Granzymes and Perforin at the Onset of Chronic Obstructive Pulmonary Disease (COPD) Exacerbations
Condition: Chronic Obstructive Pulmonary Disease
MeSH terms: Lung Diseases; Lung Diseases, Obstructive; Pulmonary Disease, Chronic Obstructive
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        COPD patients

        Subjects are recruited from a community based outpatient primary medical clinic by
        consecutive sampling.

        Control subjects

        Subjects who seek medical assistance for symptoms suggesting acute bronchitis (dyspnea,
        sputum production, purulence, wheeze, cough) are recruited from a community based
        outpatient primary medical clinic by consecutive sampling.
      
Biospecimen description: 
      sputum cells and supernatant
    
Keywords: Chronic Obstructive Pulmonary Disease; exacerbations; perforin; granzymes