Proinflammatory cytokines and sickness behavior in rheumatic diseases.

This review describes mechanisms of immune-to-brain signaling that may contribute to disease-related changes in mood, affect and behavior in chronic inflammatory rheumatic diseases. The central nervous system (CNS) modulates immune function by signaling target cells of the immune system through autonomic and neuroendocrine pathways. These immune cells relay information back to autonomic, limbic and cortical areas of the CNS to affect neural activity and consequently modify behavior, hormone release and autonomic function. In this manner, immune cells function as a sense organ, informing the CNS of peripheral events relating to infection and injury. Equally important, homeostatic mechanisms are needed at all levels to turn off the immune response when the pathogen and injurious condition are eliminated and the repair process is completed. In individuals with chronic inflammatory diseases, such as rheumatoid arthritis (RA), there is a failure of the homeostatic regulation leading to long-term immune activation that has serious health consequences. Rheumatic disorders constitute a challenge to major psychological adaptation resources leading to higher rates of psychological disorders compared with the general population. Thus the relationship between disease pathology and psychological well being is complex.

Potential use of drugs that target neural-immune pathways in the treatment of rheumatoid arthritis and other autoimmune diseases.

Many autoimmune disorders share two common features, dysregulation of the immune system and stress pathways. Two stress pathways, the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS), regulate immune system responses, through release of corticosteroids and norepinephrine (NE), respectively. These neuromediators act on immune cells via specific receptors on their surface to modulate the production of key regulatory cytokines. Glucocorticoids modulate immune responses by glucocorticoid binding to cytoplasmic glucocorticoid receptors within target cells. NE regulates immune responses through interaction with plasma membrane beta- or alpha-adrenergic receptors (AR). Both NE and glucocorticoids promote humoral immunity by altering macrophages and T cell cytokine production after an antigen challenge. Glucocorticoids and NE do this by inhibiting interleukin (IL)-12, and interferon (IFN)-gamma, which drives cell-mediated immunity. Additionally, catecholamines drive humoral immunity by stimulating macrophage IL-10 production. These catecholamine effects are mediated largely via beta(2)-AR activation. Both glucocorticoids and NE inhibit inflammation. However, under some circumstances NE promotes inflammation through interaction with macrophage alpha1-AR and subsequent increases in tumor necrosis factor alpha (TNFalpha production. Although macrophages do not normally express alpha(1)-AR, expression of this receptor on macrophages and monocytes occurs in some disease states, including rheumatoid arthritis (RA). Through these mechanisms the HPA axis and the SNS influence the course and progression of RA. Thus, the HPA axis and the SNS are likely to play key roles in the pathology of RA. Furthermore, therapeutic agents targeting the neural pathways that normally regulate immune system homeostasis may prove beneficial for treating RA and other autoimmune diseases.

Strain differences in the expression of corticotropin-releasing hormone immunoreactivity in nerves that supply the spleen and thymus.

The existence of nerve fibers containing corticotropin-releasing hormone (CRH) immunoreactivity in primary and secondary lymphoid organs from three strains of young adult male rats was examined. Spleens and thymuses from Fischer 344 (F344), Sprague-Dawley (SD) and Lewis (LEW) rats were prepared for immunocytochemistry using antisera directed against CRH. In F344 and SD rats, we were unable to demonstrate CRH-immunoreactive nerves in either the thymus or the spleen. Despite the lack of CRH-containing nerves, CRH immunoreactivity was present in pleotropic cells in the septum, cortex and medulla of the thymus, and in the red and white pulp of spleens from F344 and SD rats. In contrast, CRH+ nerves were found in thymuses and spleens from LEW rats. CRH+ nerves coursed in the interlobular septa, capsule, cortex and medulla of the LEW rat thymus. Large CRH-immunoreactive nerve bundles were present in the hilar region of the LEW rat spleen, and individual CRH+ fibers coursed in the capsule, trabeculae, red pulp, venous sinuses and marginal zone of the white pulp of the spleen. These findings indicate strain differences in neurotransmitter-specific nerves that innervate the rat spleen and thymus under basal conditions.

Effects of interleukin-2 on the expression of corticotropin-releasing hormone in nerves and lymphoid cells in secondary lymphoid organs from the Fischer 344 rat.

This study examined the influence of interleukin (IL)-2 on corticotropin releasing hormone (CRH) immunoreactivity in the Fischer 344 (F344) rat spleen. Rats were given either vehicle or 1, 10, 25, 50, 100, or 200 ng of human recombinant (hr)IL-2 by intraperitoneal (i.p.) injection, and were sacrificed 0.5, 1, 4, 12, or 24 h after treatment. Spleens and mesenteric lymph nodes were prepared for immunocytochemistry to localize CRH. In spleens from vehicle-treated animals, CRH immunoreactivity was present in several types of cells of the immune system, but CRH(+) nerves were not observed in either spleens or lymph nodes from vehicle-treated animals. Treatment with IL-2 induced CRH expression in nerves in the spleen in a dose- and time-dependent manner. CRH(+) nerves were not found in the mesenteric lymph nodes after IL-2 treatment, instead a dramatic time- and dose-dependent accumulation of CRH(+) cells (resembling small lymphocytes and large granular mononuclear cells) in the cortex and medulla. These findings indicate that IL-2 stimulates the synthesis of CRH in nerves that innervate the F344 rat spleen, and promote the appearance of CRH(+) immunocytes into draining mesenteric lymph nodes.

Chemotactic-like receptors and Abeta 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 Alzheimer's 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.

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.

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 Freund's 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.

Amyloid-beta induces chemokine secretion and monocyte migration across a human blood--brain barrier model.

BACKGROUND: Aside from numerous parenchymal and vascular deposits of amyloid beta (A beta) peptide, neurofibrillary tangles, and neuronal and synaptic loss, the neuropathology of Alzheimer's disease is accompanied by a subtle and chronic inflammatory reaction that manifests itself as microglial activation. However, in Alzheimer's disease, alterations in the permeability of the blood-brain barrier and chemotaxis, in part mediated by chemokines and cytokines, may permit the recruitment and transendothelial passage of peripheral cells into the brain parenchyma. MATERIALS AND METHODS: Human monocytes from different donors were tested for their capacity to differentiate into macrophages and their ability to secrete cytokines and chemokines in the presence of A beta 1-42. A paradigm of the blood-brain barrier was constructed utilizing human brain endothelial and astroglial cells with the anatomical and physiological characteristics observed in vivo. This model was used to test the ability of monocytes/macrophages to transmigrate when challenged by A beta 1-42 on the brain side of the blood-brain barrier model. RESULTS: In cultures of peripheral monocytes, A beta 1-42 induced the secretion of proinflammatory cytokines TNF-alpha, IL-6, IL-1 beta, and IL-12, as well as CC chemokines MCP-1, MIP-1 alpha, and MIP-1 beta, and CXC chemokine IL-8 in a dose-related fashion. In the blood-brain barrier model, A beta 1-42 and monocytes on the brain side potentiated monocyte transmigration from the blood side to the brain side. A beta 1-42 stimulated differentiation of monocytes into adherent macrophages in a dose-related fashion. The magnitude of these proinflammatory effects of A beta 1-42 varied dramatically with monocytes from different donors. CONCLUSION: In some individuals, circulating monocytes/macrophages, when recruited by chemokines produced by activated microglia and macrophages, could add to the inflammatory destruction of the brain in Alzheimer's disease.

Norepinephrine content in primary and secondary lymphoid organs is altered in rats with adjuvant-induced arthritis.

Chemical sympathectomy of secondary lymphoid organs with sparing of the hind limbs exacerbates adjuvant-induced arthritis (AA) in Lewis rats supporting a role for noradrenergic (NA) innervation of the immune system in AA pathology. The present study examines sympathetic innervation of lymphoid organs from Lewis rats 32 days after treatment with complete Freund's adjuvant (CFA) or vehicle using fluorescence histochemistry for localization of catecholamines (CA) and high-performance liquid chromatography with electrochemical detection (LCEC) for measurement for norepinephrine. The thymus from AA rats was significantly reduced in size, while secondary lymphoid organs, i.e., spleen and draining lymph nodes (DLN), were significantly enlarged compared with that seen in vehicle-treated controls. Fluorescence histochemistry revealed no apparent differences in the density of NA innervation, or the intensity of staining in sympathetic nerves in any of the secondary lymphoid organs from AA rats compared with that observed in control animals. However, there was an apparent increase in the density of NA nerve fibers in the thymus of AA rats. Norepinephrine (NE) concentration (pmol NE per g or mg wet weight), in the thymus from AA rats was significantly increased. Conversely, a significant decrease in splenic and lymph node NE concentration was measured in adjuvant-treated animals compared with that seen in vehicle-treated rats. Total NE content (pmol NE per whole organ weight) in lymphoid organs was not altered, except in popliteal lymph nodes (PLN), where it was increased. Collectively, our findings suggest that changes in NA innervation of lymphoid organs from AA rats result largely from increases or decreases in organ mass. Since NE released from NA nerves acts in a paracrine fashion, changes in lymphoid tissue volume that result from enhanced proliferation, migration, or cell death can make a significant difference in the availability of NE for interaction with immune target cells in these organs, even in the absence of a change in NE metabolism. Decreased thymic weight and increased spleen and lymph node weight should increase and decrease NE availability for interaction with target cells, respectively. Additionally, in PLN (a site where the highest concentration of antigen is encountered) an increase in total NE content suggests compensatory changes in NE metabolism.

beta-Amyloid-induced IL-1 beta release from an activated human monocyte cell line is calcium- and G-protein-dependent.

The proinflammatory cytokine, interleukin-1 (IL-1) is elevated in the Alzheimer's disease (AD) brain. Studies from our laboratory have demonstrated that beta-amyloid (A beta) 1-42, fibrillar A beta 1-40 and A beta 25-35 induce the release of IL-1 beta from activated THP-1 cells, a human monocyte cell line. A beta also is chemotactic for primary rodent microglia and peritoneal macrophages. We hypothesize that A beta is a chemokine and induces these responses by interaction with chemotactic receptors. If this is true, then these A beta-induced responses should be calcium-dependent and require activation of pertussis toxin-sensitive G-proteins. To test this hypothesis, THP-1 cells were grown in culture with lipopolysaccharide (LPS) and incubated with A beta 1-42 (5 muM) in the presence and absence of a calcium chelator, an inhibitor of intracellular calcium mobilization, a calcium channel blocker, or pertussis toxin, a bacterial endotoxin which uncouples G proteins from receptors by catalyzing the ADP ribosylation of cysteine near the carboxy-terminus of the alpha subunit. The media was collected and IL-1 beta present in the media was measured using an ELISA. Treatment of LPS-activated THP-1 cells with A beta 1-42 significantly elevated IL-1 beta released into the media as previously shown. Addition or ethylene glycol-bis (beta-aminothyl ether) N,N,N'N'-tetraacetic acid (EGTA) (0.5 mM), a calcium chelator, to the media blocked A beta-induced IL-1 beta release, but had no effect on LPS-activated THP-1 cell release of IL-1 beta. The presence of 3,4,5-trimethoxybenzoic acid 8-(diethyl amino)-octyl ester (TMB-8), an inhibitor of intracellular calcium mobilization, as well as nickel chloride, a non-specific calcium channel blocker, in the media also inhibited A beta-induced IL-1 release from LPS-activated THP-1 cells. IL- 1 beta release from activated THP-1 monocytes incubated with TMB-8 and nickel chloride without A beta remained at baseline values. Pretreatment of THP-1 monocytes with pertussis toxin for 4 h, followed by LPS activation and incubation with A beta, antagonized the release of IL-1 beta from these cells, but did not alter IL-1 beta release from activated THP-1 monocytes. These data suggest that A beta-induced IL-1 beta release from these cells is calcium-dependent and requires the activation of specific G-proteins. These findings are consistent with known second messengers that are activated following stimulation of chemotactic receptors.

Vasoactive intestinal polypeptide (VIP) innervation of rat spleen, thymus, and lymph nodes.

In the thymus, VIP-positive (+) fibers were found in the capsular/septal system, cortex, and medulla. In the spleen, VIP+ nerves coursed along large arteries and central arterioles, and in the white pulp, venous/trabecular system, and red pulp. Splenic VIP innervation was more robust in Long-Evans hooded rats than in Fischer 344 rats. VIP+ nerves in mesenteric lymph nodes were found in the cortex, and along the cortical vasculature and medullary cords. No VIP innervation was observed in popliteal lymph nodes. Immunocytes also were VIP+, suggesting that both neural and cellular synthesis of VIP contributes to VIP concentration in lymphoid organs. Surgical sympathectomy did not alter splenic or thymic VIP content, respectively, and VIP innervation of these organs was not altered, suggesting an origin for VIP+ nerves other than the sympathetic nervous system.

beta-Amyloid induces increased release of interleukin-1 beta from lipopolysaccharide-activated human monocytes.

Previous reports have demonstrated that IL-1 is elevated in the Alzheimer's disease brain. We propose that beta-amyloid (A beta) in senile plaques triggers microglial interleukin-1(IL-1) release. Since microglia and monocytes have similar lineage and functions, the human monocyte cell line, THP-1, was used to determine whether A beta peptides can stimulate release of IL-1 beta. THP-1 cells were grown in culture with LPS and incubated with various A beta peptides (0.5-10 microM). IL-1 released into the medium was measured using either an IL-1 beta ELISA or an IL-1 bioassay. Treatment of activated THP-1 cells with A beta 25-35, fibrillar A beta 1-40, or A beta 1-42 significantly elevated IL-1 beta release. A beta 25-35 with a scrambled sequence or non-fibrillar A beta 1-40 did not significantly change IL-1 beta release from activated THP-1 cells. The A beta 25-35- and fibrillar A beta 1-40 induced IL-1 beta release was dose-dependent. IL-1 released following treatment with A beta 25-35 and measured using an IL-1 bioassay gave similar results. The present report provides evidence that A beta is capable of elevating release of functional IL-1 beta, a potent pro-inflammatory cytokine, from macrophages/microglia and provides support that a chronic local inflammatory response is an ongoing phenomenon within and surrounding senile plaques.

Application of 6-hydroxydopamine into the fatpads surrounding the draining lymph nodes exacerbates adjuvant-induced arthritis.

Adjuvant-induced arthritis (AA) was examined in Lewis rats following local injection of 6-hydroxydopamine (6-OHDA) into the fatpads of the popliteal and inguinal lymph nodes which drain the hindlimbs (DLN). This method of 6-OHDA treatment resulted in noradrenergic (NA) denervation of DLN, spleen, and other organs in the peritoneal cavity, while sparing NA nerve fibers in the hindlimbs. Sympathectomy exacerbated the inflammation and osteopathic destruction of arthritic joints. Significant increases in dorsoplantar width in arthritic rats following denervation were observed by day 27 following immunization compared to nondenervated arthritic animals. Radiographic evaluation on day 27 after immunization confirmed the inflammation of soft tissue and revealed deterioration of bones of the ankle joint in both AA groups compared with the control groups; more extensive joint damage was apparent in arthritic rats following denervation compared to nondenervated arthritic rats. These findings suggest that the NA innervation of DLN and spleen (and possibly other organs of the peritoneal cavity) plays a regulatory role in the expression of AA. These data supports the hypothesis that absence of NA innervation in lymphoid organs during initiation, onset, and progression of the disease results in exacerbation of AA.

The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation.

Evidence for VIP influences on immune function comes from studies demonstrating VIP-ir nerves in lymphoid organs in intimate anatomical association with elements of the immune system, the presence of high-affinity receptors for VIP, and functional studies where VIP influences a variety of immune responses. Anatomical studies that examine the relationship between VIP-containing nerves and subpopulations of immune effector cells provide evidence for potential target cells. Additionally, the presence of VIP in cells of the immune system that also possess VIP receptors implies an autocrine function for VIP. The functional significance of VIP effects on the immune system lies in its ability to help coordinate a complex array of cellular and subcellular events, including events that occur in lymphoid compartments, and in musculature and intramural blood circulation. Clearly, from the work described in this chapter, the modulatory role of VIP in immune regulation is not well understood. The pathways through which VIP can exert an immunoregulatory role are complex and highly sensitive to physiological conditions, emphasizing the importance of in vivo studies. Intracellular events following activation of VIP receptors also are not well elucidated. There is additional evidence to suggest that some of the effects of VIP on cells of the immune system are not mediated through binding of VIP to its receptor. Despite our lack of knowledge regarding VIP immune regulation, the evidence is overwhelming that VIP can interact directly with lymphocytes and accessory cells, resulting in most cases, but not always in cAMP generation within these cells, and a subsequent cascade of intracellular events that alter effector cell function. VIP appears to modulate maturation of specific populations of effector cells, T cell recognition, antibody production, and homing capabilities. These effects of VIP are tissue-specific and are probably dependent on the resident cell populations within the lymphoid tissue and the surrounding microenvironment. Different microenvironments within the same lymphoid tissue may influence the modulatory role of VIP also. Effects of VIP on immune function may result from indirect effects on secretory cells, endothelial cells, and smooth muscle cells in blood vessels, ducts, and respiratory airways. Influences of VIP on immune function also may vary depending on the presence of other signal molecules, such that VIP alone will have no effect on a target cell by itself, but may greatly potentiate or inhibit the effects of other hormones, transmitters, or cytokines. The activational state of target cells may influence VIP receptor expression in these cells, and therefore, may determine whether VIP can influence target cell activity. Several reports described in this chapter also indicate that VIP contained in neural compartments is involved in the pathophysiology of several disease states in the gut and lung. Release of inflammatory mediators by cells of the immune system may destroy VIP-containing nerves in inflammatory bowel disease and in asthma. Loss of VIPergic nerves in these disease states appears to further exacerbate the inflammatory response. These studies indicate that altered VIP concentration can have significant consequences in terms of health and disease. In addition, the protective effects of VIP from tissue damage associated with inflammatory processes described in the lung also may be applicable to other pathological conditions such as rheumatoid arthritis, anaphylaxis, and the swelling and edema seen in the brain following head trauma. While VIP degrades rapidly, synthetic VIP-like drugs may be developed that interact with VIP receptors and have similar protective effects. Synthetic VIP-like agents also may be useful in treating neuroendocrine disorders associated with dysregulation of the hypothalamic-pituitary-adrenal axis, and pituitary release of prolactin.

Acetylcholinesterase staining and choline acetyltransferase activity in the young adult rat spleen: lack of evidence for cholinergic innervation.

Acetylcholinesterase (AChE) staining in spleens from young adult Sprague-Dawley rats was examined following several denervation paradigms to determine the source of splenic AChE+ nerve fibers. In spleens from all control groups, AChE+ neural-like profiles were present along the vasculature and in the trabeculae. AChE+ reactivity also was present in lymphoid and reticular cells in the spleen, and in neuronal cell bodies in the superior mesenteric-coeliac ganglion (SM-CG). Neurochemical analysis revealed no significant choline acetyltransferase activity in spleens from control animals. Surgical removal of the SM-CG resulted in a total loss of both noradrenergic (NA) and AChE+ nerve profiles, as well as a loss of AChE staining in nonneural compartment in the spleen. On Days 1 and 3 after treatment, chemical sympathectomy with 6-hydroxydopamine also resulted in a loss of both NA and AChE nerve profiles in the spleen, except for a few resistant fibers in the hilar region. AChE reactivity in nonneural compartments also was diminished in chemically denervated regions of the spleen. AChE staining in both neural and nonneural profiles progressively increased from 10 to 56 days after chemical sympathectomy, with a time course and distribution pattern similar to NA fibers reinnervating the spleen. AChE+ staining was preserved following bilateral vagal nerve transection. The miniscule splenic levels of choline acetyltransferase suggest that at best, only a small density of cholinergic nerves distribute to the rat spleen. Further, what cholinergic innervation is present does not arise from the vagus nerve as suggested in the earlier literature. Collectively, the overlapping distribution of AChE+ and NA nerve profiles in spleen and parallel loss of both population of nerve fibers following surgical and chemical sympathectomy support the presence of AChE in NA nerves colocalized with norepinephrine, and thus make AChE+ staining an inappropriate marker for cholinergic innervation in the rat spleen.

Noradrenergic and peptidergic innervation of secondary lymphoid organs: role in experimental rheumatoid arthritis.

Noradrenergic (NA) and peptidergic nerve fibres are present in both primary and secondary lymphoid organs, distributing with the vasculature, trabecular and capsular smooth muscle, and within the parenchyma among cells of the immune system. NA nerve terminals directly abut lymphocytes and macrophages in spleen and lymph nodes. In these organs, norepinephrine has fulfilled the basic criteria for neurotransmission with cells of the immune system as targets. In vitro and in vivo studies have demonstrated NA modulation of primary and secondary antibody responses, cytotoxic T cell responses, natural killer cell activity, and proliferation and differentiation of both T and B lymphocytes. Substance P (SP) has been shown to modulate inflammatory responses, lymphocyte proliferation, and other immunologic reactivity. We investigated the role of NA and SP nerve fibres within lymph nodes in experimental allergic auto-immune arthritis in Lewis rats. Denervation of NA nerve fibres in popliteal and inguinal lymph nodes with 6-hydroxy-dopamine resulted in earlier onset and enhanced severity of arthritic changes as well as inflammation in bilaterally induced experimental arthritis, while denervation of SP nerve fibres in popliteal and inguinal lymph nodes with capsaicin resulted in delayed onset and diminished severity of the inflammatory changes ipsilaterally, and prevention of contralateral arthritic changes in unilaterally induced experimental arthritis. These findings suggest that NA and SP nerve fibres in lymph nodes can modulate the time course of onset and the severity of experimental arthritis in Lewis rats. These modulatory effects are distinctly different from the effects of NA and SP nerve fibres in the joints themselves.

Innervation of lymphoid organs and implications in development, aging, and autoimmunity.

We now have substantial evidence demonstrating noradrenergic sympathetic and peptidergic innervation of both primary and secondary lymphoid organs. We have established criteria for norepinephrine, and some of the neuropeptides, as neurotransmitters, and have found changes in immune responsiveness following pharmacological manipulation of noradrenergic sympathetic or peptidergic nerves. Classic receptor binding studies have demonstrated a wide variety of target cells that possess beta-adrenoceptors and receptors for neuropeptides on cells of the immune system, including lymphocyte subsets, macrophages, accessory cells, or stromal elements. In this chapter we describe noradrenergic and peptidergic innervation of primary and secondary lymphoid organs in development, at maturation and during the normal aging process, and discuss possible functional implications of direct neural signals onto cells of the immune system at critical time points in the lifespan of an animal. Further, we examine for involvement of noradrenergic sympathetic and peptidergic innervation in the development and progression of several autoimmune disorders, including adjuvant-induced arthritis, New Zealand mice strains as a model for hemolytic anemia and lupus-like syndrome, and the experimental allergic encephalomyelitis model for multiple sclerosis.

Substance P innervation of spleen in rats: nerve fibers associate with lymphocytes and macrophages in specific compartments of the spleen.

We investigated the distribution of SP+ nerve fibers in the spleen of adult male Fischer 344 rats. SP+ nerve fibers entered the spleen with the splenic artery in the hilar region, arborized along the venous sinuses, and extended from these larger plexuses into trabeculae and the surrounding red pulp. In the white pulp, SP+ nerve fibers were found in the marginal zone, and in the outer regions of the PALS among T lymphocytes. No SP+ nerve fibers were observed in association with the splenic capsule, the central arteries of the white pulp, or the follicles. SP levels in rat spleen were 5.7 +/- 0.4 ng/g wet wt. On the basis of the present findings of SP presence in nerve fibers in the spleen, and published evidence for SP receptors on lymphocytes and macrophages, we suggest that SP derived from nerve fibers in the spleen can act as a neurotransmitter with cells of the immune system as targets. These SP nerve fibers may be an important neural link between the nervous system and the immune system and may participate in modulation of immune reactivity and inflammatory responses.

Substance P innervation of the rat thymus.

Immunohistochemistry for substance P (SP) in the rat thymus revealed fine varicose neural profiles in specific regions of the thymus. Thymic SP innervation was abundant within the capsule and interlobular septa. The majority of SP+ nerve fibers within the septa were free of vascular association, although some fibers were associated with the vasculature deep within the septa. SP+ nerve fibers entered the thymic cortex from the septa and distributed among cortical thymocytes and mast cells. Along the corticomedullary junction, SP+ nerve fibers were found in association with the vasculature. The medullary region of the thymus received only a sparse innervation of SP+ fibers. In addition, SP+ nerve fibers coursed adjacent to OX-8+ cells and mast cells in the extrathymic connective tissue surrounding the thymus. The present study provides evidence that SP is present in nerve fibers in the thymus, and may be available to interact with thymocytes, mast cells, and other cells in the thymus, and affect their development and function.

Noradrenergic reinnervation of the rat spleen following chemical sympathectomy with 6-hydroxydopamine: pattern and time course of reinnervation.

The time course and pattern of reinnervation of noradrenergic (NA) sympathetic nerve fibers into the spleen following acute chemical sympathectomy with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) was examined in young adult male Fischer 344 rats using glyoxylic acid fluorescence histochemistry and neurochemical measurement of NE. Reinnervation proceeds initially along the splenic artery as it enters the hilus (1-5 days), extends into the hilar region (5-10 days), and later proceeds into the regions distal to the hilus (21-56 days), suggestive of orderly growth from the hilar region to distal regions. In all 6-OHDA-treated spleens, the compartmentation of NA innervation was similar to that observed in saline-injected controls, but the density of NA nerve fibers in these compartments differed according to the distance from the hilus. By 56 days postlesion, regions distal to the hilar blood vessels contained fewer NA profiles than their age-matched controls, suggesting that the anatomical process of reinnervation does not restore the density of fibers in a fashion identical to that of nondenervated controls. In contrast, splenic NE concentration at 56 days postdenervation did not differ from the concentration seen in nondenervated control spleens. We suggest that functional restoration of splenic innervation may involve metabolic and receptor compensation for the lack of complete fiber regrowth into regions of white pulp distal from the hilus.