Chronic Obstructive Pulmonary Disease Clinical Trial
Official title:
Granzymes and Perforin at the Onset of COPD Exacerbations
COPD exacerbations are characterized by an excessive accumulation and activation of
inflammatory cells in the airways. It is not known whether this phenomenon represents a risk
for for lung damage via the release in the extracellular environment of potent cytolitic
cellular granular contents such as granzymes and perforin.
The investigators assess the intracellular expression of granzymes and perforin in
neutrophils and large granular lymphocytes (LGL) at the onset of exacerbations compared to
stable disease.
The investigators hypothesize that a greater release of intracellular perforin and granzymes
from neutrophils and LGL into the extracellular environment occur at exacerbations compared
to stable condition and that these changes are more pronounced in COPD patients than in
subjects without COPD who undergo respiratory infection.
The course of Chronic Obstructive Pulmonary Disease (COPD) is characterized by exacerbations
which lead to significant clinical deterioration and morbidity.COPD exacerbations are
characterized by an augmentation of the inflammation that is observed in the airways in
stable condition, particularly by excessive accumulation of polymorphonuclear neutrophils
and activation of lymphocytes.
It has been suggested that this abnormal increase of inflammatory cells in the airways of
COPD patients during exacerbations, might represent a significant risk for excessive
proteolytic activity in the airways via the release of their granule contents into the
extracellular environment. Large granular lymphocytes (LGL) which include NK and cytotoxic
T- cells and neutrophils contain cytolytic molecules such as, granzymes and perforin.
Therefore, the inflammatory process during exacerbations might be potentially toxic to the
lung. Notably, it was showed that sputum T-lymphocytes (CD8+) from patients with stable COPD
express more perforin and are more cytotoxic compared to normal subjects.
However, few previous investigations questioned the role of these cytolytic molecules in the
pathophysiology of COPD. Moreover, despite the considerable progress that has been made in
understanding the underlying mechanisms in COPD, there have been no studies investigating
the expression of perforin or granzymes during COPD exacerbations.
In the present prospective investigation we assess the intracellular expression of granzymes
and perforin in neutrophils and LGL cells at the onset of sputum of COPD patients by using
flow cytometry.
Subjects and protocol:
COPD subjects will be examined at the onset of an exacerbation and in stable condition. The
definition of an exacerbation will be based on the criteria described by Anthonisen,
requiring either the presence of at least two major symptoms (increase in dyspnea, sputum
production, purulence) or of one major symptom in addition to at least one minor (wheeze,
cough, nasal discharge, sore throat, fever), for two consecutive days.
In addition we will assess the expression of granzymes and perforin in neutrophils and LGL
cells of subjects without COPD(controls) who will be recruited by consecutive sampling among
those subjects who seek medical assistance at the clinic during the study period and they
satisfy all the following criteria: i) absence of COPD, Asthma, pneumonia, evidence of lung
cancer, bronchiectasis, interstitial lung disease or other respiratory disease ii)
initiation of symptoms diagnostic for respiratory infection in the past 72 hours, iii)
abstention from any new therapeutic intervention, and iv) absence of any signs suggestive of
condition requiring hospitalization. Control subjects will be examined at the onset of
respiratory infections and at stable condition. The definition of a respiratory infection
require the presence of one of the following symptoms such as dyspnea, sputum production,
purulence, cough, ± fever, for two consecutive days. Stable condition for controls will be
defined as absence of respiratory infection/exacerbation based on symptoms.
The study is approved by the Research Ethics Committee of the Hospital . Sputum induction
and processing Sputum will be induced at exacerbation or respiratory infection and in stable
condition (at least 8 weeks after exacerbation) of the same patient and control subject
respectively; samples will be processed for analysis of inflammatory cells and cytokines and
microbiology. Sputum will be induced via inhalation of a hypertonic saline solution aerosol
(Ultraneb 2000; DeVilbiss; Somerset, PA).
Antibody labeling:
The following mouse anti-human monoclonal antibodies will be used for labeling sputum cells:
phycoerythrin-conjugated antiCD3 (PE-CD3), phycoerythrin-5-conjugated (antiCD4-PCy5),
fluorescein isothiocyanate-conjugated anti-perforin-antibody, (antiperforin-FITC) and
fluorescein isothiocyanate-conjugate will be also used for competitive detection of
intracellular perforin and granzymes. The "a intraprep kit" (Beckman-Coulter Inc Fullerton
Ca, USA). Isotyping antibodies mouse antimouse IgG will be used as controls (Immunotech;
Marseille, France). For labeling cell surface antigens 1x106 cells in suspension will be
incubated with normal bovine serum to block the unspecific binding (blocking reagent), and
then the monoclonal antibody/ antibodies will be added in excess (according to
manufacturer's instruction) for 45 min at 10°C in the dark and washed three times with PBS
plus 10% FCS. For intracellular perforin and granzymes staining, cells will be subsequently
incubated in a permeabilization reagent (Intraprep; Beckman Coulter; Fullerton, CA) for 10
min. As a control, unlabeled antiperforin (or antigranzyme) at molar excess will be used
after permeabilization and then antiperforin (or antigranzyme) FITC will be added without
washing and incubated for 30 min at room temperature (competitive labeling). After staining,
the samples will be washed with PBS plus 10% FCS and will be immediately analyzed using flow
cytometry.
Flow Cytometric analysis:
The sputum samples prepared as described above will be analyzed on a fluorescence activated
cytometer (EPICS ELITE; Coultronics; Louton, UK). Cells will be tightly gated by volume and
complexity on a forward (0o) and side-light scattering (90o) mode. At least 105 cells were
analyzed in each session. PCy-5-conjugated anti-human CD45 monoclonal antibodies (Dako; Ely,
UK) will be used as pan-leukocyte stain to exclude non leukocyte events by logical gating.
The percentage of one-color, two-color, and three-color positive cells will be measured and
the mean channel value as well as the relative fluorescence intensity (RFI) corresponding to
the antigen density will be estimated. The QC-Combo Kit (FCSC; San Jun, Puerto Rico) will be
used for quantification of antibody binding and day-to-day instrument calibration
(amplification and compensation settings of the flow cytometer) will be routinely carried
out. Gating will be performed according to forward and side scattering and CD45 expression.
Positive cells within the neutrophilic and lymphocytic population will be measured as
percentages of each cell population.
Following the above methodology we will asses the expression of perforin in neutrophils and
LGL cells and the expression of granzymes in neutrophils and LGL cells both in exacerbation
and in stable COPD.
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Observational Model: Case Control, Time Perspective: Prospective
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