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(Chest. 2002;121:519-526.)
© 2002 American College of Chest Physicians

Elevated Levels of {alpha}-Defensins in Plasma and BAL Fluid of Patients With Active Pulmonary Tuberculosis*

Jun-ichi Ashitani, MD; Hiroshi Mukae, MD; Takeaki Hiratsuka, MD; Masamitsu Nakazato, MD; Kenshi Kumamoto, MD and Shigeru Matsukura, MD

* From the National Sanatorium Miyazakihigashi Hospital (Drs. Ashitani and Kumamoto), and Third Department of Internal Medicine (Drs. Mukae, Hiratsuka, Nakazato, and Matsukura), Miyazaki Medical College, Miyazaki, Japan.

Correspondence to: Hiroshi Mukae, MD, Second Department of Internal Medicine, Nagasaki University School of Medicine, Sakamoto 1–7-1, Nagasaki, Nagasaki 852-8501, Japan; e-mail: hmukae{at}post.miyazaki-med.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: To investigate the role of neutrophil peptides named {alpha}-defensins in patients with pulmonary tuberculosis (TB).

Patients: Thirty-seven patients with TB and 25 healthy subjects.

Measurements and results: Concentrations of {alpha}-defensins (human neutrophil peptide [HNP]-1, HNP-2, and HNP-3) were measured by radioimmunoassay in plasma and BAL fluid (BALF). Concentrations of {alpha}-defensins were significantly higher in plasma and BALF of patients with TB than in healthy subjects. In BALF of patients with TB, the concentration of {alpha}-defensins correlated positively with the levels of interleukin 8, and higher concentrations of {alpha}-defensins in BALF were also detected in patients with cavitary lesions. There was an inverse relationship between plasma {alpha}-defensins and FEV1/FVC ratio before treatment, and between plasma concentrations of {alpha}-defensins before treatment and the improvement in percentage of vital capacity after treatment. Plasma {alpha}-defensin concentrations returned to the normal range after treatment.

Conclusion: Our data suggest that {alpha}-defensins released from neutrophils may play an important role in the pathogenesis of TB, and that plasma {alpha}-defensin concentration may be a useful marker of disease severity and deterioration of pulmonary function.

Key Words: {alpha}-defensins • BAL fluid • human neutrophil peptides • pulmonary tuberculosis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Accumulating evidence suggests that the host defense against Mycobacterium tuberculosis depends mainly on effective interactions between macrophages and T lymphocytes.1 Neutrophils may also contribute to the control of M tuberculosis because they exert a bactericidal activity against M tuberculosis.2 3 4 In addition, neutrophils stimulated by M tuberculosis release an array of cytokines and chemokines that attract other inflammatory cells.5 However, superoxides and neutrophil granule proteins released from neutrophils might be associated with lung injury in patients with pulmonary tuberculosis (TB). In this context, Barnes and colleagues6 demonstrated that a high percentage of neutrophils on the differential WBC count is most strongly associated with an unfavorable short-term outcome (respiratory failure or death) in patients with TB. It is also known that patients with advanced TB have a higher percentage of neutrophils in BAL fluid (BALF).7 8 9 However, less is known about the role of neutrophils in the pathogenesis of TB so far compared to that of macrophages and T lymphocytes.

{alpha}-Defensins are cationic proteins with antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and enveloped viruses.10 11 In addition, studies3 4 have described a similar activity for {alpha}-defensin against M tuberculosis. Six {alpha}-defensins have so far been recognized in humans, including four forms that are exclusive to neutrophils (human neutrophil peptide [HNP]-1, HNP-2, HNP-3, and HNP-4) and two others found in Paneth cells in the small intestine.10 11 Because HNP-1, HNP-2, and HNP-3 constitute 5 to 7% of the total protein content of the human neutrophil and 30 to 50% of the total protein content of the azurophilic granules, it is thought that they are the most abundant antimicrobial proteins present in the neutrophil. In contrast, HNP-4, which has only 32% amino-acid sequence homology with other HNPs, shows anticorticotropin activity and is less potent than HNP-1, HNP-2, and HNP-3.10 11 In fact, work from our laboratory as well as others have previously demonstrated the presence of elevated levels of {alpha}-defensins in plasma and body fluids in patients with various infections.12 13 Moreover, through their cytotoxic effects on human lung cells,14 15 {alpha}-defensins are thought to be related to various neutrophil-dominated inflammatory disorders, such as cystic fibrosis, diffuse panbronchiolitis, and ARDS.15 16 17 In the present study, we determined {alpha}-defensin levels in plasma and BALF of patients with TB to clarify their role in TB.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
We studied 37 consecutive patients with active TB who were admitted to our hospital between July 1998 and November 1999. They consisted of 12 women and 25 men, aged 49 ± 3 years (mean ± SEM). Fifteen smokers, 5 ex-smokers, and 17 persons who never smoked were included. TB was diagnosed by chest radiography, clinical pictures, and at least one positive sputum smear finding for acid-fast bacilli and a positive sputum culture finding for M tuberculosis. Patients with a history of other lung diseases, recurrent TB, miliary tuberculosis, extrapulmonary tuberculosis, or coinfection with HIV were exclude from this study. Furthermore, patients with suspicious bacterial infection in sputum, stool, blood, or BALF samples were also excluded from the study. Laboratory data of the patients are summarized in Table 1 . All subjects were treated daily for 2 months with a standard four-antituberculosis-drug regimen of isoniazid (400 mg/d), rifampicin (450 mg/d), ethambutol (750 mg/d), and pyrazinamide (1.0 g/d), followed for additional 4 months of treatment with daily isoniazid and rifampicin. We also selected 25 age-matched or sex-matched healthy volunteers as control subjects (8 smokers, 2 ex-smokers, and 15 never smokers). All normal volunteers had normal chest radiographic findings, were free of symptoms, and were not receiving any medications. For each subject, blood samples were obtained at baseline (normal subjects) and before the commencement of antituberculous chemotherapy (for patients). Plasma sampling was repeated after 6 months of treatment.


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  		Table 1.. Effect of Treatment on Laboratory Data in Patients With TB*

 
Chest Radiography and CT Scan
Before treatment, chest radiography and CT were performed in all patients. Patients were classified into two groups according to the presence or absence of a cavitary lesion based on the findings of CT scan. In addition, chest radiographic findings were also scored according to the extent of the lesions as recommended by the Japan Society of Chemotherapy. Scoring was done by chest radiologists as follows: 0, no abnormal shadow; 1, shadow in expanse less than one intercostal space; 2, shadow in expanse between 1 and 3; 3, shadow in expanse of one tenth of one-sided lung; 4, shadow in expanse between 3 and 5; 5, shadow in expanse of one third of one-sided lung; 6, shadow in expanse between 5 and 7; 7, shadow in expanse of two thirds of one-sided lung; 8, shadow in all one-sided lung; 9, shadow in expanse between 8 and 10; and 10, shadow spread in both lungs. This scoring system was also used for chest radiographs obtained after the 6-month treatment protocol.

BAL
With informed consent, BAL was performed as previously described16 18 using a flexible fiberoptic bronchoscope (Olympus P-20; Olympus; Tokyo, Japan) in 12 of 37 patients with TB before treatment (5 smokers, 1 ex-smoker, and 6 never smokers) and 10 of 25 healthy subjects (3 smokers and 7 nonsmokers). Briefly, the bronchoscope was wedged into one of the segmental or subsegmental bronchi of the most heavily involved lobe, as seen on the chest CT. Then, 50 mL of sterilized saline solution at body temperature was instilled through the bronchoscope. The fluid was immediately retrieved by gentle suction using a sterile syringe, and the procedure was repeated three times. BALF was passed through two sheets of gauze and then centrifuged at 500g for 10 min at 4°C. After washing twice with phosphate-buffered saline solution free of calcium and magnesium (Gibco; Paisley, United Kingdom), the remaining cells were suspended in phosphate-buffered saline solution supplemented with 10% heat-inactivated fetal calf serum and were counted using a hemocytometer. An aliquot was then diluted to a concentration of 2 x 105 cells/mL, and a 0.2-mL cell suspension was spun down onto a glass slide at 1,100 revolutions per minute for 2 min using a cytocentrifuge (Cytospin 2; Shandon Instruments; Sewickley, PA). The remaining fluid was centrifuged at 500g for 5 min, and supernatant was stored at - 80°C until examined. The slides were dried, fixed, and then stained using a May-Giemsa method. More than 200 cells were identified using a photomicroscope.

Pulmonary Function Tests
Routine spirometry was performed in accordance with recommended standards.19 FVC and FEV1 were obtained from maximal expiratory flow-volume curves (Masterscreen; Jaeger; Breda, the Netherlands). The highest value from at least three spirometric maneuvers was used. Values are expressed as percentage of predicted values. Pulmonary function tests were repeated 6 months after treatment. Based on the results of pulmonary function tests, we calculated the difference in percentage of vital capacity (%VC) before and after treatment, where %VC = %VC after treatment -%VC before treatment.

Measurement of {alpha}-Defensins and Interleukin-8
The concentrations of {alpha}-defensins in plasma and BALF samples were measured by radioimmunoassay (RIA) established by our laboratory.20 We synthesized full-length HNP-1 using a peptide synthesizer (Model 430; Applied Biosystems; Foster City, CA), then purified by reverse-phase, high-performance liquid chromatography (RP-HPLC). In RP-HPLC, synthetic HNP-1 was eluted at a position identical to that of native HNP-1 isolated from human leukocytes. Synthetic HNP-1 was used for immunizing New Zealand white rabbits by multiple intracutaneous and subcutaneous injections. HNP-1 was radio-iodinated and the 125I-labeled peptide was purified by RP-HPLC on a TSK ODS 120A column (Tosoh Company; Tokyo, Japan). A diluted sample or standard peptide solution (100 µL) was incubated for 24 h with 100 µL of antiserum diluent (final dilution of 1/21,000). The 125I-labeled HNP-1 solution (16,000 cpm in 100 µL) was added, and the mixture was incubated again for another 24 h. In the next step, normal rabbit serum and antirabbit IgG goat serum was added and stored for 16 h. Bound and free ligands were separated by centrifugation. All procedures were performed at 4°C, and duplicate assays were performed. We used 0.5 µL of plasma and 1 to 10 mL of BALF to determine the levels of {alpha}-defensins. The antiserum equally recognized HNP-1, HNP-2, and HNP-3 on a molar basis; thus, the RIA data were expressed as the sum of HNP-1, HNP-2, and HNP-3 and their precursor proteins (pro-defensins), the presence of which were confirmed by simultaneous measurements using RP-HPLC and RIA.21 The intra-assay and interassay coefficients of variation were 3.5% and 8% at 50% binding, respectively. The concentration of interleukin (IL)-8 was measured by a commercially available kit (Toray Fuji Bionics; Tokyo, Japan).

Chromatographic Characterization of {alpha}-Defensins
Samples of plasma, whole-blood sample, and BALF were prepared from TB patients. RP-HPLC was performed using a 10-µL whole-blood sample and 100 µL of plasma or BALF on a TSK ODS SIL 120A column (Tosoh Company). A linear gradient of acetonitrile (CH3CN) from 10 to 60% in 0.1% trifluoroacetic acid (pH 2.0) was used at a flow rate of 0.5 mL/min for 40 min. All fractions of {alpha}-defensins were measured by RIA.

Statistical Analysis
Data were expressed as mean ± SEM. Differences between groups were examined using the Mann-Whitney U test. Correlations between two groups were determined using the Spearman’s rank correlation analysis. For comparison of plasma {alpha}-defensin levels measured before and after treatment, a paired-samples Wilcoxon test was used to determine statistical significance. A p value < 0.05 denoted the presence of a statistically significant difference.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pulmonary Function, and Radiographic and BALF Findings
All TB patients responded favorably to therapy, and their sputum findings were 100% culture negative after treatment. The numbers of circulating WBCs, neutrophils, and band cells (Table 1) were higher in TB patients before treatment compared with control subjects (WBC count, 5,500 ± 420/µL, p < 0.01; neutrophils, 2,610 ± 90/µL, p < 0.01; and band cells, 274 ± 17/µL, p < 0.01). These counts decreased significantly after treatment in TB patients (Table 1) . Erythrocyte sedimentation rate and C-reactive protein levels also decreased after treatment. There were no significant differences between %VC or FEV1/FVC ratio before and after treatment. However, pulmonary function worsened in some patients (11 of 37 patients [29.7%] in %VC, and 17 of 37 patients (45.9%) in FEV1/FVC ratio) during the period of therapy (Fig 1 , top, A). Before treatment, the radiographic score (see "Materials and Methods") was 4.3 ± 0.4 (range, 1 to 10) and cavitary formation was seen in 15 of 37 patients with TB by chest CT analysis. The score before treatment correlated positively with the Gaffky number (r = 0.43, p < 0.01), but did not with lung function (data not shown). The score decreased to 2.2 ± 0.5 after treatment (p < 0.01). The total number of cells recovered in BALF was significantly higher in TB patients compared with control subjects (Table 2 ), as were the differential cell counts and numbers of neutrophils and lymphocytes (Table 2) .



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  		Figure 1.. Top, A: Effect of treatment on %VC predicted in TB patients (n = 37). %VC worsened in 11 of 37 patients (29.7%). Bottom, B: Plasma levels of {alpha}-defensins in healthy subjects (n = 25) and patients with active TB (n = 37) before and after 6 months of treatment. Data represent mean ± SEM.

 

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  		Table 2.. Cell Counts in BALF in Patients With TB*

 
Plasma and BALF Levels of {alpha}-Defensins
Before treatment, plasma concentrations of {alpha}-defensins were higher in patients with TB (437 ± 38 ng/mL) than in control subjects (230 ± 28 ng/mL, p < 0.005), and returned to the normal range after treatment (245 ± 57 ng/mL; Fig 1 , bottom, B). Plasma concentrations of {alpha}-defensin correlated negatively with FEV1/FVC ratio in patients with TB before treatment (r = - 0.65, p < 0.0001; Fig 2 , top, A), but not with WBC and neutrophil counts in peripheral blood, radiographic score, or other pulmonary function tests (data not shown). There was no difference in plasma concentrations of {alpha}-defensins between patients with a cavitary lesion (377.5 ± 56.7 ng/mL) and those without a cavitary lesion (477.7 ± 49.2 ng/mL). In addition, there was a significant negative correlation between plasma concentrations of {alpha}-defensins before treatment and %VC (r = - 0.66, p < 0.0001; Fig 2 , bottom, B).



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  		Figure 2.. Top, A: Correlation between plasma levels of {alpha}-defensins and FEV1/FVC ratio before treatment in TB patients. Bottom, B: Correlation between plasma levels of {alpha}-defensins before treatment and %VC (%VC after treatment - %VC before treatment) in patients with TB.

 
BALF {alpha}-defensins concentrations before treatment (1,245 ± 314 ng/mL) were also higher than those in control subjects (14 ± 4 ng/mL, p < 0.0001). There was no correlation between plasma and BALF {alpha}-defensin concentrations in TB patients (data not shown). Six of 12 patients showed a cavity on the chest CT. High concentrations of BALF {alpha}-defensins were detected in patients with cavitary lesions (1,992 ± 422 ng/mL) than those without cavitary lesions (498 ± 179 ng/mL, p < 0.01), but BALF {alpha}-defensins concentrations before treatment did not correlate with the radiologic score and %VC (data not shown).

BALF IL-8 concentrations in TB patients (353 ± 81 pg/mL) were significantly higher than in control subjects (4 ± 4 pg/mL, p < 0.001). There was a positive correlation between BALF {alpha}-defensins and IL-8 (r = 0.88, p < 0.0005; Fig 3 ).



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  		Figure 3.. Correlation between {alpha}-defensins and IL-8 levels in BALF of patients with TB.

 
Chromatographic Characterization of {alpha}-Defensins
Mature {alpha}-defensins and pro-defensins were present in the plasma of normal representative subjects in a 3:1 ratio as detected by RP-HPLC (Fig 4 , top, A).21 22 However, in the plasma sample obtained from a representative patient with TB, mature {alpha}-defensins and pro-defensins were present in a ratio of 4:9 before treatment (Fig 4 , middle, B). However, mature {alpha}-defensins increased again after treatment to 77% of total molecules (Fig 4 , bottom, C), and became equivalent to that of the normal subject. In a whole-blood sample and BALF from a representative patient with TB before treatment, mature {alpha}-defensins accounted for 98% and 100%, respectively, of total {alpha}-defensins (Fig 5 ).



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  		Figure 4.. Representative RP-HPLC profiles of plasma {alpha}-defensin immunoreactivity. Arrows indicate the elution position of mature {alpha}-defensins and pro-defensins. {alpha}-Defensins and pro-defensins in normal plasma (top, A), and in plasma of a representative TB patient before (middle, B) and after treatment (bottom, C).

 


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  		Figure 5.. Representative RP-HPLC profiles of whole-blood and BALF {alpha}-defensin immunoreactivity in patients with TB. Arrows indicate the elution position of mature {alpha}-defensins and pro-defensins. {alpha}-Defensin and pro-defensin levels are shown in a whole-blood sample (top, A) and BALF of a representative TB patient with a cavity (bottom, B).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of the present study was that patients with active TB showed high concentrations of {alpha}-defensins in both plasma and BALF. Other bacterial infections also induce high plasma concentrations of {alpha}-defensins,12 and TB patients may have concurrent bacterial infection such as anaerobic infections. To clarify the real role of {alpha}-defensins in TB, patients with suspected coinfection by other pathogens in serum, blood, sputum, and BALF samples or patients receiving antibiotics were excluded from this study. Several investigators7 8 9 23 24 have shown increased percentage and number of neutrophils in BALF of TB patients. This increase of neutrophils was only found in the active stage7 23 and in affected lesions of the lung.7 24 Condos and colleagues8 also demonstrated that neutrophils were the predominant BALF cell type in smear-positive patients. These findings suggest that neutrophils also participate in the host defense system in patients with TB like macrophages and T lymphocytes1 because neutrophils can kill M tuberculosis.2 3 4 This conclusion is supported by the finding reported by Pedrosa and associates,25 who demonstrated a protective role for neutrophils in the host defense against M tuberculosis via a nonphagocytic mechanism in a murine model of TB. Our finding showing high {alpha}-defensin levels in plasma and BALF of TB patients suggests that {alpha}-defensins, which are also known to kill M tuberculosis3 4 like other neutrophil granule proteins,26 may contribute at least in part to the antibacterial activity of neutrophils against M tuberculosis.

However, Barnes and associates6 have shown a close relationship between neutrophilia in peripheral blood and unfavorable short-term outcome in TB patients. This finding suggests that neutrophils may also act disadvantageously to the host with TB. The present study showed elevated levels of {alpha}-defensins in BALF of TB patients with cavitary disease. This finding expands a previous finding by Condos et al8 demonstrating that TB patients with cavitary disease show an increased number of neutrophils in BALF. Combined together, these findings suggest that neutrophils may injure through the cytotoxic ability of {alpha}-defensin the lungs in TB. This conclusion is supported by our finding showing an inverse relationship between plasma levels of {alpha}-defensins and FEV1/FVC ratio before treatment.

Our finding of a positive correlation between IL-8 and {alpha}-defensins in BALF of TB patients fits well with previous reports of the relationship between IL-8 and {alpha}-defensins. IL-8 is a potent neutrophil attractant that can induce the release of {alpha}-defensins from neutrophils.22 M tuberculosis or its cellular components are capable of stimulating both gene expression and protein secretion of IL-8 from alveolar macrophages and epithelial cells.27 28 {alpha}-Defensins, released by activated neutrophils, also stimulate IL-8 synthesis by airway epithelial cells.29 Thus, IL-8 may be a key cytokine that contributes to the high BALF {alpha}-defensin level in this disease.

Several studies with long follow-up periods have shown that a large percentage of treated TB patients show evidence of permanent airflow obstruction and restrictive impairment.30 31 Hnizdo and colleagues30 estimated that approximately 18% of patients with one episode of active TB had chronic lung function impairment. In another study,31 lung function deteriorated or was unchanged in 46% of TB patients during the course of treatment for 6 months. This impairment was associated with the extent of the disease on the original chest radiograph.31 32 In our study, there was no significant difference in lung function before and after treatment. However, a certain number of patients showed a decrease in pulmonary function after treatment (Fig 1 , top, A), which was consistent with previous reports. In addition, plasma levels of {alpha}-defensins before treatment correlated inversely with improvement of %VC after treatment in TB patients. This finding suggests that {alpha}-defensins also participate in lung function impairment after treatment in patients with TB and they may be good markers to predict the outcome of pulmonary function in this disease.

The biosynthesis of {alpha}-defensins appears first in the promyelocyte stage and is restricted to cells of neutrophil lineage.32 These peptides are initially synthesized as 94 amino-acid precursors that produce 75 amino-acid and 73 amino-acid pro-defensins by the cleavage of signal peptides. The majority of two pro-defensins is processed to 56 amino-acid intermediates by preaspartate proteolytic cleavage in neutrophil precursor cells in the bone marrow, and then to mature {alpha}-defensins in peripheral blood neutrophils.21 Some portions of pro-defensins are secreted from neutrophil precursor cells into the plasma, where they make up 25% of the plasma {alpha}-defensin molecules in normal subjects.21 22 By contrast, peripheral blood neutrophils of healthy subjects contain pro-defensin < 0.1%,21 and pro-defensins are not detected in the culture medium of neutrophils incubated with phorbol myristate acetate.22 Our RP-HPLC analysis also showed that the major molecular form of {alpha}-defensins in TB patients was the mature form in peripheral blood neutrophils and BALF (Fig 5) . In contrast, pro-defensin levels were extremely high in plasma of TB patients compared with control subjects (Fig 4 , middle, A, and bottom, B). Thus, the high BALF {alpha}-defensin levels probably result from the release of mature form from activated neutrophils at local sites of inflammation. In contrast, the rise in plasma {alpha}-defensin levels in patients is mainly derived from neutrophil precursor cells in the bone marrow. Mediators released from alveolar macrophages and other inflammatory cells stimulated by M tuberculosis may stimulate the bone marrow,9 24 33 and also facilitate the release of precursor neutrophils from the bone marrow into circulation.34 Plasma levels of {alpha}-defensins may directly reflect the degree of bone marrow stimulation in patients with TB. This conclusion is supported by our finding that peripheral neutrophil count as well as band cell count were higher in TB patients compared with control subjects, and these numbers returned to the normal range after treatment.

In conclusion, we demonstrated that plasma and BALF concentrations of {alpha}-defensins were elevated in patients with active TB. Our findings suggest that neutrophils may cause pulmonary dysfunction through {alpha}-defensins in patients with TB, and that these peptides could serve as new parameters of disease severity and outcome in this disease.


   Footnotes
 
Abbreviations: BALF = BAL fluid; HNP = human neutrophil peptide; IL = interleukin; RIA = radioimmunoassay; RP-HPLC = reverse-phase, high-performance liquid chromatography; TB = pulmonary tuberculosis; %VC = percentage of vital capacity

Received for publication March 21, 2001. Accepted for publication August 6, 2001.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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