A total of 56 male DBA/1 mice (weight, 20 g; age, 6–8 weeks; 24 mice died prematurely whilst the model was established) were purchased from the SJA Laboratory Animal Co. (Shanghai, China) for use in the CIA studies. The mice were housed under specific-pathogen-free conditions at 24–28°C, 40–70% humidity, 12 h light/dark cycle and free access to food and water. The animal experiments were performed in accordance with the institutional guidelines for animal care, and were approved by the committee for the use and care of animals at Central South University (Changsha, China).

The mice were randomly divided into four groups (n=8): Control group [sham vagotomy + phosphate-buffered saline (PBS)], model group (sham vagotomy + CIA + PBS), vagotomy group (vagotomy + CIA + PBS), and nicotine group (sham vagotomy + CIA + nicotine). To inhibit the CAP, mice in the vagotomy group were subjected to left-side cervical vagotomy 4 days prior to CIA induction (14). In sham-operated mice, the left vagus nerve was exposed and isolated from the surrounding tissue but was not transected. In the nicotine group, the peripheral segment of the CAP was stimulated by intraperitoneal pretreatment with nicotine daily (250 µg/kg; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), beginning 4 days prior to CIA induction. The other groups were injected with PBS as a control.

CIA was induced in the DBA/1 mice as previously described (14). Bovine type II collagen (2 mg/ml; Chondrex, Inc., Redmond, WA, USA) was emulsified in an equal volume of Freund's complete adjuvant (containing 4 mg/ml Mycobacterium tuberculosis; Chondrex, Inc.) at 4°C by magnetic stirring until fully emulsified. The final concentration of collagen was 1 mg/ml. All male DBA/1 mice, with the exception of the control mice, were initially immunized intracutaneously at the base of the tail with 0.1 ml of this emulsion (100 µg collagen). On day 21, the mice were administered booster injections of 0.1 ml emulsion in Freund's incomplete adjuvant (Chondrex, Inc.) in the same manner.

Clinical signs of arthritis in the joints were visually assessed every 3 days by two observers. All animals were regularly examined for signs of arthritis, and the disease severity for each paw was graded on a scale between 0 and 4, according to the levels of erythema, swelling and induration. Arthritis index score criteria were as follows: 0, no redness or swelling of the joints; 1, red or slight swelling; 2, moderate swelling; 3, severe swelling; and 4, severe swelling and unloadable. Assessment of incidence and macroscopic score were carried out by two independent observers, without knowledge of the experimental groups. The total arthritic score per mouse was derived from the sum of the individual scores of four paws. Two independent observers without knowledge of the experimental groups assessed the incidence and macroscopic score.

The mice were sacrificed by cervical dislocation on day 42, and their hind paws were collected. The joint tissues were fixed in 4% paraformaldehyde overnight at room temperature, decalcified in 13% ethylenediamine tetra-acetic acid for 2–3 weeks and embedded in paraffin and cut into 5 µm sections. The sections (2.5×2.5 cm) were then dewaxed using xylene, dehydrated using an alcohol gradient, and were stained with hematoxylin and eosin (H&E) for 1 min at room temperature for histological assessment. Arthritis severity in histological samples was determined by cumulative assessment of synovial inflammation. The scoring was: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. This was completed by two independent observed, without knowledge of the experimental groups. Histopathological sections of the hind paws were examined for hyperplasia of the synovial membrane, infiltration with mononuclear cells, and cartilage and bone damage (14).

For immunofluorescence detection of macrophage infiltration, sections were dewaxed and dehydrated as aforementioned. Slides were retrieved using a heat-mediated retrieval method, and endogenous peroxidase activity was quenched with 3% H₂O₂ for 5 min at room temperature. The sections were then blocked with 3% bovine serum albumin (Sijiqing, Hanzhou, China) for 1 h at room temperature and were incubated overnight at 4°C with an anti-CD11b primary antibody (1:200; cat. no. 101213; BioLegend, San Diego, CA, USA). Subsequently, the sections were incubated with Alexa Fluor 555-conjugated goat anti-rat immunoglobulin G (IgG; cat. no. A-21434; 1:150; Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h at room temperature. The number of Alexa Fluor 555-labeled CD11b-positive macrophages was counted under a confocal microscope (LSM780; Carl Zeiss Jena GmbH, Jena, Germany) using a ×63 oil immersion objective by a blinded observer.

For staining of CCR2 and ICAM-1, the sections were pretreated with proteinase K for CCR2 retrieval for 20 min at 37°C, and were then treated with citric buffer (pH 6) for heat-mediated antigen retrieval of ICAM-1 at 80°C for 20 min. Sections were incubated with rabbit anti-mouse CCR2 (1:200; cat. no. ab32144; Abcam, Cambridge, MA, USA), rabbit anti-mouse ICAM-1 monoclonal antibody (1:400; cat. no. ab124759; Abcam) or normal rabbit IgG (1:200; cat. no. A7016; Biyuntian, Shanghai, China) at 4°C overnight. Subsequently, the samples were washed three times for 5 min in PBS and were incubated with biotin-conjugated goat anti-rabbit IgG (ABC kit; cat. no. PK-6100; Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature. After three 5-min washes in PBS, the sections were incubated with an avidin-biotinylated enzyme complex (ABC kit; Vector Laboratories) for 2 h at room temperature. Positive signals were visualized using a diaminobenzidine kit (Vector Laboratories), the sections were counterstained with hematoxylin, images were captured using an Olympus microscope (IMT-2, Olympus Corporation, Tokyo, Japan) and were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

On experimental day 42, the mice were sacrificed, and serum samples were harvested. Whole blood samples (0.8–1 ml each) were centrifuged at 12,000 × g for 20 min at 4°C. The serum levels of MIP-1α (cat. no. MMA00; R&D Systems, Inc., Minneapolis, MN, USA), MCP-1 (cat. no. 81-BMS6005; eBioscience, Inc., San Diego, CA, USA) and MIP-2 (cat. no 70-EK21422; Multiscience Biotech, Co., Ltd., Hangzhou, China) were measured using ELISA kits, according to the manufacturers' protocols.

Spleen samples were obtained from the sacrificed mice. Splenocytes were separated from the spleen using lymphocyte-separation medium (Histopaque^®-1119; Sigma-Aldrich; Merck Millipore) after filtering the samples with a cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, the cells were incubated for 1 h at 4°C with phycoerythrin-conjugated anti-CD11b (eBioscience, Inc.) or the respective isotype control (cat. no. 12-4031; eBioscience, Inc.). The cells were then washed with PBS and resuspended in 100 µl PBS for flow cytometric analysis (BD FACS Canto II; BD Biosciences). The data were analyzed using FlowJo software version 9.9.4 (FlowJo, LLC, Ashland, OR, USA).

Data were analyzed using GraphPad Prism software version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) and are presented as the mean ± standard deviation. The experiments were repeated twice. The significance of differences between groups was determined by one-way analysis of variance followed by a multiple comparisons test (Student-Newman-Keuls). The Kruskal-Wallis test was used to determine the heterogeneity of variance. P<0.05 was considered to indicate a statistically significant difference.

The CIA model was induced as aforementioned. The arthritis index score was recorded by two researchers every 3 days after the second immunization until the mice were sacrificed. Slight swelling could be observed in the vagotomy group beginning at day 24, and reaching a peak at day 42. The model group began to present with arthritic symptoms at day 27, with symptoms peaking after 42 days. Arthritis developed slowly and was milder in the nicotine group (Fig. 1A). The histopathological features of the four groups were assessed by H&E staining. The results were consistent with the aforementioned findings, in that the histopathological changes in the nicotine group were reduced, as detected by decreased synovial proliferation, inflammatory cell infiltration and bone destruction (Fig. 1B). Semi-quantitative analysis of the histopathological features is presented in Fig. 1C.

Since the present study suggested that nicotine attenuates clinical arthritis and restrains cytokine production, such as TNF-α in our previous study (14), it was determined whether nicotine can modulate macrophage infiltration in the synovium and spleen. Macrophage infiltration serves a critical role in modulating the immune responses. In mice, the CD11b antigen, as a marker of monocytes/macrophages, is highly expressed on monocytes, macrophages, and to a lesser extent, on granulocytes and a subset of dendritic cells. The present study detected the distribution of CD11b-positive macrophages in the synovium and spleen. Immunofluorescence analysis detected an increase in infiltrated CD11b-positive macrophages in the synovium of the model, vagotomy and nicotine groups, as compared with in the control group. However, among the model, vagotomy and nicotine groups, the percentage of CD11b-positive macrophages was lowest in the nicotine group (Fig. 2A and B). In addition, the percentage of CD11b-positive cells was detected among the splenic mononuclear cells in the four groups, using flow cytometry (Fig. 2C and D). In the model group, the percentage of CD11b-positive cells was increased compared with in the control group. Treatment with nicotine significantly reduced the percentage of CD11b-positive cells in the spleen, as compared with in the model and vagotomy groups.

Chemokines serve a key role in the process of leukocyte extravasation. To determine whether the CAP affects chemotaxis of macrophages in the CIA model, the present study detected the expression levels of the macrophage-associated chemokines MIP-1α, MCP-1 and MIP-2, which have been reported to have major roles in the trafficking of monocytes toward synovial tissue, in the serum of the four groups by ELISA (7,17). Compared with its release in the model and vagotomy groups, nicotine, a classical cholinergic agonist, significantly attenuated release of the chemokines MIP-1α and MCP-1 (Fig. 3A and B), but not MIP-2 (Fig. 3C).

Chemokines control the directed movement of cells expressing their respective receptors. The chemokine receptor CCR2 is bound by MCP-1, thus contributing to the migration of monocytes from the bloodstream into the tissue (18). The present study examined CCR2 expression in the synovial tissue by immunohistochemistry. The results of a semi-quantitative analysis indicated that CCR2 was highly expressed on synoviocytes in mice with CIA but not in normal mice, and nicotine treatment reduced CCR2 expression in the CIA mice (Fig. 4A-C).

ICAM-1 has classically been considered to serve a role in intercellular adhesion. To evaluate the effects of nicotine on the expression of adhesion molecules on vascular endothelial cells, ICAM-1 production was measured in the synovium. Immunohistochemical analysis of mouse synovial sections revealed that ICAM-1 was highly expressed in the model and vagotomy groups. Conversely, ICAM-1 expression was significantly suppressed in nicotine-treated mice (Fig. 5A-C).