Abstract
Invasive pulmonary aspergillosis is a devastating complication of immunosuppression that usually occurs in neutropenic patients. In this setting, augmentation of the antifungal activity of available immune cells may improve the outcome of the infection. Macrophage inflammatory protein-1α (MIP-1α) is a CC chemokine with potent chemotactic activity for various subsets of mononuclear leukocytes. We therefore tested the hypothesis that the influx of mononuclear cells into the lung in invasive pulmonary aspergillosis is in part mediated by MIP-1α, and the manipulation of this ligand alters the outcome of the infection. We found that in both immunocompetent and neutropenic mice, MIP-1α was induced in the lungs in response to intratracheal administration of Aspergillus fumigatus conidia. In neutrophil-depleted mice challenged with intratracheal conidia, there was evidence of invasive fungal pneumonia associated with a predominantly mononuclear leukocyte infiltrate. Ab-mediated depletion of MIP-1α resulted in a 6-fold increase in mortality in neutropenic mice, which was associated with a 12-fold increase in lung fungal burden. Studies of single-cell suspensions of whole lungs revealed a 36% decrease in total lung leukocyte infiltration as a result of MIP-1α neutralization. Flow cytometry on whole lung suspensions showed a 41% reduction in lung monocyte/macrophages as a result of MIP-1α neutralization, but no difference in other lung leukocyte subsets. These studies indicate that MIP-1α is a critical mediator of host defense against A. fumigatus in the setting of neutropenia and may be an important target in devising future therapeutic strategies against invasive aspergillosis.
Invasive pulmonary aspergillosis is a devastating pneumonia afflicting immunocompromised hosts. Its incidence has increased steadily in recent decades as more patients are treated with potent immunosuppressive agents as part of therapy for organ transplantation and cancer chemotherapy (1, 2, 3). Current treatment for invasive aspergillosis centers on direct antifungal agents, but the crude mortality of the disease exceeds 85% despite the best available therapy (4, 5, 6). Because invasive aspergillosis is extremely rare in immunocompetent individuals, therapy aimed at improving the defect in the host’s immune response to the organism offers a promising new approach in the treatment of this disease.
The commonest and best characterized risk factor for development of invasive aspergillosis is neutropenia (7, 8). The role of neutrophils in the innate host defense against Aspergillus species has been well documented in both in vitro (9, 10) and in vivo (11, 12) experiments. Other immune cells also play a role in host defense against Aspergillus species, however. In experimental animals, alveolar and peritoneal macrophages bind and engulf Aspergillus fumigatus conidia in vitro (10, 13, 14, 15) and produce IL-1 and TNF when exposed to either conidia or hyphae (16). In vitro studies of human peripheral monocytes (17, 18), monocyte-derived macrophages (19), and alveolar macrophages (20, 21) have also shown these cells to phagocytose conidia and kill both conidial and hyphal forms of A. fumigatus. One study has suggested a role for splenic macrophages in acquired immunity against i.v. administered A. fumigatus conidia in vivo (22). Lung histopathology specimens from immunocompromised mice (23) and neutropenic patients (24) with invasive aspergillosis also show prominent infiltration of mononuclear cells in association with fungal hyphae. Because the patients who develop invasive aspergillosis have a limited pool of available neutrophils to combat the pathogen, studies aimed at augmentation of the antifungal activity of other immune cells, such as the mononuclear phagocytes, may be relevant in devising new therapeutic strategies against this infection.
Macrophage inflammatory protein-α (MIP-1α),3 a member of the CC family of chemokines, exhibits potent chemotactic and activating properties for several populations of leukocytes, including monocytes, various lymphocyte subsets, mast cells, and eosinophils (reviewed in Refs. 25 and 26). MIP-1α is induced in patients with diverse inflammatory diseases, including bacterial sepsis (27, 28), malaria (29), and interstitial lung diseases (30), and in vivo administration of MIP-1α to laboratory animals results in localized infiltration of monocytes (31, 32, 33). In animal models of infections, MIP-1α has been shown to play a critical role in the cellular recruitment and inflammatory response to coxsackie and influenza infections (34), endotoxemia (35), bacterial pneumonia (36), and cryptococcosis (37, 38). Two previous studies have examined the expression of MIP-1α in response to Aspergillus species; one showed that rat alveolar macrophages express MIP-1α mRNA when exposed to A. fumigatus conidia in vitro (39), and another demonstrated that MIP-1α is induced in the lungs after intrapulmonary challenge with conidia (40). We have previously shown that neutralization of TNF in experimental pulmonary aspergillosis results in increased mortality and decreased fungal clearance, which was associated with decreased levels of lung MIP-1α (41). The role and mechanism of action of MIP-1α in host defense against A. fumigatus have not, however, been characterized.
In this study we hypothesized that the influx of mononuclear phagocytes into the lung in invasive pulmonary aspergillosis is in part mediated by MIP-1α, and that the manipulation of this chemokine alters the outcome of the infection. We tested this hypothesis by evaluating the outcome and severity of the infection in neutropenic mice in the setting of MIP-1α depletion and by determining the effect of MIP-1α neutralization on inflammatory cellular influx into the lungs.
Materials and Methods
Reagents
42, 43). Abs were purified over a protein A column, and endotoxin contamination was excluded by Limulus lysate assay (ICN Biomedical, Costa Mesa, CA). In neutralization experiments, 0.5 ml of rabbit anti-murine MIP-1α serum or control rabbit serum was administered i.p. 2 h before A. fumigatus inoculation, and 0.25 ml was given on days 2 and 4 after inoculation.
Animals
Specific pathogen-free C57BL/6 mice (6- to 8-wk-old females; The Jackson Laboratory, Bar Harbor, ME) were used in all experiments. 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 (44, 45, 46, 47, 48). TSD BioServices (Germantown, NY) produced the Ab by i.p. injection of RB6-8C5 hybridoma into nude mice and ascites collection. The resultant ascitic fluid was pooled, and the concentration of IgG was quantified by HPLC. One hundred microliters of a 1/200 dilution of the pooled ascites was administered i.p. 1 day before 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
A. fumigatus strain 13073 (American Type Culture Collection, Manassas, VA) was used in all studies, as this strain has previously been shown to induce invasive aspergillosis in immunocompromised mice (49). The organism was grown on Sabouraud dextrose agar plates (Becton Dickinson, Cockeysville, MD) for 7–10 days at 37°C. The surface of each plate was then washed with 100 ml of sterile 0.1% Tween 80 (Sigma, 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 using a particle counter (Z2 particle analyzer, Coulter, Hialeah, FL). The suspension was then diluted to the desired concentration, and the concentration was again measured before administration. In preliminary experiments the number of particles determined by the particle counter was in close agreement with the number of viable CFU found by serial dilution and plating of the suspension. On the day of inoculation, each animal was anesthetized with 1.8–2 mg of pentobarbital i.p. Using standard aseptic technique, the trachea was exposed, and a 30-μl inoculum (A. fumigatus suspension or 0.1% Tween 80 vehicle) 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 × 106 to 1–2 × 107 conidia in various experiments. In preliminary studies i.t. administration of the Tween vehicle to immunocompetent animals did not result in changes in histology or influx of neutrophils (data not shown).
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 of 4% paraformaldehyde in PBS, inflated with 1 ml of 4% paraformaldehyde in PBS via the trachea, and then excised en bloc. Lungs for 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 various assays were homogenized in 1 ml of 2× complete protease inhibitor cocktail buffer (Roche, 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 × g for 10 min. Supernatants were passed through a 0.45-μ pore size filter (Gelman Sciences, Ann Arbor, MI) and stored at 4°C for ELISA.
Single-cell suspensions of the lungs were prepared as previously described (50). Briefly, freshly resected lungs were minced with scissors to a fine slurry and incubated at 37°C for 30 min in an RPMI solution (Sigma) containing 1 mg/ml type A collagenase (Roche) and 30 U/ml DNase (Sigma). The solutions were then drawn up and down 20 times in 10-ml syringes (Becton Dickinson, Franklin Lakes, NJ) to disperse the cells mechanically. The resulting cell suspensions were pelleted, resuspended, passed through Nitex mesh filters (Tetko, Kansas City, MO), and passed through a 20% Percoll gradient (Sigma) before cell counting under a hemocytometer. Cytospin slides of this suspension were then prepared and stained (Diff-Quik Stain Set, Dade Behring, Newark, DE), and differential cell counts were determined using a high power microscope. The absolute number of a leukocyte type was determined by multiplication of the percentage of that cell type by the total number of lung leukocytes in that sample.
Flow cytometric analyses
Total lung leukocytes were isolated from animals as described above. For identification of total lymphocyte population, isolated leukocytes were stained with FITC-labeled anti-αβ-TCR, anti-γδ-TCR, anti-CD19, and anti-NK1.1. For identification of monocyte/macrophage populations, isolated leukocytes were stained with FITC-labeled anti-CD11b (all reagents from PharMingen, San Diego, CA). In addition, cells were stained with anti-CD45-Tricolor (Caltag, South San Francisco, CA), allowing discrimination of leukocytes from nonleukocytes and thus eliminating any nonspecific binding of cell surface markers on nonleukocytes. The samples were washed in staining buffer and fixed in 1% paraformaldehyde (Sigma) in PBS. Stained samples were kept in the dark at 4°C until analyzed on a FACScan cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson). Lymphocyte and monocyte/macrophage populations were analyzed by first gating on CD45-positive cells of the appropriate light scatter characteristics and were then examined for FL1 and FL2 fluorescence expression. The absolute number of a leukocyte type was determined by multiplication of the percentage of that cell type by the total number of lung leukocytes in that sample.
Lung chitin assay
Given that molds (including Aspergillus species) do not reliably form reproductive units in tissue, we employed a previously described assay for chitin to measure the burden of organisms in the lungs (41). Chitin, a component of the hyphal wall, is absent from conidia and mammalian tissue. The level of chitin, as detected by this assay, directly correlates with the weight of hyphae (51). Lungs were homogenized, centrifuged, and resuspended in sodium lauryl sulfate (3%, w/v), heated at 100°C for 15 min, washed and resuspended in KOH (120%, w/v), heated at 130°C for 60 min, cooled, incubated on ice after the addition of 8 ml of ice-cold 75% ethanol, pelleted after the addition of 0.3 ml of Celite suspension, washed, and resuspended in 0.5 ml of distilled water. NaNO2 (5%, w/v; 0.5 ml) and KHSO4 (5%, w/v; 0.5 ml) were added to each sample and to standards (consisting of distilled water and 10 μg/ml glucosamine). Aliquots (0.6 ml) of each were transferred to separate tubes, 0.2 ml of ammonium sulfamate and then 0.2 ml of 3-methyl-2-thiazolone hydrazone HCl monohydrate (50 mg in 10 ml of distilled water) were added, and samples were heated to 100°C for 3 min and cooled. FeCl3·6H2O (0.83%, w/v; 0.2 ml) was then added to each, and OD was measured at 650 nm after 25 min. Chitin content, in glucosamine equivalents, was then calculated as follows: chitin content = {[5 × (OD of organ − OD of control organ)]/(OD of glucosamine − OD of water)}.
Lung myeloperoxidase (MPO) activity
Lung MPO activity was measured as a marker of neutrophil sequestration, as described previously (52). 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% hexadecyltrimthylammonium bromide, and 5 mM EDTA. Samples were sonicated for 10 s and centrifuged at 3000 × 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% H2O2, and o-dianisidine hydrochloride, and read at 490 nm. MPO units were calculated as the change in absorbency over time.
MIP-1α ELISA
Murine MIP-1α level was quantified using a modification of a double-ligand method as described previously (36). Briefly, flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F, Nunc, Copenhagen, Denmark) were coated with 50 μl/well of rabbit anti-MIP-1α 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, and 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 (undiluted and 1/10) cell-free 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 anti-MIP-1α Abs (3.5 μg/ml in PBS (pH 7.5), 0.05% Tween 20, and 2% FCS), and plates were 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 3 M H2SO4 solution. Plates were read at 490 nm in an ELISA reader. Standards were half-log dilutions of recombinant murine MIP-1α from 1 pg/ml to 100 ng/ml. This ELISA method consistently detected MIP-1α at concentrations >25 pg/ml. The ELISAs did not cross-react with IL-1, IL-2, IL-4, IL-6, IL-10, or IFN-γ. In addition, the 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 the InStat version 2.01 statistical package (GraphPad Software, San Diego, CA). Survival data were compared using Fisher’s exact test. All other data were expressed as the mean ± SEM and compared using an unpaired two-tailed Mann-Whitney (nonparametric) test. Values of p < 0.05 were considered statistically significant.
Results
Involvement of mononuclear cells in host defense against A. fumigatus in neutropenic mice
We first examined the influx of inflammatory cells into the lungs in response to intratracheal challenge with A. fumigatus conidia in both normal and neutrophil-depleted C57BL/6 mice. Lungs were harvested at various time points after challenge for histology. In normal animals, histology showed evidence of a patchy peribronchial infiltration of inflammatory cells within both the interstitial and alveolar compartments on day 2, which consisted predominantly of neutrophils. By day 4 after challenge, the cellular infiltrate had decreased and consisted of both neutrophils and mononuclear cells (Figs. 1⇓, A and B). As previously described, fungal hyphae did not form in tissues of immunocompetent animals, and the inflammatory cellular infiltrates were associated with areas of conidia deposition (41, 53).
Lung histopathology after A. fumigatus challenge. A and B, Representative lung hematoxylin and eosin (H&E) stains in immunocompetent mice 2 and 4 days after inoculation with 1–2 × 107 A. fumigatus conidia, respectively (magnification, ×200). There was a dense inflammatory cellular infiltrate, consisting predominantly of neutrophils, in peribronchial areas. By day 4 the cellular infiltrate was less dense and consisted of both neutrophils and mononuclear cells. C and D, Representative lung H&E stains in mice treated with RB6-8C5 2 and 4 days after inoculation with 1–2 × 106 A. fumigatus conidia, respectively (magnification, ×200). The cellular infiltrate on day 2 consisted predominantly of mononuclear cells, which are more clearly visible under high power (magnification, ×400, inset). By day 4, the cellular infiltrate was very dense and consisted of both neutrophils and mononuclear cells. The data shown are representative of three experiments.
Neutrophil depletion was induced by a single i.p. injection of RB6-8C5 1 day before i.t. challenge with 1–2 × 106 A. fumigatus conidia (day −1). This resulted in peripheral blood neutropenia (absolute circulating neutrophil count, <50 cells/μl) by days 0 and 2 after i.t. challenge, with a return of peripheral counts to pretreatment levels by day 4. Lung histology on day 2 showed a predominantly mononuclear cellular infiltrate (Fig. 1⇑, C and inset). These inflammatory cells occurred in association with fungal hyphae as previously described (41, 53). By day 4 after challenge, the patchy cellular infiltrate was extremely dense and consisted of both neutrophils and mononuclear cells associated with fungal hyphae (Fig. 1⇑D).
Expression of MIP-1α in the lungs in response to A. fumigatus
To address the mechanism of influx of mononuclear cells into the lungs in response to challenge with A. fumigatus in normal and neutropenic animals, we next measured concentrations of MIP-1α in lung homogenates at various times after i.t. administration of conidia. MIP-1α was induced in the lungs of both normal and neutropenic animals after inoculation with conidia compared with animals challenged with vehicle. MIP-1α levels reached a higher peak in neutropenic animals compared with normal animals (Fig. 2⇓), but were undetectable in plasma in both groups, indicating a compartmentalized response.
Time-dependent production of MIP-1α protein in lungs from normal and neutrophil-depleted mice after the i.t. administration of vehicle or A. fumigatus conidia. A, Lung MIP-1α levels after challenge with 1–2 × 107 A. fumigatus conidia in immunocompetent mice. B, Lung MIP-1α levels after challenge with 1–2 × 106 A. fumigatus conidia or vehicle in immunocompetent and neutropenic mice. The lung levels of MIP-1α in immunocompetent animals receiving this dose of intratracheal conidia (♦) were not significantly different from those in uninfected animals. Af, A. fumigatus; RB6-8C5, rat anti-mouse Ly-6G IgG2b mAb. Data shown represent the mean ± SEM of six animals for each group at each time point. ∗, p < 0.05 compared with animals receiving i.t. vehicle. The data shown are representative of three separate experiments.
Effect of MIP-1α neutralization on the course of infection in neutropenic mice challenged with A. fumigatus
To assess the contribution of MIP-1α to host defense against A. fumigatus in neutropenic animals, we next assessed the survival of animals pretreated with rabbit anti-MIP-1α serum. In normal (nonneutropenic) animals, administration of anti-MIP-1α serum followed by i.t. challenge with 1–2 × 107 conidia did not result in any deaths. However, administration of anti-MIP-1α serum to neutropenic animals 2 h before challenge with 1–2 × 106 conidia resulted in a >6-fold increase in mortality compared with neutropenic animals receiving control serum (Fig. 3⇓).
Effect of MIP-1α neutralization on survival after A. fumigatus challenge in neutropenic mice. Mice were treated with i.p. RB6-8C5 mAb 1 day before i.t. challenge with A. fumigatus (1–2 × 106 conidia). NRS, normal rabbit serum; RB6-8C5, rat anti-mouse Ly-6G IgG2b mAb (n = 14 or 15 animals/group). ∗, p < 0.05 compared with animals receiving i.p. saline. The data shown were pooled from two separate experiments.
To determine the cause of increased lethality in anti-MIP-1α-treated neutropenic animals, mice were sacrificed on day 2 after challenge with conidia, and lungs were harvested for histology and chitin content. Compared with those in animals treated with control serum, lungs of anti-MIP-1α-treated animals contained a substantially greater number of hyphal forms, as determined by histologic examination (Fig. 4⇓). To quantify the burden of hyphae present in the two groups, lung chitin levels were measured 2 days after challenge with conidia in neutropenic animals receiving anti-MIP-1α or control serum. A 12-fold increase in lung chitin content was noted in animals treated with anti-MIP-1α compared with control animals (Fig. 5⇓). Chitin levels were undetectable in nonneutropenic animals treated with anti-MIP-1α or control serum (data not shown).
Effect of MIP-1α neutralization on lung histopathology after A. fumigatus challenge. A and B, Representative lung Gomori methanamine silver (GMS) stain in neutrophil-depleted animals 2 days after inoculation with 1–2 × 106 A. fumigatus conidia in animals pretreated with normal rabbit serum and anti-MIP-1α serum, respectively (magnification, ×200). The data shown are representative of two experiments.
Effect of MIP-1α neutralization on lung chitin in neutropenic mice 2 days after intratracheal challenge with 1–2 × 106 A. fumigatus conidia. Negative control, lungs of uninfected immunocompetent animals; RB6-8C5, rat anti-mouse Ly-6G IgG2b mAb; NRS, normal rabbit serum. The data shown represent the mean ± SEM of six experimental animals for each group. ∗, p < 0.05 compared with animals receiving i.p. normal rabbit serum. The data shown are representative of two separate experiments.
Effect of MIP-1α depletion on influx of immune cells into the lungs in neutropenic mice challenged with A. fumigatus
To determine whether impaired fungal clearance in MIP-1α-depleted animals occurred as a result of changes in lung neutrophil influx, lungs from neutropenic animals treated with anti-MIP-1α or control serum were harvested for estimation of myeloperoxidase activity, a surrogate measure of neutrophil presence. On the basis of studies showing time of maximum expression of MPO (not shown), MPO levels were evaluated 2 days after inoculation in RB6-8C5-treated animals. There was no significant difference in the lung MPO content of neutropenic animals treated with anti-MIP-1α or control serum 2 days after challenged with 1–2 × 106 conidia, indicating that differences in neutrophil influx did not explain the worse outcome with MIP-1α neutralization.
We next assessed whether the poor outcome of infection in MIP-1α-depleted neutropenic animals was attributable to differences in the influx of other cell populations into the lungs, by examining whole lung single cell suspensions. Normal animals, neutropenic animals pretreated with control serum, and neutropenic animals pretreated with anti-MIP-1α serum were sacrificed 2 days after i.t. challenge with 1–2 × 106 A. fumigatus conidia, and lung cell suspensions were prepared and compared with those from normal uninfected animals. The total number of cells in the lungs of neutropenic animals pretreated with anti-MIP-1α was 36% less than the number in neutropenic mice treated with control serum (Fig. 6⇓A). The number of lung neutrophils, however, did not differ significantly between the two groups (Fig. 6⇓B), thus confirming the findings of earlier experiments that employed MPO as a surrogate for neutrophil presence. Flow cytometry of lung homogenates showed that anti-MIP-1α-treated neutropenic animals had 41% fewer lung monocyte/macrophages compared with neutropenic animals treated with control serum (Fig. 6⇓C), while the total numbers of lung lymphocytes were not significantly different in the two groups (mean, 1.16 × 107 and 7.49 × 106 cells, respectively; p = 0.39).
Effect of MIP-1α neutralization on cell influx into the lungs 2 days after intratracheal challenge with 1–2 × 106A. fumigatus conidia. A, Total cell content of lung suspensions. B, Number of lung neutrophils, as measured by total lung cell suspension count and differentials. C, Number of lung monocyte/macrophages, as measured by total lung cell suspension count and FACS analysis. Af, A. fumigatus; RB6-8C5, rat anti-mouse Ly-6G IgG2b mAb; NRS, normal rabbit serum. The data shown represent the mean ± SEM of six experimental animals for each group. ∗, p < 0.05 compared with neutropenic animals treated with normal rabbit serum. The data shown are representative of two separate experiments.
Discussion
Invasive aspergillosis is a devastating infection of immunocompromised hosts that carries a very high mortality. Because the vast majority of affected patients have a reduced number of available neutrophils, we investigated the role of mononuclear cells and mediators of their recruitment and activation in host defense against this infection in the setting of neutropenia. Neutrophil depletion in humans most often results from the administration of cytotoxic chemotherapy in the setting of malignancy or bone marrow transplantation, which also results in depletion or dysfunction of multiple other immune cells. We employed a murine model of transient Ab-mediated neutrophil depletion, which we have characterized previously (53). This model closely resembles human disease clinically and histologically and is unaffected by the pleotropic influence of chemotherapeutic agents on various other immunologically active cells. Furthermore, the dynamics of recovery of neutrophil counts in this model and the accumulation of available neutrophils in the lungs as the counts recover mimic the clinical scenario in patients recovering from cytotoxic chemotherapy.
We have previously shown that depletion of TNF in murine invasive pulmonary aspergillosis resulted in increased mortality and lung fungal burden, which was associated with decreased lung levels of the CXC chemokine, MIP-2, and the CC chemokines, MIP-1α and JE (41). We subsequently demonstrated that neutralization of the common murine receptor for the glutamic acid-leucine-arginine-positive CXC chemokines, CXCR-2, resulted in a markedly increased susceptibility to this infection in nonneutropenic animals, which occured in association with a decreased influx of neutrophils into the lungs. In addition, the intrapulmonary transgenic expression of the glutamic acid-leucine-arginine-positive CXC chemokine, KC, reduced the susceptibility to this infection (53). In the present study we demonstrated that in the setting of pulmonary aspergillosis in transiently neutropenic mice, MIP-1α plays a critical role in host defense. Specifically, neutralization of MIP-1α resulted in a reduction of lung mononuclear cell influx, identified as monocyte/macrophages by flow cytometry. Given the short duration of the response (2 days), this increase in lung monocyte/macrophages almost certainly represents active recruitment rather than proliferation. In addition to changes in the numbers of lung mononuclear phagocytes after MIP-1α neutralization, the activation state of these cells is also likely to have changed. Although we did not observe qualitative changes in total lung lymphocytes, a similar change in the activation state cannot be excluded. With regard to neutrophils, we found that neutralization of MIP-1α in nonneutropenic mice did not result in mortality or invasive disease. Furthermore, in transiently neutrophil-depleted animals, we found no difference in the numbers of neutrophils in the lungs of MIP-1α-depleted and control animals either by direct cell count or lung MPO activity. Collectively, these observations suggest that the effects of MIP-1α in this model are not mediated through alterations in neutrophil recruitment.
Like many chemokines, MIP-1α exhibits receptor promiscuity, binding to both CCR-1 and CCR-5. A study of mice with targeted deletion of CCR-1 has shown increased susceptibility of these animals to i.v. injected A. fumigatus, a finding that was interpreted as evidence of abnormal neutrophil-mediated host defense (54). Importantly, we did not observe any increase in susceptibility to intratracheal A. fumigatus in CCR-1 knockout animals compared with wild-type controls (data not shown). Our data therefore indicate that CCR-1 is not essential for protective innate immunity against A. fumigatus in the lung. The discrepancy between our findings and those of Gao et al. may be explained by several considerations, which are not mutually exclusive. First, the i.v. administration of Aspergillus conidia, as employed by Gao et al. (55), has previously been shown to result in foci of infection predominantly in the kidney and brain rather than the lung. Second, in the setting of disseminated and overwhelming infection in a normal host, the innate immune response is likely to differ from the response to intrapulmonary challenge in a neutropenic host and may absolutely require CCR-1 expression by neutrophils and possibly CCR-1 ligands other than MIP-1α.
A caveat to all studies of chemokines in murine models is the potential for dissimilarities in chemokine biology between human and murine systems; such differences have been well characterized in the CXC chemokines (56). This study has identified MIP-1α as a critical mediator of host defense against invasive aspergillosis in the setting of neutropenia. Because the beneficial effect of MIP-1α in invasive pulmonary aspergillosis is most likely to be observed with compartmentalized overexpression, studies aimed to define the therapeutic potential of lung-specific overexpression of MIP-1α are in progress.
Footnotes
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1 This work was supported in part by National Institutes of Health Grants 1K08HL04220-01, HL57243, HL58200, and P50HL60289.
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2 Address correspondence and reprint requests to Dr. Borna Mehrad, University of Michigan Medical Center, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0360. E-mail: bmehrad{at}umich.edu
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↵3 Abbreviations used in this paper: MIP-1α, macrophage inflammatory protein-1α; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; i.t., intratracheal; MCP, monocyte chemoattractant protein; MPO, myeloperoxidase.
- Received December 29, 1999.
- Accepted April 25, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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