Long-Term Risk of Mortality Associated With Selective and Combined Elevation in Office, Home, and Ambulatory Blood Pressure
the Clinica Medica (G.M., R.F., M.B., G.G., R.S.), Università Milano-Bicocca, Ospedale San Gerardo
Centro Interuniversitario di Fisiologia Clinica e Ipertensione (G.M., R.F., M.B., G.G., R.S.), and Centro Auxologico Italiano (G.M., G.G.), Milan, Italy.
Abstract
In the Pressioni Arteriose Monitorate e Loro Associazioni (PAMELA) study, office, home, and ambulatory blood pressure (BP) values were measured contemporaneously between 1990 and 1993 in a large population sample (n=2051). Cardiovascular (CV) and non-CV death certificates were collected over the next 148 months, which allowed us to assess the prognostic value of selective and combined elevation in these 3 BPs over a long follow-up. There were 69 CV and 233 all-cause deaths. Compared with subjects with normal office and 24-hour BP, the hazard ratio for CV death showed a progressive increase in those with a selective office BP elevation (white-coat hypertension), a selective 24-hour BP elevation (masked hypertension), and elevation in both office and 24-hour BP. This was the case also when the above conditions were identified by office versus home BP values. Selective elevation in home versus ambulatory BP or vice versa also carried an increased risk. There was indeed a progressive increase in both CV and all-cause mortality risk from subjects in whom office, home, and ambulatory BP were all normal to those in whom 1, 2, or all 3 BPs were elevated, regardless of which BP was considered. The trends remained significant after adjustment for age and gender, as well as, in most instances, after further adjustment for other cardiovascular risk factors. Thus, white-coat hypertension and masked hypertension, both when identified by office and ambulatory or by office and home BPs, are not prognostically innocent. Indeed, each BP elevation (office, home, or ambulatory) carries an increase in risk mortality that adds to that of the other BP elevations.
Key Words: blood pressure monitoring, ambulatory hypertension, white-coat cardiovascular diseases
Introduction
Use of ambulatory and home blood pressure (BP) measurements has allowed us to discover 2 conditions that were unknown when BP was mainly measured in the clinic environment, that is: (1) isolated office (or white-coat) hypertension (HT), in which BPs obtained in the office are 140 mm Hg systolic or 90 mm Hg diastolic, whereas ambulatory or home BP values remain within their normal range; and (2) masked HT, in which office BP is normal, whereas ambulatory or home BPs are elevated.1 The clinical significance of these conditions is still a matter of debate, because cross-sectional studies aimed at examining whether white-coat HT is accompanied by a greater incidence of HT-related organ damage have not provided univocal results.2–4 In addition, and more importantly, the results have not been univocal in the few studies that have addressed this issue prospectically and have reported white-coat HT (as diagnosed by office BP elevation and ambulatory BP nor-mality) to carry no increase in the incidence of cerebral or cardiovascular (CV) disease5–9 or to have a greater or delayed CV risk as compared with that of normotensive subjects.10–13 Furthermore, masked HT has been reported to have a greater prevalence of organ damage14–16 and a prognostically greater risk than that of normotensive individuals and possibly of white-coat hypertensives.13,17,18 However, the evidence is largely confined to a few studies based on ambulatory BP in populations with selected ages, limited follow-up periods, and variable definitions of ambulatory BP normality over 24 hours or the day only. Finally, limited evidence is available as to the prognostic significance of the 2 above-mentioned clinical conditions, when identified by home BP measurements, that is, procedures of large and growing use in clinical practice.
The Pressioni Arteriose Monitorate e Loro Associazioni (PAMELA) study examined cross-sectionally a large sample representative of the population of Monza (a town in the northeast outskirt of Milan) from 1990 to 1993 to establish the normality range of ambulatory and home BPs.19 From recruitment to October 2004, contact with participants was maintained by mail and phone, and a copy of the death certificate was obtained for all of the subjects who died. This allowed a number of issues to be examined (some for the first time) in the context of a population and over a long follow-up. First, it allowed the prognostic value of white-coat or masked HT to be assessed over a long observational period in a general population rather than in selected cohorts of subjects, as in most previous studies. Second, it studied the prognostic value of white-coat or masked HT when the diagnosis is based on home BP vis a vis that based on ambulatory BP. Third, it studied the prognosis of individuals in whom a discrepancy exists between home and ambulatory BP, that is, one is elevated, whereas the other is normal and vice versa. Last, it studied whether and how the elevation of 1, 2, or all 3 BPs has a progressively adverse impact on prognosis, independent of their "in-office" or "out-of-office" nature.
Methods
The methodology used in the PAMELA Study has been reported in detail elsewhere.19 Briefly, 3200 individuals were randomly selected from the residents of the town of Monza to be representative of the population for gender and age decades (25 to 74 years), according to the criteria used in the World Health Organization-MONItoring trends and determinants on CArdiovascular diseases (MONICA) Study conducted in the same geographic area. The overall participation rate was 64% (n=2051) consistently in each age and sex stratum. The demographic characteristics of nonparticipants were similar to those of participants. This was also the case for CV risk factors based on information collected via phone interviews.
Entry Data
Participants were invited to come to the outpatient sector of the local hospital (San Gerardo) in the morning of a working day (Monday through Friday), where several data were collected. Those relevant to the present study are (1) 3 sphygmomanometric BP measurements with the subject in the sitting position, starting 10 minutes after the beginning of the medical visit, (2) a 24-hour ambulatory BP monitoring through an oscillometric device (Spacelabs 90207, Spacelabs) with the BP readings set at 20-minute intervals (the subjects were sent home after checking for the device accuracy with the instruction to attend at their usual activities and to come back the next morning for the device removal), (3) 2 home BP measurements (approximately at 7:00 AM and 7:00 PM) through a validated semiautomatic device (Model HP 5331, Philips) using the arm contralateral to that used for ambulatory monitoring, (4) 3 additional sphygmomanometric sitting BP measurements, after removal of the ambulatory BP device, and (5) information (history and physical examination) on CV risk factors including overweight, smoking habit, serum cholesterol, blood glucose, diabetes mellitus, and history of previous CV morbid events. Total serum cholesterol and blood glucose levels were measured in all of the subjects by standard radioenzymatic method.
Follow-Up
the time of the medical visit to October 1, 2004, a copy of the death certificate was obtained for all of the subjects who died. The causes of death reported in the certificate were coded according to the Tenth International Classification of Diseases. Over an average follow-up period amounting to 148 months, there were 233 deaths, of which 69 were of a CV nature.
Data Analysis
The 3 office and the 2 home BP measurements obtained at the initial visit were separately averaged. As reported in detail in the article describing the PAMELA data,19 ambulatory BP values were edited from artifacts according to preselected criteria20 and averaged for the 24 hours, the day (7:00 AM to 11:00 PM), and the night (11:00 PM to 7:00 AM). Valid ambulatory BP readings were 95.9% of those planned (ie, 72 readings over 24 hours) with an homogeneous distribution (2.9 per hour) throughout the entire recording period and a similar percentage of valid readings compared with the expected ones over the day (95.7%) and the night (96.5%). Based on office (pooled data) and 24-hour ambulatory BP values, subjects were divided into 4 groups: (1) normal office (<140/90 mm Hg) and 24-hour (<125/79 mm Hg) systolic and diastolic BP; (2) elevation in office systolic or diastolic BP with normal 24-hour ambulatory BP, that is, isolated office or white-coat HT; (3) normal office BP with elevation in 24-hour home systolic or diastolic BP, that is, masked HT; and (4) elevated office and 24-hour systolic or diastolic BP. A similar subdivision into 4 groups was made based on office versus home BP values normality (<135 mm Hg systolic or <83 mm Hg diastolic) or elevations (135 mm Hg systolic or 83 mm Hg diastolic). Subjects were divided into 4 groups also according to the normal and/or elevated 24-hour and home BP values. The upper normality values of 24-hour and home BPs were derived from the cross-sectional data obtained in the whole PAMELA population based on their correspondence with office BPs equal to 140/90 mm Hg on the regression line linking the 3 BPs.19 These values are superimposable to the normality values reported by other studies using different approaches and mentioned by international guidelines as the most likely cutoffs dividing out-of-office HT from normoten-sion.1,21 The incidence of events was calculated via a logistic model. The hazard ratio was calculated by the Cox proportional hazard model, the assumption of BP proportionality being assessed by proper statistical test. The 2 test was used to evaluate the trends in death incidence or hazard ratio: (1) from the normotensive to white-coat hypertensive, masked hypertensive, and in-office and out-of-office hypertensive group; (2) from the group with normal 24-hour and home BP, the group with selective 24-hour BP elevation, the group with selective home BP elevation, and the group with elevation in both 24-hour and home BP; and (3) from the group with no BP elevation to the groups with elevation in 1, 2, or all 3 of the BPs, regardless of whether they were measured. Groups were ordered in the above fashion because of the suggestion from previous studies that: (1) in white-coat HT, CV damage and risk may be greater than in "true" normotension but less in true HT2,3,5,6–13; (2) masked HT may be clinically worse than white-coat HT because of the superior prognostic significance of ambulatory versus office BP14; and (3) the ability of home BP to predict the risk of death compares favorably with that of ambulatory BP.14 Data were adjusted for age and gender. Further adjustments were also made for history of CV disease, smoking prevalence, blood glucose, and serum total cholesterol, which were included as covariates into the model. Throughout the text, values in parentheses refer to the SD of the mean. A 2-tailed P value <0.05 was considered to be statistically significant.
Results
As shown in Table 1, compared with normotensive subjects, subjects with white-coat HT, masked HT, and elevation, both in-office and out-of-office BP showed a greater male prevalence, age, body mass index, serum total cholesterol, and blood glucose. This was the case either when BP normality and elevation were identified by office versus 24-hour and by office versus home BP criteria (Table 1, bottom). As shown in Figure 1A and 1B, compared with normotensive subjects, age- and gender-adjusted incidence of CV and all-cause death were usually greater in the remaining 3 groups. There was a statistically significant trend toward a progressively greater unadjusted and age- and gender-adjusted risk of CV from the entirely normotensive to the white-coat HT, masked HT, and entirely hypertensive group, regardless of whether BP normality or elevation was identified by office versus ambulatory or by office versus home BP criteria (Figure 2A and 2B). A similar trend was observed for unadjusted hazard ratios for all-cause mortality (Figure 3A and 3B), as well as for further adjustment of the risk of CV mortality and history of CV disease, smoking, serum total cholesterol, and blood glucose (Table 2).
Discussion
Our study allows several conclusions to be made. First, in the PAMELA population, the incidence and risk of CV death showed a progressive increase from subjects in which in-office and out-of-office BPs were both normal to subjects with white-coat HT, masked HT, and in-office and "out-of-office" HT, independent of age and gender. Second, the progressive increase in mortality from the entirely normotensive to the entirely hypertensive group occurred regardless of whether the above conditions were identified based on office versus ambulatory or office versus home BP. Thus, white-coat and masked HT are not clinically innocent conditions, but they rather indicate a transition toward a greater risk that reached the maximum when in-office and out-of-office BP are both increased. This has 2 implications for the practice of medicine. First, physicians should not lightly decide to dismiss treatment in patients with white-coat HT. Second, normal office BP values should not be taken as a guarantee that there is no increase in risk because of the possibility of an elevation in out-of-office BP. This implies that out-of-office BP values should be more frequently collected than is recommended by current guidelines.1 The noticeable prevalence of masked HT in the normotensive fraction of the PAMELA population (14.5% and 15.5% when assessed by ambulatory and office BP, respectively; Reference14) scores in this direction.
Our study provides new evidence on other clinically relevant issues. Although in our subjects only 2 home BP measurements were available, a selective elevation in home BP increased the age- and gender-adjusted risk of CV and all-cause mortality to an extent that was, if anything, greater than the increase associated with a selective elevation in 24-hour BP. This confirms the importance of BP values self-measured in the home environment9,18 of which the prognostic significance may favorably compare with that of 24-hour BP even when the potential of this approach (daily measurements for weeks) is only partially used. The above, however, does not mean that home BP should be considered just as a substitute of ambulatory BP monitoring, because, in our study, an elevation in only 1 of these out-of-office BPs was accompanied by a risk of CV or all-cause death, which was less than that seen when both of these out-of-office BPs were elevated. Thus, home and ambulatory BP do not represent a duplicate of the same type of information. This is also made clear by the observation that age- and gender-adjusted risk of CV and all-cause death increased progressively in subjects in whom office, home, and 24-hour BP were all normal to subjects in whom 1, 2, or all 3 of these BPs were elevated, regardless of which BP showed the elevation. This leads to the conclusion that office, home, and 24-hour BPs have an individual prognostic value that may add to the prognostic value of the others BPs. Thus, obtaining information on office, home, and 24-hour BP may represent the ideal clinical procedure.
Confirming previous findings,22–26 white-coat hypertensives, masked hypertensives, and hypertensives with in-office and out-of office BP elevations of the PAMELA study all showed body mass index, serum total cholesterol, and blood glucose values that were greater the those of individuals with in-office and out-of-office BP normality. In addition, however, they show that body mass index, total serum cholesterol, and plasma glucose all displayed a progressive increase from individuals in whom office, home, and ambulatory BPs were all normal to those in which 1, 2, or all 3 BPs were elevated (Table 1, P<0.0001 for all trends). This emphasizes that there is a close quantitative relationship between metabolic and BP abnormalities, regardless of where and how BP is measured, with each BP offering a specific contribution to the overall dysmetabolic profile. It also makes it obvious that the progressive increase in risk from normotension to white-coat HT, masked HT, and true HT, as well as from subjects with no elevation to subjects with 3 BP elevations may have a multifactorial nature, that is, it may also be because of alterations in glucose and lipid metabolism. BP alterations, per se, however, are likely to be involved, because in all of the above conditions, the progressive increase in CV mortality remained significant after adjustment for differences in metabolic, as well as other risk factors between groups, suggesting that in-office and out-of-office BPs, per se, play a role. This role may be accounted for by the data shown in Tables 1 and 3. That is, in white-coat HT, the office BP elevation was accompanied by ambulatory or home BP values that, although normal, were higher than the values seen in subjects without white-coat HT. Conversely, in masked HT, office BP values, although normal, were higher than those observed in subjects without masked HT. Finally, moving from the condition of no BP elevation to that of 1, 2, or all 3 BP elevations was associated with a progressive increase in all of the BPs, that is, both in the BPs that reached the HT range and in those that remained in the normotensive range (Table 3, bottom). This may have prognostic relevance, because office, home, and 24-hour BP have all been shown to have a continuous relationship with CV risk.1,2,9,10
Our study has a number of favorable characteristics but also 2 limitations. The favorable characteristics include the long follow-up, as well as the objective nature (death) of the events. One limitation is that the number of CV events was small, given the low CV risk of Mediterranean populations, leading to hazard ratios with large confidence limits. However, the results were often supported by the data on all-cause death, which was >3 times as large as CV death. This was, for example, the case for age- and gender-adjusted risk of all-cause death in subjects with no or 1, 2, or 3 BP elevations. The other limitation is that the observational nature of our study did not allow us to assess the effect on the prognosis of antihypertensive treatment and therapeutic corrections of glucose and lipid abnormalities. Antihypertensive treatment, for example, might have been more common in white-coat than in masked hypertensives, because in the clinical practice treatment is usually guided by office BP elevations, contributing to the better prognosis of the former versus the latter condition.
Perspectives
Our study provides long-term prognostic evidence that white-coat or masked HT are not innocent conditions. It also provides evidence that office, home, and 24-hour BP may each have an adverse prognostic value, which adds to that of the other BPs.
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Giovanni Candiano, Maurizio Bruschi, Nicoletta Pedemonte, Emanuela Caci, Sabrina Liberatori, Luca Bini, Carlo Pellegrini, Mario Viganò, Brian J. O'Connor, Tak H. Lee, Luis J. V. Galietta and Olga Zegarra-Moran
Laboratories of Uremic Physiopathology and Molecular Genetics, Istituto G. Gaslini, Genoa; Laboratory of Functional Proteomics, Molecular Biology Department, Università degli Studi di Siena, Siena; Cardiosurgery Section, IRCCS Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy; and Department of Asthma, Allergy, and Respiratory Science, GKT School of Medicine, King's College London, London, United Kingdom
ABSTRACT
Rationale: The airway surface liquid, the thin layer of liquid covering the airways, is essential for mucociliary clearance and as a barrier against microbial and other noxious agents. Proteins secreted into the airway surface liquid by epithelial and nonepithelial cells may be important in innate immunity and to improve the fluidity of mucous secretions.
Objectives: We aimed to identify proteins specifically secreted into the airway surface liquid by human bronchial epithelial cells, in resting conditions and after treatment with interleukin 4 (IL-4), a cytokine released in asthma.
Methods and Main Results: By using a proteomics approach, we found that one of the most abundant proteins was gelsolin, which breaks down actin filaments. Gelsolin mRNA and protein secretion were increased threefold in the airway surface liquid of epithelia treated with IL-4. These results were confirmed at the functional level by measuring actin depolymerization using a fluorescence assay. Gelsolin protein was also upregulated in the airways of subjects with asthma.
Conclusions: Our findings indicate that gelsolin is released by epithelial cells into the airways and that its secretion is increased by IL-4 in vitro. In addition, we found that the concentration of both IL-4 and gelsolin were raised in the bronchoalveolar lavage of patients with asthma. These results suggest that gelsolin might improve the fluidity of airway surface liquid in asthma by breaking down filamentous actin that may be released in large amounts by dying cells during inflammation.
Key Words: actin epithelium mucociliary clearance
Allergic asthma is an inflammatory disorder of the airways associated with bronchial hyperresponsiveness and variable airflow obstruction. In addition, individuals with asthma frequently suffer from mucus overproduction, which is believed to contribute to airway obstruction (1). There is compelling evidence that T-helper type 2 (Th2) lymphocytes, and the cytokines that they produce (i.e., interleukin 4 [IL-4], IL-5, IL-9, and IL-13), may be involved in the orchestration of airway inflammation. IL-4 is the major factor regulating IgE production by B cells, and is required for optimal Th2 lymphocyte differentiation (2). IgE-mediated immune responses are further enhanced by IL-4 through upregulation of IgE receptors on the surface of B cells. IL-4 contributes to airway obstruction in asthma through the induction of mucin gene expression and the hypersecretion of mucus (3). IL-4 promotes cellular inflammation in the asthmatic lung by the induction of vascular cell adhesion molecule-1 on vascular endothelium (3). In patients with asthma, T cells producing IL-4, IL-5, IL-9, and IL-13 have been identified in bronchoalveolar lavage fluid (BALF) (2, 4, 5). Furthermore, concentration of IL-4 is elevated in BALF from patients with asthma, although the levels demonstrate considerable variability (6–8).
The airway surface is covered by a thin fluid layer, the airway surface liquid (ASL), which has an essential role as a barrier to protect the underlying epithelium (9). Air contains particulate material, such as pollen, ash, mineral dust, bacteria, and viruses, all of which are capable of damaging the lungs either directly or by predisposing the lung to infection. The airways must be able to handle this load by neutralizing, and discarding, injuring agents, as well as repairing any resultant damage to cells. In addition, recent studies have demonstrated that airway epithelial cells can produce a wide variety of cytokines that promote the differentiation of inflammatory cells and other multifunctional factors that initiate and amplify inflammatory events (2). We and other authors have previously shown that IL-4 and IL-13 (10, 11) modify the ion transport properties of cultured cells by changing the expression and activity of ion channels favoring Cl– secretion over Na+ absorption. The consequent changes in volume and composition of ASL secretions could have a considerable influence on the mucociliary clearance and viscosity of mucous secretions. We hypothesized that some of the proteins secreted into the ASL to improve mucociliary clearance, or increase antimicrobial defense in asthma, could be modulated by IL-4. Recently, a database of proteins recovered by BAL has been created using two-dimensional electrophoresis (12). Moreover, Magi and colleagues (13), using a proteomics approach to study the BALF obtained from patients with sarcoidosis and idiopathic pulmonary fibrosis, have identified a number of proteins that may have pathogenic roles in these diseases. However, interpreting the source of proteins present in BALF is difficult because they may have many different origins, including diffusion from serum, production by pulmonary T cells, synthesis by alveolar cells or by alveolar macrophages, or secretion by surface bronchial epithelial cells or submucosal gland cells.
To overcome the problem of multiple cellular sources of secreted proteins in this study, we have used a proteomics strategy to identify the proteins secreted specifically by airway epithelial cells in resting and stimulated conditions. Analysis of the protein content from the ASL of polarized human bronchial epithelial cell cultures shows that one of the most abundant proteins is gelsolin, which breaks down actin filaments. We also find that gelsolin protein levels are increased in cell cultures treated with IL-4 and in BALF of patients with asthma. This finding suggests an important role for gelsolin in the ASL barrier function and indicates that exogenous gelsolin might be considered for therapeutic use in diseases characterized by chronic inflammation with significant levels of filamentous actin release, such as cystic fibrosis (CF) (14).
Some of the results of these studies have been previously reported in the form of an abstract (15).
METHODS
Human bronchi were obtained from lung resections or lobectomies and cultured as previously described (10, 16, 17). The collection and processing of human cells were approved by the local ethics committee. To obtain polarized epithelia, human bronchial epithelial cells were plated on transwell clear permeable supports (Corning Costar; Celbio, Milan, Italy). The apical culture medium was removed, and epithelia maintained in an air–liquid interface (i.e., with culture medium only on the basolateral side). Under these conditions, the epithelia maintain a thin layer of liquid similar to the ASL in vivo (18). IL-4, 10 ng/ml, was added daily to the basolateral medium and was maintained for 24 to 120 h. Collection of ASL proteins was achieved by washing the apical side of polarized epithelium with Ringer's solution.
BALF Samples
BALF samples were obtained from six nonasthmatic, nonatopic subjects (mean age, 32 ± 3 yr) on one occasion, and from six subjects with atopic asthma (mean age, 26 ± 1 yr) on two occasions, before and 24 h after inhaled allergen challenge (house dust mite, cat hair dander, or grass pollen, as appropriate). All subjects provided informed consent, and the study was approved by the local research ethics committee of King's College Hospital, London. Bronchoscopy was performed according to the standard operating procedure of King's College Hospital.
Two-Dimensional Gel Electrophoresis
BALF and ASL samples were treated as previously reported (19, 20). Samples were separated in the first dimension on soft, immobilized pH gradient, in a nonlinear pH 3–10 interval (21, 22). In the second dimension, proteins were separated based on their molecular weight in polyacrylamide gels, 180 x 160 x 1.5 mm. Proteins were visualized by a double staining procedure: first, the methyl-trichloroacetate negative staining (23), followed by the silver staining (24) for the analytic image, or colloidal Coomassie staining (25) for mass spectrometry analysis. Additional details on the methods are provided in an online supplement.
Mass Spectrometry
After visualization with colloidal Coomassie blue staining, electrophoretic spots were excised and proteins digested as previously described (26). Peptide mass fingerprinting spectra were acquired using an Ettan MALDI-ToF mass spectrometer (Amersham Biosciences, Uppsala, Sweden). Peptide sequencing was performed using an LCQDeca ESI-Ion Trap mass spectrometer (Thermo, San Jose, CA). Database searching for protein identification was performed using available online software.
Gelsolin Functional Assay
Rabbit muscle actin was dissolved in a low-ionic-strength solution, diluted with a polymerizing buffer, and stored in small aliquots at –20°C. Rhodamine phalloidin was dissolved in dimethyl sulfoxide at a concentration of 500 μM and stored at –20°C. Human plasma gelsolin was dissolved and stored at –80°C at a final concentration of 400 nM. Fluorescence intensity was measured with a fluorescence microplate reader (FluoStar Galaxy; BMG LabTechnologies, Germany) using 96-well, black, clear-bottom plates. We used high-quality filters (Chroma Technology, Rockingham, VT) with maximal transmittance at 540 and 590 nm for excitation and emission, respectively. Additional details are provided in the online supplement.
Statistics
Results are presented as representative data or as arithmetic means plus SEM. Significance was determined on unpaired groups of data using two-tailed Student's t test, if not otherwise indicated, or by nonparametric Kruskal-Wallis followed by Dunn's multiple comparison test.
RESULTS
Two-Dimensional Gel Analysis of Cultured Cells' ASL
Analytic gels prepared with the proteins recovered from the apical surface of cultured airway epithelia showed hundreds of silver-stained spots (Figure 1, top panel). Side-by-side comparison of gels prepared with samples from cultures treated with and without IL-4 revealed changes in intensity and/or mobility of some of the spots. Particularly evident were the modifications of two clusters of spots or charge trains (circled in Figure 1, bottom panels) with apparent molecular weights of 85 and 45 kD. For each molecular weight, up to seven distinct spots could be distinguished that were positioned between an isoelectric point range of 5.6 to 6.2 for the 85-kD cluster, and 5 to 5.2 for the 45-kD cluster. As shown in Figure 1 (bottom panels), the spots in the two clusters were significantly increased in intensity when the cells were treated with IL-4. Preparative gels were generated for protein identification. Recovery of the single spots and subsequent mass spectrometric analysis revealed that the polypeptides in both spot clusters belonged to gelsolin. The presence of charge trains of the same protein indicates various degrees of in vivo modifications, such as glycosylation, phosphorylation, or acetylation, which affect electrophoretic mobility (27). The sequences of fragments from the low-molecular-weight spots matched with the carboxyl-terminal half of the human gelsolin sequence (Figure 2). Conversely, sequences obtained from the high-molecular-weight cluster matched with the whole gelsolin. Spots that were expected to correspond to actin were confirmed by mass spectrometry. In contrast to gelsolin, the intensity of actin charge trains was not modified by IL-4 (underlined in Figure 1). None of the spots recovered from two-dimensional gels corresponded to the amino-terminal half of gelsolin.
Quantification of Gelsolin Protein and mRNA
We performed densitometric analysis of protein spots from two-dimensional gels from untreated and IL-4–treated cells (Figure 3A). Our results demonstrated that secreted gelsolin increased threefold at 24 h after IL-4 stimulation and remained upregulated for the following 96 h. We confirmed the presence of 85- and 45-kD bands by Western blot using an antibody against human gelsolin (Figure 3B; for details on method, see online supplement). Intensity of both bands increased after IL-4 treatment, by approximately three- and twofold for the high- and low-molecular-weight material, respectively. We then reasoned that the upregulation of gelsolin protein was caused by an increase in gelsolin mRNA. We therefore quantified gelsolin mRNA using real-time reverse transcriptase–polymerase chain reaction (Figure 3C; for details on the method used, see online supplement). Our data showed that gelsolin transcript was significantly increased at 6 h after IL-4 addition, reached a peak at 24 h, and then started to decrease.
Functional Analysis of Gelsolin
Gelsolin cleaves actin filaments (28). To evaluate the presence and upregulation of gelsolin at the functional level in the ASL of cultured cells, we used an assay based on the properties of rhodamine-labeled phalloidin to increase its fluorescence emission when bound to polymerized but not to depolymerized actin (29). In the absence of gelsolin, addition of polymerized actin caused a fuorescence enhancement of 233 ± 15.8% (from 3,490 ± 203 to 8,121 ± 609 arbitrary units, n = 14). This effect was slow, requiring approximately 7 to 8 min to reach a stable fluorescence level (not shown). The addition of human gelsolin to the reaction mixture caused a dose-dependent reduction of actin-dependent signal (Figures 4A and 4B). The gelsolin response was normalized by taking the fluorescence with phalloidin plus actin and that with phalloidin alone as 100 and 0%, respectively (see Figure 4A). The fit of experimental data to a Michaelis-Menten function yielded a half-effective concentration of 24.7 nM. After generating a calibration curve, we tested the effect of ASL samples from cultured cells. The ASL lavage from untreated epithelia did not change the fluorescence of the phalloidin-actin complex (Figure 4C). In contrast, 20 μl ASL lavage from IL-4–treated epithelia caused a clear fluorescence reduction of 38.1 ± 12% (Figure 4D; n = 6; p < 0.05). By interpolation to the calibration curve, this change corresponds to that elicited by approximately 25 nM gelsolin (see Figure 4B, filled circle).
Detection of Gelsolin in BALF
To verify whether gelsolin is secreted into the airways in vivo, we analyzed BALF samples from six nonasthmatic and from six patients with asthma (Figure 5). Protein spots corresponding to the molecular weights of the two forms of gelsolin and actin were identified. The high-molecular-weight spots of gelsolin were partially covered by the big spot corresponding to albumin. However, two-dimensional gels showed that gelsolin spots of low molecular weight in patients with asthma were more intense than in control patients, whereas actin spots were unchanged (Figure 5, bottom). These findings were confirmed in Western blots using the antihuman gelsolin antibody and extended to the high-molecular-weight form of gelsolin as well (Figure 6). The 85- and the 45-kD bands, particularly the latter one, were more intense when obtained from patients with asthma than from nonasthmatic individuals. We also analyzed the BALF from patients with asthma after an allergen challenge (see METHODS). We found that the abundance of the high-molecular-weight gelsolin was similar before and after allergen, whereas the low-molecular-weight band intensity decreased to values similar to those of nonasthmatic patients.
Detection of IL-4 in BALF
We wanted to establish whether there was a correlation in BALF between gelsolin and IL-4 levels. We therefore measured IL-4 concentration by ELISA (see the online supplement) in the BALF of the same control subjects and subjects with asthma from which gelsolin was determined. The BALF from control patients contained low levels of IL-4 (0.68 ± 0.12 pg/ml). In contrast, the IL-4 content from patients with asthma was 14-fold higher (9.68 ± 1.7 pg/ml). After exposure to allergen, patients with asthma displayed IL-4 values similar to those of nonasthmatic subjects (0.76 ± 0.26 pg/ml).
DISCUSSION
Proteomics is a powerful approach to analyze the protein content in biological fluids. Comparison of two samples of protein mixtures (normal vs. affected, treated vs. untreated) by two-dimensional gel electrophoresis, followed by identification of the proteins by mass spectrometry, can detect the presence of novel proteins, reveal new roles for detected proteins, and discover patterns of regulated post-translational modifications (30). Regarding the airways, a database of BAL proteins has recently been created using two-dimensional electrophoresis (12). A variety of soluble components from human lung have been identified, including approximately 80 proteins. Analysis of BAL protein content is complicated by the presence of proteins derived from many different sources, including leukocytes, serum, submucosal glands, and surface epithelium. This study therefore adopted a simplified approach by using an in vitro model of differentiated airway epithelium. We have found that the ASL of polarized human bronchial epithelia contains gelsolin whose secretion is significantly stimulated by treatment with IL-4.
Gelsolin is the most potent protein that severs actin filament identified to date (28). This protein weakens noncovalent bonds between actin filaments so that they can then be broken. This effect is rapid, stoichiometric, and highly efficient. Interaction of gelsolin with actin is strongly Ca2+-dependent (31). However, when gelsolin is cleaved by caspase-3, the effector caspase in apoptosis (28), or by other proteases, it becomes Ca2+-independent. Indeed, gelsolin is formed by two tandem homologous halves connected by a long linker where the protease site is located. The isolated C-half can bind a single actin molecule but only when Ca2+ concentration is higher than 1 μM. The isolated N-half binds two actin molecules even in the absence of Ca2+ (28, 32). Recently, evidence that gelsolin is a substrate of metalloproteinase 14 has been provided (33). Gelsolin might also be cleaved by proteases involved in epithelial remodeling that are expressed in the airways, in particular, by metalloproteinases 2 and 9, which have been found increased in the lung in inflammatory lung diseases, including asthma (34).
We did not observe the amino-terminal half of gelsolin in our two-dimensional gels. Possible explanations are as follows: first, gelsolin is usually cleaved inside the cell, and only the complete and the C-half forms are secreted into the ASL; second, that the N-half is secreted into the ASL but it is further digested by other proteases; third, given that it is Ca2+-independent, the N-half of gelsolin is bound to actin filaments present in the ASL and not loaded into the two-dimensional gels because of their big size. Besides being involved in the dynamic remodeling of actin filaments inside cells, gelsolin is also secreted into the extracellular space. Considerable amounts of actin are released into the extracellular space during acute lung injury, and circulating actin–gelsolin complexes can be detected in the peripheral blood (35). In the plasma, gelsolin acts as an actin-scavenging protein to prevent increases in blood viscosity due to actin leakage from dying cells. Gelsolin may serve a similar role in the ASL. Because actin is one of the most abundant intracellular proteins, cell death that may occur during inflammatory processes can release large amounts of filamentous actin in the airways, thus increasing the viscosity of mucous secretions. Evidence for this comes from studies on patients with CF. In CF, disruption of chloride secretion, mediated by the CFTR protein, causes dehydration of airway surface and inflammation (9). Mucociliary clearance is severely impaired and the airways become susceptible to mucus plugging and infection. It is well known that one of the major sources of sputum viscosity in CF comes from the DNA released by dying cells. Vasconcellos and colleagues (14) have shown that sputum samples from patients with CF also contain large amounts of filamentous actin, and that this contributes significantly to sputum viscosity. Indeed, the authors demonstrated that addition of gelsolin in vitro to CF sputum decreased the viscosity, thus emphasizing the possible use of exogenous gelsolin as a mucolytic agent. The presence of endogenous gelsolin in the airways was not investigated in that study.
Our results now demonstrate that gelsolin is a protein normally secreted into the ASL by bronchial epithelial cells, and that its levels are high and significantly upregulated by stimulation with IL-4. Quantification by real-time reverse transcriptase–polymerase chain reaction shows an increase of gelsolin mRNA in IL-4–treated cells, which is consistent with upregulation of the corresponding protein in the ASL. Therefore, our results are consistent with an increase in gelsolin protein synthesis due to enhanced gene transcription or enhanced stability of gelsolin mRNA.
We performed functional assays to check whether the gelsolin protein found in the cultured cells' ASL retains its ability to depolymerize actin filaments. We found no gelsolin-like activity in untreated cells. This could be due to lack of sensitivity of our assay or, more probably, to the fact that gelsolin was already complexed to endogenous actin. In contrast, in IL-4–treated cells we found significant depolymerization of exogenous actin, which could be due to excess of free gelsolin. Our findings suggest that the stimulation by IL-4 can create a reserve of gelsolin in airway periciliary fluid and this may in turn degrade filamentous actin that is leaked from dying cells.
IL-4 is a cytokine particularly important in asthma. It is part of a cascade that causes release of chemoattractant molecules, recruitment of leukocytes into the airway lumen, and consequent release of proinflammatory mediators, proteases, and mucus hypersecretion (36). Significant release of actin filaments would be expected as a consequence of the resulting inflammation and cell damage. We therefore speculate that IL-4–induced gelsolin upregulation may be part of a complex host antiinflammatory response.
Patients with asthma had increased amounts of gelsolin protein in their airways with respect to control subjects. It is tempting to conclude that, in agreement with our in vitro results, this upregulation is a result of the enhanced levels of IL-4 in the airways of patients with asthma. Indeed, we found that IL-4 was increased in the BALF of patients with asthma by 14-fold with respect to the level found in the BALF of nonasthmatic subjects. The low IL-4 values found in control subjects (0.68 ± 0.12 pg/ml) are similar to those reported by others (6, 7, 37). IL-4 levels in BALF from patients with asthma are more controversial, probably depending on patients' selection criteria and disease severity. Some authors reported IL-4 levels in patients with asthma similar to control subjects (6). Others reported undetectable IL-4 levels but using an assay with a lower sensitivity (7). Finally, other investigators have found increased IL-4 in the BALF of allergic patients with asthma (38) in agreement with our results. We found a correlation between IL-4 and gelsolin levels: both are low in control subjects and increased in patients with asthma. These data further support the notion that gelsolin secretion in asthma is regulated by IL-4.
Surprisingly, after stimulation with allergens, patients with asthma showed a decrease of the low-molecular-weight form of gelsolin. This finding and the role of the two gelsolin forms is not clear. The IL-4 level in the BALF was also reduced after allergen exposure and this result is in contrast with previous reports (6, 7). We have no explanation for this difference, but it is possible that allergen caused release of mast cell proteases and this might have cleaved the cytokine.
In conclusion, our study reports for the first time the expression and secretion of gelsolin by airway epithelial cells and its regulation by IL-4, supporting the view that ASL has an important role as a first line of host defense. A recent report reveals an important additional function that gelsolin may play in the airways (39). In that study, the authors show that the activity of some antimicrobial peptides, which have the characteristic of being polyvalent cations, is inhibited by filamentous actin through the formation of aggregates. The filament association, and thus the antimicrobial activity inhibition, is reversed by gelsolin. Therefore, gelsolin can be important not only to control mucus viscosity but also to preserve the innate antimicrobial activity in the airway surface. Given the stoichiometric nature of the interaction between gelsolin and actin, massive actin release, which happens during severe inflammatory processes as in CF, may overwhelm the capacity of endogenous gelsolin. This may perhaps be an indication for the therapeutic use of exogenous gelsolin or of other actin-severing agents in diseases characterized by massive airway inflammation with significant levels of filamentous actin release. Nevertheless, caution must be taken because, in another report, actin was found to bind IL-8 in the airways, preventing this chemokine from binding to neutrophil receptors (40). These authors found that gelsolin releases IL-8 from actin, increasing the proportion of free cytokine, and therefore the possible level of inflammation.
FOOTNOTES
Supported by a grant from the Italian Cystic Fibrosis Research Foundation. The Pediatrics Department, Università degli Studi di Genova, has received support from Fondo per gli Investimenti della Ricerca di Base (FIRB) funds.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Conflict of Interest Statement: G.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.J.O. serves on advisory boards for GlaxoSmithKline (GSK), Boehringer Ingelheim, Atlana, Trinity Chiesi, and Celgene, and has received compensation for this activity. He has also received lecture fees from GSK and Pfizer. He runs a clinical academic research unit, which receives grants from major respiratory pharmaceutical companies, including GSK, Aventis, UCB, Pfizer, and Nicox to evaluate potential new therapies for asthma. He also received unrestricted grants to support Ph.D. studentships from GSK and Pfizer. T.H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.J.V.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. O.Z.-M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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Centro Interuniversitario di Fisiologia Clinica e Ipertensione (G.M., R.F., M.B., G.G., R.S.), and Centro Auxologico Italiano (G.M., G.G.), Milan, Italy.
Abstract
In the Pressioni Arteriose Monitorate e Loro Associazioni (PAMELA) study, office, home, and ambulatory blood pressure (BP) values were measured contemporaneously between 1990 and 1993 in a large population sample (n=2051). Cardiovascular (CV) and non-CV death certificates were collected over the next 148 months, which allowed us to assess the prognostic value of selective and combined elevation in these 3 BPs over a long follow-up. There were 69 CV and 233 all-cause deaths. Compared with subjects with normal office and 24-hour BP, the hazard ratio for CV death showed a progressive increase in those with a selective office BP elevation (white-coat hypertension), a selective 24-hour BP elevation (masked hypertension), and elevation in both office and 24-hour BP. This was the case also when the above conditions were identified by office versus home BP values. Selective elevation in home versus ambulatory BP or vice versa also carried an increased risk. There was indeed a progressive increase in both CV and all-cause mortality risk from subjects in whom office, home, and ambulatory BP were all normal to those in whom 1, 2, or all 3 BPs were elevated, regardless of which BP was considered. The trends remained significant after adjustment for age and gender, as well as, in most instances, after further adjustment for other cardiovascular risk factors. Thus, white-coat hypertension and masked hypertension, both when identified by office and ambulatory or by office and home BPs, are not prognostically innocent. Indeed, each BP elevation (office, home, or ambulatory) carries an increase in risk mortality that adds to that of the other BP elevations.
Key Words: blood pressure monitoring, ambulatory hypertension, white-coat cardiovascular diseases
Introduction
Use of ambulatory and home blood pressure (BP) measurements has allowed us to discover 2 conditions that were unknown when BP was mainly measured in the clinic environment, that is: (1) isolated office (or white-coat) hypertension (HT), in which BPs obtained in the office are 140 mm Hg systolic or 90 mm Hg diastolic, whereas ambulatory or home BP values remain within their normal range; and (2) masked HT, in which office BP is normal, whereas ambulatory or home BPs are elevated.1 The clinical significance of these conditions is still a matter of debate, because cross-sectional studies aimed at examining whether white-coat HT is accompanied by a greater incidence of HT-related organ damage have not provided univocal results.2–4 In addition, and more importantly, the results have not been univocal in the few studies that have addressed this issue prospectically and have reported white-coat HT (as diagnosed by office BP elevation and ambulatory BP nor-mality) to carry no increase in the incidence of cerebral or cardiovascular (CV) disease5–9 or to have a greater or delayed CV risk as compared with that of normotensive subjects.10–13 Furthermore, masked HT has been reported to have a greater prevalence of organ damage14–16 and a prognostically greater risk than that of normotensive individuals and possibly of white-coat hypertensives.13,17,18 However, the evidence is largely confined to a few studies based on ambulatory BP in populations with selected ages, limited follow-up periods, and variable definitions of ambulatory BP normality over 24 hours or the day only. Finally, limited evidence is available as to the prognostic significance of the 2 above-mentioned clinical conditions, when identified by home BP measurements, that is, procedures of large and growing use in clinical practice.
The Pressioni Arteriose Monitorate e Loro Associazioni (PAMELA) study examined cross-sectionally a large sample representative of the population of Monza (a town in the northeast outskirt of Milan) from 1990 to 1993 to establish the normality range of ambulatory and home BPs.19 From recruitment to October 2004, contact with participants was maintained by mail and phone, and a copy of the death certificate was obtained for all of the subjects who died. This allowed a number of issues to be examined (some for the first time) in the context of a population and over a long follow-up. First, it allowed the prognostic value of white-coat or masked HT to be assessed over a long observational period in a general population rather than in selected cohorts of subjects, as in most previous studies. Second, it studied the prognostic value of white-coat or masked HT when the diagnosis is based on home BP vis a vis that based on ambulatory BP. Third, it studied the prognosis of individuals in whom a discrepancy exists between home and ambulatory BP, that is, one is elevated, whereas the other is normal and vice versa. Last, it studied whether and how the elevation of 1, 2, or all 3 BPs has a progressively adverse impact on prognosis, independent of their "in-office" or "out-of-office" nature.
Methods
The methodology used in the PAMELA Study has been reported in detail elsewhere.19 Briefly, 3200 individuals were randomly selected from the residents of the town of Monza to be representative of the population for gender and age decades (25 to 74 years), according to the criteria used in the World Health Organization-MONItoring trends and determinants on CArdiovascular diseases (MONICA) Study conducted in the same geographic area. The overall participation rate was 64% (n=2051) consistently in each age and sex stratum. The demographic characteristics of nonparticipants were similar to those of participants. This was also the case for CV risk factors based on information collected via phone interviews.
Entry Data
Participants were invited to come to the outpatient sector of the local hospital (San Gerardo) in the morning of a working day (Monday through Friday), where several data were collected. Those relevant to the present study are (1) 3 sphygmomanometric BP measurements with the subject in the sitting position, starting 10 minutes after the beginning of the medical visit, (2) a 24-hour ambulatory BP monitoring through an oscillometric device (Spacelabs 90207, Spacelabs) with the BP readings set at 20-minute intervals (the subjects were sent home after checking for the device accuracy with the instruction to attend at their usual activities and to come back the next morning for the device removal), (3) 2 home BP measurements (approximately at 7:00 AM and 7:00 PM) through a validated semiautomatic device (Model HP 5331, Philips) using the arm contralateral to that used for ambulatory monitoring, (4) 3 additional sphygmomanometric sitting BP measurements, after removal of the ambulatory BP device, and (5) information (history and physical examination) on CV risk factors including overweight, smoking habit, serum cholesterol, blood glucose, diabetes mellitus, and history of previous CV morbid events. Total serum cholesterol and blood glucose levels were measured in all of the subjects by standard radioenzymatic method.
Follow-Up
the time of the medical visit to October 1, 2004, a copy of the death certificate was obtained for all of the subjects who died. The causes of death reported in the certificate were coded according to the Tenth International Classification of Diseases. Over an average follow-up period amounting to 148 months, there were 233 deaths, of which 69 were of a CV nature.
Data Analysis
The 3 office and the 2 home BP measurements obtained at the initial visit were separately averaged. As reported in detail in the article describing the PAMELA data,19 ambulatory BP values were edited from artifacts according to preselected criteria20 and averaged for the 24 hours, the day (7:00 AM to 11:00 PM), and the night (11:00 PM to 7:00 AM). Valid ambulatory BP readings were 95.9% of those planned (ie, 72 readings over 24 hours) with an homogeneous distribution (2.9 per hour) throughout the entire recording period and a similar percentage of valid readings compared with the expected ones over the day (95.7%) and the night (96.5%). Based on office (pooled data) and 24-hour ambulatory BP values, subjects were divided into 4 groups: (1) normal office (<140/90 mm Hg) and 24-hour (<125/79 mm Hg) systolic and diastolic BP; (2) elevation in office systolic or diastolic BP with normal 24-hour ambulatory BP, that is, isolated office or white-coat HT; (3) normal office BP with elevation in 24-hour home systolic or diastolic BP, that is, masked HT; and (4) elevated office and 24-hour systolic or diastolic BP. A similar subdivision into 4 groups was made based on office versus home BP values normality (<135 mm Hg systolic or <83 mm Hg diastolic) or elevations (135 mm Hg systolic or 83 mm Hg diastolic). Subjects were divided into 4 groups also according to the normal and/or elevated 24-hour and home BP values. The upper normality values of 24-hour and home BPs were derived from the cross-sectional data obtained in the whole PAMELA population based on their correspondence with office BPs equal to 140/90 mm Hg on the regression line linking the 3 BPs.19 These values are superimposable to the normality values reported by other studies using different approaches and mentioned by international guidelines as the most likely cutoffs dividing out-of-office HT from normoten-sion.1,21 The incidence of events was calculated via a logistic model. The hazard ratio was calculated by the Cox proportional hazard model, the assumption of BP proportionality being assessed by proper statistical test. The 2 test was used to evaluate the trends in death incidence or hazard ratio: (1) from the normotensive to white-coat hypertensive, masked hypertensive, and in-office and out-of-office hypertensive group; (2) from the group with normal 24-hour and home BP, the group with selective 24-hour BP elevation, the group with selective home BP elevation, and the group with elevation in both 24-hour and home BP; and (3) from the group with no BP elevation to the groups with elevation in 1, 2, or all 3 of the BPs, regardless of whether they were measured. Groups were ordered in the above fashion because of the suggestion from previous studies that: (1) in white-coat HT, CV damage and risk may be greater than in "true" normotension but less in true HT2,3,5,6–13; (2) masked HT may be clinically worse than white-coat HT because of the superior prognostic significance of ambulatory versus office BP14; and (3) the ability of home BP to predict the risk of death compares favorably with that of ambulatory BP.14 Data were adjusted for age and gender. Further adjustments were also made for history of CV disease, smoking prevalence, blood glucose, and serum total cholesterol, which were included as covariates into the model. Throughout the text, values in parentheses refer to the SD of the mean. A 2-tailed P value <0.05 was considered to be statistically significant.
Results
As shown in Table 1, compared with normotensive subjects, subjects with white-coat HT, masked HT, and elevation, both in-office and out-of-office BP showed a greater male prevalence, age, body mass index, serum total cholesterol, and blood glucose. This was the case either when BP normality and elevation were identified by office versus 24-hour and by office versus home BP criteria (Table 1, bottom). As shown in Figure 1A and 1B, compared with normotensive subjects, age- and gender-adjusted incidence of CV and all-cause death were usually greater in the remaining 3 groups. There was a statistically significant trend toward a progressively greater unadjusted and age- and gender-adjusted risk of CV from the entirely normotensive to the white-coat HT, masked HT, and entirely hypertensive group, regardless of whether BP normality or elevation was identified by office versus ambulatory or by office versus home BP criteria (Figure 2A and 2B). A similar trend was observed for unadjusted hazard ratios for all-cause mortality (Figure 3A and 3B), as well as for further adjustment of the risk of CV mortality and history of CV disease, smoking, serum total cholesterol, and blood glucose (Table 2).
Discussion
Our study allows several conclusions to be made. First, in the PAMELA population, the incidence and risk of CV death showed a progressive increase from subjects in which in-office and out-of-office BPs were both normal to subjects with white-coat HT, masked HT, and in-office and "out-of-office" HT, independent of age and gender. Second, the progressive increase in mortality from the entirely normotensive to the entirely hypertensive group occurred regardless of whether the above conditions were identified based on office versus ambulatory or office versus home BP. Thus, white-coat and masked HT are not clinically innocent conditions, but they rather indicate a transition toward a greater risk that reached the maximum when in-office and out-of-office BP are both increased. This has 2 implications for the practice of medicine. First, physicians should not lightly decide to dismiss treatment in patients with white-coat HT. Second, normal office BP values should not be taken as a guarantee that there is no increase in risk because of the possibility of an elevation in out-of-office BP. This implies that out-of-office BP values should be more frequently collected than is recommended by current guidelines.1 The noticeable prevalence of masked HT in the normotensive fraction of the PAMELA population (14.5% and 15.5% when assessed by ambulatory and office BP, respectively; Reference14) scores in this direction.
Our study provides new evidence on other clinically relevant issues. Although in our subjects only 2 home BP measurements were available, a selective elevation in home BP increased the age- and gender-adjusted risk of CV and all-cause mortality to an extent that was, if anything, greater than the increase associated with a selective elevation in 24-hour BP. This confirms the importance of BP values self-measured in the home environment9,18 of which the prognostic significance may favorably compare with that of 24-hour BP even when the potential of this approach (daily measurements for weeks) is only partially used. The above, however, does not mean that home BP should be considered just as a substitute of ambulatory BP monitoring, because, in our study, an elevation in only 1 of these out-of-office BPs was accompanied by a risk of CV or all-cause death, which was less than that seen when both of these out-of-office BPs were elevated. Thus, home and ambulatory BP do not represent a duplicate of the same type of information. This is also made clear by the observation that age- and gender-adjusted risk of CV and all-cause death increased progressively in subjects in whom office, home, and 24-hour BP were all normal to subjects in whom 1, 2, or all 3 of these BPs were elevated, regardless of which BP showed the elevation. This leads to the conclusion that office, home, and 24-hour BPs have an individual prognostic value that may add to the prognostic value of the others BPs. Thus, obtaining information on office, home, and 24-hour BP may represent the ideal clinical procedure.
Confirming previous findings,22–26 white-coat hypertensives, masked hypertensives, and hypertensives with in-office and out-of office BP elevations of the PAMELA study all showed body mass index, serum total cholesterol, and blood glucose values that were greater the those of individuals with in-office and out-of-office BP normality. In addition, however, they show that body mass index, total serum cholesterol, and plasma glucose all displayed a progressive increase from individuals in whom office, home, and ambulatory BPs were all normal to those in which 1, 2, or all 3 BPs were elevated (Table 1, P<0.0001 for all trends). This emphasizes that there is a close quantitative relationship between metabolic and BP abnormalities, regardless of where and how BP is measured, with each BP offering a specific contribution to the overall dysmetabolic profile. It also makes it obvious that the progressive increase in risk from normotension to white-coat HT, masked HT, and true HT, as well as from subjects with no elevation to subjects with 3 BP elevations may have a multifactorial nature, that is, it may also be because of alterations in glucose and lipid metabolism. BP alterations, per se, however, are likely to be involved, because in all of the above conditions, the progressive increase in CV mortality remained significant after adjustment for differences in metabolic, as well as other risk factors between groups, suggesting that in-office and out-of-office BPs, per se, play a role. This role may be accounted for by the data shown in Tables 1 and 3. That is, in white-coat HT, the office BP elevation was accompanied by ambulatory or home BP values that, although normal, were higher than the values seen in subjects without white-coat HT. Conversely, in masked HT, office BP values, although normal, were higher than those observed in subjects without masked HT. Finally, moving from the condition of no BP elevation to that of 1, 2, or all 3 BP elevations was associated with a progressive increase in all of the BPs, that is, both in the BPs that reached the HT range and in those that remained in the normotensive range (Table 3, bottom). This may have prognostic relevance, because office, home, and 24-hour BP have all been shown to have a continuous relationship with CV risk.1,2,9,10
Our study has a number of favorable characteristics but also 2 limitations. The favorable characteristics include the long follow-up, as well as the objective nature (death) of the events. One limitation is that the number of CV events was small, given the low CV risk of Mediterranean populations, leading to hazard ratios with large confidence limits. However, the results were often supported by the data on all-cause death, which was >3 times as large as CV death. This was, for example, the case for age- and gender-adjusted risk of all-cause death in subjects with no or 1, 2, or 3 BP elevations. The other limitation is that the observational nature of our study did not allow us to assess the effect on the prognosis of antihypertensive treatment and therapeutic corrections of glucose and lipid abnormalities. Antihypertensive treatment, for example, might have been more common in white-coat than in masked hypertensives, because in the clinical practice treatment is usually guided by office BP elevations, contributing to the better prognosis of the former versus the latter condition.
Perspectives
Our study provides long-term prognostic evidence that white-coat or masked HT are not innocent conditions. It also provides evidence that office, home, and 24-hour BP may each have an adverse prognostic value, which adds to that of the other BPs.
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Giovanni Candiano, Maurizio Bruschi, Nicoletta Pedemonte, Emanuela Caci, Sabrina Liberatori, Luca Bini, Carlo Pellegrini, Mario Viganò, Brian J. O'Connor, Tak H. Lee, Luis J. V. Galietta and Olga Zegarra-Moran
Laboratories of Uremic Physiopathology and Molecular Genetics, Istituto G. Gaslini, Genoa; Laboratory of Functional Proteomics, Molecular Biology Department, Università degli Studi di Siena, Siena; Cardiosurgery Section, IRCCS Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy; and Department of Asthma, Allergy, and Respiratory Science, GKT School of Medicine, King's College London, London, United Kingdom
ABSTRACT
Rationale: The airway surface liquid, the thin layer of liquid covering the airways, is essential for mucociliary clearance and as a barrier against microbial and other noxious agents. Proteins secreted into the airway surface liquid by epithelial and nonepithelial cells may be important in innate immunity and to improve the fluidity of mucous secretions.
Objectives: We aimed to identify proteins specifically secreted into the airway surface liquid by human bronchial epithelial cells, in resting conditions and after treatment with interleukin 4 (IL-4), a cytokine released in asthma.
Methods and Main Results: By using a proteomics approach, we found that one of the most abundant proteins was gelsolin, which breaks down actin filaments. Gelsolin mRNA and protein secretion were increased threefold in the airway surface liquid of epithelia treated with IL-4. These results were confirmed at the functional level by measuring actin depolymerization using a fluorescence assay. Gelsolin protein was also upregulated in the airways of subjects with asthma.
Conclusions: Our findings indicate that gelsolin is released by epithelial cells into the airways and that its secretion is increased by IL-4 in vitro. In addition, we found that the concentration of both IL-4 and gelsolin were raised in the bronchoalveolar lavage of patients with asthma. These results suggest that gelsolin might improve the fluidity of airway surface liquid in asthma by breaking down filamentous actin that may be released in large amounts by dying cells during inflammation.
Key Words: actin epithelium mucociliary clearance
Allergic asthma is an inflammatory disorder of the airways associated with bronchial hyperresponsiveness and variable airflow obstruction. In addition, individuals with asthma frequently suffer from mucus overproduction, which is believed to contribute to airway obstruction (1). There is compelling evidence that T-helper type 2 (Th2) lymphocytes, and the cytokines that they produce (i.e., interleukin 4 [IL-4], IL-5, IL-9, and IL-13), may be involved in the orchestration of airway inflammation. IL-4 is the major factor regulating IgE production by B cells, and is required for optimal Th2 lymphocyte differentiation (2). IgE-mediated immune responses are further enhanced by IL-4 through upregulation of IgE receptors on the surface of B cells. IL-4 contributes to airway obstruction in asthma through the induction of mucin gene expression and the hypersecretion of mucus (3). IL-4 promotes cellular inflammation in the asthmatic lung by the induction of vascular cell adhesion molecule-1 on vascular endothelium (3). In patients with asthma, T cells producing IL-4, IL-5, IL-9, and IL-13 have been identified in bronchoalveolar lavage fluid (BALF) (2, 4, 5). Furthermore, concentration of IL-4 is elevated in BALF from patients with asthma, although the levels demonstrate considerable variability (6–8).
The airway surface is covered by a thin fluid layer, the airway surface liquid (ASL), which has an essential role as a barrier to protect the underlying epithelium (9). Air contains particulate material, such as pollen, ash, mineral dust, bacteria, and viruses, all of which are capable of damaging the lungs either directly or by predisposing the lung to infection. The airways must be able to handle this load by neutralizing, and discarding, injuring agents, as well as repairing any resultant damage to cells. In addition, recent studies have demonstrated that airway epithelial cells can produce a wide variety of cytokines that promote the differentiation of inflammatory cells and other multifunctional factors that initiate and amplify inflammatory events (2). We and other authors have previously shown that IL-4 and IL-13 (10, 11) modify the ion transport properties of cultured cells by changing the expression and activity of ion channels favoring Cl– secretion over Na+ absorption. The consequent changes in volume and composition of ASL secretions could have a considerable influence on the mucociliary clearance and viscosity of mucous secretions. We hypothesized that some of the proteins secreted into the ASL to improve mucociliary clearance, or increase antimicrobial defense in asthma, could be modulated by IL-4. Recently, a database of proteins recovered by BAL has been created using two-dimensional electrophoresis (12). Moreover, Magi and colleagues (13), using a proteomics approach to study the BALF obtained from patients with sarcoidosis and idiopathic pulmonary fibrosis, have identified a number of proteins that may have pathogenic roles in these diseases. However, interpreting the source of proteins present in BALF is difficult because they may have many different origins, including diffusion from serum, production by pulmonary T cells, synthesis by alveolar cells or by alveolar macrophages, or secretion by surface bronchial epithelial cells or submucosal gland cells.
To overcome the problem of multiple cellular sources of secreted proteins in this study, we have used a proteomics strategy to identify the proteins secreted specifically by airway epithelial cells in resting and stimulated conditions. Analysis of the protein content from the ASL of polarized human bronchial epithelial cell cultures shows that one of the most abundant proteins is gelsolin, which breaks down actin filaments. We also find that gelsolin protein levels are increased in cell cultures treated with IL-4 and in BALF of patients with asthma. This finding suggests an important role for gelsolin in the ASL barrier function and indicates that exogenous gelsolin might be considered for therapeutic use in diseases characterized by chronic inflammation with significant levels of filamentous actin release, such as cystic fibrosis (CF) (14).
Some of the results of these studies have been previously reported in the form of an abstract (15).
METHODS
Human bronchi were obtained from lung resections or lobectomies and cultured as previously described (10, 16, 17). The collection and processing of human cells were approved by the local ethics committee. To obtain polarized epithelia, human bronchial epithelial cells were plated on transwell clear permeable supports (Corning Costar; Celbio, Milan, Italy). The apical culture medium was removed, and epithelia maintained in an air–liquid interface (i.e., with culture medium only on the basolateral side). Under these conditions, the epithelia maintain a thin layer of liquid similar to the ASL in vivo (18). IL-4, 10 ng/ml, was added daily to the basolateral medium and was maintained for 24 to 120 h. Collection of ASL proteins was achieved by washing the apical side of polarized epithelium with Ringer's solution.
BALF Samples
BALF samples were obtained from six nonasthmatic, nonatopic subjects (mean age, 32 ± 3 yr) on one occasion, and from six subjects with atopic asthma (mean age, 26 ± 1 yr) on two occasions, before and 24 h after inhaled allergen challenge (house dust mite, cat hair dander, or grass pollen, as appropriate). All subjects provided informed consent, and the study was approved by the local research ethics committee of King's College Hospital, London. Bronchoscopy was performed according to the standard operating procedure of King's College Hospital.
Two-Dimensional Gel Electrophoresis
BALF and ASL samples were treated as previously reported (19, 20). Samples were separated in the first dimension on soft, immobilized pH gradient, in a nonlinear pH 3–10 interval (21, 22). In the second dimension, proteins were separated based on their molecular weight in polyacrylamide gels, 180 x 160 x 1.5 mm. Proteins were visualized by a double staining procedure: first, the methyl-trichloroacetate negative staining (23), followed by the silver staining (24) for the analytic image, or colloidal Coomassie staining (25) for mass spectrometry analysis. Additional details on the methods are provided in an online supplement.
Mass Spectrometry
After visualization with colloidal Coomassie blue staining, electrophoretic spots were excised and proteins digested as previously described (26). Peptide mass fingerprinting spectra were acquired using an Ettan MALDI-ToF mass spectrometer (Amersham Biosciences, Uppsala, Sweden). Peptide sequencing was performed using an LCQDeca ESI-Ion Trap mass spectrometer (Thermo, San Jose, CA). Database searching for protein identification was performed using available online software.
Gelsolin Functional Assay
Rabbit muscle actin was dissolved in a low-ionic-strength solution, diluted with a polymerizing buffer, and stored in small aliquots at –20°C. Rhodamine phalloidin was dissolved in dimethyl sulfoxide at a concentration of 500 μM and stored at –20°C. Human plasma gelsolin was dissolved and stored at –80°C at a final concentration of 400 nM. Fluorescence intensity was measured with a fluorescence microplate reader (FluoStar Galaxy; BMG LabTechnologies, Germany) using 96-well, black, clear-bottom plates. We used high-quality filters (Chroma Technology, Rockingham, VT) with maximal transmittance at 540 and 590 nm for excitation and emission, respectively. Additional details are provided in the online supplement.
Statistics
Results are presented as representative data or as arithmetic means plus SEM. Significance was determined on unpaired groups of data using two-tailed Student's t test, if not otherwise indicated, or by nonparametric Kruskal-Wallis followed by Dunn's multiple comparison test.
RESULTS
Two-Dimensional Gel Analysis of Cultured Cells' ASL
Analytic gels prepared with the proteins recovered from the apical surface of cultured airway epithelia showed hundreds of silver-stained spots (Figure 1, top panel). Side-by-side comparison of gels prepared with samples from cultures treated with and without IL-4 revealed changes in intensity and/or mobility of some of the spots. Particularly evident were the modifications of two clusters of spots or charge trains (circled in Figure 1, bottom panels) with apparent molecular weights of 85 and 45 kD. For each molecular weight, up to seven distinct spots could be distinguished that were positioned between an isoelectric point range of 5.6 to 6.2 for the 85-kD cluster, and 5 to 5.2 for the 45-kD cluster. As shown in Figure 1 (bottom panels), the spots in the two clusters were significantly increased in intensity when the cells were treated with IL-4. Preparative gels were generated for protein identification. Recovery of the single spots and subsequent mass spectrometric analysis revealed that the polypeptides in both spot clusters belonged to gelsolin. The presence of charge trains of the same protein indicates various degrees of in vivo modifications, such as glycosylation, phosphorylation, or acetylation, which affect electrophoretic mobility (27). The sequences of fragments from the low-molecular-weight spots matched with the carboxyl-terminal half of the human gelsolin sequence (Figure 2). Conversely, sequences obtained from the high-molecular-weight cluster matched with the whole gelsolin. Spots that were expected to correspond to actin were confirmed by mass spectrometry. In contrast to gelsolin, the intensity of actin charge trains was not modified by IL-4 (underlined in Figure 1). None of the spots recovered from two-dimensional gels corresponded to the amino-terminal half of gelsolin.
Quantification of Gelsolin Protein and mRNA
We performed densitometric analysis of protein spots from two-dimensional gels from untreated and IL-4–treated cells (Figure 3A). Our results demonstrated that secreted gelsolin increased threefold at 24 h after IL-4 stimulation and remained upregulated for the following 96 h. We confirmed the presence of 85- and 45-kD bands by Western blot using an antibody against human gelsolin (Figure 3B; for details on method, see online supplement). Intensity of both bands increased after IL-4 treatment, by approximately three- and twofold for the high- and low-molecular-weight material, respectively. We then reasoned that the upregulation of gelsolin protein was caused by an increase in gelsolin mRNA. We therefore quantified gelsolin mRNA using real-time reverse transcriptase–polymerase chain reaction (Figure 3C; for details on the method used, see online supplement). Our data showed that gelsolin transcript was significantly increased at 6 h after IL-4 addition, reached a peak at 24 h, and then started to decrease.
Functional Analysis of Gelsolin
Gelsolin cleaves actin filaments (28). To evaluate the presence and upregulation of gelsolin at the functional level in the ASL of cultured cells, we used an assay based on the properties of rhodamine-labeled phalloidin to increase its fluorescence emission when bound to polymerized but not to depolymerized actin (29). In the absence of gelsolin, addition of polymerized actin caused a fuorescence enhancement of 233 ± 15.8% (from 3,490 ± 203 to 8,121 ± 609 arbitrary units, n = 14). This effect was slow, requiring approximately 7 to 8 min to reach a stable fluorescence level (not shown). The addition of human gelsolin to the reaction mixture caused a dose-dependent reduction of actin-dependent signal (Figures 4A and 4B). The gelsolin response was normalized by taking the fluorescence with phalloidin plus actin and that with phalloidin alone as 100 and 0%, respectively (see Figure 4A). The fit of experimental data to a Michaelis-Menten function yielded a half-effective concentration of 24.7 nM. After generating a calibration curve, we tested the effect of ASL samples from cultured cells. The ASL lavage from untreated epithelia did not change the fluorescence of the phalloidin-actin complex (Figure 4C). In contrast, 20 μl ASL lavage from IL-4–treated epithelia caused a clear fluorescence reduction of 38.1 ± 12% (Figure 4D; n = 6; p < 0.05). By interpolation to the calibration curve, this change corresponds to that elicited by approximately 25 nM gelsolin (see Figure 4B, filled circle).
Detection of Gelsolin in BALF
To verify whether gelsolin is secreted into the airways in vivo, we analyzed BALF samples from six nonasthmatic and from six patients with asthma (Figure 5). Protein spots corresponding to the molecular weights of the two forms of gelsolin and actin were identified. The high-molecular-weight spots of gelsolin were partially covered by the big spot corresponding to albumin. However, two-dimensional gels showed that gelsolin spots of low molecular weight in patients with asthma were more intense than in control patients, whereas actin spots were unchanged (Figure 5, bottom). These findings were confirmed in Western blots using the antihuman gelsolin antibody and extended to the high-molecular-weight form of gelsolin as well (Figure 6). The 85- and the 45-kD bands, particularly the latter one, were more intense when obtained from patients with asthma than from nonasthmatic individuals. We also analyzed the BALF from patients with asthma after an allergen challenge (see METHODS). We found that the abundance of the high-molecular-weight gelsolin was similar before and after allergen, whereas the low-molecular-weight band intensity decreased to values similar to those of nonasthmatic patients.
Detection of IL-4 in BALF
We wanted to establish whether there was a correlation in BALF between gelsolin and IL-4 levels. We therefore measured IL-4 concentration by ELISA (see the online supplement) in the BALF of the same control subjects and subjects with asthma from which gelsolin was determined. The BALF from control patients contained low levels of IL-4 (0.68 ± 0.12 pg/ml). In contrast, the IL-4 content from patients with asthma was 14-fold higher (9.68 ± 1.7 pg/ml). After exposure to allergen, patients with asthma displayed IL-4 values similar to those of nonasthmatic subjects (0.76 ± 0.26 pg/ml).
DISCUSSION
Proteomics is a powerful approach to analyze the protein content in biological fluids. Comparison of two samples of protein mixtures (normal vs. affected, treated vs. untreated) by two-dimensional gel electrophoresis, followed by identification of the proteins by mass spectrometry, can detect the presence of novel proteins, reveal new roles for detected proteins, and discover patterns of regulated post-translational modifications (30). Regarding the airways, a database of BAL proteins has recently been created using two-dimensional electrophoresis (12). A variety of soluble components from human lung have been identified, including approximately 80 proteins. Analysis of BAL protein content is complicated by the presence of proteins derived from many different sources, including leukocytes, serum, submucosal glands, and surface epithelium. This study therefore adopted a simplified approach by using an in vitro model of differentiated airway epithelium. We have found that the ASL of polarized human bronchial epithelia contains gelsolin whose secretion is significantly stimulated by treatment with IL-4.
Gelsolin is the most potent protein that severs actin filament identified to date (28). This protein weakens noncovalent bonds between actin filaments so that they can then be broken. This effect is rapid, stoichiometric, and highly efficient. Interaction of gelsolin with actin is strongly Ca2+-dependent (31). However, when gelsolin is cleaved by caspase-3, the effector caspase in apoptosis (28), or by other proteases, it becomes Ca2+-independent. Indeed, gelsolin is formed by two tandem homologous halves connected by a long linker where the protease site is located. The isolated C-half can bind a single actin molecule but only when Ca2+ concentration is higher than 1 μM. The isolated N-half binds two actin molecules even in the absence of Ca2+ (28, 32). Recently, evidence that gelsolin is a substrate of metalloproteinase 14 has been provided (33). Gelsolin might also be cleaved by proteases involved in epithelial remodeling that are expressed in the airways, in particular, by metalloproteinases 2 and 9, which have been found increased in the lung in inflammatory lung diseases, including asthma (34).
We did not observe the amino-terminal half of gelsolin in our two-dimensional gels. Possible explanations are as follows: first, gelsolin is usually cleaved inside the cell, and only the complete and the C-half forms are secreted into the ASL; second, that the N-half is secreted into the ASL but it is further digested by other proteases; third, given that it is Ca2+-independent, the N-half of gelsolin is bound to actin filaments present in the ASL and not loaded into the two-dimensional gels because of their big size. Besides being involved in the dynamic remodeling of actin filaments inside cells, gelsolin is also secreted into the extracellular space. Considerable amounts of actin are released into the extracellular space during acute lung injury, and circulating actin–gelsolin complexes can be detected in the peripheral blood (35). In the plasma, gelsolin acts as an actin-scavenging protein to prevent increases in blood viscosity due to actin leakage from dying cells. Gelsolin may serve a similar role in the ASL. Because actin is one of the most abundant intracellular proteins, cell death that may occur during inflammatory processes can release large amounts of filamentous actin in the airways, thus increasing the viscosity of mucous secretions. Evidence for this comes from studies on patients with CF. In CF, disruption of chloride secretion, mediated by the CFTR protein, causes dehydration of airway surface and inflammation (9). Mucociliary clearance is severely impaired and the airways become susceptible to mucus plugging and infection. It is well known that one of the major sources of sputum viscosity in CF comes from the DNA released by dying cells. Vasconcellos and colleagues (14) have shown that sputum samples from patients with CF also contain large amounts of filamentous actin, and that this contributes significantly to sputum viscosity. Indeed, the authors demonstrated that addition of gelsolin in vitro to CF sputum decreased the viscosity, thus emphasizing the possible use of exogenous gelsolin as a mucolytic agent. The presence of endogenous gelsolin in the airways was not investigated in that study.
Our results now demonstrate that gelsolin is a protein normally secreted into the ASL by bronchial epithelial cells, and that its levels are high and significantly upregulated by stimulation with IL-4. Quantification by real-time reverse transcriptase–polymerase chain reaction shows an increase of gelsolin mRNA in IL-4–treated cells, which is consistent with upregulation of the corresponding protein in the ASL. Therefore, our results are consistent with an increase in gelsolin protein synthesis due to enhanced gene transcription or enhanced stability of gelsolin mRNA.
We performed functional assays to check whether the gelsolin protein found in the cultured cells' ASL retains its ability to depolymerize actin filaments. We found no gelsolin-like activity in untreated cells. This could be due to lack of sensitivity of our assay or, more probably, to the fact that gelsolin was already complexed to endogenous actin. In contrast, in IL-4–treated cells we found significant depolymerization of exogenous actin, which could be due to excess of free gelsolin. Our findings suggest that the stimulation by IL-4 can create a reserve of gelsolin in airway periciliary fluid and this may in turn degrade filamentous actin that is leaked from dying cells.
IL-4 is a cytokine particularly important in asthma. It is part of a cascade that causes release of chemoattractant molecules, recruitment of leukocytes into the airway lumen, and consequent release of proinflammatory mediators, proteases, and mucus hypersecretion (36). Significant release of actin filaments would be expected as a consequence of the resulting inflammation and cell damage. We therefore speculate that IL-4–induced gelsolin upregulation may be part of a complex host antiinflammatory response.
Patients with asthma had increased amounts of gelsolin protein in their airways with respect to control subjects. It is tempting to conclude that, in agreement with our in vitro results, this upregulation is a result of the enhanced levels of IL-4 in the airways of patients with asthma. Indeed, we found that IL-4 was increased in the BALF of patients with asthma by 14-fold with respect to the level found in the BALF of nonasthmatic subjects. The low IL-4 values found in control subjects (0.68 ± 0.12 pg/ml) are similar to those reported by others (6, 7, 37). IL-4 levels in BALF from patients with asthma are more controversial, probably depending on patients' selection criteria and disease severity. Some authors reported IL-4 levels in patients with asthma similar to control subjects (6). Others reported undetectable IL-4 levels but using an assay with a lower sensitivity (7). Finally, other investigators have found increased IL-4 in the BALF of allergic patients with asthma (38) in agreement with our results. We found a correlation between IL-4 and gelsolin levels: both are low in control subjects and increased in patients with asthma. These data further support the notion that gelsolin secretion in asthma is regulated by IL-4.
Surprisingly, after stimulation with allergens, patients with asthma showed a decrease of the low-molecular-weight form of gelsolin. This finding and the role of the two gelsolin forms is not clear. The IL-4 level in the BALF was also reduced after allergen exposure and this result is in contrast with previous reports (6, 7). We have no explanation for this difference, but it is possible that allergen caused release of mast cell proteases and this might have cleaved the cytokine.
In conclusion, our study reports for the first time the expression and secretion of gelsolin by airway epithelial cells and its regulation by IL-4, supporting the view that ASL has an important role as a first line of host defense. A recent report reveals an important additional function that gelsolin may play in the airways (39). In that study, the authors show that the activity of some antimicrobial peptides, which have the characteristic of being polyvalent cations, is inhibited by filamentous actin through the formation of aggregates. The filament association, and thus the antimicrobial activity inhibition, is reversed by gelsolin. Therefore, gelsolin can be important not only to control mucus viscosity but also to preserve the innate antimicrobial activity in the airway surface. Given the stoichiometric nature of the interaction between gelsolin and actin, massive actin release, which happens during severe inflammatory processes as in CF, may overwhelm the capacity of endogenous gelsolin. This may perhaps be an indication for the therapeutic use of exogenous gelsolin or of other actin-severing agents in diseases characterized by massive airway inflammation with significant levels of filamentous actin release. Nevertheless, caution must be taken because, in another report, actin was found to bind IL-8 in the airways, preventing this chemokine from binding to neutrophil receptors (40). These authors found that gelsolin releases IL-8 from actin, increasing the proportion of free cytokine, and therefore the possible level of inflammation.
FOOTNOTES
Supported by a grant from the Italian Cystic Fibrosis Research Foundation. The Pediatrics Department, Università degli Studi di Genova, has received support from Fondo per gli Investimenti della Ricerca di Base (FIRB) funds.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Conflict of Interest Statement: G.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.J.O. serves on advisory boards for GlaxoSmithKline (GSK), Boehringer Ingelheim, Atlana, Trinity Chiesi, and Celgene, and has received compensation for this activity. He has also received lecture fees from GSK and Pfizer. He runs a clinical academic research unit, which receives grants from major respiratory pharmaceutical companies, including GSK, Aventis, UCB, Pfizer, and Nicox to evaluate potential new therapies for asthma. He also received unrestricted grants to support Ph.D. studentships from GSK and Pfizer. T.H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.J.V.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. O.Z.-M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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