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(Chest. 2006;129:527-535.)
© 2006 American College of Chest Physicians

Treatment With AM3 Restores Defective T-Cell Function in COPD Patients*

Eduardo Reyes, PhD; Alfredo Prieto, PhD; Antonio de la Hera, MD, PhD; Pilar de Lucas, MD, PhD; Rudolfo Alvarez-Sala, MD, PhD; José Luis Alvarez-Sala, MD, PhD and Melchor Alvarez-Mon, MD, PhD

* From the Laboratory of Immune System Diseases and Oncology (Drs. Reyes, Prieto, and de la Hera), CNB-CSIC Research and Development Associated Unit, Department of Medicine, University of Alcalá; Pulmonary Service (Dr. Lucas), Hospital Universitario Gregorio Marañón; Pulmonary Service (Dr. R. Alvarez-Sala), Hospital Universitario La Paz; Pulmonary Service (Dr. J Alvarez-Sala), Hospital Universitario San Carlos; and Immune System Diseases and Oncology Service (Dr. Alvarez-Mon), Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Madrid, Spain.

Correspondence to: Melchor Alvarez-Mon, MD, PhD, Departamento de Medicina, Universidad de Alcalá, Carretera Madrid-Barcelona, Km 33,600, E-28871 Alcalá de Henares, Madrid, Spain; e-mail: eduardo.reyes{at}uah.es

Abstract

Background: Lymphocyte alterations have been associated with an increased prevalence of acute respiratory infections in COPD patients. AM3 is an oral immunomodulator that normalizes the defective functions of peripheral blood natural killer and phagocytic cells in COPD patients and improves their health-related quality of life.

Objectives: To characterize putative systemic abnormalities of the T-cell compartment in COPD patients, and to investigate whether AM3 can restore such abnormalities.

Design: The study was a randomized, prospective, double-blind, placebo-controlled trial in a cohort of COPD patients. The results were also compared to those of nonsmoker and ex-smoker healthy control subjects.

Setting: Outpatient departments of four hospitals.

Patients: Seventy COPD patients were randomized to receive either AM3 or a placebo orally for 90 consecutive days. Populations of 36 healthy nonsmokers and 36 healthy ex-smokers were used as control subjects.

Measurements: Peripheral blood mononuclear cell (PBMC) proliferation and production of interleukin (IL)-2, IL-4, IL-12p40, tumor necrosis factor-{alpha}, and interferon (IFN)-{gamma} proteins in response to the T-cell polyclonal mitogens were assessed at baseline and at the end of treatment.

Results: The proliferative response was significantly decreased in COPD patients. Decreased production of IFN-{gamma} was the only defect in the profiles of the cytokine measures, and was selectively observed in COPD patients, but not in nonsmoker and ex-smoker healthy control subjects. Treatment with AM3 significantly restored the PBMC proliferative response to polyclonal mitogens and significantly promoted stimulated IFN-{gamma} production in these patients. The normalization of these proliferative responses was not related to significant variations in the numbers of peripheral blood monocytes, CD3+, CD4+, CD8+ cells or of any major naïve/memory/activated T-cell subset. The increased IFN-{gamma} production in the AM3 study arm was associated with an increase in the mean of number of IFN-{gamma} molecules produced per CD8+ T cells.

Conclusions: PBMCs of COPD patients showed clear functional T-lymphocyte abnormalities that are rescued by AM3 treatment.

Key Words: COPD • immunity • Inmunoferon • interferon-{gamma} • T-lymphocyte subsets

COPD is the fourth-most-common cause of death worldwide.12 A clinically defined respiratory disease, COPD is associated with several lung and systemic immune disturbances.345 Several factors may contribute to this immunologic impairment, including cigarette smoking,26 age,7 and relapsing viral and bacterial respiratory infections.8910

Monocytes and T-lymphocytes play a pivotal role in the regulation of the immune response to microorganisms.311 These regulatory effects are mediated by direct cell-to-cell interactions and by the secretion of cytokines.1112 This secretion includes inflammatory molecules such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-12p40, and regulatory cytokines such as IL-2 and interferon (IFN)-{gamma}.31112131415 The progression of chronic airway obstruction has been associated with higher rates of acute respiratory infection.8911 Alterations in lymphocyte function and cytokine production patterns have been implicated in the increased prevalence of acute respiratory infections in COPD patients,1116 but no causal association has been definitively established. The rationale for treatment with immunomodulators in COPD is the normalization of the impaired immune system parameters, with reduced risk of respiratory infections that would result in improved health-related quality of life.813171819

AM3 is a commercially available immunomodulator with a low toxicity profile; its active ingredient is a polysaccharide/protein compound purified from Candida utilis.19 AM3 is a clinically used oral immunomodulator with a wide range of regulatory effects on innate and adaptive immunity in mice and humans.162021 In COPD patients, AM3 is able to normalize deficient effector functions in natural killer and phagocytic cells, which are involved in the innate immune response.316 Herein, the aim of this study was to address whether systemic immune defects also occur in PBMC T-lymphocytes, responsible for acquired immunity,3 and whether they could be normalized by AM3 treatment. We have found proliferative and cytokine production T-lymphocyte defects in COPD patients. The rational to study the reported cytokines was as follows: IFN-{gamma} was chosen as the accepted prototype T-helper type 1 cytokine and reported relevance in fighting systemic dissemination of bacteria colonizing the lung, IL-12 as key cytokine in IFN-{gamma} polarization, IL-4 as prototype T-helper type 2 cytokine, TNF-{alpha} as key proinflammatory cytokine with systemic effects potentially relevant in COPD patients, and IL-2 was chosen because it has a pivotal role in the proliferation of T-lymphocytes in vitro.345678 Moreover, in the present study, a randomized, prospective, placebo-controlled, double-blind clinical trial, we observed immunomodulatory effects of AM3 treatment on the functional characteristics of T-lymphocytes from the peripheral blood mononuclear cells (PBMCs) of COPD patients.

Materials and Methods

Study Subjects
Seventy patients with COPD were randomized to receive AM3 (n = 35) or an indistinguishable placebo (n = 35). Subject characteristics are shown in Table 1 . All subjects received extensive information about the study and gave informed, witnessed consent to participate. Neither patients nor control subjects were unavailable for follow-up.


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  		Table 1. Characteristics of Patients With COPD Randomized to Placebo or AM3*

 
Two groups of age- and sex-matched control subjects were included for immunologic comparison: one group was comprised of 36 healthy nonsmokers (mean age, 61 ± 8 years [± SD]), and the other group was comprised of 36 healthy ex-smokers with a smoking history of 50.8 pack-years, and a mean of 9.9 years (range, 2 to 30 years) years since quitting smoking (mean age, 62 ± 4 years). These subjects showed no signs of COPD in their clinical history, in physical examinations, or in spirometry results. The study was approved by the Research and Ethics Committee of Alcalá University and followed the principles of the Declaration of Helsinki.

Study Design
The study was undertaken in Spain before the onset of winter. All COPD patients received either AM3 or placebo (starch) orally for 90 consecutive days during the winter months. Peripheral blood for immunologic assessment was collected at baseline and again within the last 6 days of treatment. No deaths occurred during the study.

Inclusion and Exclusion Criteria
All patients enrolled in this study were a homogeneous group of patients with moderate COPD (Global Initiative for Chronic Obstructive Lung Disease stage II), a clinically and epidemiologically important subgroup of patients. The study subjects were white men and women aged 40 to 80 years with a diagnosis of COPD,22 all of whom had a Karnofsky score > 70.

Inclusion Criteria:
The diagnosis of COPD was defined using the following spirometry parameters: FEV1/FVC < 70% with FEV1 < 70% to > 35% with no evidence of a significant reversible component as demonstrated by a < 15% (200 mL) change from baseline after bronchodilator challenge. For inclusion, patients also had to have had at least two episodes of acute respiratory exacerbation type 1 during the previous year.9 Those included were required to have a history of smoking at least one pack of cigarettes per day for 20 years and to have quit at least 6 months prior to the study.

Exclusion Criteria:
The following patients were excluded: those unable or unlikely to comply with the study protocol, pregnant or breast-feeding women, women who declined to use a standard birth control method, patients with evidence of respiratory exacerbations during the 3 months prior to the study, those with congenital or acquired immunodeficiencies or autoimmune disease, and those with malabsorption syndrome, hypercalcemia, bronchiectasis, active pulmonary tuberculosis, neoplasm, cystic fibrosis, restrictive pulmonary fibrosis, cardiac insufficiency (class III or worse), advanced renal or liver disease. To avoid a T-helper type 2 bias in the immunologic studies, patients with clinical and laboratory criteria (eosinophilia in peripheral blood and/or elevated serum IgE) of atopia were excluded. Also excluded were those who in the 3 months prior to the study had received immunosuppressants, immunomodulators, cimetidine, or other medications considered to modify the immune response (except inhaled corticosteroids; maximum dose, 800 µg of beclomethasone or an equivalent dose of other inhaled corticosteroids); those who received systemic corticosteroid therapy within the 2 weeks prior to the study; those who had an acute respiratory exacerbation treated with antibiotics within the month prior to the study; and those who had clinical destabilization within the 15 days prior to the start of the study.

Treatments
AM3 is an orally administered immunomodulator with a low toxicity profile.16192021 Previous dose-finding and kinetic studies16171922 in humans have demonstrated that 3 g/d is the optimal dose for maximal immunomodulation. Six capsules per day (2 x 500-mg capsules tid) of either AM3 or placebo were therefore administered po for 90 consecutive days.

Cell Separation
PBMCs were obtained by density gradient centrifugation (Ficoll-Hypaque; Lymphoprep Nyegaard; Oslo, Norway).23 Cell viability was checked by Trypan blue exclusion.

Mitogen Stimulation Assays
Triplicate cultures of 5 x 104 PBMCs per well were stimulated with 10 µg/mL of phytohemagglutinin (Difco Laboratories; Detroit, MI), 12.5 ng/mL soluble anti-CD3 antibodies (Orthomune; Orthodiagnostic System; Raritan, NJ), with or without 1.6 ng/mL anti-CD28 (Orthomune), or 100 IU/mL IL-2 in complete medium in 96-well, flat-bottomed microtiter plates (Nunc Corporation; Roskilde, Denmark). The amount of [methyl-3H]thymidine (Radiochemical Center; Amersham, UK) incorporated into the cultures was determined as previously described.24

Immunophenotype Studies
PBMCs were incubated with combinations of fluorescein, phycoerythrin, and phycoerythrin-cyanine-5 conjugate-labeled monoclonal antibodies against CD3, CD4, CD8, CD14, CD19, CD28, CD45RA, and CD45RO (all by BD Biosciences; San Jose, CA) as previously described.16

Intracellular Cytokine Assays
For intracellular cytokine assays, 2.5 x 106 PBMCs (106/mL) were stimulated with 50 ng/mL phorbol myristate acetate (Sigma Chemical; St. Louis, MO) plus 1 µg/mL of ionomycin (Calbiochem-Novabiochem; La Jolla, CA) and 2 µmol/L of monensin (Sigma Chemical) in complete medium. After 6 h of culture, the cells were harvested and counted and then stained with combinations of anti-CD3-phycoerythrin and anti-CD8-phycoerythrin-cyanine-5 conjugate monoclonal antibodies. After cell surface staining, cells were fixed, permeabilized, and the cytokine stained with anti-IFN-{gamma}-fluorescein (BD Biosciences) as previously described.25 Quantitative three-color immunofluorescence was performed with a FACSCalibur flow cytometer (BD Biosciences; San Jose, CA) using Cell Quest software (BD Biosciences) as previously described.2425

Cytokine Production Assays
For the determination of cytokines, PBMCs were adjusted to 2.5 x 106/mL and cultured in 24-well plates (Becton Dickinson Labware; Franklin Lakes, NJ) for 72 h in the presence or absence of 10 µg/mL of phytohemagglutinin. The supernatants were then collected, aliquoted, and frozen at – 20°C until use. The IFN-{gamma}, IL-2, IL-4, IL-12p40, and TNF-{alpha} were measured by enzyme-linked immunosorbent assay using matched-pair antibodies (R&D Systems; Minneapolis, MI) according to the recommendations of the manufacturer. Notably, the kinetics of the three above cytokine allows to quantify them in parallel. The lower limits of detection were 8.0, 7.0, 10.0, 15.0, and 1.6 pg/mL, respectively. We have not observed any cross-reactivity of AM3 in the IFN-{gamma}, or other cytokines, enzyme-linked immunosorbent assay, or intracellular-staining assays used in this report.

Statistical Analysis
All analysis was performed using statistical software (SPSS for Windows version 11.0; SPSS; Chicago, IL). Since the normality of variables failed, statistical analysis was performed with the Mann Whitney U test for nonparametric data to analyze the differences between the groups at baseline. Analysis of variance followed by Wilcoxon signed-rank test was used to analyze the effects of AM3 and placebo. Significance was set at p < 0.05.

Results

PBMCs From COPD Patients Show Defects in Proliferation and IFN-{gamma} Production in Response to T-Lymphocyte Mitogens
The proliferative response of PBMCs from COPD patients, as well as those of nonsmoker and ex-smoker healthy control subjects, to stimulation with phytohemagglutinin and anti-CD3 monoclonal antibodies was investigated. The kinetics of the proliferative response of PBMCs to the mitogens were analyzed at 3 days, 5 days, and 7 days of culture. The maximum response was found at 5 days of culture for both sets of control subjects and patients (data not shown); this culture period was therefore used for all analyses. PBMCs from COPD patients showed significantly poorer proliferative responses to phytohemagglutinin and anti-CD3 than did those from healthy nonsmoker and ex-smoker control subjects (Fig 1 ). The addition of stimulatory signals such as IL-2 or anti-CD28 monoclonal antibodies to the culture provoked significant increases in the proliferative responses of PBMCs from both groups of control subjects and the COPD patients (Fig 1). However, the addition of IL-2 did not normalize the proliferative response of PBMCs from COPD patients (Fig 1).


Figure 1
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  		Figure 1. Proliferative response in COPD patients. PBMC proliferative response to mitogens is significantly reduced in COPD patients (white bars) compared to both groups of healthy control subjects included (nonsmokers, black bars; ex-smokers, gray bars). Data are shown in counts per minute (cpm) as mean ± SEM. *Indicates a significantly lower proliferative response in the COPD group (p < 0.0001; Mann Whitney U test). **Indicates a significantly lower proliferative response in the COPD group (p < 0.001; Mann Whitney U test). {dagger}Indicates a significantly higher proliferative response compared to absence of IL-2 or anti-CD28 stimulation (p < 0.001, Wilcoxon signed-rank test). {dagger}{dagger}Indicates a significantly higher proliferative response compared to absence of IL-2 or anti-CD28 stimulation (p < 0.0005, Wilcoxon signed-rank test).

 
As shown in Table 2 , there were no significant differences in the percentages of either peripheral blood monocytes, helper T-cells, cytotoxic T-cells, or naïve or memory T-cells between COPD patients and healthy nonsmoker or ex-smoker control subjects. Furthermore, the expression of CD28 by CD4 and CD8 lymphocytes was similar in all three subject populations.


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  		Table 2. Nonsignificant Effect of AM3 or Placebo on Peripheral Blood Lymphocyte Phenotypic Profile in COPD Patients*

 
The production of a panel of inflammatory and regulatory cytokines by PBMCs from COPD patients and nonsmoker and ex-smoker healthy control subjects was also investigated. Table 3 shows that IFN-{gamma} production by phytohemagglutinin-stimulated PBMCs from COPD patients was significantly lower than in nonsmoker and ex-smoker healthy control subjects. However, there were no significant differences in the production of IL-2, IL-4, IL-12p40, and TNF-{alpha} (data not shown).


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  		Table 3. Production of IFN-{gamma} by Stimulated PBMCs and Intracellular Cytokine Staining of CD4 and CD8 Populations*

 
The defective secretion of IFN-{gamma} by PBMCs from COPD patients was further characterized by analyzing its production in T-lymphocytes at the intracytoplasmic level. The expression of IFN-{gamma} by the PBMCs was analyzed in optimal stimulation conditions.242526 The percentage of IFN-{gamma}–producing CD4 and CD8 T-lymphocytes from COPD patients was significantly lower than in both control groups (Table 3). However, the mean number of IFN-{gamma} molecules expressed by IFN-{gamma} CD4- and CD8-producing cells was similar in the three groups of subjects.

Effect of AM3 Treatment on T-Lymphocyte Function
Immunologic variables in COPD patients were analyzed before and after treatment with placebo or AM3. At baseline, no significant differences between placebo and AM3 treatment groups for any of the variables assessed were found (Fig 2 ; Tables 2, 4 ). Whereas COPD patients treated with placebo showed no significant changes from baseline to the end of treatment, treatment with AM3 significantly increased PBMC proliferative responses to phytohemagglutinin, anti-CD3, and anti-CD3 plus anti-CD28 (Fig 2). After treatment with AM3, the PBMC proliferative responses to all mitogens tested were significantly greater than in the placebo-treated group (Fig 2). This functional effect was not related to significant changes in the percentages of CD3+, CD4+, CD8+, or any of the major naïve/memory/activated T-cell subsets analyzed (Table 2). In addition, the AM3-treated group was functionally undistinguishable from the healthy reference groups in terms of proliferative responses to phytohemagglutinin, anti-CD3, and anti-CD3 plus anti-CD28 (Fig 2). The decreased proliferative responses of PBMCs from COPD patients receiving placebo were not normalized by the addition of stimulatory factors such as IL-2 (data not shown).


Figure 2
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  		Figure 2. Proliferative responses of PBMCs from COPD patients after adjuvant therapy; PBMC proliferative responses after 90 consecutive days of either placebo (white bars) or AM3 (black bars) treatment. The dotted lines indicate the control group levels. Data are shown in counts per minute (cpm) as mean ± SEM. *Indicates a significant improvement between before and after post-treatment in the proliferative responses of COPD subjects treated with AM3 (p < 0.05, Wilcoxon signed-rank test). {dagger}Indicates a significantly higher proliferative response after treatment in the placebo group than in the AM3 group (p < 0.05, Mann Whitney U test).

 

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  		Table 4. Effect of Placebo or AM3 Treatment on the Production of IFN-{gamma} Production by Stimulated PBMCs, and Intracellular Cytokine Staining of CD4 and CD8 Populations at Baseline and After Treatment*

 
Finally, treatment with AM3 significantly increased the defective IFN-{gamma} production by phytohemagglutinin-stimulated PBMCs from COPD patients (Table 4). This immunoregulatory effect was associated to a significant increase in the mean number of IFN-{gamma} molecules produced by CD8 T-lymphocytes. In contrast, the latter parameter remained unchanged in CD4 T-cells.

Discussion

This article shows that COPD patients have a defective PBMC proliferative response to polyclonal T-lymphocyte mitogens and that the percentage of CD4 and CD8 T-lymphocytes producing IFN-{gamma} is reduced compared to nonsmoker and ex-smoker healthy subjects. The treatment with the immunomodulator AM3 is able to restore these defects.

We and others81116 have previously reported decreased natural killer cell activity and impaired monocyte and polymorphonuclear chemotactic and phagocytic activities in COPD patients (smokers and nonsmokers). COPD patients have moderate-to-severe alterations in several mechanisms involved in the control of innate immunity that are restored by the immunomodulator AM3.16 This article address the systemic defects in the T-cell compartment in COPD patients and its modification by AM3.

The analysis of the immune system in COPD patients is made difficult by the confounding influence of multiple pathogenic factors such as genetics,6813 tobacco,2811 infectious exacerbation,289 seasonal variation,27 and patient age.7 Given this level of complexity, it was decided to study a group of homogeneous moderate COPD Global Initiative for Chronic Obstructive Lung Disease stage II, nonatopic, 40- to 80-year-old white patients, mostly men, with a history of smoking at least one pack of cigarettes a day for 20 years, but who had quit at least 6 months prior to the study, and who had a history of acute respiratory exacerbations but who were free of such in the 3 months prior to the study (which began in the autumn) [Table 1]. For control subjects, we analyzed two age- and sex-matched populations of healthy nonsmoker and ex-smoker subjects. The study of the healthy ex-smoker population ruled out that the immunologic results of the ex-smoker COPD patients might be related to potential residual effects of smoking.

Our results show a clearly defective proliferative response to T-cell–specific activation signals (monoclonal antibodies against CD3 and/or CD28) in COPD patients. This defect in the selective activation of T-cells can neither be restored by adding IL-2 to the medium nor associated to a marked redistribution in any of the major peripheral blood immune cell subsets measured in this study. In agreement with previous results, a proliferative deficiency of PBMCs after lectin stimulation (which has multiple interactions with PBMCs) occurs in COPD and is not normalized even when saturating doses of exogenous IL-2 are administered.28 These data are consistent with the notion that the T-cells of COPD patients have a functional deficiency.82930

IFN-{gamma} and its specific receptor play a pivotal role in the body defense under circumstances of respiratory exposure to microorganism.111231323334 CD4 T-cells cooperate with phagocytic cells to increase to their antimicrobial effector capabilities via the secretion of IFN-{gamma}.3 CD8 T-cells also use IFN-{gamma} as an antiviral effector moiety.353637 The present results show that the percentage of T-cells secreting IFN-{gamma} is reduced in both CD4 and CD8 subsets in COPD patients, compared to nonsmoker and ex-smoker healthy control subjects. However, no difference was seen between either the number of IFN-{gamma} molecules in the cytoplasm of IFN-{gamma}–producing T-lymphocytes from COPD patients and healthy nonsmoker and ex-smoker control subjects, or their IL-4, IL-12p40, and TNF-{alpha} secreted levels. The mechanisms that regulate the signal transduction, transcriptional control of gene expression, and translational and posttranslational control of protein secretion are different for IL-2, IL-4, IL-12p40, IFN-{gamma}, and TNF-{alpha}.3 It allows a room for differential regulation (ie, IFN-{gamma} expression can be promoted by IL-12, but additional cytokines such as IL-18 can also modulate IFN-{gamma} levels).3 Also, the cell sources producing the indicated cytokines, and immune cell recirculation patterns, in response to the stimuli used can differ.3 Selective functional defects in given subpopulations may thus render selective split reduction of IFN-{gamma} production without defect in the secretion IL-2, IL-12p40, and TNF-{alpha}.

The functional systemic defects in circulating immune cells from COPD patients do not need to be superimposable or identical to those occurring in the lung. However, studies have shown that IFN-{gamma} producing CD8 T-lymphocytes are significantly reduced in sputum,38 and a relative deficiency in the serum levels of IFN-{gamma},39 of COPD patients as compared to healthy control subjects. The increased number of T-cells in lung parenchyma and airways of COPD patients with greatest elevation in CD8 cells has been correlated with the amount of alveolar destruction and severity of airflow obstruction. However, the role of T-cells in pathophysiology is not yet certain.40 Acute exacerbation may be prolonged and may have a profound effect on the quality of life.841 It is now evident that many exacerbation in COPD, as in asthma, are due to upper respiratory tract viral infections (such as rhinovirus infection) among and another factors.842 IFN-{gamma} is known to reduce the expression of intracellular adhesion molecule-1, the receptor for airway epithelial cell entry of rhinovirus,43 which provides a novel noncytopathic IFN-{gamma}–mediated antiviral effect.43 The functional heterogeneity of type 1 effector T-lymphocytes in response to pulmonary infection has been related to the potential for IFN-{gamma} expression, with high IFN-{gamma} T-lymphocytes homing preferentially to the lung.37 We show here that AM3 selectively augments IFN-{gamma} expression in CD8 T-lymphocytes, and not CD4 T-lymphocytes, from COPD patients. Our finding is reminiscent of the differential modulation of IFN-{gamma} gene expression in CD4 and CD8 T-cell subsets, and the selective increase of IFN-{gamma} in CD8 T-lymphocytes after forskolin-induced cyclic adenosine monophosphate increase, found by Aune and coworkers44 in an in vitro mouse model. Altogether, the functional recovery of T-cell proliferation, IFN-{gamma} production, natural killer cytotoxic, and phagocytic activities in peripheral blood induced by AM3 treatment in COPD patients16 is consistent with the observed improvement in quality of life of these patients, and a trend toward a reduction in the COPD exacerbation rate.1945 These immunomodulatory effects of AM3 in COPD patients are not associated to induction of secondary effects.161945 Further studies are required to clarify the significance of T-cell compartment systemic defects, AM3 mechanism of action, and the balance between the proposed beneficial and the pathogenic roles of IFN-{gamma} in COPD patients and their potential value as targets for therapy.

Acknowledgements

The placebo and AM3 used in this study were provided by the manufacturer (I. F. Cantabria; Madrid, Spain). The authors thank the Immune System Involvement in Respiratory Disease Research Group (J. Jareño, Hospital Del Aire, Madrid; J. M. Rodriguez, Hospital Universitario Gregorio Marañon, Madrid; M. Calle, Hospital Universitario Clinico San Carlos, Madrid; J.L. Izquierdo, Pulmonary Unit, Hospital General Universitario, Guadalajara; and E. Sanz, Department of Medicine, University of Alcalá, Madrid) for help in developing the study protocol and recruiting patients for inclusion. We also thank Adrian Burton for linguistic assistance.

Footnotes

Abbreviations: IFN = interferon; IL = interleukin; PBMC = peripheral blood mononuclear cell; TNF = tumor necrosis factor

All of the data acquisition and analyses were completed in the Department of Medicine, Alcalá University.

This work was partially supported by grants from the Ministerio de Ciencía y Tecnología (FIT-2003–090000–0105, SAF-2004–08138), the Instituto de Salud Carlos III (PI021909 and GO3/075) and the Chronic Inflammatory Disease Group of Castilla-La Mancha, Spain.

Received for publication March 8, 2005. Accepted for publication September 3, 2005.

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