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* From the Clinical Research Branch, Human Studies Division, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, NC.
Correspondence to: Yuh-Chin T. Huang, MD, MHS, FCCP, Campus Box 7315, Human Studies Division, US EPA, 104 Mason Farm Rd, Chapel Hill, NC 27599-7315; e-mail: huang.tony{at}epa.gov
Abstract
Study objectives: Bronchoscopy with BAL is being used increasingly in the investigation of acute and chronic lung inflammation. The scope of the acute phase response induced by the procedure is not fully evaluated. The purpose of the study is to characterize the acute phase response induced by bronchoscopy with BAL.
Design: Observational study.
Setting: A human study research facility.
Participants: Normal nonsmoking volunteers.
Intervention: A total of 28 subjects were recruited. Under local anesthesia, the subjects underwent bronchoscopy with a videofiberoptic bronchoscope. One subsegment of the lingular segment of the left upper lobe and the right middle lobe were lavaged each with 170 to 270 mL of sterile normal saline solution.
Measurements and results: CBC count, serum levels of indexes of iron homeostasis, fibrinogen, C-reactive protein (CRP), and plasma mediators related to neutrophil migration and endothelial cell activation, including interleukin (IL)-8, angiotensin converting enzyme (ACE), soluble intercellular adhesion molecule (sICAM)-1, and nitrite/nitrate, were measured. Measurements of these plasma markers were done immediately before, immediately after, and 24 h after bronchoscopy. Changes in acute phase response were detected primarily at 24 h after the procedure. WBCs, primarily neutrophils, increased by approximately 50%. Fibrinogen increased by 25% while CRP increased by more than sevenfold. Serum ferritin increased by 25% while serum iron, total iron-binding capacity, and transferrin saturation decreased, indicating dysregulation of iron homeostasis. There were no changes in IL-8, ACE, sICAM-1, or nitrite/nitrate plasma levels.
Conclusions: Bronchoscopy with BAL induces a variety of acute phase responses that includes peripheral neutrophilia, dysregulation of iron homeostasis, and increased levels of fibrinogen and CRP. Human research that employs BAL may need to consider the biological effects induced by the procedure-related acute phase response.
Key Words: C-reactive protein • fibrinogen • inflammation • inflammation mediators • interleukin
The acute phase response is a syndrome of acute and chronic inflammation accompanied by systemic metabolic and neuroendocrine changes.1 These changes can include fever, sleep, adrenocorticotropin and cortisol release, B-cell and T-cell activation, and altered hepatic synthesis of a number of plasma proteins, the acute phase proteins. The acute phase response is considered to be an adaptive process allowing the progression of inflammation so that optimal healing occurs, although the precise role of each reactant in the processes has not been determined. The acute phase response is observed following a wide variety of injuries, including infections, neoplasms, arthritides, acute trauma, infarction, surgery, burns, and heatstroke. Normal physiologic processes such as exercise and pregnancy have also been demonstrated to elicit an acute phase response.
Exposure to ambient air pollution particles can be associated with changes in acute phase reactants. Inhalation of elevated concentrations of particulate matter increases the plasma level of both C-reactive protein (CRP)2 and fibrinogen.234 Elevations in WBC counts have also been described.25 Abnormalities in these end points support an inflammatory response to ambient air pollution particles, which has been described in the lower respiratory tract using BAL.3
Bronchoscopy with BAL has been associated with recruitment of lung neutrophils and increases in plasma levels of neutrophil chemoattractants, interleukin (IL)-6, and granulocyte-colony stimulating factor.6 To fully understand the lung inflammation and the significance of acute phase reactants induced by ambient air pollution particles, it is necessary to better characterize the various aspects of acute phase response induced by bronchoscopy with lavage. In this study, the acute phase response was assessed in a group of healthy, nonsmoking volunteers before, immediately after, and 24 h after bronchoscopy with BAL. The parameters of acute phase response measured included changes in blood counts, iron homeostasis, and a variety of acute phase proteins and inflammatory mediators related to neutrophil trafficking and endothelial cell activation.
Materials and Methods
Study Population
Volunteers responding to a newspaper advertisement were prescreened over the telephone using the following criteria: age between 18 and 40 years; nonsmokers for at least 5 years prior to study; no history of allergies or respiratory diseases (food allergy, hay fever, dust allergies, rhinitis, asthma, chronic bronchitis, COPD, tuberculosis, hemoptysis, or recurrent pneumonia); and not presently receiving any medication prescribed by a physician (except birth control pills). A urine pregnancy test was performed on all female subjects, and a positive result excluded the subject from further participation in this study.
Prior to participation in the study, subjects were informed of the procedures and potential risks, and each signed a statement of informed consent. The protocol and consent form were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. The screening procedures for each subject included a medical history, physical examination, and routine hematologic and biochemical tests. Two subject populations were studied. The subject population was recruited between August 2003 and December 2004.
Bronchoscopy
Using a standard protocol,7 we performed bronchoscopy with lavage using a fiberoptic bronchoscope with an outer diameter of 4.8 mm (BF-P160; Olympus America; Melville, NY). The subject was administered lidocaine by aerosolization and nasal gel before bronchoscopy, and by endobronchial instillation during the procedure. Some subjects were also premedicated with atropine (0.4 mg IV). The bronchoscope was wedged into a segmental bronchus of the lingula. Between four and six aliquots of sterile saline solution (170 to 270 mL) were instilled and immediately aspirated. The first aliquot was 20 mL, and this fraction was labeled the bronchial lavage sample. The remaining three to five aliquots were 50 mL each and were designated the alveolar samples. The procedure was repeated on the right middle lobe using 170 to 270 mL of saline solution.
Venipuncture and Assays
Venous blood was sampled from an antecubital site immediately before, 1 to 2 h after, and 24 h after the procedure. Blood cell values included hemoglobin, platelet count, WBC count, and the percentage of neutrophils and lymphocytes. These values were quantified by cell impedance using an automated hematology system (Model Gen-S; Coulter; Miami, FL). Fibrinogen levels were determined using a centrifugal chemical analyzer (F. Hoffmann-La Roche Ltd; Basel, Switzerland). CRP was quantified using a latex immunoturbidometric assay on a chemistry analyzer (Roche Integra 400; Roche Diagnostics Corporation; Indianapolis, IN). Iron and total iron-binding capacity (TIBC) were measured by a colorimetric assay (Roche Modular Analyzer; Roche Diagnostics Corporation). Transferrin and ferritin were quantified using immunoprecipitin analysis and immunochemiluminometric assays, respectively (Roche Integra 400). Interleukin (IL)-8, angiotensin-converting enzyme (ACE), and soluble intercellular adhesion molecule (sICAM)-1 concentrations in the blood were measured using commercially available enzyme-linked immunosorbent assay kits (R&D Systems; Minneapolis, MN). Nitrite and nitrate were measured using a colorimetric Greiss reaction kit (R&D Systems).
Statistical Analysis
Data are expressed as mean ± SD except where specified otherwise. The two-tailed paired t test was used to evaluate for significant changes immediately after and 24 h after the procedure, adjusted for multiple comparisons using the Bonferroni correction.
To determine acute phase reactants associated with changes in WBC count, stepwise multiple linear regression was used to correlate changes in WBC count after the procedure (dependent variable) with changes in CRP, fibrinogen, iron, ferritin, and iron saturation (independent variables). Only linear terms of the independent variables were considered. Both R2 and forward elimination (exit 
of 0.10) model selection procedures were used to screen the independent variables for significant associations with the dependent variable. The R2 values are adjusted for the number of parameters in the model to overcome the objection to the traditional R2, that the value can be driven to one simply by adding superfluous variables to the model with no improvement in fit. The general goal of this analysis was to identify independent variables showing significant association with the dependent variable. All statistical analysis was conducted using software (StatView for Windows, version 5.0.1; SAS Institute; Cary, NC). Significance was assumed at p < 0.05.
Results
Study Population
The population included 28 volunteers (14 men and 14 women; mean age, 24.8 ± 4.8 years). The average height was 169.4 ± 9.2 cm, and the average weight was 74.3 ± 14.1 kg. The average duration of bronchoscopy was 19.3 ± 3.1 min. The amount of saline solution removed during BAL was approximately 70% of the injected volume.
Changes in Blood Cell Values
Hemoglobin, platelet count, WBC counts, percentage of neutrophils, and percentage of lymphocytes before, immediately after, and 24 h after bronchoscopy were measured in 15 subjects, and the results are summarized in Table 1
. Relative to the value obtained prior to the procedure, there were small but statistically significant decreases in hemoglobin and platelet counts immediately after bronchoscopy with BAL but not 24 h after the procedure. In contrast, the WBC count and the percentage of neutrophils in the blood showed no immediate change following the bronchoscopy, but both were significantly increased 24 h later. The percentage of lymphocytes was not affected by bronchoscopy with BAL.
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Discussion
Characteristic features of the acute phase response include fever; neutrophilia; changes in lipid metabolism; hypoferremia and hypozincemia; increased gluconeogenesis; increased muscle protein catabolism and transfer of amino acids from muscle to liver; activation of the complement and coagulation pathways; hormonal changes; and induction of acute phase proteins.8 An acute phase protein is defined as a protein with a plasma concentration either increased or decreased by at least 25% in the first 7 days following tissue damage, and there are > 30 of such proteins identified.9 This group of acute phase proteins includes those that increase with inflammation (CRP, serum amyloid A protein, 2-macroglobulin, 1-antitrypsin, fibrinogen, haptoglobin, hemopexin, ceruloplasmin, and complement C3) and those that decrease (albumin and transferrin). The concentrations of these proteins have been shown to correlate with the severity of tissue injury. Discordance between concentrations of different acute phase proteins is common. The acute phase proteins showing the greatest increase and the most rapid rise following inflammation are CRP and serum amyloid A.9 Both may increase in relatively minor inflammation, such as the common cold and with exercise or smoking.10
A systemic inflammatory response is reported to occur following bronchoscopy with lavage in both healthy volunteers and patients.6 This has included fever, tachycardia, tachypnea, and leukocytosis 4 to 24 h after the procedure. Fever has been reported in 13%11 to > 30%12 of individuals undergoing bronchoscopy with lavage. Both the WBC count and blood neutrophils can be elevated in patients having the procedure.6 It has been our experience that fever is a very rare event following bronchoscopy with BAL in healthy subjects (< 1%). Based on the small cohort in this study, however, an acute phase response was likely quite prevalent and diverse following bronchoscopy with BAL. These responses included increases in WBC count, neutrophils, CRP, fibrinogen, and altered indexes of iron homeostasis mainly at 24 h following bronchoscopy with BAL. Our results confirmed and expanded the scope of acute phase response induced by bronchoscopy with BAL in healthy subjects reported by a previous study.6
The acute phase response is associated with cytokine release. These cytokines originate from a variety of cells, but macrophages and T-cells are the major sources. The IL-6 family of cytokines is considered to be the major inducer of most acute phase proteins.111213 Other cytokines that contribute to the systemic response, but deemed subsidiary roles, include tumor necrosis factor and IL-1ß.14 Serum concentrations of IL-8, a potent neutrophil chemotactic factor, however, showed no changes in our cohort and a group of healthy subjects in a previous study.6 Other inflammatory mediators that are related to neutrophil migration and endothelial cell activation, including ACE, sICAM-1, and nitrite/nitrate, also did not change. Changes in the WBC and neutrophil counts correlate with serum concentrations of IL-6 and granulocyte-colony stimulating factor,6 and our stepwise regression analysis identified increases in CRP as the most significant factor associated with increases in WBC. These latter results indicate that these two acute phase responses (increases in neutrophil and CRP) may share common mechanisms.
Several indexes of iron metabolism also demonstrated significant changes following bronchoscopy with BAL including iron, saturation, TIBC, and ferritin. Decrements in iron, saturation, and TIBC have been previously described in the acute phase response.115 Ferritin has also been observed to increase in numerous injuries and exposures.116 The hypoferremia shown in the acute phase response has been considered to help host defense against invading microorganisms by depleting the metal required for microbial proliferation. The utility of such an acute phase reaction following noninfective stimuli (including bronchoscopy with BAL) is not clear.
We conclude that bronchoscopy with BAL induced a wide range of acute phase responses. This response includes changes in WBC count, especially neutrophils, acute phase proteins, and indexes of iron homeostasis. The magnitude of the acute phase response induced by bronchoscopy with BAL is much greater than that seen with long-term air pollution and other noninfectious stimuli.234 Human studies that include bronchoscopy with BAL as an intervention may need to consider the potential biological effects induced by the acute phase response associated with the procedure.
Footnotes
Abbreviations: ACE = angiotensin-converting enzyme; CRP = C-reactive protein; IL = interleukin; sICAM = soluble intercellular adhesion molecule; TIBC = total iron-binding capacity
This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Received for publication June 16, 2005. Accepted for publication December 9, 2005.
References
after bronchoscopy and bronchoalveolar lavage. Chest 1991;99,1529-1530
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