Although tumor necrosis factor- (TNF- ) has

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1 Imbalances Between Tumor Necrosis Factor- and Its Soluble Receptor Forms, and Interleukin-1 and Interleukin-1 Receptor Antagonist in BAL Fluid of Cavitary Pulmonary Tuberculosis* Thomas C. Y. Tsao, MD, FCCP; Ji-hong Hong, MD, PhD; Li-Fu Li, MD; Meng-Jer Hsieh, MD; Shuen-Kuei Liao, PhD; and Kenneth S. S. Chang, MD, PhD Objectives: We investigated the possibility that the large pulmonary cavity in tuberculosis (TB) lesions might result from imbalances between tumor necrosis factor- (TNF- ) and soluble TNF- receptor forms (stnf-ri and stnf-rii), and interleukin- (IL-1 ) and IL-1 receptor antagonist (IL-1RA) in sites of local inflammation. Patients and methods: BAL was performed in 32 patients with active pulmonary TB, and the recovered BAL fluid (BALF) was examined for concentrations of TNF- and its soluble receptor forms, IL-1, and IL-1RA. Patients were classified into two groups: group 1, patients with a large cavity (> 4 cm) on chest radiographs (n 15); and group 2, patients with a small cavity (< 4 cm; n 3) or no cavity (n 14) on chest radiographs. Results: The concentrations of TNF-, IL-1, and IL-1RA in BALF were significantly higher in group 1 patients than in group 2 patients before standardization. The difference was also statistically significant for TNF- and IL-1 after standardization with urea. Furthermore, group 1 patients had significantly higher ratios of TNF- to stnf-ri and stnf-rii and IL-1 to IL-1RA compared with group 2 patients. Conclusions: These findings suggest that the relative abundance of TNF- and IL-1 associated with imbalances of secretion of soluble TNF- receptor forms and IL-1RA may have caused tissue necrosis leading to cavity formation in patients with active pulmonary TB. (CHEST 2000; 117: ) Key words: interleukin-1 ; interleukin-1 receptor antagonist; pulmonary tuberculosis; soluble tumor necrosis factor- receptor forms; tumor necrosis factor- Abbreviations: BALF BAL fluid; ELF epithelial lining fluid; IL-1 interleukin-1; IL-1RA interleukin-1 receptor antagonist; stnf-ri soluble tumor necrosis factor receptor form I; stnf-rii soluble tumor necrosis factor receptor form II; TB tuberculosis; TNF- tumor necrosis factor- Although tumor necrosis factor- (TNF- ) has harmful effects, such as acute-phase pathophysiologic events including fever and tissue necrosis, 1,2 it *From the Division of Pulmonary and Critical Care Medicine (Drs. Tsao, Hong, Li, and Hsieh), Chang Gung Memorial Hospital, Taipei, and the Graduate Institute of Clinical Medicine (Drs. Liao and Chang), Chang Gung University, Linko, Taiwan. Supported by a research grant from Chang Gung University Medical Research Foundation, CMRP521. Manuscript received February 24, 1999; revision accepted July 8, Correspondence to: Thomas C.Y. Tsao, MD, Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, 199 Tun-Hwa North Rd, Taipei, Taiwan; drtsao@ adm.cgmh.com.tw also plays a protective role against mycobacterial infection. When mice were infected with bacillus Calmette-Guérin, bactericidal granulomas developed in the liver and coincided with local TNF- synthesis. 3 Treatment with anti-tnf antibody not only interferes with the development of granulomas but also enhances the experimental infection of mycobacterium in mice. 4,5 Soluble TNF receptor forms, stnf-ri (55 kd) and stnf-rii (75 kd), have been found in body fluid as well as tissue and are thought to regulate the bioavailability and limit the toxic effects of TNF- in the body. 6 8 stnf-ripretreated mice or TNF-RI-knockout mice devel- CHEST / 117 / 1/ JANUARY,

2 oped fewer and smaller granulomas than did control mice after injection with bacillus Calmette-Guérin. 9 These facts strongly suggest that TNF-RI function is necessary for granuloma development. Chensue and colleagues 10 showed that peripheral blood mononuclear cells from active tuberculosis (TB) patients and patients with acute inflammatory disease spontaneously released more interleukin-1 (IL-1) when compared with those from healthy control subjects. IL-1 receptor antagonist (IL-1RA) competitively blocked the binding of IL-1 and IL-1 to type I and type II IL-1 receptors, but exerted no agonist activity. 11,12 As a naturally occurring anti-inflammatory protein, IL- 1RA is elevated in the serum of animals with sepsis 13 and in patients with autoimmune diseases. 14,15 Increased amounts of TNF-, 16,17 soluble TNF- receptor forms, 18,19 and IL-1RA 12,13,20 were found in serum of patients with active pulmonary TB. We have shown increased concentrations of TNF- and IL-1 in the BAL fluid (BALF) with the upregulation of their messenger RNA in macrophages lavaged from patients with active pulmonary TB. 21 These findings suggest that patients with active pulmonary TB could have bronchoalveolitis in the lower respiratory tracts. The cytokine concentrations in BALF would be a good index of the severity of local TB inflammation. Because release of TNF- and IL-1 at sites of inflammation can cause both beneficial and adverse effects, the balance between these cytokines and their naturally occurring inhibitors, ie, TNF- and its soluble receptor forms, as well as IL-1 and IL-1RA, would be important for the outcome of inflammation. Girardin et al 22 described that the ratios of TNF- /TNF-RI and TNF- /TNF-RII were higher in patients with fatal outcome from severe meningococcemia than in survivors. They proposed that the higher ratios of TNF- /TNF-RI and TNF- /TNF-RII result in a relative abundance of biologically active TNF-, which may lead to a detrimental outcome. In staging of pulmonary TB, patients with a large cavity ( 4 cm) in the lungs were thought to have more severe disease and are classified as far advanced TB. 23 The possibility that a large pulmonary cavity in TB lesions might result from an imbalance between TNF- and soluble TNF receptor forms and between IL-1 and IL-1RA at sites of local inflammation was examined in this study. We collected BALF from group 1 (patients with a large cavity [ 4 cm]) and group 2 (patients with a small [ 4 cm] or no cavity on chest radiographs), and compared the ratios between TNF- and soluble TNF receptor forms and between IL-1 and IL-1RA in BALF as well as serum of these two groups of patients. Patient Selection Materials and Methods We studied 32 patients with active pulmonary TB. All patients were admitted to Chang Gung Memorial Hospital in Taipei, a university medical center. Twenty-two patients were male and 10 were female; the mean age was 50 5 years (range, 22 to 77 years). Pulmonary TB was diagnosed when there was at least a positive sputum smear for acid-fast bacilli and a positive sputum culture for Mycobacterium tuberculosis. All patients had pulmonary TB symptoms such as cough, fever, hemoptysis, and so forth, and typical chest radiographic presentations. No patients had accompanying miliary or extrapulmonary involvement. To assess the presence and size of the TB cavity in the lungs, all 32 patients received plain posteroanterior and lateral chest radiographs. To avoid observer bias, the radiographs were initially assessed independently by two pulmonary physicians before the laboratory studies. To obtain a clearer image for patient classification, chest CT scans were performed in 20 patients whose cavity sizes were in doubt. Patients were classified into two groups: group 1, patients with large cavity ( 4 cm) on chest radiographs (n 15, including five patients who had accompanying smaller cavities); and group 2, patients with a small cavity ( 4 cm; n 3, including one with two small cavities) or no cavity (n 14) on chest radiographs. The previous duration of TB disease varied from 2 weeks to 4 months, and it was not significantly correlated with the size of the cavity. None of the patients were immunocompromised. BAL BAL was performed with methods described previously. 24 In brief, after determination of the location of the TB lesions by chest radiographs, we wedged an 5.0-mm fiberoptic bronchoscope (Olympus; Tokyo, Japan) into a fourth or fifth subsegmental bronchus with local lidocaine anesthesia. All patients were pretreated with codeine phosphate (5 mg, IM) 30 min before the procedure, and some patients were also treated with midazolam (2 to 3 mg, IV slowly) just before the procedure. A total of 300 ml of saline solution was infused sequentially, and the return fluid was obtained through the same syringe. Any subject who could not tolerate the entire procedure or whose returned fluid was 60% of the total infused volume was excluded from further study. In 19 patients, bronchoscopy was performed within 3 days of antituberculous chemotherapy, 11 patients within 5 days, and 2 patients within 7 days. All returned fluid was filtered through sterile four-layer gauze, pooled, and chilled immediately for experiments. The pooled fluid was spun at 4 C at 400g for 15 min, and then the supernatant was centrifuged at 80,000g for 30 min at 4 C to remove the surfactant-rich fraction. The resultant supernatant was concentrated 10-fold on a 10,000-molecular weight cutoff filter (Amicon; Danvers, MA) under nitrogen. The concentrated supernatant was then divided into 200- L aliquots and rapidly frozen at 70 C. One aliquot of resultant supernatant was not concentrated, but stored for determination of urea and albumin concentrations. Serum was obtained by drawing whole blood through standard procedures on the same day before the lavage. Determination of Cytokine Concentrations Sandwich enzyme-linked immunosorbent assay was used to detect the concentrations of TNF-, stnf-ri, and stnf-rii in the BALF and serum. The kits for TNF- and IL-1 assays were purchased from Medgenix (Fleurus, Belgium), and those for 104 Clinical Investigations

3 stnf-ri, stnf-rii, and IL-1RA, from Quantikine (R&D; Minneapolis, MN). The frozen aliquots from BALF and serum were thawed at room temperature for each assay, and for stnf-ri and stnf-rii assays the samples were diluted 1:10. For kits from Medgenix, samples were added to wells of microtiter plates coated with monoclonal antibody to human TNF-. The horseradish peroxidase-conjugated antibody against the corresponding cytokine was then added. After incubation, the excess horseradish peroxidase-conjugated antibody was removed by washing. The revealing solution was added, and the reaction was stopped by H 2 SO 4. The color intensity was measured with a microtiter plate reader. For kits from Quantikine, similar procedures with slightly different reagents were used. The minimal detectable dose using a standard curve generated with calibrator diluent was 3 pg/ml for TNF-, 10 pg/ml for TNF-RI, 5 pg/ml for TNF-RII, 0.06 pg/ml for IL-1, and 6.5 pg/ml for IL-1RA. Standardization of Cytokine Concentrations in BALF With Urea The technique of BAL is based on the concept that aliquots of sterile normal saline solution infused through the bronchoscope mix with epithelial lining fluid (ELF). When the saline solution is recovered by aspiration, the ELF and its components are recovered along with it. However, the recovered BALF is a variable mixture of saline solution, ELF, and ELF components. Therefore, it has been difficult to estimate the actual concentration of recovered molecules in the ELF in situ. Because of the low molecular weight of urea and its high permeability across membranes, it is likely that the urea concentration in ELF is equal to that in serum. Because the concentration of urea can be quantified in plasma and the total amount of urea recovered by lavage can be measured, the total volume of the recovered ELF can be calculated by using the formulas: 1 Total amount of urea recovered by lavage total Urea balf Urea balf volume of total recovered lavage fluid Vol balf 2 Urea elf total Urea balf /Vol elf 3 Urea serum total Urea balf /Vol elf 4 Urea serum Urea balf Vol balf /Vol elf 5 Vol elf Urea balf Vol balf / Urea serum Urea balf / Urea serum Vol balf The cytokine concentrations in the ELF were then calculated by the following formulas and are shown as picograms per milliliter of ELF: 6 cytokine balf Vol balf cytokine elf Vol elf 7 cytokine elf cytokine balf Vol balf /Vol elf (I) (II) Although variations in the concentration of urea in BALF have been reported, these standardization methods remove the variable of dilution and allow comparison between data in different subjects and from different investigators Urea concentration was determined using a modified urease Berthelot reaction in microtiter plates (Sigma; St. Louis, MO). 27 Urea concentration was measured on unconcentrated BALF. Statistical Analysis Values were expressed as mean SEM (range). All data were compared by nonparametric Mann-Whitney U and Wilcoxon Rank Sum W tests, and the correlation analysis was by nonparametric Spearman test. The null hypothesis was rejected at p Results The BALF differential cell counts in patients with pulmonary TB are shown in Table 1. The number of cells per volume of recovered BALF increased in group 2 patients, but the difference was not statistically significant. Cell differentials performed on BALF revealed significant percentage increases in neutrophils but a decrease in macrophages in group 2 patients with TB. The assays we used could detect the total amount of soluble receptors present in the samples, including free receptors and receptors bound to TNF-. In 13 normal control subjects (age- and sex-matched), the respective concentrations of stnf-ri and stnf-rii were pg/ml and pg/ml in serum, as well as pg/ml and pg/ml in BALF. After standardization with urea, the BALF concentrations for stnf-ri were 50, ,412.4 pg/ml ELF, and for stnf-rii were 40, ,234.7 pg/ml. In the normal control subjects, the concentrations of TNF- in serum were undetectable in 10 subjects and 5 pg/ml in 3 subjects, and concentrations of TNF- in the BALF were undetectable in 11 subjects and 10 pg/ml in 2 subjects. The ages of TB patients in these two groups were comparable (49 4 years and 52 5 years, respectively). Table 2 shows the average concentrations of TNF-, soluble TNF- receptor forms, IL-1, and IL-1RA in BALF before and after standardization with urea as well as in serum from the two groups of patients with active pulmonary TB. The concentrations of TNF-, IL-1, and IL-1RA in BALF were significantly Table 1 BALF Differential Cell Counts From Patients With Active Pulmonary TB* Variables Group 1 (n 15) Group 2 (n 17) p Value BALF recovery, % NS Cell number, 10 3 / L NS Cell viability, % NS Macrophages, % Lymphocytes, % NS Neutrophils, % *Values are expressed as mean SEM. Group 1, patients with large cavity ( 4 cm) on chest radiographs; group 2, patients with small cavity ( 4 cm) or without cavity on chest radiographs; NS not significant. CHEST / 117 / 1/ JANUARY,

4 Table 2 Concentrations of TNF-, TNF- Soluble Receptor Forms, IL-1, and IL-1RA in BALF and Serum From Patients With Active Pulmonary TB Before and After Standardization With Urea* BALF Serum Cytokine Group 1 (n 15) Group 2 (n 17) Group 1 (n 15) Group 2 (n 16) TNF ( ) ( ) ( ) ( ) Corr. TNF- 35, , , (7, ,689.6) (2, ,448.2) stnf-ri 3, , , , , ,168.2 ( ,449.1) ( ,611.6) ( ,117.6) ( ,979.8) Corr. stnf-ri 324, , , ,308.7 (38, ,058,682.5) (20, ,986.0) stnf-rii 4, , , , , , ( ,439.4) ( ,148.2) ( ,731.2) (1, ,660.1) Corr. stnf-rii 334, , , ,106.8 (72, ,061,712.6) (38, ,300.6) IL (15.5 1,064.7) (0 39.7) (0 3.52) (0 20.0) Corr. IL-1 20, , (1, ,305.4) (0 3,374.5) IL-1RA 4, , (3, ,165.6) ( ,681.9) ( ) (40.6 3,486.6) Corr. IL-1RA 346, , , ,138.1 (242, ,241.6) (32, ,597.7) *Values are expressed as mean SEM (range). Results are given as picograms per milliliter of BALF or serum, or for the corrected (Corr.) cytokines, after standardizing to urea, as picograms per milliliter of ELF. p with respect to group 1. p 0.05 with respect to group 1. higher in group 1 patients than in group 2 patients before standardization. The difference was also statistically significant for TNF- and IL-1 after standardization with urea. No significant differences were detected for those in serum. Although TNF- may play a role in stimulating the production of soluble TNF- receptor forms, the balance between the amount of TNF- and each of the two receptor forms is also important. The distribution of individuals according to the concentrations of their TNF- and soluble receptor forms is shown in Figure 1. Group 1 patients tended to show a correlation, although statistically nonsignificant, between the concentrations of TNF- and its soluble receptor forms (Fig 1, left). Group 2 patients tended to show low concentrations of TNF- in spite of high concentrations of its soluble receptors (Fig 1, middle) and failed to show any correlation between the concentrations of TNF- and its soluble receptor forms (Fig 1, Figure 1. Relationship between TNF- and its soluble receptors (stnf-ri and stnf-rii) in group 1(left) and group 2 (middle). Group 1 patients have a large cavity ( 4 cm) on chest radiographs (n 15), and group 2 patients have no (n 14) or a small cavity ( 4 cm; n 3) on chest radiographs. Left and middle graphs are shown at the same scale on the x- and y-axes to demonstrate the differences of concentrations of cytokines between group 1 and group 2 patients. Right: the middle (group 2) is replotted with a smaller scale of the x- and y-axes to show the distribution of TNF- and its receptor forms, and their correlation. 106 Clinical Investigations

5 Figure 2. Relationship between IL-1 and IL-1RA in group 1 (left) and group 2 (middle). Group 1 patients have a large cavity ( 4 cm) on chest radiographs (n 15), and group 2 patients have no (n 14) or a small cavity ( 4 cm; n 3) on chest radiographs. Left and middle graphs are shown at the same scale on the x- and y-axes to demonstrate the differences of concentrations of cytokines between group 1 and group 2 patients. Right: the middle (group 2) is replotted with a smaller scale of the x- and y-axes to show the distribution of IL-1 and IL-1RA and their correlation. right). The distribution of individuals according to their concentrations of IL-1 and IL-1RA is shown in Figure 2, with similar findings found in TNF- and it soluble receptor forms. We found a significant difference in the ratios of TNF- and soluble TNF- receptor forms between groups 1 and 2. For group 1 patients, the ratios of TNF- /stnf-ri and TNF- /stnf-rii were (0.012 to 0.549) and (0.026 to 0.618), respectively, compared with (0.014 to 0.251; p 0.005) and (0.009 to 0.144; p 0.001), respectively, for group 2 patients (Fig 3). There was also a significant difference in the ratio of IL-1 and IL-1RA between groups 1 and 2. For group 1 patients, the ratio of IL-1 /IL-1RA was (0.003 to 0.259) compared with (0 to 0.049; p 0.001) for group 2 patients (Fig 3). Thus, group 1 patients who had a big cavity ( 4 cm) in the lungs had a relatively larger portion of biologically active TNF- and IL-1 in BALF than did group 2 patients. We failed to find any differences between samples from patients in groups 1 and 2 in the ratios of TNF- and soluble TNF- receptor forms and between IL-1 and IL-1RA in serum. Discussion TB is still one of the most important infectious diseases in the world and causes about 3 million deaths annually. 28 TB patients who have large cavities ( 4 cm) in the lungs are considered to be far advanced cases 23 ; these are more difficult to treat and have a poor prognosis. 29 In patients with ARDS, persistent elevation of TNF- in BALF was shown to be a predisposing factor for mortality. 30 In a previous Figure 3. Ratios of TNF- and soluble TNF- receptor forms and of IL-1 and IL-1RA for patients of groups 1 and 2. Group 1 patients have a large cavity ( 4 cm) on chest radiographs (n 15), and group 2 patients have no (n 14) or a small cavity ( 4 cm; n 3) on chest radiographs. CHEST / 117 / 1/ JANUARY,

6 study, we demonstrated increased concentrations of TNF- and IL-1 in BALF with the upregulation of their messenger RNA in macrophages lavaged from patients with active pulmonary TB. 21 In the current study, the average concentrations of TNF- in BALF of group 1 patients were nearly nine times higher before and eight times higher after standardization with urea than those of group 2 patients despite the fact that there was no such difference in serum. The difference in BALF was more significant for IL-1, at nearly 30 times higher before and 27 times higher after standardization with urea. These findings suggest that local release of large amounts of TNF- and IL-1 may have caused significant tissue necrosis in lung lesions of group 1 patients. Also, it is possible that the release of these cytokines is a consequence of the necrosis. TNF- and IL-1 are possibly released by pulmonary macrophages or other cells and/or accumulate in the lung in response to mycobacterial infection, which may have a certain similarity to the findings observed in ARDS. 31 Besides, more aggressive TB may be associated with a more important pulmonary inflammation, with higher local cytokine concentrations, and with larger amounts of proteases, the latter also being responsible for tissue destruction and cavity formation. The effects of standardization of concentrations of TNF- and its soluble receptor forms using urea or albumin tend to negate the increase in TNF-, its soluble receptor forms, IL-1, and IL-1RA in group 1 patients when compared with group 2 patients. These effects are caused by higher values of ELF volume calculated by urea and by a lower recovered BALF volume in group 1 than in group 2 patients. Also, the normal control subjects had lower values of ELF volume and higher recovered BALF volume than those of the two patient groups. Soluble TNF- receptor forms may be one component of an autocrine and paracrine regulatory system that limits the toxic effects of systemically circulating TNF-. 7,8,32 In patients with ARDS, elevated concentrations of stnf-ri and stnf-rii in BALF were found to be correlated with an increase of TNF-. 31 In the current study, we found significantly higher concentrations of TNF- and IL-1 in group 1 as compared with group 2 patients. After comparison of the distribution of TNF- and soluble TNF- receptor forms in BALF from each group, we found that the ratios between TNF- and soluble TNF- receptor forms in BALF were significantly higher in group 1 patients than in group 2 patients. Otherwise, there was no significant correlation between TNF- and its soluble receptor forms. The reason for the relatively lower concentrations of soluble TNF- receptor forms in group 1 patients is unclear. In a previous study, elevated concentrations of soluble TNF- receptor forms were found in humans after infusion with TNF-. 33 Girardin et al 22 reported that two types of correlations were found between TNF- and its soluble receptor forms in serum from patients with severe meningococcemia. When TNF- concentrations were low ( 500 pg/ ml), the increase in TNF- was proportional to that of soluble TNF- receptor forms. However, when TNF- concentrations were 500 pg/ml, the concentrations of stnf-ri and stnf-rii did not increase proportionally at the early stage of the disease. In view of the ninefold or eightfold increase in the amount of TNF- concentrations in BALF from group 1 as compared with group 2 patients, high TNF- concentrations in BALF resulting in inadequate production of soluble TNF- receptor forms in the former might be one of the reasons for the imbalance between TNF- and its soluble receptors. Increased clearance of soluble TNF- receptor forms might be another possible reason. Rydberg et al 34 described an imbalance between TNF- and its soluble receptor forms in TB infection of the CNS. In that study, patients with TB meningitis had significantly lower ratios of TNF- to stnf-ri and stnf-rii, at and 0.038, respectively, in cerebrospinal fluid as compared with those from patients with bacterial meningitis, at and 0.250, respectively. They ascribed these findings to the acute infectious course of bacterial meningitis. In our study, the ratios of TNF- to stnf-ri and stnf- RII in BALF from group 2 patients were and 0.045, respectively, similar to their results from patients with TB meningitis, and might indicate a more chronic infectious course. Nevertheless, the ratios of TNF- to stnf-ri and stnf-rii in BALF from group 1 patients were and 0.157, respectively, which are closer to their results from patients with bacterial meningitis, indicating a more acute and severe infectious course. The above findings were also found for IL-1 and IL-1RA between group 1 and group 2 patients. In conclusion, we demonstrated a significantly larger amount of TNF- accompanied by a relative paucity of stnf-ri and stnf-rii in BALF from patients with active pulmonary TB carrying large cavities, compared with those from TB patients with small or no cavities. Similar results were also found for IL-1 and IL-1RA. This indicates that a high concentration of biologically active TNF- and IL-1 in the former condition may have caused severe tissue necrosis, ie, a larger cavity. The reason for these findings remains unclear. Inadequate production and/or increased clearance of these soluble receptor forms or IL-1 antagonists may reasonably account for this imbalance. 108 Clinical Investigations

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