Nicotine primarily suppresses lung Th2 but not goblet cell and muscle cell responses to allergens

Abstract

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C(4); and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

FIGURE 1. Nicotine inhibits RW-induced inflammation into the lung

BN rats were treated with saline or NT for a week followed by RW sensitization. Animals were sensitized with RW (i.p.) on day 0 and day 4 and challenged (i.t.) with RW on day 11. Three days after the challenge, lung tissues from all the three groups were fixed and embedded in paraffin. Five-micron thick sections were stained with H&E and examined microscopically. A & B) unsensitized control rats; C & D) RW sensitized rats pre-treated with saline; E & F) RW sensitized rats pre-treated with NT. NT pre-treated rats (E) show less lung inflammation than saline pre-treated rats (C) after RW sensitization. Note the increased numbers of lymphocytes present in the inflammatory infiltrates in (D, small arrow) compared to the more histiocytic infiltrates in (F, large arrow). The results represent data from control (CON, n = 4), RW-sensitized/challenged (RW, n = 5), and NT-treated and RW-sensitized/challenged (NRW, n = 5).

FIGURE 2. Nicotine inhibits the RW-induced emigration of leukocytes into the lung

BAL cells from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals were stained, and the total number of leukocytes (A) and eosinophils (B) were counted microscopically. Data are presented as means ± SE for each group (n = 4-6).

FIGURE 3. Nicotine suppresses allergen-specific IgE

Rats were sensitized/challenged with RW or HDM as described in materials and methods. Total IgE from lavage (A) and serum (C), and RW-specific IgE from lavage (B) and serum (D) from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) rats were determined by ELISA. Total IgE from lavage (E) and serum (F) from HDM sensitized/challenged BN rats were also determined. Data are presented as means ± SE for each group (n = 4-6).

FIGURE 4. Nicotine does not suppress total or RW-specific IgG and IgG subclasses

RW-specific lavage (A) and serum (B) IgG, serum IgG1 and IgG2a levels were measured in BN rats, either control (CON), RW-sensitized/challenged (RW), or NT-treated and RW-sensitized/challenged (NRW) groups. Data are presented as means ± SE for each group (n = 4-6).

FIGURE 5. Nicotine blocks RW-induced leukotriene secretion

LTC₄ content in the BAL of control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals was determined by an enzyme immunoassay as described in materials and methods. Data are presented as means ± SE for each group of 4-6 rats.

FIGURE 6. Nicotine down regulates allergen-induced Th2 cytokine expression in the lung

Rats were sensitized with RW as described in Figure 1 and challenged with RW i.t. on 3 consecutive d (i.e., d 11-13). At 24 h after the last challenge, RNA from lung tissues from control (CON), RW-challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) groups was analyzed by qPCR. Relative abundance of mRNAs of IL-4 (A), IL-5 (B), and IL-13 (C) is shown. The results are representative of two independent experiments with 3 animals per group.

FIGURE 7. Nicotine inhibits the allergen-induced Th2 cytokine production in the BAL

Rats were sensitized and challenged with RW as described in Figure 6, and BALFs were collected from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals. The amounts of IL-4 (A) and IL-13 (B) were determined by ELISA as described in Methods. Data are presented as means ± SE from 4-6 animals per group.

FIGURE 8. Nicotine down regulates the expression of RW-induced IL-25 protein in the lung

Rats were sensitized and challenged with RW as described in Figure 6. At 24 h after the last RW challenge, lung sections from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals were examined by IHC as described in Methods. The experiment was repeated twice with 3 animals per group.

FIGURE 9. Nicotine down regulates the expression of RW-induced eotaxin in the lung

Rats were sensitized and challenged with RW as described in Figure 6. At 24 h after the last RW challenge, lung tissues from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals were examined for eotaxin protein expression by IHC (A) and eotaxin mRNA expression by qPCR (B). The results are representative of two independent experiments with 3 animals in each group.

FIGURE 10. Nicotine does not decrease RW-induced Muc5ac expression in the lung but suppresses Muc5ac mucin secretion in the bronchoalveolar lavage fluid

Rats were sensitized and challenged with RW as described in Figure 6, and lung tissues and BALFs were collected from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals the BAL content of Muc5ac protein (A) was determined by ELISA, and Muc5ac mRNA expression (B) by qPCR as described in Methods.

FIGURE 11. Nicotine decreases the number of mucous cells but increases the volume of mucus substances in goblet cells

Rats were sensitized and challenged with RW as described in Figure 6. At 24 h after the last challenge, lung tissues from control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) were fixed. Tissue sections were stained with AB/PAS (A), and the volume of intraepithelial mucosubstances and the number of mucous cells per millimeter basal lamina (B) were determined as described in materials and methods.

FIGURE 12. Nicotine treatment does not decrease the airway resistance to methacholine

Rats were sensitized and challenged with RW as described in Figure 1. Three days after the challenge, the airway resistance of control (CON), RW-sensitized/challenged (RW), and NT-treated and RW-sensitized/challenged (NRW) animals in response to increasing doses of methacholine was determined using Flexivent plethysmography as described in Methods. Pooled data from three experiments using the peak resistance value for each dose of methacholine were used to plot the data (n = 4).