Human Peripheral Blood Eosinophils Research Articles

The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia

BackgroundEosinophilic asthma is increasingly recognized as one of the most severe and difficult-to-treat asthma subtypes. The JAK/STAT pathway is the principal signaling mechanism for a variety of cytokines and growth factors involved in asthma. However, the direct effect of JAK inhibitors on eosinophil effector function has not been addressed thus far. ObjectiveHere we compared the effects of the JAK1/2 inhibitor baricitinib and the JAK3 inhibitor tofacitinib on eosinophil effector function in vitro and in vivo. MethodsDifferentiation of murine bone marrow-derived eosinophils. Migratory responsiveness, respiratory burst, phagocytosis and apoptosis of human peripheral blood eosinophils were assessed in vitro. In vivo effects were investigated in a mouse model of acute house dust mite-induced airway inflammation in BALB/c mice. ResultsBaricitinib more potently induced apoptosis and inhibited eosinophil chemotaxis and respiratory burst, while baricitinib and tofacitinib similarly affected eosinophil differentiation and phagocytosis. Of the JAK inhibitors, oral application of baricitinib more potently prevented lung eosinophilia in mice following allergen challenge. However, both JAK inhibitors neither affected airway resistance nor compliance. ConclusionOur data suggest that the JAK1/2 inhibitor baricitinib is even more potent than the JAK3 inhibitor tofacitinib in suppressing eosinophil effector function. Thus, targeting the JAK1/2 pathway represents a promising therapeutic strategy for eosinophilic inflammation as observed in severe eosinophilic asthma.

Differential expression of Triggering Receptor Expressed on Myeloid cells 2 (Trem2) in tissue eosinophils.

No longer regarded simply as end-stage cytotoxic effectors, eosinophils are now recognized as complex cells with unique phenotypes that develop in response stimuli in the local microenvironment. In our previous study, we documented eosinophil infiltration in damaged muscle characteristic of dystrophin-deficient (mdx) mice that model Duchenne muscular dystrophy. Specifically, we found that eosinophils did not promote the generation of muscle lesions, as these persisted in eosinophil-deficient mdx.PHIL mice. To obtain additional insight into these findings, we performed RNA sequencing of eosinophils isolated from muscle tissue of mdx, IL5tg, and mdx.IL5tg mice. We observed profound up-regulation of classical effector proteins (major basic protein-1, eosinophil peroxidase, and eosinophil-associated ribonucleases) in eosinophils isolated from lesion-free muscle from IL5tg mice. By contrast, we observed significant up-regulation of tissue remodeling genes, including proteases, extracellular matrix components, collagen, and skeletal muscle precursors, as well as the immunomodulatory receptor, Trem2, in eosinophils isolated from skeletal muscle tissue from the dystrophin-deficient mdx mice. Although the anti-inflammatory properties of Trem2 have been described in the monocyte/macrophage lineage, no previous studies have documented its expression in eosinophils. We found that Trem2 was critical for full growth and differentiation of bone marrow-derived eosinophil cultures and full expression of TLR4. Immunoreactive Trem2 was also detected on human peripheral blood eosinophils at levels that correlated with donor body mass index and total leukocyte count. Taken together, our findings provide important insight into the immunomodulatory and remodeling capacity of mouse eosinophils and the flexibility of their gene expression profiles in vivo.

Functionally Active Eosinophil Purification from Peripheral Blood.

The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress® Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.

An in vitro study to explore the modulation of eosinophil effector function in human allergic peripheral blood eosinophils using enzymatically extracted salmonid oil

Background: The consumption of oily fish in early childhood diets has been correlated to a reduction in the progression of asthma during adolescence and young adulthood. Extending these results to studies on supplementation with omega-3 fish oils has led to equivocal results, with no clear results. One subset of asthma patients is classified as steroid-treatment resistant, which may be attributable to eosinophil dysfunction. This in-vitro study investigated the prophylactic effect of enzymatically extracted salmon oil, as a proxy for eating fresh whole oily fish, on eosinophil function using allergic human peripheral blood eosinophils.Methods: We measured three eosinophil effector functions in allergic human peripheral blood eosinophils after prophylactic treatment with enzymatically extracted salmonid oil. We measured changes in: i) eosinophil shape change in normal PMNL ii) integrin upregulation in normal PMNL and iii) eosinophil apoptosis. We further measured changes in these functions after prophylactic treatment with omega-3 fish oil, krill oil, and fractions of the salmon oil subjected to several commercial fish oil processing conditions.Results: Prophylactic treatment of allergic human peripheral blood eosinophil with 100 ug/ml of enzymatically extracted salmonid oil showed modulation of eosinophil effect functions and apoptosis comparable to APOA-IV at 3ug/ml. Vigorous processing conditions applied to the salmon oil degraded the observed eosinophil modulation. Krill oil and standard fish oil showed no to low eosinophil modulation.Conclusions: Enzymatically extracted salmonid oil demonstrates potential therapeutic promise for the treatment of eosinophil effector malfunction-based allergic and inflammatory conditions, particularly certain types of asthma and COPD. Treatment of the oil with commercial marine oil processing conditions significantly reduces or eliminates this eosinophil effector function modulation.Keywords: Eosinophils, asthma, salmon oil, APOA-IV, apoptosis

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

Cytokine Diversity in Human Peripheral Blood Eosinophils: Profound Variability of IL-16.

Eosinophilic leukocytes develop in the bone marrow and migrate from peripheral blood to tissues, where they maintain homeostasis and promote dysfunction via release of preformed immunomodulatory mediators. In this study, we explore human eosinophil heterogeneity with a specific focus on naturally occurring variations in cytokine content. We found that human eosinophil-associated cytokines varied on a continuum from minimally (coefficient of variation [CV] ≤ 50%) to moderately variable (50% < CV ≤ 90%). Within the moderately variable group, we detected immunoreactive IL-27 (953 ± 504 pg/mg lysate), a mediator not previously associated with human eosinophils. However, our major finding was the distinct and profound variability of eosinophil-associated IL-16 (CV = 103%). Interestingly, eosinophil IL-16 content correlated directly with body mass index (R 2 = 0.60, ***p < 0.0001) in one donor subset. We found no direct correlation between eosinophil IL-16 content and donor age, sex, total leukocytes, lymphocytes, or eosinophils (cells per microliter), nor was there any relationship between IL-16 content and the characterized -295T/C IL-16 promoter polymorphism. Likewise, although eosinophil IL-1β, IL-1α, and IL-6 levels correlated with one another, there was no direct association between any of these cytokines and eosinophil IL-16 content. Finally, a moderate increase in total dietary fat resulted in a 2.7-fold reduction in eosinophil IL-16 content among C57BL/6-IL5tg mice. Overall, these results suggest that relationships between energy metabolism, eosinophils, and IL-16 content are not direct or straightforward. Nonetheless, given our current understanding of the connections between asthma and obesity, these findings suggest important eosinophil-focused directions for further exploration.

Proteomics of Eosinophil Activation.

We recently identified and quantified >7,000 proteins in non-activated human peripheral blood eosinophils using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) and described phosphoproteomic changes that accompany acute activation of eosinophils by interleukin-5 (IL5) (1). These data comprise a treasure trove of information about eosinophils. We illustrate the power of label-free LC–MS/MS quantification by considering four examples: complexity of eosinophil STATs, contribution of immunoproteasome subunits to eosinophil proteasomes, complement of integrin subunits, and contribution of platelet proteins originating from platelet–eosinophil complexes to the overall proteome. We describe how isobaric labeling enables robust sample-to-sample comparisons and relate the 220 phosphosites that changed significantly upon treatment with IL5 to previous studies of eosinophil activation. Finally, we review previous attempts to leverage the power of mass spectrometry to discern differences between eosinophils of healthy subjects and those with eosinophil-associated conditions and point out features of label-free quantification and isobaric labeling that are important in planning future mass spectrometric studies.

Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study.

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

Involvement of the α/β Isoform of p38 MAP Kinase in Chemotactic Responses of Human Eosinophils to Eotaxin (CCL11) and RANTES (CCL5)

Eosinophils are the principal effector cells for allergic inflammation in a variety of diseases, in which they contribute to tissue damage and remodelling processes via the secretion of cytotoxic granular proteins and cytokines. The intracellular mechanisms that control the activation, recruitment and survival of eosinophils are fundamental in understanding these disease processes. Phosphoinositide 3-kinase (PI3K) has been shown previously to be essential for eosinophil chemotactic responses to some stimuli but not others. Human blood neutrophils have been shown to utilize two antagonistic signalling pathways for chemotaxis: PI3K and p38 mitogen-activated protein kinase (p38 MAPK). In the present study, the role of p38 MAPK in chemotactic responses of an eosinophil-differentiated myeloid leukaemia cell line (EOL-1) and human peripheral blood eosinophils to a range of stimuli - platelet-activating factor (PAF), eotaxin 1 (CCL11), RANTES (CCL5), interleukin 8 (IL8, CXCL8) and IL16 - was explored through the use of the p38 MAPK α/β isoform inhibitor, SB 203580. SB 203580 caused significant inhibition of chemotactic responses of both EOL-1 cells and blood eosinophils to eotaxin 1 and RANTES (≥75% inhibition at 1 μM SB 203580, p<0.01) but had no effect on the migration induced by PAF and IL16 (<25%) and little or no effect on responses to IL8. Responses to PAF - but not eotaxin - have been shown previously to be suppressed by PI3K inhibition. The complementary pattern of inhibition observed in the present study provides evidence that distinct PI3K-dependent and p38 MAPK-dependent chemoattractants may also exist for eosinophils.

Evidence for a role of eosinophils in blister formation in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune bullous disease of the skin characterized by subepidermal blister formation due to tissue-bound and circulating autoantibodies to the hemidesmosomal antigens BP180 and BP230. Although eosinophils and their toxic mediators are found abundantly in BP lesions, their role in blister formation has remained unclear. To investigate the role of eosinophils in the pathogenesis of BP with a specific focus on blister formation and to define conditions inducing dermal-epidermal separation (DES). In an ex vivo human model of BP, normal human skin cryosections were incubated with purified human peripheral blood eosinophils with or without activation in the presence or absence of BP autoantibodies, brefeldin A, diphenyleneiodonium, DNase or blocking F(ab')2 fragments to CD16, CD18, CD32 and CD64. Dermal-epidermal separation was assessed by light microscopy studies and quantified using Fiji software. Following activation with IL-5 and in the presence of BP autoantibodies, eosinophils induced separation along the dermal-epidermal junction of ex vivo skin. Dermal-epidermal separation was significantly reduced by blocking any of the following: Fcγ receptor binding (P = 0.048), eosinophil adhesion (P = 0.046), reactive oxygen species (ROS) production (P = 0.002), degranulation (P < 0.0001) or eosinophil extracellular trap (EET) formation (P = 0.048). Our results provide evidence that IL-5-activated eosinophils directly contribute to BP blister formation in the presence of BP autoantibodies. Dermal-epidermal separation by IL-5-activated eosinophils depends on adhesion and Fcγ receptor activation, requires elevated ROS production and degranulation and involves EET formation. Thus, targeting eosinophils may be a promising therapeutic approach for BP.

Neutrophil autophagy and extracellular DNA traps contribute to airway inflammation in severe asthma.

Autophagy and neutrophil extracellular DNA traps (NETs) are implicated in asthma; however, their roles in asthma pathogenesis have not been elucidated. We compared autophagy and NET production levels from peripheral blood neutrophils (PBNs) of patients with severe asthma (SA) and non-severe asthma (NSA). Additionally, we investigated the inflammatory effects of NETs on human airway epithelial cells (AECs) and peripheral blood eosinophils (PBEs). Peripheral blood neutrophils from patients with SA (n=30) and NSA (n=38) were treated with interleukin (IL)-8 (100ng/mL). Autophagy (light chain 3-II expression) and NET production levels were evaluated by Western blot, immunofluorescence microscopy, and PicoGreen assay. The effects of NETs on AECs were assessed by investigating cell death, cell detachment, expression of occludin and claudin-1, and IL-8 production; the effects of NETs on PBEs were examined by investigating the activation and release of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Untreated and IL-8-treated PBNs from the SA group produced higher autophagy and NET levels compared with those from the NSA group (P<0.01). IL-8 increased autophagy and NET levels in PBNs from the SA group, but not from the NSA group. NET levels were correlated with autophagy levels in PBNs (P<0.001). IL-8-induced NET production levels negatively were correlated with FEV1/FVC (r=-0.700, P=0.016). NETs induced cell death, detachment, degradation of occludin and claudin-1, and IL-8 production from AECs. Higher levels of NET-induced ECP and EDN were released from PBEs in SA compared with NSA groups. Neutrophil autophagy and NETs could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and PBEs. Modulating neutrophil autophagy and NET production may be a new target therapy for SA.

Effective antigen presentation to helper T cells by human eosinophils.

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4+ T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4+ Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.

Olig2 is expressed late in human eosinophil development and controls Siglec-8 expression.

Oligodendrocyte transcription factor 2, a basic helix-loop-helix transcription factor that binds to E-box motifs, is known to have a key role in determining lineage specification of oligodendrocytes and motor neurons. In the present study, we report that oligodendrocyte transcription factor 2 is expressed in human eosinophils and involved in transcriptional activation of the gene encoding sialic acid binding immunoglobulin-like lectin 8 (Siglec-8), a late eosinophil-differentiation marker known to exert eosinophil apoptosis. When cord blood CD34+ hematopoietic stem cells differentiated toward eosinophils during a 24-d culture period, oligodendrocyte transcription factor 2 protein was expressed in cord blood eosinophils on d 24, a time when cord blood eosinophils are considered fully differentiated, whereas it was not detectable on d 18 or at earlier time points. Oligodendrocyte transcription factor 2 protein was also abundantly expressed in human peripheral-blood eosinophils but not in neutrophils, monocytes, lymphocytes, or cord blood mast cells. RNA sequencing analysis showed that numerous genes, especially those encoding eosinophil surface molecules, were highly up-regulated along with OLIG2 Among the genes examined, SIGLEC-8 messenger RNA and protein were markedly down-regulated in parallel with OLIG2 by an oligodendrocyte transcription factor 2 small interfering RNA or a short hairpin RNA, as evidenced by real-time polymerase chain reaction, fluorescence-activated cell sorting, and Western blot analyses. In reporter gene and chromatin immunoprecipitation experiments, an E-box in the first intron was found to stimulate SIGLEC-8 gene transcription and to bind oligodendrocyte transcription factor 2. Hence, at least one important aspect of eosinophil differentiation is regulated by oligodendrocyte transcription factor 2, a transcription factor that has not previously been reported, to our knowledge, in normal granulocytes.

Expression and function of Olig2 in eosinophils

Abstract Eosinophils are granulocytes that are associated with homeostasis and type 2 immune responses, allergy and parasitic infection. Olig2 is a basic helix loop helix transcription factor that is known to play a key role in cell fate decision and subsequent differentiation of oligodendrocytes. Herein, we report the expression and possible function of Olig2 in eosinophils. When cord blood (CB) CD34+ hematopoietic stem cells differentiated toward eosinophils during a 24 day culture period, Olig2 protein was expressed only in CB eosinophils at day 24, a time when these cells were considered fully differentiated and matured, while Olig2 protein was not detected at day 18 or at earlier time points. Olig2 protein was also abundantly expressed in human peripheral blood eosinophils, but not neutrophils, monocytes, lymphocytes or CB mast cells. RNA-seq analysis showed that numerous genes, especially those encoding eosinophil surface molecules, were highly upregulated along with OLIG2. Among the genes examined, SIGLEC-8 mRNA and protein were downregulated in parallel with OLIG2 by an Olig2 siRNA or a shRNA. In reporter gene and chromatin immunoprecipitation experiments, an E-box in the first intron was found to stimulate SIGLEC-8 gene transcription and bind Olig2. Occupancy of the E-box by Olig2 was also seen upon differentiation of immature eosinophilic AML14.3D10 cells. Hence, at least one important aspect of eosinophil differentiation is driven by Olig2, a transcription factor that has not previously been reported in normal granulocytes.

Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen-induced pulmonary inflammation in mice.

BackgroundAccumulation of activated eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid 2‐arachidonoylglycerol (2‐AG) has been proposed to elicit eosinophil migration in a CB 2 receptor/Gi/o‐dependent manner. However, it has been claimed recently that this process may also involve other mechanisms such as cytokine priming and the metabolism of 2‐AG into eicosanoids. Here, we explored the direct contribution of specific CB 2 receptor activation to human and mouse eosinophil effector function in vitro and in vivo.Methods In vitro studies including CB 2 expression, adhesion and migratory responsiveness, respiratory burst, degranulation, and calcium mobilization were conducted in human peripheral blood eosinophils and mouse bone marrow‐derived eosinophils. Allergic airway inflammation was assessed in mouse models of acute OVA‐induced asthma and directed eosinophil migration.Results CB 2 expression was significantly higher in eosinophils from symptomatic allergic donors. The selective CB 2 receptor agonist JWH‐133 induced a moderate migratory response in eosinophils. However, short‐term exposure to JWH‐133 potently enhanced chemoattractant‐induced eosinophil shape change, chemotaxis, CD11b surface expression, and adhesion as well as production of reactive oxygen species. Receptor specificity of the observed effects was confirmed in eosinophils from CB 2 knockout mice and by using the selective CB 2 antagonist SR144528. Of note, systemic application of JWH‐133 clearly primed eosinophil‐directed migration in vivo and aggravated both AHR and eosinophil influx into the airways in a CB 2‐specific manner. This effect was completely absent in eosinophil‐deficient ∆dblGATA mice.ConclusionOur data indicate that CB 2 may directly contribute to the pathogenesis of eosinophil‐driven diseases. Moreover, we provide new insights into the molecular mechanisms underlying the CB 2‐mediated priming of eosinophils. Hence, antagonism of CB 2 receptors may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophilic disorders.

SCD48 Is a Novel Eosinophil Derived Decoy Receptor That Decreases Seb Activity in Vitro and In Vivo

CD48, a GPI-anchored receptor (that may exist in a soluble form (sCD48)) as expressed by eosinophils (mCD48) is a central player in allergy and mediates eosinophils interaction with S. aureus/toxins. We investigated in vitro S. aureus enterotoxin B (SEB) regulation of eosinophils CD48 and the role of sCD48 in SEB mouse eosinophilic peritonitis. Human peripheral blood eosinophils were incubated with SEB (up to 18hr): sCD48 release (ELISA), CD11b expression (FC), phospholipases (PL) C/D activity (inhibitors), transport (brefeldinA), protein synthesis (RT-PCR, cycloheximide) were assessed (0-18 hrs). SEB peritonitis was induced in mice (100ug/mouse) and peritoneal cells (FC) and lavage examined. Eosinophils expressed mCD48 and intracellular CD48 that partly co-localized with ECP/EDN. SEB activated eosinophils released significant sCD48 that directly correlated to CD11b expression. Eosinophils activated by SEB in the presence of PL inhibitors or cycloheximide or brefeldin A, displayed decreased sCD48 indicating that PLs (C and D) are involved in the cleavage of mCD48 that is maintained by new synthesis and transport. sCD48 binds to SEB (ELISA) and eosinophils cultured with SEB pre-incubated with sCD48 released significantly less EPO and IL-8 and had a reduced chemotaxis. In SEB peritonitis, peritoneal sCD48 was elevated and correlated to eosinophils and lymphocytes. SEB pretreatment with sCD48 significantly downregulated peritonitis' various inflammatory parameters. SEB regulates CD48 dynamics on eosinophils causing sCD48 formation that acts as a SEB decoy receptor. This highlights the importance of CD48 as a central player in eosinophil regulation in diseases such as allergy in which S. aureus is a primary pathogen.

Integrins are Mechanosensors That Modulate Human Eosinophil Activation

Eosinophil migration to the lung is primarily regulated by the eosinophil-selective family of eotaxin chemokines, which mobilize intracellular calcium (Ca2+) and orchestrate myriad changes in cell structure and function. Eosinophil function is also known to be flow-dependent, although the molecular cognate of this mechanical response has yet to be adequately characterized. Using confocal fluorescence microscopy, we determined the effects of fluid shear stress on intracellular calcium concentration ([Ca2+]i) in human peripheral blood eosinophils by perfusing cells in a parallel-plate flow chamber. Our results indicate that fluid perfusion evokes a calcium response that leads to cell flattening, increase in cell area, shape change, and non-directional migration. None of these changes are seen in the absence of a flow stimulus, and all are blocked by chelation of intracellular Ca2+ using BAPTA. These changes are enhanced by stimulating the cells with eotaxin-1. The perfusion-induced calcium response (PICR) could be blocked by pre-treating cells with selective (CDP-323) and non-selective (RGD tripeptides) integrin receptor antagonists, suggesting that α4β7/α4β1 integrins mediate this response. Overall, our study provides the first pharmacological description of a molecular mechanosensor that may collaborate with the eotaxin-1 signaling program in order to control human eosinophil activation.

Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

The CD48 receptor mediates Staphylococcus aureus human and murine eosinophil activation.

Allergy is characterized by eosinophilia and an increased susceptibility to microbial infection. Atopic dermatitis (AD) is typically associated with Staphylococcus aureus (SA) colonization. Some of the mechanisms by which SA and its exotoxins interact with eosinophils remain elusive. CD48, a glycosylphosphatidylinositol-anchored receptor belonging to the CD2 family, participates in mast cells-SA stimulating cross-talk, facilitates the formation of the mast cell/eosinophils effector unit and as expressed by eosinophils, mediates experimental asthma. To investigate the role of CD48 expressed on human peripheral blood and mouse bone marrow-derived eosinophils (BMEos) in their interaction with heat-killed SA and its three exotoxins, Staphylococcal enterotoxin B (SEB), protein A (PtA) and peptidoglycan (PGN). Eosinophils were obtained from human peripheral blood and BM of WT and CD48-/- mice. SA was heat killed and eosinophils-SA/exotoxins interactions were analyzed by confocal microscopy, adhesion and degranulation, cell viability, cytokine release and cell signalling. In addition, peritonitis was induced by SEB injection into CD48-/- and WT mice. CD48 expression was studied in AD patients' skin and as expressed on their leucocytes in the peripheral blood. We provide evidence for the recognition and direct physical interaction between eosinophils and SA/exotoxins. Skin of AD patients showed a striking increase of eosinophil-associated CD48 expression while on peripheral blood leucocytes it was down-regulated. SA/exotoxins enhanced CD48 eosinophil expression, bound to CD48 and caused eosinophil activation and signal transduction. These effects were significantly decreased by blocking CD48 on human eosinophils or in BMEos from CD48-/- mice. We have also explored the role of CD48 in a SEB-induced peritonitis model in CD48-/- mice by evaluating inflammatory peritoneal cells, eosinophil numbers and activation. These data demonstrate the important role of CD48 in SA/exotoxins-eosinophil activating interactions that can take place during allergic responses and indicate CD48 as a novel therapeutic target for allergy and especially of AD.

Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.

Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs), commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos) would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway), while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD), which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal) or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery); subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2), and an IL-5-dependent increase in interleukin-1β (IL-1β) mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, MβCD+Chol pretreatment attenuated an IL-5-induced increase in cell survival at 48 hours, measured as total cellular metabolism. The reduction in cell survival following cholesterol addition despite unaltered STAT phosphorylation contradicts the current dogma in which JAK/STAT activation is sufficient to promote eosinophil survival, and suggests an additional, unidentified mechanism critically regulates IL-5-mediated human PBEos survival.