Neuronal control of localized inflammation through expressed nicotinic acetylcholine receptors : a study carried out in mice

Introduction Although the local inflammatory reactions are known to be regulated through cholinergic antiinflammatory pathways, the exact subtypes of nicotinic acetylcholine receptors involved in neuroimmune modulation are not well identified. Objectives Immunohistochemical localisation of a 1 subunit of nicotinic acetylcholine receptors (a1 nAChR) in sites of localised inflammation induced by injecting turpentine to the hind limbs of Balb/C mice. Methods Localised inflammation and subsequent development of sterile abscesses was induced by injecting sterile turpentine subcutaneously into thighs of Balb/C mice. Sterile saline was used in the control.. Skin and muscle tissues of inflammatory sites were recovered from the animals after 48 hours and were stained with hematoxylin and eosin. Indirect immunohistochemistry was done using anti-a1nAChR as the primary antibody and biotinylated anti-rat IgG as the secondary antibody. Labeled streptavidin biotin (LSAB) technique was used with diaminobenzedene to detect the immunoreactivity (IR). Intensity of immunostaining was determined based upon a score of 0 3+ by qualitative computerised image analysis using FSX 100 Olympus microscope. Results Hand E stained slides showed polymorphonuclear leukocytes (PNL) infiltration at the abscess sites while the saline injected control tissue sections did not show PNL infiltration. A 2+ immunoreactivity (IR) of a 1nAChRs was visible at peripheral zones of sterile abscesses where PNL infiltrations were high while the central area with necrotic tissue did not show IR. A subcutaneous lymph node found within the inflammatory region expressed IR of a 1nAChR in its capsular sinuses, subcapsular sinuses and trabecular regions. Conclusions The findings suggest the possible role of controlling localised inflammatory response by parasympathetic cholinergic nerves through a 1nAChRs of inflammation sites. Ceylon Medical Journal 2015; 60: 143-147 Introduction The central nervous system is known to interact with the immune system through the autonomic nervous system and modify immune responses. Though the sympathetic system is the main component which controls the functions of lymphoid tissue, recent evidence shows significant influence by the cholinergic nervous system as well [1,2]. Accordingly cholinergic anti-inflammatory mechanisms can inhibit the activation of macrophages and release of cytokines [2]. The cholinergic system, which uses acetylcholine as a neurotransmitter, is one of the excitatory pathways of the central nervous system [2]. Acetylcholine and multiple nicotinic acetylcholine receptor (nAChR) subunits are present on many cell types including endothelial cells and cells of the immune system [3, 4]. Thymocytes, thymic epithelial and myoid cells of immune organs express nAChRs on their cell surface [5]. Peripheral T cells express an unusual profile of muscle type nicotinic subunits, but their nAChR expression has. not been identified [6]. In addition, expression of mRNA of as, a9, a10, ~1, ~2, ~4 nAChR subunits in the freshly isolated CD4 and CD8 T cells have been reported [7]. Gene expression of o.l , a7 and o l O nAChRs also has been shown in mRNAs of primary human macrophages isolated from blood mononuclear cells [8]. Majority of the evidence shows the expression of a7nAChRs on monocytes and macrophages. [3). a7nAChR is involved in the anti-inflammatory pathway [8]. Similarly, several studies show the crucial role of a7nAChR in the inhibition of excessive inflammation of many organs ip. the neuroimmune network [9, 10]. But the anatomicai distribution of other nAChRs or their functional effects has not been studied. A recent study shows that the parasympathetic cholinergic innervations are found predominantly in lymph nodes and spleen parenchyma in the supporting framework of reticulin fibres and consists mainly of alnAChRs with minimum of a7nAChR in T cell and macroph,:ge abundant regions of these tissues [11]. 'Depertmeni of Human Biology, Faculty of Health-Care Sciences, Eastern University of Sri Lanka and -Deoertment of Anatomy, Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka. Correspondence: SGY, e-mail: . Received 5 November and revised version accepted 2 October 2015. October 2015. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Vol. 60, No. 4, December 2015 143

Introduction Although the local inflammatory reactions are known to be regulated through cholinergic antiinflammatory pathways, the exact subtypes of nicotinic acetylcholine receptors involved in neuroimmune modulation are not well identified.
Objectives Immunohistochemical localisation of a 1 subunit of nicotinic acetylcholine receptors (a1 nAChR) in sites of localised inflammation induced by injecting turpentine to the hind limbs of Balb/C mice.

Methods
Localised inflammation and subsequent development of sterile abscesses was induced by injecting sterile turpentine subcutaneously into thighs of Balb/C mice.Sterile saline was used in the control.. Skin and muscle tissues of inflammatory sites were recovered from the animals after 48 hours and were stained with hematoxylin and eosin.Indirect immunohistochemistry was done using anti-a1nAChR as the primary antibody and biotinylated anti-rat IgG as the secondary antibody.Labeled streptavidin biotin (LSAB) technique was used with diaminobenzedene to detect the immunoreactivity (IR).Intensity of immunostaining was determined based upon a score of 0 -3+ by qualitative computerised image analysis using FSX 100 Olympus microscope.
Results Hand E stained slides showed polymorphonuclear leukocytes (PNL) infiltration at the abscess sites while the saline injected control tissue sections did not show PNL infiltration.A 2+ immunoreactivity (IR) of a 1nAChRs was visible at peripheral zones of sterile abscesses where PNL infiltrations were high while the central area with necrotic tissue did not show IR.A subcutaneous lymph node found within the inflammatory region expressed IR of a 1nAChR in its capsular sinuses, subcapsular sinuses and trabecular regions.

Conclusions
The findings suggest the possible role of controlling localised inflammatory response by parasympathetic cholinergic nerves through a 1nAChRs of inflammation sites.

Introduction
The central nervous system is known to interact with the immune system through the autonomic nervous system and modify immune responses.Though the sympathetic system is the main component which controls the functions of lymphoid tissue, recent evidence shows significant influence by the cholinergic nervous system as well [1,2].Accordingly cholinergic anti-inflammatory mechanisms can inhibit the activation of macrophages and release of cytokines [2].
The cholinergic system, which uses acetylcholine as a neurotransmitter, is one of the excitatory pathways of the central nervous system [2].Acetylcholine and multiple nicotinic acetylcholine receptor (nAChR) subunits are present on many cell types including endothelial cells and cells of the immune system [3,4].Thymocytes, thymic epithelial and myoid cells of immune organs express nAChRs on their cell surface [5].Peripheral T cells express an unusual profile of muscle type nicotinic subunits, but their nAChR expression has.not been identified [6].In addition, expression of mRNA of as, a9, a10, ~1, ~2, ~4 nAChR subunits in the freshly isolated CD4 and CD8 T cells have been reported [7].
Gene expression of o.l , a7 and ol O nAChRs also has been shown in mRNAs of primary human macrophages isolated from blood mononuclear cells [8].
Majority of the evidence shows the expression of a7nAChRs on monocytes and macrophages.
[3). a7nAChR is involved in the anti-inflammatory pathway [8].Similarly, several studies show the crucial role of a7nAChR in the inhibition of excessive inflammation of many organs ip. the neuroimmune network [9,10].But the anatomicai distribution of other nAChRs or their functional effects has not been studied.
A recent study shows that the parasympathetic cholinergic innervations are found predominantly in lymph nodes and spleen parenchyma in the supporting framework of reticulin fibres and consists mainly of alnAChRs with minimum of a7nAChR in T cell and macroph,:ge abundant regions of these tissues [11].