CC chemokine receptor 5 and CXC chemokine receptor 6 expression by lung CD8+ cells correlates with chronic obstructive pulmonary disease severity

Abstract

Chronic obstructive pulmonary disease (COPD) is a progressive disease associated with a cellular inflammatory response. CD8(+) T cells are implicated in COPD pathogenesis, and their numbers significantly correlate with the degree of airflow limitation. Dendritic cells (DCs) are important sentinel immune cells, but little is known about their role in initiating and maintaining the CD8 T-cell response in COPD. To investigate the mechanisms for CD8(+) T-cell recruitment to the lung, we used resected human lung tissue to analyze chemokine receptor expression by CD8(+) T cells and chemokine production by CD1a(+) DCs. Among 11 surveyed chemokine receptors, only CC chemokine receptor (CCR5), CXC chemokine receptor (CXCR) 3, and CXCR6 correlated with COPD severity as defined by criteria from the Global Initiative for Chronic Obstructive Lung Disease. The CD8(+) T cells displayed a Tc1, CD45RA(+) effector memory phenotype. CD1a(+) DCs produced the respective ligands for CCR5 and CXCR3, CCL3 and CXCL9, and levels correlated with disease severity. CD1a(+) DCs also constitutively expressed the CXCR6 ligand, CXCL16. In conclusion, we have identified major chemokine elements that potentially mediate CD8(+) T-cell infiltration during COPD progression and demonstrated that CD1a(+) mucosal-associated DCs may sustain CD8(+) T-cell recruitment/retention. Chemokine targeting may prove to be a viable treatment approach.

Figure 1

Immunohistochemical localization of CD1a⁺ (left) and CD8⁺ (right) cells in the lungs of smokers with GOLD stage 0 (A and B), stage 1 (C and D), stage 2 (E and F), and stage 3 (G and H) COPD. Stain is immunoperoxidase based with diaminobenzidine as the chromagen substrate. Arrows point to CD1a⁺ cells. Magnification: ×200 (A, B, D, F, and H); ×400 (C, E, and G).

Figure 2

Lung CD8⁺ T cells from COPD patients express a Tc1 effector memory phenotype. CD8⁺ T cells isolated from human lung samples by immunomagnetic beads were used for RNA analysis. CD8⁺ T cells expressed transcripts for IFNγ (A). Transcripts are expressed as arbitrary units and were measured by quantitative real-time RT-PCR. Surface expression of CD45RA was determined by flow cytometry (B) and is shown as the percentage of CD8⁺ cells among total leukocytes. Correlation statistical analysis is shown.

Figure 3

Lung CD1a⁺ DCs do not express CD83. Surface expression of CD83 was determined by flow cytometry and is expressed as the percentage of CD1a⁺ cells among total leukocytes. Correlation statistical analysis is shown.

Figure 4

Chemokine receptors CCR5, CXCR3, and CXCR6 are expressed by lung CD8⁺ T cells and correlate with COPD severity. Chemokine receptor expression was profiled in dispersed lungs by flow cytometry (A, B, and C) and in preparations of enriched CD8⁺ T cells by real-time RT-PCR (D, E, and F). CCR5 (A and D), CXCR3 (B and E), and CXCR6 (C and F) expression was correlated to COPD severity, as determined by GOLD stage. Correlation analysis is shown.

Figure 5

Flow cytometric histograms of CCR5, CXCR3, and CXCR6 in GOLD stage 0 and GOLD stage 4 patients. Representative histograms show expression by gated CD8⁺ cells. The white profiles show receptor-specific antibody staining, and the shaded profiles show staining with isotype-matched control antibodies.

Figure 6

Chemokine expression in lungs of COPD patients. Real-time PCR was used to measure chemokine transcripts for CCL3 (A), CXCL9 (B), and CXCL16 (C) among mRNA isolated from whole lung samples. Results are expressed in arbitrary units. CCL3 (D) and CXCL9 (E) protein levels were measured using Luminex xMAP microsphere technology. Results are expressed as picograms of chemokine per milligram of total protein. Levels were correlated to COPD severity, as determined by GOLD stage. Correlation analysis is shown.

Figure 7

CCL3, CXCL9, and CXCL16 transcript expression by purified CD1a⁺ DCs. CD1a⁺ DCs were isolated from human lungs by immunomagnetic beads and used for RNA analysis. Chemokine transcripts for CCL3 (A), CXCL9 (B), and CXCL16 (C) are shown. Transcripts were measured by real-time PCR, and results are expressed as arbitrary units. Levels were correlated to COPD severity, as determined by GOLD stage. Correlation statistical analysis is shown.