THE ROLE OF α4β1 INTEGRIN (VLA-4) IN RECRUITMENT OF MYCOBACTERIUM TUBERCULOSIS-SPECIFIC TH1-LIKE RECALL RESPONSES TO THE HUMAN LUNG by JESSICA R. WALRATH Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Richard Silver Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of ______________________________________________________ candidate for the Master of Science Degree*. 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TABLE OF CONTENTS TABLE OF CONTENTS…………………………………….………………………….ii LIST OF FIGURES………………………………………………..……………………iii LIST OF ABBREVIATIONS…………………………………..………………………iv ABSTRACT………………………………………………………...…………………….v INTRODUCTION……………………………………………………………………….1 Overview……………………………………………………………………………...1 Pathogenesis of infection with M. tuberculosis……………….……………………...2 Lymphocyte homing and localization of protective immune responses……………..4 Dendritic cells in the development of tissue-specific homing……………………......6 Limitations of BCG vaccination……………………………………………………...7 Assessment of local immunity in humans following respiratory exposure to M. tuberculosis……………………………………………………………………….8 MATERIALS AND METHODS………………………………………………………11 RESULTS……………………………………………………………………………….21 Bronchoscopic challenge with PPD results in development of localized lymphocytic inflammation in challenged BAL segments…………………………..21 Skewing of homing molecule expression on CD4+ T-cells in baseline BAL………22 Role of homing molecule expression in recruitment of antigen-specific Th1 cells to the lung in response to bronchoscopic challenge with PPD…………...24 Assessment of the role of lymphocyte proliferation in increasing BAL CD4+ T-cells in response to PPD challenge…………………………………….…..26 DISCUSSION………………………………………………………………………...…28 FIGURES…………………………………………………………………………..……36 REFERENCES……………………………………………………………………….…43 LIST OF FIGURES Figure 1 Technique of bronchoscopic segmental antigen challenge………………..36 Figure 2 Lymphocyte subset recruitment in response to PPD bronchoscopic challenge of PPD+ subjects…………………………….....37 Figure 3 Analysis of homing molecule expression on CD4+ T-cells in baseline BAL, PBMC, and 48hrs post challenge BAL of a PPD+ subject………....38 Figure 4 A Percent expression of homing molecules on CD4+ T-cells in baseline BAL and PBMC of PPD+ subjects……………………………39 Figure 4B Percent expression of homing molecules on CD4+ T-cells in baseline BAL and PBMC of PPD- subjects.……………………………39 Figure 5A Percent expression of homing molecules on CD4+ T-cells in baseline BAL and 48hrs post challenge BAL of PPD+ subjects……….40 Figure 5B Total number of CD4+ T-cell expression of homing molecules in baseline BAL and 48hrs post challenge BAL of PPD+ subjects…...…..40 Figure 6A Percent expression of homing molecules on PPD-specific Th1-like CD4+ T-cells in baseline BAL and 48hrs post challenge BAL of PPD+ subjects…………………………………….41 Figure 6B Total number of PPD-specific Th1-like CD4+ T-cell expression of homing molecules in baseline BAL and 48hrs post challenge BAL of PPD+ subjects…….........................................................................41 Figure 7 In vitro and in vivo Ki67 assay of baseline BAL, PBMC and 48hrs post challenge BAL of PPD+ subjects……………………...….42 ii LIST OF ABBREVIATIONS γδ gamma delta α4β1 alpha 4 beta 1 α4β7 alpha 4 beta 7 APC allophycocyanin AS autologous serum BAL bronchoalveolar lavage BCG Mycobacterium bovis strain Bacillus of Calmette and Guerin BrdU bromodeoxyuridine CCR chemokines (C-C motif) receptor CLA cutaneous lymphocyte antigen CXCR chemokines (C-X motif) receptor DC dendritic cell DTH delayed-type hypersensitivity EDTA ethylenediaminetetraacetic acid FITC fluorescein isothicoyanate HIV human immunodeficiency virus IFNγ interferon gamma IL interleukin IMDM Iscove’s Modified Dulbecco’s Medium IP-10 interferon gamma-inducible protein 10 MAdCAM-1 mucosal addressin cell adhesion molecule 1 Mig monokine induced by interferon gamma MIP-1α macrophage inflammatory protein-1-alpha NK natural killer NS normal saline PBMC peripheral blood mononuclear cells PBS phosphate buffer saline PE phycoerythrin PerCP peridinin chlorophyll protein PFA paraformaldyde PPD purified protein derivative of Mycobacterium tuberculosis RANTES regulated-upon activation, normal T-cell expressed and secreted RML right middle lobe SEB staphylococcal enterotoxin B Th1 T-helper (aka CD4+ T-cell), type 1, IFNγ and IL-2 producing Th2 T-helper (aka CD4+ T-cell), type 2, IL-4 producing TU tuberculin units VCAM-1 vascular cell adhesion molecule 1 VLA-4 very late antigen 4 (also known as α4β1) iii The Role of α4β1 Integrin (VLA-4) in Recruitment of Mycobacterium tuberculosis- Specific Th1-like Recall Responses to the Human Lung Abstract by JESSICA R. WALRATH Tuberculosis remains a major international health threat. Although BCG vaccination does not clearly protect against pulmonary tuberculosis, Mycobacterium tuberculosis- exposed individuals are relatively protected against subsequent re-infection. We have utilized bronchoscopic antigen challenge with PPD to model immune responses of infected individuals following re-exposure. Currently, we assessed the role of homing molecules in recruitment of M. tuberculosis-specific recall responses to the human lung. Intracellular staining for IFNγ was performed, as was surface staining for α4β1 and α4β7 integrins and CLA. Baseline bronchoalveolar lavage (BAL) is enriched for α4β1- expressing CD4+ T-cells. This skewing continues following PPD-induced lymphocyte recruitment, and is even more pronounced for M. tuberculosis-specific CD4+ T-cells. Expression of α4β1 integrin therefore appears to mediate optimal localization of recall responses to the lung. Comparison with vaccinated subjects may clarify whether intradermal BCG is as effective as natural infection in inducing recall responses that can be localized to the lung. iv INTRODUCTION Overview: Mycobacterium tuberculosis currently infects over 1/3 of the world’s population. There are 8.4 million new cases of tuberculosis annually, and it is estimated that 1.5-2 million people die of tuberculosis each year [1]. Multiple factors contribute to the continued importance of tuberculosis as an international threat to public health. Although tuberculosis is largely a preventable and treatable disease, effective antibiotic therapy requires months of daily treatment. Delivering this therapy is difficult in underdeveloped countries where government infrastructure is often weak and tuberculosis control programs are poorly funded. Not surprisingly, these countries account for 95% of the tuberculosis cases and tuberculosis-related deaths worldwide [1]. The onset of the HIV epidemic has been associated with increased incidence of tuberculosis due both to the marked susceptibility of HIV-infected individuals to development of active disease, and to the prominence of HIV infection in the same underdeveloped regions where M. tuberculosis infection is endemic [2, 3]. In addition, the development of multi-drug resistant and extensively drug resistant isolates of M. tuberculosis raise further concern for the prospects of the return to an era in which tuberculosis was not a treatable disease [1, 4]. All of these circumstances point to the potential importance of effective vaccination in the control of tuberculosis. The current tuberculosis vaccine, the Bacillus of Calmette 1 and Guerin (BCG), is an empirically attenuated strain of Mycobacterium bovis developed in the 1920’s and used extensively since that time. In most of the world, BCG is given to newborns via intradermal injection. BCG vaccination is known to be effective at preventing disseminated tuberculosis in very young children, but does not clearly prevent the development of pulmonary tuberculosis in adults [5]. Because pulmonary tuberculosis is the contagious form of the disease, however, it represents the manifestation of M. tuberculosis infection for which prevention is most needed in order to improve world-wide control of the disease [6]. Pathogenesis of infection with Mycobacterium tuberculosis: The normal course of pulmonary infection with M. tuberculosis may provide insight into how to improve upon the protection induced by BCG. Mycobacterium tuberculosis is transmitted from patients with pulmonary tuberculosis via aerosolized “droplet nuclei” generated by coughing. Individuals who are in close proximity to a contagious patient then inhale these droplets. Once inhaled, the bacilli travel to the alveoli of the lungs whether they are phagocytosed by alveolar macrophages [7]. Because M. tuberculosis bacilli are uniquely well-suited to survival within alveolar macrophages, these cells cannot themselves control the intracellular pathogen. Instead, intracellular replication continues until infected macrophages die, releasing viable bacilli. Containment of infection then requires the development of an antigen-specific lymphocyte response to the organism. This is initiated by the action of specialized tissue dendritic cells (DC) that travel to the alveoli, phagocytose