Donald A. Chambers

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

Abstract: 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 α2‐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 α‐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.

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.

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-dosel-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 membranepenetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The β-adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal orl-PAM TuB spleen cells. Since the curative effectiveness of low-dosel-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.

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.

Plasminogen activator in human periodontal health and disease

Plasminogen activators, proteases associated with the fibrinolytic system, also play a major part in extravascular processes such as tissue remodelling, cell migration and activation of prohormones, growth factors and other proteases. It is likely that plasminogen activators participate in the pathophysiology of periodontal disease. Plasminogen activator has been identified in human gingival crevicular fluid in a concentration 100-fold greater than in plasma. The local activity of plasminogen activator in gingival tissues was examined and changes detected in its distribution in relation to the extent of disease. Frozen sections from human gingival biopsies were overlaid on fibrin-coated slides; tissue-type plasminogen activator activity was found in all samples. Focal activity was observed in healthy tissue, originating from the most superficial cells of the junctional epithelium. Biopsies of clinically healthy sites obtained 6 weeks after treatment for periodontitis also showed epithelial plasminogen activator activity localized to this area. In contrast, in diseased tissue the entire epithelium lining the periodontal pocket showed activity. This differential pattern of activity in health and disease is consistent with the hypothesis that plasminogen activator is a modulator of periodontal homeostasis.

Mindful immunology: neuroimmunomodulation.

Abstract A recent congress**The 4th International Congress of the International Society for Neuroimmunomodulation was held at Lugano, Switzerland on 29 September–2 October, 1999 on neuroimmunomodulation revealed how the technologies of contemporary molecular sciences have been used to explore and define signal transduction and cross-talk between the nervous and immune systems.

Activation of p38 mitogen-activated protein kinase by norepinephrine in T-lineage cells.

The catecholamine norepinephrine (NE) stimulates T lymphocytes through a beta‐adrenergic receptor (βAR)/adenylyl cyclase (AC)/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, leading to altered cell responsiveness and apoptosis. p38 Mitogen‐activated protein kinase (MAPK), a major intracellular signalling mediator for cellular and environmental stressors, is involved in the production of immune modulators and in the regulation of T‐cell development, survival and death. In these studies we investigated the relationship among NE signalling, p38 MAPK activity and T‐cell death. We showed that NE stimulation of BALB/c mouse thymocytes and S49 thymoma cells selectively increases the dual phosphorylation and activity of p38α MAPK. p38 MAPK activation involves the βAR, Gs protein, AC, cAMP and PKA, as determined through the use of a βAR antagonist, activators of AC and cAMP, and S49 clonal mutants deficient in Gs and PKA. Dual phosphorylation of p38 MAPK is also dependent on its own catalytic activity. Inhibition of p38 MAPK activity revealed its involvement in cAMP‐mediated activating transcription factor‐2 (ATF‐2) phosphorylation, Fas ligand messenger RNA (mRNA) up‐regulation, and cell death. These results identify a mechanism through which NE stimulation of the βAR/Gs/PKA pathway activates p38 MAPK, which can be potentiated by autophosphorylation, and leads to changes in T‐cell dynamics, in part through the regulation of Fas ligand mRNA expression.

Molecular characterization of plasminogen activator in human supragingival plaque

Extravascular participation of the serine protease plasminogen activator (PA) in tissue remodelling and cell migration may be relevant in the regulation of periodontal homeostasis and the pathogenesis of periodontal disease. The molecular nature of authentic PA in crevicular fluid has been characterized, and this study has sought to determine whether human supragingival plaque also contains PA; if so, of which molecular species and from what source. Thirty samples of supragingival plaque plaque from 10 individuals, extensively washed to remove adherent saliva, were all found by substrate analysis to have tissue-type PA (tPA) activity. Unstimulated parotid or mixed saliva also showed tPA activity, but submandibular saliva had no measurable PA activity. Freshly isolated plaque microbes cultured under aerobic or anaerobic conditions contained no PA activity (preliminary investigation). PA activity was restricted to individual epithelial cells suggesting that the origin of PA activity in human supragingival plaque is in part from plaque-adherent epithelial cells.

Plasminogen Activator in Periodontal Health and Disease

Plasminogen activators (PAS) are best known for playing a key role in the initiation of the fibrinolytic cascade. However, much current interest is fbcusing on the extravascular functions of PAS, which have been shown to participate in numerous biological processes including cell migration, wound heahg, inflammatory cell chemotaxis, nerve regeneration, and proenzyme activation. 1 Given the multiplicity of biological processes with which PAS are associated, PA activity may participate in many of the regulatory events ongoing in the periodontium, the bone and gingival tissues that sup port the teeth. Since the most common form of periodontal disease, which affects the majority of the adult population, is a chronic bacterial disease, understanding the relationships between PAS and their inhibitors, PAIs, and the pathognesis of periodontal disease may suggest broader significance for PAs/PAIs in other inflammatory/immune processes. These experiments 1) characterized PAs/PAIs in the gingival crevicular fluid which bathes the periodontiurn and 2) localized PA activity in healthy and diseased tissues. Fluid collected on filter paper strips placed in the healthy gingival crevices of 12 adults (30 sites assayed) was eluted overnight in Xis-Triton X100. SDS PAGE zymography revealed that PA activity of gingival crevicular fluid comigrated with purified human tissue-type PA (tPA) and was inhibited by antibodies directed against tPA but not urokinase-type PA (uPA) (FIG. 1). The mean concentration of tPA antigen, measured by ELISA (Immunobind 122, American Diagnostica, Greenwich, cr) was 2.0 ng/p1, a concentration 200-fold greater than that of plasma. Immunoblots of 26 samples assayed for PAI-1 and PA13 demonstrated the presence of both inhibitors in all samples (FIG. 2). PAI-1 was detected as the 60 kDa active molecule and 120 kDa activator-inhibitor complexes; occasionally a 55 kDa latent PAI-1 band was also identified. All samples contained a t least one PA13 band in the m g e of 60-63 kDa, probably representing the glycosylated form of the molecule, and a 120 kDa activatorinhibitor band. In addition, a band of 55 kDa was often seen and 3 of 26 samples showed a 40 kDa band that migrated with the unglycosylated PA13 standard. Frozen sections of gingiva were placed on fibrin-coated slides (in the presence or

Dynamics of Langerhans cells in genetically defined murine epidermal cell culture

Unlike keratinocytes, Langerhans cells express both surface ATPase activity and Ia (HLA‐DR) ANTIGENS. A well‐characterized in vitro system containing Langerhans cells would be of great use in elucidating their functions. Thus, epidermal cell cultures derived from neonatal Balb/c mice were examined for the presence of Langerhans cells. Twenty‐four hours after initiation of culture, ATPase‐ and Ia‐positive cells were seen to be associated with cell aggregates. By day 3, Langerhans cells migrated on to the substratum and, as the cultures matured and stratified, were seen both in groups and as single cells for the duration of the cultures (day 14). During culture, although the total number of cells increased, the percentage of cells expressing Ia antigen and ATPase activity remained constant, suggesting that Langerhans cells increase in number during cell culture. Such a situation could arise from actual division of Langerhans cells during culture or from latent expression of Ia antigen and ATPase activity by pre‐existing cells. This is the first study of the dynamics of Langerhans cells in a cell culture system and shows that Langerhans cells are present throughout the lifespan of the cultures.