1999 Progress Report: Chemokines in the Pathogenesis of Asthma (Asthma Chemokines)EPA Grant Number: R826710C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R826710
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Michigan Center for the Environment and Children’s Health
Center Director: Israel, Barbara A.
Title: Chemokines in the Pathogenesis of Asthma (Asthma Chemokines)
Investigators: Remick, Daniel , Philbert, Martin
Current Investigators: Israel, Barbara A. , Brown, Randall , Keeler, Gerald J. , Lin, Xihong , Parker, Edith , Philbert, Martin , Remick, Daniel , Robins, Thomas
Institution: University of Michigan
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1998 through January 1, 2002
Project Period Covered by this Report: January 1, 1998 through January 1, 1999
Project Amount: Refer to main center abstract for funding details.
RFA: Centers for Children's Environmental Health and Disease Prevention Research (1998) RFA Text | Recipients Lists
Research Category: Children's Health , Health Effects , Health
Asthma represents a serious health problem particularly for inner city children. Recent studies have identified that many of the asthmatic attacks are triggered by exposure to cockroaches. It is not exposure to the entire cockroach, but only small fragments or residue. These small fragments are responsible for the allergic response and they are called allergens. In the normal allergic, or asthmatic response, the body reacts to the allergens to start inflammation. This inflammation is initiated by having the cells of the body produce specific chemicals or mediators. With asthma, most of the mediators are produced directly in the lung. While many potential mediators have been examined previously, asthma still exacts a toll on children and adults. More specific, targeted therapy has the potential to improve the treatment of asthma by identifying those mediators directly responsible for the causing disease. This research project will test the hypothesis that asthma-like pulmonary injury is mediated by the local production of specific mediators, which are called chemokines. Chemokines are small molecular weight proteins which induce the movement and recruitment of inflammatory cells. The chemokines are powerful mediators with long lasting and potent biological activities. The first specific aim is to determine the acute and chronic pulmonary inflammation that develops after direct injection of the chemokines into the lung. The assessment of the injury will include a microscopic analysis of the lung as well as an assessment of the nerves within the airways. The second specific aim is to develop a mouse model of asthma-like pulmonary inflammation in response to cockroach allergens. For this specific aim, a model in mice which is similar to humans will be set up to attempt to decipher the specific mediators that cause the lung injury. This model will be established by locating households with high levels of cockroach allergens and using this material to immunize the mice. The mice will be challenged by exposure to aerosols containing the dust with the cockroach allergens and the pulmonary injury carefully quantitated including an analysis of innervation of the airways. The third specific aim is to investigate the signals responsible for inducing the cells to make the chemokines. This will be done in cells sensitized and then challenged with cockroach allergens. This will focus on reactive oxygen and reactive nitrogen intermediates since they have been demonstrated to increase the synthesis of chemokines. The last specific aim is to rigorously test the central hypothesis that chemokines are important in causing asthma. This will be tested by blocking the biological activity of the chemokines with specific therapies to inhibit the chemokines and then determining if there is a reduction in the pulmonary inflammation induced by repeated exposures to the cockroach allergens. Successful completion of this project will both delineate the underlying mechanisms of disease and identify potential novel targets for intervention.
Specific Aims: There have been no changes in the specific aims. Investigations into the immuno-pathologic alterations which occur in asthma have been initiated. Studies began shortly after the initial grant award was given, and started with the first specific aim of the grant.
The first specific aim is to determine if recombinant chemokines placed in the lungs will induce an inflammatory response. To achieve this aim it was necessary to ensure that there would be sufficient amounts of purified recombinant murine chemokines on hand to complete all of the planned studies. Since these chemokines are prepared from bacteria, it was also necessary to confirm that endotoxin was not present in the preparations. The 255 bp insert for the MIP-2 protein had previously been ligated into a PGEX-2T plasmid in DH5. The protein was then produced using IPTG induction followed by purification with GST-sepharose column chromatography. SDS-page gel separation was done to confirm the protein size followed by an ELISA to confirm the protein identity with an anti-MIP-2 antibody.
Similarly KC was produced from HB101 that contained PGEX-2T plasmids with the 280 bp insert for KC. These KC and MIP-2 stocks were adsorbed for endotoxin and tested for residual endotoxin levels. These levels were found to be extremely low, and below the level that would cause any acute inflammation. The acute and chronic pulmonary inflammation characterization experiments were then initiated with the new chemokine stocks.
To begin to test the ability of these recombinant proteins to induce inflammation, they were injected into the lungs. A modification to the original proposal was made to incorporate a new method for administering agents into the lung. For this method, the mice are anesthetized and suspended vertically. The tongue is gently pulled forward from the mouth and then 50 microliters of fluid containing the recombinant chemokine is placed in the posterior portion of the hypopharynx. Traction is kept on the tongue for 30 seconds, which closes the epiglottis and forces all of the fluid to be drawn into the lungs with the next breath.
The first experiments were done by injecting the recombinant chemokines as described above and sacrificing the mice 4 hours later. The lungs were then lavaged and the total number of neutrophils in the lavage fluid was determined. Measurements were taken of the constituents of the peripheral blood and the numbers of neutrophils, lymphocytes and monocytes were quantitated. There were no substantial changes in the peripheral blood, indicating that the recombinant chemokines were exerting an effect solely on the lung. This was the desired outcome, inflammation in the lung with minimal to no systemic toxicity or effect. The utility of silver degeneration stains in skeletal muscles obtained from control mice has been confirmed. However, the staining patterns obtained in trachea from control animals is capricious and unpredictable. Although adequate staining of neuromuscular junctions for cholinesterase activity has been achieved, optimizing the staining protocol for visualization of the terminals innervating tracheal muscle will be continued.
Significance: These data indicate that the recombinant chemokines will induce substantial inflammation and lung injury. The inflammation is indicated by the increase in the number of neutrophils and the albumin concentration demonstrates that lung injury has occurred. Identification of altered neuronal morphology, and possibly hyperinnervation following inflammation, at the level of the neuromuscular junction will provide insights into the role of the peripheral nervous system in the etiology and progression of asthma-like disease.
Investigations will continue as planned in the original proposal. During the next 2 months, household dust will be collected to measure cockroach allergens, and the kinetics studies of the pulmonary inflammatory response to recombinant murine chemokines will be completed. Staining procedures developed to date to investigate the utility of novels stains such a s fluorojade in the visualization of nerve terminals will be optimized.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other subproject views:||All 3 publications||1 publications in selected types||All 1 journal articles|
|Other center views:||All 45 publications||24 publications in selected types||All 22 journal articles|
||Clark NM, Brown RW, Parker E, Robins TG, Remick Jr. DG, Philbert MA, Keeler GJ, Israel BA. Childhood asthma. Environmental Health Perspectives 1999;107(Suppl 3):421-429.||
Supplemental Keywords:asthma, ambient air, indoor air, exposure, health effects, children, stressor, pathogens, community-based, social science, pathology, monitoring, Detroit., RFA, Health, Scientific Discipline, Air, Toxicology, Environmental Chemistry, Health Risk Assessment, Susceptibility/Sensitive Population/Genetic Susceptibility, Allergens/Asthma, Children's Health, genetic susceptability, indoor air, Biology, asthma, asthma triggers, chemokines, health effects, sensitive populations, home, lung, exposure, airway disease, biological response, respiratory problems, children, airway inflammation, Human Health Risk Assessment, inhalation, pulmonary, assessment of exposure, childhood respiratory disease, children's vulnerablity, asthma chemokines, allergic response, indoor air quality, mediators, respiratory, cockroaches, indoor environment, allergen, disease, environmental hazard exposures
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R826710 Michigan Center for the Environment and Children’s Health
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R826710C001 Indoor and Outdoor Air Contaminant Exposures and Asthma Aggravation Among Children (Asthma Exposure)
R826710C002 Chemokines in the Pathogenesis of Asthma (Asthma Chemokines)
R826710C003 A Community-Based Intervention to Reduce Environmental Triggers for Asthma Among Children (Asthma Intervention)