Peripheral elimination of the sympathetic nervous system stimulates immunocyte retention in lymph nodes and ameliorates collagen type II arthritis.

Brain, Behavior, and Immunity xxx (2016) xxx–xxx

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Peripheral elimination of the sympathetic nervous system stimulates immunocyte retention in lymph nodes and ameliorates collagen type II arthritis Susanne Klatt, Hubert Stangl, Julia Kunath, Torsten Lowin, Georg Pongratz, Rainer H. Straub ⇑ Laboratory of Experimental Rheumatology and Neuroendocrine Immunology, Department of Internal Medicine, University Hospital Regensburg, Germany

a r t i c l e

i n f o

Article history: Received 27 March 2015 Received in revised form 1 February 2016 Accepted 8 February 2016 Available online xxxx Keywords: Sympathetic nervous system Leukocyte egress Leukocyte entry Secondary lymphoid organ Arthritis

a b s t r a c t Objectives: In collagen type II-induced arthritis (CIA), early activation of the sympathetic nervous system (SNS) is proinflammatory. Here, we wanted to find new target organs contributing to proinflammatory SNS effects. In addition, we wanted to clarify the importance of SNS-modulated immunocyte migration. Methods: A new technique termed spatial energy expenditure configuration (SEEC) was developed to demonstrate bodily areas of high energy demand (to find new targets). We studied homing of labeled cells in vivo, lymphocyte expression of CCR7, supernatant concentration of CCL21, and serum levels of sphingosine-1-phosphate (S1P) in sympathectomized control/arthritic animals. Results: During the course of arthritis, SEEC identified an early marked increase of energy expenditure in draining lymph nodes and spleen (nowhere else!). Although early sympathectomy ameliorated later disease, early sympathectomy increased energy consumption, organ weight, and cell numbers in arthritic secondary lymphoid organs, possibly a sign of lymphocyte retention (also in controls). Elimination of the SNS retained lymph node cells, elevated expression of CCR7 on lymph node cells, and increased CCL21. Serum levels of S1P, an important factor for lymphocyte egress, were higher in arthritic than control animals. Sympathectomy decreased S1P levels in arthritic animals to control levels. Transfer of retained immune cells from draining lymph nodes of sympathectomized donors to sympathectomized recipients markedly increased arthritis severity over weeks. Conclusions: By using the SEEC technique, we identified draining lymph nodes and spleen as major target organs of the SNS. The data show that the SNS increases egress of lymphocytes from draining lymph nodes to stimulate arthritic inflammation. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction The sympathetic nervous system (SNS) plays an important role in the course and development of autoimmune diseases like arthritis (Bellinger et al., 2008; Schaible and Straub, 2014). In collagen type II-induced arthritis (CIA) in mice, early activation of the SNS was proinflammatory, but the SNS suppressed inflammation in later stages of the disease (Härle et al., 2005). Similarly, a dual role of the SNS was described in adjuvant arthritis (Lubahn et al., 2004). Proinflammatory effects in the initial phase of the disease are partly due to a SNS-dependent upregulation of IL-17 secretion from lymph node and spleen cells (Ebbinghaus et al., 2012). During the first hours of inflammation, the SNS is responsible for an ⇑ Corresponding author at: Lab. of Exp. Rheumatology and NeuroendocrinoImmunology, Division of Rheumatology, Department of Internal Medicine, University Hospital, 93042 Regensburg, Germany. E-mail address: [email protected] (R.H. Straub).

increase in vessel leakage, stimulation of sensory nerve fibers, plasma extravasation, migration and chemotaxis of leukocytes to inflamed tissue (Levine et al., 1987, 1988), which are proinflammatory stimuli. In addition, the SNS might influence inflammation by provision of energy-rich fuels because catecholamines via b2-adrenoceptor signaling liberate glucose and free fatty acids needed by the energy-consuming immune system (reviewed in (Bartness et al., 2010; Frauwirth and Thompson, 2004; Ghesquière et al., 2014; Nonogaki, 2000; Straub et al., 2010)). An autoimmune response is energy-demanding with differentiation and proliferation of clonal immune cells and the subsequent tissue-directed inflammatory process (symptomatic phase). As recent calculations demonstrate, a moderately activated immune system in a normally sized human needs approximately 2000 kJ/day, which is 20% of the basal metabolic rate (Frauwirth and Thompson, 2004; Ghesquière et al., 2014; Straub et al., 2010). This demonstrates that bodily energy regulation and cellular bioenergetics are important to serve the stimu-

http://dx.doi.org/10.1016/j.bbi.2016.02.006 0889-1591/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: Klatt, S., et al. Peripheral elimination of the sympathetic nervous system stimulates immunocyte retention in lymph nodes and ameliorates collagen type II arthritis. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.02.006

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S. Klatt et al. / Brain, Behavior, and Immunity xxx (2016) xxx–xxx

lated immune system (Frauwirth and Thompson, 2004; Ghesquière et al., 2014; Straub et al., 2010). In arthritis, we expect that secondary lymphoid organs need much energy, because they are sites of immune cell clonal proliferation. However, in arthritis, energy expenditure in different organs at different time points has never been investigated. In arthritis, energy expenditure might be different in the asymptomatic phase of an autoimmune disease or during apparent inflammation (the symptomatic phase of the disease). Energy expenditure can be studied by measuring oxygen consumption of viable cells in vitro (Buttgereit and Brand, 1995; Ma and Wu, 2008). Moreover, the SNS has a well-known influence on leukocyte mobilization in humans, which was demonstrated under stressful situations or catecholamine infusion (Schedlowski et al., 1996). Leukocyte migration is essential for promoting a crosstalk between cells to ensure adequate immune responses. Homing to lymph nodes and egress is a dynamic process that requires recognition between specific adhesion molecules expressed by leukocytes and cells of postcapillary high endothelial venules (reviewed in Ref. (von Andrian and Mempel, 2003)). The chemokine (C-C motif) receptor 7 (CCR7) and its ligand chemokine (C-C motif) ligand 21 (CCL21) play an important role in cell recruitment to lymph nodes. These molecules promote homing of lymphocytes and dendritic cells to lymphoid tissues but they are also important for egress of cells from lymphoid organs. In contrast to lymph nodes, in the spleen, leukocyte entry seems to be independent of chemokines (Comerford et al., 2013). In addition, sphingosine-1-phosphate (S1P) has emerged as a central mediator of lymphocyte egress (Cyster and Schwab, 2012), and S1P is involved in many cellular processes, including angiogenesis, proliferation and apoptosis (Melendez, 2008; Spiegel and Milstien, 2003). Furthermore, elevated expression of S1P and the S1P type 1 receptor have been demonstrated in the synovium of patients with rheumatoid arthritis (Kitano et al., 2006; Pi et al., 2006), and S1P signaling via S1P type 1 receptor induced synoviocyte proliferation (Pi et al., 2006). Our first aim of the study was the development of a new high throughput method for the measurement of energy consumption in different organs and tissues of arthritic animals during the course of the disease. In a next step, we wanted to characterize the influence of the SNS on energy consumption during the course of asymptomatic and symptomatic disease. This method may show the critically involved target organs in experimental arthritis that increase or decrease normal energy consumption. With the information gathered, we hypothesized that the SNS is an important player for homing and egress of leukocytes to/from lymph nodes and spleen, and we studied the influence of the SNS on CCR7, CCL21 and S1P in control and arthritic animals. 2. Materials and methods 2.1. Animals Male DBA/1J mice (6–8 weeks old) were purchased from Janvier (France). They were housed in specific pathogen-free environment with unrestricted access to chow and water. Five animals lived in one cage, and they were acclimated to the environment during 1 week before commencement of experiments. The Government of the Oberpfalz approved all experiments according to institutional and governmental regulations for animal use (AZ 542531.1-24/06, Az. 54-2532.1-04/13). 2.2. Induction and assessment of arthritis and scoring Arthritis was induced on day 0 by a single injection of 100 lg of bovine type II collagen (CII, Seattle, WA) emulsified in an equal vol-

ume of complete Freund adjuvant (Sigma, Deisenhofen, Germany) intradermally at the base of the tail. Animals were inspected at various time points after the induction of arthritis (days 0, 5, 14, 21, 25, 28, 35, 40, and 55) for joint swelling of toes and ankles. Score points were assigned for inflamed toes, midfeet and ankles. Each limb was scored separately and scored as follows: 0, no swelling; 0.5, mild ankle swelling; up to 2.0, severe ankle swelling. Scores for all toes and ankles were totaled for each mouse. The maximum arthritis score amounted to 12 points per extremity (2 points for each of four toes, 2 points for midfeet and ankle) and 48 per animal. Scoring was performed in a blinded manner. 2.3. Sympathectomy and cell isolation Early sympathectomy was induced by injection of 80 mg 6hydroxydopamine (Sigma) intraperitoneally per kg body weight in 0.1% ascorbic acid on three consecutive days starting on day 8 before immunization (immunization was day 0). Inducing sympathectomy in this manner leads to a reduction of noradrenaline content in the spleen by 75% and a nearly complete loss of tyrosine hydroxylase-positive sympathetic nerve fibers (Härle et al., 2005). Sympathectomy was maintained with a single injection of 80 mg 6-hydroxydopamine per kg body weight on day 14 and day 28 after immunization. In some experiments in non-immunized animals, observation time lasted until day 0 in relation to sympathectomy (on days -8, -7, and -6). This allowed us to investigate the pure effect of the SNS under healthy conditions. In these experiments, control and sympathectomized, non-immunized animals were killed on day 0 after sympathectomy, and cells from spleens and draining inguinal lymph nodes were isolated by meshing the tissue through a 70 lm cell strainer (BD Biosciences, Heidelberg, Germany). Cells were kept cool in complete RPMI 1640 medium. In order to induce erythrocyte lysis, spleen cell suspensions were incubated in 0.84% NH4Cl for 10 min. After a final washing step, cells were counted and viability was determined by trypan blue exclusion. 2.4. Spatial energy expenditure configuration The Sensor DishÒ Reader allows a non-invasive and continuous oxygen measurement in cell and tissue culture in 24-well culture dishes (PreSens GmbH, Regensburg, Germany). A sensor spot that contains a luminescent dye is fixed on the bottom of each well (Fig. 1A). The sensor spot is excited by light from the Sensor DishÒ Reader placed under the 24-well culture dish. Measuring of dissolved oxygen in cell culture medium is based on the principle of dynamic luminescence quenching by oxygen (PreSens GmbH). The luminescence lifetime of the dye depends on the oxygen partial pressure and is converted to oxygen values by using calibration parameters (PreSens GmbH). The Sensor DishÒ Reader was used in the incubator at 37 °C and 5% CO2. Oxygen values were calculated every 3 min for 4–6 h. At around 3.5 h oxygen concentrations were stable and used for calculations. For the determination of oxygen consumption of different organs, mice were sacrificed at different time points after immunization (day 0, 5, 14, 21, 25, 28, 35, 40, and 55) and immediately organs were placed on ice. We investigated spleen, thymus, heart, draining inguinal lymph nodes, adrenals, liver, kidney, brain, gut (rectum, colon, small intestine), thyroid gland, and fat tissue surrounding the draining inguinal lymph nodes. To measure oxygen consumption in different organs as a high throughput method, pieces of a defined size of 4 mm were cut with biopsy punches (GlaxoSmithKline GmbH, Munich, Germany) from every investigated organ (except inguinal lymph nodes and adrenals), weighed and put into one well with 1 ml medium in the oxygen measurement multidishes. For inguinal lymph nodes and adrenal glands,



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Fig. 1. Development of a new high-throughput technique for the measurement of energy expenditure in an experimental model of arthritis. (A) Non-invasive and continuous oxygen measurement in 24-well culture dishes making use of an oxygen-sensitive sensor in the bottom of the well. (B) Oxygen levels in dishes with splenic tissue from 3 control and 3 arthritic mice 33 days after immunization (3 lines = 3 animals). From these raw data, the oxygen consumption was calculated using the time point of 3.5 h. (C) Oxygen consumption in different organs of control and arthritic mice. Each dot represents the oxygen consumption of one organ piece (n = 120, for lymph nodes n = 60, for adrenals n = 30) taken from different mice at different time points throughout the experimental time (day 0, 5, 14, 21, 25, 28, 35, 40, and 55). Box plots represent 10th, 25th, 50th (median), 75th, and 90th percentiles. The Mann Whitney test was used to compare groups. Abbreviations: CIA, collagen type II arthritis.

the entire organ was used. In preliminary studies, we tested the best measure for oxygen consumption. It turned out that oxygen consumption can be given either in lmol/l/h/mg organ (as in Fig. 1) or can be calculated as oxygen consumption of the whole organ corrected by body weight (lmol/l/h/kg b.w. as in Fig. 2). Assay variation from day to day, from animal to animal, organ to organ, cell suspension to cell suspension was below 5%. The composition of cell culture medium was as follows. For measurement of tissue from spleen, thymus, inguinal lymph node, heart, thyroid gland, and inguinal lymph node fat tissue, complete RPMI 1640 was used supplemented with 10% fetal calf serum, 1% penicillin/streptomycin (100 units/ml), 10 mM HEPES, 0.57 mM ascorbic acid, and 1.75 lM 2-mercaptoethanol (all from Sigma). For liver and kidney, DMEM was used supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. For gut, RPMI 1640 medium was used supplemented with 2% fetal calf serum, 1% penicillin/streptomycin, 10 mM HEPES and 8 lg/ml Ciprofloxacin. Oxygen measurement of brain tissue was done in F12 and IMDM medium (1:1) supplemented with 1% penicillin/streptomycin and 10% fetal calf serum (all from Sigma).

RPMI 1640 medium was added to neutralize free dye solution and to stop the staining process. Cells were then washed twice and viability was checked with trypan blue. Before intravenous (i.v.) injection into recipient mice, staining efficacy was determined by FACS analysis (was P95%). A total of 25 106 splenocytes or lymphocytes were injected into control and sympathectomized nonimmunized mice and allowed to migrate for 24 h. Draining inguinal lymph nodes and spleens were then harvested and the frequency of labeled cells in spleens and inguinal lymph nodes was determined by FACS analysis. In order to study the influence of draining inguinal lymph node cells on arthritis, these cells were removed from donors and reinjected i.v. into recipients. Both, donors and recipients, were sympathectomized, immunized, and arthritic. Sympathectomy was performed as described above. Donors and recipients developed mild arthritis from day 21 onwards. On day 33, donors were sacrificed, and 25 106 draining inguinal lymph node cells were injected intravenously into recipient mice. In these experiments a control group with intact SNS was additionally investigated. Arthritis was scored from day 28 until day 55.

2.5. FACS analysis

2.7. Determination of sphingosine-1-phosphate and CCL21

For flow cytometric analysis, 2 106 cells were stained with phycoerythrin (PE) Rat anti-Mouse CD197 (CCR7, Clone 4B12; BD Biosciences) for 30 min in the dark at 4 °C. The respective isotype control PE Rat IgG2a (BD Biosciences) was used as a control for unspecific binding. We analyzed CCR7 surface expression using a FACS Calibur (Becton Dickinson Immunocytometry Systems, San Jose, California), and FACS data were analyzed with FlowJo software (Tree Star Inc, Ashland, Oregon).

Serum was collected on day 18 post immunization by cardiac puncture (a time point shortly before disease outbreak when cell migration is expected and antibody response is observable). Venous blood was obtained from the tail vein (and allowed to clot), centrifuged, and stored at 20 °C before S1P levels were assayed by ELISA. S1P levels in serum from DBA/1 mice were analyzed with a S1P competitive ELISA kit (Echelon Biosciences via Mobitec, Göttingen, Germany). Serum samples were diluted to 1:1000. The sensitivity of the assay was 30 nM. Murine CCL21 concentrations in cell culture supernatants of splenocytes and lymphocytes of control and sympathectomized donors were analyzed by mouse CCL21/6CKine DuoSet ELISA (R&D Systems, Wiesbaden, Germany, assay sensitivity 15 pg/ml).

2.6. Cell labeling and adoptive transfer studies Spleen cells and lymph node immunocytes from nonimmunized control donor Male DBA/1J mice were isolated as described under Section 2.3. They were washed with PBS at room temperature and then stained with PKH26 cell linker kit for general membrane labeling according to instructions (Sigma). Briefly, 3 107 cells were incubated with 4 ll of PKH26 in one milliliter of diluent. After three minutes of gentle shaking, serum containing

2.8. Statistical analysis Results are presented as box plots with the 10th, 25th, 50th (median), 75th, and 90th percentile. The non-parametric


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Fig. 2. Early sympathectomy (SYX) ameliorates disease outcome of collagen induced arthritis (CIA) and enhances oxygen consumption in secondary lymphoid organs. (A) Course of clinical arthritis score over time. (B) Oxygen consumption in draining lymph nodes during the course of arthritis. In (A) and (B) data are given as median with 25th and 75th percentile, and 6 animals were investigated at each time point. (C and D) Oxygen consumption in draining lymph nodes and spleen summarized from all investigated time points (n at least 30 organ pieces per condition). (E and F) Oxygen consumption in liver and kidney tissue summarized from all investigated time points (n at least 30 organ pieces per condition). Each dot represents one organ piece (whole organ in the case of lymph nodes). Abbreviations: b.w., body weight; CIA, collagen type II – induced arthritis; CIA-SYX, CIA plus sympathectomy; Co, control; SYX, sympathectomy.

Mann-Whitney test was used to compare two groups (Sigma Plot, Version 11, Systat Software GmbH, Erkrath, Germany). For comparison of arthritis scores in entire animal groups over time (in Figs. 2A, B and 6), the general linear model with Bonferroni correction for multiple comparisons was used. We considered p-values below 0.05 as statistically significant.

3. Results 3.1. Collagen-induced arthritis enhances energy consumption in draining inguinal lymph nodes and spleen The measurement of oxygen consumption in vitro can be regarded as a measure of local energy expenditure and cell activation. In normal culture medium, oxygen concentration is

approximately 180 lmol/l after 3 h (Fig. 1B, black line). Further experiments with one million cells in the culture dish revealed, that arthritic spleen cells (approximately 40 lmol/l of oxygen, red lines in Fig. 1B) consume more oxygen than control spleen cells (approximately 100 lmol/l of oxygen, green lines, Fig. 1B). Oxygen readings were constant over time and very reproducible from day to day, week to week, and animal to animal (data not shown). From these preliminary experiments onwards, oxygen readings were taken at 3.5 h when oxygen concentration curves were unchanging (Fig. 1B). The SEEC technique allows for parallel investigation of multiple organs of one animal using several 24-well multidishes and several Sensor Dish ReadersÒ. Out of 13 organs investigated, the arthritic spleen and draining inguinal lymphoid nodes showed higher oxygen consumption when compared to control organs (Fig. 1C). In contrast, liver of arthritic animals demonstrated a decreased



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oxygen consumption compared to controls (Fig. 1C). All other organs did not differ in energy consumption in control versus arthritic animals. Lowest oxygen consumption was determined in adipose tissue (Fig. 1C). 3.2. Early sympathectomy ameliorates disease severity and enhances energy consumption in secondary lymphoid organs Early sympathectomy before immunization leads to a milder disease outcome in experimental arthritis (Härle et al., 2005; Levine et al., 1987). Here, we studied whether energy consumption in lymphoid organs changes after sympathectomy. First manifestations of arthritis occurred on day 21 in the CIA group, whereas in the sympathectomy group with CIA joint swelling became obvious on day 28 post immunization (Fig. 2A). This demonstrates a less severe arthritis in sympathectomized animals compared to SNSintact animals (Fig. 2A; p < 0.05 for the comparison of the entire curves of the two groups between day 21 and day 55). Similar results are described later in independent experiments (see below in Section 3.5). The highest score was 25 in the CIA group, and 12 in the sympathectomy group with CIA (Fig. 2A). There were no differences in the incidence of experimental arthritis between the groups (data not shown), and sympathectomy without immunization did not lead to the development of the disease (data not shown). We further monitored oxygen consumption during the course of disease in control animals, arthritic animals, and animals that underwent prior sympathectomy (control-sympathectomy and CIA-sympathectomy) in draining inguinal lymph nodes (Fig. 2B). In arthritic inguinal lymph nodes (red line in Fig. 2B) when compared to control inguinal lymph nodes (green line in Fig. 2B), oxygen consumption was markedly elevated from 6 h after immunization (on day 0) until day 55 (p < 0.001 for comparison of green and red curve). In the two groups that underwent sympathectomy compared to controls, oxygen consumption of inguinal lymph nodes was markedly elevated throughout the entire observation period until day 55 (p < 0.001, blue lines versus green line in Fig. 2B). Using all data from all investigated time points of draining inguinal lymph nodes and spleen, we were able to show that oxygen consumption was higher in CIA and in both sympathectomy groups when compared to controls (Fig. 2C and D). In contrast, we observed reduced energy expenditure in liver in sympathectomized animals compared to SNS-intact controls (Fig. 2E). In addition, CIA reduced energy expenditure in the liver, which was not further reduced by sympathectomy (Fig. 2E). In addition, we observed reduced energy expenditure in kidneys in sympathectomized animals compared to controls, and energy expenditure was reduced in sympathectomized animals with CIA compared to SNS-intact CIA (Fig. 2F). Importantly, draining inguinal lymph nodes and spleen also became heavier in CIA and in both sympathectomy groups (Fig. 3A and B). 3.3. Sympathectomy increased frequency of adoptively transferred inguinal lymph node cells and splenocytes in secondary lymphoid organs Since results of Fig. 3A and B were interpreted as a probable sign of lymphocyte retention in lymphoid organs in control sympathectomized, arthritic, and arthritic sympathectomized animals, we wanted to study if sympathectomy influences frequency of adoptively transferred cells in secondary lymphoid organs. For this purpose, we labeled splenocytes and draining inguinal lymph node cells with the fluorescent dye PKH26 and re-injected labeled cells intravenously.

Fig. 3. Organ weights of lymph nodes (A) and spleen (B). One dot stands for one animal (n at least 15). The weights were measured in different animals at three different time points (day 35, 40, and 55) and demonstrated in one box plot. Box plots represent 10th, 25th, 50th (median), 75th, and 90th percentiles. The Mann Whitney test was used to compare groups. Abbreviations: see legend to Fig. 2.

In sympathectomized compared to control non-immunized animals, significantly more adoptively transferred inguinal lymph node cells were observed in inguinal lymph nodes and spleens (Fig. 4A). In contrast, significantly more adoptively transferred splenocytes were only observed in the spleen of sympathectomized than control non-immunized animals but not in inguinal lymph nodes (Fig. 4B). This phenomenon was paralleled by an increase in cell numbers in draining inguinal lymph nodes in sympathectomized non-immunized animals compared to nonimmunized controls (Fig. 4C), which was not observed in the spleen (data not shown). These results indicate that the SNS, particularly, in draining inguinal lymph nodes decreases cell numbers. 3.4. Early sympathectomy modulates lymph node immunocyte CCR7 expression and CCL21 secretion, and decreases S1P serum concentration in arthritis to control levels The two molecules CCR7 and CCL21 are critical for entry of dendritic cells, T cells, and B cells into draining inguinal lymph nodes, while S1P is critical for the egress of cells from lymph nodes (Comerford et al., 2013). In sympathectomized animals compared to controls, we found elevated expression of CCR7 on inguinal


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Fig. 4. Sympathectomy increases numbers of cells in secondary lymphoid organs of non-immunized healthy mice. (A) PKH26-labeled cells from inguinal lymph nodes recovered in inguinal lymph nodes (LN) and spleen. A total of 25 106 cells from inguinal lymph nodes were labeled with the fluorescent dye PKH26 and injected intravenously into control (Co, n = 10) and sympathectomized animals (SYX, n = 10). After 24 h, inguinal lymph nodes and spleen were removed and cells studied by flow cytometry. (B) PKH26-labeled cells from spleen recovered in draining inguinal lymph nodes (LN) and spleen. A total of 25 106 cells from spleen were labeled with the fluorescent dye PKH26 and injected intravenously into control (Co, n = 10) and sympathectomized animals (SYX, n = 10). After 24 h, inguinal lymph nodes and spleen were removed and cells studied by flow cytometry. (C) Cell numbers in inguinal lymph nodes (n = 18). Viability was determined by trypan blue exclusion test. For all panels: All data are given as box plots with 10th, 25th, 50th (median), 75th, and 90th percentiles. The Mann Whitney test was used to compare groups. Each dot represents one investigated organ.

lymph node cells but not on splenocytes (Fig. 5A). Similarly, CCL21 levels in supernatants of inguinal lymph node cells were increased, which was not observed using splenocytes (Fig. 5B). Both experi-

Fig. 5. Modulation of CCR7 expression, CCL21 secretion, and sphingosine-1phosphate serum concentration by sympathectomy in non-immunized animals. (A) Flow cytometric analysis of CCR7 expression from draining lymph node and spleen cells in control and sympathectomized mice (n = 20 each). (B) CCL21 secretion of in vitro cultured cells from control and sympathectomized mice (n = 20 each). In (A) and (B), cells for analyses were taken on day 0 after prior sympathectomy on days -8, -7, and -6. (C) Influence of collagen type II – induced arthritis and sympathectomy on serum levels of S1P. On day 18 after immunization serum was collected and assayed by ELISA. Abbreviations: CIA, collagen type II – induced arthritis; Co, control mice; LN, draining lymph nodes; S1P, sphingosine-1phosphate; SYX, sympathectomy.

ments indicate that the SNS inhibits important lymph node entry signals. In order to study egress signals, S1P serum concentrations were investigated. Animals with CIA demonstrated increased serum S1P levels when compared to controls (Fig. 5C). Sympathectomy of CIA animals decreased serum S1P concentrations to control levels (Fig. 5C). This indicates that the SNS supports the egress via an increase of serum S1P in the arthritic situation.



3.5. Simulation of release of sympathectomy-retained cells from draining inguinal lymph nodes aggravate arthritis in sympathectomized arthritic recipients Sympathectomy ameliorates arthritis and retains cells in draining inguinal lymph nodes, which might be mediated by CCR7, CCL21, and S1P. However, it is unclear whether sympathectomyretained immune cells have proinflammatory capacities that would aggravate clinical arthritis when released from these lymph nodes. In order to simulate release of immune cells, immune cells were harvested from draining inguinal lymph nodes of sympathectomized and arthritic donor animals, and 25 106 cells were reinjected i.v. into sympathectomized arthritic recipients. The design of experiments is given in Fig. 6A. Sympathectomized animals demonstrated markedly milder arthritis as compared to SNS-intact animals (Fig. 6B, red and green lines). When sympathectomized arthritic animals received draining inguinal lymph node cells of sympathectomized arthritic donor animals, arthritis was markedly aggravated (Fig. 6B, red and black line). This indicates that transferred lymph node cells possess proinflammatory capacities.

4. Discussion This study demonstrates that secondary lymphoid organs like draining inguinal lymph node and spleen consume more energy in arthritic compared to control animals. The other investigated organs did not increase energy consumption, and the liver even demonstrated a decrease presently unexplained. Unexpectedly, removal of the SNS increased energy consumption in draining inguinal lymph nodes and spleen of control animals. This was also observed in arthritic controls and arthritic sympathectomized animals, although sympathectomy markedly reduced disease severity. This was paralleled by heavier secondary lymphoid organs, and higher inguinal lymph node cellularity. This suggested an SNS influence on cell entry into or cell egress from secondary lymphoid organs. The SNS markedly decreased entry signals (CCR7 and CCL21) in inguinal lymph nodes and increased egress signals under inflammatory conditions (S1P), which explains the sympathetic interference with cell migration. Injection of inguinal lymph node cells from sympathectomized arthritic donors into sympathectomized arthritic recipients markedly increased clinical arthritis. In our earlier work, we demonstrated an ameliorating effect of early sympathectomy on arthritis outcome when sympathectomy was maintained by repeated injections of 6-hydroxydopamine until day 45 (Härle et al., 2005). After cessation of 6hydroxydopamine injections at day 45, arthritis scores gradually increased to reach the levels of arthritic animals without sympathectomy (Härle et al., 2005). This demonstrates that the SNS fosters later arthritis when animals had undergone early sympathectomy. Others found a similar anti-inflammatory influence of early sympathectomy in different arthritis models, but they did not observe the animals long enough to see a similar increase in arthritis scores after sympathectomy termination (Bellinger et al., 2008; Ebbinghaus et al., 2012; Levine et al., 1987). Cutaneous immunization experiments with/without sympathectomy support the proinflammatory influence of the SNS (Madden et al., 1989). From this point of view, we can summarize that the SNS has a proinflammatory influence in the early immunization phase of arthritis but reasons for this phenomenon are only partly known. Since the SNS is important in energy provision to the activated immune system (b2-adrenergically driven gluconeogenesis, glycolysis, and lipolysis as reviewed in (Bartness et al., 2010; Frauwirth and Thompson, 2004; Ghesquière et al., 2014; Nonogaki, 2000; Straub et al., 2010)), energy consumption in the body might show

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us some new involved target organs or pathways during the development of arthritis. Thus, we developed a new technique to investigate the spatial energy expenditure configuration (SEEC) in the mouse body. Oxygen consumption measurement as a way to study energy expenditure has been applied in cell suspensions, for example, with the Clark electrode (Buttgereit and Brand, 1995; Dziurla et al., 2010). However, Clark electrodes are difficult to miniaturize, they consume oxygen, they are limited to discrete measurements and difficult to use in high throughput applications (Nagl et al., 2007). Oxygen consumption might also be measured in vivo according to recently published methods (Diepart et al., 2009; Ma and Wu, 2008). However, the in vivo technique is more sensitive to overall changes in oxygen consumption, barometric pressure, temperature, and similar physical aspects. In this project, we managed to set up a new method to measure energy utilization in form of oxygen consumption in vitro. The SEEC method enables us to identify the bodily areas of high energy demand during the course of chronic inflammatory diseases like arthritis. Although we investigated several organs like thymus, liver, kidney, heart, gut, thyroid gland, and brain, we detected a marked increase in energy expenditure only in the draining inguinal lymph nodes and spleen. This was expected since these secondary lymphoid organs are the sites of immune cell clonal proliferation and need considerable energy for activation and proliferation. In further studies, we investigated the influence of early sympathectomy on the outcome of oxygen consumption in draining inguinal lymph nodes and spleen. From an energetic point of view, elimination of the influence of the SNS on reallocation of energy rich fuels should lead to an overall reduced energy expenditure. This reduction may also affect the immune reaction in immunologically active tissue and, thus, the severity of inflammatory reactions. This has been observed in liver and kidneys in this study, which was expected due to the lower availability of energy-rich substrates after loss of the sympathetic influence on metabolism (reviewed in (Bartness et al., 2010; Nonogaki, 2000)). However, it is demonstrated that early sympathectomy unexpectedly increases oxygen consumption in draining inguinal lymph nodes and spleens of arthritic animals but, importantly, also in non-immunized control animals. Since this phenomenon is paralleled by increased weight of these secondary lymphoid organs and increased cellularity in draining inguinal lymph nodes, the SNS might play an important role in cell entry into and cell egress from secondary lymphoid organs. We hypothesized that sympathectomy leads to retention of activated and energy-consuming immune cells that cannot leave to the site of inflammation. For many years, it is known that the SNS influences the numbers of circulating blood cells, which has been nicely demonstrated in humans under stress of parachute jumping and intravenous catecholamine infusion (Moore, 1984; Schedlowski et al., 1996). Under clinical conditions in the emergency unit, one often recognizes leukocytosis in stressful events of acute non-inflammatory diseases such as myocardial infarction, pulmonary embolism, and similar ones. The subject of SNS-driven increase of circulating leukocyte was summarized recently (Dhabhar et al., 2012). Leukocyte recruitment seems to be SNS-driven in a circadian fashion (Scheiermann et al., 2012). In otherwise healthy BALB/C mice, Madden and colleagues demonstrated increased migration of cells from normal mice to lymph nodes of sympathectomized animals using the 51Cr-labeled lymphocyte technique (Madden et al., 1994a). In a first set of experiments, we studied entry of labeled cells into secondary lymphoid organs. We observed that labeled reinjected inguinal lymph node cells were more prevalent in inguinal lymph nodes and spleen of sympathectomized compared to control animals, which confirms earlier work in BALB/C mice (Madden et al., 1994a). Similarly, labeled re-injected spleen cells


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Fig. 6. Immune cells from sympathectomized mice aggravate arthritis in sympathectomized recipients. (A) Schematic representation of transfer experiments. One arthritic group (+SNS) remained sympathetically intact throughout the entire observation period. The other group was sympathectomized prior to immunization (days -8, -7, -6) and 14 and 28 days after immunization (SYX). Of this second group, three independent subgroups were generated: One subgroup (SYX) received a control injection of sodium on day 33. One subgroup was sacrificed in order to remove draining inguinal lymph node cells (indicated by a black cross). Cells of these sympathectomized donors were reinjected on day 33 into the third subgroup of sympathectomized recipients (SYX+). Arthritis was scored from day 28 until day 55. (B) Results of transfer experiments. The explanatory colors are given in panel A. The p-values given were derived from statistical analyses with the general linear model with Bonferroni correction for multiple comparisons using the entire curves from day 35 until day 55. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

were more frequent in spleens of sympathectomized compared to control animals. This was a first indication that the SNS is responsible for blockade of entry or increased egress of labeled cells. The differential effect of either lymph node cells that preferentially home to lymph nodes or splenocytes that preferentially home to spleen is known in the literature (Gallatin et al., 1986; MacKay, 1991). Entry and egress of cells into/from secondary lymphoid organs depends on key molecules such as CCL21/CCR7 (entry) and S1P/

S1P type 1 receptor (egress) as demonstrated in Fig. 7. CCR7 is mainly expressed on various subsets of immune cells and promotes migration towards its ligands CCL19 and CCL21, which are expressed on blood vessel high endothelial venules (HEVs) and also fibroblastic reticular cells in the T cell zone (Comerford et al., 2013). We observed an elevated expression of CCR7 on lymph node immunocytes in sympathectomized mice in comparison to control animals. In addition, CCL21 supernatant levels of cultured inguinal lymph node cells were markedly increased in sympathectomized



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Fig. 7. Entry and egress of cells into and from lymph nodes. The figure was re-structured on the basis of a recent publication (Comerford et al., 2013). Entry of leukocytes happens in peripheral lymphatic vessels (left rectangular area) and over high endothelial venules in lymph nodes (right upper rectangular area). In both situations, the pair of CCR7 and CCL21 is important for entry. Within the lymph nodes, migration into the paracortex (right lower rectangular area) and migration of helper T cells to the blue B cell zone (not shown in a rectangular area) also depend on CCR7/CCL21. Within the blue B cell area, mainly CXCR5/CXCL13 prevail over other factors. Egress of leukocytes depends on a gradient of sphingosine-1-phosphate (sphingosine-1P) generated between low tissue levels in the lymph node and high intraluminal levels in lymphatic veins and efferent lymphatic vessels (right middle rectangular area and central lymph node area at the hilum). Abbreviations: CCL21, chemokine (C-C motif) ligand 21; CCR7, chemokine (C-C motif) receptor 7; CXCL13, chemokine (C-X-C motif) ligand 13; CXCR5, chemokine (C-X-C motif) receptor 5.

compared to control mice. Both mechanisms contribute to a higher entry of T cells, B cells, and dendritic cells in sympathectomized lymph nodes. Since we have not investigated different immune cell subtypes separately, we are unable to say which cell type is mainly responsible for the observed clinical effects demonstrated. The essential molecule for immunocyte egress from secondary lymphoid organs is S1P, which is secreted from endothelial cells into the lumen of the vessel. Since concentrations of S1P in tissue are a 100 times lower than in the serum, a gradient is generated along which leukocytes with the S1P type 1 receptor can migrate towards the vessel lumen (egress) (Fig. 7) (Comerford et al., 2013; Cyster and Schwab, 2012). CCR7 has been shown to counteract this egress signal and sequestrates cells within secondary lymphoid organs (Pham et al., 2008). In our study, the SNS increased S1P serum levels in arthritic mice since sympathectomy reduced increased serum S1P concentration to control levels. The SNS had no influence under control conditions in these experiments, which suggests that an additional pathway is switched-on in arthritic animals only. Such an additional pathway might be endothelial nitric oxide-TNF pathway (De Palma et al., 2006). We think that more proximate measurements of S1P concentrations directly in lymph node veins or efferent lymphatic vessels would have resulted in a clearer picture but retrieving fluid from these sites is technically difficult. In addition, we studied the clinical effect of isolated draining inguinal lymph node cells from arthritic sympathectomized animals by injecting these cells into sympathectomized arthritic recipients. We expected a short-lived aggravation of arthritis, but these experiments demonstrated a marked worsening of clinical arthritis severity over the entire observation period. It is important to mention that the anti-inflammatory effect of sympathectomy in

sympathectomized control animals and in sympathectomized recipient animals declines from day 42 onwards because sympathectomy was only maintained until day 28 (by repeated injections of 6-hydroxydopamine). The reduced effect of 6-hydroxydopamine is typical after 2–3 weeks as demonstrated earlier (Härle et al., 2005). These experiments prove that retained draining inguinal lymph node cells possess proinflammatory capacities. In addition, experiments with sympathectomy show that immune cells become retained and that arthritis severity is strongly reduced. Although we have not tested the transfer of control cells from arthritic SNS-intact animals to arthritic sympathectomized animals, all data suggest that the SNS is responsible for the egress of proinflammatory cells from draining lymph nodes to stimulate a proinflammatory process in the joints and, probably, elsewhere. Yet, we cannot exactly describe the immune cell subtype necessary for these proinflammatory effects, which needs to be studied in future experiments. In our study in DBA/1 mice, we did not investigate proliferation of cells after sympathectomy although others demonstrated an influence on cell proliferation and DNA synthesis (Madden et al., 1989, 1994a). From these earlier studies in BALB/C mice, it seems that sympathectomy increases cell proliferation of entire lymph node cells and also transiently of spleen and bone marrow cells in vivo. Such an influence of sympathectomy seems to depend on the cell type because T cell proliferation was reduced while B cell proliferation was increased (Madden et al., 1994b). Since we have not tested cellular energy consumption either for proliferation compared to cell entry/retention, we are unable to define the more important mechanism for energy expenditure in these organs. Technically it might be very difficult to separate the two possible


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aspects of increased energy expenditure. However, increased retention must play a decisive role because it explains clinical amelioration of arthritis (see paragraph before). In conclusion, using the SEEC technique, we identified secondary lymphoid organs as target organs of the SNS. This is not new but original is the fact that no other organ was involved. The present data support the idea that arthritis amelioration after early sympathectomy depends on sequestration of immune cells within secondary lymphoid organs, particularly, draining lymph nodes. This sympathetic influence prevents recirculation of activated and proinflammatory immune cells to peripheral sites of inflammation, thereby, inducing the beneficial effect. This perfectly fits to data that demonstrate arthritis worsening after cessation of maintenance sympathectomy (Härle et al., 2005). It also fits to the beneficial effects of the immunosuppressive drug fingolimod that retains immunocytes in lymphoid organs (Pitman et al., 2012). From this point of view, drug-induced retention of lymph node immunocytes is a promising therapeutic approach in arthritis. Contribution to the study Susanne Klatt: development of parts of the concept, generation of data, generating draft figures, drafting parts of the paper, final approval. Hubert Stangl: generation of data, revising the draft paper, final approval. Julia Kunath: generation of data, revising the draft paper, final approval. Torsten Lowin: providing study tools and techniques, revising the draft paper, final approval. Georg Pongratz: providing study tools and techniques, revising the draft paper, final approval. Rainer H. Straub: development of the major concept, generating draft and final figures, drafting parts of the paper and revising the final paper, final approval. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This study was sponsored in the context of the DFG Research Unit FOR696 using overhead money. References Bartness, T.J., Shrestha, Y.B., Vaughan, C.H., Schwartz, G.J., Song, C.K., 2010. Sensory and sympathetic nervous system control of white adipose tissue lipolysis. Mol. Cell. Endocrinol. 318, 34–43. Bellinger, D.L., Millar, B.A., Perez, S., Carter, J., Wood, C., Thyagarajan, S., Molinaro, C., Lubahn, C., Lorton, D., 2008. Sympathetic modulation of immunity: relevance to disease. Cell. Immunol. 252, 27–56. Buttgereit, F., Brand, M.D., 1995. A hierarchy of ATP-consuming processes in mammalian cells. Biochem. J. 312, 163–167. Comerford, I., Harata-Lee, Y., Bunting, M.D., Gregor, C., Kara, E.E., Mccoll, S.R., 2013. A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system. Cytokine Growth Factor Rev. 24, 269–283. Cyster, J.G., Schwab, S.R., 2012. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu. Rev. Immunol. 30, 69–94. De Palma, C., Meacci, E., Perrotta, C., Bruni, P., Clementi, E., 2006. Endothelial nitric oxide synthase activation by tumor necrosis factor alpha through neutral sphingomyelinase 2, sphingosine kinase 1, and sphingosine 1 phosphate receptors: a novel pathway relevant to the pathophysiology of endothelium. Arterioscler. Thromb. Vasc. Biol. 26, 99–105. Dhabhar, F.S., Malarkey, W.B., Neri, E., McEwen, B.S., 2012. Stress-induced redistribution of immune cells–from barracks to boulevards to battlefields: a tale of three hormones–Curt Richter Award winner. Psychoneuroendocrinology 37, 1345–1368.

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Gallium nitrate ameliorates type II collagen-induced arthritis in mice.

Water-soluble undenatured type II collagen ameliorates collagen-induced arthritis in mice.

Eupatilin ameliorates collagen induced arthritis.

Functional anatomy of lymph nodes. II. Peripheral lymph-borne mononuclear cells.

CD5 positive B cells in peripheral blood and lymph nodes in rheumatoid arthritis.

Contribution of the spleen, lymph nodes and bone marrow to the antibody response in collagen-induced arthritis in the rat.

The sympathetic nervous system and heart failure.

Stress, the sympathetic nervous system and hypertension.

Substance P ameliorates collagen II-induced arthritis in mice via suppression of the inflammatory response.

Adjuvant arthritis pretreatment with type II collagen and Mycobacterium butyricum.

FTY720 Abrogates Collagen-Induced Arthritis by Hindering Dendritic Cell Migration to Local Lymph Nodes.

Cannabinoid-based drugs targeting CB1 and TRPV1, the sympathetic nervous system, and arthritis.

Proceedings: Effects of ketamine on the peripheral sympathetic nervous system of the rat.

Autoimmunity to type II collagen an experimental model of arthritis.

Humoral and cellular sensitivity to collagen in type II collagen-induced arthritis in rats.

Immunization of adult rats against 2.5 S NGF: effects on the peripheral sympathetic nervous system.

Exposure of female mice to type II collagen reduces susceptibility to collagen-induced arthritis in offspring.

Interactions between ANG II, sympathetic nervous system, and baroreceptor reflexes in regulation of blood pressure.

Locus coeruleus lesions and PCOS: role of the central and peripheral sympathetic nervous system in the ovarian function of rat.

Cannabis and the peripheral nervous system.

Characteristics of function-specific pathways in the sympathetic nervous system.

Fasting, feeding and regulation of the sympathetic nervous system.

Trophic mechanisms in the peripheral nervous system.

Selective response of rat peripheral sympathetic nervous system to various stimuli.