| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Guest Access | Sign In via User Name/Password
|
|
|
|||||||
Guest Access | Sign In via User Name/Password |
|||||||||
* From the Departments of Immunology and Respiratory Medicine, Division of Immunology, Infection and Inflammation (Drs. McSharry, Chaudhuri, and Thomson and Ms. Fraser), University of Glasgow, Glasgow; Department of Medicine (Dr. Anderson), Crosshouse Hospital, Kilmarnock; Department of Respiratory Medicine (Dr. Bourke), Royal Victoria Infirmary, Newcastle upon Tyne; and Department of Medicine (Dr. Boyd), Stobhill Hospital, Glasgow, UK.
Correspondence to: Charles P. McSharry, PhD, Department of Immunology, Western Infirmary, Glasgow G11 6NT, UK; e-mail: cms4q{at}clinmed.gla.ac.uk
Abstract
Background: Nerve growth factor (NGF) is a neurotrophic cytokine with immunomodulatory activity. NGF contributes to neurogenic inflammation and has been described in asthma and idiopathic pulmonary fibrosis.
Objectives: To identify and quantify NGF in serum and peripheral blood lymphocyte cultures from pigeon fanciers, and to investigate an association with the immune response to inhaled avian antigens, and with symptoms of acute hypersensitivity pneumonitis (HP).
Methods: NGF was quantified and compared with serum IgG antibody against inhaled avian antigens, with serum C-reactive protein (CRP), and with KL-6, a marker of lung interstitial inflammation. These were measured using enzyme-linked immunoassay. Levels were compared with symptom history in 55 pigeon fanciers (26 subjects with acute HP but symptom-free at the time of testing) and 15 subjects with no avian exposure.
Results: Pigeon fanciers had higher-than-normal serum IgG antibody, CRP, and KL-6 levels (p < 0.01 each). These measures were unrelated to HP symptom category; instead, in all pigeon fanciers, the concentrations of CRP and KL-6 correlated with each other and with the antibody titers (p < 0.01 each). Serum NGF levels were normal; however, NGF production by mitogen-activated lymphocytes was higher than normal, and correlated with IgG antibody titer (p < 0.05) and with serum CRP (p < 0.05).
Conclusions: Serum NGF was normal in pigeon fanciers; however, their blood lymphocytes ex vivo synthesized increased NGF in concentrations that correlated with the titer of serum IgG antibody to inhaled avian antigens. These also correlated with CRP and KL-6 levels, suggesting that antigen exposure in seropositive subjects is associated with subclinical inflammation involving coordinated synthesis of neurotrophin and immune mediators.
Key Words: C-reactive protein • hypersensitivity pneumonitis • KL-6 • nerve growth factor
Hypersensitivity pneumonitis (HP) [synonym: extrinsic allergic alveolitis] describes an interstitial and bronchioloalveolar disease of the lungs caused by an immune-mediated hypersensitivity response. It is associated with repeated exposure to a variety of inhaled organic dusts. One common example occurs among pigeon fanciers and is caused by inhaling avian antigens in the dust of pigeon lofts.1 Only a proportion of these subjects become affected, but the factors that determine susceptibility are not completely understood. For example, antigen-specific antibody and lymphocyte sensitization seem to be necessary but not sufficient for the development of HP; therefore, other inflammatory or immunomodulatory factors may contribute to symptoms. Hope-Gill et al2 suggest that functional up-regulation of lung sensory neurones contributes to the pathogenesis and symptoms of idiopathic pulmonary fibrosis. In that context, we examined whether nerve growth factor (NGF) was a likely mediator for acute HP. NGF is the prototype for the neurotrophin family of polypeptides that are essential in the development and survival of certain sympathetic and sensory neurons. NGF is associated with cough2 and hyperalgesia associated with inflammation,345 and these are among the symptoms associated with acute HP.
Investigations show that an increasing range of nonneuronal tissues can produce NGF; most notably immune-related human hematopoietic cells—primarily mast cells6 and T-lymphocytes7—and an increase in both these cell types is typical of the bronchioloalveolar cytology of HP.18 NGF has been found in increased quantities in the serum of patients with asthma910 and has been postulated to contribute to the pathogenesis.111213 Our aims were to identify and quantify NGF in serum and immune cells of pigeon fanciers, and to investigate the association of NGF with immunologic sensitization to avian antigens and with symptoms of acute HP.
Materials and Methods
Study Subjects and Control Group
Four hundred volunteers at a convention of pigeon fanciers completed a questionnaire supervised by respiratory physicians who recorded the nature, frequency, and severity of respiratory and systemic symptoms specifically associated with avian exposure. Individuals were categorized as symptomatic for acute HP if they described the occurrence of at least three systemic and three respiratory symptoms occurring together from 4 to 8 h after avian exposure on at least three occasions within the last 3 months.1415 Twenty-six subjects had symptoms of acute HP (symptomatic category), and 29 similarly exposed but healthy individuals formed the asymptomatic category. The rest were excluded because their symptoms were indeterminate or were due to other medical conditions. Ever-smokers were excluded. A control group of 15 research staff in attendance had no significant avian exposure. All subjects signed an informed consent form and donated an 8-mL blood sample collected in preservative-free heparin. The ethics committee of Stobhill Hospital, Glasgow, approved the study, and signed informed consent was obtained.
Measurement of Immunologic and Inflammatory Mediators
NGF immunoreactivity was measured by enzyme-linked immunoassay (EIA) [Promega UK Ltd; Southampton, UK]. The performance characteristics were coefficient of variation < 4.0% with sensitivity of 16 pg/mL. Assay performance was validated by demonstrating full recovery of biologically active exogenous ß-NGF (Sigma Ltd; Poole, UK) added to serum samples.
Serum IgG antibody activity against pigeon antigen was quantified by an EIA developed in this laboratory.16 Pigeon serum, which is the cleanest and most complete source of antigen, was used at 5 µg/mL in the EIA, and this showed no cross-reactivity with dietary egg antigens. Total IgE was measured by commercial EIA (UniCAP; Pharmacia UK Ltd; Milton Keynes, UK). C-reactive protein (CRP) was measured by commercial EIA (
Diagnostic International; Autogen Bioclear; Wiltshire, UK). KL-6, a high-molecular-weight sialoglycoprotein derived from proliferating alveolar type 2 epithelial cells and raised levels in serum reflects lung inflammation in interstitial lung disease,17 was measured by EIA kindly provided by Sanko Junyaku Co. Ltd., Tokyo, Japan.
Total WBC Count and Peripheral Blood Lymphocyte Phenotype and Function
WBCs were counted automatically (Coulter CBC5; Coulter Electronics; Hialeah, FL), and the lymphocyte and T-cell subset differential count was established using fluorochrome-labeled antibodies CD14-phycoerythrin and CD45-fluorescein isothiocyanate, and anti-CD3, CD4, CD8, CD19, and CD16/56 (Sigma Ltd) and flow cytometry (FACScan; Becton Dickenson UK Ltd; Abingdon, UK). Lymphocyte cultures were prepared using 20% volume/volume heparinized blood with Iscove medium containing 50 U/mL of penicillin and 50 µg/mL of streptomycin (Gibco Ltd; Paisley, UK). Cells were incubated with or without phytohemagglutinin at 5 µg/mL for 3 days. The proliferative responses were measured by [3H] thymidine incorporation (15 kilobecquerel for the last 6 h) and expressed for each subject as counts per minute (cpm). All blood samples were collected at the same time and processed within 36 h of phlebotomy, and all cultures and serologic tests were set up at the same time and under identical conditions to minimize variation.
Statistical Analysis
Data were analyzed using statistical software (Minitab v.14; Minitab Ltd; Coventry, UK). Variables with a skewed distribution were summarized by median and interquartile range (IQR). Mann-Whitney U test was used for between-group comparisons and correlations by Spearman 
, after log transformation. The comparison between NGF and symptoms was made by cluster analysis. Statistical significance was accepted at the 95% level.
Results
Age and Indexes of Avian Antigen Exposure
Twenty-six pigeon fanciers described a symptom profile consistent with acute HP specifically associated with avian contact, which resolved between episodes. Patients described individual symptoms as chest tightness (n = 22), shivering (n = 18), rhinitis (n = 18), dry cough (n = 18), sweating (n = 17), fever (n = 16), myalgia (n = 15), wheeze (n = 15), tiredness (n = 14), and conjunctivitis (n = 9). Each subject reported a combination of symptoms (median, 9; IQR, 6–10). There was no difference in the age distribution between pigeon fanciers and control subjects, and no difference in avian exposure between those pigeon fanciers with and without symptoms of acute HP (Table 1
).
|
In the study group of pigeon fanciers as a whole, there were significant quantitative correlations between the indexes of specific and nonspecific inflammation. The IgG antibody titer correlated with CRP (r = 0.36, p < 0.01) and KL-6 concentrations (r = 0.38, p < 0.01). The CRP and KL-6 concentrations also correlated (r = 0.35, p < 0.01). The total serum IgE level was significantly lower in the pigeon fanciers than control subjects (median, 61 IU/mL [IQR, 14–134] and 134 IU/mL [IQR, 64–636], respectively; p = 0.026), but there was no difference in the concentration between pigeon fanciers with or without symptoms of acute HP.
Total WBC Count, Lymphocyte Phenotype, and Function
There were no significant differences in the peripheral blood WBC count and in the absolute counts and relative proportions of the major (T-cell, B-cell, and natural killer) and minor (CD4+ T-helper and CD8+ T-cytotoxic) lymphocyte populations between the different groups, and all were within the normal range. The in vitro proliferative response to phytohemagglutinin (PHA) mitogen was significantly lower in the pigeon fanciers than normal subjects (median, 29.1 cpm x 103 [IQR, 16.0–48.4] and 42.5 cpm x 103 [IQR, 22.7–88.2], respectively; p < 0.01), but was not different between pigeon fanciers with or without acute HP (median, 28.3 cpm x 103 [IQR,18.3–45.0] and 29.4 cpm x 103 [IQR, 16.0–56.4]; p = not significant). The lymphocyte proliferation was inversely correlated with the titer of serum IgG antibody against avian antigen (r = – 0.31, p = 0.012).
NGF
The serum NGF concentration in pigeon fanciers was not different from control subjects (median, 37.0 pg/mL [IQR, 27.6–46.8] and 40.4 pg/mL [IQR, 28.8–64.4], respectively; p = 0.72). Nor was there any difference between serum NGF concentration in pigeon fanciers with or without symptoms of acute HP (median, 31.7 pg/mL [IQR, 27.0–48.8] and 39.4 pg/mL [IQR, 30.3–45.2], respectively; p = 0.51).
The amount of NGF produced by lymphocytes in vitro either constitutively or after mitogen stimulation is shown in Figure 1 . The constitutive (p = 0.005) or stimulated levels (p = 0.047) were significantly higher than normal in the pigeon fanciers. The mean stimulation index of NGF production in vitro (inducible/constitutive) for the pigeon fanciers was significantly higher than in control subjects (mean, 51.7 [SD 41.3] vs mean, 29.2 [SD 21.8], p = 0.02); however, there were no significant differences between the amounts of lymphocyte NGF produced by pigeon fanciers with or without symptoms of acute HP, nor was there any association between the NGF concentration and any individual symptom or symptom cluster.
|
|
Discussion
We have demonstrated that the constitutive and mitogen-stimulated NGF synthesis by peripheral blood lymphocytes from pigeon fanciers was higher than in normal control subjects, and that the concentration of NGF correlated with serum IgG antibody against inhaled avian antigens, and with serum CRP, suggesting that immune sensitization to inhaled avian antigen and systemic inflammation was associated with neurotrophin production. Despite this, the serum NGF concentration in all pigeon fanciers was normal. Serum NGF concentration in other conditions such as asthma,910 systemic lupus erythematosus,18 and Kawasaki disease,19 is higher than normal during active disease, whereas at the time of phlebotomy the pigeon fanciers in the present study were symptom free.
Lymphocytes are known to synthesis NGF constitutively in vitro,7 and the spontaneous release of NGF by lymphocytes from pigeon fanciers was higher than normal, and was negatively associated with serum NGF. One explanation for this might be that the lymphocytes had been deactivated in vivo, and had lower residual synthetic capacity for this mediator ex vivo. Alternatively, the different conditions prevailing in culture that resulted in altered NGF synthesis might be due to an inhibitory or modulatory serum factor. We plan to trace this experimentally by using different proportions of control and patients serum in vitro.
The mitogen-induced production of NGF by lymphocytes in vitro was also higher than normal and was quantitatively related to the serum levels of IgG antibody and CRP. Despite the overlap with normal levels, the absolute concentration of lymphocyte-derived NGF correlated with the other indexes suggesting a coordinated immune, inflammatory and neurotrophin response.
Despite the close association of these responses, none of these indexes reflected symptom category. One explanation for this might be that the subjects were symptom free when the blood samples were taken. However, the raised CRP and KL-6 levels suggested that there was subclinical inflammation that was independent of symptoms. There is a precedent for the separation of symptoms and physiologic responses; we have previously identified increased pulmonary clearance of inhaled 99mTc-diethylenetriamine penta-acetic acid in pigeon fanciers, and this occurred in subjects who were seropositive to avian antigen rather than in those with symptoms of acute HP.20 This is an important consideration because HP commonly presents in a chronic form, and identification of subclinical changes may be an important early indicator of disease.1
Confirmation of subclinical changes using radiology, scans, and lung function would have been very useful but was not feasible because of the number of pigeon fanciers screened and the location where the study took place. However, the observations from this cross-sectional study merit confirmation by a prospective longitudinal study using the pigeon fanciers initially selected as above, followed up with appropriate lung function and scans to identify acute, subacute, and chronic categories of disease. Sampling of the airway neurotrophin levels in induced sputum would be a less invasive alternative to BAL and be applicable both before and after natural exposure or laboratory challenge.
In asthma, the concentrations of NGF in serum and in lung lavage fluid were higher than normal, correlated with total IgE, and reflected disease severity.1112 In the present study, we were careful to exclude subjects with symptoms of asthma, and this was partly confirmed by the IgE levels being lower than normal. IgE did not correlate with any measure of NGF. Lymphocytes721 and mast cells22 can both produce and respond to NGF. HP is characterized by an intense lymphocytic and mast cell infiltrate1; therefore, this indicates a potential route between NGF and airway inflammation, and places NGF centrally in an autocrine amplification loop of inflammation in HP.
We have shown that NGF production by lymphocytes increased in parallel with serum CRP and KL-6, and all were quantitatively related to the antibody response to the inhaled antigens in pigeon fanciers. This profile of inflammatory mediators was not uniquely a feature of those with symptoms of acute HP, and suggested subclinical inflammation in those with no symptom history. This is important because HP commonly presents in chronic form, and those individuals with raised NGF and inflammatory markers should be monitored. Further clarification of the role of neurotrophins in conjunction with other inflammatory or fibrogenic mediators may identify new pathways for therapeutic intervention.
Footnotes
Abbreviations: cpm = counts per minute; CRP = C-reactive protein; EIA = enzyme-linked immunoassay; HP = hypersensitivity pneumonitis; IQR = interquartile range; NGF = nerve growth factor; PHA = phytohemagglutinin
The authors have no conflict of interest financial or otherwise regarding this manuscript.
Received for publication October 10, 2005. Accepted for publication January 3, 2006.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
