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. RESULTS: Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. CONCLUSIONS AND CLINICAL RELEVANCE: We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.

Segmental allergen challenge enhances chitinase activity and levels of CCL18 in mild atopic asthma.

BACKGROUND: Allergic airway inflammation contributes to the airway remodelling that has been linked to increased obstruction and morbidity in asthma. However, the mechanisms by which allergens contribute to airway remodelling in humans are not fully established. CCL18, chitotriosidase (CHIT1) and YKL-40 are readily detectable in the lungs and contribute to remodelling in other fibrotic diseases, but their involvement in allergic asthma is unclear. OBJECTIVE: We hypothesized that CCL18, YKL-40 and CHIT1 bioactivity are enhanced in allergic asthma subjects after segmental allergen challenge and are related to increased pro-fibrotic and Th2-associated mediators in the lungs. METHODS: Levels of CCL18 and YKL-40 protein and chitotriosidase (CHIT1) bioactivity in bronchoalveolar lavage (BAL) fluid, as well as CCL18, YKL-40 and CHIT1 mRNA levels in BAL cells were evaluated in patients with asthma at baseline and 48 h after segmental allergen challenge. We also examined the correlation between CCL18 and YKL-40 levels and CHIT1 activity with the levels of other pro-fibrotic factors and chemokines previously shown to be up-regulated after allergen challenge. RESULTS: Chitotriosidase activity and YKL-40 and CCL18 levels were elevated after segmental allergen challenge and these levels correlated with those of other pro-fibrotic factors, T cell chemokines, and inflammatory cells after allergen challenge. CCL18 and YKL-40 mRNA levels also increased in BAL cells after allergen challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that CCL18 and YKL-40 levels and CHIT1 activity are enhanced in allergic airway inflammation and thus may contribute to airway remodelling in asthma.

Human eosinophils release IL-1ß and increase expression of IL-17A in activated CD4+ T lymphocytes.

BACKGROUND: Differentiation and activation of CD4(+) T cells is controlled by various cytokines produced by innate immune cells. We have shown that eosinophils (EOS) have the potential to influence Th1 and Th2 cytokine generation by CD4(+) cells, but their influence on IL-17A (IL-17) has not been established. OBJECTIVE: The purpose of this study is to determine the effect of EOS on IL-17 production by lymphocytes. METHODS: Pre-activated CD4(+) T cells were cultured in the presence of either autologous EOS or EOS culture supernatants. Expression of IL-17 was determined by real-time quantitative PCR (qPCR) after 5 h and protein level was measured after 48 h. To determine the effect of allergen-induced airway EOS on IL-17, subjects with mild allergic asthma underwent bronchoscopic segmental bronchoprovocation with allergen (SBP-Ag) after a treatment with an anti-IL-5 neutralizing antibody (mepolizumab) to reduce airway eosinophilia. IL-17 mRNA was measured in bronchoalveolar lavage (BAL) cells by qPCR. RESULTS: In vitro, EOS significantly increased IL-17 production by CD4(+) T cells. Addition of exogenous IL-1ß increased expression of IL-17 mRNA by CD4(+) T cells. EOS expressed and released IL-1ß. Furthermore, levels of IL-1ß in EOS supernatants highly correlated with their ability to increase IL-17 expression by CD4(+) T cells, and neutralizing antibody to IL-1ß reduced expression of IL-17 mRNA. In vivo, reduction of EOS in the airway using mepolizumab was associated with diminished IL-17 expression after SBP-Ag. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate that EOS can promote IL-17 production through the release of IL-1ß. Enhanced IL-17 cytokine production is another mechanism by which EOS may participate in pathogenesis of allergic airway inflammation in asthma.

Minute quantities of granulocyte-macrophage colony-stimulating factor prolong eosinophil survival.

Allergic asthma is characterized by pulmonary infiltration and accumulation of eosinophils, which is enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). T cells, fibroblasts, and eosinophils themselves produce GM-CSF, suggesting it functions in the lung microenvironment as a survival factor. However, the amounts and the mechanism by which GM-CSF supports eosinophil survival remain poorly understood. We have previously reported that human peripheral blood eosinophils (PBEo) can be transfected with GM-CSF mRNA using particle-mediated gene transfer (PMGT). Using this technology, GM-CSF mRNA was introduced into resting PBEo, and GM-CSF production and cell survival were assessed. GM-CSF protein was undetectable (< 1 pg/ml) in the supernatant but present intracellularly at very low levels. Unexpectedly, the in vitro survival of transfected PBEo was 4-fold greater than that of controls. Neutralizing anti-GM-CSF but not anti-interleukin-5 (anti-IL-5) antibody added up to 24 h after transfection abolished enhanced survival, demonstrating that the continuous presence of GM-CSF was required. Conditioned medium prepared from transfected PBEo prolonged the survival of naive cells. Comparable survival activity was mimicked by a single dose of 100-500 pg/ml or multiple administrations of 0.1 pg/ml recombinant human GM-CSF (rHuGM-CSF). Survival was completely inhibited by a Jak2 inhibitor, suggesting that GM-CSF-mediated survival involved signaling through the Jak-Stat pathway. Thus, autocrine production of low levels of GM-CSF by a minority of PBEo can block apoptosis of the entire culture by a minute but sustained GM-CSF release.

[Sleep and dreams in Korsakoff's amnesia due to alcoholism]. / Sommeil et rêves dans l'amnésie de Korsakoff d'origine alcoolique.

Sleep and dreams in 15 chronic alcoholic patients with amnesia were compared with sleep and dreams of 15 age- and sex-adjusted normal subjects. The patients were subjected to psychological tests in order to determine their I.Q. and their memory disturbances. All subjects had two nights of polygraphic recordings; the first tested the natural sleep organization. During the second night, they were awakened 7 min after the onset of each REM sleep episode, and, at least once, 20 min after the onset of a stage II episode, in order to record on a tape their dream reports according to a standardized protocol. The sleep patterns of the amnesic patients did not show any significant alteration. However, after wakening during the night, patients exhibited a higher tendency to return to REMS than controls. There was still some dream activity in those patients, although noticeably less frequently, and their dream activity had a very poor verbal expression. However, there was no change with respect to the spatio-temporal organization, sensorial perceptions, motor activity and verbalizations during their dreams.

[Lesion of the deep branch of the ulnar nerve caused by fracture of the hook of the hamate]. / Atteinte de la branche profonde du cubital par fracture de l'os crochu.

We present a case of lesion of the deep branch of ulnar nerve at the wrist caused by fracture of the hook of the hamate. According to Wu's classification (1985) based on clinical and electromyographic findings and the clinicoanatomic correlations, our case corresponds to type IV of this classification with a pure motor ulnar neuropathy with sparing of hypothenar muscles. Improvement was observed after surgery.

[Long-term effects of treatment with SMS 201-995 on sleep apnea syndrome associated with acromegaly]. / Effets à long terme du traitement par SMS 201-995 sur un syndrome d'apnées du sommeil associé à une acromégalie.

A 60-year-old woman with acromegaly associated with sleep apnea was treated with the somatostatin analogue SMS 201-995 (Sandoz) for several months. Growth hormone levels were normalized and a rapid improvement in sleep apnea was controlled with polygraphic nocturnal monitoring. Hypophysectomy seems to have variable effects on sleep apnea in acromegaly. The origin of obstructive apnea in acromegaly is therefore unclear.

Frequency of obstructive sleep apnoea syndrome detected by means of a questionnaire in patients with coronary heart disease.

As previous studies have suggested an association between obstructive sleep apnea syndrome (OSA) and ischaemic heart disease, the aim of the present study was to evaluate the frequency of clinically relevant OSA in this selected population. From September 1992 to April 1993, 136 patients referred to the Cardiology Unit for suspected angina pectoris were asked to participate in the study. The main inclusion criterion was a stable coronary heart disease, diagnosed by angiography. A sleep questionnaire was administered by a trained physician. Patients who experienced one of the following symptoms: association of body mass index (-weight/height2) above 27.5 and heavy snoring, breathing stops, or daytime hypersomnolence, were selected for a nocturnal oxygen saturation recording. The occurrence of at least one desaturation, defined as a 4% fall from baseline, led to a full night-time polysomnography. One hundred and eight patients (78 males), satisfied the entry criteria. A pulse oximetry was performed in 15, and three patients experienced a significant desaturation. Finally, one patient satisfied the criteria of OSA. The estimated proportion of OSA amounted to 0.92%, in accordance with the prevalence of OSA described in the general population. Clinically relevant OSA does not appear to be more frequent in patients suffering from stable coronary heart disease. Systematic sleep investigations in such patients do not appear to be useful in daily clinical practice.

[Axonal neuropathy and chronic Codobromyl poisoning]. / Neuropathie axonale et intoxication chronique au Codobromyl.

We report a 56 year-old woman with a severe axonal neuropathy due to a chronic intoxication by a cough soothing (Codobromyl). Symptoms of codeine and alcohol intoxication were present. The problems of self medication are considered.

[Regressive leukoencephalopathy induced by an overdose of cyclosporine A]. / Leucoencéphalopathie régressive au cours d'un surdosage en cyclosporine A.

We report a case of leucoencephalopathy with tremor and generalized motor seizures a few days after introduction of cyclosporin A (Cy A) in a kidney recipient. There was a relationship between acute neurological symptoms and high and sudden blood level of Cy A (1 260 ng/ml; therapeutic range: 120-275 ng/ml). White-matter hypodensities at CT scanning and severe slow-waves abnormalities at EEG were present. All neurological symptoms were reversible after Cy A level was lowered. Three months later, a blood Cy A level at 217 ng/ml due to nicardipine co-administration resulted in severe tremor and was reversed by decreasing blood Cy A level to 126 ng/ml.

[Sleep and dreams in patients with parietal and frontal lobe lesions]. / Sommeil et rêve chez des patients atteints de lésions partiétales et frontales.

Polygraphic recordings, psychological tests, and analyses of dreams during paradoxical sleep were conducted in 9 patients with parietal lobe, in 7 with frontal lobe lesions and in a control group. No significant differences in sleep organization were observed in the parietal group, but there was a considerable reduction in oneiric activity and alterations in results of some psychological tests. Several correlations based on statistical data are discussed. Oneiric activity was disorganized to a much lesser degree in patients with frontal lesions.

[Radiation induced femoral palsy (author's transl)]. / Paralysie crurale post-radique.

We report four cases of femoral palsy due to compressive fibrosis, after pelvic radiation therapy. Three patients had Hodgkin's disease, and one testicular seminoma. Prominent clinical features include major groin induration and underlying swelling. Unlike what is usually seen in tumor relapse, little or no pain is associated with these neuropathies. The femoral post-radiation palsy develops earlier and faster than brachial plexus palsy of same aetiology. In one case, progressive aggravation led to surgical neurolysis which resulted in dramatic and long lasting improvement. The principal preventive and therapeutic managements are discussed: since compressive fibrosis is related to the use of isolated and massive electron beam therapy, various association of cobalt and electron beam therapy are designed to best prevent the side effects of each of these methods. The early treatment of developing fibrosis by D. penicillamine is discussed.

Granulocyte macrophage-colony-stimulating factor mRNA is stabilized in airway eosinophils and peripheral blood eosinophils activated by TNF-alpha plus fibronectin.

Airway eosinophils show prolonged in vitro survival compared with peripheral blood eosinophils (PBEos). Recent studies have shown that autocrine production and release of GM-CSF is responsible for enhanced survival, but the mechanisms controlling cytokine production remain obscure. We compared GM-CSF mRNA decay in eosinophils from bronchoalveolar lavage (BALEos) after allergen challenge or from PBEos. BALEos showed prolonged survival in vitro (60% at 4 days) and expressed GM-CSF mRNA. The enhanced survival of BALEos was 75% inhibited at 6 days by neutralizing anti-GM-CSF Ab. Based on transfection studies, GM-CSF mRNA was 2.5 times more stable in BALEos than in control PBEos. Treatment of PBEos with fibronectin and TNF-alpha increased their in vitro survival, GM-CSF mRNA expression, and GM-CSF mRNA stability to a comparable level as seen in BALEos. These data suggest that TNF-alpha plus fibronectin may increase eosinophil survival in vivo by controlling GM-CSF production at a posttranscriptional level.

Primary peripheral blood eosinophils rapidly degrade transfected granulocyte-macrophage colony-stimulating factor mRNA.

Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV-driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t1/2 in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t1/2 of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded, suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM-CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow detailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.

Stabilization of granulocyte-macrophage colony-stimulating factor RNA in a human eosinophil-like cell line requires the AUUUA motifs.

Human eosinophils activated by calcium ionophore produce granulocyte-macrophage colony-stimulating factor (GM-CSF). In T lymphocytes GM-CSF messenger RNA (mRNA) stability is regulated by 3' untranslated region (UTR) adenosine-uridine-rich elements (AREs). We show endogenous GM-CSF mRNA is rapidly induced in an eosinophil cell-line (AML14.3D10) after activation with ionomycin. To calculate the decay rate of GM-CSF mRNA in activated cells, eosinophils were transfected with wild-type, full-length GM-CSF mRNA or a mutant version lacking the AUUUA motifs. In unstimulated cells, wild-type GM-CSF mRNA decayed with a half-life time (t1/2) of 6+/-2 min while the mutant decayed with a t1/2 of 20+/-4 min, demonstrating the dominant, destabilizing effect of multiple AUUUA motifs. Within 1 hr of activation by ionomycin, the half-life of transfected wild-type mRNA increased by 2.5-fold, which increased up to 4-fold after 2 hr of activation. The half-life of the mutant GM-CSF was unaffected by ionomycin, demonstrating that ionophore-mediated stabilization requires intact AUUUA motifs. Actinomycin D (ActD) stabilized wild-type GM-CSF mRNA as well, causing poly(A) tail elongation and translation inhibition. These data show that in eosinophil-like cell lines, GM-CSF mRNA is exquisitely unstable but can be markedly stabilized by calcium ionophore. Both effects require intact 3' UTR AREs.

Expression of IL-17 in human memory CD45RO+ T lymphocytes and its regulation by protein kinase A pathway.

In the present study, the authors compared the interleukin 17 (IL-17 expression of human naive and phenotypically defined memory T cells as well as its regulation by cAMP pathway. Our data showed that IL-17 mRNA was highly expressed in memory human peripheral CD8(+)45RO+T cells and CD4(+)45RO+T cells when peripheral blood mononuclear cells were first stimulated with ionomycin/PMA. IL-17 expression in memory CD8(+)T cells required accessory signals since culture of ionomycin/PMA-activated CD8(+)45RO+T cells alone did not result to IL-17 expression. In contrast, memory CD4(+)T cell population seems to be more independent. IL-17 and interferon gamma(IFN-gamma) mRNA were both inhibited in the presence of PGE2 or the cAMP analogue (dibutyryl-cAMP), while the anti-inflammatory cytokine IL-10 was highly increased. In contrast, naive CD45RA+T cells were unable to express IL-17 whatever the culture conditions. Naive CD4(+)and CD8(+)T cells were sensitive to the PKA regulatory pathway since they represent a significant source of IL-10 when PBMC were first cultured with ionomycin/PMA in the presence of either PGE2 or db-cAMP. The authors showed that naive cells are highly dependent to their microenvironment, since culture of ionomycin/PMA-activated CD45RA+T cells alone did not result in detectable levels of cytokines even in the presence of PGE2. Results also showed that PGE2 induced quite the same levels of intracellular cAMP in naive and memory cells suggesting that these cell populations are equally sensitive to PGE2. However, we suggest that PGE2 may be more efficient in blocking both IL-17 and IFN-gamma expression in already primed memory T cells, rather than in suppressing naive T cells that could represent a significant source of IL-10. Data suggest that PKA activation pathway plays a critical role in the regulation of cytokine profiles and consequently the functional properties of both human naive and memory CD4(+) and CD8(+)T cells during the immune and inflammatory processes.