Marsha D. W. Ward

Induction of Asthma and the Environment: What We Know and Need to Know

The prevalence of asthma has increased dramatically over the last 25 years in the United States and in other nations as a result of ill-defined changes in living conditions in modern society. On 18 and 19 October 2004 the U.S. Environmental Protection Agency and the National Institute of Environmental Health Sciences sponsored the workshop “Environmental Influences on the Induction and Incidence of Asthma” to review current scientific evidence with respect to factors that may contribute to the induction of asthma. Participants addressed two broad questions: a) What does the science suggest that regulatory and public health agencies could do now to reduce the incidence of asthma? and b) What research is needed to improve our understanding of the factors that contribute to the induction of asthma and our ability to manage this problem? In this article (one of four articles resulting from the workshop), we briefly characterize asthma and its public health and economic impacts, and intervention strategies that have been successfully used to prevent induction of asthma in the workplace. We conclude with the findings of seven working groups that focus on ambient air, indoor pollutants (biologics), occupational exposures, early life stages, older adults, intrinsic susceptibility, and lifestyle. These groups found strong scientific support for public health efforts to limit in utero and postnatal exposure to cigarette smoke. However, with respect to other potential types of interventions, participants noted many scientific questions, which are summarized in this article. Research to address these questions could have a significant public health and economic impact that would be well worth the investment.

A murine model for low molecular weight chemicals: differentiation of respiratory sensitizers (TMA) from contact sensitizers (DNFB)

Exposure to low molecular weight (LMW) chemicals contributes to both dermal and respiratory sensitization and is an important occupational health problem. Our goal was to establish an in vivo murine model for hazard identification of LMW chemicals that have the potential to induce respiratory hypersensitivity (RH). We used a dermal sensitization protocol followed by a respiratory challenge with the evaluation of endpoints typically associated with RH in human disease. Trimellitic anhydride (TMA) was used as a prototype respiratory sensitizer and was compared to the dermal sensitizer; 2,4-dinitrofluorobenzene (DNFB), along with vehicle controls. BALB/c mice were dermally sensitized using two exposure protocols. Mice in both protocols were dermally exposed on experimental days; D-18 and D-17 (abdomen), and D-13 (ear). On D 0 mice received an intratracheal (IT) challenge. The mice in Protocol 2 were abdominally exposed twice with the addition of exposures on D-25 and D-24. Results indicate that mice required the additional dermal sensitization and the IT challenge (Protocol 2) to significantly elevate total IgE in serum and bronchoalveolar lavage fluid (BALF). Additional responses suggestive of RH were seen following Protocol 2, including increases in BALF cell numbers and neutrophils post IT with TMA (but not DNFB). These data suggest that the dermal sensitization and IT challenge followed by evaluation of serum antibodies and lung parameters are a reasonable and logistically feasible approach towards the development of a model for RH responses to LMW chemicals.

Comparison of respiratory responses to Metarhizium anisopliae extract using two different sensitization protocols

Metarhizium anisopliae, an entomopathogenic fungus, is a prototypic microbial pesticide licensed for indoor control of cockroaches, a major source of allergens. We have previously demonstrated allergy and asthma-like responses in BALB/c mice intraperitoneally (IP) sensitized in the presence of adjuvant and intratracheally (IT) challenged with the soluble factors from M. anisopliae crude antigen (MACA) (Ward et al., 1998, 2000). This protocol has been used frequently to establish animal models of allergenicity. However, the sensitization protocol is artificial and not representative of an environmental exposure. Concern has been raised that this protocol might produce allergic responses that would not occur under normal environmental exposure conditions. The objective of this study was to compare responses in mice to MACA by two exposure protocols: (1) exclusive respiratory exposures without adjuvant (representative of environmental exposures) and (2) intraperitoneal sensitization in the presence of adjuvant followed by IT challenge (the traditional approach). The intratracheal protocol consisted of four IT exposures of 10 microg MACA in 50 microl HBSS each over a 4-week period. A vehicle control group of mice was exposed IT to HBSS. The intraperitoneal protocol consisted of IP sensitization with 25 microg MACA in 0.2 ml of 1.3% alhydrogel (aluminum hydroxide) followed 14 days later with an IT challenge (10 microg MACA/50 microl HBSS). Airway reactivity responsiveness to methacholine was assessed, serum and bronchoalveolar lavage fluid (BALF) samples were obtained, and the lungs were fixed for histopathology at 1, 3, and 8 days following the last MACA IT challenge. Both groups exhibited immune and pulmonary responses typical of allergic asthma. In general, local responses in the lung, including inflammatory responses (eosinophils, lymphocytes, and macrophages), BALF IgE, and functional responses to methacholine were greater in the IT sensitized group compared to the IP sensitized group, whereas the systemic IgE response was greater in the IP sensitized group. The BALF IL-5 cytokine levels were elevated before and throughout the eosinophil influx. IL-4 was detected in the BALF of IP sensitized, but not IT sensitized mice. Histopathologic changes in the two groups were similar in nature but more severe in the IT mice. The results suggest that the IP sensitization protocol does not induce the level of respiratory responsiveness that results from sensitization by a physiologically relevant route of exposure. Thus total serum IgE levels, which were greater following IP sensitization, may not be the best indicator of allergen potency, at least with respect to respiratory responses.

A comparison of the allergic responses induced by Penicillium chrysogenum and house dust mite extracts in a mouse model

UNLABELLED A report by the Institute of Medicine suggested that more research is needed to better understand mold effects on allergic disease, particularly asthma development. We compared the ability of the fungal Penicillium chrysogenum (PCE) and house dust mite (HDM) extracts to induce allergic responses in BALB/c mice. The extracts were administered by intratracheal aspiration (IA) at several doses (0, 2.5, 5, 10, 20, 40, and 80 μg) four times over a 4-week period. Three days after the last IA exposure, serum and bronchoalveolar lavage fluid (BALF) were collected. The relative allergenicity of the extracts was evaluated based on the lowest dose able to induce a significant response compared to control (0 μg) and the robustness of the response. PCE induced the most robust response at the lowest dose for most endpoints examined: BALF total, macrophage, neutrophil, and eosinophil cell counts, and antigen-specific IgE. Taken together, our data suggest that PCE may induce a more robust allergic and inflammatory response at lower doses than HDM. PRACTICAL IMPLICATIONS Our data suggest that Penicillium chrysogenum is a robust allergen and may be a more potent allergen source than house dust mite (HDM) in this mouse model. Two critical factors in the development of human allergic disease, exposure levels and sensitization thresholds, are unknown for most allergens including molds/fungi. Human exposure levels are not within the scope of this article. However, the data presented suggest a threshold dose for the induction of allergic responsiveness to P. chrysogenum. Additionally, P. chrysogenum as well as other molds may play an important role in asthma development in our society.

Differential allergy responses to Metarhizium anisopliae fungal component extracts in BALB/c mice

Intratracheal aspiration (IA) exposure to Metarhizium anisopliae crude antigen (MACA), which is composed of equal protein amounts of mycelium (MYC), conidia (CON) and inducible proteases/chitinases (IND) extracts/filtrates, has resulted in responses characteristic of human allergic asthma in mice. The study objective was to evaluate the potential of each component extract to induce allergic/asthma-like responses observed in this mouse model. BALB/c mice received 4 IA exposures to MACA, CON, MYC, IND, or bovine serum albumin (BSA; negative control) or appropriate vehicle control or inflammatory control over a 4-wk period. Mice were assessed by whole-body plethysmography for immediate airway responses and airway hyperresponsiveness to methacholine (Mch) challenge (PenH). Serum and bronchoalveolar lavage fluid (BALF) were collected 3 d after the final exposure. Additionally, BALF neurotrophin levels and extract protease and chitinase activity levels were evaluated. Western blot analysis showed that each component contained different IgE-reactive proteins. All fungal extract exposures resulted in elevated BALF total and differential cell counts, IgE and IgA and total serum IgE compared to HBSS and BSA controls. MYC-exposed mice had the highest responses except for neutrophil influx, which was highest in MACA and IND exposures. However, the MYC-exposed mice had significantly lower PenH values compared to other treatments. By comparison IND and MACA induced significantly higher PenH values. Additionally, IND had substantially higher protease activity levels but induced the lowest neurotrophin levels compared to the other fungal exposures. In this allergic asthma model extract chitinase activity was not associated with allergic responses. In summary, multiple exposures to any of the M. anisopliae component extracts induced allergic/asthma-like responses in BALB/c mice but the response magnitude was different for each component and each appears to contain unique IgE-reactive proteins. Therefore, hazard identification and/or risk assessment for molds must test both mycelia and conidia.

Assessing the allergenic potential of molds found in water-damaged homes in a mouse model

Abstract Damp/moldy indoor environments, which have resulted from flooding events and may increase as a result of climate change, have been associated with asthma exacerbation. Certain molds found in significantly higher or lower concentrations in asthmatics’ homes compared to control homes have been categorized as Group 1 (G1) and Group 2 (G2) molds, respectively. We have compared the allergic potential of selected G1/G2 molds to house dust mite (HDM) in a mouse model. BALB/c mice were exposed to mold (0–80 µg) or HDM (20 µg) extract by intratracheal aspiration either 4X over 4 weeks (allergenicity) or 1X (non-specific responses). Airflow limitation (methacholine challenge) was measured (Day 1) and serum and bronchoalveolar lavage fluid were collected (Day 2) after the final exposure. The G1 molds induced low-to-moderate responses and required higher doses to achieve antigen-specific IgE results similar to those induced by HDM. Compared to HDM responses, the G2 mold in this study required lower doses to induce a similar response. Acute exposure responses suggest some molds may exacerbate asthmatic responses. These studies demonstrate the differing capacities of molds to induce responses associated with allergic asthma, including differences in the threshold dose for allergy induction. Therefore, molds must be evaluated individually for allergic/asthmatic potential. These studies along with our previous studies with G1 (Stachybotrys chartarum)/G2 (Penicillium chrysogenum) molds suggest that the G1/G2 categorization is not indicative of allergic potential but they do not preclude this categorization’s utility in determining unhealthy building dampness.

Animal Models to Assess the Effects of Air Pollutants on Allergic Lung Disease

Abstract: Animal models provide toxicologists with useful tools for assessing risks associated with respiratory allergy. Both the mouse and BN rat models described exhibit many of the features of human allergic asthma. It is clear that environmental contaminants can exacerbate the expression of these features. Work is under way to explore underlying mechanisms and to develop methods for applying these data to human health risk assessment.

Biomarkers of acute respiratory allergen exposure: Screening for sensitization potential

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens following an acute exposure in naïve individuals. Female BALB/c mice received a single intratracheal aspiration exposure to Metarhizium anisopliae crude antigen (MACA) or bovine serum albumin (BSA) in Hanks Balanced Salt Solution (HBSS) or HBSS alone. Mice were terminated after 1, 3, 6, 12, 18 and 24 h. Bronchoalveolar lavage fluid (BALF) was evaluated to determine total and differential cellularity, total protein concentration and LDH activity. RNA was isolated from lung tissue for microarray analysis and qRT-PCR. MACA administration induced a rapid increase in BALF neutrophils, lymphocytes, eosinophils and total protein compared to BSA or HBSS. Microarray analysis demonstrated differential expression of genes involved in cytokine production, signaling, inflammatory cell recruitment, adhesion and activation in 3 and 12 h MACA-treated samples compared to BSA or HBSS. Further analyses allowed identification of approximately 100 candidate biomarker genes. Eleven genes were selected for further assessment by qRT-PCR. Of these, 6 demonstrated persistently increased expression (Ccl17, Ccl22, Ccl7, Cxcl10, Cxcl2, Saa1), while C3ar1 increased from 6-24 h. In conclusion, a single respiratory exposure of mice to an allergenic mold extract induces an inflammatory response which is distinct in phenotype and gene transcription from the response to a control protein. Further validation of these biomarkers with additional allergens and irritants is needed. These biomarkers may facilitate improvements in screening methods.

Maternal respiratory sensitization and gestational allergen exposure does not affect subsequent pup responses to homologous or heterologous allergen

Evidence suggests that the predisposition towards atopy begins early in life. Maternal allergy has been associated with an increased risk of the development of allergic disease in offspring. Some studies suggest that the development of childhood atopy may also be influenced by prenatal allergen exposure. In this study, a respiratory allergen exposure model was used to determine the impact of maternal sensitization (with or without additional exposures during pregnancy) on subsequent pup responses to homologous or heterologous allergen. Female BALB/c mice received two intratracheal aspiration (IA) exposures to Metarhizium anisopliae crude antigen (MACA) or Hank’s buffered salt solution (HBSS) prior to breeding. Some mice also received three additional exposures during pregnancy. Control mothers did not receive treatment. Young adult offspring received three IA exposures to MACA, house dust mite extract (HDM) or HBSS. Offspring sensitized as young adults to either HDM or MACA developed an airway inflammatory response, including increased bronchoalveolar lavage fluid lactate dehydrogenase activity, total protein and total and differential cell counts compared to offspring exposed to HBSS. Increased airway responsiveness to methacholine was observed in pups treated with HDM but not with MACA. Maternal sensitization status (with or without gestational allergen exposure) had no effect on offspring response to either MACA or HDM. In conclusion, this study demonstrates that IA administration of MACA or HDM extract to young adult BALB/c mice induces the development of an inflammatory airway response. In contrast to previous reports, neither maternal sensitization nor gestational allergen exposure could be demonstrated to have a clear effect on offspring sensitization. This discrepancy may be a function of the respiratory sensitization and exposure protocol used in this study, which mimics natural sensitization more closely than do parenteral routes of exposure.

The effects of pregnancy on the exacerbation and development of maternal allergic respiratory disease.

The T-helper 2 (TH2) bias associated with pregnancy may predispose the pregnant mother to the development or exacerbation of allergic disease. To determine the effects of pregnancy on pre-existing maternal sensitization, we sensitized BALB/c mice before breeding by two intratracheal aspiration (IA) exposures to the fungal allergen, Metarhizium anisopliae crude antigen (MACA). Some mice also received three IA exposures to MACA on gestational days 11, 15, and 19. After weaning, all mice were challenged IA with MACA before killing. To determine the effects of pregnancy on susceptibility to future sensitization, naïve parous and nulliparous BALB/c mice were sensitized by three IA exposures to MACA or to Hank’s buffered salt solution vehicle control. Pregnancy did not have a significant effect on individual inflammatory parameters (airway responsiveness to methacholine, total serum and bronchoalveolar lavage fluid (BALF) IgE, BALF total protein, lactate dehydrogenase activity, and total and differential cell counts) following allergen challenge in sensitized mice, regardless of post-breeding allergen exposure. In conclusion there was a weak inhibition of the overall response in mice exposed to allergen during pregnancy compared to identically treated nulliparous mice. In contrast, parous mice that did not encounter allergen post-breeding tended to have exacerbated responses. Parity had no significant impact on future susceptibility to sensitization.