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Biomedical subjects

R F Vrtis

Publications and source records attributed to R F Vrtis.

8 recordsLinked to original sources
PubMed2000

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.

Antibodies↗
PubMed1999

Engagement of alpha4beta7 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro.

Asthma is characterized by an airway inflammatory infiltrate that is rich in eosinophilic leukocytes. Cellular fibronectin and VCAM-1, ligands for alpha4 integrins, are enriched in the fluid of airways of allergic patients subjected to Ag challenge. We therefore hypothesized that ligands of alpha4 integrins can promote eosinophil survival independent of cell adhesion. Cellular fibronectin and VCAM-1 increased viability of human peripheral blood eosinophil in a dose- and time-dependant manner whether the ligand was coated on the culture well or added to the medium at the beginning of the assay. Eosinophils cultured with cellular fibronectin were not adherent to the bottom of culture wells after 3 days. Treatment with mAb Fib 30 to beta7, but not mAb P4C10 or TS2/16 to beta1, increased eosinophil survival. The increased survival of eosinophils incubated with Fib 30 was blocked by Fab fragments of another anti-beta7 mAb, Fib 504. Eosinophils incubated with soluble cellular fibronectin or mAb Fib 30 for 6 h demonstrated a higher level of GM-CSF mRNA than eosinophils incubated with medium alone. Addition of neutralizing mAb to GM-CSF during incubation, but not mAbs to IL-3 or IL-5, reduced the enhancement of eosinophil survival by soluble cellular fibronectin or mAb Fib 30 to control levels. Thus, viability of eosinophils incubated with cellular fibronectin or VCAM-1 is due to engagement, probably followed by cross-linking, of alpha4beta7 by soluble ligand (or mAb) that stimulates autocrine production of GM-CSF and promotes eosinophil survival.

Antibodies, Monoclonal↗
PubMed1992

Comparison of airway and blood eosinophil function after in vivo antigen challenge.

Eosinophils (EOS) are important effector cells in allergic diseases and asthma. However, functional characteristics of the EOS have been derived primarily from studies of blood cells, and it is unlikely that such assessments reflect events occurring in tissues or airways. To establish more precisely the function of airway EOS, segmental Ag challenge was used to elicit and isolate large numbers of these cells. Airway, as well as blood, EOS were isolated from allergic patients 48 h after segmental Ag challenge. Both blood and bronchoalveolar lavage (BAL) EOS were fractionated over Percoll density gradients; by using this protocol, three density-distinct populations of pure (>90%) EOS were obtained from BAL fluid (1.100, 1.095, and 1.090 g/ml) and one from blood (1.100 g/ml). The functions of these various populations were compared by measuring superoxide generation, adherence to collagen and endothelial cell monolayers, cell surface receptors, and in vitro survival. BAL EOS of all three densities had greater superoxide generation and adherence with FMLP activation than did corresponding blood EOS. In contrast, blood and airway EOS responded similarly to PMA. BAL EOS also had increased expression of CD11b/CD18 and HLA-DR. The intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent marker indo-1/acetoxymethyl ester. FMLP caused a greater and more sustained increase in [Ca2+]i with BAL than blood EOS. EGTA blocked the sustained component of the [Ca2+]i response to FMLP. Our findings indicate that BAL EOS have an enhanced [Ca2+]i response to activation that may contribute to their functional up-regulation.

Adult↗
PubMed1992

Platelet-activating factor primes human eosinophil generation of superoxide.

Platelet-activating factor (PAF) is a potent inflammatory mediator that can cause airway obstruction and hyperresponsiveness; these processes are also associated with pulmonary eosinophilia, suggesting a link between these two events. Thus, PAF's interaction with eosinophils may provide a mechanism for airway damage. However, direct in vitro activation of eosinophils by PAF requires concentrations that are likely higher than those achieved in vivo. As a result, we investigated whether lower, more physiologic concentrations of PAF could prime eosinophils for subsequent activation to another receptor-stimulated factor, in this case formylmethionylleucylphenylalanine (FMLP). To test this hypothesis, eosinophils were preincubated (1 and 15 min) with low concentrations of PAF (1 x 10(-8) and 1 x 10(-10) M); this exposure to PAF resulted in enhanced generation of superoxide anion to FMLP stimulation. Moreover, similar concentrations of PAF decreased eosinophil density and increased expression of cell surface CR3 receptors. Finally, low, nonactivating concentrations of PAF (1 x 10(-10) to 1 x 10(-8) M) caused transient increases in eosinophil cytosolic free Ca2+ concentrations. Collectively, these responses are consistent with the hypothesis that short-term exposure to low concentrations of PAF primes eosinophils to cause an enhanced inflammatory response upon subsequent activation to another receptor agonist. The consequences of this PAF-associated phenomenon can produce an enhanced inflammatory response and airway injury.

Adult↗
PubMed1991

In vitro incubation with influenza virus primes human polymorphonuclear leukocyte generation of superoxide.

Viral respiratory illnesses exacerbate asthma, increase airway responsiveness, and enhance the frequency of late asthmatic reactions. A number of mechanisms have been identified to explain how respiratory viral illnesses provoke wheezing, including enhanced inflammatory activity of leukocytes. To further understand how respiratory virus-caused illnesses promote leukocyte-dependent airway injury, the following study evaluated the effect of an in vitro incubation of influenza A virus on human polymorphonuclear leukocyte (PMN) generation of superoxide (O2-). PMNs were isolated from anticoagulated human blood following density gradient centrifugation; purified PMNs were then incubated (37 degrees C x 30 min) with influenza virus (PMN:virus ratio of 5:1 [egg-infective dose 50%] and 10:1) in the presence of 10% autologous serum. After incubation, the viable PMNs (greater than 95% exclusion of trypan blue) were activated, by the chemotactic peptide formyl-methionine-leucine-phenylalanine (fMLP), calcium ionophore A23187, or phorbol myristate acetate (PMA), and O2- generation was then measured. Generation of O2- to fMLP and A23187 was significantly enhanced from PMNs that had been incubated with influenza virus. Although influenza virus itself did not generate O2-, it caused a transient increase in intracellular calcium ([Ca2+]i), when measured with Indo-1-loaded cells. These results suggest that influenza virus primes PMNs to generate increased amounts of O2- and that the priming effect is associated with a transient increase in [Ca2+]. Consequently, we postulate that influenza virus priming produces PMNs of enhanced inflammatory potential to cause greater airway injury, obstruction, and responsiveness during a viral respiratory infection.

Adult↗
PubMed1989

Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions.

Although viral upper respiratory infections (URIs) provoke wheezing in many asthma patients, the effect of these illnesses on the airway response to inhaled antigen is not established. The following study evaluated the effect of an experimental rhinovirus (RV) illness on airway reactivity and response to antigen in 10 adult ragweed allergic rhinitis patients. Preinfection studies included measurements of airway reactivity to histamine and ragweed antigen. Furthermore, the patients were also evaluated for late asthmatic reactions (LARs) to antigen (a 15% decrease in forced expiratory volume of the first second approximately 6 h after antigen challenge). 1 mo after baseline studies, the patients were intranasally inoculated with live RV16. All 10 patients were infected as evidenced by rhinovirus recovery in nasal washings and respiratory symptoms. Baseline FEV1 values were stable throughout the study. During the acute RV illness, there was a significant increase in airway reactivity to both histamine and ragweed antigen (P = 0.019 and 0.014, respectively). Before RV inoculation, only 1 of the 10 subjects had an LAR after antigen challenge. However, during the acute RV illness, 8 of 10 patients had an LAR (P less than 0.0085 compared with baseline); the development of LARs was independent of changes in airway reactivity and the intensity of the immediate response to antigen. Therefore, we found that not only does a RV respiratory tract illness enhance airway reactivity, but it also predisposes the allergic patient to develop LARs, which may be an important factor in virus-induced bronchial hyperresponsiveness.

Adult↗
PubMed1988

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.

Adolescent↗