Acute exacerbations of chronic obstructive pulmonary disease are associated with decreased CD4+ & CD8+ T cells and increased growth & differentiation factor-15 (GDF-15) in peripheral blood

Abstract

Background: Although T cells, especially CD8+, have been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, their role during acute exacerbations (AE-COPD) is uncertain.

Methods: We recruited subjects with COPD and a history of previous AE-COPD and studied them quarterly to collect blood and spontaneously expectorated sputum while stable. During exacerbations (defined by a change in symptoms plus physician diagnosis and altered medications), we collected blood and sputum before administering antibiotics or steroids. We used flow cytometry to identify leukocytes in peripheral blood, plus Luminex® analysis or ELISA to determine levels of inflammatory biomarkers in serum and sputum supernatants.

Results: Of 33 enrolled subjects, 13 participated in multiple stable visits and had ≥1 AE-COPD visit, yielding 18 events with paired data. Flow cytometric analyses of peripheral blood demonstrated decreased CD4+ and CD8+ T cells during AE-COPD (both absolute and as a percentage of all leukocytes) and significantly increased granulocytes, all of which correlated significantly with serum C-reactive protein (CRP) concentrations. No change was observed in other leukocyte populations during AE-COPD, although the percentage of BDCA-1+ dendritic cells expressing the activation markers CD40 and CD86 increased. During AE-COPD, sICAM-1, sVCAM-1, IL-10, IL-15 and GDF-15 increased in serum, while in sputum supernatants, CRP and TIMP-2 increased and TIMP-1 decreased.

Conclusions: The decrease in CD4+ and CD8+ T cells (but not other lymphocyte subsets) in peripheral blood during AE-COPD may indicate T cell extravasation into inflammatory sites or organized lymphoid tissues. GDF-15, a sensitive marker of cardiopulmonary stress that in other settings independently predicts reduced long-term survival, is acutely increased in AE-COPD. These results extend the concept that AE-COPD are systemic inflammatory events to which adaptive immune mechanisms contribute.

Trial registration: NCT00281216 , ClinicalTrials.gov.

Fig. 1

AE-COPD confirmed by increases in BCSS total score and serum CRP levels. AE-COPD events were identified by physician diagnosis (after exclusion of pneumonia by chest radiograph) plus the clinical decision to prescribe oral steroids or antibiotics. Although these decisions were blinded to biomarker results, the diagnosis of AE-COPD was retrospectively confirmed by highly significant increases in (a) BCSS total scores and (b) serum CRP levels. Open circles represent individual subjects; mean ± SEM of the grouped data are shown by the single red circles. The Wilcoxon matched-pairs signed rank test was used to determine significant differences between visits

Fig. 2

Peripheral blood CD4+ and CD8+ T cells decreased during AE-COPD while CD15+ granulocytes increased. Peripheral blood was collected during stable visits and during AE-COPD, prior to medication (n = 18 paired samples). Cells were stained and analyzed by flow cytometry. Results are shown for the individual cell type as a percentage of all CD45+ leukocytes (a, c, e) and as absolute cell numbers per μL (b, d, f). A, B. CD4+ T cells. C, D. CD8+ T cells. E, F. CD15+ cells; note difference in scale of panels E & F. Open circles represent individual subjects; mean ± SEM of the grouped data are shown by the single red circles. The Wilcoxon matched-pairs signed rank test was used to determine significant differences between visits

Fig. 3

Representative graphs from individual subjects tracking percentages of CD4+ and CD8+ T cells during stable and exacerbation visits. Peripheral blood was collected during stable visits and during AE-COPD, prior to medication. Cells were stained and analyzed by flow cytometry. Panels a-e each depicted the data for a single subject; CD4+ T cells (upper panels, blue) and CD8+ T cells (lower panels, red) shown as a percentage of all CD45+ leukocytes. a, b. Two subjects with multiple AE-COPD; c, d. two subjects with a single AE-COPD; and e, subject with no AE-COPD. Stable visits are shown by open circles and AE-COPD are shown in solid circles labeled with “Ex”

Fig. 4

CD40 and CD86 increased on BDCA-1+ DCs from peripheral blood during AE-COPD. Peripheral blood was collected during stable visits and during AE-COPD, prior to medication (n = 10 paired samples). Cells were stained and analyzed by flow cytometry. After gating on BDCA-1+ DCs, the percent of cells expressing (a) CD40 and (b) CD86 was determined. Open circles represent individual subjects; mean ± SEM of the grouped data are shown by the single red circles. The Wilcoxon matched-pairs signed rank test was used to determine significant differences between visits

Fig. 5

Soluble adhesion molecules and inflammatory mediators increased while CCL11 decreased in the serum during AE-COPD. Serum was collected during stable visits and during AE-COPD, prior to medication (n = 18 paired samples). Samples were stored at −80 °C prior to analysis by Luminex® assay, except as noted. (a) sICAM-1, (b) sVCAM-1, (c) IL-10, (d) IL-15, (e) GDF-15 (by ELISA), (f) CCL11 are shown. Open circles represent individual subjects; mean ± SEM of the grouped data are shown by the single red circles. Note differences in units of measurement. The dotted line represents the minimal detectable concentration. The Wilcoxon matched-pairs signed rank test was used to determine significant differences between visits

Fig. 6

CRP and TIMP-2 increased in sputum during AE-COPD, whereas TIMP-1 decreased. Sputum was collected during stable visits and during AE-COPD, prior to medication. After processing, sputum supernatants were stored at −80 °C prior to analysis by Luminex. (a) CRP, (b) TIMP-1, (c) TIMP-2 are shown (n = 10 paired samples for CRP and n = 7 paired samples for TIMP-1 and TIMP-2). Open circles represent individual subjects; mean ± SEM of the grouped data are shown by the single red circles. The dotted line represents the minimal detectable concentration. The Wilcoxon matched-pairs signed rank test was used to determine significant differences between visits