Targeting α- and ß-Adrenergic Receptors Differentially Shifts Th1, Th2, and Inflammatory Cytokine Profiles in Immune Organs to Attenuate Adjuvant Arthritis.

The sympathetic nervous system (SNS) regulates host defense responses and restores homeostasis. SNS-immune regulation is altered in rheumatoid arthritis (RA) and rodent models of RA, characterized by nerve remodeling in immune organs and defective adrenergic receptor (AR) signaling to immune cell targets. The SNS typically promotes or suppresses inflammation via α- and ß2-AR activation, respectively, and indirectly drives humoral immunity by blocking Th1 cytokine secretion. Here, we investigate how ß2-AR stimulation and/or α-AR blockade at disease onset affects disease pathology and cytokine profiles in relevant immune organs from male Lewis rats with adjuvant-induced arthritis (AA). Rats challenged to induce AA were treated with terbutaline (TERB), a ß2-AR agonist (600 µg/kg/day) and/or phentolamine (PHEN), an α-AR antagonist (5.0 mg/kg/day) or vehicle from disease onset through severe disease. We report that in spleen, mesenteric (MLN) and draining lymph node (DLN) cells, TERB reduces proliferation, an effect independent of IL-2. TERB also fails to shift T helper (Th) cytokines from a Th1 to Th2 profile in spleen and MLN (no effect on IFN-γ) and DLN (greater IFN-γ) cells. In splenocytes, TERB, PHEN, and co-treatment (PT) promotes an anti-inflammatory profile (greater IL-10) and lowers TNF-α (PT only). In DLN cells, drug treatments do not affect inflammatory profiles, except PT, which raised IL-10. In MLN cells, TERB or PHEN lowers MLN cell secretion of TNF-α or IL-10, respectively. Collectively, our findings indicate disrupted ß2-AR, but not α-AR signaling in AA. Aberrant ß2-AR signaling consequently derails the sympathetic regulation of lymphocyte expansion, Th cell differentiation, and inflammation in the spleen, DLNs and MLs that is required for immune system homeostasis. Importantly, this study provides potential mechanisms through which reestablished balance between α- and ß2-AR function in the immune system ameliorates inflammation and joint destruction in AA.

Altered sympathetic-to-immune cell signaling via ß2-adrenergic receptors in adjuvant arthritis.

Adjuvant-induced arthritic (AA) differentially affects norepinephrine concentrations in immune organs, and in vivo ß-adrenergic receptor (ß-AR) agonist treatment distinctly regulates ex vivo cytokine profiles in different immune organs. We examined the contribution of altered ß-AR functioning in AA to understand these disparate findings. Twenty-one or 28 days after disease induction, we examined ß2-AR expression in spleen and draining lymph nodes (DLNs) for the arthritic limbs using radioligand binding and western blots and splenocyte ß-AR-stimulated cAMP production using enzyme-linked immunoassay (EIA). During severe disease, ß-AR agonists failed to induce splenocyte cAMP production, and ß-AR affinity and density declined, indicating receptor desensitization and downregulation. Splenocyte ß2-AR phosphorylation (pß2-AR) by protein kinase A (pß2-AR(PKA)) decreased in severe disease, and pß2-AR by G protein-coupled receptor kinases (pß2-AR(GRK)) increased in chronic disease. Conversely, in DLN cells, pß2-AR(PKA) rose during severe disease, but fell during chronic disease, and pß2-AR(GRK) increased during both disease stages. A similar pß2-AR pattern in DLN cells with the mycobacterial cell wall component of complete Freund's adjuvant suggests that pattern recognition receptors (i.e., toll-like receptors) are important for DLN pß2-AR patterns. Collectively, our findings indicate lymphoid organ- and disease stage-specific sympathetic dysregulation, possibly explaining immune compartment-specific differences in ß2-AR-mediated regulation of cytokine production in AA and rheumatoid arthritis.

Preclinical efficacy of sodium narcistatin to reduce inflammation and joint destruction in rats with adjuvant-induced arthritis.

Current therapies for the treatment of rheumatoid arthritis (RA) do not work for all patients, can lose efficacy over time, and can have significant side effects. The discovery of new, effective therapies for RA remains an unmet medical need. The Amaryllidaceae isocarbostyril narciclasine was previously shown to prophylactically reduce paw swelling in rats with adjuvant-induced arthritis (AA). In this study, the efficacy of sodium narcistatin (SNS), a water-soluble cyclic phosphate pro-drug of narciclasine, was assessed in AA rats for anti-inflammatory and bone-sparing properties after disease onset. AA rats were given daily intraperitoneal injections of SNS (1.75, 3.5, or 5 mg/kg/day, in 500 µl sterile endotoxin-free saline) or saline from disease onset through severe disease stages. Footpad widths and radiographic scoring were used as indicators of inflammation and joint destruction, respectively. Ex vivo cytokine production by peripheral blood mononuclear cells (PMBC), splenocytes, and draining lymph node (DLN) cells were determined using ELISAs. SNS treatment dose-dependently reduced joint inflammation (~70%) and bone loss (~50%) compared with AA controls. SNS treatment also reduced spleen weight (without affecting body weight), pro-inflammatory cytokine production by PMBC, splenocytes, and DLN cells, and site-dependently altered T-helper (Th)1-/Th2-type and anti-inflammatory cytokine profiles. SNS dramatically reduces inflammation and has bone-sparing properties, possibly by reducing immune cell pro-inflammatory cytokine production. Our findings support the development of SNS as a therapeutic for RA.

Adjuvant-induced arthritis induces c-Fos chronically in neurons in the hippocampus.

Chronic pain, sickness behaviors, and cognitive decline are symptoms in rheumatoid arthritis. In the adjuvant-induced arthritis Lewis rat model, we examined the dynamics of c-Fos expression in the hippocampus, a brain region important for these symptoms. Brain sections were stained for c-Fos using immunohistochemistry. c-Fos-positive nuclei were counted in CA1, CA2, CA3 and the dentate gyrus of the dorsal hippocampi from rats receiving no treatment or base-of-the-tail injections of (1 or 2) incomplete or complete Freund's adjuvant (low- or high-dose), (3), Mycobacterium butyricum cell wall suspended in saline, or (4) saline, and sacrificed 4, 14, 21, or 126days post-immunization. Disease severity was evaluated by dorsoplantar foot pad widths and X-ray analysis. We report sustained dose- and subfield-dependent c-Fos expression with arthritis, but transient expression in nonarthritic groups, suggesting long-term genomic changes in rheumatoid arthritis that may be causal for behavioral changes, adaptation to chronic pain and/or cognitive decline associated with disease.

Differences in the injury/sprouting response of splenic noradrenergic nerves in Lewis rats with adjuvant-induced arthritis compared with rats treated with 6-hydroxydopamine.

Sympathetic nerves in the spleen undergo an injury and sprouting response with development of adjuvant-induced arthritis (AA), a model of rheumatoid arthritis (RA). The objective of the present study was to determine whether this injury and sprouting response is disease-specific or occurs in a non-specific manner similar to injury and sprouting responses following sympathectomy with specific neurotoxins. Changes in noradrenergic (NA) innervation in spleens from Lewis rats 28 days following adjuvant treatment to induce arthritis and/or local 6-hydroxydopamine (6-OHDA) treatment to destroy NA nerves were examined using immunocytochemistry for tyrosine hydroxylase (TH). We observed significant increases in sympathetic innervation of hilar regions, sites of nerve entry into the spleen, and a striking decline in innervation of splenic regions distant to the hilus in arthritic compared to non-arthritic rats. While increased hilar and decreased distal NA innervation in arthritic rats was strikingly similar to that of non-arthritic 6-OHDA-treated rats, there were differences in splenic compartments innervated by sympathetic nerves between these groups. In 6-OHDA-treated rats, NA nerves re-innervated splenic compartments normally innervated by sympathetic nerves. In arthritic rats, sympathetic nerves returned to normally innervated splenic compartments, but also abundantly innervated red pulp. These findings suggest that splenic sympathetic nerves undergo a disease-associated injury/sprouting response with disease development that alters the normal pattern and distribution of NA innervation. The altered sympathetic innervation pattern is likely to change NA signaling to immune cell targets, which could exert long-term regulatory influences on initiation, maintenance, and resolution of immune responses that impact disease pathology.

Changes in the density and distribution of sympathetic nerves in spleens from Lewis rats with adjuvant-induced arthritis suggest that an injury and sprouting response occurs.

Previously we demonstrated reduced norepinephrine concentrations in spleens from Lewis rats with adjuvant-induced arthritis (AA), an animal model of rheumatoid arthritis. This study extends these findings, examining the anatomical localization and density of sympathetic nerves in the spleen with disease development. Noradrenergic (NA) innervation in spleens of Lewis rats was examined 28 days following adjuvant treatment to induce arthritis or vehicle for the adjuvant by using fluorescence histochemistry for catecholamines, with morphometric analysis and immunocytochemistry for tyrosine hydroxylase. In AA rats, sympathetic nerve density in the hilar regions, where NA nerves enter the spleen, was increased twofold over that observed in vehicle-treated rats. In contrast, there was a striking twofold decline in the density of NA nerves in splenic regions distal to the hilus in arthritic rats compared with nonarthritic rats. In both treatment groups, NA nerves distributed to central arterioles, white pulp regions, trabeculae, and capsule. However, NA nerve density was reduced in the white pulp but was increased in the red pulp in AA rats compared with non-AA rats. These findings indicate an injury/sprouting response with disease development whereby NA nerves die back in distal regions and undergo a compensatory sprouting response in the hilus. The redistribution of NA nerves from white pulp to red pulp suggests that these nerves signal activated immune cells localized in the red pulp in AA. Although the mechanisms of this redistribution of NA nerves into the red pulp are not known, it may be due to migration from white pulp to red pulp of target immune cells that provide trophic support for these nerves. The redistribution of NA nerves into the red pulp may be critical in modulating immune functions that contribute to the chronic inflammatory stages of arthritis.

The importance of timing of adrenergic drug delivery in relation to the induction and onset of adjuvant-induced arthritis.

Stressful events often precede onset and exacerbate established rheumatic diseases. There are numerous reports of abnormal autonomic function in rheumatoid arthritis (RA) patients. Targeting the sympathetic nervous system (SNS) with adrenergic receptor (AR) drugs in RA patients and animal models of the disease have revealed mixed results, with treatments inhibiting and exacerbating disease pathology. We tested the hypothesis that variability in disease outcome following adrenergic drug treatment is due to different roles played by the SNS at different disease stages. The contribution of beta2- and alpha-AR subtypes to disease pathology was studied at different disease stages in adjuvant-induced arthritis (AA), an animal model of RA. Lewis rats were given twice-daily intraperitoneal (i.p.) injections of an alpha-AR antagonist (phentolamine: 500 microg/kg) or a beta2-AR agonist (terbutaline: 1200 microg/day), initiated at adjuvant challenge or disease onset, and continued through severe disease. Both adrenergic therapies, when initiated at adjuvant challenge exacerbated disease pathology. In contrast, SH1293, an adrenergic drug that targets both alpha- and beta-AR (300 microg/day; twice-daily), initiated at adjuvant challenge did not exacerbate disease severity. Additionally, the same treatment regimen of phentolamine, terbutaline or SH1293 initiated at disease onset attenuated joint-inflammation and dramatically reduced bone destruction in the arthritic hind limbs. These data support the SNS playing different roles in disease pathology preclinically and after disease onset. Given current drug therapies are not effective in preventing bone destruction, these data support using adrenergic drugs as bone sparing treatments in RA.