Introduction

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The lung inflammatory response is regulated by coordinated functions of cytokines, chemokines, and adhesion molecules. Acute lung injury induced by intrapulmonary deposition of immunoglobulin G (IgG) immune complexes (IgG-ICs) in rats has allowed delineation of a variety of mediators in the inflammatory process. In this model, production of tumor necrosis factor- (TNF-) and interleukin (IL)-1 is required for upregulation of vascular adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, on pulmonary vascular endothelial cells (1, 2). These vascular adhesion molecules, in cooperation with neutrophil chemotaxins (i.e., CXC chemokines and complement activation products), mediate the recruitment of neutrophils into the alveolar space (3). Immunostaining techniques have suggested that alveolar macrophages are the primary source of these cytokines and chemokines (4, 6). In this inflammatory model, damage to lung parenchyma results from the generation and release of oxidants and proteases from recruited neutrophils and activated residential macrophages (7, 8).

Using the IgG-IC model of lung injury, we have recently demonstrated that the transcription factor nuclear factor (NF)-B is an important signal transduction element in the development of the lung inflammatory response (9). NF-B consists of a heterodimer of p50 and p65 subunits, which in most cells is retained in the cytoplasm in complex with inhibitory proteins of the IB family (10). Upon cell stimulation, IB is degraded in a process requiring the function of the 26S proteasome. The free "activated" heterodimeric NF-B then translocates to the nucleus, where it binds to specific promoter elements and induces gene transcription. NF-B is known to regulate gene transcription of many cytokines (i.e., TNF-, IL-1), CXC chemokines (of the IL-8 family), and endothelial adhesion molecules (ICAM-1, E-selectin) involved in acute lung inflammation (11). We have recently shown in vivo that nuclear translocation of NF-B occurs in alveolar macrophages long before any evidence of lung inflammation (9), suggesting that NF-B activation in these cells triggers pulmonary production of the early proinflammatory mediators.

Depletion of alveolar macrophages by intratracheal instillation of liposomes containing the compound dichloromethylene diphosphonate (Cl₂MDP) has been used to study alveolar macrophage functions in vivo (15, 16). The liposome-encapsulated Cl₂MDP is ingested only by phagocytic cells and results in selective depletion of alveolar macrophages without damaging other cell types in the lung (17). Using this technique, it has been shown that depletion of alveolar macrophages reduces lung production of TNF- and neutrophil recruitment in a model of lipopolysaccharide-induced lung inflammation (15). Unexpectedly, in a model of bacterial pneumonia, depletion of alveolar macrophages by Cl₂MDP-liposomes caused increased lung production of TNF- and increased lung neutrophil recruitment (16). Thus, although the nature of the stimulus may dictate the function of alveolar macrophages, the latter studies suggest that under special circumstances there may be non-alveolar macrophage sources of TNF- that may be relevant during the development of lung inflammatory injury.

Although nuclear translocation of NF-B in alveolar macrophages is known to occur early in lung inflammation (9), it is unclear whether the products of alveolar macrophages are required for NF-B activation in other lung cells. In the current studies, we sought to delineate further the role of alveolar macrophages in lung NF-B activation and inflammatory injury induced by IgG-ICs. The data to be presented indicate that depletion of alveolar macrophages with Cl₂MDP-liposomes suppresses NF-B activation in lung tissues in this inflammatory model. These effects were accompanied by reduced pulmonary production of TNF- and macrophage inflammatory protein-2 (MIP-2), diminished recruitment of neutrophils, and protection against lung injury. Furthermore, lung instillation of TNF- in alveolar macrophage-depleted rats restored NF-B activation in whole-lung tissues. These data suggest that NF-B activation triggered by intrapulmonary deposition of IgG-ICs is initiated by products of alveolar macrophages. In this model, NF-B activation may then be propagated to other lung cells by products of activated alveolar macrophages such as cytokines and chemokines.

	Materials and Methods

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Materials

Liposomes composed of egg phosphatidylcholine and cholesterol and containing either phosphate-buffered saline (PBS), pH 7.4 or Cl₂MDP (a gift from Boehringer Mannheim GmbH, Mannheim, Germany) were synthesized as described previously (17). Rabbit polyclonal IgG anti-bovine serum albumin (BSA) was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). Rabbit polyclonal IgG antirat- ICAM-1 and nonspecific rabbit IgG were obtained from Accurate Chemical Company (Westbury, NY). Recombinant murine TNF- was purchased from R&D Systems, Inc. (Minneapolis, MN).

Alveolar Macrophage Depletion

Specific pathogen-free male Long-Evans rats (275 to 300 g; Harlan Sprague-Dawley, Indianapolis, IN) were anesthetized with ketamine HCl (150 mg/kg, intraperitoneally). A suspension of Cl₂MDP-liposomes in PBS (100 µl of liposomes in a total volume of 500 µl) was administered intratracheally during inspiration. As a control, PBS-liposomes were administered in a similar fashion. All subsequent interventions occurred 24 h after liposome instillation.

IgG-IC-Induced Alveolitis

A total of 1.5 mg rabbit polyclonal IgG anti-BSA in a volume of 0.3 ml PBS was instilled via an intratracheal catheter during inspiration. Immediately thereafter, 10 mg BSA in 0.5 ml PBS were injected intravenously. For analysis of pulmonary vascular permeability, trace amounts of ¹²⁵I- labeled BSA were injected intravenously. Control rats received PBS intratracheally. At 4 h after IgG-IC deposition, rats were exsanguinated, the pulmonary circulation was flushed with 10 ml saline by pulmonary artery injection, and the lungs were surgically dissected. For analysis of NF-B activation and lung myeloperoxidase (MPO) content, lungs were immediately frozen in liquid nitrogen. The extent of lung injury was quantified by calculating the lung permeability index (amount of radioactivity [¹²⁵I-labeled BSA] in the perfused lungs divided by the amount of radioactivity in 1.0 ml of blood obtained at the time of death).

Lung NF-B Activation

Nuclear extracts of whole-lung tissues were prepared by the method of Deryckere and Gannon (18) and analyzed by electrophoretic mobility shift assay (EMSA). Briefly, double-stranded NF-B consensus oligonucleotide (5'-GTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) was end-labeled with [³²P]adenosine triphosphate (3,000 Ci/mmol at 10 mCi/ml; Amersham Co., Arlington Heights, IL). Binding reactions containing equal amounts of nuclear protein extract (10 µg) and 35 fmol (~ 50,000 cpm, Cherenkov counting) of oligonucleotide were performed for 30 min in binding buffer (4% glycerol, 1 mM MgCl₂, 0.5 mM ethylenediamenetetraacetic acid [EDTA] [pH 8.0], 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris [pH 7.6], and 50 µg/ml poly [dI-dC]; Pharmacia, Piscataway, NJ). Reaction volumes were held constant to 15 µl. Reaction products were separated in a 4% polyacrylamide gel in 0.25× TBE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) and analyzed by autoradiography. NF-B activation was quantitated from digitized autoradiography films using image analysis software (Adobe Systems, Inc., San Jose, CA).

Bronchoalveolar Lavage Fluid (BALF) Cytokine Content

BALF was collected by instilling and withdrawing 5 ml of sterile PBS three times from the lungs via an intratracheal cannula. BALF content of TNF- was measured using a standard WEHI cell cytotoxicity assay as previously reported (19). Measurement of MIP-2 in BALF was by enzyme-linked immunosorbent assay (ELISA) as described elsewhere (4).

Lung Vascular Expression of ICAM-1

Rats were injected intravenously with 0.5 µCi of ¹²⁵I-labeled anti-ICAM-1 3.75 h after induction of lung injury. To control for nonspecific binding and potential accumulation of anti-ICAM-1 antibody in lung parenchyma due to injury, 0.5 µCi ¹²⁵I-labeled nonspecific rabbit IgG was administered in a separate set of rats. Fifteen minutes later (4 h after induction of lung injury), rats were killed and the lung vasculature was flushed with 10 ml PBS. Lung vascular ICAM-1 expression (binding index) was calculated by dividing the amount of radioactivity (¹²⁵I-labeled antibody) in perfused lungs by the amount of radioactivity in 1.0 ml of blood obtained at the time of death.

Lung MPO Content

Whole-lung MPO activity was quantitated as described previously (20). Briefly, whole-lung homogenates were diluted in 50 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide, pH 6.0. After sonication and two freeze-thaw cycles, samples were centrifuged at 4,000 × g for 30 min. The supernatants were reacted with H₂O₂ (0.3 mM) in the presence of tetramethylbenzidine (1.6 mM). MPO activity was assessed by measuring the change in absorbance at 655 nm.

Statistical Analysis

All values are expressed as means ± SEM. Data were analyzed with a one-way analysis of variance, and individual group means were then compared with a Student-Newman-Keuls test. Differences were considered significant when P < 0.05. For calculations of percent change, negative control values were subtracted from positive control and treatment group values.

	Results

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Depletion of Alveolar Macrophages with Cl₂MDP-Liposomes

To determine the role of alveolar macrophages in the development of IgG-IC-induced lung inflammatory injury, experiments were initiated to deplete lungs of alveolar macrophages by intratracheal administration of Cl₂MDP-liposomes. Instillation of 100 µl Cl₂MDP-liposomes in a total volume of 1 ml has been shown to result in greater than 70% depletion of alveolar macrophages within 24 h (15). In our studies, lung instillation of 1 ml of the liposome suspension was not tolerated. Therefore, we tested the effects of lower dose-volumes containing 100 µl of liposomes. Rats received Cl₂MDP-liposomes (100 µl) in total volumes of either 300 or 500 µl PBS. Twenty-four hours later the number of alveolar macrophages was assessed by BAL. Administration of Cl₂MDP-liposomes in a total volume of 300 µl produced approximately 53% depletion of alveolar macrophages (data not shown). When the dose volume was increased to 500 µl, Cl₂MDP-liposomes decreased the number of alveolar macrophages obtained by BAL by 74% (P < 0.001) when compared with rats receiving PBS-liposomes (Figure 1). Administration of PBS-liposomes did not reduce the number of alveolar macrophages compared with pretreatment with 500 µl PBS alone. For all subsequent experiments in which alveolar macrophage depletion was carried out, a dose of 100 µl Cl₂MDP-liposomes in a total volume of 500 µl PBS was used.

View larger version (32K): [in this window] [in a new window]   Figure 1.   Effects of PBS- or Cl2MDP-liposomes on alveolar macrophage numbers. Rats received lung instillations of PBS, PBS- liposomes (100 µl), or Cl2MDP-liposomes (100 µl) in a total volume of 500 µl. Twenty-four hours later, alveolar macrophages were harvested by BAL and quantitated by microcytometry. Values represent means ± SEM with n = 3 per group.

Effects of Alveolar Macrophage Depletion on IgG-IC-Induced Activation of NF-B in Lung

Our recent studies in the IgG-IC model of lung injury have suggested that lung NF-B activation is prerequisite for the development of lung injury (9). Because these studies demonstrated that NF-B activation in alveolar macrophages in vivo occurs hours before NF-B activation in whole-lung tissues, we examined whether alveolar macrophage depletion could alter NF-B activation in whole-lung tissues. Nuclear extracts of whole lungs obtained 4 h after onset of injury were analyzed by EMSA. Lungs from rats pretreated with PBS- or Cl₂MDP-liposomes and subsequently challenged intratracheally with PBS displayed normal baseline levels of NF-B activation (Figure 2A). In rats pretreated with PBS-liposomes and challenged with IgG-ICs, the expected increase in lung NF-B activation was found. Depletion of alveolar macrophages with Cl₂MDP-liposomes markedly reduced the extent of lung NF-B activation induced by intrapulmonary deposition of IgG-ICs. When EMSA blots were subjected to image analysis (Figure 2B), alveolar macrophage depletion was found to reduce IgG-IC-induced lung NF-B activation by 74% (P = 0.031).

View larger version (27K): [in this window] [in a new window]   Figure 2.   Effects of alveolar macrophage depletion on NF-B activation in whole lungs. Rats received PBS- or Cl2MDP-liposomes 24 h before receiving intratracheal challenge with either PBS or IgG-ICs. Four hours after PBS or IgG-IC challenge, NF-B binding activity of whole-lung nuclear extracts was assessed by EMSA (A). The EMSA blots were digitized and quantitated using image analysis software (B). Values represent means ± SEM with n = 4 per group.

Effects of Alveolar Macrophage Depletion on BALF Levels of TNF- and MIP-2 and Lung Vascular Expression of ICAM-1

Because it is known that NF-B regulates the gene transcription of both TNF- and MIP-2, and in view of the fact that we have shown alveolar macrophage depletion to greatly reduce lung NF-B activation (Figure 2), we examined the effects of alveolar macrophage depletion on BALF levels of TNF- and MIP-2 proteins. Using a WEHI cell cytotoxicity assay for TNF-, we observed very low levels of TNF- in BALF from rats pretreated with PBS- or Cl₂MDP-liposomes and then challenged intratracheally with PBS (Figure 3A). Intrapulmonary deposition of IgG-ICs caused a dramatic increase in BALF TNF- levels in rats treated with PBS-liposomes. However, depletion of alveolar macrophages with Cl₂MDP-liposomes caused a 48% reduction (P = 0.006) in BALF TNF- content. Similar effects were observed when MIP-2 was measured by ELISA. There were low levels of MIP-2 in BALF from rats that had been pretreated with PBS- or Cl₂MDP-liposomes and subsequently challenged with PBS (Figure 3B). In response to IgG-IC deposition, MIP-2 levels were greatly increased (> 5-fold) in BALF from rats pretreated with PBS-liposomes. In contrast, in rats depleted of alveolar macrophages by pretreatment with Cl₂MDP-liposomes, IgG-IC-induced increases in BALF MIP-2 were reduced by 78% (P = 0.004).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effects of alveolar macrophage depletion on BALF content of TNF- (A), MIP-2 (B), and lung vascular ICAM-1 expression (C). Rats received PBS- or Cl2MDP-liposomes 24 h before receiving intratracheal challenge with either PBS or IgG-IC. Analysis of BALF and measurements of lung vascular ICAM-1 were performed 4 h after challenge with PBS or IgG-IC. Values represent means ± SEM with n = 5 (BALF TNF- and MIP-2) or n = 10 (lung vascular ICAM-1) per group.

Upregulation of pulmonary vascular ICAM-1 has been shown to be required for lung neutrophil accumulation and full development of tissue injury in this model (3). Because products of activated alveolar macrophages (including TNF-) are known to be related to ICAM-1 expression (1), we determined the effects of alveolar macrophage depletion on lung vascular expression of ICAM-1 using ¹²⁵I-labeled antibody to ICAM-1. Pretreatment of rats with either PBS- or Cl₂MDP-liposomes had no effect on baseline levels of ICAM-1 expression after intratracheal challenge with PBS (Figure 3C). In rats pretreated with PBS-liposomes and challenged with IgG-ICs, lung vascular ICAM-1 expression showed the expected increase (nearly 3-fold). This increase was not due to nonspecific binding or sequestration of antibody because binding indexes for nonspecific rabbit IgG were not different between PBS-challenged and IgG-IC-challenged rats (0.17 ± 0.05 and 0.20 ± 0.07, respectively). In contrast, in the rats depleted of alveolar macrophages with Cl₂MDP-liposomes, IgG-IC- induced ICAM-1 expression was reduced by 67% (P = 0.035) (Figure 3C). As will be shown subsequently, this correlates with reduced accumulation of neutrophils in the inflamed lung.

Effects of Alveolar Macrophage Depletion on Neutrophil Recruitment and Development of Lung Injury

Because depletion of alveolar macrophages suppressed IgG-IC-induced increases in lung NF-B activation (Figure 2), BALF content of TNF- and MIP-2 (Figures 3A and 3B), and lung vascular expression of ICAM-1 (Figure 3C), we examined the extent to which these effects were associated with reduced neutrophil recruitment into lung and development of lung injury. Neutrophil recruitment into lung was quantitated by lung content of MPO activity. In rats pretreated with PBS- or Cl₂MDP-liposomes and challenged with PBS, there was no evidence of neutrophil accumulation in lung (Figure 4A). In rats pretreated with PBS-liposomes and then challenged with IgG-ICs, lung MPO content was, as expected, significantly increased from rats pretreated with PBS-liposomes and challenged with PBS. As shown in Figure 4A, depletion of alveolar macrophages with Cl₂MDP-liposomes decreased IgG-IC-induced MPO values by 59% (P = 0.002). Lung injury was quantitated by changes in vascular permeability as measured by leakage of ¹²⁵I-albumin into lung parenchyma. As shown in Figure 4B, there was no evidence of lung injury in rats challenged with PBS after pretreatment with either PBS- or Cl₂MDP-liposomes. As expected, intrapulmonary deposition of IgG-ICs caused significant lung injury in rats pretreated with PBS-liposomes, with a 5.5-fold increase in the permeability index. Rats depleted of alveolar macrophages with Cl₂MDP-liposomes incurred significantly less lung injury (55% reduction, P = 0.015) compared with positive control rats pretreated with PBS-liposomes.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Effects of alveolar macrophage depletion on lung MPO content (A) and lung vascular permeability (B) in the IgG-IC model of lung injury. Rats received PBS- or Cl2MDP-liposomes 24 h before receiving intratracheal challenge with either PBS or IgG-IC. Measurements of lung MPO and permeability were performed 4 h after challenge with PBS or IgG-IC. Values represent means ± SEM with n = 5 per group.

TNF- Restores NF-B Activation in Lung

Because depletion of alveolar macrophages suppressed lung production of TNF- and reduced lung NF-B activation, experiments were designed to determine whether exogenous administration of TNF- would lead to NF-B activation in lung of macrophage-depleted rats. Alveolar macrophage-depleted rats received lung instillation of PBS or 500 ng of murine TNF-, and lung NF-B activation was assessed by EMSA 4 h later. In macrophage-depleted rats, lung instillation of PBS did not cause nuclear translocation of NF-B (Figure 5). However, lung instillation of TNF- resulted in a marked increase in nuclear NF-B. These data suggest that products of activated alveolar macrophages, such as TNF-, may be responsible for NF-B activation in the inflamed lung.

View larger version (91K): [in this window] [in a new window]   Figure 5.   Effects of lung instillation of PBS or TNF- on lung NF-B activation in alveolar macrophage-depleted rats. Twenty-four hours after intratracheal administration of Cl2MDP-liposomes, rats received lung instillations of PBS or 500 ng murine TNF- in a total volume of 300 µl. Four hours later, lung NF-B activation was assessed by EMSA.

	Discussion

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Increasing evidence suggests an important role for NF-B in the pathogenesis of acute lung inflammation. Studies in vitro have shown that NF-B regulates gene expression of cytokines (TNF-, IL-1), chemokines (MIP-2, CINC), and adhesion molecules (ICAM-1, E-selectin), which play important roles in lung inflammatory injury (11). These in vitro findings are supported by studies in humans with acute respiratory distress syndrome showing enhanced NF-B activation in alveolar macrophages recovered by BAL (21). In addition, studies in vivo have shown an association between NF-B activation and expression of cytokines, chemokines, and vascular adhesion molecules (22). More recent studies in vivo have demonstrated that lung NF-B activation is suppressed by antioxidants (23) or anti-inflammatory cytokines (9). This results in decreased proinflammatory mediator expression and reduced inflammatory injury. Thus, it appears that activation of NF-B may be central to the development of pulmonary inflammation.

The mechanism of NF-B activation during lung inflammatory injury is known to require both TNF- and IL-1, which seem to operate as autocrine/paracrine stimulators of alveolar macrophages (24). Alveolar macrophage activation is an initial event in the genesis of lung inflammatory reactions. In the rat model of injury induced by intrapulmonary deposition of IgG-ICs, we have shown that early activation of alveolar macrophages occurs in an NF-B-dependent manner (9). Furthermore, NF-B activation in alveolar macrophages in vivo occurs prior to NF-B activation in whole-lung tissues, suggesting that products of activated alveolar macrophages are required to stimulate nuclear translocation of NF-B in other lung-cell types. The present data support that hypothesis because depletion of alveolar macrophages attenuated NF-B activation in whole-lung tissues and decreased BALF content of proinflammatory mediators. In addition, lung instillation of TNF- in alveolar macrophage-depleted rats induced NF-B activation in whole lungs.

Although alveolar macrophages are considered a major source of TNF- in the lung, alveolar macrophage depletion reduced BAL TNF- by only 48%. Similarly, IgG-IC- induced increases in pulmonary vascular permeability were decreased by 55% in rats depleted of alveolar macrophages. These data suggest the presence of other important sources of TNF- in lung, such as interstitial macrophages and perhaps nonmacrophage cell types, that may cooperatively contribute to tissue injury. Alternatively, the small amount of alveolar macrophages remaining after Cl₂MDP-liposome administration (~ 26%) may have produced a disproportionately large amount of TNF-. Treatment with Cl₂MDP-liposomes, which on average resulted in a 74% reduction in the number of lavageable alveolar macrophages, almost completely abrogated BALF content of the chemokine MIP-2 (78% reduction). These data confirm our earlier report suggesting that activated alveolar macrophages are the primary source of MIP-2 (4).

Damage to lung tissues after IgG-IC deposition is mediated primarily by neutrophil-derived oxidants and proteases (7, 8, 25). In rats depleted of alveolar macrophages, pulmonary neutrophil accumulation was greatly reduced, as was the extent of lung injury. Neutrophil recruitment into lung induced by IgG-IC deposition is known to depend on vascular expression of ICAM-1 and production of CXC chemokines, which include MIP-2 and CINC (3, 4). Alveolar macrophage depletion by Cl₂MDP-liposomes significantly reduced both MIP-2 content in BALF and pulmonary vascular expression of ICAM-1. Decreased levels of MIP-2 can be ascribed to the physical removal of its cellular source, alveolar macrophages. Decreased expression of ICAM-1 is likely due to decreased availability of TNF-, which is known to regulate ICAM-1 expression (1).

Depletion of alveolar macrophages reduced IgG-IC- induced lung NF-B activation and TNF- production in a manner similar to treatment with the anti-inflammatory cytokines IL-10 and IL-13. Both IL-10 and IL-13 suppress alveolar macrophage function by preserving cytosolic expression of the inhibitory protein IB, thus preventing nuclear localization of NF-B (9). At least in the case of IL-10, suppression of NF-B activation in macrophages in vitro is known to prevent transcription of proinflammatory cytokines (26, 27). In IgG-IC-induced lung inflammation, administration of IL-10 or IL-13 greatly suppressed lung production of TNF- and subsequent lung injury (28). Thus, it appears in vivo that IL-10 and IL-13 suppress alveolar macrophage NF-B activation, preventing proinflammatory cytokine production and reducing lung inflammation and injury. The findings of those studies, combined with the data presented in this report, suggest that specific inhibitors of NF-B (i.e., proteasome inhibitors, IB kinase inhibitors, etc.) may be targeted for delivery to alveolar macrophages by liposome encapsulation. This could represent a potent therapeutic strategy for the treatment of acute lung inflammation in which products of lung macrophages play a major role in events leading to lung injury.