INTRODUCTION

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Pulmonary tuberculosis is one of the granulomatous diseases, and its pathogenesis has been linked to monocytes and alveolar macrophages (1). Yet there are few serological markers that keenly reflect its activity. Recently, vascular endothelial growth factor (VEGF), which induces angiogenesis in malignant tumors (2), was found to be associated with Crohn's disease (3). Crohn's disease is a granulomatous disease like pulmonary tuberculosis, but, to our knowledge, there are no reports describing serum VEGF levels in patients with pulmonary tuberculosis. We investigated 43 patients with active pulmonary tuberculosis and found increased serum levels of VEGF. We suggest a possible association of VEGF with the pathogenesis of pulmonary tuberculosis.

	METHODS

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Subjects

We investigated 43 patients with active pulmonary tuberculosis who were admitted to the Third Department of Internal Medicine (Kagoshima University School of Medicine) and the Department of Respiratory Medicine (National Minami-kyushu Hospital) from 1995 to 1999. There were 30 men and 13 women whose mean age was 62.5 ± 34.2 yr old (mean ± standard deviation). For comparison, we also investigated 29 patients with old pulmonary tuberculosis (male:female = 20:9, 66.9 ± 35.3 yr old), 8 patients with drug-resistant tuberculosis (male:female = 6:2, 68.9 ± 23.1 yr old), and 25 patients with acute bronchitis (male:female = 20:9, 67.3 ± 30.5 yr old). The diagnosis of tuberculosis was made according to the 1990 edition of Diagnostic Standards and Classification of Tuberculosis published by the American Lung Association (4). According to these criteria, patients classified as class 3 were diagnosed as having active pulmonary tuberculosis, and patients classified as class 4 were diagnosed as having old pulmonary tuberculosis. A diagnosis of acute bronchitis was made for patients with respiratory symptoms (e.g., cough and sputum) and inflammatory findings (e.g., fever and increased serum level of C-reactive protein) in the absence of acid-fast bacteria and abnormal chest X-ray shadows. Thirty-six patients with active tuberculosis had positive acid-fast smears and cultures, whereas seven with active tuberculosis had positive cultures only. All patients with old tuberculosis or acute bronchitis had negative acid-fast smears and cultures. We excluded patients with rheumatoid arthritis, diabetes mellitus, acute or chronic liver disease, and immunological abnormalities that predispose to opportunistic infection. The smoking index (number of cigarettes smoked per day × smoking years) and the presence of systemic hypoxia of all patients were also determined.

Measurement of VEGF

In the patients with active pulmonary tuberculosis and acute bronchitis, we measured serum levels of VEGF before the patients underwent therapy. In seven patients with active pulmonary tuberculosis, serum VEGF levels were determined every 3 mo (before, and 3 and 6 mo after the beginning of therapy). In patients with old tuberculosis, serum VEGF levels were determined when they were in good health, after they gave written consent to participate in this study.

VEGF concentrations in sera were measured in duplicate for each sample using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) that recognizes the soluble isoforms (VEGF₁₂₁ and VEGF₁₆₅. This assay is sensitive to 9 pg/ ml (0.2 pM) VEGF and does not cross-react with platelet-derived growth factor (PDGF) or other homologous cytokines. Optical density at 450 nm was measured on a Titertek Multiskan MC plate reader (Flow Laboratories, Helsinki, Finland), and VEGF concentration was determined by linear regression from a standard curve using GraphPad software (San Diego, CA) for analysis.

Immunohistochemical Staining for VEGF

Three patients with active pulmonary tuberculosis underwent open lung biopsy for diagnosis. Immunohistochemical staining for VEGF was performed using a rabbit polyclonal antibody (Santa Cruz, CA) employing the DAB method. Four-micrometer-thick sections were cut consecutively from formalin-fixed and paraffin-embedded tissue samples mounted on poly-L-lysine-coated slides and dried overnight at 58° C. Paraffin sections were dewaxed using xylene, rehydrated with graded concentrations of ethanol, and washed in Tris-buffered saline (pH 7.4) for 10 min. For optimal antigen retrieval, sections were pressure cooked in 0.01 M citrate buffer (pH 6.0) for 90 s. Endogenous peroxidase activity was blocked using a 3% hydrogen peroxide solution in methanol for 10 min. After washing twice in phosphate-buffered saline (PBS) 1% saponin, endogenous avidin and biotin were blocked using the Vector Labs Blocking Kit. Sections were again washed in PBS 1% saponin. Sections were incubated with primary antibody solution for 2 h at room temperature using a 1:150 concentration working dilution of the antibody. Negative control slides were incubated with rabbit polyclonal antibody (Super Sensitive Rabbit; Biogenex). Slides were rinsed twice in PBS 1% saponin. Secondary biotynated anti-immunoglobulin antibody (Biogenex) was added, and the mixture was incubated for 30 min at room temperature. The sections were again rinsed twice with PBS 1% saponin. Streptavidin conjugated to horseradish peroxidase (Biogenex) was incubated for 30 min and then rinsed off with deionized water. DAB substrate solution was then added, and the mixture was incubated for 10 min. A brown color reaction represented a positive result.

Statistical Analysis

We used one-way factorial analysis of variance (ANOVA) with the Bonferroni-Dunn test. A p value below 0.05 was considered significant. Most values were expressed as mean ± standard deviation (SD). Some data were presented with appropriate 95% confidence intervals (CI).

	RESULTS

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The serum VEGF levels in patients with active pulmonary tuberculosis were significantly higher than in patients with old tuberculosis or acute bronchitis (active tuberculosis: mean ± SD = 722.6 ± 362.2 pg/ml; 95% CI = 614.3-830.7 pg/ml; old tuberculosis: mean ± SD = 185.1 ± 158.4 pg/ml, 95% CI = 127.4-242.7 pg/ml; acute bronchitis: mean ± SD = 189.3 ± 183.3 pg/ml, 95% CI = 117.4-260.1 pg/ml, Figure 1). The sensitivity of VEGF levels for the diagnosis of active tuberculosis was 93%, and specificity was 62.1% (cutoff value = 250 pg/ml). The serum VEGF levels of the eight patients with drug-resistant tuberculosis were elevated (mean ± SD = 681.4 ± 193.8 pg/ml, 95% CI = 547.1-815.7 pg/ml) despite the therapy (their mean treatment periods were 4.2 ± 1.8 mo). The serum VEGF levels in all patients were 445.4 ± 375.8 pg/ml (mean ± standard deviation). The serum VEGF levels of seven patients with active pulmonary tuberculosis decreased in parallel with improvement in clinical course (Figure 2). This difference was significant by one-way factorial ANOVA, and the serum VEGF levels before therapy were significantly higher than at 3 mo or 6 mo after the beginning of therapy (Bonferroni- Dunn test, p < 0.001). There were no significant differences between the values at 3 mo and 6 mo after the beginning of therapy. All these patients improved with antituberculosis drugs, and their sputum cultures became negative after 6 mo. Alveolar macrophages around the lesions stained intensely positive (Figure 3). The smoking index was 983 ± 643 (mean ± SD) in patients with active pulmonary tuberculosis, 1,091 ± 712 (mean ± SD) in patients with old tuberculosis, and 921 ± 611 (mean ± SD) in patients with acute bronchitis. There was no significant difference in smoking index among these groups. No patients in this study showed systemic hypoxia.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Serum VEGF levels in the three groups. Serum VEGF levels were significantly higher in patients with active pulmonary tuberculosis than in patients with old tuberculosis and acute bronchitis. Patients with active tuberculosis were studied prior to the start of therapy. Closed circles indicate mean values, and bars indicate standard deviations in each group.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Serum VEGF levels in seven patients with active pulmonary tuberculosis. The decrease in VEGF levels paralleled the improvement of the disease. Bars indicate mean values of each group.

View larger version (149K): [in this window] [in a new window]   View larger version (138K): [in this window] [in a new window]   Figure 3.   Lung biopsy specimen of a patient with active pulmonary tuberculosis showing expression of VEGF in macrophages. The arrows indicate macrophages. (Top panel: original magnification: ×200; bottom panel: original magnification: ×400.)

	DISCUSSION

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Our results showed significantly higher levels of serum VEGF in active pulmonary tuberculosis patients than in old tuberculosis patients. Although detailed immunological mechanisms of pulmonary tuberculosis are still unclear, alveolar macrophages are reported to play an important role through the cytokine interaction with T-cells (5, 6). Additionally, intense angiogenesis has been found in active pulmonary tuberculosis lesions (7). VEGF is known to be associated with angiogenesis (8). Our study showed positive staining of alveolar macrophages around active tuberculosis lesions. Macrophages have the potential to produce VEGF (9). Based on these findings, we suggest the possibility that alveolar macrophages in pulmonary tuberculosis may release VEGF together with several cytokines and contribute to the recruitment of T-cells to the lesion.

Heavy smoking (10) and systemic hypoxia (11) affect serum VEGF levels. In this study, no patients showed systemic hypoxia, and there were no significant differences in the smoking index among the three groups of patients. The serum VEGF levels of seven patients with active pulmonary tuberculosis decreased parallel to improvement of the disease. Additionally, the serum VEGF levels of drug-resistant tuberculosis patients were high despite therapy, excluding drug effect as a possible cause of decreasing serum VEGF levels. The significant difference in serum VEGF levels between active pulmonary tuberculosis and old tuberculosis patients may reflect the activity of pulmonary tuberculosis. The sensitivity and specificity of VEGF levels of 250 pg/ml for diagnosis of active tuberculosis were 93% and 62.1%, respectively. VEGF may be a useful screening marker for active tuberculosis, as a negative result greatly reduces the likelihood of tuberculosis. However, a positive result requires confirmation as the specificity is relatively low.