The effect of smoking on chronic inflammation, immune function and blood cell composition

Abstract

Smoking is the number one risk factor for cancer mortality but only 15-20% of heavy smokers develop lung cancer. It would, therefore, be of great benefit to identify those at high risk early on so that preventative measures can be initiated. To investigate this, we evaluated the effects of smoking on inflammatory markers, innate and adaptive immune responses to bacterial and viral challenges and blood cell composition. We found that plasma samples from 30 heavy smokers (16 men and 14 women) had significantly higher CRP, fibrinogen, IL-6 and CEA levels than 36 non-smoking controls. Whole blood samples from smokers, incubated for 7 h at 37 °C in the absence of any exogenous stimuli, secreted significantly higher levels of IL-8 and a number of other cytokines/chemokines than non-smokers. When challenged for 7 h with E. coli, whole blood samples from smokers secreted significantly lower levels of many inflammatory cytokines/chemokines. However, when stimulated with HSV-1, significantly higher levels of both PGE₂ and many cytokines/chemokines were secreted from smokers' blood samples than from controls. In terms of blood cell composition, red blood cells, hematocrits, hemoglobin levels, MCV, MCH, MCHC, Pct and RDW levels were all elevated in smokers, in keeping with their compromised lung capacity. As well, total leukocytes were significantly higher, driven by increases in granulocytes and monocytes. In addition, smokers had lower NK cells and higher Tregs than controls, suggesting that smoking may reduce the ability to kill nascent tumor cells. Importantly, there was substantial person-to person variation amongst smokers with some showing markedly different values from controls and others showing normal levels of many parameters measured, indicating the former may be at significantly higher risk of developing lung cancer.

Figure 1

Smokers have elevated plasma levels of CRP and fibrinogen but not PGE₂. Plasma samples were analysed for (a) CRP, (b) fibrinogen and (c) PGE₂ ● = non-smoking, controls; All the smokers were given a unique identifying symbol so that each smoker could be tracked from one assay to another. As well, all males were symbolized by a square and all females by a circle. The mean is shown as a horizontal line within each group. * indicates a statistically significant difference (P < 0.05) between smokers and the non-smoking control group.

Figure 2

IL-6 and CEA are elevated in smokers. Plasma samples were analyzed for 15 cytokines/chemokines using Luminex technology. Only those cytokines/chemokines showing a trend towards different levels in smokers are shown. The mean is shown as a horizontal line within each group. * indicates a statistically significant difference (P < 0.05) between smokers and non-smoking controls.

Figure 3

Incubation of whole blood samples from smokers yields higher levels of cytokines/chemokines in the presence or absence of HSV-1 but lower levels with E. coli. Whole fresh blood from smokers and controls were incubated for 7 h at 37 °C in the absence or presence of E. coli or HSV-1 and the conditioned plasma analyzed for the cytokine/chemokine levels using Luminex beads. (a) A comparison of plasma IL-8 levels from whole blood samples taken at time 0 or after 7 h of incubation in the absence of stimulation from smokers and controls. (b) A comparison of the plasma levels for 11 of the 15 cytokines/chemokines tested (selected on the basis of having a significant difference (P < 0.05) between smokers and controls). Shown are the levels obtained after 7 h of incubation without (C) or with stimulation by E. coli or HSV-1.

Figure 4

Whole blood samples from smokers secrete significantly more PGE₂ in response to HSV-1 stimulation and more IFNγ in response to T cell stimulation than controls. (a) Whole fresh blood from smokers and controls were incubated for 7 h at 37 °C ± E. coli or HSV-1 and the conditioned plasma analyzed for PGE₂ levels. (b) Whole fresh blood from smokers and controls were diluted tenfold with RPMI and incubated for 4 days at 37 °C in the absence (left panel) or presence (right panel) of anti-CD3 + anti-CD28 antibodies. (C) PBMCs from smokers and controls were incubated without T cell stimulation for 4 days in RPMI + 10% autologous plasma and the levels of IFNγ production determined. Each smoker is identified with a unique symbol. * indicates a statistically significant difference (P < 0.05) between controls and smokers.

Figure 5

Smoking increases hematocrits, HgB, MCV, MCH, MCHC, RDW and Pct but not RBC levels. Smokers were compared to controls for RBC, Hct, HgB, MCV, MCH, MCHC, RDW and Pct. Each smoker is identified with a unique symbol. * Indicates a statistically significant difference (P < 0.05) between smokers and non-smoking controls.

Figure 6

Smoking increases monocytes and granulocytes but does not affect CD4 and CD8 T cell populations. Smokers were compared with non-smokers for (a) total white blood cells, lymphocytes, monocytes and granulocytes. Results are expressed as the mean ± SEM. (b) Smokers were compared with non-smokers for monocytes and granulocytes. (c) Smokers were compared with non-smokers for CD4 + and CD8 + T cells and their CD4/CD8 ratio. Each smoker is identified with a unique symbol in panels B and C. * indicates a statistically significant difference (P < 0.05) between smokers and non-smokers.

Figure 7

Smoking reduces natural killer cells and increases regulatory T cells. (a) Smokers and non-smokers were compared for CD56/CD45, CD56Bright/CD45 and CD56Dim/CD45 populations. (b) Smokers and non-smokers were compared for Treg levels. Each smoker is identified with a unique symbol. * indicates a statistically significant difference (P < 0.05) between smokers and controls.