Relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection.

To test the hypothesis that rhinovirus (RV)-induced immune responses influence the outcome of RV infections, we inoculated 22 subjects with allergic rhinitis or asthma with RV16. Nasal secretions and induced sputum were repeatedly sampled over the next 14 d. RV16 infection increased nasal granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-8, which was accompanied by neutrophilia in blood and nasal secretions. Nasal G-CSF correlated closely with increased blood neutrophils (r(s) = 0.69, p < 0.005), whereas nasal neutrophils correlated with both G-CSF (r(s) = 0.87, p < 0.001) and IL-8 (r(s) = 0.75, p < 0.001). Although similar relationships were present in sputum, changes in sputum neutrophils and G-CSF with RV16 infection were relatively modest. In addition, virus-induced changes in the sputum interferon-gamma-to-IL-5 messenger RNA ratio were inversely related to both peak cold symptoms (r(s) = -0.60, p < 0.005) and the time to viral clearance (undetectable picornavirus RNA). These results indicate that airway IL-8 and G-CSF are closely associated with virus-induced neutrophilic inflammation during an experimental RV infection in atopic volunteers. In addition, the balance of airway T-helper cell type 1 (Th1)- and Th2-like cytokines induced by RV infection may help determine the clinical outcome of common cold infections, raising the possibility that the individual subject's immune response, rather than atopic status per se, is important in this regard.

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.

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.

Endothelial cells upregulate eosinophil superoxide generation via VCAM-1 expression.

BACKGROUND: In vitro eosinophil (EOS) adhesion to recombinant human (rh)-vascular cell adhesion molecule (VCAM)-1 stimulates superoxide anion (O2-) generation and enhances formyl-methionyl-leucyl phenylalanine (FMLP)-activated O2- generation. Therefore, EOS adhesion via VLA-4 to VCAM-1 expressed on endothelium may be instrumental in the selective recruitment and function of EOS in airway inflammation. OBJECTIVE: We hypothesized that EOS interaction with endothelial cells expressing VCAM-1 will undergo an enhancement in inflammatory function. METHODS: To determine this possibility, human umbilical vein endothelial cells (HUVEC) were stimulated with either a combination of interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha (100 pM) or medium alone for 24 h; the expression of adhesion proteins on HUVEC and their effect on EOS O2- generation was subsequently determined. RESULTS: As determined by both enzyme-linked immunosorbent assay and flow cytometry, IL-4 and TNFalpha acted synergistically to induce VCAM-1 expression on HUVEC. Treating HUVEC with IL-4/TNFalpha also increased EOS adhesion and primed subsequent FMLP (0.1 microM) activated EOS O2- generation. Although EOS adhesion was partially inhibited by both antialpha4 and antibeta2 monoclonal antibodies (MoAbs), O2- generation was completely inhibited by either antialpha4 integrin MoAb (HP1/2) or anti-VCAM MoAb (BBIG-V1). Furthermore, enhanced O2- generation, but not adhesion, associated with IL-4 + TNFalpha-treatment of HUVEC was inhibited when EOS were treated with the platelet activating factor (PAF)-antagonist WEB 2086 (20 microM), thus suggesting an involvement of PAF in priming EOS. However, paraformaldehyde fixation of IL-4/TFN-alpha treated HUVEC did not significantly alter EOS function. CONCLUSIONS: These results suggest EOS adhesion to endothelial cells via an VLA-4/VCAM-1 interaction may be important in the development of the function of this cell. Furthermore, our results suggest that modulation of EOS function involves two priming factors: EOS adhesion to HUVEC expressing VCAM-1 and PAF.

Eosinophils bind rhinovirus and activate virus-specific T cells.

Episodes of virus-induced exacerbations of asthma are accompanied by increased eosinophils (EOS) in respiratory secretions and evidence of EOS degranulation. Although rhinoviruses (RV) are the viruses most often implicated in exacerbations of asthma in both children and adults, little is known about the immune response to this group of viruses and, in particular, EOS-RV interactions. To define such interactions, we incubated human rhinovirus type 16 (RV16), a serotype using ICAM-1 as a receptor, with EOS purified from PBMC, and measured EOS-RV binding, EOS-mediated Ag presentation and T cell activation, and EOS cell surface marker expression and superoxide production. Significant RV16 binding occurred to EOS that were pretreated with granulocyte-macrophage CSF, and this binding was inhibited by anti-ICAM-1 mAb. EOS also presented viral Ags to RV16-specific T cells, causing T cell proliferation and secretion of IFN-gamma. RV16 induced a significant shift from CD18dim to CD18bright, but did not affect EOS expression of CD54, CD69, or HLA-DR. Finally, RV16 did not induce superoxide production from peripheral blood EOS. These findings suggest that RV16 also binds to airway EOS, which resemble granulocyte-macrophage CSF-treated blood EOS in terms of high expression of ICAM-1. Furthermore, our findings suggest that EOS could participate in RV-induced immune responses through Ag presentation and T cell activation. By activating RV-specific T cells, EOS may play an important role in the initiation of antiviral T cell responses, and these effects could also contribute to enhanced airway inflammation and increased asthma symptoms in susceptible individuals.

Rhinovirus-specific T cells recognize both shared and serotype-restricted viral epitopes.

To characterize rhinovirus (RV)-specific T cells, RV16- and RV49-specific CD4 T cells were cloned from peripheral blood, and cytokine secretion and serotype specificity were defined. Each RV-specific clone secreted high levels of interferon-gamma, and several also produced interleukin-4 and -5. To test serotype specificity, each clone was incubated separately with five different RV serotypes. Although 2 of 31 clones proliferated only in response to the virus used in cloning, the rest had significant proliferation in response to 2-5 different serotypes. Thus, RV-specific T cells can be activated by either serotype-specific or shared viral epitopes, raising the possibility that repeated activation of T cells by shared viral determinants in vivo could induce potent recall T cell responses. It is likely that enhanced T cell responses to shared viral epitopes contribute to antiviral activity, airway inflammation, or both.

Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism.

There is evidence that rhinovirus (RV) infections are frequent causes of increased asthmatic symptoms and can specifically enhance allergic inflammation in the airway. To further define effects of RV infection on cellular immunity, we have begun to develop in vitro models for study. When human PBMC were incubated with 35S-labeled RV16, specific binding via ICAM-1 on monocytes was observed. Incubation of PBMC with RV also led to a dose-related increase in the expression of the early activation marker CD69 on 30 to 70% of T cells. The RV16-induced increases in CD69 were blocked by anti-ICAM-1 mAb, and were not elicited by UV-inactivated (noninfectious) virus. The degree of CD69 enhancement correlated with the number of monocytes in mixtures of PBMC, did not occur in monocyte-depleted cultures, and was mediated by one or more soluble factor(s). RV also induced secretion of IFN-gamma from both peripheral blood T cells and NK cells, and IFN-gamma mRNA was greatest in T cells that were CD69+. Finally, supernatant from RV-activated CD3+CD69+ cells had biologic activity that promoted eosinophil survival in vitro; this RV16-associated activity was blocked when co-incubations were performed with IFN-gamma mAbs. These observations suggest that RV nonspecifically activates a large proportion of T cells through a monocyte-dependent mechanism. Such changes in vivo could enhance airway inflammation, and this may include effects on inflammatory cells in the airways of allergic individuals.

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.

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.

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.

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.

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.

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.

The presence of hypodense eosinophils and diminished chemiluminescence response in asthma.

Although peripheral blood eosinophilia is a prominent feature of asthma, the contribution of eosinophils to asthma has yet to be fully comprehended. Furthermore, study of isolated eosinophil function in asthma has been complicated by difficult purification methods and, now, the presence of hypodense eosinophils. In our study, eosinophils were isolated from normal subjects and patients with asthma. Two principal evaluations were performed: (1) a comparison of the density-gradient profiles on peripheral blood leukocytes from normal subjects and patients with asthma and (2) a comparison of the chemiluminescence (CL) response with normal dense eosinophils from these two study groups. Granulocyte preparations were initially isolated from Ficoll-Hypaque gradients and were then applied to a continuous Percoll density gradient. In asthma, 40.8 +/- 5.8% of the peripheral blood eosinophils were hypodense (defined as a density less than 1.081 gm/ml), whereas normal subjects had only 9.1 +/- 1.9% of this subpopulation (p less than 0.01). Functional assessment of purified (greater than 90%) normal dense eosinophils was made by measurement of CL to opsonized zymosan particles and the soluble stimulus phorbol myristate acetate. In asthma, eosinophil CL to zymosan, but not phorbol myristate acetate, was significantly less. Differences in eosinophil CL between normal subjects and subjects with asthma did not correlate with the severity of airway obstruction or the peripheral blood eosinophil count. The reasons for the appearance of hypodense eosinophils and diminished metabolic activity in asthma are not established but raise the possibility that their presence represents previous eosinophil activation.