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*
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033
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Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; and
§
Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Invasive aspergillosis has been noted to occur in the setting of agranulocytosis since its original description (7), and deficiencies in neutrophil number or function have since been recognized to be strongly associated with the development of invasive aspergillosis in diverse populations of patients (8, 9, 10, 11). In vitro studies have shown that normal neutrophils efficiently damage Aspergillus hyphae (12, 13). In contrast, dysfunctional neutrophils, such as those treated with corticosteroids or those isolated from patients with myeloperoxidase (MPO)3 deficiency or chronic granulomatous disease are inefficient at killing Aspergillus hyphae (14, 15). In vivo animal studies have established a causative relationship between neutrophil dysfunction or deficiency and the development of invasive aspergillosis (16, 17). However, factors that regulate neutrophil recruitment and activation in invasive aspergillosis have not been characterized.
CXC chemokines are a family of related 8- to 10-kDa polypeptide molecules that are potent chemotactic factors for numerous populations of immune cells and are characterized by four conserved cysteine residues at the N terminus, the first two of which are separated by a nonconserved amino acid. The CXC chemokines are further subdivided into those that contain the amino acid sequence glutamic acid-leucine-arginine (the ELR motif) immediately preceding the CXC sequence, and those that do not. The human ELR+ CXC chemokines include IL-8, the growth-related oncogene (GRO) chemokines, epithelial neutrophil-activating protein-78 (ENA-78), neutrophil-activating peptide-2 (NAP-2), and granulocyte chemotactic peptide-2 (GCP-2). Murine ELR+ CXC chemokines include macrophage inflammatory protein-2 (MIP-2), KC, GCP-2, LPS-induced CXC chemokine (LIX), and Lungkine (18, 19). ELR+ CXC chemokines have been shown to mediate neutrophil chemotaxis and activation in vitro (20, 21, 22, 23, 24) and in diverse in vivo settings (25, 26, 27). Several ELR+ CXC chemokines have been shown to be expressed in murine models of bacterial pneumonia (28), but the neutralization of specific chemokines has not altered survival dramatically (29).
Two human ELR+ CXC chemokine receptors have been identified, and are designated CXC chemokine receptor-1 (CXCR1) and CXC chemokine receptor-2 (CXCR2). These receptors are exclusively expressed on the surface of human granulocytes, and are involved in neutrophil chemotactic and degranulation responses (30, 31, 32). CXCR1 binds IL-8 and GCP-2, while CXCR2 binds all ELR+ CXC chemokines with equal avidity (33, 34). The CXCR1 is not expressed in the mouse, but the murine CXCR2 is highly homologous to human CXCR2. Like its human counterpart, murine CXCR2 is promiscuous and has been shown to bind to several murine ELR+ CXC chemokines. Neutrophils from mice with targeted deletion of CXCR2 fail to respond to macrophage inflammatory protein-2 (MIP-2) or KC in vitro, but exhibit normal chemotaxis to C5a (35). In in vivo studies, CXCR2 knockout mice fail to develop tissue neutrophilic infiltrates in response to s.c. urate crystals (36), suggesting that ELR+ CXC chemokines are the major mediator of neutrophil influx in this model, and that CXCR2 is the sole receptor for these ligands. The role of CXCR2 in the setting of infections has not been examined. The presence of multiple ligands and the promiscuity of the receptors have made the study of human ELR+ CXC chemokines difficult. In this context, the mouse system, which lacks CXCR1, provides a simplified system for studying the ligand-receptor interaction of ELR+ CXC chemokines.
In this study, we hypothesized that the interaction of ligands with CXCR2 is a major component of neutrophil-dependent pulmonary host defense against A. fumigatus by mediating neutrophil deployment in response to the organism. To test this hypothesis, we evaluated the outcome and severity of infection in the setting of CXCR2 neutralization, and in the setting of compartmentalized overexpression of KC in the lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal antimurine MIP-2 and KC Abs used in the ELISAs were produced by immunization of rabbits with carrier-free murine rt MIP-2 and KC (R&D Systems, Minneapolis, MN) in multiple intradermal sites with CFA, as previously described (37, 38). Polyclonal goat antimurine CXCR2 Abs used in in vivo neutralization studies were produced by immunization of a goat with murine rt CXCR2 peptide in multiple intradermal sites with CFA. The peptide sequence, Met-Gly-Glu-Phe-Lys-Val-Asp-Lys-Phe-Asn-Ile-Glu-Asp-Phe-Phe-Ser-Gly, is a portion of the seven transmembrane receptor that resides on the cell surface and has previously been shown to be the binding site for ligands (39). Abs were purified over a protein A column and endotoxin contamination excluded by Limulus lysate assay (ICN Biomedical, Costa Mesa, CA). In CXCR2 neutralization experiments, 0.5 ml goat antimurine CXCR2 serum, or control goat serum, was administered i.p. 2 h prior to A. fumigatus administration. In preliminary studies, administration of this Ab did not affect the number of circulating neutrophils over the subsequent 4 days (data not shown). In separate experiments, administration of anti-CXCR2 abrogated the influx of neutrophils into the peritoneum of normal mice in response to exogenous KC (data not shown).
Animals
Specific pathogen-free C57BL/6 mice (6 to 8-wk-old females, The Jackson Laboratories, Bar Harbor, ME) were used in all experiments except those involving KC transgenic animals. Specific pathogen-free transgenic KC mice CC51+/+ (6 to 8-wk-old females) were generated on a B6D2 background by microinjection of fertilized eggs carrying the KC transgene in their genome. The expression of the KC transgene was localized to the lung by incorporating regulating elements of the CC10 gene, which encodes a 10-kDa protein produced by nonciliated bronchial epithelial cells (Clara cells) (40, 41, 42). Age-matched wild-type B6D2 mice served as controls. All mice were housed in specific pathogen-free conditions within the animal care facility at the University of Michigan until the day of sacrifice.
Preparation and use of RB6-8C5 mAb for in vivo neutrophil depletion
RB6-8C5 is a rat anti-mouse monoclonal IgG2b directed against Ly-6G, previously known as Gr-1, an Ag on the surface of murine granulocytes. The Ag expression increases with cell maturity, and is absent from precursor cells. The Ab, originally used for flow cytometry, is a complement-fixing isotype, well suited for in vivo cell depletion (43, 44, 45, 46, 47). The Ab produced by TSD BioServices (Germantown, NY) by i.p. injection of RB6-8C5 hybridoma into nude mice and ascites collection. One hundred micrograms of RB6-8C5 was administered i.p. 1 day prior to challenge with intratracheal (i.t.) A. fumigatus or vehicle. This resulted in peripheral blood neutropenia (absolute circulating neutrophil count <50 cells/µl) by days 1 and 3 after Ab administration in both infected and control animals, with a return of peripheral counts to pretreatment levels by day 5.
Preparation and administration of A. fumigatus conidia
We chose to use A. fumigatus strain 13073 (American Type Culture Collection, Manassas, VA) in our studies, as this strain has previously been shown to induce invasive aspergillosis in immunocompromised mice (48). The organism was grown on Sabouraud dextrose agar plates (Becton Dickinson, Cockeyville, MD) for 7 to 10 days at 37°C. The surface of each plate was then washed with 100 ml of sterile 0.1% Tween 80 (SigmaUltra, St. Louis, MO) in normal saline. The resulting suspension of conidia was filtered through sterile gauze to remove clumps and hyphal debris, and then washed once and resuspended in 4 ml of 0.1% Tween-80. The concentration of Aspergillus conidia in the suspension was determined by a particle counter (Z2 particle analyzer, Coulter, Hialeah, FL). The suspension was then diluted to the desired concentration, and the concentration was again measured prior to administration. In preliminary experiments, the number of particles determined by the particle counter was in close agreement with the number of viable CFUs found by serial dilution and plating of the suspension. On the day of inoculation, each animal was anesthetized with 1.8–2 mg pentobarbital i.p. Using standard aseptic technique, the trachea was exposed and a 30-µl inoculum (A. fumigatus suspension or 0.1% Tween-80) was administered via a sterile 26-gauge needle. The skin incision was closed with surgical staples. Animals were challenged with inocula ranging from 1–2 x 106 to 1–2 x 107 conidia in various experiments.
Lung harvest
At designated time points, the mice were sacrificed by CO2 asphyxiation. The chest cavity was opened aseptically, and the pulmonary vasculature was perfused with PBS via the right ventricle. For histologic examination, lungs were perfused with 1 ml 4% paraformaldehyde in PBS, inflated with 1 ml 4% paraformaldehyde in PBS via the trachea, and then excised en bloc. Lungs for various assays were perfused with 1 ml of PBS containing 5 mM EDTA, removed, frozen in liquid nitrogen, and stored at -20°C until the day of the assay. Lungs for cytokine and MPO assays were homogenized in 1 ml of 2x complete protease inhibitor cocktail buffer (Boehringer Mannheim, Mannheim, Germany) in PBS, using a tissue homogenizer (Biospec Products, Bartlesville, OK). A 900-µl aliquot of PBS was added to 900 µl from each sample, sonicated for 10 s, and centrifuged at 500 x g for 10 min. Supernatants were passed through a 0.45-micron filter (Gelman Sciences, Ann Arbor, MI), and stored at 4°C for cytokine ELISA.
Lung chitin assay
Given that molds (including Aspergillus species) do not reliably form reproductive units in tissue, we employed an assay for chitin to measure the burden of organisms in lungs. Chitin is a component of the hyphal wall that is absent from mammalian cells and conidia. The assay was adapted from a previously described method, which demonstrated a direct correlation between the weight of hyphae and level of chitin, as detected by assay (49). Lungs were homogenized in 5 ml distilled water and centrifuged (1500 x g, 5 min, 20°C). The supernatants were discarded, pellets resuspended in sodium lauryl sulfate (3% w/v), and heated at 100°C for 15 min. Samples were then centrifuged (1500 x g, 5 min, 20°C), pellets washed with distilled water, and resuspended in 3 ml KOH (120% w/v). Samples were then heated at 130°C for 60 min. After cooling, 8 ml of ice-cold ethanol (75% v/v) was added to each sample, and tubes were shaken until ethanol and KOH made one phase. Samples were incubated on ice for 15 min, and 0.3 ml of Celite suspension (supernatant of 1 g of Celite 545 (Fisher Scientific, Pittsburgh, PA) added to 75% ethanol and allowed to stand for 2 min) was added to each. Samples were centrifuged (1500 x g, 5 min, 4°C), and supernatants were discarded. Pellets were washed once with ethanol (40% v/v) and twice with distilled water, and resuspended in 0.5 ml distilled water. Standards, consisting of 0.2 ml distilled water and 0.2 ml glucosamine (10 µg/ml), were made up. A total of 0.2 ml NaNO2 (5% w/v) and 0.2 ml KHS04 (5% w/v) was added to each standard, and 0.5 ml NaNO2 (5% w/v) and 0.5 ml KHS04 (5% w/v) was added to each tissue preparation; all samples were mixed gently for 15 min, and then centrifuged (1500 x g, 2 min, 4°C). Two 0.6-ml aliquots of supernatant from each tissue preparation were transferred to separate tubes. A total of 0.2 ml ammonium sulfamate was added to each tube, and all tubes were shaken vigorously for 5 min. A fresh solution of 3-methyl-2-thiazolone hydrazone HCl monohydrate (50 mg in 10 ml distilled water) was made, and 0.2 ml was added to each tube. Samples were then heated to 100°C for 3 min and cooled. A total of 0.2 ml of FeCl3 . 6H2O (0.83% w/v) was added to each, and OD was measured at 650 nm after 25 min. Chitin content, measured in glucosamine equivalents, was measured by the following formula: chitin content = {[5 x (OD of organ - OD of control organ)]/(OD of glucosamine - OD of water)}.
Lung MPO activity
Lung MPO activity was measured as a marker of neutrophil sequestration, as described previously (50). Briefly, a 100-µl aliquot of each lung homogenate was added to 100 µl of a buffer containing 50 mM potassium phosphate (pH 6.0), 5% hexadecyltrimethylammonium bromide, and 5 mM EDTA. Samples were sonicated for 10 s and centrifuged at 3000 x g for 15 min. The supernatant was mixed 1:15 with a buffer containing 1 M monobasic potassium phosphate, 1 M dibasic potassium phosphate, 3% H202, and o-dianisidine hydrochloride, and read at 490 nm. MPO units were calculated as the change in absorbency over time.
Chemokine ELISA
Murine MIP-2 and KC levels were quantified using a modification
of a double-ligand method, as described previously (51).
Briefly, flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F,
Nunc Roskilde, Denmark) were coated with 50 µl/well of rabbit
anti-cytokine Ab (1 µg/ml in 0.6 M NaCl, 0.26 M
H3BO4, and 0.08 N NaOH, pH 9.6) for 16 h
at 4°C and then washed with PBS, pH 7.5, 0.05% Tween-20 (wash
buffer). Microtiter plate nonspecific-binding sites were blocked with
2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed
four times with wash buffer, and diluted (neat and 1:10) cellfree
supernatants (50 µl) in duplicate were added, followed by incubation
for 1 h at 37°C. Plates were washed four times, followed by the
addition of 50 µl/well biotinylated rabbit amti-cytokine Abs (3.5
µg/ml in PBS, pH 7.5, 0.05% Tween-20, and 2% FCS), and plates
incubated for 30 min at 37°C. Plates were washed four times,
Streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added,
and the plates were incubated for 30 min at 37°C. Plates were washed
again four times and chromogen substrate (Bio-Rad) was added. The
plates were incubated at room temperature to the desired extinction,
and the reaction was terminated with 50 µl/well of 3M
H2SO4 solution. Plates were read at 490 nm in
an ELISA reader. Standards were 1/2 log dilutions of recombinant
murine MIP-2 or KC, from 1 pg/ml to 100 ng/ml. This ELISA method
consistently detected the relevant cytokine at concentrations above 25
pg/ml. The ELISAs did not cross-react with IL-1, IL-2, IL-4, IL-6,
IL-10 or IFN-
. In addition, each ELISA did not cross-react with
other members of the murine CXC or CC chemokine families.
Statistical analysis
Data were analyzed by a Power Macintosh 8600/300 computer using InStat version 2.01 statistical package (GraphPad Software, San Digeo, CA). Survival data were compared using the Fisher’s exact test. All other data were expressed as mean ± SEM and compared using an unpaired two-tail Mann-Whitney (nonparametric) test. Probability values were considered statistically significant if they were less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first determined the time course of neutrophil influx into the
lungs of normal C57BL/6 mice in response to challenge with 1–2 x
107 A. fumigatus conidia. Lungs were harvested
at various time points after challenge for histology and measurement of
MPO activity, a surrogate measure of neutrophil presence. Lung MPO
levels were elevated over baseline at 8 h after inoculation, and
reached a plateau at 24 h (Fig. 1
).
Histology showed evidence of a patchy peribronchial infiltration of
inflammatory cells within both the interstitial and alveolar
compartments at 24 h, which predominantly consisted of
neutrophils, and to a lesser extent, of mononuclear cells (data not
shown). The cellular infiltrate was resolving by day 4 after
inoculation, and predominantly consisted of mononuclear cells (data not
shown).
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In order to assess the contribution of neutrophils to host defense
against A. fumigatus, we next challenged neutrophil-depleted
mice with i.t. conidia. Neutrophil depletion was achieved by i.p.
administration of 100 µg of RB6-8C5 mAb i.p. 1 day before i.t.
A. fumigatus. In preliminary studies, we found that
administered of this dose of RB6-8C5 resulted in peripheral blood
neutropenia (absolute circulating neutrophil count <50 cells/µl) by
days 1 and 3 after Ab administration in both infected and control
animals, with a restoration of peripheral counts to pretreatment levels
by day 5; administration of control serum did not affect the peripheral
counts or lung histology (data not shown). We found a dose-dependent
mortality in mice pretreated with RB6-8C5, depending on the size of
A. fumigatus inoculum (Fig. 2). Importantly, pretreatment with
RB6-8C5 leads to greater than 90% mortality in mice challenged with
1.89 x 106 conidia, while no deaths occurred in
control animals challenged with the same inoculum.
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, A and C). The
cellular infiltrate consisted predominantly of neutrophils in normal
animals, and predominantly mononuclear cells in RB6-8C5-treated mice.
Conidia were visible in normal mice on day 2 after inoculation, which
were generally cleared by day 4 (Fig. 3B), whereas
neutrophil-depleted animals, hyphae were noted on days 2 and 4 (Fig. 3D). In order to quantify the presence of the invasive
hyphal form of A. fumigatus, we then measured the lung
burden of chitin in neutrophil-depleted animals. Lung chitin levels
were undetectable in control animals, but were elevated on both days 2
and 4 after inoculation of conidia in neutrophil-depleted animals,
returning to pretreatment levels in animals that survived to day 8
(Fig. 4).
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A). Normal animals had
maximal lung MPO levels 1 day after challenge, with gradual return to
pretreatment levels over days 2 to 4. As compared with normal animals,
RB6-8C5-treated animals had lesser quantities of lung MPO at day 1
after challenge. However, RB6-8C5-treated animals had increased levels
of lung MPO by day 2 after challenge with A. fumigatus, as
compared with immunocompetent animals. In order to correlate these
findings with the number of cells present, we performed cell counts and
differentials on fluid obtained by bronchoalveolar lavage 2 days after
challenge with 1–2 x 106 conidia. Normal animals
challenged with conidia had an increased number of recovered cells,
which were predominantly neutrophils. RB6-8C5-treated animals had a
greater total number of recovered cells, the majority of which were
neutrophils (Fig. 5B). The numbers of neutrophils counted on
bronchoalveolar lavage (BAL) in various experimental groups were in
agreement with lung MPO levels measured on day 2.
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To address the mechanism of neutrophil influx into the lungs in
response to challenge with A. fumigatus, we next measured
concentrations of the ELR+ CXC chemokines MIP-2 and KC in
lung homogenates at various times after i.t. administration of conidia.
KC and MIP-2 levels were elevated 1 day after inoculation with conidia
in both normal animals and animals treated with RB6-8C5 as compared
with animals challenged with vehicle. Both MIP-2 and KC reached higher
levels in animals treated with RB6-8C5 as compared with normal animals
(Fig. 6). MIP-2 and KC were undetectable
in plasma in both normal and RB6-8C5-treated animals, indicating a
compartmentalized response.
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To assess the contribution of the ELR+ CXC chemokines
to host response against A. fumigatus, we next assessed
survival of animals pretreated with anti-CXCR2 Abs. In preliminary
studies, administration of this Ab to normal animals did not affect the
number of circulating neutrophils over the subsequent 4 days (data not
shown). Administration of anti-CXCR2 serum to normal animals 2
h before challenge with 1–2 x 107 conidia resulted
in a marked increase in mortality, with >90% mortality in animals
treated with anti-CXCR2, as compared with minimal mortality in
immunocompetent animals treated with a similar inoculum of A.
fumigatus (Fig. 7).
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, A and B). Hyphae
were not present. However, lungs of animals treated with anti-CXCR2
serum contained patchy peribronchial mononuclear cellular infiltration
and fibrin deposition, with a paucity of neutrophils as compared with
anti-CXCR2-treated animals (Fig. 8C). Importantly, fungal
stains showed few conidia and large numbers of hyphae, indicating the
development of invasive disease (Fig. 8D).
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A). Furthermore, lungs of
animals treated with anti-CXCR2 contained 63% less myeloperoxidase
at 2 days after challenge with A. fumigatus, as compared
with animals treated with control serum (Fig. 9B),
corroborating the histologic observation of paucity of neutrophils.
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Having demonstrated the neutralization of the common receptor for ELR+ CXC chemokines results in marked impairment of host defense against A. fumigatus, we sought to determine if the overexpression of the ligand KC results in enhanced clearance of the pathogen. Since immunocompetent animals are naturally resistant to infection with Aspergillus species, and patients with defects in neutrophil numbers or function are at highest risk of developing Aspergillus infections, we utilized transiently neutrophil-depleted mice to assess the effect of KC overexpression in this infection. Transient neutrophil depletion was achieved in wild-type and transgenic animals with lung-specific overexpression of KC by i.p. injection of 100 µg of RB6-8C5 mAb. This resulted in peripheral blood neutropenia (absolute neutrophil count <50 cells/µl) on days 1 and 3 after injection in all animals, with restoration of peripheral counts to pretreatment levels by day 5 (data not shown). Animals were challenged with 1–2 x 106 i.t. A. fumigatus conidia after 24 h. Lung histology of uninfected wild-type and transgenic animals was normal and was not affected by administration of RB6-8C5 mAb.
In survival studies, KC transgenic animals had a 46% reduction in
mortality compared with wild-type animals (Fig. 10A). Lung chitin levels
measured 2 days after challenge with conidia showed KC transgenic
animals to have a 67% decrease in lung chitin content as compared with
wild-type controls (Fig. 10B). Lung histology obtained on
day 2 showed a greater accumulation of neutrophils associated with
areas of fungal hyphae in KC transgenic mice, as compared with
wild-type animals (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In order to evaluate the role of neutrophils in host defense against A. fumigatus in an animal model, we developed a model of transient in vivo neutrophil depletion. The Ab-mediated depletion of the available pool of mature neutrophils allowed the organism to establish tissue invasion, which was not observed in animals with intact neutrophil number and function. The dynamics of reduction and early recovery of neutrophil numbers in this model, induced by a single dose of RB6-8C5 1 mAb on day -1, are analogous to the clinical recovery of neutrophils in patients after chemotherapy or bone marrow transplantation. Two days after i.t. challenge with Aspergillus, the number of neutrophils in the lungs of RB6-8C5-treated animals was larger than that in normal controls challenged with the same inoculum. This greater influx of neutrophils in RB6-8C5-treated animals was in response to fungal tissue invasion and occurred in the context of peripheral blood neutropenia, representing maximal deployment of the limited pool of newly produced neutrophils to the site of infection. An advantage of this model is that it is unaffected by the pleotropic influence of chemotherapeutic agents on various other immunologically active cells. Furthermore, clinical and histological features of invasive aspergillosis in this model resembled those seen in human disease.
Given that the ELR+ CXC chemokines have been shown to
mediate neutrophil recruitment and activation in diverse conditions, we
elected to study their contribution to neutrophil-mediated host defense
against A. fumigatus. Other studies have suggested a role
for chemokines in host defense against A. fumigatus. For
example, isolated rat alveolar macrophages have been shown to produce
MIP-1
, MIP-2, and KC, as well as TNF- in response to A.
fumigatus conidia in vitro (52), and knockout mice
lacking CC chemokine receptor-1 (CCR1), a receptor for MIP-1 and
RANTES, developed disseminated infection when administered A.
fumigatus i.v. (53). In a previous study, we showed
that neutralization of TNF resulted in reduced lung neutrophil influx
associated with attenuated levels of lung CXC and CC chemokines,
including MIP-2, MIP-l, and JE (54). In this context,
we examined the importance of ELR+ CXC chemokines as a
group against A. fumigatus, by blocking their common
receptor in the mouse. This approach allowed us to overcome the
difficulties in simultaneous neutralization of the biologic effects of
multiple ligands, which may have redundant and overlapping functions.
The effects of ELR+ CXC chemokines in humans are mediated
via both CXCR-1 and CXCR-2. While the relative simplicity of the murine
model allowed us to address the role of these mediators in host defense
against A. fumigatus, the dissimilarities between the murine
and human systems represent a limitation of the present study. We
employed a goat anti-murine serum, which did not deplete
circulating neutrophils but prevented in vivo influx of neutrophils
into the peritoneum in response to exogenous KC. The pneumonia that
resulted from Aspergillus inoculation in the setting of
CXCR2 neutralization was indistinguishable from that caused by
neutrophil depletion. The residual lung accumulation of neutrophils in
the setting of CXCR2 neutralization indicates that other mechanisms,
such as other chemotactic factors or differential expression of
adhesion molecules, may play a role in neutrophil influx in response to
A. fumigatus. However, the magnitude of effect of CXCR2
neutralization highlights the essential role of CXCR2 ligands in host
defense against A. fumigatus, nearly to the exclusion of
other chemotactic mediators.
The two best-studied murine ELR+ CXC chemokines, MIP-2 and KC, are functional homologues of the human ELR+ CXC chemokines, IL-8 and Gro- chemokines (55, 56). MIP-2 and KC have been shown to mediate neutrophil-dependent host defense in other models of pneumonia. Specifically, in a murine model of Pseudomonas pneumonia, MIP-2 and KC levels were associated with presence of neutrophils in lung (57), and in murine Klebsiella pneumonia, MIP-2 was shown to be involved in recruitment of neutrophils, but had only modest effects on clearance of the pathogen and survival of animals (29). In preliminary studies, we found that Ab-mediated depletion of MIP-2 resulted in minimal change in survival in animals challenged with Aspergillus (data not shown).
We found markedly greater levels of KC and MIP-2 in the lungs of transiently neutrophil-depleted animals, as compared with immunocompetent animals, challenged with A. fumigatus. This difference may be due to the greater burden of organisms in animals with transient neutrophil depletion. Other mechanisms may include local negative feedback on the production of the chemokines, or their receptor-mediated uptake by neutrophils. The present study showed that lung-specific overexpression of KC resulted in augmented host defense against Aspergillus, as manifested by reduced mortality and lung fungal burden. Given that immunocompetent mice are intrinsically resistant to Aspergillus infection, and that neutropenic patients are at greatest risk of developing invasive aspergillosis, we used a mAb to transiently deplete neutrophils to assess the effect of KC overexpression in the lung. The limited pool of available neutrophils induced in these animals is analogous to the clinical setting in which invasive aspergillosis occurs. The mechanism of the protective effect of KC against A. fumigatus is likely via enhanced recruitment of this limited pool of available neutrophils to the lung, as suggested by histology, and may also involve greater neutrophil antifungal activity. These findings are consistent with the prior observation of a protective role for KC in murine bacterial pneumonia, which was associated with an augmented neutrophil influx (58).
The substantial effect of CXCR2 neutralization suggests that multiple ligands may be involved. Other candidate murine ELR+ CXC chemokines are less well characterized. A newly described murine chemokine, LIX, has been shown to share structural homology with the human chemokines ENA-78 and GCP-2 (18), and is chemotactic for neutrophils in vitro (59). In a murine model of systemic endotoxemia, the expression of the ELR+ CXC chemokines was found to differ markedly in various organs (19), with prominent expression of MIP-2 in the lung, LIX in the heart, and equal expression of KC in lung, heart, and liver. Another recently described murine ELR+ CXC chemokine, Lungkine, has also been shown to be chemotactic for neutrophils in vitro, and is constitutively expressed by lung epithelial cells, but not expressed in other organs. The preferential expression of chemokines in specific organs is of interest, and suggests that the various ELR+ CXC chemokines may have distinct biological roles.
This study has identified CXCR2 ligands as required components of host defense against A. fumigatus. This family of mediators may be important targets in devising future therapeutic strategies against invasive aspergillosis. We have shown that transgenic overexpression of KC in the lungs is feasible and is not complicated by detrimental effects (58). Future studies will evaluate the effect of overexpression or augmentation of other ELR+ CXC chemokines on the outcome of invasive aspergillosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Theodore J. Standiford, University of Michigan Medical Center, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0360. E-mail address:
3 Abbreviations used in this paper: MPO, myeloperoxidase; CCR1, CC chemokine receptor-1; CXCR2, CXC chemokine receptor-2; GMS, Gomori methanamine silver; ELR, glutamic acid-leucine-arginine; ENA-78, epithelial neutrophil activating protein-78; GCP-2, granulocyte chemotactic protein-2; GRO, growth-related oncogene; H&E, hematoxylin and eosin; i.t., intratracheal; LIX, LPS-induced CXC chemokine; MIP-1; macrophage inflammatory protein-1; MIP-2, macrophage inflammatory protein-2; NAP-2, neutrophil activating peptide-2; BAL, bronchoalveolar lavage; GMS, Gomori methanamine silver.
Received for publication July 20, 1999. Accepted for publication September 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  A. J. Hartigan, J. Westwick, G. Jarai, and C. M. Hogaboam CCR7 Deficiency on Dendritic Cells Enhances Fungal Clearance in a Murine Model of Pulmonary Invasive Aspergillosis J. Immunol., October 15, 2009; 183(8): 5171 - 5179. [Abstract] [Full Text] [PDF]
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 S. J. Park and B. Mehrad Innate Immunity to Aspergillus Species Clin. Microbiol. Rev., October 1, 2009; 22(4): 535 - 551. [Abstract] [Full Text] [PDF]
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 Y. Li, Y. Jia, M. Pichavant, F. Loison, B. Sarraj, A. Kasorn, J. You, B. E. Robson, D. T. Umetsu, J. P. Mizgerd, et al. Targeted deletion of tumor suppressor PTEN augments neutrophil function and enhances host defense in neutropenia-associated pneumonia Blood, May 14, 2009; 113(20): 4930 - 4941. [Abstract] [Full Text] [PDF]
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J. L. Werner, A. E. Metz, D. Horn, T. R. Schoeb, M. M. Hewitt, L. M. Schwiebert, I. Faro-Trindade, G. D. Brown, and C. Steele Requisite Role for the Dectin-1 {beta}-Glucan Receptor in Pulmonary Defense against Aspergillus fumigatus J. Immunol., April 15, 2009; 182(8): 4938 - 4946. [Abstract] [Full Text] [PDF]
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S. J. Park, M. A. Hughes, M. Burdick, R. M. Strieter, and B. Mehrad Early NK Cell-Derived IFN-{gamma} Is Essential to Host Defense in Neutropenic Invasive Aspergillosis J. Immunol., April 1, 2009; 182(7): 4306 - 4312. [Abstract] [Full Text] [PDF]
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 V. Balloy, J.-M. Sallenave, Y. Wu, L. Touqui, J.-P. Latge, M. Si-Tahar, and M. Chignard Aspergillus fumigatus-induced Interleukin-8 Synthesis by Respiratory Epithelial Cells Is Controlled by the Phosphatidylinositol 3-Kinase, p38 MAPK, and ERK1/2 Pathways and Not by the Toll-like Receptor-MyD88 Pathway J. Biol. Chem., November 7, 2008; 283(45): 30513 - 30521. [Abstract] [Full Text] [PDF]
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E. J. Cornish, B. J. Hurtgen, K. McInnerney, N. L. Burritt, R. M. Taylor, J. N. Jarvis, S. Y. Wang, and J. B. Burritt Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages J. Immunol., May 15, 2008; 180(10): 6854 - 6867. [Abstract] [Full Text] [PDF]
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 T. Carlson, M. Kroenke, P. Rao, T. E. Lane, and B. Segal The Th17-ELR+ CXC chemokine pathway is essential for the development of central nervous system autoimmune disease J. Exp. Med., April 14, 2008; 205(4): 811 - 823. [Abstract] [Full Text] [PDF]
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 T. Shea-Donohue, K. Thomas, M. J. Cody, Aiping Zhao, L. J. DeTolla, K. M. Kopydlowski, M. Fukata, S. A. Lira, and S. N. Vogel Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-{alpha}), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis Innate Immunity, April 1, 2008; 14(2): 117 - 124. [Abstract] [PDF]
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K. De Filippo, R. B. Henderson, M. Laschinger, and N. Hogg Neutrophil Chemokines KC and Macrophage-Inflammatory Protein-2 Are Newly Synthesized by Tissue Macrophages Using Distinct TLR Signaling Pathways J. Immunol., March 15, 2008; 180(6): 4308 - 4315. [Abstract] [Full Text] [PDF]
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 C. Bretz, G. Gersuk, S. Knoblaugh, N. Chaudhary, J. Randolph-Habecker, R. C. Hackman, J. Staab, and K. A. Marr MyD88 Signaling Contributes to Early Pulmonary Responses to Aspergillus fumigatus Infect. Immun., March 1, 2008; 76(3): 952 - 958. [Abstract] [Full Text] [PDF]
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 N. Kesteman, G. Vansanten, B. Pajak, S. M. Goyert, and M. Moser Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines J. Leukoc. Biol., March 1, 2008; 83(3): 640 - 647. [Abstract] [Full Text] [PDF]
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 J. P. Mizgerd and S. J. Skerrett Animal models of human pneumonia Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L387 - L398. [Abstract] [Full Text] [PDF]
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T. E. Rodriguez, N. R. Falkowski, J. R. Harkema, and G. B. Huffnagle Role of Neutrophils in Preventing and Resolving Acute Fungal Sinusitis Infect. Immun., December 1, 2007; 75(12): 5663 - 5668. [Abstract] [Full Text] [PDF]
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 T. M. Hohl and M. Feldmesser Aspergillus fumigatus: Principles of Pathogenesis and Host Defense Eukaryot. Cell, November 1, 2007; 6(11): 1953 - 1963. [Full Text] [PDF]
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 S. S. Shaftel, T. J. Carlson, J. A. Olschowka, S. Kyrkanides, S. B. Matousek, and M. K. O'Banion Chronic Interleukin-1{beta} Expression in Mouse Brain Leads to Leukocyte Infiltration and Neutrophil-Independent Blood Brain Barrier Permeability without Overt Neurodegeneration J. Neurosci., August 29, 2007; 27(35): 9301 - 9309. [Abstract] [Full Text] [PDF]
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K. B. Walsh, R. A. Edwards, K. M. Romero, M. V. Kotlajich, S. A. Stohlman, and T. E. Lane Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease J. Immunol., July 15, 2007; 179(2): 1155 - 1165. [Abstract] [Full Text] [PDF]
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M. Nguyen, A. J. Pace, and B. H. Koller Mice lacking NKCC1 are protected from development of bacteremia and hypothermic sepsis secondary to bacterial pneumonia J. Exp. Med., June 11, 2007; 204(6): 1383 - 1393. [Abstract] [Full Text] [PDF]
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 A. P. Phadke, G. Akangire, S. J. Park, S. A. Lira, and B. Mehrad The Role of CC Chemokine Receptor 6 in Host Defense in a Model of Invasive Pulmonary Aspergillosis Am. J. Respir. Crit. Care Med., June 1, 2007; 175(11): 1165 - 1172. [Abstract] [Full Text] [PDF]
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F. Rios-Santos, J. C. Alves-Filho, F. O. Souto, F. Spiller, A. Freitas, C. M. C. Lotufo, M. B. P. Soares, R. R. dos Santos, M. M. Teixeira, and F. de Queiroz Cunha Down-regulation of CXCR2 on Neutrophils in Severe Sepsis Is Mediated by Inducible Nitric Oxide Synthase-derived Nitric Oxide Am. J. Respir. Crit. Care Med., March 1, 2007; 175(5): 490 - 497. [Abstract] [Full Text] [PDF]
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C. R. Bonnett, E. J. Cornish, A. G. Harmsen, and J. B. Burritt Early Neutrophil Recruitment and Aggregation in the Murine Lung Inhibit Germination of Aspergillus fumigatus Conidia Infect. Immun., December 1, 2006; 74(12): 6528 - 6539. [Abstract] [Full Text] [PDF]
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D. Okutani, M. Lodyga, B. Han, and M. Liu Src protein tyrosine kinase family and acute inflammatory responses Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L129 - L141. [Abstract] [Full Text] [PDF]
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 K. Takemoto, Y. Yamamoto, Y. Ueda, Y. Sumita, K. Yoshida, and Y. Niki Comparative study on the efficacy of AmBisome and Fungizone in a mouse model of pulmonary aspergillosis J. Antimicrob. Chemother., April 1, 2006; 57(4): 724 - 731. [Abstract] [Full Text] [PDF]
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B. H. Segal and T. J. Walsh Current Approaches to Diagnosis and Treatment of Invasive Aspergillosis Am. J. Respir. Crit. Care Med., April 1, 2006; 173(7): 707 - 717. [Abstract] [Full Text] [PDF]
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B. Mehrad, S. J. Park, G. Akangire, T. J. Standiford, T. Wu, J. Zhu, and C. Mohan The lupus-susceptibility locus, sle3, mediates enhanced resistance to bacterial infections. J. Immunol., March 1, 2006; 176(5): 3233 - 3239. [Abstract] [Full Text] [PDF]
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S. J. Park, M. T. Wiekowski, S. A. Lira, and B. Mehrad Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease J. Immunol., February 15, 2006; 176(4): 2538 - 2545. [Abstract] [Full Text] [PDF]
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K. J. Carpenter, K. F. Buckland, Z. Xing, and C. M. Hogaboam Intrapulmonary, Adenovirus-Mediated Overexpression of KARAP/DAP12 Enhances Fungal Clearance during Invasive Aspergillosis Infect. Immun., December 1, 2005; 73(12): 8402 - 8406. [Abstract] [Full Text] [PDF]
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J. A. Belperio, M. P. Keane, M. D. Burdick, B. N. Gomperts, Y. Y. Xue, K. Hong, J. Mestas, D. Zisman, A. Ardehali, R. Saggar, et al. CXCR2/CXCR2 Ligand Biology during Lung Transplant Ischemia-Reperfusion Injury J. Immunol., November 15, 2005; 175(10): 6931 - 6939. [Abstract] [Full Text] [PDF]
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K. J. Carpenter and C. M. Hogaboam Immunosuppressive Effects of CCL17 on Pulmonary Antifungal Responses during Pulmonary Invasive Aspergillosis Infect. Immun., November 1, 2005; 73(11): 7198 - 7207. [Abstract] [Full Text] [PDF]
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V. Balloy, M. Si-Tahar, O. Takeuchi, B. Philippe, M.-A. Nahori, M. Tanguy, M. Huerre, S. Akira, J.-P. Latge, and M. Chignard Involvement of Toll-Like Receptor 2 in Experimental Invasive Pulmonary Aspergillosis Infect. Immun., September 1, 2005; 73(9): 5420 - 5425. [Abstract] [Full Text] [PDF]
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H. Echchannaoui, K. Frei, M. Letiembre, R. M. Strieter, Y. Adachi, and R. Landmann CD14 deficiency leads to increased MIP-2 production, CXCR2 expression, neutrophil transmigration, and early death in pneumococcal infection J. Leukoc. Biol., September 1, 2005; 78(3): 705 - 715. [Abstract] [Full Text] [PDF]
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T. H. Thatcher, N. A. McHugh, R. W. Egan, R. W. Chapman, J. A. Hey, C. K. Turner, M. R. Redonnet, K. E. Seweryniak, P. J. Sime, and R. P. Phipps Role of CXCR2 in cigarette smoke-induced lung inflammation Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L322 - L328. [Abstract] [Full Text] [PDF]
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M. H. Tarlowe, A. Duffy, K. B. Kannan, K. Itagaki, R. F. Lavery, D. H. Livingston, P. Bankey, and C. J. Hauser Prospective Study of Neutrophil Chemokine Responses in Trauma Patients at Risk for Pneumonia Am. J. Respir. Crit. Care Med., April 1, 2005; 171(7): 753 - 759. [Abstract] [Full Text] [PDF]
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V. Balloy, M. Huerre, J.-P. Latge, and M. Chignard Differences in Patterns of Infection and Inflammation for Corticosteroid Treatment and Chemotherapy in Experimental Invasive Pulmonary Aspergillosis Infect. Immun., January 1, 2005; 73(1): 494 - 503. [Abstract] [Full Text] [PDF]
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R. A. Johnston, J. P. Mizgerd, and S. A. Shore CXCR2 is essential for maximal neutrophil recruitment and methacholine responsiveness after ozone exposure Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L61 - L67. [Abstract] [Full Text] [PDF]
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 G.-H. Fan, L. A. Lapierre, J. R. Goldenring, J. Sai, and A. Richmond Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis Mol. Biol. Cell, May 1, 2004; 15(5): 2456 - 2469. [Abstract] [Full Text] [PDF]
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R. D. Sue, J. A. Belperio, M. D. Burdick, L. A. Murray, Y. Y. Xue, M. C. Dy, J. J. Kwon, M. P. Keane, and R. M. Strieter CXCR2 Is Critical to Hyperoxia-Induced Lung Injury J. Immunol., March 15, 2004; 172(6): 3860 - 3868. [Abstract] [Full Text] [PDF]
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 M. Pold, L. X. Zhu, S. Sharma, M. D. Burdick, Y. Lin, P. P. N. Lee, A. Pold, J. Luo, K. Krysan, M. Dohadwala, et al. Cyclooxygenase-2-Dependent Expression of Angiogenic CXC Chemokines ENA-78/CXC Ligand (CXCL) 5 and Interleukin-8/CXCL8 in Human Non-Small Cell Lung Cancer Cancer Res., March 1, 2004; 64(5): 1853 - 1860. [Abstract] [Full Text] [PDF]
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M. P. Keane, J. A. Belperio, Y. Y. Xue, M. D. Burdick, and R. M. Strieter Depletion of CXCR2 Inhibits Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer J. Immunol., March 1, 2004; 172(5): 2853 - 2860. [Abstract] [Full Text] [PDF]
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T. L. Ness, C. M. Hogaboam, R. M. Strieter, and S. L. Kunkel Immunomodulatory Role of CXCR2 During Experimental Septic Peritonitis J. Immunol., October 1, 2003; 171(7): 3775 - 3784. [Abstract] [Full Text] [PDF]
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M. J. Becker, S. de Marie, M. H. A. M. Fens, H. A. Verbrugh, and I. A. J. M. Bakker-Woudenberg Effect of amphotericin B treatment on kinetics of cytokines and parameters of fungal load in neutropenic rats with invasive pulmonary aspergillosis J. Antimicrob. Chemother., September 1, 2003; 52(3): 428 - 434. [Abstract] [Full Text] [PDF]
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 J. T. Souto, J. C. Aliberti, A. P. Campanelli, M. C. Livonesi, C. M.L. Maffei, B. R. Ferreira, L. R. Travassos, R. Martinez, M. A. Rossi, and J. S. Silva Chemokine Production and Leukocyte Recruitment to the Lungs of Paracoccidioides brasiliensis-Infected Mice Is Modulated by Interferon-{gamma} Am. J. Pathol., August 1, 2003; 163(2): 583 - 590. [Abstract] [Full Text] [PDF]
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 H. L. Van Epps, M. Feldmesser, and E. G. Pamer Voriconazole Inhibits Fungal Growth without Impairing Antigen Presentation or T-Cell Activation Antimicrob. Agents Chemother., June 1, 2003; 47(6): 1818 - 1823. [Abstract] [Full Text] [PDF]
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A. L. Miller, R. M. Strieter, A. D. Gruber, S. B. Ho, and N. W. Lukacs CXCR2 Regulates Respiratory Syncytial Virus-Induced Airway Hyperreactivity and Mucus Overproduction J. Immunol., March 15, 2003; 170(6): 3348 - 3356. [Abstract] [Full Text] [PDF]
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E. Garcia-Ramallo, T. Marques, N. Prats, J. Beleta, S. L. Kunkel, and N. Godessart Resident Cell Chemokine Expression Serves as the Major Mechanism for Leukocyte Recruitment During Local Inflammation J. Immunol., December 1, 2002; 169(11): 6467 - 6473. [Abstract] [Full Text] [PDF]
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B. Mehrad, M. Wiekowski, B. E. Morrison, S.-C. Chen, E. C. Coronel, D. J. Manfra, and S. A. Lira Transient Lung-Specific Expression of the Chemokine KC Improves Outcome in Invasive Aspergillosis Am. J. Respir. Crit. Care Med., November 1, 2002; 166(9): 1263 - 1268. [Abstract] [Full Text] [PDF]
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D. R. Chung, T. Chitnis, R. J. Panzo, D. L. Kasper, M. H. Sayegh, and A. O. Tzianabos CD4+ T Cells Regulate Surgical and Postinfectious Adhesion Formation J. Exp. Med., June 3, 2002; 195(11): 1471 - 1478. [Abstract] [Full Text] [PDF]
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B. Endlich, D. Armstrong, J. Brodsky, M. Novotny, and T. A. Hamilton Distinct Temporal Patterns of Macrophage-Inflammatory Protein-2 and KC Chemokine Gene Expression in Surgical Injury J. Immunol., April 1, 2002; 168(7): 3586 - 3594. [Abstract] [Full Text] [PDF]
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R. Hayashi, N. Kitamoto, Y. Iizawa, T. Ichikawa, K. Itoh, T. Kitazaki, and K. Okonogi Efficacy of TAK-457, a Novel Intravenous Triazole, against Invasive Pulmonary Aspergillosis in Neutropenic Mice Antimicrob. Agents Chemother., February 1, 2002; 46(2): 283 - 287. [Abstract] [Full Text] [PDF]
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J. M. Schuh, K. Blease, and C. M. Hogaboam CXCR2 Is Necessary for the Development and Persistence of Chronic Fungal Asthma in Mice J. Immunol., February 1, 2002; 168(3): 1447 - 1456. [Abstract] [Full Text] [PDF]
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S. W. Chensue Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835. [Abstract] [Full Text] [PDF]
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D. M. Lindell, T. J. Standiford, P. Mancuso, Z. J. Leshen, and G. B. Huffnagle Macrophage Inflammatory Protein 1{alpha}/CCL3 Is Required for Clearance of an Acute Klebsiella pneumoniae Pulmonary Infection Infect. Immun., October 1, 2001; 69(10): 6364 - 6369. [Abstract] [Full Text] [PDF]
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M. Akkoyunlu, S. E. Malawista, J. Anguita, and E. Fikrig Exploitation of Interleukin-8-Induced Neutrophil Chemotaxis by the Agent of Human Granulocytic Ehrlichiosis Infect. Immun., September 1, 2001; 69(9): 5577 - 5588. [Abstract] [Full Text] [PDF]
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K. Tateda, T. A. Moore, M. W. Newstead, W. C. Tsai, X. Zeng, J. C. Deng, G. Chen, R. Reddy, K. Yamaguchi, and T. J. Standiford Chemokine-Dependent Neutrophil Recruitment in a Murine Model of Legionella Pneumonia: Potential Role of Neutrophils as Immunoregulatory Cells Infect. Immun., April 1, 2001; 69(4): 2017 - 2024. [Abstract] [Full Text] [PDF]
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T. Kielian, B. Barry, and W. F. Hickey CXC Chemokine Receptor-2 Ligands Are Required for Neutrophil-Mediated Host Defense in Experimental Brain Abscesses1 J. Immunol., April 1, 2001; 166(7): 4634 - 4643. [Abstract] [Full Text] [PDF]
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L. R. Hall, E. Diaconu, R. Patel, and E. Pearlman CXC Chemokine Receptor 2 But Not C-C Chemokine Receptor 1 Expression Is Essential for Neutrophil Recruitment to the Cornea in Helminth-Mediated Keratitis (River Blindness) J. Immunol., March 15, 2001; 166(6): 4035 - 4041. [Abstract] [Full Text] [PDF]
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K. Blease, B. Mehrad, N. W. Lukacs, S. L. Kunkel, T. J. Standiford, and C. M. Hogaboam Antifungal and Airway Remodeling Roles for Murine Monocyte Chemoattractant Protein-1/CCL2 During Pulmonary Exposure to Asperigillus fumigatus Conidia J. Immunol., February 1, 2001; 166(3): 1832 - 1842. [Abstract] [Full Text] [PDF]
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M. Chignard and V. Balloy Neutrophil recruitment and increased permeability during acute lung injury induced by lipopolysaccharide Am J Physiol Lung Cell Mol Physiol, December 1, 2000; 279(6): L1083 - L1090. [Abstract] [Full Text] [PDF]
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B. Mehrad, T. A. Moore, and T. J. Standiford Macrophage Inflammatory Protein-1{alpha} Is a Critical Mediator of Host Defense Against Invasive Pulmonary Aspergillosis in Neutropenic Hosts J. Immunol., July 15, 2000; 165(2): 962 - 968. [Abstract] [Full Text] [PDF]
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W. C. Tsai, R. M. Strieter, B. Mehrad, M. W. Newstead, X. Zeng, and T. J. Standiford CXC Chemokine Receptor CXCR2 Is Essential for Protective Innate Host Response in Murine Pseudomonas aeruginosa Pneumonia Infect. Immun., July 1, 2000; 68(7): 4289 - 4296. [Abstract] [Full Text] [PDF]
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T. A. Moore, M. W. Newstead, R. M. Strieter, B. Mehrad, B. L. Beaman, and T. J. Standiford Bacterial Clearance and Survival Are Dependent on CXC Chemokine Receptor-2 Ligands in a Murine Model of Pulmonary Nocardia asteroides Infection J. Immunol., January 15, 2000; 164(2): 908 - 915. [Abstract] [Full Text] [PDF]
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