Eosinophil-associated Diseases Research Articles

The consequences of not having eosinophils

Several lines of evidence suggest that deficiency of eosinophils is not associated with any characteristic abnormality. Patients lacking eosinophils, in the setting of immunodeficiency or as a consequence of IgG-mediated eosinophil precursor destruction, do not display any distinguishing abnormalities related to eosinophil reduction. The observation that eosinophil-deficient mice do not display any distinctive syndrome or failure of their health is evidence that, under ordinary laboratory conditions, the eosinophil does not play a critical role in the well-being of mammals. Observations that monoclonal antibodies to interleukin-5 (IL-5) are well tolerated appear unsurprising in light of these findings. For example, patients with the hypereosinophilic syndrome have received mepolizumab, an anti-IL-5 monoclonal antibody, for as long as 6 years and have not developed any characteristic set of adverse events. Safety data for reslizumab, another anti-IL-5 monoclonal antibody, and benralizumab, a monoclonal antibody to the IL-5 receptor α-chain, are comparatively limited, especially for benralizumab, although reports of administration of these antibodies to humans suggest that they are well tolerated. Thus, data to the present suggest that reduction of eosinophils appears to have no characteristic ill effects on normal health, and monoclonal antibodies that deplete eosinophils have the potential to be widely employed in the treatment of eosinophil-associated diseases.

Negative Regulation of Eosinophil Production by Toll-Like Receptors

Abstract 1237Eosinophilia is a common clinical problem associated with numerous disorders such as atopic diseases, parasitic infections, hypereosinophilic syndrome (HES), and cancer. A marked decrease in circulating eosinophils, or eosinopenia, has long been associated with acute bacterial infections. Eosinopenia has been shown to be a sensitive and reliable marker for distinguishing between non-infectious and infection-associated sepsis in the intensive care unit setting. Eosinopenia with acute infection had been assumed to be secondary to the release of adrenal glucocorticoids in response to the stress of infection. However, animal studies performed in the 1970s demonstrated that inhibition of parasite-associated eosinophilia with acute bacterial infection was independent of adrenal stimulation; the investigator speculated that it was due to a direct effect on the eosinophil precursor. To date, the mechanism of infection-associated eosinopenia, and its potential for suppressing IL-5-mediated eosinophilia, has not been investigated.To study eosinophil development, we established an ex vivo liquid culture system in which we can follow the differentiation of murine eosinophil lineage-committed progenitors (EoPs) to mature functional effector eosinophils that express known eosinophil surface markers and granule proteins and respond to eosinophil-active chemoattractants. We demonstrate that murine eosinophil precursors express mRNA for six TLRs. Ligands for TLR2 heterodimers, TLR4 and TLR7, but not TLR3, inhibited eosinophil ex vivo growth by attenuating EoP proliferation in response to IL-5 stimulation. Exposure to heat-killed E. coli also resulted in diminished EoP proliferation. The developing eosinophils further responded to TLR activation with production of a subset of inflammatory cytokines, including IL-6, IL-10, TNF-alpha and CXCL10. Co-culturing of untreated EoPs with LPS-exposed EoPs resulted in reduced IL-5-mediated proliferation of the untreated EoPs, suggesting that EoPs secrete mediators in response to TLR activation that regulate their proliferation in an autocrine/paracrine manner. Neutralization of IFN-beta, but not IFN-alpha or IFN-gamma, partially protected developing eosinophils from the inhibitory effects of LPS. In vivo studies revealed that endotoxemia and bacteremia reduced numbers of EoPs in the bone marrow of wild-type mice. Further, LPS dose-dependently reduced hypereosinophilia in IL-5 transgenic mice, highlighting the potential therapeutic value of this approach even in an extreme IL-5-driven clinical state. Suppression of eosinophil production by TLR4 ligands is conserved between mice and humans as yield of eosinophils from human EoPs was reduced following LPS exposure. Taken together, these findings identify a mechanistic explanation for eosinopenia following bacterial infections and identify a novel therapeutic strategy for inhibiting eosinophil production and peripheral eosinophilia in eosinophil-associated diseases. Disclosures:No relevant conflicts of interest to declare.

Workshop report from the National Institutes of Health Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD)

Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiologic roles that eosinophils may serve in other human "eosinophil-associated" diseases remain obscure. National Institutes of Health (NIH) Institutes and the Office of Disease Prevention assembled an international taskforce of clinical and basic scientists with the charge to propose and prioritize unmet research needs in eosinophil-associated diseases. The taskforce used an organ system approach to identify the different and common themes of eosinophil cell involvement in these diseases. In early 2012, a draft document was circulated for review. The document was amended and the prioritizations were set at a NIH-organized workshop in June 2012. The taskforce identified significant research needs. These needs cross disease entities but some are disease specific. There are substantial shortcomings to the various preclinical animal models, as well as significant gaps in our epidemiologic, pathophysiologic, diagnostic, prognostic, and therapeutic knowledge. The taskforce recognized that recent efforts by patient advocacy groups have played instrumental roles in improving the identification and characterization of these disorders. However, communications among the eosinophil-interested communities, for example, governmental funding and regulatory agencies, and industry and clinician scientists need to be more comprehensive. Significant efforts are required to address our knowledge gaps to improve the outcomes of eosinophil-associated diseases. NIH Institutes, other federal agencies, lay organizations, and the pharmaceutical industry should consider the taskforce's recommendations in their future research activities.

Biomarkers of eosinophil involvement in allergic and eosinophilic diseases: Review of phenotypic and serum markers including a novel assay to quantify levels of soluble Siglec-8

There remains considerable controversy in the management of eosinophilic disorders, mainly due to a paucity of information regarding the clinical interpretation of total blood eosinophil counts versus surface activation markers versus eosinophil-derived or eosinophil-influencing mediator levels. Regrettably, few tests have been validated that define a unique clinical or prognostic phenotype that is more useful than simply monitoring total blood eosinophil counts. In this manuscript, phenotypic (cell surface) markers, along with serum and tissue-based markers that have been examined in the context of disease activity, are reviewed. We also report the development of a novel assay for detecting soluble Siglec-8 (sSiglec-8), a protein likely derived largely from eosinophils, as a potential serum biomarker. The assay consists of a competitive ELISA using a recombinant Siglec-8-Fc fusion protein. The goal of this preliminary study was to determine if sSiglec-8 is a useful biomarker that differentiates among patients with various eosinophil-associated diseases. In the final analysis, it is fair to say that further research is sorely needed to fully understand and validate the utility of various biomarkers, including sSiglec-8, before their use in clinical practice can be recommended with confidence.

P38 MAPK but not ERK is required for eosinophil migration and degranulation (172.29)

Abstract Eosinophil-associated RNases (EARs); the human eosinophil-derived neurotoxin and eosinophilic cationic protein and their murine orthologs, are stored in eosinophil cytoplasmic granules and secreted upon inflammation as part of innate immune responses. In eosinophil-associated diseases, such as hyper eosinophilic syndrome, allergy and asthma, EARs play crucial roles in disease pathology and can be used as biomarkers for disease severity. However, secretion mechanisms of EARs are not fully understood, especially in mouse eosinophils, the major animal model for eosinophil-associated diseases. Our study aimed to understand the mechanisms of eosinophil EAR secretion following chemokine stimulation. Both ERK and p38 MAPK activation were found to increase in mouse eosinophils in response to CCL11. CCR3-mediated EAR secretion was blocked by p38 inhibitor (SB202190), confirming previous data in human eosinophils. However, a specific inhibitor for ERK, a MEK1-derived peptide, did not inhibit CCR3-mediated secretion or migration. These results are in contrast to previous studies in human eosinophils and our studies in mouse eosinophils, showing ERK is crucial for CCR3-mediated EARs secretion and migration using PD98059, a less specific inhibitor of the ERK-activating enzyme, MEK. Collectively, these data suggest a signaling pathway for CCR3-mediated EAR secretion that includes: PI3K and p38 but not ERK. These findings are pertinent to host defense and eosinophil-associated diseases.

CCL11 elicits secretion of RNases from mouse eosinophils and their cell‐free granules

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.

SIRPα/CD172a Regulates Eosinophil Homeostasis

Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein α (SIRPα)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRPα/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRPα/CD172a that lacks most of its cytoplasmic domain (SIRPα Cyto(-/-)). The SIRPα Cyto(-/-) eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRPα Cyto(-/-) mice showed increased frequencies of Annexin V-binding eosinophils and free MBP(+)CD63(+) extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRPα/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRPα Cyto(-/-) mice compared with wild-type mice. Collectively, our results indicated that SIRPα/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRPα/CD172a is a potential therapeutic target for eosinophil-associated diseases.

Estradiol administration controls eosinophilia through estrogen receptor-α activation during acute peritoneal inflammation

Estrogens influence the incidence and the course of numerous immune or inflammatory diseases in humans and in experimental models. For instance, estrogens prevent the accumulation of granulocytes in acute inflammatory murine models, but the respective actions on neutrophil and eosinophil trafficking remain to be clarified. We demonstrate here that in a model of TGC-induced sterile peritonitis in ovx mice, chronic E2 administration electively and strongly inhibited peritoneal eosinophil accumulation. E2 decreased BM eosinophil number, contributing to a marked prevention of the TGC-induced eosinophil blood mobilization. These effects on eosinophil mobilization and peritoneal accumulation were abolished in ER-α(-/-) mice, demonstrating the crucial role of this nuclear receptor. Grafting ER-α(-/-) mice with ER-α(+/+) BM cells restored the suppressive effect of E2 on peritoneal eosinophilia, although the action on eosinophil blood mobilization was still abrogated. We therefore explored additional mechanisms and found that E2 reduced the peritoneal concentrations of key eosinophil prosurvival factors (IL-5, IL-9, and IL-25) and enhanced eosinophil apoptosis during the inflammatory process. Furthermore, this proapoptotic effect of E2 was abrogated in IL-5-overexpressing Tg mice. To conclude, we demonstrate for the first time that ER-α activation by exogenous E2 administration strongly inhibits eosinophil accumulation during acute inflammation in a nonreproductive target site for estrogen through combined actions on eosinophil mobilization and apoptosis. This specific, suppressive effect of chronic E2 replacement therapy on eosinophils has to be integrated to further understand the evolution of eosinophil-associated diseases in menopausal women.

Negative regulation of eosinophil production by TLR4 ligand (153.26)

Abstract It has long been appreciated that a drop in eosinophils is associated with acute bacterial infection while, in contrast, blood levels of monocytes and neutrophils rise. The initial eosinopenic response is believed to be secondary to margination of circulating eosinophils to blood vessel walls, but the mechanism for prolonged eosinophil depletion with bacterial infection remains undefined. In a culture system developed to differentiate mature eosinophils from low density bone marrow progenitors, IL-5 stimulation of progenitors resulted in induced expression of six TLRs with highest expression of TLR2 and TLR4 noted throughout eosinophil development. Surface expression of TLR4 was confirmed on CD34+ progenitors by FACS. Stimulation of progenitors with LPS markedly inhibited IL-5-mediated eosinophil production. Further, LPS administration in vivo specifically reduced numbers of eosinophil progenitors in the bone marrow. Treatment of developing eosinophils with LPS induced the generation of pro-inflammatory mediators, including IL-6, IL-10, IFN-γ and TNF-α, but mechanistic studies demonstrated that suppression of eosinophil production by LPS was independent of these mediators. Taken together, these findings suggest a direct effect of LPS on eosinophil progenitors as an explanation for eosinopenia following bacterial infections and a potentially novel therapeutic strategy for depleting eosinophil progenitors and inhibiting peripheral eosinophilia in eosinophil-associated diseases.

Cytoskeletal and microtubule machineries in concert with integrins are required for the secretion of eosinophil-associated RNases (110.13)

Abstract Eosinophils, both as end-stage effector cells and as collaborative interacting components of innate immune responses, have the capacity to secrete varied cytotoxic proteins that are stored in their cytoplasmic secretory granules, termed specific granules. Among them are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein. The mouse EAR family includes at least 13 members, six of which were found to be present in specific granules. In the last decade several studies, both in vitro and in vivo, have revealed a role for eosinophils and their EARs in anti-viral defense. However, the secretion mechanism of EARs is not fully understood, especially in mouse eosinophils. Therefore, our study aimed to understand the mechanism of EAR secretion following physiological stimulation with chemokines. We found that dynamic polymerization of the actin cytoskeleton and microtubules, as well as functional integrins were critical for CCR3-mediated secretion of EARs. However, inhibition of regulatory components of the cytoskeletal machinery, such as Rac1 and ROCK-I and -II, increased EARs secretion in mouse and human eosinophils, suggesting a negative regulation of these effectors on EARs secretion. Collectively, these data suggest a mechanism by which EARs are secreted from eosinophils under physiological stimulation, findings that are pertinent to host defense against RNA viruses and eosinophil-associated diseases.

CCR3-mediated degranulation and eosinophil-associated RNases secretion from murine eosinophils; capacity of intact cells and free granules (86.21)

Abstract Piecemeal degranulation (PMD), in which intact eosinophils completely or partially empty granules and selectively secrete performed mediators, is the principal mechanism for human eosinophil degranulation. Unlike extensively studied human eosinophils, both the capacity and mechanisms underlying eosinophil degranulation responses of murine eosinophils have been poorly investigated and remain controversial. We applied a new methodology to detect degranulation by measuring secretion of eosinophil-associated RNases (mEARs) and found that murine eosinophils “degranulated” following stimulation with CCL11 and CCL24, as detected by an increase in RNase A and EPO activities in cell supernatants. TEM revealed signs of PMD including significant increase in the number of emptying granules. CCL11 and CCL24-mediated mouse eosinophil degranulation was Gαi and PI3K-dependent. Moreover, intact beta 1 and 2 integrins as well as dynamic cytoskeleton are required for this PMD. Unexpectedly, we found that inhibition of Rho-GTPase kinase increases PMD. Moreover, cell-free granules purified from mouse eosinophils were found to express functional CCR3 and secreted mEARs as well as EPO in response to CCL11, suggesting a unique mechanism of degranulation in cell-free context. Collectively, these data demonstrate that murine eosinophils undergo PMD in response to physiological stimulation with CCL11 and CCL24, findings that are pertinent to murine models of eosinophil-associated diseases.

Eosinophil Ribonucleases and Their Cutaneous Lesion-Forming Activity

Eosinophil granule proteins are deposited in cutaneous lesions in many human diseases, but how these proteins contribute to pathophysiology is obscure. We injected eosinophil cationic protein (ECP or RNase 3), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 (MBP1) intradermally into guinea pig and rabbit skin. ECP and EDN each induced distinct skin lesions at >or=2.5 microM that began at 2 days, peaking at approximately 7 days and persisting up to 6 wk. These lesions were ulcerated (ECP) or crusted (EDN) with marked cellular infiltration. EPO and MBP1 (10 microM) each produced perceptible induration and erythema with moderate cellular infiltration resolving within 2 wk. ECP and EDN localized to dermal cells within 2 days, whereas EPO and MBP1 remained extracellular. Overall, cellular localization and RNase activity of ECP and EDN were critical for lesion formation; differential glycosylation, net cationic charge, or RNase activity alone did not account for lesion formation. Ulcerated lesions from patients with the hypereosinophilic syndrome showed ECP and EDN deposition comparable to that in guinea pig skin. In conclusion, ECP and EDN disrupt skin integrity and cause inflammation. Their presence in ulcerative skin lesions may explain certain findings in human eosinophil-associated diseases.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Establishment of humanized anti-interleukin-5 receptor alpha chain monoclonal antibodies having a potent neutralizing activity

Human interleukin-5 is the key cytokine involved in regulating the production and function of human eosinophils. IL-5 binds to its specific receptor composed of two heterogeneous alpha and beta polypeptide chains (hIL-5Ralpha and betac) that are expressed on the cell surface. The hIL-5Ralpha specifically binds IL-5 without involvement of the betac. It has been suggested that neutralizing antibodies to hIL-5Ralpha could serve as a therapeutic agent in eosinophil-associated diseases. We describe here the creation and biologic activities of a mouse monoclonal antibody against hIL-5Ralpha that blocks the following IL-5 dependent activities (a) binding of the IL-5 ligand to its receptor, (b) IL-5 dependent growth of hIL-5R expressing cells, and (c) IL-5-induced adhesion of human eosinophils. We also describe the process for humanization of the mouse Mab towards development of a therapeutic MAb. The humanized version of the monoclonal antibody also displayed potent neutralizing activity against IL-5 dependent activities.

Mepolizumab in the treatment of eosinophilic esophagitis

Mepolizumab is a humanized monoclonal antibody that binds specifically to and inactivates a protein cell messenger termed IL-5, which plays a major role in the proliferation, maturation, activation and survival of eosinophils. As a result, mepolizumab has been studied in a number of eosinophil-associated diseases, such as asthma, hypereosinophilic syndrome and eosinophilic esophagitis (EE). EE is a clinical entity characterized by eosinophilic infiltration of the esophagus (≥15 eosinophils/high power field in one or more fields) in the absence of gastroesophageal reflux. EE is on the rise, and this can not be fully accounted for by increased recognition. Currently, available therapies, although successful in many cases, can be disappointing in some patients, whether from lack of response, negative impact on quality of life, or significant toxicity. In order to better define EE therapy, investigators are evaluating novel drugs, including anti-IL-5 agents such as mepolizumab. Mepolizumab has been demonstrat...

Eosinophil granules function extracellularly as receptor-mediated secretory organelles

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-gamma, and G protein-coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Airway Fibrosis and Angiogenesis due to Eosinophil Trafficking in Chronic Asthma

Asthma is characterized by the presence of increased numbers of inflammatory cells in the airway in particular eosinophils and Th2 lymphocytes. In addition to the presence of inflammatory cells, the airways of patients with asthma exhibit varying levels of structural changes termed airway remodeling. These structural changes include subepithelial fibrosis, smooth muscle hypertrophy/hyperplasia, epithelial cell mucus metaplasia, and increased angiogenesis. This review focuses on the potential role of the eosinophil in promoting features of airway remodeling including fibrosis and neovascularization in chronic asthma. Eosinophils may potentially contribute to airway remodeling through release of eosinophil derived mediators such as TGFbeta which act directly upon target fibroblasts to promote fibrosis. In addition to the potential importance of the eosinophil to remodeling in asthma, eosinophilic esophagitis (EE) is another eosinophil associated disease that is associated with increased levels of esophageal eosinophils, increased levels of TGFbeta expression, and increased levels of fibrosis, suggesting that a similar mechanism of remodeling may contribute to both of these eosinophil associated diseases. However, remodeling in both asthma and EE is likely complex involving both eosinophil dependent and eosinophil independent pathways. Further studies in both humans and animal models will help to increase our knowledge of the contribution of the eosinophil to remodeling in asthma as well as EE.

Major Basic Protein Homolog (MBP2): A Specific Human Eosinophil Marker

Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.

The inhibitory receptor IRp60 (CD300a) suppresses the effects of IL-5, GM-CSF, and eotaxin on human peripheral blood eosinophils

Allergic, inflammatory, and immune responses carried out by eosinophils are regulated by the cross talk between activatory and inhibitory signals. While much data has been obtained on activatory signals, inhibitory receptors on these cells have received scant attention. Therefore, we screened the surface of human peripheral blood eosinophils for inhibitory receptors using monoclonal antibodies (mAbs) previously generated to recognize receptors on human natural killer cells. Eosinophils from all of the donors examined expressed the inhibitory receptors IRp60, LIR3/ILT5, FcgammaRIIB, and p75/AIRM but not LIR1/ILT2, p58.1, p58.2, p70, or NKG2A/CD94 (n = 15). Interestingly, 25% of the donors expressed p140. IRp60 cross-linking inhibited eotaxin-dependent transmigration of eosinophils in a calcium-independent fashion. In addition, cross-linking of IRp60 on the eosinophils in the presence of IL-5/GM-CSF inhibited the antiapoptotic effect of these cytokines and blocked the release of TNF-alpha, IL-1beta, IFN-gamma, IL-4, and 3T3 fibroblast proliferation. Cross-linking of IRp60 inhibited IL-5-mediated JAK2 phosphorylation as well as eotaxin- and IL-5/GM-CSF-mediated ERK1/2 and p38 phosphorylation. Furthermore, upon cross-linking, IRp60 underwent tyrosine phosphorylation and recruited SHP-1 but not SHP-2. These findings demonstrate a novel pathway for suppressing the activity of human eosinophils, thus indicating IRp60 as a future potential target for the treatment of allergic and eosinophil-associated diseases.

Major Basic Protein Homolog (MBP2): A Specific Human Eosinophil Marker

Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.