Normal Alveolar Macrophages Research Articles

Molecular pathogenesis in tuberculosis complicated with AIDS

HIV-1 infection is a major cause of worldwide epidemic of tuberculosis. There is increasing clinical evidence that coinfection with M. tuberculosis accelerates progression of AIDS. We found that, in vivo, HIV-1 load and mutation increase in involved lung segments in patients with pulmonary tuberculosis. We also reported that Mycobacterium tuberculosis stimulates HIV-1 replication by enhancing transcription on the 5' LTR in a macrophage cell line, THP-1, in vitro. In contrast, HIV-1 replication is suppressed by M. tuberculosis infection of monocytes derived macrophages (MDM) or differentiated monocytic THP-1 cells. We observed that HIV-1 5' LTR function was repressed in PMA differentiated THP-1 cells after co-infection with M. tuberculosis. Point mutations in C/EBP beta binding domains of the HIV-1 LTR negative regulatory element (NRE) abolished promoter repression. Monocyte-derived macrophages and differentiated THP-1 cells increased expression of the 16kDa inhibitory form of C/EBP after M. tuberculosis co-infection. Bronchoalveolar lavage cells obtained from normal controls and alveolar macrophages from uninflamed lung of tuberculosis patients also expressed the 16kDa inhibitory form of C/EBP. However, alveolar macrophages from lung segments involved with pulmonary tuberculosis had markedly reduced C/EBP expression. These data suggest that 16kDa isoform of C/EBP plays an important role for the control of HIV-1 replication in macrophages. We propose derepression of HIV-1 LTR mediated transcription as one mechanism for enhanced HIV-1 replication observed in pulmonary tuberculosis. Since the cellular immune response in pulmonary tuberculosis requires lymphocyte/macrophage interaction, a model system was developed in which lymphocytes were added to AM. Contact between lymphocytes and AM reduced inhibitory C/EBP beta, activated NF-kappaB and enhanced HIV-1 replication. If contact between lymphocytes and macrophages was prevented, inhibitory C/EBP beta expression was maintained and the HIV-1 long terminal repeat (LTR) was not maximally stimulated although NF-kappaB was activated. Antibodies which cross-linked macrophage expressed B-7, VCAM and CD-40 were used mimic lymphocyte contact. Cross-linking antibodies abolished inhibitory C/EBP beta expression; however, the HIV-1 LTR was not maximally stimulated and NF-kappaB was not activated. Maximal HIV-1 LTR stimulation required both lymphocyte derived soluble factors and cross-linking of macrophage expressed co-stimulatory molecules. These results demonstrate that neither contact nor soluble factor(s) are sufficient to maximally enhance HIV-1 LTR activity in macrophages. Contact between activated lymphocytes and macrophages is necessary to downregulate inhibitory C/EBP beta, thereby derepressing the HIV-1 LTR. Lymphocyte derived soluble factor(s) activate NF-kappaB, further enhancing the HIV-1 LTR.

Mechanisms for impaired effector function in alveolar macrophages from marijuana and cocaine smokers

Lung macrophages provide a first line of host defense against inhaled pathogens and their function is impaired in the lungs of inhaled substance abusers. In order to investigate the mechanism for this impairment, alveolar macrophages (AM) were recovered from nonsmokers (NS), regular tobacco smokers (TS), marijuana smokers (MS), or crack cocaine smokers (CS), and evaluated for their production of nitric oxide (NO) and the role of NO as an antimicrobial effector molecule. AM from NS and TS efficiently killed Staphylococcus aureus and their antibacterial activity correlated closely with the production of nitrite and the expression of mRNA encoding for inducible nitric oxide synthase (iNOS). In contrast, AM collected from MS and CS exhibited limited antimicrobial activity that was not affected by an inhibitor of iNOS, or associated with expression of iNOS. Treatment with either granulocyte/macrophage colony-stimulating factor (GM-CSF) or interferon-γ restored the ability of these cells to produce NO and to kill bacteria. These findings confirm a significant role for NO as an antibacterial effector molecule used by normal human AM and suggest that this host defense mechanism is suppressed by habitual exposure to inhaled marijuana or crack cocaine in vivo.

Pneumocystis carinii factor responsible for reduced phagocytic activity in alveolar macrophages.

Alveolar macrophages in hosts that are immunosuppressed and infected with Pneumocystis have a greatly reduced phagocytic activity [5]. Incubation of normal alveolar macrophages with bronchoalveolar lavage (BAL) fluid from P. carinii infected rats also leads to a decrease in phagocytosis [3]. The defect in phagocytosis has been linked to a down-regulation in transcription of the GATA-2 transcription factor [5]. Inhibition ofGATA-2 transcription in normal alveolarmacrophages leads to a reduction in phagocytic ability, and expression of GATA-2 in alveolar macrophages from P. carinii infected rats restores their phagocytic activity [5]. It is not known how P. carinii infection leads to down-regulation of GATA-2 or the decrease in phagocytosis. The current study represents the initial effort to identify factors from P. carinii responsible for the decrease in phagocytosis of alveolar macrophages.

Surfactant protein A enhances apoptotic cell uptake and TGF-beta1 release by inflammatory alveolar macrophages.

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.

Morphologic detection and functional assessment of reconstituted normal alveolar macrophages in the lungs of SCID mice.

Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for approximately 50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense.

Oxidant-mediated increases in redox factor-1 nuclear protein and activator protein-1 DNA binding in asbestos-treated macrophages.

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. We have previously shown that normal human alveolar macrophages exhibit decreased DNA binding activity of the transcription factor, AP-1, compared with monocytes. Furthermore, this decrease in AP-1 DNA binding appears to be due to a decrease in the redox active protein, redox factor (Ref)-1. Ref-1 is an important redox regulator of a number of transcription factors, including NF-kappaB and AP-1. In this study we evaluated the role of asbestos, a prototypic model of chronic fibrotic lung disease, in Ref-1 expression and activity. We found that incubation with low concentrations of crocidolite asbestos (0.5-1.25 microg/cm(2)) resulted in an increase in nuclear Ref-1 protein after 5 min, with a persistent elevation in protein up to 24 h. Additionally, an increase in nuclear Ref-1 could be induced by treating the cells with an oxidant-generating stimulus (iron loading plus PMA) and inhibited by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. The asbestos-induced accumulation of nuclear Ref-1 was associated with an increase in AP-1 DNA binding activity. These findings suggest that an exposure associated with fibrotic lung disease, i.e., asbestos, modulates accumulation of nuclear Ref-1 in macrophages, and that this effect is mediated by an oxidant stimulus.

Beta-chemokines are induced by Mycobacterium tuberculosis and inhibit its growth.

Chemokines (CK) are potent leukocyte activators and chemoattractants and aid in granuloma formation, functions critical for the immune response to Mycobacterium tuberculosis. We hypothesized that infection of alveolar macrophages (AM) with different strains of M. tuberculosis elicits distinct profiles of CK, which could be altered by human immunodeficiency virus (HIV) infection. RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and MIP-1 beta were the major beta-CK produced in response to M. tuberculosis infection. Virulent M. tuberculosis (H37Rv) induced significantly less MIP-1 alpha than did the avirulent strain (H37Ra), while MIP-1 beta and RANTES production was comparable for both strains. MIP-1 alpha and MIP-1 beta were induced by the membrane, but not cytosolic, fraction of M. tuberculosis. M. tuberculosis-induced CK secretion was partly dependent on tumor necrosis factor alpha (TNF-alpha). AM from HIV-infected individuals produced less TNF-alpha and MIP-1 beta than did normal AM in response to either M. tuberculosis strain. We tested the functional significance of decreased beta-CK secretion by examining the ability of beta-CK to suppress intracellular growth of M. tuberculosis. MIP-1 beta and RANTES suppressed intracellular growth of M. tuberculosis two- to threefold, a novel finding. Thus, beta-CK contribute to the innate immune response to M. tuberculosis infection, and their diminution may promote the intracellular survival of M. tuberculosis.

Lung macrophage-epithelial cell interactions amplify particle-mediated cytokine release.

Interactions between alveolar macrophages (AMs) and epithelial cells may promote inflammatory responses to air pollution particles. Normal rat AMs, the alveolar type II epithelial cell line RLE-6TN (RLE), or cocultures of both cell types were incubated with various particles (0-50 microg/ml) for 24 h, followed by assay of released TNF-alpha and MIP-2. The particles used included titanium dioxide (TiO2), alpha-quartz (SiO2), residual oil fly ash (ROFA), or urban air particles (UAP). For all particles, a dose-dependent increase in TNF-alpha and MIP-2 release was observed in AM+RLE co-cultures but not in RLE or AM monoculture. AM+RLE co-culture also synergistically enhanced basal levels of tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2. In contrast, when AMs were co-cultured with fibroblasts, basal and particle-induced TNF-alpha and MIP-2 were similar to levels found in AM monoculture. Particle uptake by AMs was similar in mono- or AM+RLE co-culture. Increased basal and particle-induced cytokine release were not observed when the AMs were physically separated from the RLE. This contact-dependent cytokine potentiation could not be blocked with anti-CD18/anti-CD54, arginine-glycine-aspartate (RGD) peptide, or heparin. We conclude that in vitro inflammatory responses to particles are amplified by contact-dependent interactions between AMs and epithelial cells. AM-epithelial co-culture may provide a useful model of in vivo particle effects.

In situ expression of B7 and CD40 costimulatory molecules by normal human lung macrophages and epithelioid cells in tuberculoid granulomas.

Normal alveolar macrophages (AM) are not efficient in inducing the proliferation of resting T lymphocytes, and, rather, tend to inhibit pulmonary immune responses. In contrast, epithelioid cells (EC), activated macrophages that play an essential role in the course of granulomatous responses, appear to stimulate T cell proliferation efficiently. The inability of macrophages to deliver potent costimulatory signals through the B7/CD28 and CD40/CD40L pathways could explain their weak accessory cell activity. Using MoAbs and immunohistochemical techniques, however, we found that essentially all AM in normal human lung tissue expressed B7-1, B7-2 and CD40 molecules, and most of these cells were strongly positive. Pulmonary macrophages in other compartments also expressed these costimulatory molecules; no differences in expression were observed comparing macrophages from smokers and non-smokers. Most AM recovered by bronchoalveolar lavage from normal lung segments also strongly expressed B7-1, B7-2 and CD40 molecules. In comparison, resting blood monocytes were B7-1- and only moderately positive for B7-2. Activation of monocytes with lipopolysaccharide (LPS) induced expression of these costimulatory molecules to levels similar to that of AM from the control subjects. EC in granulomatous lesions also expressed easily detectable levels of B7-1, B7-2 and CD40. T lymphocytes within and surrounding the granulomas expressed CD28, the counter-receptor for B7, and many of these T cells also expressed B7-1 and B7-2. These findings suggest that both AM and EC can deliver costimulatory signals through B7-1, B7-2 and CD40 molecules, and indicate that the impairment in accessory cell activity observed for normal AM cannot be attributed to the absence of expression of these costimulatory molecules.

GM-CSF increases AP-1 DNA binding and Ref-1 amounts in human alveolar macrophages.

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. A characteristic feature of many of the chronic lung diseases is that the types of macrophages in the lung change, and in most instances, the cells resemble monocyte-like cells. We have previously shown that normal human alveolar macrophages have a decreased capacity to express protein kinase C (PKC)-induced DNA binding activity of the transcription factor activator protein (AP)-1 compared with monocytes. This decrease in AP-1 DNA binding appears to be due to a defect in redox regulation of AP-1 proteins via a decrease in the redox active protein Ref-1. The hypothesis for this study is that there are factors generated during the development of chronic lung disease that increase AP-1 DNA binding activity and Ref-1 production in human alveolar macrophages. We have focused specifically on granulocyte-macrophage colony-stimulating factor (GM-CSF) as a prototype mediator that can be released by alveolar macrophages and is related to the fibrotic process in the lung. We found that after a 24-h incubation with GM-CSF, AP-1 DNA binding was significantly increased in both unstimulated, interleukin (IL)-13, and phorbol myristate acetate (PMA)-stimulated alveolar macrophages and that there was a corresponding increase in Ref-1 protein by Western blot analysis in the PMA-stimulated group. This suggests that disease-related cytokines such as GM-CSF and IL-13 may modulate AP-1 DNA binding activity in alveolar macrophages.

Human immunodeficiency virus type 1 infection of alveolar macrophages impairs their innate fungicidal activity.

Impaired adaptive immunity is the hallmark of AIDS, but the effects of human immunodeficiency virus type 1 (HIV-1) infection on innate immunity are less clear. Cryptococcus neoformans (CN) is a common AIDS-related fungal pathogen acquired by inhalation. Alveolar macrophages (AM) comprise the initial host defense in cryptococcosis and they may arrest infection before dissemination occurs. We hypothesized that HIV-1 infection of AM impairs their anti-cryptococcal activity. This was tested by infection of normal AM with the M-tropic strain HIV-1(Bal). Two weeks postinfection we measured fungistatic activity against CN by colony counting, binding, and internalization of CN by confocal microscopy and AM cell viability by Alamar Blue assay. Uninfected AM from most donors demonstrated innate fungicidal activity against CN. In HIV-1-infected AM, there was a significant reduction, and in most cases loss, of fungicidal activity compared with the uninfected AM. The reduced antifungal activity was not due to any cytotoxic effect of HIV-1, and HIV-1 infection did not impair binding or internalization of yeast by AM. Thus, the innate fungicidal activity of primary human AM is impaired after HIV-1 infection in vitro by a mechanism involving a defect of intracellular antimicrobial processing.

Insoluble Components of Concentrated Air Particles Mediate Alveolar Macrophage Responses in Vitro

We sought to characterize the bioactive constituents of concentrated ambient air particles (CAPs) through correlation of alveolar macrophage (AM) biological responses (production of TNF, macrophage inflammatory protein (MIP-2), nitrite; cell viability) to components of particle samples. CAPs samples collected on different days showed a range of bioactivity and a strong correlation was found between AM cytokine release and increased AM light scatter, a flow cytometric measure of relative particle load. Evaluation of soluble and insoluble fractions of CAPs suspensions indicate that 1) most biological effects on AMs are mediated by insoluble components and certain particle adsorbed factors such as endotoxin; 2) the variable bioactivity of CAPs collected on different days arises primarily from differences in the relative proportion of insoluble and soluble mass present in particle suspensions; and 3) the activation state of the AM influences which insoluble components are most bioactive. Use of endotoxin neutralizing agents (e.g., polymyxin-B) showed particle-adsorbed endotoxin in CAPs suspensions causes activation of normal (control) AMs while other (nonendotoxin) components are predominantly responsible for the enhanced cytokine release observed by primed AMs incubated with CAPs. The AM biological response did not correlate with any of a panel of elements quantified within insoluble CAPs samples (Al, Cd, Cr, Cu, Fe, Mg, Mn, Ni, S, Ti, V). These data demonstrate an important role for cell activation and phagocytosis of insoluble particulate matter in the response of AMs to CAPs suspensions.

ALVEOLAR MACROPHAGE CYTOKINE PRODUCTION IN RESPONSE TO AIR PARTICLES IN VITRO: ROLE OF ENDOTOXIN

The interaction of air particles and alveolar macrophages (AMs) may result in the release of proinflammatory cytokines. Normal mouse AMs were treated with concentrated air particle (CAPs) suspensions in vitro. After 5 h, cytokine release [macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor- a (TNF-a)] and phagocytosis of ambient air particles were measured. CAPs samples collected from urban air (Boston) on different days were used. The CAPs samples and their soluble and solid components caused significant MIP-2 and TNF- a production. Variability in the potency of samples collected on different days was observed. Trace endotoxin was measured in CAPs samples (EU/mg: 2.3 +/- 0.7, mean +/- SE, n = 10). A majority of biologic activity (cytokine induction) and endotoxin content was associated with the solid components. Neutralization of endotoxin by polymyxin B abrogated >80% of TNF- a induction by CAPs samples, but inhibited MIP-2 production by only 40%. The trace endotoxin present in CAPs caused much more MIP-2 production than predicted by concentration alone (28 +/- 8-fold increase, n = 9), indicating synergistic interaction with other AM-activating components of the particles. Data suggest that low levels of endotoxin may interact with air particles to activate lung macrophages.

ASBESTOS STIMULATION TRIGGERS DIFFERENTIAL CYTOKINE RELEASE FROM HUMAN MONOCYTES AND ALVEOLAR MACROPHAGES

Inhalation of asbestos fibers results in a variety of lung diseases, including pulmonary fibrosis. Various animal models have demonstrated the importance of cytokines in the pathogenesis of pulmonary fibrosis. Alveolar macrophages from patients exposed to asbestos spontaneously release increased amounts of cytokines. The purpose of these studies was to determine whether asbestos directly stimulates cytokine release from human alveolar macrophages after in vitro exposure. We demonstrate that, although asbestos triggers cytokine release from blood monocytes, normal alveolar macrophages do not respond to asbestos stimulation with cytokine release. However, normal alveolar macrophages are activated by asbestos particles, in vitro, as determined by the upregulation of mRNAs for cytokines, and activation of the p38 kinase, which has been shown to be important in the translation of cytokine message into protein. These studies demonstrate that asbestos stimulates both normal blood monocytes and normal alveolar macrophages, but that there is a block in translation of cytokine mRNAs in the macrophages.

Tuberculosis in patients with acquired immune deficiency syndrome

HIV-1 infection is a major cause of worldwide epidemic of tuberculosis. In Japan, the cumulative number of the patients reported is 131 by the end of 1999 with 10 to 20 annual new cases. Most of Japanese cases are advanced AIDS patients with low CD4 number less than 100/microliter. The peak age of Japanese patient is 40 to 60 years old, whereas that of foreigners is 20-30 years old, suggesting that most Japanese cases are recurrent tuberculosis. There is increasing clinical evidence that coinfection with M. tuberculosis accelerates progression of AIDS. We found that, in vivo, HIV-1 load and mutation increase in involved lung segments in patients with pulmonary tuberculosis. We also reported that Mycobacterium tuberculosis stimulates HIV-1 replication by enhancing transcription on the 5' LTR in a macrophage cell line, THP-1, in vitro. In contrast, HIV-1 replication is suppressed by M. tuberculosis infection of monocytes derived macrophages (MDM) or differentiated monocytic THP-1 cells. We observed that HIV-1 5' LTR function was repressed in PMA differentiated THP-1 cells after co-infection with M. tuberculosis. Point mutations in C/EBP-beta binding domains of the HIV-1 LTR negative regulatory element (NRE) abolished promoter repression. Monocyte-derived macrophages and differentiated THP-1 cells increased expression of the 16 kDa inhibitory from of C/EBP-beta after M. tuberculosis coinfection. Bronchoalveolar lavage cells obtained from normal controls and alveolar macrophages from uninflamed lung of tuberculosis patients also expressed the 16 kDa inhibitory form of C/EBP-beta. However, alveolar macrophages from lung segments involved with pulmonary tuberculosis had markedly reduced C/EBP-beta expression. These data suggest that 16 kDa isoform of C/EBP-beta plays an important role for the control of HIV-1 replication in macrophages. We propose derepression of HIV-1 LTR mediated transcription as one mechanism for enhanced HIV-1 replication observed in pulmonary tuberculosis.

Lipopolysaccharide Priming Amplifies Lung Macrophage Tumor Necrosis Factor Production in Response to Air Particles

Elevated concentrations of ambient air particles can result in increased mortality and morbidity, especially in people with preexisting pulmonary disease. We postulate that in the inflammatory milieu of diseased lungs, alveolar macrophages (AMs) may be primed for enhanced responses to stimuli such as inhaled air particles. To test this hypothesis in vitro, we first cultured normal AMs with or without lipopolysaccharide (LPS). We then incubated the cells with particle suspensions (urban air particles (UAP, Washington, D.C.), residual oil fly ash (ROFA), concentrated respirable-size (PM2.5) air particles (CAPs), and inert TiO2.) and compared rat and human AM production of the critical proinflammatory mediator, tumor necrosis factor (TNF). LPS priming amplified TNF production by both rat and human AMs in response to UAP and CAPs but not inert TiO2. There were also differences observed between rat and human AM responses to particle suspensions. Striking changes seen only in rat were cytotoxic effects of ROFA and diminished particle uptake in response to LPS priming. The potency of CAPs samples (which are collected on separate days) varied when comparing one day's sample with another. When centrifuged, the majority of bioactivity seen in particle suspensions (TNF release) remained within the pelleted fraction while the supernatant showed minimal bioactivity. The data suggest that AMs activated by extant pulmonary inflammation may promote further inflammation by an enhanced cytokine response to inhaled ambient air particles.

Regulation of alveolar macrophage-T cell interactions during Th1-type sarcoid inflammatory process.

The accessory function of antigen-presenting cells depends on the presence of a number of costimulatory molecules, including members of the B7 family (CD80 and CD86) and the CD5 coligand CD72. The aim of this study was to evaluate the regulation of T cell-antigen-presenting cell costimulatory pathways in the lung of patients with a typical Th1-type reaction, i.e., sarcoidosis. Although normal alveolar macrophages (AMs) did not bear or bore low levels of costimulatory molecules, AMs from sarcoid patients with CD4 T-cell alveolitis upmodulated CD80, CD86, and CD72 and expressed high levels of interleukin (IL)-15; lymphocytes accounting for T-cell alveolitis expressed Th1-type cytokines [interferon (IFN)-gamma and/or IL-2] and bore high levels of CD5 and CD28 but not of CD152 molecules. In vitro stimulation of AMs with Th1-related cytokines (IL-15 and IFN-gamma) upregulated the expression of CD80 and CD86 molecules. However, stimulation with IL-15 induced the expression of Th1-type cytokines (IFN-gamma) and CD28 on sarcoid T cells, suggesting a role for this macrophage-derived cytokine in the activation of the sarcoid T-cell pool. The hypothesis that CD80 and CD86 molecules regulate the sarcoid T-cell response was confirmed by the evidence that AMs induced a strong proliferation of T cells that was inhibited by pretreatment with CD80 and CD86 monoclonal antibodies. To account for these data, it is proposed that locally released cytokines provide AMs with accessory properties that contribute to the development of sarcoid T-cell alveolitis.

Pulmonary immunity to Listeria is enhanced by elimination of alveolar macrophages.

To determine how resident alveolar macrophages (AM) regulate the antigen-presenting-cell (APC) activities of pulmonary dendritic cells (DC) in the response to particulate antigen, we pretreated Lewis rats intratracheally with liposomes containing clodronate (LIP-CLOD), which eliminated AM in vivo. Controls received saline encapsulated in liposomes (LIP-SAL) or saline alone intratracheally. At Day 3, rats were injected intratracheally with 1 x 10(7) heat-killed Listeria (HKL) and DC purified from lung were examined for their ability to stimulate HKL-immune T cells without added HKL. Only DC from LIP-CLOD-treated rats displayed enhanced APC activities for HKL. A second intratracheal HKL challenge at Day 14 yielded lymphocytic cuffing of the microvasculature in LIP-CLOD-treated lungs only. Intratracheal adoptive transfer of normal syngeneic AM into LIP-CLOD-treated rats suppressed APC activities of DC in vitro and the lymphocytic response in vivo. Bronchoalveolar macrophages from rats treated with LIP-CLOD and HKL showed decreased production of nitric oxide (NO), a potent suppressor of DC and T-helper 1 lymphocyte activities as compared with those of controls. We conclude that eliminating AM in vivo reduces local production of NO and promotes pulmonary cell-mediated immunity to HKL.

Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR-/-) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 +/- 5, 77 +/- 2, and 85 +/- 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of approximately 70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1-reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 +/- 5 and 47 +/- 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected approximately 70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 approximately 40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

Intracellular oxidant production and cytokine responses in lung macrophages: evaluation of fluorescent probes.

The fluorescent probes dichlorofluorescin (DCFH), dihydrorhodamine (DHR), and hydroethidine (HE) allow convenient assay of alveolar macrophage (AM) oxidant responses to enviromental particulates and pathogens. We sought to more precisely define the relationship of these measures of oxidant stress to production of pro-inflammatory cytokines. Normal AMs were challenged in vitro with a panel of soluble or particulate stimuli in the presence of DCFH, HE, or DHR. Flow cytometry measured cell-associated fluorescence and relative particle uptake. Tumor necrosis factor alpha and macrophage inflammatory protein 2 expression were quantitated in the same experiments. We observed variable and complex correlations between intracellular oxidant production as reported by these probes and subsequent cytokine response, including examples of striking discordance (e.g., lipopolysaccharide induced large cytokine responses with minimal probe oxidation, whereas fly ash particles caused marked oxidation of DCFH but trivial TNF release; TiO2 caused oxidation of DHR and HE, but not DCFH, and also did not increase cytokine production). Although fluorescent probes offer many advantages in analysis of intracellular oxidant responses, the data indicate that they cannot be used reliably as quantitative predictors of AM cytokine responses to environmental particulates or other stimuli.