(b) Proliferation of spleen cells from 7nAChR-deficient and wild-type mice. (Ach) is a classical neurotransmitter involved in both cerebral and autonomous nerve transmission. Moreover, downstream cholinergic action can control the immune system via the vagus nerve (examined in [1,2]). This central cholinergic anti-inflammatory pathway is dependent upon the 7 subunit of the nicotinic acetylcholine receptor (7nAChR), which is known to mediate efficient inhibition of proinflammatory cytokine synthesis from macrophages [3]. In addition, there is accumulating evidence suggesting an additional important part for non-neuronal cholinergic mechanisms in c-Fms-IN-1 immune regulation (examined in [4,5]). T cells, macrophages along with other immune cells are capable of generating Ach and both muscarinic and nicotinic cholinergic receptors are indicated on their surfaces, including 7nAChR. Ach regulates the function of immune cells, including B and T cells, via different cholinergic receptors (examined in [5,6]). Concerning muscarinic receptors, m2 receptors have been demonstrated to possess a role in immunoregulation [7]. Of the known nicotinic cholinergic receptors, 7nAChR is considered to be the most important for immune regulatory actions [3,5]. Mice missing 7nAChR have been demonstrated to create an excess of proinflammatory cytokines in response to lipopolysaccharide (LPS) activation [3]. Increased production of antigen-specific antibodies has also been reported in 7nAChR-deficient mice [8]. However, the effects of 7 gene depletion may show different patterns concerning innate and adaptive immunity, and recently a decreased severity of autoimmune encephalitis was reported in 7 gene knock-out mice [9]. 7nAChR manifestation is c-Fms-IN-1 pronounced in the synovium of rheumatoid arthritis (RA) individuals [1012] and Ach-producing enzymes have been detected in the joint [13].In vitro, cholinergic stimulation can decrease MDNCF cytokine production from RA synovial fibroblasts [11]. In addition, vagus and parasympathetic activity is usually decreased in systemic inflammatory conditions such as rheumatoid arthritis (RA), also becoming correlated to systemic levels of proinflammatory cytokines [14]. Although Ach can mediate both central and peripheral cholinergic immune-regulating mechanisms, the part of Ach in chronic swelling and RA has not been studied in detail. Herein we demonstrate the importance of 7nAChR for development of collagen-induced arthritis and immune activation. == Materials and methods == == Animals == Mice deficient in the 7nAChR (chrna7tm1Bay, The Jackson Laboratory, Bar Harbor, Me personally, USA) (knock-out) and wild-type C57BL/6 mice were housed and bred in specific pathogen-free facilities. The mice were managed under climate-controlled conditions having a 12-h light/dark cycle and fed standard rodent chow and waterad libitum. All experiments were authorized by the Stockholm North Ethical Committee, Sweden. == Arthritis == Chicken collagen type II (CII) (Morwell Diagnostics GmbH, Zumikon, Switzerland) (2 mg/ml) was emulsified 1:1 with a mixture of the same volumes incomplete and total Freund’s adjuvant (Difco, Detroit, MI, USA) at 4C. One hundred l of the emulsion was immunized subcutaneously at the base of the tail under isoflurane anesthesia (Abbot Scandinavia Abdominal, Solna, Sweden). After 28 days the c-Fms-IN-1 mice were given a booster immunization with the same quantities of CII (2 mg/ml) and incomplete Freund’s adjuvant in an emulsion. Arthritis score was assessed for each and every animal every day from onset and onwards and the cumulative score of individual animals was compared on a group basis. The intraphalangeal important joints of digits, metacarpophalangeal important joints and the wrist in the forepaw and ankle joint in the hindpaw were each considered as one category of joint. Each paw was given a score of 03, as follows: 0 = unaffected, 1 = one type of joint affected, 2 = two types of joint affected and 3 = the entire paw affected. Evaluation of arthritis was performed by staff members blinded to the identity of the animals. The individual mice were killed 14 days after onset of disease. Inside a earlier experiment the day of maximal arthritis score had been identified as day time 14. Blood and paws were collected and the paws were fixed in zamboni answer at 4C.