Infection Nocardia Asteroides Ligands in a Murine Model of Pulmonary Dependent on CXC Chemokine Receptor-2 Bacterial Clearance A

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Bacterial Clearance and Survival Are Dependent on CXC Chemokine Receptor-2 Ligands in a Murine Model of Pulmonary Nocardia asteroides Infection This information is current as of September 29, 2021. Thomas A. Moore, Michael W. Newstead, Robert M. Strieter, Borna Mehrad, Blaine L. Beaman and Theodore J. Standiford J Immunol 2000; 164:908-915; ; doi: 10.4049/jimmunol.164.2.908 Downloaded from http://www.jimmunol.org/content/164/2/908

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Bacterial Clearance and Survival Are Dependent on CXC Chemokine Receptor-2 Ligands in a Murine Model of Pulmonary Nocardia asteroides Infection1

Thomas A. Moore,2* Michael W. Newstead,* Robert M. Strieter,* Borna Mehrad,* Blaine L. Beaman,† and Theodore J. Standiford*

Survival from murine pulmonary nocardiosis is highly dependent on CXC chemokine receptor-2 (CXCR2) ligand-mediated neutrophil chemotaxis and subsequent clearance of the infectious agent Nocardia asteroides. Intratracheal inoculation of N. asteroides rapidly up-regulated the CXC chemokines macrophage inﬂammatory protein-2 (MIP-2) and KC within 24 h, with levels remaining elevated through day 3 before returning to near baseline levels by day 7. Coinciding with elevated MIP-2 and KC were

the rapid recruitment of neutrophils and clearance of the organism. Anti-Ly-6G Ab-mediated neutrophil depletion before bac- Downloaded from terial challenge resulted in strikingly increased mortality to N. asteroides infection. The relative contribution of MIP-2 in neutrophil recruitment was examined by anti-MIP-2 Ab treatment before nocardial infection. MIP-2 neutralization had no detrimental effects on survival, neutrophil recruitment, or bacterial clearance, suggesting the usage of additional or alternative CXCR2- binding ligands. The importance of the CXC family of chemokines was determined by the administration of an anti-CXCR2 Ab capable of blocking ligand binding in vivo. Anti-CXCR2 treatment greatly increased mortality by preventing neutrophil migration into the lung. Paralleling this impaired neutrophil recruitment was a 100-fold increase in lung bacterial burden. Combined, these http://www.jimmunol.org/ observations indicate a critical role for neutrophils and CXC chemokines during nocardial pneumonia. These data directly link CXCR2 ligands and neutrophil recruitment and lend further support to the concept of CXC chemokine redundancy. For infections highly dependent on neutrophils, such as nocardial pneumonia, this is of critical importance. The Journal of Immunology, 2000, 164: 908–915.

ulmonary nocardiosis can be an acute or chronic infection The rigorous characterization of Ͼ50 strains of N. asteroides characterized by a suppurative response with pathology has greatly facilitated the study of bacterial-host interactions (re- ranging from mild, diffuse peribronchial infiltration to lo- viewed in Ref. 2). Virulent strains of N. asteroides have evolved P by guest on September 29, 2021 bar or multilobar consolidation (1, 2). Granulomatous lesions may unique mechanisms of evading host macrophage-mediated killing. form with or without central necrosis. Lesions usually consist of a Specifically, virulent strains can inhibit phagosome-lysosome fu- mixed cellular response of neutrophils, macrophages, and lympho- sion, decrease lysosomal enzyme activity, and neutralize phago- cytes. Dissemination beyond the primary pulmonary site of infec- somal acidification (3–7). These evasive mechanisms result in the tion may occur, with nearly one-half of the patients with systemic ability of the organism to grow intracellularly within alveolar mac- nocardiosis having CNS involvement. Whereas mortality is low rophages. During experimental pulmonary nocardiosis, the inflam- (Ͻ20%) in patients with no predisposing underlying illness, mor- matory response is predominantly neutrophilic, subsequently re- tality rates of Ͼ50% exist in patients with disseminated disease. placed by a mononuclear infiltration by day 7. However, the Although Nocardia asteroides is considered an opportunistic precise role of neutrophils during infection is unresolved. In vitro pathogen, 30–40% of published patient cases have no predispos- data indicate that neutrophils and monocytes only marginally kill ing underlying illness. phagocytized N. asteroides (8–11). In contrast, in vivo depletion of neutrophils using a polyclonal anti-neutrophil Ab or monocytes using silica resulted in increased nocardial burden (12, 13). Chemokines are a growing collection of chemotactic cytokines *Department of Internal Medicine, Division of Pulmonary and Critical Care Medi- divided into four families (14). The CXC chemokine family can be † cine, University of Michigan Medical Center, Ann Arbor, MI 48109; and Depart- further subdivided based on the presence or absence of a three- ment of Medical Microbiology and Immunology, University of California School of Medicine, Davis, CA 95616 amino acid motif termed ELR (glutamic acid-leucine-arginine). ϩ Received for publication August 23, 1999. Accepted for publication October ELR CXC chemokines, including IL-8, epithelial neutrophil-ac- 29, 1999. tivating protein-78, macrophage inflammatory protein-2 (MIP-2),3 The costs of publication of this article were defrayed in part by the payment of page and KC, have potent neutrophil chemotactic activity (15–17). Two charges. This article must therefore be hereby marked advertisement in accordance receptors for ELRϩ CXC chemokines have been identified in hu- with 18 U.S.C. Section 1734 solely to indicate this fact. mans, CXC chemokine receptor-1 and -2 (CXCR1 and CXCR2) 1 This research was supported in part by a research grant from the American Lung Association (T.A.M.), Grant 1KO8 HL04220–01 (B.M.), Grant P50HL60289 (18, 19). Murine CXCR2, like its human counterpart, binds all (R.M.S.), and Grants HL57243, HL58200, P50HL60289 (T.J.S.) from the National Institutes of Health. T.A.M. is a Parker B. Francis Fellow in Pulmonary Research and an Edward Livingston Trudeau Fellow of the American Lung Association. 2 Address correspondence and reprint requests to Dr. Thomas A. Moore, University 3 Abbreviations used in this paper: MIP-2, macrophage inflammatory protein-2; of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, CXCR1 and 2, CXC chemokine receptor-1 and -2; LIX, LPS-induced CXC chemo- 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642. kine; BHI, brain-heart infusion; MPO, myeloperoxidase; H&E, hematoxylin and eo- E-mail address: [email protected] sin; GMS, Gomori methenamine-silver.

ELRϩ CXC chemokines (20–23). Interestingly, mice lack expres- Whole lung homogenization for CFU, myeloperoxidase (MPO), sion of CXCR1 (20). These receptors, like all chemokine receptors and cytokine analysis identified to date, are seven-transmembrane spanning, G-protein- At designated time points, the mice were euthanized by inhalation of CO2. coupled receptors. Neutrophils from CXCR2 knockout mice fail to The lungs were perfused with 1 ml PBS, 5 mM EDTA and removed for migrate in response to MIP-2 and KC in vitro (24), confirming the analyses as previously described (34). Briefly, lungs were homogenized exclusive utilization by CXC chemokines of this receptor on with a tissue homogenizer (Biospec Products, Bartlesville, OK) in 1 ml neutrophils. PBS-complete protease inhibitor mixture (Boehringer Mannheim Bio- chemical, Chicago, IL). For lung CFU determination, a small aliquot of Here, we detail the dependence of CXCR2 ligand-mediated neu- lung homogenate was serially diluted and plated on blood agar plates and trophil recruitment for effective host defense in pulmonary infec- incubated at 37°C, and colonies were counted. tions due to N. asteroides. These data directly link CXCR2-bind- Lung MPO activity, as an indirect measurement of total neutrophil num- ing chemokines and neutrophil recruitment during N. asteroides bers, was quantitated by a method described previously (34). Briefly, 100 ␮l lung homogenate were mixed with 100 ␮l MPO homogenization buffer pulmonary infection and indicate a critical role for neutrophils and (0.5% hexadecyltrimethylammonium bromide and 5 mM EDTA) and vor- CXC chemokines during the initial phases of pulmonary texed. The mixture was sonicated and centrifuged at 12,000 ϫ g for 15 nocardiosis. min. The supernatant was then mixed 1:15 with assay buffer and read at 490 nm. MPO units were calculated as the change in absorbance over time. Materials and Methods For total lung cytokine ELISA analyses, lung homogenates were sonicated briefly to ensure complete cellular disruption, then centrifuged at Mice 2500 rpm for 10 min. The supernatants were collected and assessed for Female BALB/c mice were purchased from The Jackson Laboratory (Bar cytokine levels by ELISA. Murine MIP-2 and KC were quantitated by a Harbor, ME). Mice were used between the ages of 5–8 wk, being age- modification of a sandwich ELISA method. This methodology allows de- Downloaded from matched within a given experiment. All mice were housed under specific tection of MIP-2 and KC at concentrations of 20 pg/ml and higher. Addi- pathogen-free condition within the animal care facility at the University of tionally, assays have been shown to be specific for the indicated murine Michigan. chemokine and show no cross-reactivity with any other murine cytokines tested (34). N. asteroides growth and intratracheal inoculation Total lung leukocyte preparation N. asteroides strain GUH-2 was originally isolated from a fatal kidney infection in a renal transplant patient. It is a highly virulent strain of No- Total lung leukocytes were isolated from N. asteroides-infected mice on http://www.jimmunol.org/ cardia with growth characteristics that have been well documented (25– day 1 postinfection. Lungs were removed from euthanized animals, and 27). Briefly, brain heart infusion (BHI) broth (Difco, Detroit, MI) starter leukocytes were prepared as previously described (35). Briefly, lungs were cultures were inoculated from late stationary phase frozen stocks. Cultures minced with scissors to a fine slurry in 15 ml lung digestion buffer (RPMI, were grown for 4–6 days on an orbital shaker at 37°C. Overnight cultures 5% FCS, 1 mg/ml collagenase (Boehringer Mannheim Biochemical), 30 were then inoculated by transferring 10–30 ␮l of a late stationary phase ␮g/ml DNase (Sigma, St. Louis, MO)). Lung slurries were enzymatically starter culture into 50 ml BHI broth and shaken for 16–18 h. The bacterial digested for 30 min at 37°C. Any undigested fragments were further dis- concentration of early to mid-log phase cultures was then determined by persed by drawing the solution up and down through the bore of a 10-ml measuring absorbance at 580 nm and compared with a predetermined stan- syringe. The total lung cell suspension was pelleted, resuspended, and spun dard curve. The filamentous, branching morphology characteristic of log through a 20% Percoll gradient to enrich for leukocytes before further phase growth was confirmed for each culture by Gram stain (Difco). Bac- analysis. Cell counts and viability were determined by trypan blue exclu- by guest on September 29, 2021 teria were then diluted to the desired concentration for intratracheal inoc- sion counting on a hemacytometer. Cytospin slides were prepared and ulation. BALB/c mice were anesthetized with pentobarbital (diluted 1:7 in stained with a modified Wright-Giemsa stain to determine percent and total saline). The trachea was exposed, and 30 ␮l inoculum or saline adminis- lung neutrophil numbers. tered via a sterile 26-gauge needle. An aliquot of the inoculated nocardial suspension was serially diluted onto blood agar plates to determine actual Tissue harvesting for histological examination dose of intratracheally injected bacteria. Lungs for histochemistry were perfused with 4% paraformaldehyde in In vivo Ab administration PBS, then inflated with 4% paraformaldehyde to improve resolution of anatomic relationships, and excised at the hilum. Lung sections were then Neutrophils were depleted in vivo utilizing the pan-granulocytic Ab RB6- stained with hematoxylin and eosin (H&E) to determine inflammatory re- 8C5 (28), directed against Ly-6G. Anti-Ly-6G was produced as an ascites sponses during infection and Gomori methenamine-silver (GMS) to deter- in SCID mice by TSD BioServices (Germantown, NY) and used at a di- mine the presence of N. asteroides. lution determined to deplete both peripheral blood and resident lung neutrophils. The Ab was injected in a 0.5-ml volume i.p. 18 h before nocardial Statistical analysis infection and again 1 day postinfection. This treatment scheme has been used to deplete neutrophils in vivo in a variety of bacterial and fungal Statistical significance was determined using the unpaired, two-tailed al- models (29–31). Reagent control animals for anti-Ly-6G received normal ternate Welsh t test and the nonparametric Mann-Whitney test. Calcula- mouse serum at the same dilution used for anti-Ly-6G treatment. Poly- tions were performed using InStat For Macintosh (GraphPad Software, San clonal Ab directed against the murine CXCR2 chemokine receptor was Diego, CA). produced by immunizing goats with a peptide containing the ligand-binding sequence Met-Gly-Glu-Phe-Lys-Val-Asp-Lys-Phe-Asn-Ile-Glu-Asp- Phe-Phe-Ser-Gly. This protein sequence has been shown to contain the Results ligand-binding portion of the CXCR2 receptor (32). Anti-CXCR2 admin- Dose-dependent mortality from nocardial pneumonia induced by istration did not alter the number of circulating neutrophils in BALB/c N. asteroides strain GUH-2 mice. Additionally, i.p. injection of anti-CXCR2 Ab abrogated neutrophil recruitment into the peritoneum in response to exogenous KC (data not Murine nocardial pneumonia was induced by the intratracheal in- shown) and prevented neutrophil recruitment in a murine model of pul- jection of log growth phase cultures of the highly virulent strain monary aspergillosis (33). In CXCR2 receptor blockade experiments, mice GUH-2 of N. asteroides into BALB/c mice. To determine suscep- were injected i.p. with 0.5 ml goat anti-mouse CXCR2 Ab 2 h before tibility in our model, various doses of GUH-2 were injected and nocardial infection. For studies extending beyond 1 day postinfection, mice received another dose of Ab 36 h postinfection. Mice were treated with animals were observed for signs of illness and mortality. Inoculum 6 rabbit anti-mouse MIP-2 Ab in a manner similar to that for anti-CXCR2 doses of less than 2 ϫ 10 CFU resulted in clinical signs of illness treatment. Depletion of MIP-2 was confirmed by ELISA. Control mice in within one day post infection, which subsequently resolved with- anti-CXCR2 experiments received normal goat serum, and control mice in out any mortality observed (Fig. 1). Doses of GUH-2 ranging be- anti-MIP-2 experiments received normal rabbit serum i.p. In our hands, ϫ 6 infected mice receiving control reagents for anti-Ly-6G, anti-CXCR2, and tween 3–4 10 CFU induced significant signs of overt illness anti-MIP-2 had no detrimental effects when compared with infected ani- within one day which progressively worsened over the next 1–2 mals alone. days, resulting in significant mortality. Mice surviving the first 3–4 910 CXCR2 LIGANDS IN PULMONARY NOCARDIOSIS

inﬂammation within the lumen of distal airways 1 day after inoculation with GUH-2. Branching Nocardia organisms were still observed on day 3, although fewer in number. By day 7, few No- cardia were seen, in agreement with the paucity of recoverable CFU at this time point (ϳ0.5% of the total inoculated dose of N. asteroides, data not shown).

Effect of neutrophil depletion on nocardial pneumonia mortality The rapid increase in lung MPO activity suggested an important role for neutrophils in the resolution of N. asteroides pulmonary infection. To test this hypothesis, mice were depleted of neutrophils in vivo before intratracheal GUH-2 inoculation utilizing a mAb directed against the pan-granulocytic marker Ly-6G. Mice FIGURE 1. Dose-dependent mortality from nocardial pneumonia in- depleted of their neutrophils before infection required 100-fold duced by N. asteroides strain GUH-2. BALB/c mice were intratracheally inoculated with graded doses of early to mid-log phase nocardial bacteria. fewer inoculated N. asteroides bacteria to achieve an equivalent Percent survival was determined over the course of 7 days postinfection. lethal dose seen in non-neutrophil-depleted mice (Fig. 4, LD20, 4 6 Mice surviving the first 3–4 days continued to survive long term. Curves 3 ϫ 10 CFU in neutrophil-depleted animals vs 3 ϫ 10 in control were generated from 2–3 independent experiments per bacterial dose with mice). a total of 15–20 mice per dose. Downloaded from Chemokine production after intratracheal inoculation with N. asteroides days of infection went on to survive long-term with no overt signs Data thus far indicated a vigorous and rapid host response to no- of illness. cardial pulmonary challenge, resulting in a neutrophil dominant inflammatory response. Given that ELRϩ chemokines exhibit po- Neutrophil recruitment kinetics during nocardial pneumonia http://www.jimmunol.org/ tent neutrophil chemotactic activity in vitro and in vivo, production Pulmonary neutrophil recruitment during N. asteroides infection of the murine ELRϩ chemokines MIP-2 and KC was determined was examined by measuring total lung MPO activity over time. in lung homogenates after N. asteroides challenge (Table I). MIP-2 Lung MPO activity reached maximal levels within 1 day of infec- production was rapidly up-regulated within 24 h after nocardial tion, with elevated activity persisting through day 3 before return- challenge, with levels increasing through day 3 followed by a sig- ing to near baseline levels by day 7 (Fig. 2). nificant reduction by day 7 postchallenge. KC secretion was also The kinetics of pulmonary neutrophil recruitment and bacterial rapidly induced within 24 h. Unlike MIP-2 production, KC levels clearance were confirmed by histological examination of infected increased only slightly by day 3, followed by a rapid decrease by lungs on days 1, 3, and 7 postinfection (Fig. 3). A rapid inflam- day 7. The return to near baseline levels of both MIP-2 and KC by by guest on September 29, 2021 matory response consisting predominantly of neutrophils could be day 7 after infection is in concordance with both the reduction in seen as early as day 1. Lungs exhibited signs of perivascular in- lung MPO activity and histological examination, indicating the flammation with distinct sites of bronchopneumonia containing replacement of neutrophils within the sites of inflammation with neutrophils and proteinaceous exudates. By day 3, the inflamma- mononuclear cells. tory sites had become more consolidated, although still containing mainly neutrophils. Mononuclear cells replaced this early neutro- Effect of anti-CXCR2 treatment on nocardial pneumonia philic infiltration by day 7. A GMS stain determined the presence mortality of N. asteroides in histology sections. Abundant branching, filamentous Nocardia were observed predominantly in active sites of The rapid and sustained production of high levels of MIP-2 suggested that this chemokine may be an important component in neutrophil recruitment during N. asteroides infection. To test this hypothesis, mice were treated with an anti-MIP-2 Ab capable of inhibiting the bioactivity of MIP-2 in vivo (17). Interestingly, inhibition of MIP-2 activity before infection had no detrimental effect on survival after N. asteroides challenge (data not shown). Furthermore, anti-MIP-2 administration had no effect on the total number of pulmonary neutrophils recruited following nocardial infection or on pulmonary bacterial clearance (data not shown). This anti-MIP-2 data suggested the dependence on alternative or additional chemokines for neutrophil recruitment during pulmonary nocardiosis. Examining the role of individual CXC chemokines is compounded by the overlapping neutrophil chemotactic activity of numerous members of this family. To study the role of ELRϩ CXC chemokines as an entire family, a ligand-blocking FIGURE 2. Kinetics of total lung MPO activity during pulmonary no- polyclonal goat anti-mouse CXCR2 Ab was utilized. This Ab cardiosis. Mice were infected as described in Fig. 1, and lungs were har- ϩ blocks the binding of MIP-2, KC, and other ELR CXC chemo- vested at the indicated time points. Total lung MPO activity was determined as an indirect measurement of total lung neutrophil numbers. Mice kines to the CXCR2 receptor, thereby inhibiting neutrophil che- injected with saline were used as baseline MPO controls. MPO activity is motaxis in vitro and in vivo. Importantly, mice treated with anti- maximal by days 1 and 3, returning to near baseline levels by day 7. Each CXCR2 were significantly more susceptible to nocardial infection time point consists of 2–4 independent experiments for a total of 15–23 (Fig. 5), analogous to that observed after depletion of neutrophils animals. with anti-Ly-6G. The Journal of Immunology 911

FIGURE 3. Temporal histological examination of nocardial pneumonia. Mice were infected as described in Fig. 1, and lungs were removed on day 1 (A–C), day 3 (D–F), and day 7 (G–I) postinfection for histological analyses. Low power H&E stains (A, D, G) indicate extensive focal sites of neutrophil

dominant inflammation 1 day after infection (A) with Downloaded from gradual mononuclear cell replacement by day 7 (G). High power fields (B, E, H) more clearly show extensive neutrophil recruitment into sites of inflammation on days 1 and 3 (B, E) being replaced by mononuclear cells on day 7 (H). Examination of the same high power fields on GMS stained sections clearly show extensive filamentous nocardial growth http://www.jimmunol.org/ in sites of inflammation on day 1 (C) and day 3 (F) postinfection. By day 7, few filamentous nocardia can be seen (I, arrows), in agreement with the paucity of recoverable total lung CFU (ϳ0.5% of the total inoculated dose of N. asteroides). by guest on September 29, 2021

Effect of anti-CXCR2 or anti-Ly-6G treatment on neutrophil recruitment after N. asteroides infection To assess the effect of anti-CXCR2 or anti-Ly-6G treatment on neutrophil recruitment during N. asteroides infection, total lung neutrophil numbers were examined after infection and Ab treatment. Total pulmonary neutrophils were determined by enzymatic dissociation of lung leukocytes to observe directly any effect on neutrophil recruitment by either anti-CXCR2 or anti-Ly-6G treatment. Anti-CXCR2 and anti-Ly-6G treated mice were highly susceptible to nocardial infection; therefore the inoculum dose of GUH-2 was reduced to 1–2 ϫ 105 CFU from 2–3 ϫ 106 to ensure mouse survival 24 h postinfection. This decreased dose resulted in 100% mortality in neutrophil-depleted mice by day 2 while still FIGURE 4. Effect of neutrophil depletion on nocardial pneumonia mor- presenting a signiﬁcant challenge to nondepleted mice. Intratra- tality. Neutrophils were depleted in vivo utilizing an anti-Ly-6G mAb cheal challenge with N. asteroides resulted in a 4-fold increase in (clone RB6-8C5). Ab was injected in a 0.5-ml volume i.p. 18 h before total lung neutrophil numbers 1 day postinfection (Fig. 6). Anti- nocardial infection and again 1 day postinfection. Mice were infected with CXCR2 treatment did not decrease the baseline number of lung the indicated doses of nocardial strain GUH-2 bacteria, and effects on mor- neutrophils below that of mice challenged with intratracheal saline. tality were determined. Curves were generated from 2 independent exper- However, pretreatment of mice with anti-CXCR2 Ab prevented iments per bacterial dose with a total of 10–15 mice per dose. 912 CXCR2 LIGANDS IN PULMONARY NOCARDIOSIS

Table I. Kinetics of MIP-2 and KC production during N. asteroides pulmonary infectiona

Total Lung Cytokine Levels (ng/ml)

MIP-2 KC

Group Day 1 Day 3 Day 7 Day 1 Day 3 Day 7

Saline 0.10 (0.01) 0.09 (0.014) 0.06 (0.01) 0.06 (0.01) 0.04 (0.01) 0.02 (0.01) GUH-2 14.88 (3.07) 155.02 (56.27) 0.67 (0.09) 5.09 (1.41) 3.78 (1.23) 0.12 (0.01)

a BALB/c mice were intratracheally inoculated with 2–3 ϫ 106 CFU GUH-2 bacteria. Lungs were then harvested on the indicated time points, homogenized, and then assayed for MIP-2 and KC protein by ELISA as described in Materials and Methods. Data are presented as mean (SEM) and were generated from 2–3 independent experiments with a total of 15–20 mice. neutrophil recruitment into the lung after bacterial challenge. In dose (1–2 ϫ 105 CFU) of GUH-2, albeit at lower levels than seen contrast, mice treated with anti-Ly-6G followed by bacterial chal- with higher inoculum doses (see Table I). Both anti-CXCR2- and lenge had a dramatic decrease in total lung neutrophils compared anti-Ly-6G-treated mice had greatly enhanced production of with saline administration or after GUH-2 infection. MIP-2 (anti-CXCR2, 63 fold; anti-Ly-6G, 59 fold) and KC (anti- Histological examination of lung sections from GUH-2-infected CXCR2, 347-fold; anti-Ly-6G, 205-fold) compared with saline- mice pretreated with anti-CXCR2 or anti-Ly-6G Abs confirmed treated mice. These elevated levels are in part likely due to the Downloaded from both the extensive growth of branching chains of N. asteroides increased pulmonary bacterial burden seen in these mice (Fig. 8). localized predominantly in the distal airways and the paucity of inflammatory cell influx 24 h postinfection (Fig. 7). Filamentous Discussion N. asteroides extended into pulmonary airspaces in anti-CXCR2 or We have established a murine model of acute pulmonary nocar- anti-Ly-6G-treated mice. In comparison, airspace invasion was diosis via intratracheal inoculation of the highly virulent strain only rarely seen in control infected mice. http://www.jimmunol.org/

Effect of anti-CXCR2 or anti-Ly-6G on pulmonary bacterial burden The effect of anti-CXCR2 or anti-Ly-6G treatment on bacterial clearance was determined 24 h after inoculation. Treatment of mice with anti-CXCR2 or anti-Ly-6G before bacterial inoculation resulted in a Ͼ100 fold increase in total lung bacterial burdens when compared with control Ab-treated mice (Fig. 8). by guest on September 29, 2021 Effect of anti-CXCR2 or anti-Ly-6G treatment on pulmonary MIP-2 and KC production MIP-2 and KC are both rapidly produced at high levels during the normal course of nocardial infection. Because anti-CXCR2- or anti- Ly-6G-treated mice developed invasive disease in response to N. asteroides challenge, we reasoned that both MIP-2 and KC would be strongly up-regulated in these mice. MIP-2 (2.7-fold) and KC (3.6-fold) were induced in control mice infected with a sublethal

FIGURE 6. Total lung neutrophil numbers in anti-CXCR2- or anti-Ly- 6G-treated mice 1 day post-pulmonary nocardial infection. Because anti- Ly-6G- or anti-CXCR2-treated mice are significantly more susceptible to infection (see also Figs. 4 and 5), the dose of GUH-2 was decreased to 1–2 ϫ 105 CFU to ensure survival 1 day after infection. Lungs were harvested 24 h postinfection, and total neutrophils were determined from enzymatically digested lung leukocyte preparations. Even at this lowered inoculum dose, there is a 4-fold increase in total neutrophils in infected Ab control mice. Anti-CXCR2 treatment prevented the recruitment of neutrophils in response to infection (p Ͻ 0.005 vs Ab control group) without altering the total number of resident lung neutrophils seen in saline control animals. Anti-Ly-6G treatment dramatically reduced the total number of lung neu- FIGURE 5. Effect of CXCR2 receptor blockade on nocardial pneumo- trophils compared with saline treatment alone (p Ͻ 0.001). Statistical sig- nia mortality. Mice were injected i.p. 2 h before nocardial infection with nificance was determined by the unpaired, two-tailed Alternate Welsh t test 0.5-ml volume of a polyclonal goat anti-mouse CXCR2 Ab and again 36 h and nonparametric Mann-Whitney test. Both tests resulted in nearly iden- postinfection. Mice were infected with the indicated doses of nocardial tical significant p values. Ab control group is a composite of mice receiving strain GUH-2 bacteria and effects on mortality were determined. Curves either normal goat serum (control for anti-CXCR2) or normal mouse serum were generated from 2 independent experiments per bacterial dose with a (control for anti-Ly-6G ascites). Results are from three independent exper- total of 10–15 mice per dose. iments consisting of three mice per group per experiment. The Journal of Immunology 913

FIGURE 7. Histological examination of anti-Ly- 6G- or anti-CXCR2-treated mice. Animals were treated with anti-Ly-6G (C, D), anti-CXCR2 (E and F) or control Ab (A and B) and lungs removed 24 h post infection for histological analyses. High power H&E fields are shown in A, C, and E with corre- sponding GMS fields in B, D, and F. Extensive neutrophil infiltration (A) and filamentous nocardial growth (B) can be seen in control infected lungs. In contrast, anti-Ly-6G-treated lungs were devoid of any inflammatory cell influx (C). Anti-CXCR2- treated lungs were essentially devoid of inflammatory cell influx (E); however, some neutrophils could

be observed, in agreement with the number of total Downloaded from lung neutrophils recovered from these mice. Note the extensive nocardial growth in both anti-Ly-6G- and anti-CXCR2-treated mice (D and F) with extension of ﬁlamentous Nocardia into pulmonary airspaces. http://www.jimmunol.org/

GUH-2 of N. asteroides, thereby allowing the detailed examina- cident with rapid CXC chemokine production was neutrophil re- tion of early events during host innate immune responses to this cruitment into sites of active infection. As MIP-2 and KC levels

organism. Intratracheal infection rapidly up-regulated pulmonary decreased, neutrophils were replaced by mononuclear cells in sites by guest on September 29, 2021 MIP-2 and KC production, with levels remaining elevated for sev- of inflammatory response. Lung bacterial burden also decreased eral days before returning to near baseline levels by day 7. Coin- over time such that by day 7 fewer than 0.5% of inoculated CFU were recovered from infected lungs. The rapid up-regulation of the ELRϩ CXC chemokines MIP-2 and KC suggests an important role for this family of chemokines in neutrophil recruitment during pulmonary nocardiosis. When MIP-2 bioactivity was neutralized in vivo before infection, no detrimental effects on survival, neutrophil recruitment, or bacterial clearance were seen. CXC chemokines display functional redundancy and overlapping neutrophil chemotactic activity between numerous members of this chemokine family. Our data using a ligand blocking anti-CXCR2 Ab clearly indicates a critical role for CXCR2 ligands as a family in the recruitment of neutrophils to sites of nocardial infection. Of the murine CXCR2 ligands, MIP-2 and KC are the best studied. MIP-2 and KC have been shown to play important roles in neutrophil dependent pulmonary host de- fenses in response to other bacterial pathogens (17, 36). The inability of neutralizing anti-MIP-2 Ab to alter neutrophil recruitment during N. asteroides infection suggests additional or FIGURE 8. Effect of anti-Ly-6G or anti-CXCR2 treatment on total lung alternative CXCR2 ligand usage. The rapid and sustained produc- bacterial burden. Mice were treated with the indicated Abs as previously tion of KC during the first 3 days post inoculation would suggest described followed by intratracheal inoculation of 1–2 ϫ 105 CFU GUH-2 an important role for this chemokine during nocardiosis. We are bacteria. Lungs were analyzed 24 h later for bacterial burden. Both treat- currently attempting to generate in vivo neutralizing anti-KC Abs ments resulted in a Ͼ2-log increase in pulmonary bacterial burden when which will allow us to systematically determine the effects of anti- Ͻ compared with Ab control infected mice (p 0.001). Statistical signifi- KC alone or in combination with anti-MIP-2. Other intriguing cance was determined using the unpaired, two-tailed Alternate Welsh t test ϩ ELR CXC chemokine candidates include LPS-induced CXC and nonparametric Mann-Whitney test. Both tests resulted in nearly identical significant p values. The Ab control group is a composite of mice chemokine (LIX) and the recently described lungkine. LIX has receiving either normal goat serum (control for anti-CXCR2) or normal significant structural homology with human epithelial neutrophil- mouse serum (control for anti-Ly-6G ascites). Results are from three in- activating protein-78 and granulocyte chemotactic protein-2 (37, dependent experiments with a total of 22–25 mice per group. 38). During murine endotoxemia, prominent LIX expression was 914 CXCR2 LIGANDS IN PULMONARY NOCARDIOSIS observed in heart tissue with lesser amounts seen in lung (39). the inability of anti-MIP-2 treatment to negatively alter the out- Lungkine is of particular interest in that it is selectively produced come of infection lends further support to the concept of CXC by bronchoepithelial cells and induces neutrophil migration in vivo chemokine redundancy, resulting in the increased potential for and in vitro (40). Although not formally shown to utilize the neutrophil recruitment in the absence of any single CXC chemo- CXCR2 receptor, similarities in biological activity of lungkine kine. For infections highly dependent on neutrophils, such as pul- with known CXCR2 ligands suggest usage of this receptor. Cur- monary nocardiosis, this is of critical importance. rent studies are ongoing examining the role of lungkine and LIX in the host response to N. asteroides pulmonary infection. References Our observations point to a critical role for neutrophils in host 1. Lerner, P. I. 1996. Nocardiosis. Clin. Infect. Dis. 22:891. survival and eventual resolution of pulmonary nocardiosis. Mice 2. Beaman, B. L., and L. Beaman. 1994. Nocardia species: host-parasite relation- treated with anti-CXCR2 or anti-Ly-6G are exquisitely sensitive to ships. Clin. Microbiol. Rev. 7:213. nocardial infection, requiring 100 fold fewer inoculated bacteria to 3. Black, C. M., M. Paliescheskey, B. L. Beaman, R. M. Donovan, and E. Goldstein. 1986. Modulation of lysosomal protease-esterase and lysozyme in Kupffer cells achieve an equivalent lethal dose seen in control mice. These mice and peritoneal macrophages infected with Nocardia asteroides. Infect. Immun. are also severely impaired in their ability to clear the pulmonary 54:917. infection, displaying a 100 fold increase in bacterial burden 24 h 4. Black, C. M., M. Paliescheskey, B. L. Beaman, R. M. Donovan, and E. Goldstein. 1986. Acidification of phagosomes in murine macrophages: blockage by Nocar- after inoculation. These data extend a previous report indicating dia asteroides. J. Infect. Dis. 154:952. increased pulmonary nocardial burden in neutrophil depleted mice 5. Davis-Scibienski, C., and B. L. Beaman. 1980. Interaction of alveolar macrophages with Nocardia asteroides: immunological enhancement of phagocytosis, (12). Histological sections confirm the extensive growth of branch- phagosome-lysosome fusion, and microbicidal activity. Infect. Immun. 30:578. ing chains of N. asteroides in the absence of neutrophils (Fig. 7). 6. Black, C. M., B. L. Beaman, R. M. Donovan, and E. Goldstein. 1985. Intracel- Downloaded from Nocardial filaments were also seen extending from masses of No- lular acid phosphatase content and ability of different macrophage populations to kill Nocardia asteroides. Infect. Immun. 47:375. cardia into pulmonary airspaces. This type of airspace invasion 7. Beaman, B. L., C. M. Black, F. Doughty, and L. Beaman. 1985. Role of super- was rarely observed in nondepleted animals. Additionally, epithe- oxide dismutase and catalase as determinants of pathogenicity of Nocardia as- lial cells lining the airspaces of nocardial infected, neutrophil de- teroides: importance in resistance to microbicidal activities of human polymor- phonuclear neutrophils. Infect. Immun. 47:135. pleted mice appear enlarged and possibly disrupted. The strain of 8. Filice, G. A., B. L. Beaman, J. A. Krick, and J. S. Remington. 1980. Effects of

N. asteroides used in these studies (GUH-2) has been shown to human neutrophils and monocytes on Nocardia asteroides: failure of killing de- http://www.jimmunol.org/ specifically bind to and penetrate pulmonary epithelial cells in vivo spite occurrence of the oxidative metabolic burst. J. Infect. Dis. 142:432. 9. Filice, G. A. 1983. Resistance of Nocardia asteroides to oxygen-dependent kill- and in vitro (27, 41). It is possible that in the absence of neutro- ing by neutrophils. J. Infect. Dis. 148:861. phils, enhanced pulmonary epithelial cell invasion occurs, result- 10. Filice, G. A. 1985. Inhibition of Nocardia asteroides by neutrophils. J. Infect. ing in cellular injury and a likely worsening pathology. Dis. 151:47. ϩ 11. Beaman, B. L. 1979. Interaction of Nocardia asteroides at different phases of In addition to inducing neutrophil migration, ELR CXC che- growth with in vitro-maintained macrophages obtained from the lungs of normal mokines have been shown to activate neutrophils. Mice receiving and immunized rabbits. Infect. Immun. 26:355. 12. Filice, G. A., and D. E. Niewoehner. 1987. Contribution of neutrophils and cell- anti-CXCR2 treatment before infection still contained significantly mediated immunity to control of Nocardia asteroides in murine lungs. J. Infect. more neutrophils than did anti-Ly-6G treated mice (Fig. 6). It is Dis. 156:113. likely that anti-CXCR2 treatment, in addition to blocking neutro- 13. Pomeroy, C., and G. A. Filice. 1988. Effect of intravenous silica on the course of by guest on September 29, 2021 Nocardia asteroides pneumonia. Infect. Immun. 56:2507. phil recruitment, also prevented these resident cells from becoming 14. Rollins, B. J. 1997. Chemokines. Blood 90:909. activated by the high levels of CXC chemokines produced locally 15. Broaddus, V. C., A. M. Boylan, J. M. Hoeffel, K. J. Kim, M. Sadick, in the lung. This may in part explain why anti-CXCR2 treated A. Chuntharapai, and C. A. Hebert. 1994. Neutralization of IL-8 inhibits neutrophil influx in a rabbit model of endotoxin-induced pleurisy. J. Immunol. 152: mice displayed identical mortality rates and pulmonary bacterial 2960. burden as anti-Ly-6G mice while containing significantly more 16. Sekido, N., N. Mukaida, A. Harada, I. Nakanishi, Y. Watanabe, and neutrophils. K. Matsushima. 1993. Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature 365:654. The CXCR2 blockade studies clearly indicate the necessity of 17. Greenberger, M. J., R. M. Strieter, S. L. Kunkel, J. M. Danforth, L. L. Laichalk, CXC chemokines for neutrophil recruitment in response to N. as- D. C. McGillicuddy, and T. J. Standiford. 1996. Neutralization of macrophage teroides pulmonary challenge and that in their absence mortality inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia. J. Infect. Dis. 173:159. rates are greatly elevated. Receptor blockage or ligand neutraliza- 18. Lee, J., R. Horuk, G. C. Rice, G. L. Bennett, T. Camerato, and W. I. Wood. 1992. tion studies are useful for determining which cytokines are re- Characterization of two high affinity human interleukin-8 receptors. J. Biol. quired for the clearance of an infectious agent. A logical extension Chem. 267:16283. 19. Ahuja, S. K., and P. M. Murphy. 1996. The CXC chemokines growth-regulated of these studies would be to determine the therapeutic benefits of oncogene (GRO) ␣, GRO␤, GRO␥, neutrophil-activating peptide-2, and epithe- specific cytokine overexpression during infection. We have re- lial cell-derived neutrophil-activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor. J. Biol. Chem. 271:20545. cently shown that transgenic mice expressing the chemokine KC 20. Lee, J., G. Cacalano, T. Camerato, K. Toy, M. W. Moore, and W. I. Wood. 1995. driven by a lung specific promoter have increased resistance to Chemokine binding and activities mediated by the mouse IL-8 receptor. J. Im- Klebsiella pneumoniae infection (36). Preliminary studies using munol. 155:2158. 21. Suzuki, H., G. N. Prado, N. Wilkinson, and J. Navarro. 1994. The N terminus of these mice indicate that lung specific overexpression of KC con- interleukin-8 (IL-8) receptor confers high affinity binding to human IL-8. J. Biol. fers enhanced resistance to N. asteroides infection. Further studies Chem. 269:18263. are ongoing examining the role of KC in pulmonary nocardiosis. 22. Harada, A., K. Kuno, H. Nomura, N. Mukaida, S. Murakami, and K. Matsushima. 1994. Cloning of a cDNA encoding a mouse homolog of the interleukin-8 re- We observed enhanced production of MIP-2 and KC in anti- ceptor. Gene 142:297. CXCR2 or anti-Ly-6G treated mice suggesting that CXC chemo- 23. Bozic, C. R., N. P. Gerard, C. von Uexkull-Guldenband, L. F. Kolakowski, Jr., kine production is independent of total lung neutrophil numbers. M. J. Conklyn, R. Breslow, H. J. Showell, and C. Gerard. 1994. The murine interleukin 8 type B receptor homologue and its ligands: expression and biolog- Elevated levels of MIP-2 and KC in these treated mice is in part ical characterization. J. Biol. Chem. 269:29355. likely due to the increased pulmonary bacterial burden in these 24. Cacalano, G., J. Lee, K. Kikly, A. M. Ryan, S. Pitts-Meek, B. Hultgren, W. I. Wood, and M. W. Moore. 1994. Neutrophil and B cell expansion in mice mice (Fig. 8). Alternatively, the absence of recruited neutrophils that lack the murine IL-8 receptor homolog. Science 265:682. into the lung may result in impaired binding and uptake of CXCR2 25. Beaman, B. L., and S. Maslan. 1978. Virulence of Nocardia asteroides during its ligands via receptor internalization. growth cycle. Infect. Immun. 20:290. 26. Beaman, B. L., and S. E. Moring. 1988. Relationship among cell wall composi- Our data provide direct evidence linking CXCR2 ligands and tion, stage of growth, and virulence of Nocardia asteroides GUH-2. Infect. Im- neutrophil recruitment during nocardial pneumonia. Furthermore, mun. 56:557. The Journal of Immunology 915

27. Beaman, B. L. 1996. Differential binding of Nocardia asteroides in the murine 35. Huffnagle, G. B., M. F. Lipscomb, J. A. Lovchik, K. A. Hoag, and N. E. Street. lung and brain suggests multiple ligands on the nocardial surface. Infect. Immun. 1994. The role of CD4ϩ and CD8ϩ T cells in the protective inflammatory re- 64:4859. sponse to a pulmonary cryptococcal infection. J. Leukocyte Biol. 55:35. 28. Hestdal, K., F. W. Ruscetti, J. N. Ihle, S. E. Jacobsen, C. M. Dubois, W. C. Kopp, 36. Tsai, W. C., R. M. Strieter, J. M. Wilkowski, K. A. Bucknell, M. D. Burdick, D. L. Longo, and J. R. Keller. 1991. Characterization and regulation of RB6-8C5 S. A. Lira, and T. J. Standiford. 1998. Lung-specific transgenic expression of KC antigen expression on murine bone marrow cells. J. Immunol. 147:22. enhances resistance to Klebsiella pneumoniae in mice. J. Immunol. 161:2435. 29. Conlan, J. W., and R. J. North. 1994. Neutrophils are essential for early anti- 37. Smith, J. B., L. E. Rovai, and H. R. Herschman. 1997. Sequence similarities of Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed a subgroup of CXC chemokines related to murine LIX: implications for the by a granulocyte-depleting monoclonal antibody. J. Exp. Med. 179:259. interpretation of evolutionary relationships among chemokines. J. Leukocyte 30. Han, Y., and J. E. Cutler. 1997. Assessment of a mouse model of neutropenia and Biol. 62:598. the effect of an anti-candidiasis monoclonal antibody in these animals. J. Infect. 38. Smith, J. B., and H. R. Herschman. 1995. Glucocorticoid-attenuated response Dis. 175:1169. genes encode intercellular mediators, including a new C-X-C chemokine. J. Biol. 31. Sayles, P. C., and L. L. Johnson. 1996. Exacerbation of toxoplasmosis in neu- Chem. 270:16756. trophil-depleted mice. Nat. Immun. 15:249. 39. Rovai, L. E., H. R. Herschman, and J. B. Smith. 1998. The murine neutrophil- 32. Hebert, C. A., A. Chuntharapai, M. Smith, T. Colby, J. Kim, and R. Horuk. 1993. chemoattractant chemokines LIX, KC, and MIP-2 have distinct induction kinet- Partial functional mapping of the human interleukin-8 type A receptor. Identifi- ics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regula- cation of a major ligand binding domain. J. Biol. Chem. 268:18549. tion in endotoxemia. J. Leukocyte Biol. 64:494. 33. Mehrad, B., R. M. Strieter, T. A. Moore, W. C. Tsai, and T. J. Standiford. 1999. 40. Rossi, D. L., S. D. Hurst, Y. Xu, W. Wang, S. Menon, R. L. Coffman, and CXC chemokine receptor-2 ligands are necessary components of neutrophil-me- A. Zlotnik. 1999. Lungkine, a novel CXC chemokine, specifically expressed by diated host defense in invasive pulmonary aspergillosis. J. Immunol. 163:6086. lung bronchoepithelial cells. J. Immunol. 162:5490. 34. Greenberger, M. J., R. M. Strieter, S. L. Kunkel, J. M. Danforth, R. E. Goodman, 41. Beaman, B. L., and L. Beaman. 1998. Filament tip-associated antigens involved and T. J. Standiford. 1995. Neutralization of IL-10 increases survival in a murine in adherence to and invasion of murine pulmonary epithelial cells in vivo and model of Klebsiella pneumonia. J. Immunol. 155:722. HeLa cells in vitro by Nocardia asteroides. Infect. Immun. 66:4676. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021