Role of TNF-α in Pulmonary Host Defense in Murine Invasive Aspergillosis Borna Mehrad, Robert M. Strieter and Theodore J. Standiford This information is current as J Immunol 1999; 162:1633-1640; ; of September 24, 2021. http://www.jimmunol.org/content/162/3/1633 Downloaded from References This article cites 29 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/162/3/1633.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 24, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Role of TNF-a in Pulmonary Host Defense in Murine Invasive Aspergillosis1 Borna Mehrad, Robert M. Strieter, and Theodore J. Standiford2 Invasive pulmonary aspergillosis is a common and devastating complication of immunosuppression, whose incidence has increased dramatically in tandem with the increase in the number of immunocompromised patients. Given the role of TNF-a in other pulmonary infections, we hypothesized that TNF-a is an important proximal signal in murine invasive pulmonary aspergillosis. Intratracheal challenge with Aspergillus fumigatus conidia in both neutropenic (cyclophosphamide-treated) and nonneutropenic BALB/c mice resulted in the time-dependent increase in lung TNF-a levels, which correlated with the histologic development of a patchy, peribronchial infiltration of mononuclear and polymorphonuclear cells. Ab-mediated neutralization of TNF-a resulted in an increase in mortality in both normal and cyclophosphamide-treated animals, which was associated with increased lung fungal burden as determined by histology and as quantified by chitin content. Depletion of TNF-a resulted in a reduced lung Downloaded from neutrophil influx in both normal and cyclophosphamide-treated animals, which occurred in association with a decrease in lung levels of the C-X-C chemokine, macrophage inflammatory protein-2 and the C-C chemokines macrophage inflammatory pro- a a tein-1 and JE. In cyclophosphamide-treated animals, intratracheal administration of a TNF- agonist peptide (TNF70–80) 3 days before, but not concomitant with, the administration of Aspergillus conidia resulted in improved survival from 9% in control mice a to 55% in TNF70–80-treated animals. These studies indicate that TNF- is a critical component of innate immunity in both immunocompromised and immunocompetent hosts, and that pretreatment with a TNF-a agonist peptide in a compartmentalized http://www.jimmunol.org/ fashion can significantly enhance resistance to A. fumigatus in neutropenic animals. The Journal of Immunology, 1999, 162: 1633–1640. nvasive aspergillosis is a feared complication of immunode- and IL-1 (13, 14), as well as cytokines that promote Th-1 pheno- ficiency. Its incidence has increased dramatically over the type immune responses (15). I past 2 decades, in tandem with an increase in the number of TNF-a is a 17-kDa cytokine secreted predominantly by various immunocompromised hosts (1, 2). Invasive pulmonary aspergillo- macrophage populations, including alveolar macrophages. It has sis, the commonest form of the disease (3), carries a crude mor- shown to be a critical proximal signal in the initiation and main- tality rate of .80% with current therapy (4). Aspergillus species tenance of innate pulmonary immunity in animal models of pneu- by guest on September 24, 2021 are the second commonest fungal pathogen in immunocompro- monia (16, 17) and human pneumonia (18). In the single study mised hosts, and A. fumigatus is responsible for 90% of cases of examining the role of TNF-a in invasive aspergillosis in vivo, invasive aspergillosis. immunocompetent animals administered A. fumigatus conidia i.v. The respiratory tract is the portal of entry in the majority of had improved survival when treated with systemic murine rTNF-a cases of human invasive aspergillosis (3). It is postulated that in- (19). The role of TNF-a in pulmonary host defense against A. nate immunity is the principal pathway by which A. fumigatus is fumigatus has not been defined. cleared from the lung. This innate response consists of alveolar Given that TNF-a has been shown to be a critical mediator of macrophages, which represent the first line of defense against innate immunity against several respiratory pathogens, attempts conidia entering the alveolus, and recruited neutrophils. Neutro- have been made to administer TNF-a to augment innate and ac- phils are believed to kill conidia that have survived to form hyphae quired host responses. However, the therapeutic administration of and cause invasive infection (5). Various cells of macrophages TNF-a is limited by substantial dose-related toxicity, particularly lineage can engulf and kill Aspergillus conidia in vitro (6–8) and when this cytokine is administered systemically. A TNF-a agonist in vivo (9, 10). Both macrophages (6, 11) and polymorphonuclear peptide composed of the 11 amino acids that constitute the binding cells (5, 12) have been shown to damage hyphae in vitro. When site of native human TNF-a to its receptors (referred to as 3 exposed to conidia in vitro, cells of macrophage lineage secrete TNF70–80) has recently been characterized (20, 21). Binding of a a inflammatory mediators, including the proximal cytokines TNF- TNF70–80 to TNF- receptors (both p55 and p75) has been shown to mediate many leukocyte-activating effects of native TNF-a, including stimulation of neutrophils for enhanced pro- Department of Medicine, Division of Pulmonary and Critical Care Medicine, Uni- versity of Michigan Medical School, Ann Arbor, MI 48109 tease release and respiratory burst, and enhancement of neutro- phil phagocytic activity and killing. When administered sys- Received for publication July 6, 1998. Accepted for publication October 8, 1998. temically, TNF70–80 was associated with less toxicity than that The costs of publication of this article were defrayed in part by the payment of page a charges. This article must therefore be hereby marked advertisement in accordance observed with native TNF- , due in part to the fact that this with 18 U.S.C. Section 1734 solely to indicate this fact. peptide did not alter adhesive properties of the endothelium 1 This work was supported in part by National Institutes of Health Grants HL57243, (20). This peptide has been successfully administered directly HL58200, and P50HL60289. 2 Address correspondence and reprint requests to Dr. Theodore J. Standiford, Division 3 a a of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Abbreviations used in this paper: TNF70–80, TNF- agonist peptide; MIP-1 ; mac- 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0360. rophage inflammatory protein-1a; MIP-2, macrophage inflammatory protein-2; i.t., E-mail address: [email protected] intratracheal; MPO, myeloperoxidase; CCR-1, C-C chemokine receptor-1. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 1634 TNF-a IN MURINE INVASIVE ASPERGILLOSIS into the lungs of mice with Gram-negative bacterial pneumonia, was perfused with PBS via the right ventricle. For histologic examination, lungs were perfused with 1 ml of 4% paraformaldehyde in PBS, inflated although the beneficial effects of TNF70–80 on bacterial clear- ance and survival were only observed when the peptide was with 1 ml of 4% paraformaldehyde in PBS via the trachea, and then excised en bloc. Lungs for various assays were perfused with 1 ml of PBS con- administered 3 or 7 days before, but not concomitant with, bac- taining 5 mM EDTA, removed, frozen in liquid nitrogen, and stored at terial challenge (22). 220°C until the day of the assay. Lungs for cytokine and myeloperoxidase In the current study we hypothesized that TNF-a is an important (MPO) assays were homogenized in 1 ml of 23 complete protease inhib- proximal signal in murine invasive pulmonary aspergillosis. We itor mixture buffer (Boehringer Mannheim, Mannheim, Germany) in PBS a using a tissue homogenizer (Biospec Products, Bartlesville, OK). A 900-ml tested this hypothesis by assessing the production of TNF- in the aliquot of PBS was added to 900 ml from each sample, sonicated for 10 s, lungs of normal and immunocompromised mice challenged with and centrifuged at 500 3 g for 10 min. Supernatants were passed through A. fumigatus conidia. We next examined the effect of TNF-a de- a 0.45-mm pore size filter (Gelman, Ann Arbor, MI) and stored at 4°C for pletion on survival, the burden of fungal organisms, and the host cytokine ELISA. inflammatory response. Finally, the effects of intrapulmonary de- Lung chitin assay livery of native TNF-a or TNF on survival in cyclophos- 70–80 Given that molds (including the Aspergillus species) do not reliably form phamide-treated animals were determined. reproductive units in tissue, we employed an assay for chitin to measure the burden of organisms in lungs. Chitin, a component of the hyphal wall, is Materials and Methods absent from mammalian cells and conidia. The assay was adapted from a Reagents previously described method (24). Lungs were homogenized in 5 ml of distilled water and centrifuged (1500 3 g, 5 min, 20°C). The supernatants a a Polyclonal anti-murine TNF- , MIP-1 , JE, MIP-2, and KC Abs used in were discarded, and pellets were resuspended in sodium lauryl sulfate (3%, Downloaded from the ELISAs were produced by immunization of rabbits with murine re- w/v) and heated at 100°C for 15 min.