Kelley S. Madden

Noradrenergic Sympathetic Neural Interactions with the Immune System: Structure and Function

Histochemical studies from our (Williams & Felten 1981, Williams et al. 1981, D. Felten et al. 1981, 1984, 1985, 1987a, 1987b, Livnat et al. 1985, Ackerman et al. 1986, S. Felten et al. 1987) and other laboratories (Giron et al. 1980, Bulloch & Pomeranz, 1984, Singh 1984, Walcott & MacLean 1985) have shown the presence of autonomic nerve fibers in specific compartments of both primary and secondary lymphoid organs. These nerve fibers are associated not only with blood vessels but also with lymphocytes and macrophages. We have demonstrated that the neurotransmitter norepinephrine (NE), present in the postganglionic sympathetic fibers that richly innervate lymphoid organs, acts in the spleen as both a paracrine secretion, available to receptors on cells in the white pulp, and a localized neurotransmitter in nerve terminals that directly contact T lymphocytes in the periarteriolar lymphatic sheath (PALS) (S. Felten et al. 1986, S. Felten & Olschowka 1987). We propose that NE in lymphoid organs fulfills the criteria for neurotransmission, estabUshed in more traditional efTector tissues such as the heart, and plays a role in the modulation of immune responses. This review summarizes evidence for neurotransmission, including presence and compartmentation of NE, transmitter release, post-synaptic receptors on cells of the immune system, and functional consequences of denervation and pharmacological manipulation of NE. We also review aspects of development, aging, and plasticity of noradrenergic (NA) fibers that enhance our understanding of their role in organs of the immune system.

Sympathetic neural modulation of the immune system. I. Depression of T cell immunity in vivo and vitro following chemical sympathectomy

Chemical sympathectomy of adult mice with 6-hydroxydopamine (6-OHDA) either prior to or following epicutaneous sensitization with the trinitrophenyl (TNP) hapten decreased the delayed hypersensitivity (DH) response to ear challenge. To determine if uptake of 6-OHDA into sympathetic nerve terminals, and their subsequent destruction, was required for suppression of DH, the catecholamine uptake blocker, desipramine, was employed to block 6-OHDA-induced sympathetic denervation. Pretreatment with desipramine prevented the depression of DH. In vivo treatment with the beta blocker, propranolol, did not alter the 6-OHDA effect, eliminating the potential contribution of released catecholamines, acting on beta-adrenoceptors, to DH reduction. Sympathectomy before sensitization also diminished hapten-specific T cell reactivity of sensitized lymph node (LN) cells, as measured in vitro by IL-2 production and CTL generation. In vivo DNA synthesis in draining LN in response to immunization was modestly decreased following 6-OHDA. Thus, sympathetic denervation appears to impair T cell activity in vivo and in vitro. Overall, these results indicate the SNS plays a role in generation of cell-mediated immunity.

Sympathetic nervous system modulation of the immune system. III. Alterations in T and B cell proliferation and differentiation in vitro following chemical sympathectomy

Functional changes in lymph node (LN) and spleen lymphocytes were examined following sympathetic denervation of adult mice with 6-hydroxydopamine (6-OHDA). Sympathectomy reduced in vitro proliferation to concanavalin A (ConA) by LN cells and decreased LN Thy-1+ and CD4+ T cells. At the same time, ConA-induced interferon-gamma (IFN-gamma) production was increased, but interleukin-2 (IL-2) production was not altered. After sympathectomy, lipopolysaccharide (LPS)-stimulated proliferation of LN B cells was enhanced, in parallel with an increase in the proportion of sIgM+ cells. LPS-induced polyclonal IgM secretion was decreased, whereas polyclonal IgG secretion was dramatically enhanced. In the spleen, ConA and LPS responsiveness was reduced after sympathectomy, as was IL-2 and IFN-gamma production. The decreased proliferation was not associated with changes in splenic T and B cell populations. The uptake blocker desipramine prevented the 6-OHDA-induced changes in spleen and LN, indicating that these alterations were dependent upon neuronal destruction. These results provide evidence for heterogeneity of sympathetic nervous system regulation of T and B lymphocyte function and for organ-specific influences on immune function.

Sympathetic nervous system modulation of the immune system. II. Induction of lymphocyte proliferation and migration in vivo by chemical sympathectomy

We have used chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in adult mice to study the role of the sympathetic nervous system (SNS) in regulating cellular proliferation and migration in lymphoid organs. Following sympathectomy, an increase in inguinal and axillary lymph node (LN) weight and cellularity was observed. This increase paralleled increased cellular proliferation in vivo, as measured by uptake of [125I]deoxyuridine (125IUdR). Transient increases in cellular proliferation also were observed in spleen and bone marrow following sympathectomy. Administration of desipramine prior to 6-OHDA to prevent sympathectomy resulted in control levels of proliferation. beta-Adrenoceptor blockade just prior to or following 6-OHDA treatment did not alter the enhanced proliferation. Migration of normal 51Cr-labelled lymphocytes into inguinal and axillary LN was enhanced in sympathectomized recipients. Conversely, cells from sympathectomized animals showed diminished migration to these LN upon transfer into intact recipients. These results demonstrate that depletion of NA innervation alters cellular proliferation and lymphocyte migration in primary and secondary lymphoid organs.

Catecholamine Action and Immunologic Reactivity

This chapter discusses catecholamine action and immunologic reactivity. The cells of the immune system are capable of responding to antigen in vitro , independent of most physiologic influences present in an intact organism, and the proposal of a physiologic role for catecholamines does not imply that they can replace such requirements as antigen presentation, T-cell recognition of major histocompatibility complex products, or interleukin production. The role of the catecholamines in the effector phase of immunity must also be dissected more fully. The report of unequal distribution of β-adrenoceptors among T and B cells and among T-cell subpopulations, in conjunction with the apparent heterogeneity of lymphocyte cAMP responsiveness to β-adrenoceptor and of lymphocyte responses following a rise in intracellular cAMP, underscores the importance of identifying adrenoceptors and determining the efficiency of intracellular effector mechanism coupling in purified cell populations throughout the course of an immune response. It was suggested that these immunomodulators, which are considered external to the immune system, may participate in a cooperative effort to fine-tune the response to antigen, restraining proliferative and other effector processes where necessary to ensure that the magnitude of the response is roughly proportional to the amount of antigen encountered and that the ongoing response is appropriately terminated.

THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM IN THE MODULATION OF IMMUNE RESPONSES

Publisher Summary Studies of the effects of sympathectomy on immune responses invariably suggest that norepinephrine (NE) and the sympathetic nervous system have important roles in modulation of immune responses. Lymphoid organs, both primary and secondary, are innervated by NE-containing postganglionic sympathetic nerve fibers, as well as a variety of peptidergic fibers. At the light microscopic level, much of the innervation appears to be associated with the vasculature in these organs, although there are always fibers that appear to have no anatomic relationship to blood vessels. Immunocytochemistry of the rodent spleen at the ultrastructural level, using antibodies for tyrosine hydroxylase, has revealed that, in addition to abundant blood vessel and trabecular smooth-muscle innervation, there is direct contact between tyrosine hydroxylase-positive nerve terminals and both lymphocytes and macrophages. The presence of sympathetic nerve terminals near, or in direct contact with, lymphocytes in lymphoid organs, including thymus, spleen, lymph nodes, and bone marrow, and the presence of adrenergic receptors on lymphocytes suggest that sympathetic innervation and the transmitter NE may be important in the modulation of immune responses. Not only does NE affect cytokine production, but it is also likely that some cytokines also regulate NE release both via central nervous system sympathetic responses to circulating cytokines and by interactions with local noradrenergic nerve terminals innervating lymphoid organs and other peripheral target sites where inflammation or immune responses take place.

Regulation of the immune system by sympathetic neural mechanisms.

The immune system is made up of primary and secondary lymphoid organs, containing lymphocytes and several accessory cell types, which are the key agents of immunological reactivity. Some of the basic features of immune responses are reviewed. Several pathways from the CNS to the immune system are of potential importance in physiological regulation. The sympathetic nervous system innervates all lymphoid organs with noradrenergic fibers. Furthermore, lymphocytes have receptors for sympathetic neurotransmitters, e.g., beta adrenoceptors. Sympathetic denervation by pharmacologic treatment with 6-hydroxydopamine has a marked affect on several immune responses, including antibody production, delayed hypersensitivity, and generation of cytotoxic T lymphocytes. Norepinephrine, epinephrine, and synthetic adrenergic agonists potentiate cytotoxic T lymphocyte responses in vitro. This appears to be mediated via beta 2 adrenoceptors. Studies with adrenoceptor blockers also indicate a possible role for alpha receptors. In sum, our studies indicate that intact noradrenergic innervation is required for normal immune function. Sympathetic neural influence, via norepinephrine release, may be exerted at the cellular (activation, proliferation, secretion of products) as well as the physiologic (antigen localization, lymphocyte migration) levels.

β-Adrenergic receptors (β-AR) regulate VEGF and IL-6 production by divergent pathways in high β-AR-expressing breast cancer cell lines

Activation of β-adrenergic receptors (β-AR) drives proangiogenic factor production in several types of cancers. To examine β-AR regulation of breast cancer pathogenesis, β-AR density, signaling capacity, and functional responses to β-AR stimulation were studied in four human breast adenocarcinoma cell lines. β-AR density ranged from very low in MCF7 and MB-361 to very high in MB-231 and in a brain-seeking variant of MB-231, MB-231BR. Consistent with β-AR density, β-AR activation elevated cAMP in MCF7 and MB-361 much less than in MB-231 and MB-231BR. Functionally, β-AR stimulation did not markedly alter vascular endothelial growth factor (VEGF) production by MCF7 or MB-361. In the two high β-AR-expressing cell lines MB-231 and MB-231BR, β-AR-induced cAMP and VEGF production differed considerably, despite similar β-AR density. The β2-AR-selective agonist terbutaline and the endogenous neurotransmitter norepinephrine decreased VEGF production by MB-231, but increased VEGF production by MB-231BR. Moreover, β2-AR activation increased IL-6 production by both MB-231 and MB-231BR. These functional alterations were driven by elevated cAMP, as direct activation of adenylate cyclase by forskolin elicited similar alterations in VEGF and IL-6 production. The protein kinase A antagonist KT5720 prevented β-AR-induced alterations in MB-231 and MB-231BR VEGF production, but not IL-6 production. Conclusions β-AR expression and signaling is heterogeneous in human breast cancer cell lines. In cells with high β-AR density, β-AR stimulation regulates VEGF production through the classical β-AR-cAMP-PKA pathway, but this pathway can elicit directionally opposite outcomes. Furthermore, in the same cells, β-AR activate a cAMP-dependent, PKA-independent pathway to increase IL-6 production. The complexity of breast cancer cell β-AR expression and functional responses must be taken into account when considering β-AR as a therapeutic target for breast cancer treatment.

Neonatal sympathetic denervation alters the development of in vitro spleen cell proliferation and differentiation

The ontogeny of spleen cell proliferation to T and B cell mitogens and immunoglobulin secretion, measured in vitro, was examined in neonatally sympathectomized Fischer 344 (F344) rats, administered the neurotoxic drug 6-hydroxydopamine (6-OHDA) from 1 to 3 days of age. Compared to cells from age-matched controls, spleen cells from neonatally sympathectomized animals, aged 7-14 days, exhibited a shift in the proliferative response to the T cell mitogen, concanavalin A (Con A), with reduced proliferation in the presence of low doses of Con A, but increased proliferation with higher doses. During the same period, from 7 to 14 days, the B cell mitogen STM/DxS inhibited proliferation by spleen cells from all rats, and no effect of sympathectomy was observed. As adult-like patterns of mitogen responsiveness emerged from 21 to 42 days of age, neonatally sympathectomized rats showed reduced proliferative responses of both T and B cells. This effect dissipated by 56 days of age. Polyclonal immunoglobulin (Ig) production by B cells was assessed in vitro in the presence or absence of STM/DxS. Neonatal sympathectomy resulted in reduced spontaneous IgM production throughout development. From 28 to 42 days of age, when mitogen-triggered IgM secretion first developed, neonatal sympathectomy decreased the magnitude of the response. By 56 days of age, mitogen-induced IgM secretion was no longer affected by sympathectomy, similar to the proliferative response. Gender influenced the time course of sympathectomy-induced changes in spleen cell proliferation and differentiation; however, the magnitude and direction of these changes were similar in both males and females. Desipramine, administered prior to 6-OHDA, prevented both sympathetic denervation and the 6-OHDA-induced changes in spleen cell responsiveness. This indicates that the alterations in immune function were dependent on NA nerve fiber destruction and were not simply the result of direct 6-OHDA action on other cells. The results of this study suggest that sympathetic innervation may play an important potentiating role in the development of the lymphoid system, through effects on lymphocyte proliferation and differentiation.

Age effects on macrophage function vary by tissue site, nature of stimulant, and exercise behavior.

We explored the effects of aging on macrophage function in male BALB/c mice from three age groups: young (2 months), middle-aged (12 months), and old (21 months). Macrophages were collected from alveoli, peritonea, and spleens of each age group. Cells were cultured in vitro with LPS or LPS+IFN-gamma and assayed for production of IL-1, IL-12, NO, and TNF-alpha. Using herpes simplex virus-1, age-related changes in intrinsic antiviral resistance (plaque assay) and extrinsic antiviral resistance (NO and TNF-alpha production) were determined in alveolar and/or peritoneal macrophages. Effects of chronic exercise on age-related macrophage changes were examined. In vitro, macrophages from the alveoli and spleen of older mice generally produced more cytokine and NO compared to younger counterparts. Conversely, macrophages from the peritonea of older mice generally produced less cytokine and NO in vitro compared to younger counterparts. Alveolar macrophages from both old and young mice showed higher intrinsic antiviral resistance to HSV-1 compared to middle-aged mice, while peritoneal macrophages from young mice showed reduced intrinsic resistance compared to those from both middle-aged and old mice. When challenged with HSV-1, a trend towards decreased peritoneal macrophage production of TNF-alpha and decreased alveolar macrophage production of IL-12 with advancing age was found. Chronic moderate exercise tended to reverse age-associated changes in macrophage function in old mice.