Incidence of ligament lesions and associated degenerative changes in the elderly wrist

As the carbohydrate lacto-N-fucopentaose III (CD15 antigen or X-determinant) and its sialylated derivative sialyl-Lewis X are involved in the adhesion of cells rolling along the surface of endothelial cells, experiments were done to study the presence of these molecules on human eosinophils from patients with the idiopathic hypereosinophilic syndrome. Normal-density eosinophils from some patients showed higher levels of expression for lacto-N-fucopentaose III than light-density eosinophils. In contrast, sialyl-Lewis X was highly expressed by light-density eosinophils. Activation of normal-density eosinophils with calcium ionophore A23187 resulted in increased expression of these molecules for a short time. Monoclonal antibodies to these carbohydrates stimulated eosinophils to secrete eosinophil cationic protein, but not eosinophil peroxidase, and acted as costimulatory signals for C3b-induced degranulation of eosinophil cationic protein. It was suggested that CD15 and sialyl-Lewis X might contribute to eosinophil-mediated tissue injury in patients with the idiopathic hypereosinophilic syndrome.     

Mechanisms involved in activation of human eosinophil exocytosis by substance P: an in vitro model of sensory neuroimmunomodulation

Substance P (SP), a tachykinin with a wide range of biological activities including a priming effect on human eosinophil chemotaxis, was investigated for its influence on eosinophil cytotoxic function measured as degranulation of eosinophil-derived neurotoxin (EDN). Peripheral blood was obtained from healthy volunteers and the degranulation assays were performed using radioimmunoassay (RIA). SP and its C-terminal elicited EDN release in a time-dependent mode at a narrow range of doses with optimal activity of 10(-6) M. FK888 (NK-1 receptor antagonist) inhibited EDN release stimulated by SP in dose dependency, also a complete inhibition was observed when eosinophils were preincubated with 1000 ng/ml pertussis toxin (PTX). Pre-exposure of eosinophils to staurosporine resulted in blockage of SP-induced EDN release in a dose-dependent mode. On the other hand, SP at 10(-7) M and 10(-8) M primed eosinophils to suboptimal dose (10(-8) M) of Platelet activating factor (PAF) resulting into significant enhancement of EDN release. SP(4-11) fragment showed a similar activity while SP(1-4) fragment was not active. SP priming of eosinophils was not affected by Ca2+ depletion, however, it caused a change in the pattern of the intracellular calcium influx against the suboptimal dose of PAF. These results suggest that SP i) may induced human eosinophil matrix protein degranulation through a receptor mediated mechanism coupled to PTX sensitive G protein(s) with the probability of linkage to phospholipase C activation, and, ii) primes human eosinophils for an exalted inflammatory response through a Ca2+ independent pathway.     

The roles of adhesion molecules, cytokines and chemokines in allergic inflammation

Recently, adhesion molecules, as well as eosinophils, have been found to play an important role in the inflammatory processes in allergic disease. We demonstrated here as below. Characteristics of adhesion molecules expression on eosinophils in asthma, namely, high-intensity expression of adhesion molecules. Induction of adhesion molecule expression by PAF and RANTES and in addition induction by the supernatant of mononuclear cells from mite-allergic asthmatic patients stimulated with mite-allergen as well as with a combination of the recombinant IL-3, GM-CSF and IL-5. Elevated soluble ICAM-1 in bronchial asthma. Moreover, the presence of a large variety of membrane receptors and the identification of cytotoxic molecules (mainly granule basic proteins) have indicated that eosinophils should be considered as effector cells. We therefore investigated the possible release of granule proteins in response to signaling from ICAM-1 and its ligands. The concentrations of eosinophil cationic protein and eosinophil-derived neurotoxin in supernatants of eosinophils were significantly greater (p < 0.05) in the presence of recombinant soluble ICAM-1 than without it. These results suggest that signaling from ICAM-1 and its ligands might induce eosinophil activation and might be involved in degranulation of eosinophil granule proteins. In addition, reactive oxygen species generated by eosinophils have also been considered capable of causing airway injury at the inflamed focus. We examined the effect of recombinant soluble ICAM-1 and its ligands on eosinophil-induced radical oxygen products. Recombinant soluble ICAM-1 augmented eosinophil oxidative metabolism. It was concluded that signaling via adhesion molecules might play an important role in the pathogenesis of allergic inflammation through activation of eosinophils, such as through an increase in oxidative metabolism or degranulation of eosinophil granule proteins.     

The effect of a 50 Hz magnetic field on cognitive function in humans

Accumulation of eosinophils in the lung with concomitant tissue damage are defining histopathologic features of human asthma. Through degranulation and the release of proinflammatory proteins such as major basic protein (MBP), eosinophils may perpetuate this inflammatory response. We investigated the extent of eosinophil degranulation in a murine model of allergic pulmonary inflammation. In this paradigm, the mice develop pulmonary eosinophilia, mucus hypersecretion, tissue damage, and airway edema and hyperreactivity. To evaluate the degree of eosinophil degranulation, we used a polyclonal antibody to murine MBP (mMBP) to perform dot blot analysis of bronchoalveolar lavage (BAL) cells and fluids, and immunohistochemical fluorescent analysis of lung tissue sections. After ovalbumin antigen challenge, we were unable to detect immunoreactive mMBP in the BAL fluids from either nonsensitized or sensitized mice. However, after lysis of the recoverable BAL cells, we were able to detect mMBP by immunoblot analysis, with the levels of immunoreactive mMBP directly related to the number of recoverable eosinophils. We also examined paraffin-embedded, lung tissue sections for patterns of mMBP deposition. Whereas lung sections from allergic mice revealed prominent peribronchial eosinophilia after antigen challenge, tissue sections from nonsensitized animals rarely displayed eosinophils. Despite the presence of numerous eosinophils, no immunohistologic evidence of extracellular mMBP could be found in antigen-challenged allergic mice. Furthermore, rechallenged allergic mice displayed a significant increase in the number of recruited pulmonary eosinophils but all immunoreactive mMBP was still intracellular. We conclude that the recruited pulmonary eosinophils have not substantially degranulated. These results suggest that, in this murine model of allergic inflammation, eosinophil degranulation and release of mMBP does not contribute to the observed pulmonary inflammation and airway hyperreactivity.     

Respiratory syncytial virus-infected pulmonary epithelial cells induce eosinophil degranulation by a CD18-mediated mechanism

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the beta2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.     

Eosinophil activation in ulcerative colitis: studies on mucosal release and localization of eosinophil granule constituents

Activation of eosinophil granulocytes (eosinophils) seems to contribute to the pathophysiology of several inflammatory conditions. This process was evaluated in 18 patients with ulcerative colitis and in 18 healthy controls using intraluminal segmental perfusion of the sigmoid colon and rectum and immunoanalysis for eosinophil cationic protein (ECP) in the perfusate. Immunohistochemistry for eosinophils and neutrophils was made in simultaneously taken biopsies and in biopsies from surgical specimens taken from additional 10 patients. The mucosal release of ECP was increased severalfold in patients with UC. The bowel biopsies demonstrated a lamina propria infiltrated with eosinophils. The degree of eosinophil activation/degranulation was related to the intensity of the inflammatory reaction. Activated eosinophils and extracellular deposits of ECP were, in particular, seen in crypt abscesses and in areas with damaged surface epithelium. Since ECP is highly cytotoxic, its release at the site of inflammatory bowel lesions might reflect a potential pathophysiological mechanism.     

The effect of salmeterol on human eosinophils is both stimulus- and response-dependent

Salmeterol, a long-acting beta 2-adrenoceptor agonist, also possesses some anti-inflammatory properties, but whether eosinophils are the target of such action has been equivocal. To clarify the direct effect of salmeterol on eosinophil functions, we have studied the effect of the drug on the various responses of purified human eosinophils. Superoxide anions (O2-) release and adherence to fibronectin-coated plastic plates induced by platelet-activating factor (PAF), interleukin-5 (IL-5), leukotriene B4 (LTB4) and phorbol myristate acetate (PMA), as well as degranulation induced by C5a and formyl methionyl leucyl phenylalanine (FMLP), in the presence of cytochalasin B (CB) were studied. In the concentration range 10(-8)-10(-5) M, the drug inhibited PAF- and IL-5-induced O2- release, with an IC50 values of 3.2 +/- 1.2 x 10(-7) M and 2.2 +/- 0.4 x 10(-6) M, respectively, Superoxide anion release by LTB4 was only modestly inhibited while that due to PMA was completely unaffected. On the other hand, eosinophil adherence induced by all the 4 stimuli were significantly inhibited within the same concentration range. On eosinophil degranulation, the drug failed to significantly inhibit the release of eosinophil peroxidase (EPO) induced by either C5a or FMLP. In contrast, beta-hexoseaminidase (beta-HA) release by the same agents was significantly inhibited, the inhibition being more pronounced for FMLP-induced, than C5a-induced release. None of the effects of the drug was reversed by the selective beta 2-adrenoceptor antagonist ICI 118551 at a concentration of 10(-7) M. These results show that salmeterol may have some direct inhibitory effects on human eosinophil functions but that these effects are both stimulus- and response-dependent, and are unlikely to be mediated via beta 2 adrenoceptors.     

Induced sputum eosinophil cationic protein (ECP) measurement in asthma and chronic obstructive airway disease (COAD)

BACKGROUND: Induced sputum is a useful way to monitor airway inflammation in asthma, but cell counts are time-consuming and labour intensive. OBJECTIVE: The aim of this study was to evaluate a novel processing method using eosinophil cationic protein (ECP) as a biochemical marker of sputum eosinophil number and activation in subjects with asthma and other airway diseases. METHODS: Sputum was dispersed with dithiothreitol and centrifuged to yield cell free supernatant and a cell pellet. The pellet was treated with a cellular lysis buffer to release cell-associated ECP. ECP was measured in sputum supernatant and in the lysed cell pellet and was compared with sputum eosinophil counts in 31 adults with asthma, chronic obstructive airway disease (COAD), bronchiectasis and healthy controls. The ratio of supernatant to pellet ECP was evaluated as an index of eosinophil degranulation. The effect of sputum processing reagents and storage time on ECP measurement was also evaluated. RESULTS: ECP measured in the cell pellet lysate correlated closely with sputum absolute eosinophil counts across a range of subject groups (r = 0.72, P = 0.004). Sputum eosinophil counts were less well correlated with supernatant ECP levels (r = 0.54, P < 0.05). Incubation with dithiothreitol or lysis buffer did not influence ECP measurement and sputum ECP levels were stable over a 6-9 month period. Sputum supernatant and pellet lysate ECP concentrations were increased in stable asthma, asthma exacerbations and COAD/bronchiectasis (P < 0.05). The ratio of supernatant to pellet ECP was used as an index of eosinophil degranulation and found to be elevated in asthma exacerbations, COAD and bronchiectasis, but not in stable asthma. CONCLUSION: The measurement of ECP in the sputum cell pellet provides a reliable and efficient estimate of sputum eosinophil counts which can potentially be used in clinical trials and epidemiological surveys. The ECP ratio may be a useful marker of eosinophil activation, and was increased in asthma exacerbation and COAD. The increased ECP in COAD reflects a non-selective accumulation of eosinophils in this condition.     

Interaction with secretory component stimulates effector functions of human eosinophils but not of neutrophils

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component bound to IgA mediates transepithelial transport of IgA and confers increased stability on the resultant secretory IgA; however, the effect of secretory component on the biologic activity of IgA is unknown. Here, we report that secretory IgA and secretory component preferentially activate human eosinophils. When eosinophils were stimulated with immobilized secretory IgA, degranulation and superoxide production were two- to threefold greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to secretory IgA and serum IgA. Flow cytometric analysis showed that eosinophils bound to purified secretory component. The binding of 125I-labeled secretory component was inhibited by unlabeled secretory component or secretory IgA but not by serum IgA. Superoxide production by eosinophils stimulated with cytokines or IgG was enhanced synergistically by immobilized secretory component; secretory component showed no effect on neutrophil activation. Finally, anti-CD18 mAb abolished eosinophil superoxide production stimulated with secretory IgA or secretory component but not with serum IgA, suggesting a crucial role for beta2 integrins in eosinophil interactions with secretory IgA or secretory component. Thus, secretory component plays important roles in activating eosinophil functions but not neutrophil functions. This preferential interaction between secretory component and eosinophils may provide a novel mechanism to regulate mucosal tissue inflammation.     

Analysis of signal transduction pathways regulating cytokine-mediated Fc receptor activation on human eosinophils

Igs can be potent stimulants of eosinophil activation since interaction with IgA or IgG-coated particles can lead to eosinophil degranulation. We have investigated the comparative roles of mitogen-activated protein (MAP) kinases (MAPKs; ERK1/2 and p38) and phosphatidylinositol-3 kinase (PI3K) in the priming and regulation of Fc receptor functioning on human eosinophils utilizing a MAPK kinase (MEK) inhibitor (PD98059), a p38 inhibitor SB203580, and the widely used PI3K inhibitors wortmannin and LY294002. We demonstrate that priming of human eosinophils with Th2-derived cytokines, IL-4 and IL-5, differentially activate phosphotyrosine-associated PI3K and ERK and p38 MAP kinases. This activation can be inhibited by pre-incubation with wortmannin or LY294002, PD98059, and SB203580, respectively. Analysis of the effects of the inhibitors on rosette formation between human eosinophils and IgA- or IgG-coated beads revealed that activation of MEK was not required for IgA binding after priming with IL-4 or IL-5. However, inhibition of MEK did inhibit IL-5-primed binding of IgG-beads. The rosette formation of primed eosinophils with IgA-beads could be completely inhibited by wortmannin and LY294002 treatment, demonstrating a critical role for PI3K. Interestingly, inhibition of the p38 pathway also resulted in a complete blockade of IgA rosette formation. This work demonstrates regulatory control by inside-out signaling of Fc receptors by various cytokines on human eosinophils. Thus in vivo the local production of Th2-derived cytokines will regulate the effector functions of Fc receptors.     

Effects of PAF, FMLP and opsonized zymosan on the release of ECP, elastase and superoxide from human granulocytes

Platelet-activating factor (PAF) is a potent chemoattractant for human eosinophils and neutrophils and causes eosinophil and neutrophil recruitment into animal airways. Since eosinophils and eosinophil cationic proteins are thought to play an important role in the pathophysiology of asthma, we have examined the hypothesis that PAF may also stimulate eosinophil cationic protein (ECP) release from human granulocytes. Granulocytes (93% neutrophils, 3% eosinophils) were isolated from the blood of normal volunteers, using metrizamide density gradients, and stimulated in vitro with PAF, L-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or opsonized zymosan (OPZ). Superoxide generation was measured colorimetrically, granulocyte degranulation by a fluorimetric assay for elastase, and eosinophil activation by specific radioimmunoassay (RIA) for ECP. Granulocyte chemotaxis was also measured. Whilst both PAF and FMLP were potent chemoattractants for human mixed granulocytes (concentrations producing half the maximal effect (EC50s) ca 10 nM), PAF at concentrations below 10 microM was a poor stimulus to superoxide generation, elastase release or ECP release from the same cell population. In contrast, FMLP was a potent stimulus to both superoxide generation (EC50 48 nM) and ECP (EC50 ca 100 nM) and elastase release (EC50 ca 1 microM). OPZ was a potent stimulus to superoxide generation, but was a poor stimulus to ECP or elastase release. Thus, although PAF is a potent chemoattractant for human granulocytes, our results suggest that it alone may not stimulate their subsequent activation and release of cytotoxic products.     

Eosinophilic inflammation in cough variant asthma

Eosinophils are considered to play a central pathogenetic role in asthma. We previously reported that sputum eosinophilia was observed in patients with cough variant asthma (CVA), as well as in "classic" asthma with wheezing. This study was undertaken to further investigate the involvement of eosinophils in CVA. The serum eosinophil cationic protein (ECP) level, the percentage of eosinophils in bronchoalveolar lavage (BAL) fluid, and the number of eosinophils in bronchial biopsy specimen were examined in 14 patients with CVA, 21 with classic asthma, and in seven healthy controls. For the two asthmatic groups, the clinical severity was classified with scores of 1-3. Pulmonary function and bronchial responsiveness were not significantly different between the patients with classic asthma and those with CVA. BAL, tissue eosinophil and serum ECP were all significantly increased in both classic asthma and CVA when compared with the controls but were not different between classic asthma and CVA. In both groups of asthmatics, the clinical severity significantly correlated with serum ECP and tissue eosinophils. In conclusion, eosinophilic inflammation is involved in cough variant asthma as well as in classic asthma. Anti-inflammatory treatment may be essential in patients with CVA, as in those with classic asthma.     

Regulation of eosinophil migration by adult T cell leukemia-derived factor

Adult T cell leukemia-derived factor (ADF), originally defined as an IL-2 receptor alpha-chain (IL-2R alpha)/p55 (Tac) inducer, is a human thioredoxin homologue and has many cytokine-like activities. In this study, we examined the regulatory effect of ADF on eosinophil migration using human eosinophils and an eosinophilic subline of HL-60 human promyelocytic leukemia cells, YY-1. rADF induced migration of eosinophils from patients with hypereosinophilia, although rADF exhibited little activity on eosinophils from healthy donors. When human eosinophils were incubated with rADF (0.1-10 micrograms/ml) at 37 degrees C for 24 h, both chemotactic and chemokinetic activity of the complement anaphylatoxin peptide C5a on eosinophil migration was markedly enhanced in a dose-dependent manner. Similarly, this enhancing effect of rADF was observed in the migration assay using YY-1 cells. In contrast, rADF showed no modulation of migratory behavior of human eosinophils and YY-1 cells by IL-3, IL-5, nor granulocyte-macrophage colony-stimulating factor. Scatchard analysis of C5a receptors on YY-1 cells using 125I-C5a showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. Furthermore, mutant ADF (mADF), which had no reducing activity, had no enhancing effect on C5a-induced eosinophil migration. These results indicate a possible involvement of ADF in the recruitment of eosinophils through redox regulation by a dithiol reductase activity.     

Diltiazem compared with placebo in the prevention of myocardial ischemia during non-cardiac surgery

OBJECTIVE: Because eosinophils likely play important roles in the pathophysiology of allergic diseases, specific inhibitors of eosinophils may be desirable to treat such diseases. To evaluate the capacity of a novel compound, sulochrin, as an inhibitor of eosinophilic inflammation, we examined the effects of this compound on various effector functions of eosinophils. MATERIALS AND METHODS: We examined the effects of sulochrin on degranulation of human eosinophils stimulated with platelet-activating factor (PAF) or Sepharose 4B beads coated with secretory IgA (sIgA) or IgG. The effects of sulochrin on other effector functions of human eosinophils, including superoxide anion (O2-) production, leukotriene (LT) C4 release, and interleukin (IL)-8 production induced by sIgA-beads were also studied. Finally, using PAF and LTB4 as chemoattractants, we evaluated the potency of sulochrin to inhibit eosinophil migration in vitro and in vivo. RESULTS: Sulochrin inhibited EDN release by eosinophils stimulated with sIgA-beads. IgG-beads and PAF in a concentration-dependent manner; IC50 values were 0.75 microM, 0.30 microM and 0.03 microM. Eosinophil O2- production, LTC4 release, and IL-8 production were also inhibited by sulochrin. Furthermore, PAF-induced chemotaxis of human eosinophils and LTB4-induced chemotaxis of guinea pig eosinophils were abolished by 1 microM of sulochrin. Finally, sulochrin potently inhibited LTB4-induced infiltration of eosinophils into the skin of guinea-pig in vivo. CONCLUSIONS: These results suggest that sulochrin is a potent inhibitor of various effector functions of eosinophils. Sulochrin and its derivatives may be useful in the development of therapeutic approaches for patients with allergic diseases.     

Downregulation of the expression of intercellular adhesion molecule (ICAM)-1 on bronchial epithelial cells by fenoterol, a beta2-adrenoceptor agonist

An effect of estrogen in the uterus of rats is the invasion of eosinophil granulocytes into the endometrium and the myometrium. Progesterone prevents the estrogen induced eosinophilia. In the uterus the eosinophils degranulate, probably mediated by steroid hormones. The extent of infiltration with eosinophils may allow estimation of the estrogen activity of drugs. The present study investigates the effects of a long-term treatment with the progesterone antagonists Onapristone and ZK 112.993 on the eosinophils in the uterus of intact mature rats. The visualization of the eosinophils was done immunohistochemically with an antiserum against the Major Basic Protein (MBP) localized in the secondary granules. The localization of this protein in the extracellular matrix immediately beneath the eosinophils in Carnoy-fixed uteri was taken as a marker for their degranulation (Duchesne and Badia 1992). Onapristone caused a strong infiltration by eosinophils which corresponds to those seen in ovariectomized rats treated with estrogen, while the effect of ZK 112.993 was clearly weaker. This is in agreement with the lower antiprogestational activity of ZK 112.993 also found with other endpoints. In uteri fixed in Bouin' solution the immunoreactivity of the eosinophils was strongest and restricted to these cells. However, after Carnoy' fixation the antibody reacted with the extracellular matrix beneath the eosinophils while the staining intensity of the cells was decreased. Our results do not support the idea of a substantial extracellular deposition of MBP in the uterus of rats but speak in favour of a permeabilization of the eosinophil membranes by alcohol containing fixatives allowing granule contents to leave the cell.     

Mast cells enhance eosinophil survival in vitro: role of TNF-alpha and granulocyte-macrophage colony-stimulating factor

Mast cell-eosinophil interactions in allergy have not yet been completely defined. To determine whether mast cells influence eosinophil survival, human peripheral blood eosinophils were incubated with rat peritoneal mast cell sonicate. After 3 days, viable eosinophils in medium were 21.3% compared with 44% with mast cell sonicate. Like sonicate, supernatants of compound 48/80-activated mast cells enhanced eosinophil survival, demonstrating that the factor(s) involved is stored preformed and rapidly released. Increased eosinophil survival was due to an inhibition of apoptosis (morphologic analysis; annexin V/PI). Neutralizing Abs to granulocyte-macrophage CSF (GM-CSF), but not to IL-3 or IL-5, decreased by 61.7% the enhancing effect on eosinophil viability. Eosinophils are the source of GM-CSF since its release in the culture medium was inhibited by their incubation with the mast cell sonicate together with dexamethasone. In addition, eosinophils incubated with the sonicate expressed mRNA for GM-CSF. To partially characterize the mast cell-derived factor(s) increasing eosinophil survival, the sonicate was heated (56 degrees C/30 min or 100 degrees C/10 min) or preincubated with antihistamines or with anti-TNF-alpha-neutralizing Abs. Most of the activity was heat labile. TNF-alpha was found to be predominantly (70%) responsible, while histamine had no role. Mast cell sonicate also caused eosinophils to release eosinophil peroxidase and to display morphologic signs of activation. In conclusion, we have demonstrated that mast cells enhance eosinophil survival in part through their activation to produce and release the autocrine survival cytokine GM-CSF.     

Triple role of platelet-activating factor in eosinophil migration across monolayers of lung epithelial cells: eosinophil chemoattractant and priming agent and epithelial cell activator

Infiltration of eosinophils into the lung lumen is a hallmark of allergic asthmatic inflammation. To reach the lung lumen, eosinophils must migrate across the vascular endothelium, through the interstitial matrix, and across the lung epithelium. The regulation of this process is obscure. In this study, we investigated the migration of human eosinophils across confluent monolayers of either human lung H292 epithelial cells or primary human bronchial epithelial cells. Established eosinophil chemoattractants (IL-8, RANTES, platelet-activating factor (PAF), leukotriene B4, and complement fragment 5a (C5a)) or activation of the epithelial cells with IL-1beta induced little eosinophil transmigration (<7% in 2 h). In contrast, addition of PAF in combination with C5a induced extensive (>20%) transepithelial migration of unprimed and IL-5-primed eosinophils. Eosinophil migration assessed in a Boyden chamber assay, i.e., without an epithelial monolayer, was only slightly increased upon addition of PAF and C5a. Preincubation of eosinophils with the PAF receptor antagonist WEB 2086 only inhibited migration of unprimed eosinophils toward PAF and C5a, whereas preincubation of epithelial cells with WEB 2086 abolished migration of both IL-5-primed and unprimed eosinophils. This latter result indicated the presence of PAF receptors on epithelial cells. Indeed, addition of PAF to epithelial cells induced an increase in cytosolic free Ca2+, which was blocked by the PAF receptor antagonists WEB 2086 and TCV-309. Our results show that PAF induces permissive changes in epithelial cells, and that PAF acts as a chemoattractant and priming agent for the eosinophils.     

Hemodialysis-induced increase in serum lactoferrin and serum eosinophil cationic protein as signs of local neutrophil and eosinophil degranulation

Transient reduction in circulating polymorphonuclear granulocytes and eosinophils were observed early in hemodialysis. About a threefold increase in serum-lactoferrin occurred 2 h from the start of hemodialysis. Increments of the serum levels of eosinophil cationic protein (ECP) were observed as early as 1 h after initiation of hemodialysis, reaching maximum levels (about a fourfold increase from initial levels) 1 h later. When fresh blood was circulated through a dialyzer without having a patient in the circuit considerable increases of lactoferrin and ECP were also found. The intracellular contents of lactoferrin and ECP in granulocytes isolated from peripheral blood were unaffected throughout the dialysis period. Sera obtained at different times during dialysis induced no release of granular proteins from isolated granulocytes in vitro. The raised serum concentrations of lactoferrin and ECP during dialysis suggest that a local degranulation of neutrophils and eosinophils may take place probably in the dialyzer.     

A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo

The comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1beta-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in alphaMbeta2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of alphaMbeta2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-alpha4 and anti-beta2 integrin mAbs. In vitro, both C3a (but not C3a(desArg)) and C5a (including C5a(desArg))-dependent transmigration of eosinophils across IL-1beta-stimulated endothelial monolayer was mediated by alpha4beta1 and alphaMbeta2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.     

Intracellular expression of Fc gamma RIII (CD16) and its mobilization by chemoattractants in human eosinophils

We characterized the existence, translocation, and reabsorption during cellular activation of a constitutively expressed intracellular CD16 in the human eosinophil. By two-color flow cytometry, we showed that 6.5+/-0.3% of nonpurified eosinophils expressed surface CD16. After digestion with phosphatidylinositol-specific phospholipase C, surface CD16 on both neutrophils and eosinophils decreased substantially, suggesting that eosinophil CD16 is a glycosyl-phosphatidylinositol-linked isoform. However, CD16 was substantially expressed intracellularly in human eosinophils. Epitope-specific binding to CLB-gran11 mAb from non-NA2/NA2 donors demonstrated that intracellular eosinophil CD16 also differed from the transmembrane isoform of CD16 expressed on NK cells or macrophages. Western blot analysis performed with 3G8 or DJ130c mAb showed a broad band at approximately 65 to 80 kDa, which was the same as neutrophil CD16 from the same NA2/NA2 donors. Upon stimulation by chemoattractants C5a, FMLP, or platelet-activating-factor, eosinophilic intracellular CD16 was rapidly translocated to the eosinophil surface, expressed maximally at 30 s, and then gradually disappeared from the cell surface during the next 10 min. Intracellular flow cytometry of stimulated eosinophils and sandwich ELISA of stimulated eosinophil supernatants demonstrated that the disappearance was due to its rapid release into medium and reabsorption by the cells. Our data identify a CD16B that is consistently expressed intracellularly but only rarely on the surface of nonactivated human eosinophils. This CD16 is transiently expressed during stimulation by chemoattractants.