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Final Report: Mechanisms that Initiate, Promote, and Resolve Grain Dust/LPS Induced Inflammation
EPA Grant Number: R826711C001Subproject: this is subproject number 001 , established and managed by the Center Director under grant R826711
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: University of Iowa Children's Environmental Airway Disease Center
Center Director: Hunninghake, Gary W.
Title: Mechanisms that Initiate, Promote, and Resolve Grain Dust/LPS Induced Inflammation
Investigators: Nauseef, William , Denning, Gerene , Hunninghake, Gary W. , Moreland, Jess , Schwartz, David , Weiss, Jerry
Institution: University of Iowa
EPA Project Officer: Fields, Nigel
Project Period: January 1, 1998 through January 1, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Centers for Children's Environmental Health and Disease Prevention Research (1998)
Research Category: Children's Health , Health Effects
Description:
Objective:The hallmark of the bronchial hyperreactivity observed in children with asthma is airway inflammation, and this is also true for airway disease elicited by grain dust. In contrast to eosinophil-mediated asthma, responses stimulated by grain dust are predominately neutrophilic, suggesting that grain dust elicits proinflammatory signals largely geared toward stimulating infiltration of neutrophils. Previous studies suggest that: (1) the major biologically active component of grain dust extract is bacterial endotoxin (lipopolysaccharide [LPS]); (2) the first airway cells exposed to grain dust are alveolar macrophages and epithelial cells; (3) the inflammatory response is initiated by the release of numerous proinflammatory factors, including cytokines; and d) the inflammatory response is characterized by the influx and activation of neutrophils.
Relatively few studies have examined intact grain dust to determine its biological effects on host cells. Because this dust may contain multiple biologically active components and the association of these components with the particles themselves could influence their biological activity, the objective of this research project was to design a model system that tested the effect of dust particles on the proinflammatory responses of host immune (macrophages, neutrophils) and nonimmune (epithelial) cells. Based on these considerations, we hypothesize that exposure of alveolar macrophages to grain dust results in the release of macrophage-specific cytokines and that this release is, at least in part, a result of grain dust-associated LPS. Based on our preliminary studies, we further hypothesize that grain dust alone is not sufficient to elicit cytokine release by human airway epithelial cells but rather that macrophage cytokines (released in response to grain dust factors) are required to stimulate epithelial cell cytokine release. Lastly, we hypothesize that once recruited into the airway, neutrophils are activated during phagocytosis of grain dust particles and activation is, at least in part, a result of grain dust-associated LPS.
Summary/Accomplishments (Outputs/Outcomes):Progress in these studies was compromised by difficulties obtaining uniform characterization of grain dust samples used as agonist in the various experimental systems. For many studies performed in parallel with the animal challenge work, material eluted from the grain dust was used. Although the amount of endotoxin could be assessed in the eluate, other bioactive species were neither defined nor quantitated. Furthermore, the use of whole grain dust particles presented an additional technical problem, as uniform size of dust could not be provided. Taken together, these challenges significantly compromised the in vitro studies, although it is important to emphasize that they do not impact on the validity or strength of the in vivo work or whole animal challenges. Correction of these deficits would have entailed biophysical approaches beyond the scope of the proposed work and time frame, so most productive application of the principles derived from these experiments was applied to questions and hypotheses derived from those that initially inspired the proposed work on grain dust.
Grain Dust Elicits a Proinflammatory Response in an Animal Model
Exposure of a mouse model to aerosolized grain dust resulted in increased expression of proinflammatory cytokines. Cytokine expression by alveolar macrophages was demonstrated by RNase protection assays (RPA), and cytokine levels in lavage fluid were measured using enzyme-linked immunosorbent assays (ELISA). Although rigorous determinations of endotoxin concentrations that reach the mouse airway were not possible, overall, grain dust appeared to be less potent than the grain dust extract used as a control. The results of these studies currently are being summarized in a manuscript being prepared by Drs. Denning and Moreland.
Exposure of Human Alveolar Macrophages to Grain Dust Results in the Release of Macrophage-Specific Proinflammatory Cytokines
Macrophages in the airway respond to foreign particles by releasing immunomodulatory factors, including cytokines. In studies with isolated human alveolar macrophages, we found that:
- Grain dust stimulated an increase in the release of the cytokines TNF-α, IL-8, ENA-78, and RANTES in a dose dependent manner.
- Increased expression of these cytokines and of MCP-1 also was demonstrated by RPA.
- The effect was maximal at approximately 1-2 mg/mL of grain dust (~ 100-150 ng/mL of associated LPS).
- Higher concentrations of grain dust (10 mg/mL) actually inhibit release of all four cytokines relative to maximal levels.
These data support the hypothesis that factors present in grain dust, including endotoxin, stimulate cytokine release by human alveolar macrophages.
LPS Contributes to the Effects of Grain Dust on Release of Cytokines by Human Alveolar Macrophages
TNF-a has been identified as a key early mediator of the inflammatory response. For this reason, many of our studies have focused on release of this cytokine. Previous studies show that grain dust extract stimulates release of TNF-α by alveolar macrophages and that this activity is predominately because of LPS. In our studies, purified Escherichia coli endotoxin was used as a control. We found that:
- Both grain dust and purified LPS increased release of TNF-α by human alveolar macrophages.
- The effects of LPS, but not of grain dust, were enhanced by the addition of LPS-binding protein (LBP).
- The effects of LPS, and to a much lesser extent of grain dust, were inhibited by the endotoxin inhibitors polymyxin B and bacterial permeability-increasing protein.
- Conclusions about relative potency are confounded by several factors, including the difficulty of assessing how concentrations of LPS in grain dust extract relate to the particles themselves. We observed, however, no correlation between the levels of LPS in extract from several grain dust preparations and the increases in TNF-a release in response to each.
- Studies using radiolabeled LPS indicate that there were both hydrophilic and hydrophobic interactions between the dust particle and the LPS and that LBP did not promote release of LPS from the particles. This last finding is consistent with the hypothesis that binding to grain dust particles alters the interaction of LPS with LPS-binding proteins.
Taken together, these studies indicate that responses to purified LPS and to grain dust had some inherent differences. Thus, a more directed approach is required to address this question. Tools have recently been developed that allow us to test more directly whether LPS contributes to the biological activity of grain dust.
LPS Contributes to the Effects of Grain Dust on the Activation State of Human Neutrophils
We have examined two separate agonist-dependent responses of human neutrophils, namely priming of the NADPH-dependent oxidase for fMLP activation and IL-8 secretion. We have used precisely quantitated LPS (using acetate auxotrophs of bacteria to radiolabel the endotoxin) in these studies and focused on the roles of CD14, LBP, and TLR4 in these processes. Specifically, we currently are studying whether blocking antibodies against CD14 (required for LPS-mediated signaling) affect the response of neutrophils and macrophages to LPS. To that end, the effect of monoclonal antibodies directed against CD14 and TLR4 were used. We assessed activation of the NADPH oxidase by using lucigenin-enhanced chemiluminescence and evaluated IL-8 secretion using an ELISA. Whereas LPS elicits a direct activation at very low levels, it primes fMLP-induced oxidant release 5-10 fold in a time and concentration dependent manner. MEM18, a blocking monoclonal antibody against CD14, influenced neither the direct response of polymorphonuclear neutrophils (PMNs) to dust or the dust-dependent priming of PMNs. In contrast, LPS-dependent responses are inhibited by MEM18 but not by antibodies against TLR4. These data suggest, with the caveats mentioned previously in mind, that dust-dependent responses may not be mediated by the same pathways that are triggered by endotoxin. Such findings would dramatically alter the current understanding of dust-induced inflammation and the contribution of endotoxin to the overall clinical picture.
Grain Dust Does Not Directly Elicit Cytokine Release by Human Airway Epithelial Cells
Airway epithelial cells are exposed directly to grain dust that enters the lung. Grain dust stimulates increased expression of inflammatory cytokines, including an IL-8 homolog, by airway epithelial cells in an animal model system (Dr. Moreland). Previous studies, however, show that LPS is a poor agonist in stimulating release of inflammatory cytokines by human epithelial cells in vitro. Consistent with these previous studies, we found that relatively high concentrations of purified LPS (> 2 µg/mL) were required to stimulate cytokine release (IL-8 and ENA-78) by the human alveolar type II cell line A549 and the human bronchial epithelial cell lines HBE and Calu 3. Similarly, grain dust (≤ 1 mg/mL) elicited only small increases in cytokine release and higher concentrations actually decreased measurable cytokine levels. The effects of both LPS and grain dust were independent of serum proteins (LBP and soluble CD14) that modulate many LPS-dependent effects. As this amount of grain dust completely covers the surface of the cells, it seems unlikely that concentrations above this are achieved in the airway. These data support the hypothesis that grain dust alone is not sufficient to stimulate cytokine release by human airway epithelial cells and suggest the in vivo results are a result of cross-talk between the macrophages and epithelial cells.
We have applied many of these principles to examine PMN transmigration across human pulmonary microvascular endothelial cells, in response to purified LPS, dust, and bacterial challenge. The microbial studies have been published recently by Drs. Moreland and Nauseef (Moreland, et al., 2004).
Grain Dust Activates Human Neutrophils and This Activation is Due, at Least in Part, to LPS
A consequence of the inflammatory cascade is the influx of neutrophils. As part of our in vitro model, we compared the effects of purified LPS with those of grain dust on activation of human neutrophils using lucigenin-dependent chemiluminescence (measure of reactive oxygen species production). In preliminary studies, we observed that both purified LPS and grain dust increased chemiluminescence in a dose-dependent manner. Interestingly, the neutrophil response to both LPS and grain dust (at comparable LPS concentrations) was enhanced by LBP. These data support the hypothesis that grain dust activates neutrophils and, suggests that, in contrast to macrophage cytokine release, LPS contributes to the effect. As detailed above, further dissection of the relative contributions of CD14 and TLR4 to the overall cellular responses to dust are ongoing.
Most Significant Achievement
The major strength of these studies is that they are modeled on the physiologically relevant concept that understanding grain dust-induced asthma will require understanding the effects of intact dust on the human airway. Technical issues related the biophysical properties and composition, both chemical and microbial, of the dust need to be solved before rigorous characterization of the cell biology of the dust-dependent response can be elucidated in an in vitro system.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other subproject views: | All 14 publications | 14 publications in selected types | All 14 journal articles |
| Other center views: | All 33 publications | 32 publications in selected types | All 32 journal articles |
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Moreland JG, Bailey G, Nauseef WM, Weiss JP. Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. Journal of Immunology 2004;172(1):426-432. |
R826711 (Final) R826711C001 (Final) |
not available |
airway disease, airway inflammation, allergen, assessment of exposure, asthma, biological response, childhood respiratory disease, children, cytokines, environmentally caused disease, epidemiology, exposure, exposure assessment, grain dust, harmful environmental agents, health effects, human exposure, human health risk, lipopolysaccaride, mechanisms, rural communities, sensitive populations, tissue reactivity, children’s health,
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ENVIRONMENTAL MANAGEMENT, Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Risk Assessment, Biology, Risk Assessments, genetic susceptability, Health Risk Assessment, Epidemiology, Chemistry, Children's Health, Environmental Chemistry, Allergens/Asthma, exposure assessment, environmentally caused disease, allergen, health effects, rural communities, cytokines, assessment of exposure, childhood respiratory disease, mechanisms, LPS, harmful environmental agents, human health risk, toxics, epidemeology, agricultural community, sensitive populations, biological response, grain dust, airway disease, children, lipopolysaccaride, exposure, children's vulnerablity, tissue reactivity, asthma, human exposure, Human Health Risk Assessment
Progress and Final Reports:
2000 Progress Report
Original Abstract
Main Center Abstract and Reports:
R826711 University of Iowa Children's Environmental Airway Disease Center
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R826711C001 Mechanisms that Initiate, Promote, and Resolve Grain Dust/LPS Induced Inflammation
R826711C002 Multi-component Intervention Study of Asthma in Children from Rural Communities
R826711C003 Role of RSV Infection and Endotoxin in Airway Inflammation
R826711C004 A Model to Study the Development of Persistent Environmental Airway Disease