Jill A. Schaller

Chemotactic-like receptors and Aβ peptide induced responses in Alzheimer’s Disease

Evidence suggests that beta-amyloid (Abeta) has chemokine-like properties and may act through formyl chemotactic receptors (FPR) to induce pathophysiologically important functional changes in Alzheimers disease (AD) microglia. We have shown that Abeta 1-42, fibrillar Abeta 1-40, and Abeta 25-35 potentiate the release of interleukin-1beta (IL-1beta) from LPS activated human THP-1 monocytes [26] and LPS primed rat microglia. Moreover, Abeta-stimulated IL-1beta secretion seems to be receptor mediated because it is calcium dependent and requires activation of specific G-proteins [27]. Thus, we have evaluated the ability of Abeta 1-42 to mimic formyl chemotactic peptides in stimulating IL-1beta release from THP-1 monocytes. Several of the formyl chemotactic peptides and Abeta 1-42 significantly enhanced IL-1beta production in THP-1 monocytes. In contrast, a formyl chemotactic receptor antagonist inhibited Abeta 1-42-induced IL-1beta release from both human THP-1 monocytes and primary rat microglia. Further, primary rat microglia grown in culture expressed FPR as demonstrated by immunocytochemistry. Given the multiple pathophysiologic roles IL-1beta may play in AD, agents that block Abeta interactions with formyl chemotactic receptors on microglia might be important antiinflammatory therapeutic targets.

Dual Role for Noradrenergic Innervation of Lymphoid Tissue and Arthritic Joints in Adjuvant-Induced Arthritis

The role of noradrenergic innervation in the disease outcome of adjuvant-induced arthritis (AA) has been examined following (1) systemic administration of guanethidine and (2) local application of 6-hydroxydopamine (6-OHDA) into the lymph nodes that drain the hind limbs (DLN). Sympathetic denervation by these different neurotoxins produced directionally opposite effects on disease outcome. These conflicting findings could be explained from differential denervation of sympathetic nerves in key target tissues that result from different routes of neurotoxin administration. Alternatively, these conflicting data could be due to differences in the mechanisms by which guanethidine and 6-OHDA destroy sympathetic nerve terminals. In this study, we compared disease outcome in AA following systemic and local DLN application of 6-OHDA to determine whether the route of administration is important to the development and progression of AA. Bilateral local DLN application of 6-OHDA or vehicle was performed 1 day before injection of Freunds complete adjuvant (CFA) to induce arthritis. For systemic denervation, 6-OHDA or vehicle was given by ip injections on days 1, 3, and 5 prior to CFA challenge and then once a week. Local DLN application of 6-OHDA resulted in significant increases in dorsoplantar width in arthritic rats by 27 days following CFA treatment compared to those of non-denervated arthritic rats. In contrast, systemic denervation in arthritic rats significantly decreased dorsoplantar widths 27 days after CFA treatment compared to those in sympathetically intact arthritic animals. X-ray analysis confirmed these findings. Further, local DLN application of 6-OHDA exacerbated the disease regardless of whether the neurotoxin was administered prior to immunization with CFA or closer to the time of disease onset. Our findings indicate that the route of 6-OHDA administration for denervation of sympathetic innervation is an important parameter in determining disease outcome, presumably due to differential sympathetic denervation of target tissues that are involved in disease development and progression. 6-OHDA administration into local DLN denervated these lymph nodes, but spared sympathetic innervation of the hind limbs, a pattern of sympathetic denervation that resulted in disease exacerbation. In contrast, systemic 6-OHDA administration which denervated both the arthritic joints and the secondary lymphoid organs attenuated the severity of AA. This study supports a dual role for NA innervation in modulating the severity of AA by innervation of the arthritic joints and lymphoid organs.

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.

Local Application of Capsaicin into the Draining Lymph Nodes Attenuates Expression of Adjuvant-Induced Arthritis

Adjuvant-induced experimental arthritis (AA) was examined in adult male Lewis rats after isolated capsaicin (CAPS)-induced loss of small, nonmyelinated, afferent fibers in lymph nodes draining the site of adjuvant challenge. AA was induced by intradermal injection of Freund’s complete adjuvant (CFA) into the subplantar area of the right hind paw. Controls received similar injections of mineral oil, the vehicle for CFA. One day later, half of the CFA-treated rats and half of the mineral oil-treated rats received injections of CAPS bilaterally into the draining lymph nodes (DLN). The DLN of remaining rats were injected with 50:50 ethanol/sterile physiological saline, the vehicle for CAPS. This paradigm resulted in four groups designated: CFA/CAPS, CFA/vehicle, vehicle/CAPS and vehicle/vehicle. Since substance P (SP) is present in small, nonmyelinated, afferent fibers, the target of the neurotoxin, CAPS, a radioimmunoassay specific for SP was used to verify the loss of these nerve fibers. CAPS injections into the DLN resulted in a loss in SP concentration in the DLN, with no depletion of SP in the spleen or sciatic nerve. These findings support the destruction of SP-containing nerves, which we interpret as verification of the selective loss of small, non-myelinated afferent nerves in the DLN with no significant spread of the neurotoxin to the nearby sciatic nerves which supply small, nonmyelinated, afferent fibers to the hind limb joints. Also, preservation of SP content in spleen indicates CAPS did not circulate via the lymphatic drainage. No chronic inflammation was observed in the fore or hind limbs from rats treated with the vehicle for CFA (vehicle/vehicle, vehicle/CAPS) at any time during the study. In CFA/vehicle-treated rats, bilateral, symmetrical inflammation of the hind limbs was apparent 14 days after challenge with CFA, and became progressively more inflamed through day 20. In contrast, hind limb inflammation in arthritic rats treated with CAPS was not symmetrical. On days 14 and 20 after challenge with CFA, the inflammatory response in the left hind limb, contralateral to the site of CFA injection, was significantly (p < 0.05) attenuated compared with the response seen on the right side of CFA/CAPS-treated rats, and with the response seen in left hind limb of CFA/vehicle-treated animals. In fact, the mean dorsoplantar width of contralateral hind limbs from CFA/CAPS-treated animals was not different from that measured in non-AA control groups. These findings support a role for small, nonmyelinated, sensory nerves that modulate immune responses in DLN in the development and progression of AA in Lewis rats.

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. J. Comp. Neurol. 489:260–273, 2005.

Altered Sympathetic-to-Immune Cell Signaling via β2-Adrenergic Receptors in Adjuvant Arthritis

Adjuvant-induced arthritic (AA) differentially affects norepinephrine concentrations in immune organs, and in vivo β-adrenergic receptor (β-AR) agonist treatment distinctly regulates ex vivo cytokine profiles in different immune organs. We examined the contribution of altered β-AR functioning in AA to understand these disparate findings. Twenty-one or 28 days after disease induction, we examined β 2-AR expression in spleen and draining lymph nodes (DLNs) for the arthritic limbs using radioligand binding and western blots and splenocyte β-AR-stimulated cAMP production using enzyme-linked immunoassay (EIA). During severe disease, β-AR agonists failed to induce splenocyte cAMP production, and β-AR affinity and density declined, indicating receptor desensitization and downregulation. Splenocyte β 2-AR phosphorylation (pβ 2-AR) by protein kinase A (pβ 2-ARPKA) decreased in severe disease, and pβ 2-AR by G protein-coupled receptor kinases (pβ 2-ARGRK) increased in chronic disease. Conversely, in DLN cells, pβ 2-ARPKA rose during severe disease, but fell during chronic disease, and pβ 2-ARGRK increased during both disease stages. A similar pβ 2-AR pattern in DLN cells with the mycobacterial cell wall component of complete Freunds adjuvant suggests that pattern recognition receptors (i.e., toll-like receptors) are important for DLN pβ 2-AR patterns. Collectively, our findings indicate lymphoid organ- and disease stage-specific sympathetic dysregulation, possibly explaining immune compartment-specific differences in β 2-AR-mediated regulation of cytokine production in AA and rheumatoid arthritis.

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 Freunds 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.

Targeting α- and β-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 β2-AR activation, respectively, and indirectly drives humoral immunity by blocking Th1 cytokine secretion. Here, we investigate how β2-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 β2-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 β2-AR, but not α-AR signaling in AA. Aberrant β2-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 β2-AR function in the immune system ameliorates inflammation and joint destruction in AA.

#88 Initial safety and efficacy study demonstrates promise for the use of β2-adrenergic agonists and α-adrenergic antagonists as joint sparing treatments

#87 Androstenetriol improves survival in a trauma-hemorrhage shock model RogerM. Loria , AndreeaC.Marcu ,HansW.Carter , Wayne R. Barbee , Rao R. Ivatury , Kevin R. Ward a,f a Virginia Commonwealth University, Reanimation Engineering Schock Center, USA b Department of Microbiology, Immunology, Pathology and Emergency Medicine c Department of Biostatistics d Department of Physiology and Emergency Medicine e Department of Surgery f Department of Emergency Medicine