Increased granulocyte adherence in psoriasis and psoriatic arthritis.

Circulating polymorphonuclear leukocytes (PMN) from patients with psoriasis and psoriatic arthritis were found to be significantly more adherent to nylon fiber columns when compared with both normal and nonpsoriatic patient control groups. The increased adherence correlated positively with the extent of disease and was highest in those patients with psoriatic arthritis. Total leukocyte and PMN counts were increased in psoriasis patients and were highest in the psoriatic arthritis group. No increase in cell counts was found for mononuclear leukocytes. PMN adherence was not increased in lithium-treated patients or a nonpsoriatic patient control group although such patients did have significant granulocytosis. PMN's are frequently present in lesions of psoriasis as are activated complement components and abnormal keratinocyte cyclic nucleotide levels. These factors or others may cause a generalized activation of PMN's in psoriasis leading to migration of neutrophils into the skin lesion. The present study demonstrates a systemic effector cell alteration in psoriasis and contradicts the general concept that uncomplicated psoriasis is limited to the skin.

Increased superoxide generation by normal granulocytes incubated in sera from patients with psoriasis.

Sera from patients with untreated psoriasis were found to induce increased superoxide anion (O-2) generation when incubated with normal granulocytes (PMNs) and zymosan. Sera from patients receiving systemic chemotherapy induced O-2 generation which was similar to that of normal sera and significantly lower than sera from the untreated patients. O-2 production was measured by superoxide dismutase inhibitable ferricytochrome C reduction and was dependent on the presence of both zymosan and a heat labile serum factor. Serum C3c and C5 levels were elevated in both treated and untreated groups of psoriasis patients while C4 was elevated only in untreated patients. serum ceruloplasmin, a O-2 scavenger, was not decreased in patients with psoriasis, and consequently does not account for the increased O-2 generation. These data suggest that sera from patients with psoriasis have an increased capacity to activate PMNs. Activation of PMNs in cutaneous and joint lesions may play a pathogenic role in psoriasis.

Effect of isolation protocol on eosinophil function: Percoll gradients versus immunomagnetic beads.

Studies of in vitro eosinophil function are dependent on efficient and reliable methods of cell isolation. Protocols using Percoll or metrizamide density gradients have been of limited use in isolating peripheral blood eosinophils in sufficient numbers and purity from subjects with normal or only slightly elevated eosinophil counts, thereby restricting comparative studies to preparations from hypereosinophilic subjects. Recently, a method utilizing negative selection by anti-CD16 coated magnetic beads has greatly improved eosinophil isolation by dramatically increased yields and purity. However, little is known as to the differential effect of various isolation methods on the functional activity of eosinophils. In this study, eosinophils were isolated by either discontinuous multiple density Percoll gradients or anti-CD16-coated magnetic beads: several functional activities were then compared using cells obtained by the two methods of isolation. Compared with Percoll isolated eosinophils, anti-CD16 bead separated eosinophils had significantly increased baseline and stimulated LTC4 production, spontaneous O2- generation, and expression of specific cell surface markers. No significant difference was observed in the cells' in vitro survival and adhesion. Such differences may be due to the isolation of eosinophils of all densities by anti-CD16 beads, or the effect of neutrophils interacting with the beads to release eosinophil agonists or primers. Alternatively, the Percoll gradient method with the eosinophils' exposure to dextran and Ficoll-Hypaque may affect subsequent cell function. Therefore, comparison of eosinophil function between cells isolated by different protocols must be considered before concluding which is the true measure of in vivo cell function.

Human airway and peripheral blood eosinophils enhance Th1 and Th2 cytokine secretion.

BACKGROUND: The effector function of eosinophils involves their release of toxic granule proteins, reactive oxygen species, cytokines, and lipid mediators. Murine studies have demonstrated that eosinophils can also enhance T cell function. Whether human eosinophils, in particular, airway eosinophils, have similar immunoregulatory activity has not been fully investigated. The aim of this study was to determine whether human blood and airway eosinophils can contribute to Th1 and Th2 cytokine generation from CD4+ T cells stimulated with superantigen. METHODS: Eosinophils were obtained from blood or bronchoalveolar lavage fluid 48 h after segmental allergen bronchoprovocation. Purified eosinophils were co-cultured with autologous CD4+ blood T cells in the presence of staphylococcal enterotoxin B (SEB). Cytokine levels in the supernatant fluid were determined by enzyme-linked immunosorbent assay (ELISA). Eosinophil expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules was assessed by flow cytometry before culture, 24 h after granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, and 24 h after co-culture with CD4+ T cells and SEB. RESULTS: Interleukin (IL)-5, IL-13, and interferon (IFN)-gamma generation increased when CD4+ T cells were co-cultured with either blood or airway eosinophils in the presence of SEB. The ability of eosinophils to enhance cytokine generation was independent of their source (blood vs airway), activation by GM-CSF, or detectable expression of human leukocyte antigen (HLA)-DR, CD80, or CD86. CONCLUSION: Our data demonstrate that SEB-induced generation of Th1 and Th2 cytokines is increased in the presence of human blood and airway eosinophils. Thus, eosinophils can have an immunoregulatory function in pathogen-associated allergic diseases such as atopic dermatitis, chronic sinusitis, and asthma exacerbations.

Inhibitory effect of cetirizine on cytokine-enhanced in vitro eosinophil survival.

BACKGROUND: Cetirizine is an antihistamine that inhibits in vivo eosinophil influx into the inflamed airways following allergen challenge, and in vitro eosinophil chemotaxis and adhesion. Since eosinophils are proposed to have an important role in the pathophysiology of asthma and allergic disease, the effects of cetirizine on eosinophil function may be a mechanism of this agent's therapeutic regulation of the allergic reaction. OBJECTIVE: To determine the effect of cetirizine on in vitro eosinophil survival. METHODS: Using human eosinophils isolated from patients with allergic rhinitis, the cells were cultured in vitro for 48 to 72 hours with medium, cetirizine, or dexamethasone in the presence of IL-5, IL-3, or GM-CSF. Eosinophil survival was assessed by trypan blue exclusion. RESULTS: In the presence of IL-5, but not GM-CSF or IL-3 100 microM cetirizine significantly inhibited eosinophil survival at 48 and 72 hours; the magnitude of this inhibition was dependent on cytokine concentration. Although cetirizine significantly suppressed cytokine promotion of eosinophil survival, it was not as potent as dexamethasone. CONCLUSIONS: Although the in vitro concentration of cetirizine was required to be quite high, cetirizine may affect in vivo airway inflammation through its inhibition of IL-5-dependent eosinophil survival.

Eosinophil eicosanoid relations in allergic inflammation of the airways.

Eosinophils are prominent features of allergic inflammation and can contribute to this process through release of inflammatory enzymes, granule-associated proteins, and leukotriene products. There is considerable interest in the fact that selected cytokines enhance eosinophil generation of leukotrienes. Therefore, future directions must include efforts to identify factors that regulate eosinophil synthesis of leukotrienes and therapeutic agents that might control these specific inflammatory responses.

Eosinophils in asthma.

Eosinophils, a prominent feature of asthma, are found in increased numbers in the circulation and sputum, usually in relation to the severity of asthma. As a consequence of these clinical observations, investigators now speculate that the eosinophil has a central role in the pathogenesis of asthma. Recent evidence has begun to confirm these speculations. The allergic reaction of the airway to antigen and the development of the late asthmatic reaction have provided a clinical model to study asthma and the contribution of eosinophils to bronchial reactivity. In the late asthmatic reaction, airway eosinophilia occurs. Through a series of independent observations, the following eosinophil-related events have been noted with the development of late asthmatic reactions. With either laboratory or natural exposure to antigen, eosinophilic chemotactic factors are released. Although the sources of eosinophil chemotaxis are multicellular, this is an early step in the attraction of eosinophils to the airway. As this process is initiated, a series of events occurs to cause eosinophils to arrive in the airway and promote obstruction, injury, and bronchial hyperresponsiveness. These steps include eosinophil migration through the vascular endothelium, upregulation of eosinophils (characterized by a change in cell density), adhesion of eosinophils to airway epithelium, and release of eosinophil toxic products. This presentation will review some of the eosinophil-dependent factors that can cause asthma. Furthermore, the eosinophil may be a good target for future therapeutic interventions.

Adhesion proteins on airway eosinophils in allergy and asthma.

Adhesion to, and interaction with, airway endothelium, interstitial matrix and epithelium during migration to the allergen-challenged airways of allergic rhinitis and asthma patients may account for the observed functional upregulation (priming) of airway eosinophils compared with corresponding blood eosinophils.

Stimulus-dependent inhibition of superoxide generation by prostaglandins.

Infiltrating phagocytes generate superoxide anion (O2-) and prostaglandin (PG) at sites of inflammation. Thus PG-O2- interactions may be important to the initiation and control of inflammation. PGE1, PGE2, and PGD2 inhibit O2- generation (as measured by superoxide dismutase-inhibitable reduction of ferricytochrome c) in a dose-dependent manner (10(-6)-10(-9) M) when human peripheral blood polymorphonuclear leucocytes (PMN) are stimulated with 10(-7) M of the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). These PG did not alter O2- generation when PMN were stimulated with 0.1 microgram/ml phorbol myristate acetate (PMA) or 1 mg/ml serum-treated zymosan (STZ). Increments of cyclic AMP (cAMP) (peak: fourfold) in PGE1, PGE2, and PGD2 treated PMN stimulated with PMA or STZ (in which O2- was not reduced) were similar to those in PG-treated PMN stimulated with FMLP (in which O2- was reduced markedly). High concentrations of theophylline and dibutyryl cAMP reduced FMLP and STZ stimulated O2- generation but had no effect on PMA stimulation, suggesting that the stimuli induce different sensitivities to the effects of cellular cAMP. PGF2 alpha had little effect on O2- generation or cAMP levels regardless of the stimulus. PGE1 did not inhibit binding of FML(3H)P to PMN and did not scavenge O2- anions. Therefore the effect of PG on O2- production is dependent on the specific stimulator and an increased concentration of cAMP in activated PMN is by itself not sufficient to limit O2- generation induced by all stimuli.

Characteristics of airway eosinophils.

Eosinophils are important participants and contributors to allergic inflammation in asthma. The mechanisms by which eosinophils migrate to the airway and are activated are not clear. Moreover, there is evidence that eosinophils from the airway are functionally distinct from those cells in circulation. In initial studies, we have found distinct differences in function, cell surface markers, calcium metabolism in response to activation, and survival in eosinophils from the airway. In an attempt to ascertain what factors regulate these phenotypic changes, we have evaluated the effect of cytokines and adhesion on eosinophil function.

Differential effects of granulocyte-macrophage colony-stimulating factor on eosinophil and neutrophil superoxide anion generation.

Eosinophils (EOS) are specifically recruited to sites of allergic inflammation and parasitic infection, while neutrophil (NEUT) influx predominates in bacterial infections. This biologic selectivity suggests that granulocytes may respond differently to inflammatory mediators such as granulocyte-macrophage-CSF (GM-CSF). To establish the mechanisms of the response of granulocytes to GM-CSF, isolated human peripheral blood EOS and NEUT were obtained from allergic rhinitis patients and incubated in vitro with this cytokine. Incubation with GM-CSF (10 or 100 pM) significantly enhanced FMLP-stimulated EOS superoxide anion (O2-) generation, LTC4 release, and adhesion to tissue culture plates. Both GM-CSF-enhanced EOS adhesion and O2- generation were inhibited by an anti-beta 2 (CD18) Ab suggesting that this beta 2 integrin was associated with increased cell function. In contrast, FMLP + cytochalasin B were required to demonstrate GM-CSF enhancement of NEUT O2- and LTB4 generation; GM-CSF had no effect on NEUT adhesion. Furthermore, GM-CSF augmentation of FMLP + cytochalasin B-activated NEUT O2- generation was not affected by anti-beta 2 Ab but was blocked by a 5-lipoxygenase-activating protein antagonist, BAY x 1005. Finally, 0.1 to 10 nM LTB4 mimicked the GM-CSF-priming effect on NEUT O2- generation, thus suggesting that the augmented NEUT respiratory burst was the result of LTB4 production and its effect on the NEUT. These data demonstrate that GM-CSF promotes EOS and NEUT O2- generation to FMLP via distinct mechanisms: enhanced adhesion of EOS vs autocrine-priming by enhanced LTB4 generation of NEUT.

Synergistic activation of eosinophil superoxide anion generation by VCAM-1 and GM-CSF. Involvement of tyrosine kinase and protein kinase C.

During the development of allergic inflammation of asthma, eosinophils (EOS) are likely to interact with endothelial adhesion molecules, such as VCAM-1 and inflammatory cytokines, such as GM-CSF. To determine whether VCAM-1 and GM-CSF can interact to modify EOS superoxide anion (O2-) generation, peripheral blood EOS were incubated in either recombinant human (rh)-VCAM-1 or buffer (control)-coated 96-well plates in the presence or absence of 100 pM GM-CSF. VCAM-1 and GM-CSF acted synergistically to stimulate O2- generation which was significantly inhibited by either genistein, a tyrosine kinase inhibitor, or staurosporine, a protein kinase C inhibitor. These results indicate that interaction between VCAM-1 and GM-CSF can stimulate EOS function and its eventual contribution to the allergic inflammation process. Furthermore, our results demonstrate the involvement of tyrosine kinase and protein kinase C in this specific EOS activation.

The appearance of hypodense eosinophils in antigen-dependent late phase asthma.

Although peripheral blood eosinophils (EOS) of low density are increased in patients with asthma, the mechanisms contributing to their presence are not well established. The following study evaluated the effect of an antigen bronchoprovocation (BP) on the percentage of circulating hypodense eosinophils (HE) in asthma. EOS density was measured by centrifugation of peripheral blood granulocytes over multiple discontinuous density Percoll gradients in samples taken immediately before and 24 h after antigen BP. We found that the percentage of peripheral blood HE (density less than 1.095 g/ml) increased significantly (p less than 0.02) over baseline values (78.9 +/- 4.8% versus 53.3 +/- 8.2%, mean +/- SEM, n = 11) when evaluated 24 h after antigen BP but only in patients who experienced both an immediate (IAR) and late phase asthma reaction (LAR). No significant change in the percentage of HE was observed in asthma subjects who had only an early asthma response to inhaled antigen (n = 6). In an expanded population of allergic asthmatics (n = 38), a significant correlation was found between the percent peripheral blood HE and disease severity as represented by the percent predicted FEV1 (r = -0.56, p = 0.003). These data suggest that in vivo activation of asthma by inhaled antigen increases the proportion of peripheral blood EOS that are hypodense but only in those patients with both an IAR and LAR. Furthermore, we also conclude that the percentage of HE better reflects the severity of asthma than the concentration of total peripheral blood eosinophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide generation by hypodense eosinophils from patients with asthma.

Low density, or hypodense eosinophils (HE) are found in increased numbers in asthma. Since HE have been reported to have increased inflammatory potential and correlate with the severity of airway obstruction, it has been suggested that this subpopulation of eosinophils may contribute to the asthma process. However, investigations to define the function of HE in asthma have been limited. In this study, pure populations of both HE and normal dense eosinophils were isolated from the peripheral blood of seven patients with asthma, and functional activity of these cells was determined by measuring superoxide (O2-) generation to several activators. Compared to normal dense eosinophils, HE generated significantly more O2- when activated with the chemotactic peptide FMLP or opsonized zymosan; however, these differences were small. In contrast, no difference was observed in O2- production between normal- and low-density eosinophils from asthma patients when stimulated by phorbol myristate acetate (PMA) or the calcium ionophore A23187. Furthermore, both eosinophil populations, and corresponding neutrophil isolates, from asthma patients generated significantly more O2- than control granulocytes when activated by PMA. When stimulated by calcium ionophore, all three eosinophil populations released equivalent amounts of O2-, which were, however, higher than from both neutrophil suspensions. Compared to increased activity found in some other hypereosinophilic conditions, blood HE in asthma do not necessarily have enhanced function. This suggests that eosinophil heterogeneity extends not only to cell density but is also reflected in cell function and that these cell variations are dependent on many factors, including the function assessed, source of cells (normal versus patients with some diseases), and stimulus used.

Hypodense eosinophils in allergic rhinitis.

Based on density, function, and membrane receptors, peripheral blood eosinophils are a heterogeneous population of cells. Importantly, hypodense eosinophils (HE) are metabolically more active and likely to contribute to tissue injury. In the following study, peripheral blood from patients with allergic rhinitis (AR) was evaluated for the presence of HE. To accomplish this, blood was obtained from patients with ragweed AR, granulocytes were isolated and fractionated by continuous density Percoll gradients, and the density distribution of these cells was determined after centrifugation. A significantly higher percentage of peripheral blood eosinophils were hypodense (defined as density less than 1.081 gm/ml) in patients with AR when these patients were compared to control subjects, 30.0 +/- 5.0% versus 9.0 +/- 1.9%, p less than 0.01. Moreover, we also noted that an increased percentage of HE was found more often in patients with moderate-to-severe AR than in subjects with none-to-mild disease (p less than 0.01). These data suggest that the appearance of HE in the circulation may relate to the development of allergic symptoms. Furthermore, our observations suggest that the HE, because of its enhanced metabolic activity and now its association with more symptomatic hay fever, may participate in the pathophysiology of AR.

Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells.

In bronchial asthma, eosinophils (EOS) adhere to, and migrate across, the lung microvasculature to exert their effector functions in the airways. This study was conducted to determine the effect of cytokines on adhesion molecule expression on human pulmonary microvascular endothelial cells (HPMEC) and the influence of these molecules on EOS adhesion and transmigration in vitro. Unlike ICAM-1 expression (>80% positive cytokine-treated HPMEC by flow cytometry), VCAM-1 expression varied with the cytokine(s) pretreatment; the order of potency was: TNF-alpha + IL-4 (82.2 +/- 4.2% positive cells) > TNF-alpha (41.8 +/- 5.1%) > IL-1beta (20.8 +/- 4.7%). IL-4 alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion to cytokine-treated HPMEC followed the same order as that observed for VCAM-1 expression. Interestingly, EOS migration across cytokine-treated HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to, HPMEC; IL-1beta (21.2 +/- 1.4% migration) > TNF-alpha (12.6 +/- 2.6%) > TNF-alpha + IL-4 (9.1 +/- 2.0%). EOS adhesion was greatest with TNF-alpha + IL-4-treated HPMEC, was dependent on VCAM-1, and inhibited with anti-alpha4 integrin mAb (67.7 +/- 7.5% inhibition, p < 0.0005). In contrast, the highest EOS migration occurred across IL-1beta-treated HPMEC and was inhibited by anti-beta2 integrin mAb (40.4 +/- 2.5% inhibition, p < 0.005). Viable HPMEC were required for EOS migration but not adhesion. Our results suggest that EOS adhesion and transmigration are differentially regulated by VCAM-1 and ICAM-1 expression and the interaction of these adhesion proteins with their respective counterligands, i.e., alpha4 and beta2 integrins on EOS.

Stimulus-dependent differences in superoxide anion generation by normal human eosinophils and neutrophils.

The role of eosinophils in allergic and hypersensitivity diseases has yet to be fully established and remains limited by techniques to isolate the eosinophil in high purity. Consequently, most studies that evaluate and characterize eosinophil function are conducted with isolates from patients with hypereosinophilia. There is, however, evidence to suggest that isolates from such patients do not represent normal function. Now, with new techniques to isolate and purify eosinophils from normal subjects without eosinophilia, metabolic function of the normal eosinophil can be assessed. To accomplish this, granulocytes from healthy volunteers were separated by continuous density Percoll gradients into populations of purified eosinophils (90.3 +/- 1.9%) and neutrophils (98.2 +/- 0.4%). Superoxide (O2-) generation was measured with a microassay of superoxide dismutase-inhibitable cytochrome c reduction in response to several soluble and particulate agonists. Normal eosinophils generated significantly more O2- in response to either phorbol myristate acetate or calcium ionophore A23187 than their matched neutrophil fractions. In contrast, differences in granulocyte response to zymosan and chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, were dependent on the presence of cytochalasin B (CB) in the reaction. N-formyl-methionyl-leucyl-phenylalanine-stimulated eosinophils generated less O2- in the absence of CB but similar amounts in the presence of CB, compared to neutrophils. Activation by zymosan in the presence of 10% autologous serum generated similar amounts of O2- in all the cell populations when CB was present; however, in the absence of CB, neutrophils produced less O2- when they were compared to eosinophils. Therefore, normal eosinophils respond differently to some activators, compared to neutrophils, and these differences may prove significant as the contribution of eosinophils to inflammation becomes established.

Inhibition of eosinophil density change and leukotriene C4 generation by nedocromil sodium.

Nedocromil sodium (NS) has been shown to inhibit the late asthma response to inhaled antigen and to control symptoms in chronic asthma; in both processes the eosinophil is thought to be an important contributor. To understand the antiinflammatory actions of NS in asthma, its effects on three eosinophil functions were evaluated: (1) change in cell density during in vitro culture, (2) synthesis of leukotriene C4 (LTC4), and (3) generation of superoxide anion. In these studies normal density (greater than 1.095 gm/ml) purified human peripheral blood eosinophils were cultured for 24 hours in 50% conditioned medium from cow pulmonary arterial endothelial cells. After incubation, 45.2% +/- 8.0% of the eosinophils had a density less than 1.085 gm/ml. In the presence of NS (10 mumol/L), only 32.0% +/- 7.3% became less dense (p = 0.0393). In contrast, NS had no effect on changes in cell density after a 20-minute exposure of eosinophils to functional activators N-formyl-methionyl-leucyl-phenylalanine (0.1 mumol/L) or platelet activating factor (0.1 mumol/L). Furthermore, calcium ionophore-activated LTC4 secretion was found to be significantly inhibited by NS (3.8 +/- 0.6 ng/ml vs. 2.4 +/- 0.3 ng/ml with 1 mumol/L NS or 1.7 +/- 0.6 ng/ml with 10 mumol/L NS, p less than 0.025). However, NS did not significantly alter eosinophil superoxide anion generation. The effects of NS on eosinophil function suggest a mechanism by which this medication may be effective in asthma, particularly in the regulation of the late asthmatic response. Furthermore, the selective regulatory effects of NS may also provide insight into the biologic activities of eosinophils.

The effect of transendothelial migration on eosinophil function.

In bronchial asthma, eosinophils found in the airways have an enhanced inflammatory capacity. We hypothesized that, at least in part, changes in functional phenotype are due to the effect of transendothelial migration. To model in vivo eosinophil trafficking to the lung, we cultured human pulmonary microvascular endothelial cell (HPMEC) monolayers on Transwell filters. The HPMECs were activated with interleukin (IL)-1beta to increase cell expression of intercellular adhesion molecule (ICAM)-1 and, hence, eosinophil transmigration. Peripheral blood eosinophils from allergic patients were added to HPMEC-covered Transwell filters and incubated for 3 h at 37 degrees C. The eosinophils were collected from below (migrated cells) and above (nonmigrated cells) the HPMEC monolayer to determine surface receptor expression, in vitro survival, and oxidative burst. Eosinophils never exposed to HPMECs were used as controls. Eosinophil cell surface expression of CD69, human leukocyte-associated antigen-DR (HLA-DR), and CD54 (ICAM-1) was significantly increased after transendothelial migration through IL-1beta-treated HPMECs compared with control cells (CD69: P<0.0005; HLA-DR and CD54: P<0.05) and nonmigrated eosinophils (CD69 and HLA-DR: P<0.05). Moreover, the percent in vitro survival (48 h) of migrated eosinophils was also significantly greater (P<0.0001 by trypan blue exclusion, P< 0.05 by flow cytometry) than that of control or nonmigrated eosinophils. Prolonged survival of migrated eosinophils was inhibited by addition of anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibodies (P<0.05) to the 48-h survival culture, suggesting that autocrine production of GM-CSF was, at least partially, responsible for increased eosinophil survival. Although GM-CSF protein was not measurable in survival culture supernates, GM-CSF messenger RNA (mRNA) was expressed in both nonmigrated and migrated eosinophils but not in control cells. Similarly, the eosinophils' oxidative burst induced by platelet-activating factor, formylmethionyl leucylphenylalanine, or phorbol myristate acetate was equally, and significantly, increased in both nonmigrated and migrated eosinophils (P<0.05 versus control). Therefore, whereas exposure of eosinophils to cytokine-activated HPMECs can increase surface receptor expression, in vitro survival, GM-CSF mRNA, and the respiratory burst, transendothelial migration can further potentiate receptor expression and survival in migrated cells. These results suggest that the process of transendothelial migration selectively participates in determining the eventual phenotype of airway eosinophils.

The effect of platelet-activating factor on the generation of superoxide anion in human eosinophils and neutrophils.

The precise role of platelet-activating factor (PAF) in asthma has yet to be established. Nonetheless, the potential relationship between PAF and asthma appears to include the eosinophil (EOS) as an important link. Thus, to evaluate the effect of PAF on leukocyte-dependent inflammation, purified populations of human blood EOSs and neutrophils were isolated from the same subject. The two granulocyte populations were then incubated with PAF, and superoxide anion (O2-) generation was measured by reduction of cytochrome c in a microassay system. Both granulocyte cell types generated O2- when they were incubated with PAF. However, the generation of O2- was 3.4 times greater with EOSs (9.8 +/- 1.5 nmole of cytochrome c reduced per 5 x 10(5) cells) than neutrophils (2.9 +/- 0.4 nmole of cytochrome c reduced per 5 x 10(5) cells; p less than 0.0001). When the effect of PAF on [Ca++]i was measured with the fluorescent label, Indo-1, PAF caused similar increases in cellular fluorescence in both neutrophils and EOSs, but the increase in [Ca++]i of neutrophils occurred with lower concentrations of PAF. Furthermore, when similar experiments were conducted in the presence of an extracellular calcium chelator, ethylene glycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, there was partial suppression in both the cellular fluorescence and O2- generation to PAF; this suggests that full expression of EOS generation of O2- by PAF requires both intracellular mobilization and a transmembrane influx of Ca++. Our data indicate that PAF can stimulate leukocyte O2- generation, but this response is greater in the EOS than the neutrophil. Therefore, our findings support the observation that the EOS is more responsive to PAF activation than other granulocytes and that this difference may contribute to participation of PAF in asthma.