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. 2010 Jul 1;185(1):588-96.
doi: 10.4049/jimmunol.0902227. Epub 2010 May 26.

Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors

Neerad C Mishra  1 , Jules Rir-sima-ah, R Thomas Boyd, Shashi P Singh, Sravanthi Gundavarapu, Raymond J Langley, Seddigheh Razani-Boroujerdi, Mohan L Sopori
Affiliations

Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors

Neerad C Mishra et al. J Immunol. .

Abstract

Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

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Figures

Figure 1
Figure 1. NT inhibits LT synthesis and secretion in RBL cells
RBL cells were treated with various concentrations of NT or 1 μM cromoglycate sodium (CS) (A) or cotinine (B) for 8 h, followed by stimulation with 10 ng of anti-FcεRI antibody. LTC4 content in the supernatants was determined as described in Materials and Methods. Release of LTC4 is expressed as the percent of control RBL cells treated with anti-FcεRI alone. Data shown are the means ± SE of 4 independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 2
Figure 2. NT inhibits the expression of proinflammatory cytokines in RBL
Where indicated, RBL cells were pretreated with 1 μM NT for 8 h, followed by stimulation with 10 ng of anti-FcεRI antibody. (A & B) After 30 min, total RNA was isolated and analyzed for TNF-α and IL-1β expression by qPCR, (C) 2 hrs post anti-FcεRI stimulation release of TNF-α was measured using ELISA. The results are the means ± SE of three independent experiments and expressed as the fold change in the expression and release of these cytokines. * p < 0.05.
Figure 3
Figure 3. NT treatment does not affect degranulation in RBL cells
Cells were pretreated with various concentrations of NT for 8 h followed by stimulation with 10 ng of anti-FcεRI antibody for 20 min. Supernatants were analyzed for enzymatic activity of β-hexosaminidase (A) and histamine (B) as described in Materials and Methods. Data represent means ± SE for 4 independent experiments each performed in triplicate.
Figure 4
Figure 4. RBL cells bind α-BTX and express α7-, α9-, and α10-nAChRs
(A) RBL cells were incubated with Alexa fluor 488-conjugated α-BTX and analyzed by flow cytometry as described in Materials and Methods. Note: After α-BTX treatment there are no detectable α-BTX unbound cells as seen by lack of cells within the control (CON) region of the histogram. (B) qPCR analysis for the expression of α 7-, α9-, and α10-nAChRs on RBL. Note: On RBL cells the expression of α7-, α9-, and α10-nAChRs is approximately comparable.
Figure 5
Figure 5. NT treatment suppresses the activation of ERK1/2, Akt and NF-κB in RBL cells
Cells were treated with indicated concentrations of NT for 8 h followed by stimulation with 10 ng of anti-FcεRI antibody for 20 min. Whole cell lysates (40 μg protein) were analyzed by Western blots as described in Materials and Methods. Blots were probed by (A) anti-phospho ERK1/2 and anti-ERK1/2 (B) anti-phospho Akt and anti-Akt. ERK1/2 and Akt were used as loading controls. Western blots shown are representative of 6 experiments. (C) Nuclear extracts (8 μg protein) from control (CON) and NT-treated RBL cells were incubated with 32P-end-labeled NF- κB oligonucleotide for 30 min at 37°C. The resulting DNA-protein complexes were analyzed by electrophoretic mobility gel shift assay as described in Material and Methods. Densitometry readings are average from 4-6 independent experiments. * p < 0.05.
Figure 6
Figure 6. Nicotine treatment inhibits cPLA2 activation in RBL cells
Cells were treated with NT for 8 h followed by stimulation with 10 ng of anti-FcεRI antibody for 20 min. cPLA2 activity was measured by enzyme immunoassay following manufacturer’s instructions. Data represent means ± SE of 3 independent experiments. * p < 0.05.
Figure 7
Figure 7. Antagonists of α7 and α9-nAChRs (α-BTX and MLA) attenuate the NT-induced inhibition of ERK and Akt
Prior to NT treatment, the cells were preincubated with either 100 nM MLA or 40 nM α-BTX for 30 min. Cells were then stimulated with 10 ng of anti-FcεRI antibody for 20 min. Whole cell extracts were fractionated on SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-phospho ERK1/2 and anti-phospho Akt antibodies. Total ERK1/2 and Akt were used as loading controls. Western blots shown are representative of 3 experiments.
Figure 8
Figure 8. nAChRs α7 and α9 interact to mediate the effects of NT on mast cell function
(A) Specific siRNAs selectively knockdown α7 or α9 nAChRs. RBL cells were treated with α7- or α9-specific siRNA as described in Materials and Methods. At 48 h post siRNA treatment, α7- and α9-nAChR mRNA expression was assessed by qPCR. (B) Knockdown of either α7- or α9- nAChRs attenuate NT effects on RBL cell function. At 48 h after siRNA treatment, cells were incubated with 1 μM NT and then stimulated with anti-FcεRI antibody. Whole cell extracts were run on SDS-PAGE and probed with anti-phospho ERK1/2. Total ERK1/2 was used as loading control. The results are representative of 3 independent experiments.
Figure 9
Figure 9. nAChRs α7, α9 and α10 interact to mediate the effects of NT in RBL cells
(A) Specific siRNAs selectively knockdown α10 nAChRs. RBL cells were treated with α10-specific siRNA and 48 hrs post siRNA treatment, α7-, α9- and α10-nAChRs mRNA expression was assessed by qPCR. (B) Knockdown of α10-nAChRs attenuates NT effect of RBL cells. At 48 h after siRNA treatment, cells were incubated with 1 μM NT and then stimulated with anti-FcεRI antibody. Whole cell extracts were run on SDS-PAGE and probed with anti-phospho ERK1/2. β-actin was used as loading control. Western blot shown is representative of 3 independent experiments.
Figure 10
Figure 10. Nicotine inhibits the rise in anti-FcεRI-induced [Ca2+]i
RBL cells were preincubated with indicated concentrations of NT, and after 8 h cells were loaded with Indo-1, treated with anti-FcεRI antibody, and analyzed for changes in [Ca2+]i by spectrofluorometry as described in Materials and Methods. Results shown are representative of two independent experiments.
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