β2-Agonists inhibit TNF-α-induced ICAM-1 expression in human airway parasympathetic neurons

Abstract

Background: Major basic protein released from eosinophils to airway parasympathetic nerves blocks inhibitory M(2) muscarinic receptors on the parasympathetic nerves, increasing acetylcholine release and potentiating reflex bronchoconstriction. Recruitment of eosinophils to airway parasympathetic neurons requires neural expression of both intercellular adhesion molecular-1 (ICAM-1) and eotaxin. We have shown that inflammatory cytokines induce eotaxin and ICAM-1 expression in parasympathetic neurons.

Objective: To test whether the β(2) agonist albuterol, which is used to treat asthma, changes TNF-alpha-induced eotaxin and ICAM-1 expression in human parasympathetic neurons.

Methods: Parasympathetic neurons were isolated from human tracheas and grown in serum-free medium for one week. Cells were incubated with either (R)-albuterol (the active isomer), (S)-albuterol (the inactive isomer) or (R,S)-albuterol for 90 minutes before adding 2 ng/ml TNF-alpha for another 4 hours (for mRNA) or 24 hours (for protein).

Results and conclusions: Baseline expression of eotaxin and ICAM-1 were not changed by any isomer of albuterol as measured by real time RT-PCR. TNF-alpha induced ICAM-1 expression was significantly inhibited by (R)-albuterol in a dose dependent manner, but not by (S) or (R,S)-albuterol. Eotaxin expression was not changed by TNF-alpha or by any isomer of albuterol. The β-receptor antagonist propranolol blocked the inhibitory effect of (R)-albuterol on TNF-alpha-induced ICAM-1 expression.

Clinical implication: The suppressive effect of (R)-albuterol on neural ICAM-1 expression may be an additional mechanism for decreasing bronchoconstriction, since it would decrease eosinophil recruitment to the airway nerves.

Figure 1. β₂ receptors are identified by anti-β₂ receptors antibody on human trachea parasympathetic neurons (red, A, B–D) under high (A,C) and low (D) power.

Neurons are labeled with anti-neurofilament antibodis (B, green) and the merged image (for neuronal and β₂ receptor staining) is shown in C. Nuclei stain blue with DAPI. The insert of D is the absence of primary antibody. Magnification bars: 50 µm.

Figure 2. Pretreatment with (R)-albuterol before TNF-α significantly inhibits TNF-α-induced ICAM-1 mRNA expression in human parasympathetic neurons as detected by real-time qPCR (A).

(S)-or (R,S)-albuterol does not inhibit TNF-α induced ICAM-1 (A). The inhibitory effect of (R)-albuterol on TNF-α-induced ICAM-1 mRNA expression is dose dependent (B). Neither TNF-α nor any albuterol isomer changes eotaxin expression (C). *indicates significantly different from control. **indicates significantly different from TNF-α treatment, as analyzed by one way ANOVA.

Figure 3. ICAM-1 protein expression is measured by fluorescence intensity of a labeled anti-ICAM-1 antibody.

(R)-albuterol (A) but not S- or (R-,S)-albuterol (B and C) significantly inhibits TNF-α-induced ICAM-1 protein (p<0.005). *indicates significantly different from control and **indicates significantly different from TNF- treatment, as analyzed by paired T-test.

Figure 4. Pretreatment with β-receptor antagonist propranolol completely prevents the suppressive effect of R-albuterol on TNF-α-induced ICAM-1 protein expression that is identified by fluorescence intensity of anti-ICAM-1 antibody staining in human parasympathetic nerves.

*indicates significant difference from control, ** indicates significant difference from TNF-α treatment and *** indicates significant difference from (R)-albuterol, as analyzed by one way ANOVA.