Effect Of α2-Adrenergic Agonists And Antagonists On Cytokine Release From Human Lung Macrophages Cultured In Vitro

Abstract

The most trusted hypothesis to explain how α2-adrenergic agonists may preserve pulmonary functions in critically ill patients is that they directly act on macrophages by interfering with an autocrine/paracrine adrenergic system that controls cytokine release through locally synthetized noradrenaline and α1- and α2-adrenoreceptors. We tested this hypothesis in primary cultures of resident macrophages from human lung (HLMs). HLMs were isolated by centrifugation on percoll gradients from macroscopically healthy human lung tissue obtained from four different patients at the time of lung resection for cancer. HLMs from these patients showed a significant expression of α2A, α2B and α2C adrenoreceptors both at the mRNA and at the protein level. To evaluate whether α2 adrenoreceptors controlled cytokine release from HMLs, we measured IL-6, IL-8 and TNF-α concentrations in the culture medium in basal conditions and after preincubation with several α2-adrenergic agonists or antagonists. Neither the pretreatment with the α2-adrenergic agonists clonidine, medetomidine or dexdemetomidine or with the α2-adrenergic antagonist yohimbine caused significant changes in the response of any of these cytokines to LPS. These results show that, different from what reported in rodents, clonidine and dexdemetomidine do not directly suppress cytokine release from human pulmonary macrophages. This suggests that alternative mechanisms such as effects on immune cells activation or the modulation of autonomic neurotransmission could be responsible for the beneficial effects of these drugs on lung function in critical patients.

Keywords: clonidine; cytokines; dexmedetomidine; macrophages; medetomidine; yohimbine.

Figure 1

ADRA2A, ADRA2B, ADRA2C expression in HLMs. Panel A: RT-PCR performed with primers specific for ADRA2A, ADRA2B and ADRA2C. Gels were loaded with retrotranscripts from lung tissues of two different patients (Lines 1 and 2) and from MCF-7 cells, a breast cancer cell known to express all the three α₂-adrenoreceptor isoforms that was used as an internal reference. In all the PCR reactions bands of about 200 bp were amplified, a size corresponding to what expected for ADRA2A (211 bp; first line), ADRA2B (230 bp; second line) and ADRA2C (242 bp; third line). β actin was used as housekeeping gene product or normalization. Panel B: Western blotting performed using anti-α2A-, anti α2B- and anti- α2C-adrenoreceptor antibodies and cellular lysates from HLMs obtained from two different patients preparations. GAPDH immunoreactivity was used for normalization. In both HLM preparations, immunoreactive bands of the expected size were obtained for all the three α2-adrenoreceptor isoforms (70, 62 and 60 kDa for α2A-, α2B- and α2C-adrenoreceptors, respectively).

Figure 2

Effect of α₂-adrenergic agonists and antagonists on in vitro cytokine release from HMLs. The bar graphs show the effect of vehicle and of progressively higher concentrations of the α₂-adrenergic agonists dexmedetomidine (0.1–10 μM) (panel A), medetomidine (0.1–10 μM) (panel B) and clonidine (0,3–3 μM) (panel C) and of the α₂-adrenergic antagonist yohimbine (1–10 μM) (panel D) on LPS-induced TNF-α, IL-8 and IL-6 release in the culture medium from HLMs. HLMs were preincubated with the aforementioned drugs for 30 min before adding LPS (100 ng/ml) to induce TNF-α, IL-8 and IL-6 release. The concentrations of these cytokines in culture medium were measured by ELISA 18 hours after LPS addition. Each bar represents as the mean± SEM of the concentrations of the respective cytokine obtained in four different experimental sessions.