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Respiratory syncytial virus (RSV) is associated with bronchiolitis in infancy and the later development of asthma. Research on RSV in vitro requires preparation of a purified RSV stock. The objective for this work was to develop best methods for RSV purification, while monitoring the samples for potential contaminating proinflammatory mediators. Using polyethylene glycol concentration, and sucrose-gradient ultracentrifugation, we collected samples at each step of purification and measured the values of RSV titer, total protein (µg/mL), and proinflammatory cytokines (ELISA). We analyzed the efficacy of each step in the purification procedure. In so doing, we also determined that despite optimal purification methods, a well-known chemokine in the field of allergic disease, CCL5 (RANTES), persisted within the virus preparations, whereas other cytokines did not. We suggest that researchers should be aware that CCL5 appears to co-purify with RSV. Despite reasonable purification methods, a significant level of CCL5 (RANTES) persists in the virus preparation. This is relevant to the study of RSV-induced allergic disease.

Eosinophil degranulation is a determining factor in allergy-mediated airway pathology. Receptor-mediated degranulation in eosinophils requires vesicle-associated membrane protein 7 (VAMP-7), a principal component of the SNARE fusion machinery. The specific contribution of eosinophil degranulation to allergen-induced airway responses remains poorly understood. We generated mice with VAMP-7 gene deficiency exclusively in eosinophils (eoCRE/V7) from a cross using eosinophil-specific Cre recombinase-expressing mice crossed with VAMP-7 f/f mice. Eosinophils from eoCRE/V7 mice showed deficient degranulation responses in vitro, and responses continued to be decreased following ex vivo intratracheal adoptive transfer of eoCRE/V7 eosinophils into IL-5/hE2/EPX −/− mice. Consistent with diminished degranulation responses, reduced airway hyperresponsiveness was observed in ovalbumin-sensitized and challenged eoCRE/V7 mice following methacholine inhalation. Therefore, VAMP-7 mediates eosinophil degranulation both in vitro and ex vivo, and this event augments airway hyperresponsiveness.

Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a+ structures, some of which colocalized with CD63+ crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation.

Background 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. Objective 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. Methods 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. Results 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. Conclusions 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.

Background: Tissue recruitment and activation of eosinophils contribute to allergic symptoms by causing airway hyperresponsiveness and inflammation. Shape changes and mediator release in eosinophils may be regulated by mammalian Rho-related guanosine triphosphatases. Of these, Rac2 is essential for F-actin formation as a central process underlying cell motility, exocytosis, and respiratory burst in neutrophils, while the role of Rac2 in eosinophils is unknown.We set out to determine the role of Rac2 in eosinophil mediator release and F-actin-dependent shape change in response to chemotactic stimuli. Methods: Rac2-deficient eosinophils from CD2-IL-5 transgenic mice crossed with rac2 gene knockout animals were examined for their ability to release superoxide through respiratory burst or eosinophil peroxidase by degranulation. Eosinophil shape change and actin polymerization were also assessed by flow cytometry and confocal microscopy following stimulation with eotaxin-2 or platelet-activating factor. Results: Eosinophils from wild-type mice displayed inducible superoxide release, but at a small fraction (4–5%) of human eosinophils. Rac2-deficient eosinophils showed significantly less superoxide release (p < 0.05, 26% less than wild type). Eosinophils lacking Rac2 had diminished degranulation (p < 0.05, 62% less eosinophil peroxidase) and shape changes in response to eotaxin-2 or platelet-activating factor (with 68 and 49% less F-actin formation, respectively; p < 0.02) compared with wild-type cells. Conclusion: These results demonstrate that Rac2 is an important regulator of eosinophil function by contributing to superoxide production, granule protein release, and eosinophil shape change. Our findings suggest that Rho guanosine triphosphatases are key regulators of cellular inflammation in allergy and asthma.

Platelet-activating factor (PAF [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine]) is a phospholipid mediator released from activated macrophages, mast cells, and basophils that promotes pathophysiologic inflammation. Eosinophil responses to PAF are complex and incompletely elucidated. We show in this article that PAF and its 2-deacetylated metabolite (lysoPAF) promote degranulation (release of eosinophil peroxidase) via a mechanism that is independent of the characterized PAFR. Specifically, we demonstrate that receptor antagonists CV-3988 and WEB-2086 and pertussis toxin have no impact on PAF- or lysoPAF-mediated degranulation. Furthermore, cultured mouse eosinophils from PAFR−/− bone marrow progenitors degranulate in response to PAF and lysoPAF in a manner indistinguishable from their wild-type counterparts. In addition to PAF and lysoPAF, human eosinophils degranulate in response to lysophosphatidylcholine, but not phosphatidylcholine, lysophosphatidylethanolamine, or phosphatidylethanolamine, demonstrating selective responses to phospholipids with a choline head-group and minimal substitution at the sn-2 hydroxyl. Human eosinophils release preformed cytokines in response to PAF, but not lysoPAF, also via a PAFR-independent mechanism. Mouse eosinophils do not release cytokines in response to PAF or lysoPAF, but they are capable of doing so in response to IL-6. Overall, our work provides the first direct evidence for a role for PAF in activating and inducing degranulation of mouse eosinophils, a crucial feature for the interpretation of mouse models of PAF-mediated asthma and anaphylaxis. Likewise, we document and define PAF and lysoPAF-mediated activities that are not dependent on signaling via PAFR, suggesting the existence of other unexplored molecular signaling pathways mediating responses from PAF, lysoPAF, and closely related phospholipid mediators.

Eosinophils expressing indoleamine 2, 3-dioxygenase (IDO) may contribute to T-helper cell (Th)2 predominance. To characterize human thymus IDO+ eosinophil ontogeny relative to Th2 regulatory gene expression, we processed surgically obtained thymi from 22 children (age: 7 days to 12 years) for immunohistochemistry and molecular analysis, and measured cytokine and kynurenine levels in tissue homogenates. Luna+ eosinophils (∼2% of total thymic cells) decreased in number with age (P = 0.02) and were IDO+. Thymic IDO immunoreactivity (P = 0.01) and kynurenine concentration (P = 0.01) decreased with age as well. In addition, constitutively-expressed interleukin (IL)-5 and IL-13 in thymus supernatants was highest in youngest children. Eosinophil numbers correlated positively with expression of the Th2 cytokines IL-5, IL-13 (r = 0.44, P = 0.002), and IL-4 (r = 0.46, P = 0.005), transcription factor signal transducer and activator of transcription-6 (r = 0.68, P = 0.001), and the chemokine receptor, CCR3 (r = 0.17, P = 0.04), but negatively with IL-17 mRNA (r = −0.57, P = 0.02) and toll-like receptor 4 expression (r = −0.74, P = 0.002). Taken together, these results suggest that functional thymic IDO+ eosinophils during human infant life may have an immunomodulatory role in Th2 immune responses.

Background: Indoleamine 2,3 dioxygenase (IDO), the rate-limiting enzyme in tryptophan catabolism, is important in generating tolerance at the foetal–maternal interface. Studies using 1-methyl-tryptophan (1-MT), the specific inhibitor of IDO, showed that this enzyme is important in interferon-gamma (IFN-γ)-dependent inhibition of allergic inflammation in the respiratory airway during immunotherapy. Aims of study: We investigated the role of IDO in the development of allergic sensitization, leading to allergic inflammation and airway hyper-responsiveness (AHR). Methods: We used a mouse model to generate mucosal tolerance to lipopolysaccharide-free ovalbumin (OVA) following repeated intranasal inoculation of OVA over a 3-day period. We tested the successful induction of tolerance by subsequent intraperitoneal (i.p.) sensitization followed by intranasal challenge with OVA. A slow-release pellet of 1-MT implanted into mice was used to block IDO activity prior to repeated intranasal inoculation of OVA. We measured T-cell proliferation in response to OVA, determined airway inflammation, and measured AHR to intranasal methacholine to investigate the role of IDO in sensitization to OVA. Results: Repeated intranasal administration of OVA generated tolerance and prevented a subsequent sensitization to OVA via the i.p. route. This response was inhibited in mice receiving a slow-release pellet of 1-MT. However, we successfully reconstituted tolerance in mice receiving 1-MT following intra-peritoneal injection of a mixture of kynurenine and hydroxyanthranilic acid. Conclusion: Our data suggest that, in addition to their role in IFN-γ-mediated inhibition of allergic airway inflammation, products of tryptophan catabolism play an important role in the prevention of sensitization to potential allergens in the respiratory airway.

A recent review inAllergy, Asthma, and Clinical Immunologysuggested that eosinophils play a minor role, if any, in the inflammatory spectrum of asthma and allergic inflammation. The article that dealt with mast cells suggested that the presence of these important cells within the smooth muscle layer in asthmatic airways renders this cell type primal in asthma and an obvious and important target for therapy. This article proposes that in a complex inflammatory milieu characterizing the complex syndromes we call asthma, no single cell phenotype is responsible for the condition and thus should be a sole target for therapeutic strategies. Our reductionist approach to research in asthma and related conditions has provided us with convincing evidence for multiple roles that immune, inflammatory, and structural cell types can play in complex diseases. The next stage in understanding and ameliorating these complex conditions is to move away from the simplistic notion of one cell type being more important than another. Instead, what is needed is to acquire knowledge of intricate and exquisite biological systems that regulate such conditions in both health and disease involving various cell types, mediators, pharmacologically active products, their multifaceted capacities, and their socio-biological networking.

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-γ, 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.

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.

Background: Animal and human studies demonstrated that interleukin (IL)-12, a Th1 cytokine, reduces blood and bronchial eosinophilia, and airway hyperreactivity. According to current concepts, these effects are mediated through the release of cytokines promoting eosinophil recruitment and activation. However, the presence of IL-12 receptors on eosinophils suggests that IL-12 also acts directly on eosinophils. We postulated that IL-12 directly modulates eosinophil functions and has the capacity to regulate eosinophil degranulation, migration and survival, in vitro. Method: Effects of IL- 12 on purified human blood eosinophils were evaluated for peroxidase (EPO) release, eotaxin-induced migration through a model of basement membrane (Matrigel), and survival. Results: IL-12 inhibited 50% of PAF and secretory IgA-induced EPO release (n = 8, p < 0.001). IL-12 also reduced eotaxin-induced migration through Matrigel by 54 ±6% (n = 6, p < 0.01). These effects were not explained by an IL-12-induced impaired viability or apoptosis. Conclusion: Our results demonstrate that IL-12 directly modulates eosinophil functions without promoting apoptosis and explain, at least in part, the effects of IL-12 on eosinophils observed in in vivo studies.

Background: Clarithromycin exhibits anti-inflammatory as well as antimicrobial activity, leading to decreased symptoms of asthma and chronic sinusitis. The mode of anti-inflammatory effects of clarithromycin on inflammatory cells is not well understood. We hypothesized that clarithromycin inhibits inflammatory cell mediator release and survival. Methods: We investigated the effects of this drug on survival and mediator release from mast cells, eosinophils and neutrophils. Results: Human eosinophil and neutrophil respiratory burst was inhibited by up to 54% after 1–2 h pretreatment with 100 µg/ml clarithromycin. Similar doses of erythromycin did not affect respiratory burst responses in these cells. Clarithromycin at doses of up to 100 µg/ml had no effect on granule-derived mediators released from mast cells and neutrophils. However, we found that clarithromycin (100 µg/ml) induced cell death in mast cells and eosinophils after 16–48 h incubation. Conclusion: Clarithromycin inhibited inflammatory cell mediator release and survival, which may enhance its ability to reduce the symptoms of chronic sinusitis and asthma.

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-γ showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-γ-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-γ-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-γ alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-γ. Coculture of eosinophils with an IFN-γ-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

The production and release of eosinophil peroxidase (EPO) has been associated with human pathology. Degranulation assays with eosinophils are typically very difficult to do, with very low release values. EPO is unique for its high cationic charge. As such, it adheres to most extracellular surfaces, rendering it more difficult to measure compared with other released cellular proteins. Based on the understanding of the sticky nature of EPO, we were concerned that EPO released in vitro cannot be reproducibly measured in the supernatants of stimulated cells. Instead, we suspected that much of the released EPO was left adherent to the tube walls. We chose to investigate the measurement of EPO activity using the peroxidase substrate, O-phenylenediamine (OPD). Unlike other peroxidase substrates, OPD is soluble in aqueous physiological solutions, which do not lyse cell membranes, thereby allowing us to add OPD directly to eosinophils and exclusively measure extracellular EPO. This novel approach would remove the concerns of incorrect EPO measurements due to its adhesive nature. In addition, we developed this method to quantify EPO release in terms of EPO concentration. Finally, using this technique, we have been able to demonstrate secretory IgA (s-IgA)-induced release of EPO. By using OPD, we have developed a more sensitive and specific method to analyze the release of extracellular EPO.

Disorders of the respiratory system, such as cystic fibrosis (CF), involve the infiltration and activation of airway inflammatory cells, including neutrophils. This leads to the secretion of peroxidases, which react further with substrates in solution to produce oxidative metabolites, such as 3-chlorotyrosine. Elevated levels of modified tyrosine residues in the airways of patients with CF may be detectable by nuclear magnetic resonance (NMR) in correlation with inflammatory cell influx. In this study, high-resolution (500 MHz) 1H NMR was used to analyze the production of modified tyrosine residues resulting from in vitro stimulation of peripheral blood eosinophils and neutrophils, as well as in sputum samples from control subjects and patients with CF. Following in vitro stimulation, purified peripheral blood neutrophils generated 3-chlorotyrosine, while eosinophils produced predominantly 3-bromotyrosine and 3,5-dibromotyrosine. Chlorinated and brominated tyrosine residues were detected in sputum samples from patients with CF (N=7), but were not detected in the control group (N=9). Neutrophil counts in CF sputum correlated strongly with the presence of 3-chlorotyrosine (r2=0.869). Our findings indicate that neutrophil and eosinophil activation in CF is detectable by NMR. NMR may be a useful tool for the detection of biological markers of inflammatory processes in patient airways.

A major goal in asthma therapy is to reduce or prevent the inflammatory response associated with bronchial hyperresponsiveness, reversible airway obstruction and airway remodelling. However, because of the complex nature of the disease, a single target for such an ideal therapeutic approach remains elusive. To ensure a more rational design of anti-asthma drugs, recent investigations have attempted to elucidate the roles of inflammatory cellular components in asthma. Such studies have shown that eosinophilic infiltration is a prominent feature in the pathophysiology of asthma. Nonetheless, the role of the eosinophil in asthma has been questioned following recent human studies investigating the efficacy of a novel therapeutic strategy targeted at eosinophils.

Inflammatory cells secrete proteins from intracellular vesicles or granules by a process referred to either as exocytosis or as degranulation, which is common to all cell types. Exocytosis is a precise term that describes the process of granule or vesicular fusion with the plasma membrane and is accompanied by release of granule/vesicle contents to the cell exterior. This process is of particular significance with respect to tissue damage and remodeling in inflammatory diseases, inasmuch as these changes are the consequences of inflammatory cell activation and mediator elaboration. Despite its unifying importance to all inflammatory cell types, little is known about the precise molecular and intracellular mechanisms that regulate mobilization of secretory granules/vesicles and, ultimately, secretion of mediators from immune and inflammatory cells. This article reviews the mechanisms and molecules currently implicated at distal stages of exocytosis from eosinophils, neutrophils, mast cells, platelets, and macrophages. Conserved molecules identified among inflammatory cell types indicate a convergence of pathways leading to mediator secretion. The identification of essential molecules in the cascade of events leading to exocytosis is critical in the search for novel therapeutic targets aimed at modulating mediator secretion from these cell types.

Human eosinophils produce a large number of cytokines, including immunoregulatory cytokines. Given that eosinophils store and release interleukin (IL)-4, a key cytokine in the pathogenesis of allergic inflammation, and that IL-4 and IL-13 share common biological functions, we investigated the possibility that IL-13 may be synthesized by these cells. Using flow cytometry and immunocytochemistry, we show that eosinophils synthesize and store IL-13. Granule localization was demonstrated after subcellular fractionation, and IL-13 immunoreactivity was localized to crystalloid, granule-enriched fractions. Furthermore, electron microscopic analyses specifically localized IL-13 to the dense cores of bicompartmental secondary granules. Upon CD28 ligation, IL-13 was released by eosinophils, whereas a combination of CD28 and immunoglobulin A complexes resulted in decreased IL-13 secretion. Furthermore, eosinophil-derived IL-13 exerts a biological effect, inducing CD23 expression on B cells. By having the capacity to synthesize and release IL-13, eosinophils may participate in the development and maintenance of the T helper cell type 2 response, a prominent feature of allergic diseases.

Nitric oxide (NO) affects the function of ion channels in many cell types, but its role in the regulation of eosinophil ion channels is unknown. In this study, we used the perforated patch-clamp method to investigate the effect of endogenous and exogenous NO on eosinophil ion channels. Using the NO synthase inhibitor, N-nitro-l-arginine methyl ester, we showed that endogenous NO did not affect the whole-cell current in eosinophil. However, two NO donors, S-nitroso-glutathione and S-nitroso-N-acetyl penicillamine, activated whole-cell currents via a NO/cGMP-dependent pathway. Ion substitution and pharmacological studies showed that NO-activated currents were carried by K+ ions, likely through ATP-dependent K+ channels (KATP). Although RT-PCR studies showed the expression of several classes of K+ channels in human eosinophils, NO donors affected only KATP channel function. We conclude that NO, at concentrations likely to be encountered in vivo, could prevent eosinophil activation by opening KATP channels.

Background: Exocytosis of eosinophil granule-derived mediators is thought to be an important effector response contributing to allergic inflammation. Secretion from many cell types has been shown to be dependent on the formation of a docking complex composed of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on the vesicle (v-SNAREs) and the target membrane (t-SNAREs). The SNARE isoforms VAMP-2, SNAP-23, and syntaxin-4 have been described in secretory processes in myeloid cells. Previously, we have demonstrated that the v-SNARE VAMP-2 is a candidate v-SNARE involved in eosinophil exocytosis and is localized to a pool of RANTES-positive vesicles that translocate to the cell periphery after IFN-γ-induced degranulation. Objective: We sought to determine whether eosinophils express the t-SNARE isoforms SNAP-23 and syntaxin-4 as potential binding targets for VAMP-2 during exocytosis. Methods: Human peripheral blood eosinophils (>97%) from atopic subjects were subjected to RT-PCR and sequence analysis by using specific primers for SNAP-23 and syntaxin-4. Protein expression and localization was determined by means of Western blot analysis of eosinophil subcellular fractions and confirmed with confocal laser scanning microscopy. Results: Nucleotide sequences obtained from PCR products exhibited nearly identical (>95%) homology with reported sequences for human SNAP-23 and syntaxin-4. Both SNAP-23 and syntaxin-4 were present in plasma membranes, with some staining in endoplasmic reticulum and Golgi membranes.Negligible expression was detected in crystalloid and small secretory granules. Conclusions: The plasma membrane-associated t-SNAREs SNAP-23 and syntaxin-4 are expressed in human eosinophils and are likely candidates for association with VAMP-2 during docking, which is followed by exocytosis. These findings support a role for SNARE molecules in eosinophil mediator release. (J Allergy Clin Immunol 2002;109:299-306.)

SummaryBackground Infants that develop severe bronchiolitis due to respiratory syncytial virus (RSV) are at increased risk of developing asthma later in life. We investigated a potential immunological mechanism for the association between RSV and the development of allergic inflammation. The enzyme indoleamine 2,3-dioxygenase (IDO) has been reported to induce selective apoptosis of T helper 1 (Th1) cells and contributed to Th2-biased immune responses. Objective To determine whether RSV infection in vitro could induce IDO expression and bioactivity in human dendritic cells, leading to a Th2-biased immune response. Methods Human peripheral blood monocytes from healthy adult donors were isolated, differentiated to dendritic cells (moDC), in vitro. We studied RSV infection and mechanisms of IDO activation in moDC with subsequent effect on T-bet expression. Results We found that moDC were infected by RSV and that this induced IDO activation. RSV-induced IDO activity was inhibited by palivizumab, UV inactivation, TL4R inhibition, and ribavirin. However, blocking endosomal TLR function with chloroquine did not inhibit IDO activity. Selective inhibitors suggested that RSV-induced IDO activity was dependent on the retinoic acid-inducible gene-I (RIG-I) related pathway via NF-κB and p38 MAPK. Coculture of RSV-infected moDC with activated T cells, in a transwell system, suppressed expression of T-bet (a Th1-associated factor) but not GATA3 (a Th2 regulator). Inhibition of IDO activity with the competitive inhibitor, 1-methyl tryptophan, blocked the effect on T-bet expression. Conclusion and Clinical Relevance Our data show for the first time that RSV can induce the expression and bioactivity of IDO in human moDC, in a virus replication-dependant fashion. We suggest that RSV activation of IDO could be a potential mechanism for the development of allergic diseases.

Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

Background: Exocytosis is an integral event during IFN-γ–induced piecemeal degranulation in eosinophils. In many tissues soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including vesicle-associated membrane protein (VAMP), act as specific intracellular receptors to allow granule fusion with the membrane during degranulation. However, the mechanisms underlying eosinophil piecemeal degranulation induced by IFN-γ are not well understood. Objective: We sought to assess whether eosinophils express the vesicular SNARE protein VAMP-2 and to determine the involvement of VAMP-2 in IFN-γ–induced piecemeal degranulation. Methods: Human peripheral blood eosinophils (≥97%) from atopic subjects were subjected to RT-PCR and sequence analysis with specific primers for VAMP-2 mRNA. Western blotting and flow cytometric analysis were carried out to confirm the identity of VAMP-2 and its susceptibility to cleavage by tetanus toxin. Confocal laser scanning microscopy imaging was conducted on double-labeled cytospin preparations of eosinophils at 0, 5, 10, 30, and 60 minutes and 16 hours of IFN-γ (500 U/mL) stimulation. Results: Eosinophils expressed VAMP-2 mRNA (n = 4 donors), which exhibited 100% homology with human VAMP-2 cDNA on sequencing. Eosinophils were also found to express tetanus toxin–sensitive VAMP-2 protein. RANTES and VAMP-2 immunofluorescence were observed to colocalize to similar intracellular structures by means of confocal imaging. IFN-γ induced a rapid translocation of VAMP-2 + organelles toward the cell membrane in correlation with RANTES. Conclusions: These findings suggest that exocytosis in human eosinophils is regulated by SNAREs, with a specific role indicated for VAMP-2 in piecemeal degranulation. (J Allergy Clin Immunol 2001;107:671-8.)

Background: Epithelium is considered an active participant in allergic inflammation. Proteinase-activated receptor (PAR) 2 is expressed in a variety of cell types, including epithelial cells, and has been implicated in inflammation. Objective: PAR-2–mediated activation of airway epithelial cells induces the release of mediators that could promote eosinophil survival and mediate eosinophil recruitment. Methods: PAR-2–activating peptides were used to activate the human airway epithelial cell line A549, as well as primary cultures of small airway epithelial cells (SAECs). Human peripheral blood eosinophils were cultured in the presence or absence of epithelial cell supernatants. Survival was assessed by using an Annexin V apoptosis detection kit. GM-CSF and eotaxin were measured by using ELISA. Results: Eosinophils undergo apoptosis in the absence of growth factors. Supernatants from PAR-2–activated A549 epithelial cells increased eosinophil survival. Supernatants from resting SAECs also increased eosinophil survival, but supernatants from PAR-2–activated SAECs showed a greater effect. The effect of PAR-2–activated epithelial cell supernatants on eosinophil survival was completely inhibited by a neutralizing anti-GM-CSF antibody but not an anti-IL-5 antibody. Resting A549 cells did not release any detectable GM-CSF, whereas PAR-2–activated cells released 35 pg/10 6 cells. Resting SAECs released 754.3 pg/10 6 cells of GM-CSF, which was further increased to 1360.5 pg/10 6 cells after PAR-2–mediated activation. Budesonide inhibited this PAR-2 effect. PAR-2–activated epithelial cells also released eotaxin. Conclusion: PAR-2–mediated activation of airway epithelial cells induced release of GM-CSF, which promoted eosinophil survival and activation. It also induced release of eotaxin, which could mediate eosinophil recruitment to the airways. (J Allergy Clin Immunol 2001;107:679-85.)

The controversy surrounding the effector function of eosinophils in health and disease continues at both the clinical and basic discovery levels. Two recent studies reported that an anti-human IL-5 mAb (mepolizumab) specifically targeting eosinophils significantly reduced asthma exacerbations, providing interesting insights into the potential role of this cell in asthma. Similarly, there is increasing evidence that eosinophils play a major role in tissue remodeling within asthmatic airways. In addition, eosinophils appear to play major roles in orchestrating the TH2 bias associated with atopic conditions through production of specific chemokines, cytokines, and metabolic enzymes. Previous studies from our laboratory provided ample evidence that eosinophils synthesize, store, and release a wide range of proinflammatory mediators, including cationic proteins, cytokines, chemokines, and growth factors. The vast majority of eosinophil-derived mediators are prestored in unique granular structures known as crystalloid or secondary granules. After their release to the extracellular environment, crystalloid granule mediators are known to be widely associated with allergic inflammatory events. Exocytosis describes intracellular fusion of granules or secretory vesicles with the plasma membrane after cell activation, leading to the release of granule contents to the extracellular space. Despite the unifying importance of the process of exocytosis to all immune and inflammatory cell types, the precise molecular and intracellular mechanisms that regulate mobilization of secretory granules/vesicles are only recently becoming clear. It is generally believed that eosinophil mediators are synthesized in the Golgi compartment, processed, prepackaged, and stored in crystalloid granules. Three different mechanisms are known to facilitate mediator release, namely classical exocytosis (whereby a granule at a time fuses with the plasma membrane en route to the exterior; this is rarely observed in eosinophils)

SUMMARY Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34 + cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine mRNA profiles were analysed by the reverse transcription‐polymerase chain reaction (RT‐PCR) to determine transcription of IL-4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequences encoding IL-4 and RANTES were expressed in freshly prepared, nondifferentiated CD34 + cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2Rpositive colony cells, as assessed using in situ RT‐PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to b2-microglobulin) in rhIL-3- and rhIL-5-treated colony cells (comprising >90% eosinophil-like cells) up to day 28. Immunoreactivity for IL-4 and RANTES co-localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation.

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins.

Tumour necrosis factor (TNF) is an important inflammatory cytokine produced by several cell types. To test the hypothesis that there is cell-type-specific regulation and not redundancy of TNF production, we investigated its production by alveolar macrophages (AM) and peritoneal mast cells (PMC). Cell lysates of freshly isolated AM and PMC contained 9 +/- 3 pg and 57 +/- 17 pg of TNF/10(6) cells, respectively. Furthermore, unstimulated PMC expressed 4 x 10(3)-fold more attomols of TNF mRNA/microg total RNA compared with AM. These data may explain in part the greater TNF-dependent cytotoxicity of PMC. Furthermore, fixed PMC showed significantly higher TNF-dependent cytotoxic activity than AM (sevenfold), suggesting that PMC express more membrane TNF than AM. Although AM and PMC contain different amounts of TNF, antigen stimulation caused a similar release of TNF from sensitized rats. Interferon (IFN)-gamma, respectively, stimulated and inhibited AM and PMC TNF-dependent cytotoxicity whereas lipopolysaccharide (LPS) significantly stimulated TNF-dependent cytotoxicity in both cell types. However, TNF released (AM 400-fold and PMC threefold) and TNF mRNA expression, as measured by competitive reverse transcription-polymerase chain reaction (AM 7 x 10(3)-fold and PMC twofold), were considerably greater in LPS-stimulated AM than PMC. Our data indicate that TNF is differentially expressed in these two cell types and that its production is dependent on the nature of the stimulus. These data provide vital basis in experimental approaches aimed at modulating the effect of TNF in airway disease conditions involving both AM and mast cells.

Eosinophils induce tissue injury by releasing granule-associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane-bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP-bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP-dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription–polymerase chain reaction analysis and sequencing of the amplified product, guinea-pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane-associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-gamma (IFN-gamma) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-gamma to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 +/- 15 pg/mL per 2 x 10(6) cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-gamma stimulation. This effect was not observed in eosinophils treated with IFN-alpha, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-gamma. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

Eosinophils and their products play a major role in inflammatory reactions associated with asthma and allergic diseases. There is a growing body of evidence that eosinophils synthesize, store, and release bioactive cytokines and chemokines with the potential to contribute to local inflammatory changes. Fluorescein isothiocyanate (FITC) has been widely used as an immunofluorescent conjugate for antibodies specific for detection of these molecules. However, FITC is an ionic fluorochrome (negatively charged) which binds strongly to positively charged eosinophil granule proteins. We developed new methods to prevent charge-based interactions of ionic fluorochromes with granule proteins, and optimised immunofluorescent staining techniques for eosinophils. An antibody to interleukin-6 (IL-6) was used to optimise this procedure for eosinophil-derived granule proteins. We attempted to block nonspecific binding of FITC-labelled anti-IL-6 using normal human IgG, foetal calf serum (FCS), bovine serum albumin (BSA), and goat, horse, and normal human sera at concentrations ranging between 1-10%. Only human IgG (2%; 20 mg/ml) was able to reduce background fluorescence. These results were confirmed using Texas Red conjugates. We also used antibodies conjugated to a nonionic fluorochrome, BODIPY FL, to detect IL-6 in eosinophils. Unlike FITC, BODIPY FL-conjugated antibodies did not require strong blocking conditions (2% BSA). We recommend that a neutral fluorochrome (BODIPY FL) should be used for immunofluorescence studies in eosinophils. Alternatively, strong blocking conditions may be used to decrease background binding of FITC-conjugated antibodies.

Eosinophils, prominent cells in asthmatic inflammation, have been shown to synthesize, store, and release an array of up to 18 cytokines and growth factors, including interleukin-6 (IL-6). In this report, we show that IL-6 immunofluorescence localizes to the matrix of the crystalloid granule in peripheral blood eosinophils from atopic asthmatics using confocal laser scanning microscopy (CLSM). Granule localization of IL-6 was confirmed using dot-blot analysis and enzyme-linked immunosorbent assay (ELISA) on subcellular fractions of highly purified eosinophils produced from density centrifugation across a 0% to 45% Nycodenz gradient. IL-6 was found to coelute with eosinophil crystalloid granule marker proteins, including eosinophil peroxidase (EPO), major basic protein (MBP), arylsulfatase B, and β-hexosaminidase. Immunoreactivity to IL-6 colocalized with granule-associated IL-2 and IL-5 in subfractionated eosinophils. We also made the novel and compelling observation that interferon γ (IFNγ), a Th1-type cytokine, stimulated an early elevation in eosinophil IL-6 immunoreactivity. A 2.5-fold enhancement of IL-6 immunoreactivity in eosinophil granules was observed within 10 minutes of IFNγ treatment (500 U/mL), as determined by subcellular fractionation and CLSM. These findings suggest that IFNγ has short-term effects on human eosinophil function and imply that a physiologic role exists for Th1-type cytokine modulation of Th2-type responses in these cells.

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil´s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.

Background: The importance and specific role(s) of eosinophils in modulating the immune/inflammatory phenotype of allergic pulmonary disease remain to be defined. Established animal models assessing the role(s) of eosinophils as contributors and/or causative agents of disease have relied on congenitally deficient mice where the developmental consequences of eosinophil depletion are unknown. Methods: We developed a novel conditional eosinophil-deficient strain of mice (iPHIL) through a gene knock-in strategy inserting the human diphtheria toxin (DT) receptor (DTR) into the endogenous eosinophil peroxidase genomic locus. Results: Expression of DTR rendered resistant mouse eosinophil progenitors sensitive to DT without affecting any other cell types. The presence of eosinophils was shown to be unnecessary during the sensitization phase of either ovalbumin (OVA) or house dust mite (HDM) acute asthma models. However, eosinophil ablation during airway challenge led to a predominantly neutrophilic phenotype (>15% neutrophils) accompanied by allergen-induced histopathologies and airway hyper-responsiveness in response to methacholine indistinguishable from eosinophilic wild-type mice. Moreover, the iPHIL neutrophilic airway phenotype was shown to be a steroid-resistant allergic respiratory variant that was reversible upon the restoration of peripheral eosinophils. Conclusions: Eosinophil contributions to allergic immune/inflammatory responses appear to be limited to the airway challenge and not to the sensitization phase of allergen provocation models. The reversible steroid-resistant character of the iPHIL neutrophilic airway variant suggests underappreciated mechanisms by which eosinophils shape the character of allergic respiratory responses.

Many household products contain fragrances. Little is known about exposure to fragrances on human health, particularly within the airways. This study aimed to evaluate how common household fragrance products (i.e. air fresheners, cleaning products) affect people with asthma, who frequently report sensitivity to these products. Many of these products have volatile organic compounds or semi-volatile organic compounds. This study evaluated nine fragrance materials in an aerosol formulation to assess effects on airway physiology, airway inflammation and symptom perception in normal controls and those with asthma.The effects of fragrances were evaluated in people without asthma, people with mild asthma and people with moderate asthma in a four-way crossover placebo-controlled study. Subjects were exposed twice to a fragranced aerosol and twice to a placebo aerosol (15 and 30 min each). Subjects completed a questionnaire for 29 symptoms during and up to 3 h after each exposure scenario. Spirometry was performed prior to and 3 h post-exposure; sputum induction was conducted 3 h post-exposure.Nasal symptoms showed the greatest frequency of response in all three subject groups, and moderate asthmatics reported the greatest symptom severity and symptom types. No significant differences were noted in physiology or cellular inflammation.A trend for increased symptoms was noted in moderate asthmatics, suggesting that asthma severity may play a factor in fragrance sensitivity.

Eosinophils synthesize and store various cytokines with potential autocrine activity. We hypothesized that eosinophils synthesize and store RANTES, a CC-chemokine with potent eosinophil chemotactic activity. Expression of RANTES mRNA in highly purified eosinophil populations was detected by reverse transcription followed by polymerase chain reaction analysis. In situ hybridization (ISH) with 35S-labeled RANTES-specific riboprobes showed that 6.8-10% of peripheral blood eosinophils obtained from atopic subjects expressed RANTES mRNA, increasing to 25% after incubation (16 h) with interferon (IFN)-gamma, but not ionomycin in vitro. Peripheral blood eosinophils also showed specific immunoreactivity with an anti-RANTES monoclonal antibody, consistent with translation of the mRNA. By enzyme-linked immunosorbent assay, blood eosinophils were shown to contain a median of 7300 pg (range 5200-8800) RANTES per 10(6) cells, of which a mean of 24% was released into culture supernatants after stimulation of the cells with serum-coated particles in vitro. These culture supernatants exhibited eosinophil chemotactic activity which was inhibited (mean 68%) by a specific anti-RANTES antibody. Sequential immunocytochemistry and ISH on biopsies obtained from allergen-induced late-phase cutaneous reactions showed that 55-75% of the infiltrating RANTES mRNA+ cells were EG2+ eosinophils. Allergen, but not diluent challenge, was also associated with a time-dependent increase in the number of cells showing RANTES immunoreactivity. Of these cells, 55% were identified as eosinophils by morphological criteria. Thus, human eosinophils have the capacity to synthesize, store and secrete physiologically relevant quantities of RANTES, and may therefore be an important source of this chemokine in allergic inflammation.

There has been recent interest in eosinophils as a histological diagnostic marker of liver allograft rejection. However, the reliability of counting eosinophils in sections stained with hematoxylin and eosin (H&E) has not been evaluated previously. We quantified eosinophils in 10 day-5 protocol liver biopsy specimens in 10 patients. The control groups were 5 patients with cytomegalovirus infection, 5 patients with obscure liver dysfunction, and 6 patients with HCV infection. Eosinophil count was assessed using H&E and by specific monoclonal antibody staining using (1) an anti-ECP antibody (EG2) and (2) a monoclonal antibody against human eosinophil major basic protein (MBP) (BMK-13). The average percentage of the total inflammatory infiltrate of eosinophils in portal tracts was 9% in the moderate to severe rejection group as compared with 0.25% in the mild rejection group (P < .001) and 0% in the control group (P < .001). The eosinophil count decreased markedly after successful treatment of rejection. The H&E staining correlated with MBP+ the (BMK-13 immunoreactive) cells but were more numerous with BMK-13. BMK-13 also stained significantly more cells when compared with EG2 (P < .01). This difference may be because EG2 staining only activated eosinophils, whereas BMK-13 is a pan-eosinophilic maker, regardless of activation. This study confirms that eosinophils are a specific feature of acute cellular rejection and are an aid to its diagnosis. BMK-13 is a useful pan-eosinophilic marker that is more efficient in obtaining eosinophil count when compared with H&E.