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Effect of tobacco smoke on leukocyte adhesion in vivo

Institution: La Jolla Institute for Molecular Medicine
Investigator(s): Pragada Sriramarao, Ph.D.
Award Cycle: 1998 (Cycle 7) Grant #: 7RT-0197 Award: $754,361
Subject Area: Pulmonary Disease
Award Type: Research Project Awards
Abstracts

Initial Award Abstract
Over 1 million individuals between the ages of 45 and 60 are currently on Social Security rolls as being partially or wholly disabled on the basis of chronic obstructive pulmonary disease (COPD) The most important etiological factor in COPD is airway inflammation that is particularly associated with exposure to tobacco smoke (TS) and the recruitment of leukocytes into the airways of the lungs. This relationship appears to strengthen with the number of cigarettes smoked and the amount of smoke inhaled (i.e., both mainstream smoking and exposure to secondhand smoke). At the cellular level, pulmonary inflammation is characterized by the inducement of circulating leukocytes (e.g., certain types of white blood cells) to migrate through the cell layer lining blood vessels in the lung (pulmonary endothelium) into the “extravascular” spaces of the lung. The molecular and cellular mechanisms mediating the interaction of circulating leukocytes with pulmonary endothelial cells in smokers are not well understood. It has been suggested that exposure to TS could result in activation of circulating leukocytes and result in their recruitment in the airways. Thus we hypothesize that the inhibition of the inflammatory recruitment process (and, in turn, the prevention of the migration of activated leukocytes into the airways of humans exposed to mainstream or secondhand TS) will result in attenuation of smoking-mediated tissue damage caused by pulmonary inflammation. By expressing chemical mediators and specific molecules necessary for adhesion, endothelial cells play a central role in the various steps involved in leukocyte recruitment. These steps include leukocyte “rolling”, adhesion to the endothelium, and transmigration to sites of inflammation in the airways. These critical steps of the recruitment process are mediated by distinct “adhesion receptors”. Although several of the receptors involved in mediating leukocyte adhesion under normal physiological conditions have been identified, how this process may be altered by exposure to TS (in mainstream and second hand smokers) has not been investigated.

This project will apply the current knowledge of immunology, vascular and cell biology in understanding the cellular mechanisms that mediate leukocyte adhesion to pulmonary blood vessels. Leukocytes isolated from the peripheral blood of smokers (suffering from chronic bronchitis or asthmatic bronchitis) or non-smokers will be fluorescently labeled and the subsequent recruitment process will be closely observed using an animal model. Using state of the art microscopic techniques, it will be possible to visualize the interaction of circulating leukocytes (and any detrimental effects of TS) within pulmonary vessels. From the results obtained from these studies we hope to identify the specific receptors and adhesion molecules that contribute to leukocyte recruitment during episodes of chronic bronchitis or asthma that afflict smokers (and some individuals exposed to secondhand smoke). The knowledge derived from these studies will help in developing therapeutic strategies to mitigate TS-related pulmonary disease in humans.

Final Report
Airway inflammation is important etiological factor for tobacco smoke (TS) induced chronic obstructive pulmonary disease. At a cellular level, lung inflammation is characterized by the recruitment of circulating leukocytes into extravascular spaces of the lung. It has been suggested that exposure to TS and its components including nicotine could result in activation of circulating neutrophils or eosinophils and result in their recruitment in the airways. The overall objective of this investigation is to understand mechanisms by which eosinophils and neutrophils are activated within blood vessels due to exposure to constituents of TS and recruited into the airways of smoking individuals. We have specifically investigated how specific adhesion molecules are up regulated in response to exposure to nicotine and how circulating leukocytes roll, adhere and transmigrate across nicotine treated blood vessels. The specific aims of this project are: (1) To examine if neutrophils and eosinophils isolated from smokers preferentially roll, adhere and transmigrate in response to activation with specific chemokines IL-8 and eotaxin, and if constituents of CSC induce activation of circulating leukocytes under conditions of flow in vivo; and (2) To examine the effect of CSC on expression of P-Selectin, E-selectin, VCAM-1 and ICAM-1 by pulmonary microvessels and their role in promoting rolling, sticking and transendothelial migration of murine neutrophils and eosinophils in pulmonary microvessels in a murine model of pulmonary inflammation.

Employing techniques of intravital video microscopy (IVM) and a mouse model of lung transplantation, we have been able to visualize how circulating leukocytes traffic through lung microvessels and respond to stimuli such as nicotine. Our data indicates that nicotine plays a pro-inflammatory role in causing increased leukocyte and eosinophil rolling, adhesion and retention in lung microvessels. This increased adhesion/binding of leukocytes in the blood vessels of the lung can be significantly ameliorated by treatment with monoclonal antibodies against leukocyte expressed L-selectin and vascular E-selectin and P-selectin (that are up-regulated by lung microvessels in response to nicotine). Likewise, our studies have demonstrated that pretreatment of animals with anti-E+ anti-P-selectin mAbs prevents nicotine induced leukocyte interactions in lung microvessels. Finally, we have also demonstrated that exposure to nicotine or TS, results in the inhibition of bone marrow hematopoiesis in vivo and vitro. Overall, the TRDRP funds have facilitated in developing an in vivo model system to visualize the study the dynamics of leukocyte interactions within blood vessels of the lung tissue exposed to nicotine. The studies have identified the function of adhesion molecules, L-, P- and E-selectins that appear to participate in nicotine-mediated leukocyte trafficking and retention in the lungs. The results obtained from these studies have formed the basis of new studies that will be conducted as part of the new continuation TRDRP grant 10RT-0171 awarded to the PI. It is anticipated that results obtained from the current as well as continuation award will facilitate in developing novel therapeutic strategies to prevent cigarette smoke-induced pulmonary disease in humans.
Publications

Inhibition of eosinophilic inflammation in allergen challenged IL-1-receptor type 1 deficient mice are associated with reduced eosinophil rolling and adhesion on vascular endothelium
Periodical: Blood Index Medicus:
Authors: Broide DH, Campbell K, Gifford T, Sriramarao P ART
Yr: 2000 Vol: 95 Nbr: Abs: Pg: 263-269

Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells
Periodical: Blood Index Medicus:
Authors: Khaldoyanidi S, Orlovskaya I, Matrosova V, Koslov V, Sriramarao P ART
Yr: 2001 Vol: 98 Nbr: 2 Abs: Pg: 302-312