The dialogue between the brain and immune system involves not only the HPA-axis.

Starting out from our previous observations that defects in the immune system-brain feedback predispose to pathogenic immune responses, our interest focuses at the roles of adrenergic/cholinergic neurotransmitters in brain-immune interactions. We have shown in rodent models that 1) both catecholamines and acetylcholine are potent modulators of peripheral immune functions, 2) cholinergic signals are involved in the afferent signalling of the immune system, and 3) lymphocytes not only express functional adrenergic and cholinergic receptors, but synthesize and release neurotransmitters, such as acetylcholine, in quantitative dependence of differentiation and activation. Studies are presently being initiated to investigate the role(s) of these non-neuronal neurotransmitters within immune tissues, and to explore the relevance of excitatory amino acids as important central neurotransmitters in the brain-immune system dialogue.

In vivo immunomodulation by peripheral adrenergic and cholinergic agonists/antagonists in rat and mouse models.

Our work is devoted to defining relationships between the immune system and the adrenergic and cholinergic systems in vivo. In the rat model, we have shown that the cells of different immune compartments express the genes of a defined set of adrenergic/cholinergic receptors, and it was shown that lymphocytes are a site of non-neuronal production of norepinephrine and acetylcholine. Furthermore, using implantable slow-release tablets containing adrenergic or cholinergic agonists/antagonists, distinct and partly opposite effects were observed on peripheral immune functions. Concerning sympathetic immunoregulation, our data--in contrast to those of other studies--suggest that an enhanced adrenergic tonus leads to immunosuppression primarily via alpha 2-receptor-mediated mechanisms. Beta-blockade strongly enhances this effect, most likely by inhibition of pineal melatonin synthesis. In recent experiments on the kinetics it was found that the continuous alpha-adrenergic treatment entails a strong suppression of cellular responsiveness during the first few hours, which is increasingly followed by a general loss of lymphocytes in blood and lymphoid organs most likely due to enhanced apoptosis. More recently, we have extended our studies to the mouse model. First data obtained with RNAse protection assays suggest a biphasic effect on the gene expression of several cytokines in spleen cells due to adrenergic in vivo treatment.

Norepinephrine-mediated inhibition of antitumor cytotoxic T lymphocyte generation involves a beta-adrenergic receptor mechanism and decreased TNF-alpha gene expression.

We have previously shown that norepinephrine (NE) inhibits the in vitro generation of anti-MOPC-315 CTL activity by spleen cells from BALB/c mice rejecting a large MOPC-315 tumor as a consequence of low-dose melphalan (l -phenylalanine mustard (l -PAM)) treatment (l -PAM TuB spleen cells). Since TNF-alpha plays a key role in the generation of antitumor CTL activity in this system, we determined whether NE mediates this inhibition through inhibition of TNF-alpha production. Here, we show that NE inhibits the production of TNF-alpha protein and mRNA by l -PAM TuB spleen cells stimulated in vitro with mitomycin C-treated tumor cells. Flow cytometric analysis of intracellular expression of TNF-alpha revealed substantial NE-mediated decreases in the percentages of TNF-alpha+ cells among CD4+ and CD8+ T cells and F4/80+ activated macrophages. NE inhibition of CTL generation was largely overcome by addition of TNF-alpha to the stimulation cultures. When the beta-adrenergic antagonist propranolol was added to the stimulation cultures of l -PAM TuB spleen cells at a concentration that prevented NE-induced cAMP elevation, the NE-mediated decrease in TNF-alpha mRNA and NE-mediated inhibition of CTL generation were reversed. Collectively, these results suggest that NE inhibits antitumor CTL generation, at least in part, by inhibiting TNF-alpha synthesis through a mechanism(s) involving beta-adrenergic receptor signaling.

Mechanism of catecholamine-mediated destabilization of messenger RNA encoding Thy-1 protein in T-lineage cells.

The Ig superfamily cell surface glycoprotein Thy-1 expressed on immune cells and neurons of rodents and humans is hypothesized to function in cell adhesion and signal transduction in T cell differentiation, proliferation, and apoptosis. This study analyzes effects of cAMP and catecholamines on transcriptional Thy-1 gene expression. Incubation of murine thymocytes or S49 mouse thymoma cells with dibutyryl-cAMP, 8-bromo-cAMP, cholera toxin, norepinephrine, or isoproterenol caused time- and concentration-dependent decreases in levels of Thy-1 mRNA assayed by Northern hybridization or T2 nuclease protection. After 4 h of treatment with 500 microM dibutyryl-cAMP or 8-bromo-cAMP, 1 nM cholera toxin, 100 microM norepinephrine, or 100 microM isoproterenol, Thy-1 mRNA levels were 60 to 96% lower than those of controls. Norepinephrine-mediated decreases in Thy-1 mRNA levels were prevented by the beta-adrenergic receptor antagonist propranolol (10 microM). Dibutyryl-cAMP and norepinephrine decreased the apparent half-life of S49 cell Thy-1 mRNA from >>6 h to 2 to 3 h, whereas nuclear run-on assays showed no cAMP or norepinephrine effect on de novo transcription of the Thy-1 gene. In mutant S49 cells lacking cAMP-dependent protein kinase A, neither dibutyryl cAMP nor norepinephrine affected Thy-1 mRNA levels. These observations show that exogenous cAMP and norepinephrine can induce decreases in steady state Thy-1 mRNA levels in T-lineage cells through posttranscriptional destabilization of Thy-1 mRNA, associated with protein kinase A-mediated protein phosphorylation. Catecholamine-mediated beta-adrenergic protein kinase A-dependent Thy-1 mRNA destabilization may be an example of a more general mRNA decay system regulating cellular responses to stress.

Hydrocortisone regulates the dynamics of plasminogen activator and plasminogen activator inhibitor expression in cultured murine keratinocytes.

The plasminogen activators tPA and uPA, and their inhibitors, PAI-1 and PAI-2, have been associated with epithelial homeostasis and wound healing. In these studies, we investigate the effect of the steroid hormone hydrocortisone, a commonly used therapeutic modality for skin, on PAs/PAIs in serum- and plasminogen-free primary cultures of murine keratinocytes. SDS-PAGE fibrin zymography showed that addition of 1 microM hydrocortisone to cultures significantly reduced tPA fibrinolytic activity in both cell extracts and conditioned medium. uPA activity in conditioned medium was similarly inhibited. Cells were also cultured in the presence of dibutyryl cyclic AMP (dbcAMP). dbcAMP (5 mM) alone enhanced uPA and tPA fibrinolytic activity in conditioned medium, but this increase was diminished in the presence of 1 microM hydrocortisone. Immunoblots revealed a three- to fivefold induction of free PAI-1 by hydrocortisone which was partially blocked by dbcAMP. Northern blots showed that PAI-1 mRNA increased threefold 2 h after addition of hydrocortisone and remained elevated at least 8 h. In contrast, uPA and tPA mRNA were unchanged over the same time course. uPA, tPA, and PAI-1 mRNA increased in the presence of dbcAMP; levels remained elevated at least 8 h. HC suppressed the induction of uPA and tPA by dbcAMP. Studies directed at identifying plasminogen mRNA showed that in this culture system, keratinocytes produce no plasminogen mRNA either in the presence or in the absence of hydrocortisone or dbcAMP. Collectively, these results show that keratinocyte plasminogen activator activity is suppressed by hydrocortisone as a function of increased PAI-1 combined with an attenuation of PA induction by agents that increase intracellular cAMP. These results provide additional information to further define the mechanism by which glucocorticoids inhibit wound healing.

A multi-center clinical trial of a new chairside test in distinguishing between diseased and healthy periodontal sites. II. Association between site type and test outcome before and after therapy.

The aim of the present study was to evaluate the association between the outcome of a chairside test measuring gingival crevicular fluid (GCF) levels of the enzyme aspartate aminotransferase (AST) and other clinical measures of disease including probing depth, severity of inflammation, and GCF flow before and after therapy. We studied 91 patients with moderate to severe periodontitis. Eight sites with probing depths between 5 mm and 8 mm and obvious signs of inflammation were selected and designated diseased sites. Four sites with probing depth < or = 3 mm with no or minimal signs of inflammation were selected and designated non-diseased sites in patients. Thirty healthy individuals were enrolled and four sites in each were selected and designated healthy controls. Patients were treated with scaling and root planing and control subjects with supragingival prophylaxis. Measurements including GCF volume, gingival inflammation, and probing depth were performed at screening baseline, 1 week later at pretreatment baseline, and at weeks 2 and 4 after treatment. AST content of GCF was measured using a chairside colorometric test. It was concluded that the outcome of the test is an effective objective measure distinguishing between diseased sites and non-diseased sites in patients and control subjects when evaluated both prior to and following application of therapy. Use of this simple chairside test, when combined with other standard diagnostic procedures, provides an objective measurement permitting improved capacity to distinguish between diseased and non-diseased periodontal sites, and to better assess and monitor the outcome of therapy.

A multicenter clinical trial of PerioGard in distinguishing between diseased and healthy periodontal sites. (I). Study design, methodology and therapeutic outcome.

We designed and performed a multicenter clinical trial to determine the relationship between measurements of the level of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) to other measures used to detect periodontal disease and monitor outcome of treatment, including pocket depth and gingival inflammation. 32 periodontitis patients were enrolled at the University of Washington, Seattle, 30 at the University of Florida, Gainesville, and 34 at the University of Illinois, Chicago. 10 periodontally normal control subjects were enrolled at each location. 8 diseased and 4 healthy sites were designated for study in each patient and 8 healthy sites designated in each control subject. Measures of disease included pocket depth, severity of gingival inflammation, and GCF volume. AST levels were measured using the PerioGard test kit. Clinical measurements were made and GCF samples harvested and tested 2x before and 2x after therapy consisting of scaling and root planing under local anesthetic. Specific design and other issues are discussed, including selection of patients and control subjects, sample size, selection of experimental test sites, methods for assessment of diseased and therapeutic improvement, harvesting of GCF and selection of appropriate biostatistical methods for data analysis. Demographics of the patient populations at the 3 locations are reported. As expected, therapy induced only negligible changes in the measures of disease at healthy sites in control subjects, and relatively minor improvement in healthy sites in patients. In contrast, statistically significant improvement relative to pretreatment baseline status in all 3 measures of disease was observed for diseased sites at all 3 study locations with all p-values less than 0.0002. The magnitude of improvement was comparable to that reported previously by others. The % of PerioGard-positive sites decreased significantly between the screening baseline and both post-treatment visits for patients at all 3 locations, with p values of 0.0001 to <0.0008.

Molecular characterization of plasminogen activators in human gingival crevicular fluid.

Plasminogen activators (PAs), a family of serine proteases, and their inhibitors (PAIs) are important in fibrinolysis, wound healing and tissue remodelling. Previous studies revealed differences in the localization of PA activity between healthy and diseased gingival tissues, suggesting that PAs and PAIs could play a part in periodontal homeostasis and disease. PAs and PAIs are synthesized by most of the cells types making up the periodontium and can be identified in gingival crevicular fluid (GCF). These studies sought to characterize the molecular species of PAs and their inhibitors in GCF collected from clinically healthy sites. PA enzymatic activity in GCF samples demonstrated by fibrin zymography revealed the presence of only tissue-type PA (tPA) activity. No urokinase-type PA (uPA) enzymatic activity was detected. tPA enzymatic activity appeared predominantly as an uncomplexed 70-kDa species, although some samples contained enzyme-inhibitor complexes. Quantitation of total tPA by enzyme immunoassay showed a mean concentration of 1.6 ng/microl. Analysis of GCF samples for uPA by immunoblotting and enzyme immunoassay disclosed the presence of small amounts of uPA (0.2 ng/microl), which were present predominantly in activator-inhibitor complexes. Immunoblotting showed specific PAI-2 immunoreactivity bands in high molecular-weight complexes and low molecular-weight degradation products, but less than nanogram amounts of free PAI-2 molecules. Enzyme immunoassay revealed that PAI-2 was present in an at least a seven times greater amount than PAI-1. These observations support the hypothesis that PA-generated proteolysis and its regulation by endogenous inhibitors has a role in the diverse biochemical mechanisms underlying periodontal physiology and pathology including host-microbial interaction, polymorphonuclear leucocyte infiltration, turnover and migration of epithelial cells, connective tissue degradation and remodelling, fibrinolysis and wound healing.

Inhibition of lymphocyte activation by catecholamines: evidence for a non-classical mechanism of catecholamine action.

The effects of noradrenaline and other adrenergic agonists on lymphocyte activation were studied. Spleen and thymus cells from BALB/c mice were stimulated by mitogens and lymphocyte activation was monitored by measuring the incorporation of [methyl-3H]thymidine into DNA. Noradrenaline, adrenaline, isoproterenol and dopamine all inhibited the activation of spleen and thymus cells by concanavalin A, a T-cell specific mitogen, and the activation of spleen cells by lipopolysaccharide, a T-independent B-cell mitogen. The various catecholamines were approximately equipotent, having IC50 of approximately 10 microM. alpha-adrenergic agonists (phenylephrine, clonidine) did not inhibit lymphocyte activation. Noradrenaline, adrenaline and isoproterenol also inhibited DNA synthesis in S49 T lymphoma cells. The effects of adrenergic receptor antagonists on lymphocyte function were also studied. The inhibition of lymphocyte activation by catecholamines could not be reversed by antagonists to beta-adrenergic receptors (propranolol), alpha-adrenergic receptors (phentolamine), or dopaminergic receptors (haloperidol). Experiments with human peripheral blood leucocytes revealed that, as with murine cells, the beta-adrenergic antagonists propranolol and nadalol did not affect the catecholamine-mediated inhibition of lymphocyte activation. Although lymphocytes contain beta-adrenergic receptors that are coupled to adenylyl cyclase activity, catecholamines appear to inhibit murine lymphocyte activation by a mechanism that is independent of these or other classical adrenergic receptors.

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma.

We have previously shown that, as a consequence of low-dose melphalan (L-phenylalanine mustard (L-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was "mimicked" by the membrane penetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The beta-adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal or L-PAM TuB spleen cells. Since the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8+ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.

Neuroimmune modulation: signal transduction and catecholamines.

In recent years, much interest has centered on the commonalities and bi-directional interactions between the nervous system and the immune system. This review focuses on mechanisms through which, catecholamines, a class of neuro-endocrine molecules, modulate immune functions. Catecholamines can be immune suppressive and inhibit lymphocyte activation of both T and B cells as well as the generation of immune-mediated anti-tumor responses. Some of these catecholamine-regulated activities appear to be modulated through the second messenger, cyclic AMP, whereas others appear to be catecholamine-dependent but cyclic AMP independent. Further delineation of the interacting ligand-receptor complexes, populations of responding cells and signal transduction mechanisms leading to the activation of specifically involved genes and gene products, will lead to enhanced understanding of the integratory functions of the nervous system in immune responses, the biology of stress, the role of stress-associated molecular mechanisms in perturbations of physiological homeostasis and the development of a new biological psychiatry with accompanying rational therapeutic modalities.

Mouse hepatitis virus infection suppresses modulation of mouse spleen T-cell activation.

Natural infection by mouse hepatitis virus (MHV) can affect interpretation of immunological studies in mice. MHV, a collective term describing a group of corona viruses, is found in natural infections in over 70% of laboratory mouse populations in the U.S.A. and Canada. Natural outbreaks of MHV in our animal colony afforded us the opportunity to study MHV-induced immunosuppression as well as the effects of MHV infection on neurotransmitter immunomodulation. Concanavalin A (Con A)-stimulated DNA synthesis by spleen T lymphocytes from MHV-infected mice was 20-50% that of non-infected mice. The MHV infection also altered neurotransmitter modulation of spleen T-lymphocyte activation. In contrast to noradrenaline ablation of Con A-activated DNA synthesis by spleen lymphocytes from non-infected mice, DNA synthesis by the infected group was not inhibited by noradrenaline or dibutyryl-cAMP. These effects of MHV infection were specific for spleen T lymphocytes since MHV infection did not alter Con A stimulation of thymocytes, lipopolysaccharide stimulation of spleen B lymphocytes, or noradrenaline inhibition of thymocyte and B-cell DNA synthesis. MHV infection also did not alter spleen T-lymphocyte subset proportions. Thus, MHV infection inhibits spleen T-lymphocyte activation and blocks in vitro catecholamine and cAMP regulation of spleen T-cell activation but does not affect activation of thymic cells or spleen B cells.

Association of gingival crevicular fluid aspartate aminotransferase levels with histopathology during ligature-induced periodontitis in the beagle dog.

Previous investigations have shown a clear association between the presence of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) and clinical evidence of periodontal disease in humans, as well as in the beagle dog model. This paper describes a 26-week study that uses the beagle dog model of ligature-induced periodontitis in which GCF-AST (corrected for collection time) was correlated with microscopic evidence of tissue destruction in the periodontium at the sites of fluid collection. GCF and clinical data were collected at baseline, at optimal gingival health, during gingivitis, and after ligation. A cross-mouth design was implemented so that six premolar teeth in each dog were ligated for periods up to five weeks. Formalin-fixed tissues from the sites of GCF collection were prepared for light microscopy and evaluated for the presence of epithelial ulceration, bone resorption, and inflammatory cell infiltration. The relationship between GCF-AST levels and microscopic findings was analyzed by calculation of sensitivity and specificity and by plots of Receiver Operating Characteristics. These data revealed a correlation between elevated enzyme concentration and microscopic evidence of disease activity. Taken together with human studies, these results provide support for the use of AST as a marker of periodontal disease progression.