Chemokine Receptor Expression on Peripheral Lung Lymphocytes

<div><p>(A) Single color histograms showing expression of chemokine receptors CCR4, CCR5, and CXCR3 from representative control and emphysema participants.</p> <p>(B) Pooled data from all participants (control, <i>n =</i> 10; emphysema, <i>n =</i> 18) showing percent (median ± SD) of total lung lymphocytes expressing CCR4 and CCR5.</p> <p>(C) Pooled data from same participants showing percent (median ± SD) CCR5 expression on CD4 (top) and CD8 (middle) T cells, and CXCR3 expression on unfractionated T cells (bottom) from the same participant groups.</p> <p>(D) Analysis of total lung lymphocyte chemokine receptor (median ± SD) profiles among participants with emphysema. Participants had either (1) lung volume reduction surgery for emphysema (non-cancer, <i>n =</i> 8) or (2) lung resection for treatment of small peripheral cancer (<i>n =</i> 10). Participants showed similar inflammatory indices as determined by CCR5 expression.</p> <p>In (B) and (C), *, <i>p</i> < 0.001; †, <i>p =</i> 0.01; ‡, <i>p =</i> 0.02; ∫, <i>p =</i> 0.007 (Mann-Whitney test) for the comparison of emphysema and control groups.</p></div

IFN-γ, MIG, and IP-10 Production by Isolated Lung Lymphocytes

<div><p>(A–C) Lung lymphocytes from control individuals and participants with emphysema were cultured without additional stimulation for 3 or 4 d and assessed for secretion of (A) IFN-γ, (B) MIG, and (C) IP-10 (control, <i>n =</i> 8; emphysema, <i>n =</i> 12). Columns are median, bars represent SD. *, <i>p=</i> 0.007; †, <i>p =</i> 0.01; ‡, <i>p =</i> 0.02 for the comparison of emphysema and control participants.</p> <p>(D) The same cells from a representative ex-smoker individual with emphysema were either left unstimulated (No ST) or treated with PMA/ionomycin (PMA/I) for 24 h and assessed for surface CD8 and CD69 expression and the intracytoplasmic accumulation of IFN-γ by flow cytometry.</p> <p>(E) Production of IL-4 by lung lymphocytes. Lung lymphocytes from a representative ex-smoker individual with emphysema were cultured for 24 h with or without PMA/ionomycin stimulation (PMA/I) and assessed for intracytoplasmic IL-4 and IFN-γ accumulation by flow cytometry.</p></div

Expression of CXCR3 in Lungs of Control and Emphysematous Smoker Individuals

<div><p>(A) Representative forward and side-scatter characteristics of whole lung cells from a participant with COPD and emphysema. Anti-CD11b PE-conjugated and anti-CD14 FITC-conjugated antibodies detect lung macrophages (middle), and histogram of mean fluorescence intensity showing anti-CXCR3-Cy5 and control antibodies (cIg) detects lung macrophages in the patient with emphysema.</p> <p>(B) Pooled data from control individuals without (<i>n =</i> 5) and with (<i>n =</i> 8) emphysema. Columns are median, bars represent SD. *, <i>p =</i> 0.009 (Mann-Whitney test) for the comparison of emphysema and control participants.</p> <p>(C) Negative association between CXCR3 expression on CD3⁺ T cells and FEV1 percentage predicted based on an <i>R²</i> goodness-of-fit statistic of 0.27 (<i>p =</i> 0.0089, <i>r</i> = −0.52, <i>n =</i> 24).</p></div

Regulation of MMP12 by Type 1 Cytokines

<div><p>(A) CD14⁺, lymphocyte-depleted lung leukocytes were cultured with and without the indicated amounts of recombinant human IP-10 and IFN-γ, and supernatants were assessed for the presence of MMP12 by Western blotting.</p> <p>(B) Fold increase relative to unstimulated of MMP12 mRNA from lung macrophages stimulated without (<b>–</b>) and with (<b>+</b>) 500 ng/ml of IP-10 in the presence or absence of a function-blocking antibody to CXCR3 as determined by real-time PCR.</p> <p>(C and D) Lung tissue from a participant with emphysema (C) shows strong immune staining for MMP12 localized to macrophages (arrows), and (D) shows lung tissue from a control participant without emphysema and with undetectable MMP12. The insets show a high-power view of lung macrophages staining positive (C) and negative (D) for MMP12 (×60) *, <i>p =</i> 0.04.</p></div