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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1263-1268, (2002)
© 2002 American Thoracic Society

Original Article

Transient Lung-Specific Expression of the Chemokine KC Improves Outcome in Invasive Aspergillosis

Borna Mehrad, Maria Wiekowski, Brad E. Morrison, Shu-Cheng Chen, Elizabeth C. Coronel, Denise J. Manfra and Sergio A. Lira

Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Texas Southwestern Medical Center, Dallas, Texas; and Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey

Correspondence and requests for reprints should be addressed to Dr. Borna Mehrad, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390–9034. E-mail: borna.mehrad{at}utsouthwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Invasive aspergillosis is a common and devastating pneumonia in immunocompromised hosts. Neutrophils are critical for defense against this infection, and ELR+ CXC chemokines are potent neutrophil chemoattractants. We hypothesized that transient lung-specific overexpression of one such ligand, KC, in mice with invasive aspergillosis improves the outcome of disease. We generated mice in which transgenic expression of KC was limited to the lungs and occurred only upon exposure to tetracycline analogues, and we exposed them to doxycycline after the onset of invasive aspergillosis. Transgenic mice had a threefold greater survival, a 74% lower lung fungal burden, a greater magnitude of lung KC induction, and an earlier and higher peak of lung neutrophil influx compared with wild-type mice. In addition to a higher number of neutrophils, we found a 1.8-fold higher number of monocytes–macrophages in the lungs of transgenic mice as compared with wild-type mice. Furthermore, transgenic mice had greater lung expression of interferon-{gamma} and interleukin-12 in response to infection, suggesting that transgenic expression of KC indirectly regulated the expression of other cytokines associated with improved host defense against this pathogen. Taken together, these data suggest that overexpression of KC in the lung in the setting of established invasive aspergillosis results in improved host defense and outcome of disease.

Key Words: fungi • neutrophils • pneumonia • transgenic mice


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Among the broad spectrum of infections that occur in immunocompromised patients, invasive pulmonary aspergillosis is a devastating illness, characterized by an increasing incidence (1, 2) and a case fatality rate that remains at greater than 50% (3). Despite recent advances in available antifungal agents (4), current therapy does not prevent death from invasive aspergillosis in up to two-thirds of afflicted patients (3). Given that the vast majority of cases occur in immunocompromised hosts, therapy directed at modulating the immune response to the pathogen seems logical and represents a potentially appealing alternative or adjunctive approach to current modes of therapy, which center on administration of fungicidal antibiotics and surgical resection of infected tissues.

The CXC chemokines are a family of structurally related peptides secreted by somatic cells that are integral to the homing of various leukocyte populations to injured tissues. A subset of the CXC chemokines, defined by the presence of the amino acid sequence Glu-Leu-Arg (ELR) preceding the CXC motif, is specific and is a potent chemotactic factor for neutrophils in diverse in vitro and in vivo settings. The ELR+ CXC chemokines include human interleukin (IL)-8 (CXCL8) and growth-related oncogene (GRO)-chemokines (CXCL1–3), and their murine counterparts, KC and macrophage inflammatory protein-2. The expression of KC, in particular, is markedly induced in response to numerous inflammatory stimuli in vitro (5–8) and in the setting of tissue infection or inflammation in vivo (9–12). Importantly, in vivo transgenic expression of KC in the absence of inflammatory stimuli has been shown to mediate the influx of neutrophils without inducing neutrophil activation or degranulation (13, 14).

Neutrophil deficiency or dysfunction has long been recognized as the most important clinical risk factor for invasive aspergillosis (4), and several lines of in vitro and in vivo animal evidence have underscored the critical role of neutrophils in host defense against Aspergillus species (reviewed in 15). In this context, we have previously shown that impaired induction of the ELR+ CXC chemokines results in markedly poorer outcome in invasive aspergillosis (16). Furthermore, the neutralization of CXCR2, the sole receptor for ELR+ CXC chemokines in the mouse, resulted in dramatic increases in severity of infection and mortality, associated with a marked impairment of influx of neutrophils into the lungs (17). Although these experiments established the critical role of the ELR+ CXC chemokines in host defense against Aspergillus fumigatus, the question of whether these ligands have any therapeutic value in invasive aspergillosis remains unanswered. Specifically, therapeutic relevance requires proof that (1) the ligand in question is overexpressed above levels normally encountered in the setting of infection, (2) such overexpression has an additive physiologic effect above that seen in response to the infection, and (3) a beneficial effect on the outcome of infection is demonstrable even when the overexpression of ligand temporally antecedes the onset of infection.

In this study, we hypothesized that the transient overexpression of KC in the lungs in the setting of established invasive aspergillosis improves the outcome of the infection. We tested this hypothesis by generating conditional transgenic animals in which the transgenic expression of KC was limited to the lungs and could be tightly regulated by exposing animals to tetracycline analogs. We then examined the outcome and severity of invasive aspergillosis, as well as the components of host response to A. fumigatus, in the setting of transient and compartmentalized overexpression of KC.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Mice
The tetracycline-inducible system used in these experiments consisted of "activator" and "reporter" transgenes (18, 19) (see online data supplement for additional details of methods); the activator construct encoded the tet-activator protein, under control of the airway-specific promoter CC10, thus limiting its expression to the lungs. In the presence of the tetracycline analogue doxycycline, tet-activator protein binds the tetracycline responsive DNA element in the "reporter" construct, driving the transcription of the reporter transgenes, KC and LacZ (Figure 1A) . We chose LacZ as an indicator of reporter activation because its product, ß-galactosidase, is an intracellular enzyme and its transgenic expression has previously been shown not to result in a measurable inflammatory response (20, 21). DNA containing each transgene was microinjected into mouse eggs and transferred into foster mothers (22). Transgenic offspring were identified by polymerase chain reaction of genomic DNA. Independent lines, transgenic for either the reporter or activator constructs, were intercrossed to produce animals transgenic for both, hereafter referred to as double-transgenic animals (Figure 1B). All animals were maintained under specific pathogen-free conditions and in compliance with institutional animal care and research committees.



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  		Figure 1. The lung-specific tetracycline-inducible system. (A) Activator mice express the tet-activator protein (rtTA) under control of the Clara cell–specific CC10 promoter, which limits its expression to the lung. In the presence of doxycycline, rtTA binds to a tetracycline responsive element (TRE) on the reporter transgene and activates transcription of LacZ and KC from the cytomegalovirus (CMV) minimal promoter. (B) Transgenic animals were identified using a six-primer polymerase chain reaction protocol. Activator mice carry the rtTA gene, reporter mice the LacZ gene, and double-transgenic mice both transgenes. Primers for the endogenous ZP3 gene (ZP3) were included in each polymerase chain reaction sample as an internal control for the amplification reaction.

 
Transient Neutrophil Deficiency and Inoculation of A. fumigatus Conidia
RB6-8C5 is a rat antimouse monoclonal immunoglobulin 2b against Ly-6G, an antigen expressed on mature murine granulocytes but not their precursors. In vivo administration of RB6-8C5 induces transient neutrophil deficiency (17, 23, 24). A. fumigatus strain 13073 (ATCC, Rockville, MD) was used in all infection studies because a murine model of invasive aspergillosis using this strain has been previously characterized (16).

Transgene Activation and Tissue Harvest
Transgene expression was induced by a single injection of doxycycline. To ensure that transgene activation anteceded the onset of infection, doxycycline was administered 6 hours after intratracheal inoculation with A. fumigatus conidia; in preliminary experiments, the earliest transgene product became detectable 24 hours after inoculation with conidia, when invasive disease was histologically detectable (data not shown). To exclude any contributing antifungal or immunomodulatory effect attributable to doxycycline, both transgenic and wild-type groups were treated with doxycycline in all infection experiments. At designated time points, animals were killed, and blood and lungs were removed for various assays. KC levels were measured using a specific enzyme-linked immunosorbent assay kit, and tissues for histology were either fresh frozen for ß-galactosidase histochemistry or were fixed in paraformaldehyde for histologic stains.

Bronchoalveolar Lavage and Lung Cell Suspension
Bronchoalveolar lavage (BAL) and single-cell suspensions of the lungs were prepared as previously described (25). Cells obtained from BAL or a single-cell suspension were counted and differentials cell counts were determined. Flow cytometry was performed on single-cell lung suspensions using cell markers for T cells, B cells, and monocytes/macrophages. Absolute numbers of each leukocyte subtypes were determined by multiplication of the percentage of each type by the total number of leukocytes in that sample.

Lung Chitin Assay and Myeloperoxidase Activity
Molds, including Aspergillus species, do not reproducibly form reproductive units in tissue. Chitin, a component of the hyphal wall, is absent from fungal conidia and from mammalian tissues. We therefore employed a previously described assay for chitin to measure the burden of organisms in the lungs (16). Myeloperoxidase (MPO) activity was measured as a marker of neutrophil sequestration in the lungs, as described previously (26).

Statistical Analysis
Survival data were compared using the Fisher's exact test. All other data were expressed as mean ± SEM and were compared using a two-tail Mann-Whitney test. Probability values were considered statistically significant if less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Transgenic Mice
Double-transgenic mice were phenotypically normal and had a normal life span. To screen for the absence of reporter transgene expression at baseline and for its inducibility, double-transgenic mice were injected with doxycycline or saline, were killed after 24 hours, and had organs examined for ß-galactosidase activity. Macroscopically, lungs from doxycycline-treated animals showed strong ß-galactosidase activity, whereas no activity could be detected in organs from saline-treated animals (Figures 2A and 2B) . Lung cryosections showed ß-galactosidase activity to be limited to Clara cells and absent from other lung tissue (Figures 2C and 2D). ß-Galactosidase activity was also absent from other organs of double transgenic animals and from all organs in single-transgenic mice (data not shown).



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  		Figure 2. Lung LacZ expression in double-transgenic animals. Blue staining indicates expression of the lacZ transgene in all panels. (A and B) Lobes of lung of double-transgenic mice without doxycycline treatment (-Dox) and 24 hours after administration of doxycycline (+Dox). (C and D) Micrographs of lung cryosections of untreated (-Dox) and doxycycline-treated (+Dox) double-transgenic mice (magnification x400). ß-Galactosidase activity in D is limited to airway lining cells with abundant cytoplasm, features characteristic of Clara cells.

 
We next examined the kinetics of KC transgene induction by measuring KC levels in BAL fluid at various time points after a single dose of doxycycline (Figure 3A) . There was a dramatic 58-fold induction of BAL KC levels 24 hours after doxycycline injection, which returned to baseline by 72 hours after injection. To investigate the biologic consequences of KC induction, we next quantified BAL neutrophil numbers at various time points after doxycycline injection (Figure 3B). There was a significant increase in airspace neutrophil numbers 24 hours after doxycycline injection, which waned over the subsequent 48 hours, indicating that the induced KC was biologically active. We next evaluated lungs of double-transgenic animals histologically for evidence of lung injury induced by the influx of neutrophils (Figure 4) . Double-transgenic animals had normal lung histology after treatment with saline, whereas 24 hours after treatment with doxycycline, there was a marked accumulation of a pure population of neutrophils around bronchi and bronchioles. Importantly, there was no histologic evidence of tissue damage to the airway, alveolar, or vascular structures despite the influx of neutrophils in doxycycline-treated animals.



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  		Figure 3. Kinetics of doxycycline-induced KC expression and neutrophil accumulation in lung airspace. Concentrations of KC (A) and numbers of neutrophils (B) were measured in BAL fluid at various time points after doxycycline administration in double-transgenic mice. Data points represent mean ± SEM, experimental n = 4 to 7 per time point. *p < 0.05 compared with values at Time 0.

 


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  		Figure 4. Effect of transgenic KC expression in the lung on lung histology. Lungs of doxycycline-treated (+Dox) double-transgenic animal were collected at 24 hours after treatment and were stained with hematoxylin and eosin (magnification x400). Lungs from untreated transgenic animals (-Dox) are shown as control. Both panels show lung parenchyma and a terminal bronchiole. Clara cells are identified as nonciliated airway lining cells with abundant cytoplasm. Arrows indicate neutrophil infiltration around the airway.

 
Effect of Transient Compartmentalized Overexpression of KC on the Outcome of Invasive Aspergillosis
Having established that biologically active KC can be conditionally induced in a time-dependent and compartmentalized manner in the lungs of double-transgenic mice, we proceeded to examine the outcome of invasive aspergillosis in these animals. Because normal mice are naturally resistant to Aspergillus infection, we used a previously characterized model of invasive aspergillosis in the setting of transient antibody-mediated neutrophil deficiency, which closely resembles human disease (17, 27). To ensure that the transgenic expression of KC did not precede the onset of infection, animals were injected with 500 µg of doxycycline intraperitoneally 6 hours after intratracheal challenge with A. fumigatus conidia. To exclude any direct antimicrobial or immunomodulatory effect of doxycycline that was unrelated to transgene activation, doxycycline was administered to both wild-type and double-transgenic animals. In survival studies, double-transgenic mice had a threefold higher survival rate than wild-type infected animals (Figure 5A) . A similar reduction in mortality was observed when survival studies were repeated, comparing double-transgenic animals with or without doxycycline treatment (data not shown).



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  		Figure 5. Effect of transient transgenic expression of KC in the lung on outcome of invasive aspergillosis. Wild-type and double-transgenic mice were treated with intraperitoneal RB6-8C5 monoclonal antibody, inoculated with intratracheal A. fumigatus (2–3 x 106 conidia) 1 day later, followed by intraperitoneal doxycycline 6 hours after inoculation. (A) A survival study. Data pooled from two separate experiments (n = 20–22 animals per group). (B) Lung chitin content 2 days after fungal challenge. Data shown represent mean ± SEM of five to six animals per group. *p < 0.05 compared with wild-type infected animals.

 
To establish that this reduced lethality in doxycycline-treated transgenic mice was due to reduced severity of infection, we next measured the burden of fungal hyphae in the lungs of infected doxycycline-treated wild-type and double-transgenic mice 2 days after intrapulmonary inoculation with A. fumigatus conidia (Figure 5B). Double-transgenic animals were found to have a 74% reduction in lung fungal content, indicating substantially improved host defenses against the pathogen in transgenic animals.

Kinetics of KC Expression and Lung Neutrophil Accumulation in Response to Invasive Aspergillosis in Transgenic Animals
To address the mechanism of protection conferred to transgenic mice, we next compared the kinetics of KC expression in the lungs of transgenic and wild-type mice in the setting of invasive aspergillosis. Infected animals from both groups were given a single intraperitoneal injection of doxycycline 6 hours after infection and were killed at designated time points for measurement of KC concentration in blood and lung homogenates (Figure 6A) . Although KC was markedly induced in the lungs in both groups in response to infection, levels were more than 50% higher in double-transgenic mice as compared with wild-type mice on Day 1 after inoculation, indicating that the activation of the transgene resulted in at least an additive expression of lung KC above induction of native KC. Importantly, lung homogenate KC levels decreased successively on Days 2 and 4 after infection in transgenic mice but remained persistently elevated in wild-type lungs, presumably in response to ongoing infection. Blood KC levels remained unchanged in both groups throughout the infection, indicating a strictly compartmentalized chemokine response.



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  		Figure 6. Kinetic KC expression and MPO activity in lungs of wild-type and double-transgenic mice with invasive aspergillosis. Wild-type and double-transgenic mice were treated with intraperitoneal RB6-8C5 monoclonal antibody, inoculated with intratracheal A. fumigatus (2–3 x 106 conidia) 1 day later, followed by intraperitoneal doxycycline 6 hours after inoculation. (A) KC levels in blood and lung homogenates. (B) Lung MPO activity as a marker for neutrophil presence in the lung. Data shown represent mean ± SEM of five to six animals per group at each time point. *p < 0.05 as compared with wild-type infected animals at the same time point.

 
We next sought to quantify the kinetics of lung neutrophil influx in invasive aspergillosis in the setting of transient transgenic expression of KC. We therefore measured lung MPO activity as a surrogate for the presence of neutrophils in infected wild-type and double-transgenic animals after administration of doxycycline (Figure 6B). We found evidence of induction of lung MPO activity in both groups, but MPO activity was approximately twofold higher on Days 1 and 2 of infection in double-transgenic mice as compared with wild types. To determine the location of these neutrophils anatomically, we killed doxycycline-treated wild-type and double-transgenic mice on Day 2 after challenge with Aspergillus conidia and examined the lungs histologically. Although there was evidence of tissue invasion by fungal hyphae in both groups, double-transgenic mice had dense infiltration of inflammatory cells associated with areas of fungal invasion as compared with infected wild-type animals (Figure 7) . Interestingly, the inflammatory infiltrate in double-transgenic animals consisted of both mononuclear cells and neutrophils (Figure 7D).



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  		Figure 7. Effect of transient transgenic expression of KC on lung histopathology in invasive aspergillosis. Lungs were examined histologically on Day 2 after inoculation with 2–3 x 106 A. fumigatus conidia (magnification x400). (A and B) Successive lung sections of doxycycline-treated wild-type mice that were stained with Gomori methanamine silver and hematoxylin and eosin stains, respectively. (C and D) Similarly, stained successive lung sections of doxycycline-treated double-transgenic mice. In A and C, there is tissue invasion with fungal hyphae, recognized as black branching structures. There is a greater infiltration of neutrophils and mononuclear cells into the adjacent lung in transgenic animals (D) as compared with wild types (B).

 
Effect of Transient KC Expression in the Lung on Leukocyte Influx and Cytokine Levels in Invasive Aspergillosis
To quantify the histologic observation of increased accumulation of both neutrophils and monocytes/macrophages in the lungs of transgenic mice, we next examined whole-lung single suspensions 48 hours after infection in doxycycline-treated wild-type and double-transgenic animals. In addition to confirming the increased number of neutrophils, double-transgenic mice were found to have an 80% increase in the number of lung monocyte/macrophages as compared with wild-type mice (Figure 8) . Given that KC is not directly chemotactic for monocytes/macrophages, we sought to determine whether the KC-induced influx of neutrophils into the lungs resulted in enhancement of the inflammatory cytokine response to the infection, which may, in turn, have resulted in increased recruitment of monocytes/macrophages. We therefore examined lung homogenates for interferon-{gamma} and IL-12 levels in doxycycline-treated wild-type and double-transgenic mice with invasive aspergillosis (Figure 9) . On Day 2 of infection, we found substantially higher lung levels of both interferon-{gamma} and IL-12 in transgenic animals as compared with wild-type animals (44% and 117% increase, respectively). A possible explanation for these results is that the in vivo overexpression of KC may regulate the expression of other cytokines involved in host defense against this pathogen.



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  		Figure 8. Effect of transient transgenic expression of KC on leukocyte numbers in whole lung cell suspensions 2 days after intratracheal challenge with 2–3 x 106 A. fumigatus conidia. Data shown represent mean ± SEM of experimental n = 6 for each group. *p < 0.05 as compared with leukocyte numbers in infected wild-type animals.

 


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  		Figure 9. Effect of transient transgenic expression of KC on lung interferon-{gamma} and IL-12 levels 2 days after intratracheal challenge with 2–3 x 106 A. fumigatus conidia. Data represent mean ± SEM of experimental n = 5 for each group. *p < 0.05 as compared with the concentration in infected wild-type animals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Invasive pulmonary aspergillosis is a fairly common infection with a very poor outcome despite currently available therapy. Because neutrophils are critical to the host defense against Aspergillus species, we used a transgenic model to evaluate the effect of transient overexpression of KC, a potent neutrophil chemoattractant, on the outcome of the infection. Our results indicate that the transient transgenic expression of KC in the setting of established infection improves survival and clearance of the pathogen. This effect was associated with increased numbers of neutrophils and monocytes/macrophages in the lungs, as well as higher lung levels of IFN-{gamma} and IL-12.

The ELR+ CXC chemokines have been shown to mediate neutrophil chemotaxis in diverse in vitro and in vivo settings. In the setting of infections in particular, we have previously reported that neutralization of CXCR2, the common receptor for mouse ELR+ CXC chemokines, results in impairment of neutrophil chemotaxis and poor outcome in invasive aspergillosis (17). The interaction of ELR+ CXC chemokines with CXCR2 has since been shown to be essential for neutrophil-mediated host defenses in several other animal models of infections, including bacterial pneumonias (28, 29), experimental brain abscess (12), urinary tract infection (11), and toxoplasmosis, a protozoal infection (30). Interestingly, in a model of allergic airway response to Aspergillus species, CXCR2-/- mice were found to have reduced airway hyperreactivity and Th2 responses without evidence of invasive pneumonia as compared with wild-type mice, indicating that this receptor is also involved in orchestrating acquired immunity in the lung (31).

Prior studies of constitutive organ-specific transgenic expression of KC have shown that localized expression of KC mediates in vivo neutrophil migration to various organs, including the skin epidermis, thymus, and the brain (13, 14). In the lung, constitutive overexpression of KC resulted in an increased number of lung neutrophils throughout life (14). In the setting of infection, animals with constitutive lung-specific transgenic KC expression had improved outcomes in pneumonias as compared with wild-type animals (17, 28). Similar to the findings of this article, isolated transgenic expression of KC in the lung in these studies was associated with a modest influx of neutrophils into the lungs in the absence of inflammatory stimuli but resulted in a particularly vigorous recruitment in response to intrapulmonary pathogens. This finding is presumably attributed to the concomitant activation of other components of the inflammatory cascade in response to the pathogen, such as induction of proximal cytokines and upregulation of endothelial adhesion molecules (32).

Importantly, in studies of constitutive transgenic expression of KC in infection models, it was not possible to determine whether the improved clearance of pathogens was due to improved recruitment of neutrophils in response to the pathogens or to the pre-existing pool of neutrophils already present in the lungs before the onset of infection. As a result, the value of KC as a therapeutic modality could not be addressed by these studies. In this context, this study shows that transient transgenic KC expression, which began after the onset of invasive aspergillosis, was sufficient to improve survival and clearance of the pathogen. Furthermore, the transient nature of ligand expression achieved in this system side stepped issues of receptor desensitization or loss of ligand gradient, as occur with noncompartmentalized or prolonged transgenic expression of ligand (14, 33).

An unexpected finding in our study was that in addition to neutrophils, infected transgenic animals had a marked increase in lung monocyte/macrophage numbers. Because KC itself is not directly chemotactic for this cell population, we hypothesized that the increased number of lung neutrophils in infected transgenic animals contributed to the improved outcome of infection not only by directly attacking the pathogen, but also via immunomodulatory effects that enhanced the inflammatory response. The increase in lung levels of interferon-{gamma} and IL-12 in infected transgenic animals is of particular interest because these T-1 phenotype cytokines have recently been shown to occur in the context of neutrophil influx in response to several stimuli (34–36). In addition, these cytokines are associated with improved host defense against Aspergillus species (37–40). The observation that, as compared with wild-type infected mice, the transgenic animals had enhanced cytokine and cellular responses in response to a lower burden of pathogens (as documented by lung chitin measurements at the same time point; Figure 4B) underscores the prominent effect of augmented KC expression in host defense in this infection. Although not addressed directly by our study, we speculate that the mechanism by which KC overexpression leads to greater induction of proinflammatory cytokines and recruitment of other leukocyte subsets involves an immunomodulatory role for the neutrophils recruited by greater expression of KC. The observation that the early recruitment of neutrophils may affect the subsequent host response to an inflammatory stimulus has been made in other models of infection (31, 34) and has important implications for therapeutic use of neutrophil chemoattractants. Finally, although our study shows that the beneficial effects of KC overexpression are clearly associated with increased influx of inflammatory cells, our results do not preclude an additional direct antimicrobial activity for KC, as has been demonstrated for some members of the CXC chemokine family (41, 42).

We have previously asserted that members of the ELR+ CXC chemokine family may be important targets in devising immunomodulatory strategies against invasive aspergillosis (17). We have now shown that transient expression of one such ligand, KC, in the lung is both feasible and effective in improving the outcome of invasive aspergillosis, even when it temporally antecedes the onset of pneumonia. Future studies will evaluate means of localization of expression of such ligands to the site of infection.


   Acknowledgments
 
The authors gratefully acknowledge the excellent technical assistance of Petronio Zalamea and Brian Wilburn.


   FOOTNOTES
 
Supported in part by National Institutes of Health grant K08HL04220 (B.M.) and American Lung Association grant RG-005-N (B.M.)

This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form April 25, 2002; accepted in final form May 30, 2002


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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