Megan Stogsdill and Dr. Paul Reynolds, Physiology and Developmental Biology

Receptors for advanced glycation end products (RAGE) are receptors that can bind a variety of ligands and are members of the immunoglobin family of cell surface receptors. RAGE is found in many tissue types during inflammatory lesions but is found most abundantly in the lung, where it was subsequently discovered (1). It is known to bind to chemicals found in cigarette smoke (2) and has been implicated as a progression factor in a number of diseases including chronic obstructive pulmonary disease (COPD) (3).

COPD with emphysema is defined as airflow obstruction which may not be reversible with long term damage to the lung structure (4). It is currently the third leading cause of death in the United States (5) and is often associated with prolonged inhalation of harmful chemicals such as those found in cigarette smoke. Characteristics of COPD include an increase in inflammation and an enlargement of the alveolar ducts. Based on previous work done in the Reynolds lab (6), we hypothesized that increasing RAGE in the adult mouse lung would produce a lung that has similar characteristics as a lung suffering from COPD without inhalation of cigarette smoke.

In order to test the hypothesis that increased RAGE is a major contributing factor in the disease progression of COPD, we used a transgenic mouse model. This mouse model was used previously in the embryonic mouse to demonstrate that over expression of RAGE during development results in severe lung hypoplasia and perinatal lethality (6). This model works in conjunction with doxycycline introduced via the diet. A mouse is considered an over-expressor if it contains both transgenes and is fed doxycycline (dox). A control mouse may have one or none of the transgenes and is fed dox.

We began feeding the experimental groups and the control groups dox at postnatal (PN) day 20 at which point the pups are weaned and alveologenesis is complete. Mice were sacrificed at 50, 80, and 110 days of age, representing 30, 60, and 90 days on dox respectively. The lungs were either inflated with paraformaldehyde and removed whole or flushed with saline solution to obtain bronchoalveolar lavage fluid (BALF). We then performed analysis on the samples.

Whole lungs were processed using standard techniques and embedded in paraffin for sectioning and slide preparation. The tissue slides were then stained with hemotoxylin and eosin in order to adequately view the lung structures. In order to assess the morphological changes induced by RAGE over expression we obtained the Mean Linear Intercept (lm) for eight different mice in each time point. We photographed each slide in 10 randomized locations at 25 times magnification and overlaid the image with two intersecting lines of .5 mm each. Each time a line intersected an alveolar wall it was given a score of one. Each time a line intersected a vessel or partially intersected an alveolus it was given a score of .5. The total number of intercepts was divided by the length of the line (.5 mm) and a score was obtained for each slide. Lower scores indicated larger alveolar spaces.

BALF was analyzed to determine cell populations as an indirect measure of vascular permeability and for the presence of various cytokines associated with inflammation which are both important indicators of COPD. The BALF was placed in a centrifuge and the pellet containing the cells was resuspended in saline solution and the supernatant was saved for protein analysis. The total number of cells was counted and recorded. We determined the differential cell count by staining a slide containing cells from the BALF with a wright-giemsa stain and determined the number of polymorphonucleocytes (PMN) as compared to mononuclear cells. We also quantified total protein and performed an ELISA to determine concentrations of proinflammatory proteins.

The results of our experiment demonstrated that over-expressing RAGE in the adult mouse lung produces a lung with characteristics similar to a lung with COPD. Staining of lung tissue demonstrated that a change had occurred in the structure of the lung parenchyma as well as in the concentrations of certain cell populations. Staining with hemotoxylin and eosin enabled us to determine the lm which we found to be significantly different in the over-expressing mice lungs and the wild type lungs. The over-expressing lungs had larger lm numbers indicating enlarged alveolar ducts; similar to what is seen in COPD.

Analysis of the BALF demonstrated an increase in proinflammatory cytokines which recruit cells associated with inflammation. This increase is an indirect indicator of inflammation. There was not a significant difference between the total cell counts of wild type and over-expressing mice, however there was a difference in the ratio of cell types found in the BALF. We observed a shift in which we counted more polymorphonucleocytes (PMN) as opposed mononuclear cells. The increase in the percentage of PMN’s is indicative of the inflammation seen in COPD.

In conclusion, our preliminary findings suggest that these RAGE over-expressing mice are suitable as a smokeless model for COPD. A paper written about the results of our work has been submitted to the American Journal of Physiology and returned with several suggestions which we are currently working on to improve the paper. The Reynolds lab plans to continue the study by determining the mechanism by which increases in RAGE production result in this emphysematous phenotype and how RAGE may play a role in the pathogenesis of other lung diseases.

References

1. Hofmann, MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila C, Kambham N, Bierhaus A, Nawroth P.1999. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97,889-901.
2. Reynolds P.R., Kasteler S.D., Schmitt R.E. and Hoidal J.R. 2010. RAGE Signals Through Ras During Tobacco Smoke-Induced Pulmonary Inflammation. Am J Resp Cell Mol Biol 45:411-418.
3. Wu L., Ma L., Nicholson L.F., and Black P.N. Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD. Respir Med. 2011; 105(3), 329-36.
4. Celli BR and MacNee W. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J. 2004;23(6):932-946.
5. Jemal A, Ward E, Hao Y, Thun M. Trends in the leading causes of death in the United States, 1970-2002. Jama. Sep 14 2005;294(10):1255-1259.
6. Reynolds P.R., Stogsdill J.A., Stogsdill M.A., Heimann N.B. Up-regulation of RAGE by alveolar epithelium influences cytodifferentiation and causes severe lung hypoplasia. Am. J. Respir. Cell. Mol. Biol. 2011; 45:1195-1202.