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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 611-618, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4868

Involvement of Microbial Components and Toll-like Receptors 2 And 4 in Cytokine Responses to Air Pollution Particles

Susanne Becker, Matthew J. Fenton and Joleen M. Soukup

U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Research Triangle Park, North Carolina; and Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts

Address correspondence to: Susanne Becker, Ph.D., EPA Building, Human Studies Facility, 104 Mason Farm Road, Chapel Hill, NC 27599. E-mail: Becker.susanne{at}epa.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhalation of particulate matter (PM) may result in exacerbation of inflammatory airways disease, including asthma. Results from this laboratory have shown that the coarse inhalable particle fraction (PM2.5–10) is responsible for most of the PM effects on human airway macrophages (AM), including induction of cytokine production. Endotoxins associated with these particles account for a large part of their potency, as activity of PM can be inhibited by polymixin B and an activating moiety bound by lipopolysaccharide (LPS)-binding protein (LBP). The hypothesis behind the present study was that not only particle-bound LPS, but also Gram-negative (Gram-) and Gram-positive (Gram+) bacteria are responsible for PM-induced stimulation of AM, and therefore that PM are likely to activate receptors involved in recognition of microbes. Low level contamination of model pollution particles with environmental Staphyloccocus, Streptococcus, and Pseudomonas species was found to confer cytokine-inducing activity on inactive particles. Only one Gram- bacterium was sufficient for significant stimulatation of 100 AM, whereas at least three times more Gram+ bacteria were required for a similar level of response. Cytokine responses induced by PM as well as Gram+ and Gram- bacteria were inhibited by anti-CD14 antibody and required the presence of LBP-containing serum. The involvement of Toll-like receptor (TLR) 2 and 4 in recognition of PM2.5–10 was investigated in transfected Chinese hamster ovary cells expressing CD14 and TLR2 or TLR4. TLR4 was found to be involved in PM2.5–10 and Pseudomonas-induced activation, whereas TLR2 activation was induced by both Gram+ and Gram- bacteria and by PM. The synthetic lipid A analog E5531 fully inhibited the response to purified LPS and partially inhibited the response to PM and Pseudomonas. In contrast, E5531 had no effect on the response to Staphylococcus. Taken together, these results implicate microbial components as important players in AM-dependent inflammatory responses to PM.

Abbreviations: alveolar macrophages, AM • Chinese hamster ovary, CHO • fetal bovine serum, FBS • LPS-binding protein, LBP • lipopolysaccharide, LPS • particulate matter, PM • residual oil fly ash, ROFA • Toll-like receptor, TLR • tumor necrosis factor, TNF • volcanic ash, VA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The inhalable fraction of air pollution particulate matter (PM10) influences the respiratory health of both children and adults. Epidemiologic studies have demonstrated that increased school absenteeism, bronchitis, asthma exacerbation, and hospital emergency room visits occur during periods of increased PM10 levels (1–5). A key pulmonary cell in airways host defense and inflammation is the alveolar macrophage (AM) (6, 7). Pollution particles are phagocytized by the AM and, depending on particle composition, can evoke an inflammatory response. In in vitro studies, the coarse particle fraction (PM2.5–10) has been found to contain more than 90% of the AM-stimulating components of PM10 (8), whereas exposure to fine and ultrafine particles resulted in relatively minor stimulation.

Microbial agents, including bacteria, viruses, and fungi, are the most potent activators of AM (9). Constituents of the cell wall or membrane of both Gram-positive (Gram+) and -negative (Gram-) bacteria stimulate the release of a variety of inflammatory mediators which are involved in the recruitment of inflammatory cells to the airways (10, 11). In severe infection these mediators—e.g., tumor necrosis factor (TNF), interleukin (IL)-1, IL-8, IL-6, and various chemokines—are involved in septic shock. Ambient air has been shown to contain variable numbers of bacteria (12). However, most bacteria in the air are nonviable or do not grow on conventional substrates (13). Therefore, the actual amount of microbial material in the air is underestimated. Gram+ and Gram- bacteria and their degradation products are part of particulate matter of outdoor air, and are found to be associated mainly with inhalable PM2.5–10 (9, 14, 15). Best demonstrated in occupational settings, inhalation of dust-containing bacterial components, especially lipopolysaccharide (LPS), may cause symptoms involving airways inflammation, fever, decrements in lung function, and airways hyperreactivity (16, 17). However, ambient levels of endotoxin in homes of children with asthma have also been shown to be important determinants of asthma exacerbation (18, 19).

Cytokine production induced by pollution particles has been reported to be partially inhibited by polymixin B (8), suggesting that particle-bound LPS is involved in AM activation. The response to LPS is dependent on binding to the membrane receptor CD14 in the presence of LPS-binding protein (LBP) present in serum (20) Although CD14 was initially thought to be an exclusive receptor for LPS, recently both Gram+ cell wall peptidoglycans and lipoteichoic acid have also been shown to stimulate macrophages by a CD14-dependent mechanism (21, 22). The glycophosphatidylinositol anchored protein CD14 itself is not a signaling molecule, but requires interaction with Toll-like receptor (TLR) proteins for signal transduction resulting in nuclear factor (NF)-{kappa}B activation and cytokine production (23). When macrophages are stimulated with the purified endotoxin component LPS, TLR4 appears to be of major importance; however, other bacterial products such as Gram+ peptidoglycan and lipoteichoic acid have been shown to signal through TLR2 (24–27). Some fungal elements have also been shown to possess TLR agonist activities (28). Because airway macrophages seldom if ever encounter bacterial components individually, the responses to Gram+ and Gram- bacteria and fungal elements will be orchestrated by a combination of TLR receptors. This is likely to also be the case in the response to pollution particles.

AM are specialized to remove inorganic dusts from the airways without causing airway inflammation. On the other hand, elimination of microbes from the lung often requires a cytokine response by the AM, which leads to recruitment of granulocytes and stimulation of the immune response (29). Our working hypothesis is that microbes and their degradation products attached to air pollution particles, upon inhalation, deposit in the central and lower airways, where they influence airways host defenses and inflammation. This may cause respiratory symptoms and disease, including asthma exacerbation. The intent of this study was to define cytokine induction by environmental bacteria, in numbers likely to be associated with PM10 in inhaled air, and to identify receptors and signaling pathways involved in the cytokine response, which may be shared by PM10 and bacteria.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria
Collection/identification of bacteria in air. Duplicate petri dishes (100 x 15 mm) of nutrient, trypticase soy (TSA) (BBL 211,043; Difco Labs, Detroit, MI), sheep red blood agar (S-RBA) (TSA + 5% Sheep red blood cells [#1122]; Becton Dickinson, Sparks, MD), and sabouraud dextrose agar (S-D; Colorado Serum Co., Denver, CO) were uncovered and exposed to ambient air for 30 min, outside the EPA laboratory building in Chapel Hill, NC. One set of plates was incubated at 37°C (bacterial) or 25°C (fungal) and checked daily for colony growth of organisms while a second set of air-exposed plates was immediately sent to Aerotech Laboratories, Inc. (Phoenix, AZ) for identification of Gram-positive and negative bacteria.

Bacteria and LPS for stimulation. All bacteria were initially grown in 5 ml of nutrient broth and a 250-µl aliquot of this growth was spread onto agar plates using sterile glass spreaders or transferred to new broth tubes for further growth. Staphylococcus lentus (ATCC #49574) was cultured 2–3 d on TSA plates at 37°C in a bacterial incubator. Streptococcus sp. (group B) (ATCC #12112) was cultured 3–4 d on S-RBA plates at 37°C in a bacterial incubator. Pseudomonas sp. (ATCC #9230) was cultured in 10 ml sterile nutrient broth (Difco 233,000) for 3–4 d at 30°C in a bacterial incubator. Bacteria on agar plates were scraped off the surface using a cell scraper (#3010; CoStar, Cambridge, MA) into Dulbecco's PBS (#14190–144; Life Tech., Rockville, MD). Bacterial counts were performed using a Live/Dead Bacterial Assay Kit (L-7007; Molecular Probes, Eugene, OR). LPS (Salmonella) was obtained from Sigma Chemical Co (St. Louis, MO).

UV-irradiation. Thin films, 5 ml, of bacterial solutions were spread onto 150 x 15 mm sterile plastic petri dishes on ice and irradiated in a UV Strata-Linker 1800 (Stratagene, La Jolla, CA) for 15 min. Bacterial growth was then assessed and if growth occurred the process was repeated until all of the bacteria were UV-inactivated.

Source of Pollution Particles
The urban air dust preparation EHC-93 was obtained courtesy of Dr. R. Vincent, Health Canada. Mount St. Helens volcanic ash was sampled for the Environmental Protection Agency (EPA) from an open field at Washtucha, Washington. Residual oil fly ash (ROFA) was an oil combustion fly ash collected from an electrostatic precipitator (collection temperature 340°C) at an eastern U.S. power plant (Niagara, NY). Silica was a commercial preparation (S 5631) (Sigma). These dust have been described in detail in previous publications (30, 31). Particles were also collected in Chapel Hill, NC outside the EPA Human Studies Facility using a ChemVol High Volume Cascade Impactor (Rupprecht and Patashnick Co., Albany, NY). PM2.5–10 were collected onto polyurethane foam cleaned with methanol and water under sterile conditions. Particles were extracted from the foam by sonication for 10 min. Particles were pelleted, lyophilized, and weighed. Four different samplings, PM1, 2, 3, and 4 collected in spring of 2001 were used as stimuli in this study.

Stimulation of AM and Inhibition of Activation
Human alveolar macrophages were obtained by bronchoalveolar lavage from healthy male and female subjects, 20–35 yr of age, by a previously detailed procedure (32) under a research protocol approved by the Internal Review Board at University of North Carolina Medical School. AM at 2–3 x 105 cell in 1 ml of RPMI-1640 supplemented with 5% fetal bovine serum (FBS) (Life Tech.) were exposed overnight to stimuli in 5-ml polypropylene tubes; bacteria (103–2 x 106/tube) particulate matter (PM) (30 µg/ml) or LPS (10 ng/ml). Supernatants were harvested and stored frozen until assayed for cytokines. Polymixin B Sulfate (Sigma) at a concentration of 10 µg/ml was used to inhibit endotoxin activity in particles and bacteria. The lipid A agonist E5531 (33), a kind gift of Eisai Corporation (Boston, MA), was used at 3 and 1 µg/ml, to block LPS-induced cytokine production.

Stimulation of Chinese Hamster Ovary Cells
Clone 3E10 is an adherent Chinese hamster ovary cell (CHO)-K1 stably transfected with CD14 (34). This line contains a reporter construct with the structural gene for CD25 under the control of the human E-selectin promoter which contains an NF-{kappa}B binding site. CD25 expression in these cells is completely dependent on NF-{kappa}B translocation to the nucleus. These cells were maintained in HAM's F-12 with 5% FBS and antibiotics. 3E10 cells expressing TLR2 (CHO-TLR2) and TLR4 (CHO-TLR4) were constructed by stable transfection of the cells with human TLR2 and TLR4 cDNA (35).

Enzyme-Linked Immunosorbent Assay
TNF-{alpha} and IL-6 in supernatants of stimulated AM were quantified by enzyme-linked immunosorbent assay (cat #s DTA-50/D6050; R&D Systems, Minneapolis, MN) as per manufacturer's instructions.

Bioassay for IL-6
IL-6 in hamster supernatants were quantified using the IL-6–dependent hybridoma 7TD1 growth assay and hexosaminidase to assess cell proliferation (36). An IL-6 standard curve was generated with recombinant human IL-6 (R&D Systems).

Flow Cytometry
Fluorescent antibody to CD25 and an irrelevant control antibody of the same isotype was purchased from Becton Dickinson (Mountainview, CA). CHO/CD14, CHO-TLR2, and CHO-TLR4, at 2 x 105 cells per tube were incubated with 20 µl of antibody for 45 min whereafter cells were washed with 4 ml of cold PBS and fixed with 0.1% formaldehyde. Flow cytometry was performed with a FACSORT cytometer (Becton Dickinson, San Jose, CA) using an argon-ion laser (wavelength of 488 nm). The FACSORT was calibrated using Calibrite beads (Becton Dickinson, San Jose, CA) before use.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Demonstration of Microbes in Ambient Air
Nutrient Agar plates were exposed to outdoor air in a nonwindy location for 30 min to allow bacteria to settle if present in the air. Plates were then placed in an incubator for 16 h. In 30 min, 15–25 viable colony forming units had settled per plate. The colonies were classified by gram stain. Thirty percent of the colonies were Gram- (Pseudomonas spp. identified by morphology), and the rest of the colonies were mainly Gram+ cocci. The air also contained fungal spores detected by growth on Sabourad agar (12–16 colonies/plate). These were classified as belonging to Penicillium, Claudosporium, and Alternaria species by morphology.

Stimulation of Cytokine Production by Small Numbers of Environmental Bacteria
Two Gram+ species (Staphylococccus lentus [Staph] and Streptococcus spp. [Strep]) and Gram- Pseudomonas spp. (Pseudo) were chosen for assessment of the number of bacteria needed to stimulate 2 x 105 alveolar macrophages. Dose responses for each of the bacteria are shown in Figure 1 . The stimulation of AM with 0.1 bacteria per macrophage results in cytokine levels similar to levels induced following stimulation with freshly collected PM preparations at {bsim} 25 µg/ml (not shown). Gram- Pseudo was approximately three times more effective in stimulating the IL-6 response compared with Gram+ Staph. Strep did not effectively stimulate TNF nor IL-6 at bacteria:AM ratios less than one.



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  		Figure 1. Dose response of bacteria stimulating cytokine production by human AM. AM at 2 x 105/ml were stimulated with two bacteria/AM and 1:3 dilutions thereafter. Supernatants were collected at 18 h and assessed for cytokines by ELISA. A depicts the TNF response and B the IL-6 response. The figure shows the results of three experiments with different macrophage populations. The responses to the three bacteria were significantly different from each other (P < 0.05).

 
In the air, most bacteria are dead, or do not grow on the conventional agar plates (14). Cytokine induction by small numbers of viable and UV-killed bacteria were compared. There was a small but insignificant loss in AM stimulatory activity following UV treatment (not shown). All experiments below were performed with UV-killed bacteria.

Polymixin B and Anti-CD14 Blocking of Bacteria-Induced Cytokine Production
Our previous investigations have shown that PM10-induced IL-6 production is in part inhibited by polymixin B (8). Figure 2 shows that IL-6 production induced by Gram- bacteria was inhibited by polymixin B, whereas there was no significant effect of the endotoxin-neutralizing drug on cytokine production by the two Gram+ bacteria.



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  		Figure 2. Inhibition of bacteria-induced cytokine response by polymixin B. Pseudomonas, Staphylococcus, and Streptococcus were incubated with 10 µg/ml polymixin B for 30 min before addition to 2 x 105 AM at ratios of Pseudo 0.1:1, Staph 1:1, and Strep 1:1. Supernatants were collected at 6 h and assessed for IL-6. The bars represent the mean ± SEM of three experiments. Lightly shaded bars, before incubation; darkly shaded bars, after incubation with polymixin B. *P < 0.05.

 
On the other hand, IL-6 production in response to all three bacteria, and to PM2.5–10, was inhibited by an antibody against CD14 (Figure 3) . Furthermore, as with all CD14-dependent stimulation, the response to all three bacteria required the presence of a serum factor during stimulation (not shown).



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  		Figure 3. Inhibition of bacteria and PM-induced cytokine production with antibody against CD14. AM were incubated with 50 µg/ml anti-CD14 monoclonal antibody for 30 min, then exposed to the various bacterial stimuli. Pseudo added at 0.1:1, Staph at 1:1, and PM2.5–10 at 50 µg/ml. Supernatants were collected at 18 h and assessed for IL-6 by ELISA. The bars represent the mean ± SEM of three experiments. *P < 0.05.

 
Bacteria Drive Cytokine Responses to Pollution Particles
AM were stimulated with various sources of model pollution particles including silica, ROFA, volcanic ash (VA), and urban air particles (EHC-93) at 30 µg/ml. With the exception of EHC-93, these particles did not stimulate cytokine production as shown in Figure 4 . However, when silica, ROFA, or VA particles were contaminated with environmentally relevant numbers of bacteria (104 Staph; 104 Strep; 103 Pseudo), the response to the particles was driven by the bacteria. Furthermore, the particles did not appear to interfere with the response to the bacteria.



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  		Figure 4. Contamination of pollution particles with small numbers of dead bacteria affect cytokine responses by human alveolar macrophages. AM were stimulated with various particles at 30 µg/ml which were spiked with 104 Staph, 104 Strep, 2 x 103 Pseudo, or all three bacteria. The particles were Mt. St Helens ash, Silica, Residual Oil Fly ash, and EHC-93. Supernatants were collected at 18 h and assessed for IL-6. The bars represent the mean ± SEM of three experiments. Shaded bars, control; striped bars, silica; filled bars, volcanic ash; hatched bars, ROFA; open bars, EHC-93.

 
Involvement of TLR2 and TLR4 in Response to PM
CD14-dependent responses involve signaling through TLR2 and TLR4. Because the response to PM2.5–10 and all three bacteria was dependent on binding to CD14, we thought to investigate the involvement of TLR2 and TLR4. The CHO/CD14 cell line containing the structural gene for CD25 under the regulation of NF-{kappa}B, and stably transfected with either TLR2 or TLR4, was stimulated with 100 ug/ml of four different preparations of PM2.5–10 and bacteria in a ratio of 3 and 1 bacteria/target cell. Stimulation was measured by CD25 expression, and by release of IL-6. The results of NF-{kappa}B–dependent CD25 expression is shown in Figure 5 . The CHO/TLR4 line responded to stimulation with Pseudo and PM, but not when exposed to Strep or Staph. Activation of CHO/TLR2 was measured following stimulation with PM2.5–10 as well as Staph and Pseudo. The cells did not respond to Strep. Stimulated CHO cell supernatants were also assessed for released IL-6. A similar response pattern to bacteria and PM10 was seen with IL-6 release as in the CD25 response. In the CHO/TLR4 cells, only PM and Pseudo gave a significant IL-6 response (Figure 6A) . The CHO/TLR2 line produced IL-6 in response to PM, Staph, and Pseudo (Figure 6B). The parental CHO/CD14 cells were not activated by the concentrations of stimuli used, although a higher concentration of Pseudo could induce some IL-6 production presumably mediated by the endogenous hamster TLR4 in these cells (not shown).



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  		Figure 5. Activation of NF-{kappa}B–dependent CD25 expression in CHO-TLR4 and CHO-TLR2 cells with pollution particles and bacteria. CHO-TLR4 (A) and CHO-TLR2 cells (B) were incubated with four different air pollution particle preparations (PM1, PM2, PM3, and PM4) at 100 µg/ml, or the three bacteria at three (pse 3, strep3, staph3) or one (pse1, strep1, staph1) microbes/cell in 12-well culture plates. LPS was added at 100 ng/ml. Cells were analyzed for CD25 expression by flow cytometry at 18 h after stimulation. The bars represent means ± SD of four separate exposures from two experiments.

 


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  		Figure 6. Release of IL-6 by pollution particle and bacteria-stimulated CHO-TLR4 and CHO-TLR2. CHO-TLR4 (A) and CHO-TLR2 cells (B) were stimulated with two air pollution particle preparations (PM1, PM2) at 100 µg/ml, and by Pseudomonas, Staphylococcus, and Streptococcus species at three bacteria/cell. LPS was added at 100 ng/ml. Supernatants were collected at 18 h and assessed for IL-6 activity in bioassay. The bars represent mean ± SD of three experiments.

 
Effect of TLR4 Inhibitor E5531 on PM and Bacteria-Induced Macrophage Cytokine Production
The synthetic lipid A analog E5531 has successfully been used to inhibit TLR4-dependent NF-{kappa}B activation and cytokine secretion by LPS (33, 37). Experiments were conducted using different concentrations of E5531 added in AM cultures that were exposed to PM, soluble LPS, Pseudo, or to Staph. As can be seen in Figure 7 , E5531 at 3 and 1 µg/ml (not shown) completely abolished TNF (panel A) and IL-6 (panel B) production induced by LPS and partially inhibited activation by PM and Pseudo. E5531 had no effect on Staph-induced or Strep-induced (not shown) cytokine production.



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  		Figure 7. Inhibition of particle- and bacteria-induced cytokine production by E5531. AM were incubated with 3 µg/ml of E5531 for 30 min before addition of stimuli to the cells. Particles were added at 50 µg/ml and Pseudo at 0.1:1, and Staph at 1:1. LPS was added at 100 ng/ml. Supernatants were collected at 6 h and assessed for TNF (A) and IL-6 (B). The bars represent mean ± SD of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent investigations into health effects of pollution particles have been geared toward understanding components of particles less than 2.5 µm in diameter (PM2.5), which may be the cause of increases in human morbidity and mortality reported in numerous investigations from urban centers around the world. These fine particles are derived primarily from mobile and industrial emission sources and may be controlled by scrubbing devices. However, studies from this laboratory using airway macrophages as targets of inhaled particles have shown that these cells are affected by coarse mode particles, or so-called nuisance dusts, and only minor responses are seen with PM2.5 (8), or by ultrafine particles (Becker, unpublished results). The cytokine response to coarse particles appears to be in large part driven by endotoxins attached to the particles. The present investigation demonstrates that small numbers of whole bacteria in the outdoor environment may influence the production of cytokines by AM exposed to air particulate pollution. The ambient urban air has been shown to contain relatively high levels of bacteria (12), and bacterial components have been shown to preferentially associate with particles (13). The particles act as carriers of microbes and allergens, directing their deposition to sites in the airways where they would not by themselves deposit due to their aerodynamic properties. Coarse mode particles have in humans been shown to preferentially deposit in the small airways where asthma pathology occurs (38, 39).

Our earlier studies have implicated endotoxin as an important contaminant of PM, because approximately half of the cytokine response by macrophages stimulated with 100 µg/ml PM could be blocked by polymixin B, or water soluble activity could be removed by LBP (8). At lower stimulatory concentrations of particles (25 µg/ml), blocking with polymixin B was complete. The involvement of endotoxin in macrophage activation by fine size PM2.5 has also been reported (8, 15). These finding suggested that the particles contained LPS as well as other moieties with ability to induce cytokines. Either these moieties were present in low concentration in PM, or more material was required for stimulation of AM. Experiments that compared the cytokine-inducing potential of Gram+ and Gram- bacteria revealed that {bsim} 3 times more Gram+ Staph were needed for a comparative cytokine response as induced with Gram- Pseudo. As few as one Gram- bacterium/one hundred AM (0.01:1) caused significant IL-6 production. Approximately 30% of the viable colony-forming bacteria in air from which the particles were collected were Gram-. Although the majority were Gram+, the preferential response of AM to Gram- cells indeed suggested that these would be of major importance in the response at lower particle levels. On the other hand, at high particle concentrations it is likely that sufficient numbers of Gram+ bacteria would be present to induce a cytokine response.

Cytokine production by both PM and bacteria could be inhibited by blocking CD14, and by omitting serum from the assay, implicating involvement of "pathogen-associated molecular pattern" recognition in the response to PM. Signaling to cytokine production in response to various bacterial molecules is mediated by TLR proteins TLR2 and TLR4. TLR2 appears to be a promiscuous receptor for glycolipids, lipoteichoic acid, and proteoglycans of Gram+ and Gram- bacteria. Pure LPS lipid A signals only through TLR4 (24). Some specificity has been assigned to other TLR proteins, such as TLR6 for flagellin (40) and TLR9 for CpG DNA (41). Many of these TLRs may interact in responses to PM, which undoubtedly contain a variety of bacterial and fungal elements. Here, studies with CHO/TLR2 and CHO/TLR4 revealed that both receptors were activated by PM2.5–10, although CHO/TLR4 could distinguish between Pseudo and Staph. Streptococcus lentus was poorly recognized by both macrophages and CHO/TLR transfectants. However, in unpublished studies we have found that human monocytes respond better to Streptococcus lentus than to Staphylococcus spp. This suggests the involvement of an additional receptor for Strep-induced signaling that is poorly expressed in alveolar macrophages.

In conclusion, human airway macrophages appear to react to particulate pollution by receptors evolved to recognize foreign microorganisms. Most likely, activation of AM is mediated by bacteria or bacterial components attached to the particles. Particles which act on macrophages fall in the coarse size range, which preferentially deposit in small airways (38, 39). We suggest that these inhaled particles, which stimulate inflammatory mediator production and reduce phagocytic host defenses in the airway macrophages (8), may impact on asthma and infectious disease severity. In search for the culprit of air pollution particle–mediated health effects, the nuisance dust should not be overlooked, as it avidly binds microbes and degradation products, the concentrations of which are directly related to human activity and waste production


   Acknowledgments
 
The investigators thank the staff of the Medical Station at HSD for providing alveolar macrophages for these studies. The helpful suggestions of Dr. Ian Gilmour and Dr. David Peden in preparation of this manuscript are much appreciated.


   Footnotes
 
DISCLAIMER: The research described in this article has been supported by the United States Environmental Protection Agency. It has been subjected to Agency review and has been approved for publication. Approval does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names and commercial products does not constitute endorsement or recommendation for use.

Received in original form March 28, 2002

Received in final form May 28, 2002


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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