Vol. 58, No. 6
INFECTION AND IMMUNITY, June 1990, p. 1572-1576 0019-9567/90/061572-05$02.00/0
Copyright © 1990, American Society for Microbiology
A Role In Vivo for Tumor Necrosis Factor Alpha in Host Defense against Chlamydia trachomatis DWIGHT M. WILLIAMS,1 2* D. MITCHELL MAGEE,"2 LYNDA F. BONEWALD,2 JEFFREY G. SMITH,' CHERYL A. BLEICKER,' GERALD I. BYRNE,' AND JULIUS SCHACHTER4 Infectious Diseases Section, Audie L. Murphy Memorial Veterans' Hospital,' and Department of Medicine, University of Texas Health Science Center,2 San Antonio, Texas 78284; Department of Medical Microbiology, University of Wisconsin Medical School, Madison, Wisconsin 537063; and Department of Laboratory Medicine, University of California at San Francisco, San Francisco, California, 941324 Received 27 November 1989/Accepted 22 February 1990 In a mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent
[MoPn]), tumor necrosis factor alpha (TNF-a) antigen and bioactivity were demonstrated in vivo in the lung during MoPn infection in both athymic (nude) and heterozygous (nul+) mice. Antibody to TNF-oa that was exogenously given neutralized the TNF-a in the lung, significantly accelerated mortality, and caused a borderline increase in MoPn counts in the lung by culture in nul+ mice. Lipopolysaccharide-induced TNF-oa activity or injections of recombinant murine TNF-a significantly but modestly protected nul+ mice against MoPn-induced mortality. TNF-oa is produced in vivo during C. trachomatis infection and plays a role in host defense.
102 to 105 inclusion-forming units in a volume of 0.05 ml (4). The diluent was McCoy modified 5A medium (Difco Laboratories, Detroit, Mich.). RIA for murine TNF-a. Radioimmunoassay (RIA) was performed as described previously (24) and was specific for murine TNF-a. The algorithm used for fitting standards and calculating unknowns was the four-parameter logistic, an iterative weighted linear regression with proper weighting from accumulation of a historical curve (16, 17). Values less than 200 pg/ml were considered not significant. TNF-a cytotoxicity assay. The TNF-a cytotoxicity assay was a modification of an assay previously described (14, 18, 24). Briefly, L929 tumorigenic fibroblasts were seeded at 2 x 104 cells per well in a 96-well microdilution plate in a 200-,ul volume of Dulbecco modified Eagle medium containing 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah), 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml, for 18 to 24 h at 37°C in 5% CO2. The medium was removed, and 100 ,ul of fresh medium containing 2 ,ug of Actinomycin D per ml was added. A 100-pld aliquot was added to each assay well. Negative-control (spontaneouslysis) wells had 100 ,ul of medium added, while positivecontrol (maximum-lysis) wells had 100 ,ul of saponin-lysing solution added to lyse all L929 cells in the well. Each sample was tested in triplicate in the microdilution plate. Plates were then incubated for 24 h at 37°C in 5% CO2. A 20-pd volume of a 5-,ug/ml solution of MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (Sigma Chemical Co., St. Louis, Mo.) was added to each well of the microdilution plate. The plates were then incubated for 4 h at 37°C in 5% CO2. The medium was poured off the plates, and 100 pld of an isopropyl alcohol solution containing 0.33 ml of concentrated HCl per 100 ml of isopropyl alcohol was added. The optical density (OD) of the wells was then read at 600 nm on an enzyme immunoassay plate reader. In the assay, 1 U of TNF-a activity was defined as the point at which 50% of the L929 cells were lysed, and minimal detectable levels were 5 U/ml. The biological activity of samples was confirmed to be TNF-a by neutralizing samples with anti-TNF-ot antibody
In previous studies, we described a model of murine pneumonia that employed the mouse biovar of Chlamydia trachomatis (mouse pneumonitis agent [MoPn]) (27-29). In this model, the heterozygous nul+ mouse is more resistant to MoPn than its athymic nude littermates (nulnu), and resistance can be adoptively transferred to the nulnu mouse by T cells (27). By using this model, we showed previously that gamma interferon (IFN--y) is produced by MoPn-infected nul+ but not nulnu mice in response to MoPn antigen (4, 5) in vitro and that this T-cell-dependent cytokine plays a role in vivo in host defenses against MoPn as well (25). Sarov et al. (13, 20, 21) recently proposed that tumor necrosis factor alpha (TNF-a) is able to inhibit replication of C. trachomatis in vitro. IFN-13 appears to play a significant role in the antichlamydial effect of TNF-a (21). We showed previously, in our murine pneumonia model, that whole spleen cells from both infected and uninfected nul+ and nulnu mice are able to produce TNF-a in vitro in response to MoPn antigen (24). These data, combined with those of Sarov et al. (13, 20, 21), suggested that TNF-a might provide a nonspecific host defense mechanism in vivo against C. trachomatis. The studies reported here show that TNF-a is produced in the lung during infection with MoPn and that TNF-a plays a role in vivo in host defense against MoPn. MATERIALS AND METHODS Mice. Specific-pathogen-free nul+ and nulnu mice on a BALB/c background were bred and maintained under barrier conditions at the Audie L. Murphy Veteran's Administration Hospital, San Antonio, Tex., and were used in these and earlier studies (4, 26). These mice were free of pathogenic bacterial and all viral contamination as assessed by culture and serology (4, 26). MoPn. MoPn, the mouse biovar of C. trachomatis, was obtained and maintained in embryonated hen eggs as previously described (4, 24). Mice were infected intranasally with *
Corresponding author. 1572
VOL. 58, 1990
(Genzyme Inc., Boston, Mass.) and observing a concomitant loss of cytotoxicity in the assay. Preparation of lung samples for determination of in vivo TNF-ot production. Whole-lung homogenates were prepared
from both infected and noninfected mice. Mice were challenged with MoPn and then were killed by cervical dislocation at the appropriate times. The inoculum was diluted in McCoy modified 5A medium. Control, uninfected mice received a mock challenge of McCoy medium. The pleural cavity was exposed, and the lungs were excised. The lungs were then placed in a tissue homogenizer along with 1.5 ml of RPMI 1640 containing 10% fetal bovine serum, 100 p.g of vancomycin per ml, and 50 ,g of gentamicin sulfate (medium) per ml. The lungs were homogenized thoroughly, and the homogenates were centrifuged at 1,000 x g for 10 min. The supernatants were removed, filtered, divided into aliquots, and stored at -70°C. For some samples, the lungs were perfused and lavaged prior to homogenization. After cervical dislocation, the pleural and peritoneal cavities were exposed. The inferior vena cava was then cut just posterior to the liver. A 6-ml volume of medium with 10 U of preservative-free heparin per ml was injected into the right ventricle by using a 26-gauge needle on a 12-ml syringe. The trachea was then exposed, and the lungs were lavaged with 3 ml of medium. Approximately 2 ml of the bronchioalveolar lavage fluid was recovered, subsequently filtered, divided into aliquots, and stored at -70°C. Treatment of mice with anti-TNF-a in vivo. Initial doseresponse experiments established that 105 neutralizing units of rabbit polyclonal antibody to murine TNF-a (anti-TNF-a) (Genzyme) was the minimal effective dose. Unless otherwise stated, all mice in mortality studies were given four doses intravenously of 105 neutralizing units of anti-TNF-a on days 0, 1, 3, and 5, with MoPn infection on day 0. Control mice were given normal rabbit serum (NRS). Neither anti-TNF-a nor NRS contained azide and were not heat inactivated prior to injection. Mortality was followed daily. In some experiments, lungs were harvested for quantitative culture of MoPn or for MoPn antigen quantitation. Quantitative culture of MoPn. Quantitative culture of MoPn in the lung was performed in McCoy cell monolayers as previously described (25). Quantitation of MoPn antigen. Quantitative MoPn antigen determinations were performed by using an enzyme-linked immunosorbent assay kit (Ortho Diagnostic, Inc., Raritan, N.J.). Lungs were removed, homogenized in 1.0 ml of medium, and frozen at -70°C until assay. After thawing, the homogenates were diluted further in medium and processed for assay according to the instructions of the manufacturer. A standard curve was run with each determination by using purified MoPn elementary bodies (EBs). An EB protein dose response between 10 and 200 ng of protein per ml was detected by a linear increase in OD. The standard curve, however, varied between determinations. Therefore, for statistical evaluations, all groups to be compared were run simultaneously, and the data were expressed as a ratio of experimental OD values to the OD values of uninfected
lungs.
1573
TNF-a IN HOST DEFENSE AGAINST C. TRACHOMATIS
Treatment of mice with LPS or TNF-a in vivo. To determine the in vivo effect of bacterial lipopolysaccharide (LPS) (Escherichia coli 0111:B4; Sigma) or recombinant murine TNF-a (rm TNF-a; lot number 4296-17; 1.2 x 107 U/mg; graciously supplied by Howard Jaffe, Genentech, Inc., South San Francisco, Calif.), the following treatments were carried out. Mice were given single doses of 100 ,g of LPS
TABLE 1. TNF-a determined by RIA in mouse whole-lung preparations after MoPn infection Day postinfection
0 3 7
10 a
b
TNF-a
(pg/ml) in micea:
nu/nu
INFECTION AND IMMUNITY, June 1990, p. 1572-1576 0019-9567/90/061572-05$02.00/0
Copyright © 1990, American Society for Microbiology
A Role In Vivo for Tumor Necrosis Factor Alpha in Host Defense against Chlamydia trachomatis DWIGHT M. WILLIAMS,1 2* D. MITCHELL MAGEE,"2 LYNDA F. BONEWALD,2 JEFFREY G. SMITH,' CHERYL A. BLEICKER,' GERALD I. BYRNE,' AND JULIUS SCHACHTER4 Infectious Diseases Section, Audie L. Murphy Memorial Veterans' Hospital,' and Department of Medicine, University of Texas Health Science Center,2 San Antonio, Texas 78284; Department of Medical Microbiology, University of Wisconsin Medical School, Madison, Wisconsin 537063; and Department of Laboratory Medicine, University of California at San Francisco, San Francisco, California, 941324 Received 27 November 1989/Accepted 22 February 1990 In a mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent
[MoPn]), tumor necrosis factor alpha (TNF-a) antigen and bioactivity were demonstrated in vivo in the lung during MoPn infection in both athymic (nude) and heterozygous (nul+) mice. Antibody to TNF-oa that was exogenously given neutralized the TNF-a in the lung, significantly accelerated mortality, and caused a borderline increase in MoPn counts in the lung by culture in nul+ mice. Lipopolysaccharide-induced TNF-oa activity or injections of recombinant murine TNF-a significantly but modestly protected nul+ mice against MoPn-induced mortality. TNF-oa is produced in vivo during C. trachomatis infection and plays a role in host defense.
102 to 105 inclusion-forming units in a volume of 0.05 ml (4). The diluent was McCoy modified 5A medium (Difco Laboratories, Detroit, Mich.). RIA for murine TNF-a. Radioimmunoassay (RIA) was performed as described previously (24) and was specific for murine TNF-a. The algorithm used for fitting standards and calculating unknowns was the four-parameter logistic, an iterative weighted linear regression with proper weighting from accumulation of a historical curve (16, 17). Values less than 200 pg/ml were considered not significant. TNF-a cytotoxicity assay. The TNF-a cytotoxicity assay was a modification of an assay previously described (14, 18, 24). Briefly, L929 tumorigenic fibroblasts were seeded at 2 x 104 cells per well in a 96-well microdilution plate in a 200-,ul volume of Dulbecco modified Eagle medium containing 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah), 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml, for 18 to 24 h at 37°C in 5% CO2. The medium was removed, and 100 ,ul of fresh medium containing 2 ,ug of Actinomycin D per ml was added. A 100-pld aliquot was added to each assay well. Negative-control (spontaneouslysis) wells had 100 ,ul of medium added, while positivecontrol (maximum-lysis) wells had 100 ,ul of saponin-lysing solution added to lyse all L929 cells in the well. Each sample was tested in triplicate in the microdilution plate. Plates were then incubated for 24 h at 37°C in 5% CO2. A 20-pd volume of a 5-,ug/ml solution of MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (Sigma Chemical Co., St. Louis, Mo.) was added to each well of the microdilution plate. The plates were then incubated for 4 h at 37°C in 5% CO2. The medium was poured off the plates, and 100 pld of an isopropyl alcohol solution containing 0.33 ml of concentrated HCl per 100 ml of isopropyl alcohol was added. The optical density (OD) of the wells was then read at 600 nm on an enzyme immunoassay plate reader. In the assay, 1 U of TNF-a activity was defined as the point at which 50% of the L929 cells were lysed, and minimal detectable levels were 5 U/ml. The biological activity of samples was confirmed to be TNF-a by neutralizing samples with anti-TNF-ot antibody
In previous studies, we described a model of murine pneumonia that employed the mouse biovar of Chlamydia trachomatis (mouse pneumonitis agent [MoPn]) (27-29). In this model, the heterozygous nul+ mouse is more resistant to MoPn than its athymic nude littermates (nulnu), and resistance can be adoptively transferred to the nulnu mouse by T cells (27). By using this model, we showed previously that gamma interferon (IFN--y) is produced by MoPn-infected nul+ but not nulnu mice in response to MoPn antigen (4, 5) in vitro and that this T-cell-dependent cytokine plays a role in vivo in host defenses against MoPn as well (25). Sarov et al. (13, 20, 21) recently proposed that tumor necrosis factor alpha (TNF-a) is able to inhibit replication of C. trachomatis in vitro. IFN-13 appears to play a significant role in the antichlamydial effect of TNF-a (21). We showed previously, in our murine pneumonia model, that whole spleen cells from both infected and uninfected nul+ and nulnu mice are able to produce TNF-a in vitro in response to MoPn antigen (24). These data, combined with those of Sarov et al. (13, 20, 21), suggested that TNF-a might provide a nonspecific host defense mechanism in vivo against C. trachomatis. The studies reported here show that TNF-a is produced in the lung during infection with MoPn and that TNF-a plays a role in vivo in host defense against MoPn. MATERIALS AND METHODS Mice. Specific-pathogen-free nul+ and nulnu mice on a BALB/c background were bred and maintained under barrier conditions at the Audie L. Murphy Veteran's Administration Hospital, San Antonio, Tex., and were used in these and earlier studies (4, 26). These mice were free of pathogenic bacterial and all viral contamination as assessed by culture and serology (4, 26). MoPn. MoPn, the mouse biovar of C. trachomatis, was obtained and maintained in embryonated hen eggs as previously described (4, 24). Mice were infected intranasally with *
Corresponding author. 1572
VOL. 58, 1990
(Genzyme Inc., Boston, Mass.) and observing a concomitant loss of cytotoxicity in the assay. Preparation of lung samples for determination of in vivo TNF-ot production. Whole-lung homogenates were prepared
from both infected and noninfected mice. Mice were challenged with MoPn and then were killed by cervical dislocation at the appropriate times. The inoculum was diluted in McCoy modified 5A medium. Control, uninfected mice received a mock challenge of McCoy medium. The pleural cavity was exposed, and the lungs were excised. The lungs were then placed in a tissue homogenizer along with 1.5 ml of RPMI 1640 containing 10% fetal bovine serum, 100 p.g of vancomycin per ml, and 50 ,g of gentamicin sulfate (medium) per ml. The lungs were homogenized thoroughly, and the homogenates were centrifuged at 1,000 x g for 10 min. The supernatants were removed, filtered, divided into aliquots, and stored at -70°C. For some samples, the lungs were perfused and lavaged prior to homogenization. After cervical dislocation, the pleural and peritoneal cavities were exposed. The inferior vena cava was then cut just posterior to the liver. A 6-ml volume of medium with 10 U of preservative-free heparin per ml was injected into the right ventricle by using a 26-gauge needle on a 12-ml syringe. The trachea was then exposed, and the lungs were lavaged with 3 ml of medium. Approximately 2 ml of the bronchioalveolar lavage fluid was recovered, subsequently filtered, divided into aliquots, and stored at -70°C. Treatment of mice with anti-TNF-a in vivo. Initial doseresponse experiments established that 105 neutralizing units of rabbit polyclonal antibody to murine TNF-a (anti-TNF-a) (Genzyme) was the minimal effective dose. Unless otherwise stated, all mice in mortality studies were given four doses intravenously of 105 neutralizing units of anti-TNF-a on days 0, 1, 3, and 5, with MoPn infection on day 0. Control mice were given normal rabbit serum (NRS). Neither anti-TNF-a nor NRS contained azide and were not heat inactivated prior to injection. Mortality was followed daily. In some experiments, lungs were harvested for quantitative culture of MoPn or for MoPn antigen quantitation. Quantitative culture of MoPn. Quantitative culture of MoPn in the lung was performed in McCoy cell monolayers as previously described (25). Quantitation of MoPn antigen. Quantitative MoPn antigen determinations were performed by using an enzyme-linked immunosorbent assay kit (Ortho Diagnostic, Inc., Raritan, N.J.). Lungs were removed, homogenized in 1.0 ml of medium, and frozen at -70°C until assay. After thawing, the homogenates were diluted further in medium and processed for assay according to the instructions of the manufacturer. A standard curve was run with each determination by using purified MoPn elementary bodies (EBs). An EB protein dose response between 10 and 200 ng of protein per ml was detected by a linear increase in OD. The standard curve, however, varied between determinations. Therefore, for statistical evaluations, all groups to be compared were run simultaneously, and the data were expressed as a ratio of experimental OD values to the OD values of uninfected
lungs.
1573
TNF-a IN HOST DEFENSE AGAINST C. TRACHOMATIS
Treatment of mice with LPS or TNF-a in vivo. To determine the in vivo effect of bacterial lipopolysaccharide (LPS) (Escherichia coli 0111:B4; Sigma) or recombinant murine TNF-a (rm TNF-a; lot number 4296-17; 1.2 x 107 U/mg; graciously supplied by Howard Jaffe, Genentech, Inc., South San Francisco, Calif.), the following treatments were carried out. Mice were given single doses of 100 ,g of LPS
TABLE 1. TNF-a determined by RIA in mouse whole-lung preparations after MoPn infection Day postinfection
0 3 7
10 a
b
TNF-a
(pg/ml) in micea:
nu/nu
