Impact of tobacco smoke on interleukin-16 protein in human airways, lymphoid tissue and T lymphocytes

Abstract

CD4(+) and CD8(+) lymphocytes are mobilized in severe chronic obstructive pulmonary disease (COPD) and the CD8(+) cytokine interleukin (IL)-16 is believed to be important in regulating the recruitment and activity of CD4(+) lymphocytes. In the current study, we examined whether tobacco smoke exerts an impact not only on IL-16 in the lower airways but also in CD4(+) or CD8(+) lymphocytes or in lymphoid tissue. The concentration of IL-16 protein was measured by enzyme-linked immunosorbent assay (ELISA) in concentrated bronchoalveolar lavage fluid (BALF) collected from 33 smokers with chronic bronchitis (CB), eight asymptomatic smokers (AS) and seven healthy never-smokers (NS). The concentrations of IL-16 and soluble IL-2 receptor alpha (sIL-2Ralpha) protein were also measured in conditioned medium from human blood CD4(+) and CD8(+) lymphocytes stimulated with tobacco smoke extract (TSE) in vitro. IL-16 mRNA was assessed in vitro as well, using reverse transcription-polymerase chain reaction (RT-PCR). Finally, the intracellular immunoreactivity for IL-16 protein (IL-16IR) was assessed in six matched pairs of palatine tonsils from smokers and non-smokers. BALF IL-16 was higher in CB and AS than in NS. TSE substantially increased the concentration of IL-16 but not sIL-2Ralpha in conditioned medium from CD4(+) and CD8(+) lymphocytes. There was no corresponding effect on IL-16 mRNA. IL-16IR in tonsils was lower in smokers than in non-smokers. The current findings demonstrate that tobacco smoke exerts a wide impact on the CD8(+) cytokine IL-16, in the airway lumen, in blood CD4(+) and CD8(+) lymphocytes and in lymphoid tissue. The effect on IL-16 release may be selective for preformed IL-16 in CD4(+) lymphocytes. New clinical studies are required to evaluate whether tobacco smoke mobilizes T lymphocytes via IL-16 in the lower airways and whether this mechanism can be targeted in COPD.

Fig. 1

Concentration of IL-16 protein in bronchoalveolar lavage fluid from never-smokers (NS), from asymptomatic smokers (AS) and from smokers with chronic bronchitis (CB). For CB alone, filled triangles represent patients with an FEV₁ <80% predicted and open squares represent FEV₁ >80% predicted. Data are shown as individual plus median values (bold lines). *P < 0·05 (Kruskal–Wallis test followed by Mann–Whitney U-test).

Fig. 2

Concentration of IL-16 protein in conditioned medium from either isolated human CD4⁺ lymphocytes (Fig. 2a) or human CD8⁺ lymphocytes (Fig. 2b) cultured in vitro, with TSE (tobacco smoke extract) or without TSE (negative control) stimulation or CI plus PMA (positive control). Filled symbols represent data from co-cultures with adherent mononuclear cells (AMC). Data are shown as individual values plus median values (bold lines) *P < 0·05(Wilcoxon's signed-rank test).

Fig. 3

Expression of mRNA for IL-16 normalized to the housekeeping gene (HPRT) in isolated CD4⁺ lymphocytes cultured in vitro, with tobacco smoke extract (TSE, solid line) or without (dashed line). Data represents three different time-points (5, 10 and 20 h).

Fig. 4

Immunoreactivity for intracellular IL-16 (IL-16IR) in matched human palatine tonsils from non-smokers and from smokers (n = 6 in each group). The data is expressed as IL-16IR in percentage of total tissue area (% area). Data are shown as individual values plus median values (bold lines). *P < 0·05 (Wilcoxon's signed-rank test).

Fig. 5

Immunohistochemical double-staining from a never-smoking subject showing immunoreactivity (IR) for IL-16 in a CD4⁺ lymphocyte (Fig. 5a) and IL-16 in a CD8⁺ lymphocyte (Fig. 5b). Further, immunohistochemical double staining from a tobacco-smoking subject showing IR for IL-16 in a CD4⁺ lymphocyte (Fig. 5c) and IL-16 in a CD8⁺ lymphocyte (Fig. 5d). The IL-16IR is shown in brown and the CD4⁺ IR as well as the CD8⁺ IR is shown in blue. Arrows indicate the double-staining cells. The magnification factor was 100×.