Catecholaminergic suppression of immunocompetent cells

VIEWPOINT I M M U N O L O G Y TO D AY Catecholaminergic suppression of immunocompetent cells Jonas Bergquist, Andrej Tarkowski, Andrew Ewing and Rolf Ekman The synthesis of catecholamines by immunocompetent cells has now T clear that the connections between the imhe central nervous system been demonstrated. In addition, mune and the nervous system are essential (CNS) – the brain and spinal catecholamines have been discovered to many functions during the mammalian cord – and the peripheral nerinside the nuclear membrane and life cycle7. Several of the nerve functions are vous system (PNS) together controlled by classical neurotransmitters but regulate multiple body functions. The neurmay therefore interact in the these molecules might in fact be acting as ones communicate with other organs and transcription process. Here, Jonas immunotransmitters or even neuroimmune cell types, as well as with nervous tissue, by Bergquist and colleagues describe transmitters. neurotransmission. Neurotransmitters are generally released from the synapses into MCapillary electrophoresis has recently bethe powerful impact that the synaptic cleft, the space between the precome an effective analytical tool for neurocatecholamines exert on the immune and post-synaptic membranes, in very small biological investigations8 including interacvolumes and low concentrations. Most tions between the immune and nervous system by downregulation of neurotransmitters fall into one of three system. However, its application in improliferation and differentiation, and chemical categories: amino acids, amines or munology has not been widespread. In the induction of apoptosis. peptides, and they can be excitatory or infew studies to date, immunological properhibitory in their role of action. ties, such as antibody–antigen interactions, The mammalian immune system comhave been used as tools in capillary elecprises organized lymphoid tissue, circulating cells and soluble pro- trophoresis, but capillary electrophoresis has not been used as a tool teins, such as immunoglobulins, cytokines and complement factors, for direct immunological research until recently. Early applications that control immune homeostasis in the whole body. Communi- of capillary electrophoresis were more related to pure protein chemcation within the immune system is largely dependent on cytokines, istry, especially concerning the analyses of recombinant proteins many of which have been described along with their effects on vari- and the hydrolysis of these proteins. The purities of antigen-binding ous immunocompetent cells. F(ab9)2 regions and antibodies, as well as the thermal stability of The detailed mechanisms underlying communication between antibodies have all been assessed using capillary electrophoresis. the nervous and immune systems are presently not fully under- However, the possibility of resolving a large variety of molecules, stood. There are many examples of direct communication between from small ions to large macromolecules, is a significant advantage cells in the CNS/PNS and the immune system, involving both cy- of capillary electrophoresis, which thus becomes a valuable tool for tokines and neurotransmitters as potential signalling molecules. the separation and detection of neuroimmune transmitters in imHere we suggest that endogenous and exogenous catecholamines mune and other tissues. can act as regulators of the immune system, thus providing a novel means of communication between the nervous and the immune Capillary electrophoresis of lymphocytes systems. Since 1948, when Medawar1 presented the results of his studies Capillary electrophoresis (Fig. 1) has been used to sample single on the CNS, this compartment has been considered an immuno- lymphocytes and extracts of lymphocyte populations. At the injection privileged site. However, it is now clear that the immunoprivileged end of the capillary a microinjector is etched to allow sampling of status of the CNS is not unconditional: there is direct contact be- single cells. At the detection end of the capillary an optimized endtween the interstitial fluid of the brain and the lymph2; lymphocytes column electrochemical detector allows, in the best cases, detection can pass through the blood–brain barrier3,4; and antigen-presenting limits as low as 10221 mol. This system permits sampling of large cells (APCs) can be found in the brain parenchyma5,6. Therefore, a invertebrate cells9, cell cytoplasm10 and small mammalian cells11,12. mechanism must exist by which the immune reactivity within the Capillary electrophoresis with electrochemical detection has CNS is controlled and suppressed. The same mechanism may also been used to examine the chemical composition of lymphocytes. be pertinent in peripheral sites and be responsible for local immune Capillary electrophoresis of single lymphocytes obtained directly regulation. from human cerebrospinal fluid (CSF) has demonstrated the presNeuroimmunological interactions involve a variety of molecules, ence of endogenous catecholamines in these cells12. The represenwith a multitude of characteristics. The best studied of these are the tative electropherogram in Fig. 2, shows the analysis of a single cytokines, often called neurokines, due to their regulatory functions human lymphocyte with a volume of ~180 3 10215 l. A single cell is in nervous tissue as well as in the immune system. It is increasingly injected into an etched capillary by electroosmotic flow and, after 0167-5699/98/$ – see front matter © 1998 Elsevier Science. All rights reserved. D E C E M B E R 562 Vo l . 1 9 1 9 9 8 No.12 PII: S0167-5699(98)01367-X VIEWPOINT I M M U N O L O G Y TO D AY + High voltage - MSingle cloned CD41 T cells and B cells have also been examined by capillary electrophoresis12. The average catecholamine Carbon content of cloned CD41 T cells was found to fibre Capillary be 3 3 10217 mol cell21, with a large biologiworking electrode cal variation between the different clones, whereas a single B cell contained 3.1 3 10219 mol catecholamines. MThe level of catecholamine has also been SSCE Buffer reservoir reference quantified in extracts from cloned human B electrode cells and CD41 T cells, both populations were found to contain on average 2 3 10218 Capillary mol cell21, within the range of catecholamine levels found in single CD41 T Ground Etched microinjector tip cells12. Using a modified analytical appaOptimized end-column detection for CE ratus, the dopamine and the norepinephrine peak can be separated based on elecFig. 1. The capillary electrophoretic system used for electrochemical detection of catecholamines. At trophoretic mobility. Freshly isolated pethe injection end of the capillary, a microinjector is etched, to allow analysis of single cells. At the deripheral blood mononuclear cells (PBMCs) 39 tection end of the capillary an optimized end-column electrochemical detector is used . Abbrevihave been found to hold dopamine at a ations: CE, capillary electrophoresis; SSCE, sodium saturated calomel electrode. basal level of 1.6 3 10218 mol cell21, while basal norepinephrine levels are 1 3 10218 mol cell21 (Ref. 13). These separations detect not only the main 2 3 metabolite of dopamine (DOPAC), but also the main metabolite of norepinephrine, 3-methoxy-4-hydroxyphenylglycol (MHPG), 2 pA strongly suggesting the presence of both dopamine and norepinephrine in these cells (Fig. 3). Using a similar methodological approach, mouse spleen cells, peritoneal macrophages, as well as T- and B-cell hybridomas have been shown to contain catecholamines14. Spleen cells contain ~7 3 10217 mol cell21 dopamine and 2 3 10217 mol cell21 norepinephrine, 4 1 whereas peritoneal macrophages contain 2 3 10217 mol cell21 of dopamine and no detectable amounts of norepinephrine. The variation among different hybridoma cell clones is notable, ranging from 2 3 10218 mol of dopamine to below the detection limit (~1 3 0 10 20 30 40 10219 mol). In addition, norepinephrine levels vary from 2.8 3 10219 Time (min) mol cell21 down to undetectable levels (see Table 1). SSCE reference electrode Fig. 2. Electropherogram of a single human cerebrospinal fluid lymphocyte. Electrophoretic mobilities of the major peaks correspond to the calculated electrophoretic mobilities of dopamine (peak 1), a neutral species (peak 2), uric acid (peak 3), and 3,4-dihydroxyphenylacetic acid (peak 4). Figure reproduced, with permission, from Ref. 12. lysing the cell, a potential is applied to separate and mobilize the intracellular components towards the electrochemical detector. The cationic peak at 10.8 min (peak 1) has been identified as dopamine, based on electrophoretic mobility, and an average dopamine content of 2 3 10218 mol has been found for CSF lymphocytes. Peak 1 might also contain other electroactive amines with similar electrophoretic mobilities such as norepinephrine, epinephrine or serotonin. However, the electrophoretic mobility of peak 4 corresponds to 3,4-dihydroxyphenylacetic acid (DOPAC), a metabolite of dopamine, which strongly suggests that peak 1 contains dopamine. Lymphocyte catecholamines: passive uptake or active synthesis? One of the questions arising from the above observations is whether the catecholamines observed in lymphocytes and monocytes/ macrophages represent a passive uptake through receptor-mediated mechanisms or active synthesis. To examine this issue, human and murine T-cell clones have been incubated with dopamine, levo-dihydroxyphenylalanine (the biosynthetic precursor to dopamine) or the dopamine synthesis inhibitor a-methyl-p-tyrosine12,13. Incubation with dopamine or levo-dihydroxyphenylalanine, increased the level of dopamine per cell whereas incubation with amethyl-p-tyrosine produced a decrease. Furthermore, the uptake of dopamine into PBMCs is significantly decreased (p ,0.01) by co-incubating the cells with nomifensine, a dopamine transporter antagonist13. The corresponding incubation with dopamine in the presence of tetrabenazine, a catecholamine storage blocker, leads to similar results, with D E C E M B E R Vo l . 1 9 No.12 1 9 9 8 563 VIEWPOINT I M M U N O L O G Y TO D AY N MHPG 5 pA Capillary electrophoresis analysis of catecholamines in isolated lymphocyte nuclei DA NE UA DOPAC 0 20 Time (min) Fig. 3. Separation and detection of easily oxidized species in microextracts of isolated human peripheral blood mononuclear cells by capillary electrophoresis. The electrophoretic mobilities of the major peaks shown correspond to the calculated electrophoretic mobilities of dopamine (DA), norepinephrine (NE), a neutral species (N), 3-methoxy-4-hydroxyphenylglycol (MHPG), uric acid (UA), 3,4-dihydroxyphenylacetic acid (DOPAC). Figure reproduced, with permission, from Ref. 13. a significant decrease of intracellular catecholamine levels (p ,0.05). None of these substances affects the basal levels of catecholamines in PBMCs. The results clearly indicate that lymphocytes actively synthesize the catecholamines. These data are supported by reports of low levels of norepinephrine and epinephrine in lymphocytes, detected by a radioenzymatic assay15 and in vitro catecholamine synthesis in peripheral human lymphocytes16. Recently, we presented the first structural characterization of the endogenous catecholamines in human lymphocytes with electrospray ionization mass spectrometry17. The levels of catecholamines in isolated and extracted human lymphocyte nuclei have now been determined with capillary electrophoresis and electrochemical detection. Dopamine is present in nuclei at levels of 5.3 6 2.6 3 10221 mol and norepinephrine is present at 2.1 6 0.9 3 10221 mol. No catecholamine metabolites have been detected in the nuclear extract, however, these may be present at levels below the detection limit. In parallel, PBMC whole-cell extracts show that dopamine is present at 2.6 6 0.6 3 10218 mol cell21 and norepinephrine at 2.0 6 0.3 3 10219 mol cell21. These levels are not significantly different from those previously reported13, but they are significantly higher than the catecholamine levels found in the nuclei. The experimental data suggest that 0.1–0.2% of the total amount of catecholamine is situated inside the nuclear membrane and indicate that catecholamines exert their regulatory mechanism on lymphocytes through interaction with nuclear components. Because both monoamine oxidase and catechol-o-methyltransferase, the two major catabolic enzymes for catecholamines, are located at the outer membrane of the mitochondria or in the cytosolic compartment of catecholamine-producing cells, catecholamines located inside the nuclear membrane appear to be protected from degradation. Catechol-o-methyltransferase has been found in PBMCs (Ref. 18). In addition, a nuclear membrane transporter for catecholamines exists19 which further strengthens the concept of transnuclear passage of catecholamines. The exact role of the catecholamines inside the nuclear membrane remains unresolved, but might involve interaction with some intranuclear receptor (e.g. steroid receptors) or/and interference with the transcription process. Table 1. Summary of analysed catecholamine content in cells from the immune system Cell type Average dopamine content (mol cell21) Single cells Human CSF lymphocytes 2 3 10218 Human CD41 T cells 3 3 10217 Human B cells 3.1 3 10219 Extracted cell populations Human CD41 T cells 2 3 10218 Human B cells 2 3 10218 Human PBMCs 1.6–8.6 3 10218 Murine spleen cells 7 3 10217 Murine peritoneal 2 3 10217 macrophages Murine T cells 1.9 3 10218– ,1.1 3 10220 Murine B cells 1.7 3 10219–6.9 3 10220 Average norepinephrine content (mol cell21) Refs ND ND ND 12 12 12 ND 2 3 10218 1–11 3 10218 2 3 10217 ,9.8 3 10220 12 13 13, 17 14 14 2.8 3 10219– ,9.8 3 10220 ,9.8 3 10220 14 14 Abbreviations: CSF, cerebrospinal fluid; ND, not determined; PBMCs, peripheral blood mononuclear cells. D E C E M B E R 564 Vo l . 1 9 1 9 9 8 No.12 Catecholamine responses to mitogen or superantigen stimulation of PBMCs Catecholamine levels have been measured in isolated PBMCs after 24 and 72 hours of stimulation with the mitogens concanavalin A (ConA) and anti-CD3 monoclonal antibody (mAb), or with superantigen staphylococcal enterotoxin A (SEA). Anti-CD3 mAb induces a significant decrease in dopamine (p ,0.01 and p ,0.05 after 24 and 72 h, respectively), whereas ConA appears to increase dopamine levels over time. Cells treated with SEA show a biphasic change in dopamine levels, increasing at 24 h and decreasing at 72 h. Norepinephrine levels are consistently diminished by SEA. To date, no obvious explanation for these results can be provided. One hypothesis is that different stimuli are mediated by different intracellular activation mechanisms that lead to different VIEWPOINT I M M U N O L O G Y TO D AY (a) catecholamine synthesis, but it is not known how this synthesis is regulated nor how the catecholaminergic inhibitory effect is mediated. However, these data further emphasize the complexity of neuroimmunological interactions, indicating that immunocompetent cells might change their catecholamine content due to a variety of exogenous stimuli. (b) Proliferation 5a Differentiation Proliferation 5b Differentiation Apoptosis 4a 4b 2a 1a 2b 3a 1b 3b 6 A model of catecholamine-induced suppression of immune cells The discovery of catecholamines in lympho- Fig. 4. A schematic drawing of a lymphocyte (a) with normal proliferative rate and differentiation, cytes has generated many questions, espe- and (b) exposed to high exogenous levels of catecholamines, for example, by being near a synaptic tercially in light of the dramatic effects that cat- minal or a sympathetic varicosity. Normally, there is a small uptake of exogenous catecholamines echolamines have on proliferation and across the cellular membrane (step 1a) that can be blocked with nomifensine. A higher concentration differentiation of lymphocytes. Because of exogenous catecholamines leads to an increased uptake by the cell (step 1b). Once inside the cell, lymphocytes can actively produce cat- the catecholamines are susceptible to degradation via enzymes in the mitochondrial membrane (step echolamines, both autocrine and paracrine 2a), but at higher concentrations may also have direct toxic effects on the mitochondria, which affects routes for catecholamine-mediated immune the total energy supply for the cell (step 2b). The intracellular catecholamines could also be protected regulation might exist. Catecholamines have by storage in vesicle-like compartments (step 3a), a mechanism that augments with increasing levels been found to trigger apoptosis of immuno- of intracellular catecholamines (step 3b), and that can be blocked with tetrabenazine. Some of the catcompetent cells13,14 which might explain echolamines might be transported through the nuclear envelope via a specific transporter (step 4a) their inhibition of lymphocytic function. and interact directly with the intranuclear regulatory mechanisms. This uptake is also increased by The route by which exogenous cat- higher concentrations of exogenous catecholamines (step 4b). An inhibitory effect of increasing levecholamines affect different lymphocytes is els of exogenous catecholamines was found on proliferation and differentiation, in combination with still somewhat unclear. However, as lym- an induction of apoptosis (step 5). Finally, an endogenous production of catecholamines in normal phocytes express both a- and b-adreno- lymphocytes has been found (step 6). This synthesis, which can be blocked by a-methyl-p-tyrosine, receptors20–22, a receptor-mediated effect is may provide an autocrine loop, whereby the endogenous catecholamines under normal conditions can possible. Previous studies demonstrated re- regulate cellular functions. ceptor-mediated effects on migration and activation of lymphocytes. Receptor antagonists have direct effects on cell function, thus it is difficult to study be mediated both via endogenous catecholamines as an autocrine the catecholamine-mediated regulation of lymphocyte function if it regulatory loop, or by exogenous catecholamines from immune and is only an effect of receptor interaction. As described below, other non-immune tissue as a paracrine regulation. The ability to produce catecholamines was earlier believed to be restricted to neuronal mechanisms may be involved. The downregulatory effects triggered by catecholamines are also cells. However, low levels have been found in different non-neurinduced by in vitro addition of levo-dihydroxyphenylalanine13,14,23. onal cells, such as from the gastrointestinal system, the renal system These effects are probably not receptor mediated due to the lack of and the immune system. By comparison, single cell analysis of giant any known levo-dihydroxyphenylalanine-sensitive receptor to date. dopamine nerve cells from Planorbis corneus (water pond snail) reIn addition to synthesis, lymphocytes accumulate exogenous vealed catecholamine levels of 344–461 3 10215 mol cell21 (Ref. 30), dopamine, indicating the presence of an endogenous dopamine whereas a mammalian PC12 cell (rat adrenal pheochromocytoma transporter24,25. This is further supported by the finding that cell) contains on average 2.9 3 10215 mol cell21 (Ref. 8). The systemic concentrations of the catecholamines is rather low dopamine uptake can be blocked by the specific dopamine transport inhibitor nomifensine13. In the cell cytoplasm, catecholamines (~1 nM)31, but may reach high local concentration in the immediate are degraded by monoamine oxidase and catechol-o-methyltrans- vicinity of the synapse or close to a sympathetic varicosity. To evaluferase, unless they are protected via storage in vesicles. Indeed, in- ate the concentration of catecholamines at a synapse a simplistic tracellular catecholamines in lymphocytes are apparently stored in calculation is provided. A typical synaptic vesicle has a diameter in vesicle-like compartments, as incubation with tetrabenazine de- the range of 20–100 nm (Refs 32–34) (50 nm would give a volume pletes these vesicles and consequently reduces the intracellular of 6.5 3 10220 l) and a catecholamine concentration of 0.05–0.5 M (Ref. 35). This would result in ~2000–20 000 molecules per 50 nm levels of catecholamines13. The regulatory effect of catecholamines on immunocompetent vesicle. If one vesicle fuses with the presynaptic membrane and recells is a well studied phenomenon12–14,23,26–29. This regulation may leases its contents into the synapse (with an approximate volume of D E C E M B E R Vo l . 1 9 No.12 1 9 9 8 565 VIEWPOINT I M M U N O L O G Y TO D AY 1 3 10217 l, calculated as a cylinder with a height of 50 nm and a diameter of 500 nm), this would result in a concentration of 0.3– 3 mM. This concentration could be even higher if many vesicles were released during the same time period (i.e. 3–30 mM for 10 vesicles). These concentrations are very rough estimates, and re-uptake and degradation mechanisms have been ignored36. However, these calculations demonstrate that lymphocytes could encounter high concentrations of catecholamines in a local environment, for example near synapses in the brain or close to sympathetic varicosities in the lymphoid organs (e.g. gut associated lymphoid tissue, lymph nodes or spleen) known to be richly innervated by the sympathetic nerves. Together, these data suggest new mechanisms by which the central and peripheral nervous systems might downregulate, or drain, the immune system locally. This would be especially apparent in brain regions containing catecholamine synapses. Furthermore, it has been shown that dopamine can be released outside the synapse in some cell systems and can travel ~10 mm (Refs 37, 38). Thus, downregulation of lymphocyte activity via this mechanism seems even more likely. Catecholamines appear to act by suppressing the activity of immunocompetent cells, possibly by inducing apoptosis. This has been assessed by flow cytometry to measure genomic DNA fragmentation, and by quantitating apoptotic marker proteins, such as Bcl2/Bax and Fas/Fas ligand using western blot techniques. Both methods provide evidence for a specific induction of apoptosis in human PBMCs by catecholamines13. In a parallel study, the presence and effects of catecholamines in cells of murine origin, have been examined with similar results14. Thus, we propose that the immunological privilege of the CNS (as well as in peripheral sites) is at least in part due to catecholamines in the extracellular fluid of the tissue: these can be transported into the nuclei of immunocompetent cells resulting in apoptosis. Although the exact details of regulation have yet to be determined, this represents a novel mechanism for the role of catecholamines as messengers in the neuroimmune axis (see Fig. 4). The authors gratefully acknowledge their co-workers who have contributed to this work, and M. Verdrengh and R. Persson for skilful technical assistance. This work was supported by grants from the Swedish Medical Research Council, the Swedish Society for Medical Research, the Swedish Natural Science Research Council, the Fredrik and Ingrid Thuring Foundation, the Magnus Bergvall Foundation, the Gamla Tjänarinnors Foundation, the Clas Groschinsky Foundation, the Wilhelm and Martina Lundgren Foundation, the Alzheimer Foundation, the National Institutes of Health and the National Science Foundation. Jonas Bergquist (jonas.bergquist@ms.se) and Rolf Ekman are at the Institute of Clinical Neuroscience, Dept of Psychiatry and Neurochemistry and Andrej Tarkowski is at the Dept of Rheumatology, Göteborg University, Sahlgrenska University Hospital/Mölndal, S-431 80 Mölndal, Sweden; Andrew Ewing is at the Dept of Chemistry, Penn State University, University Park, PA 16802, USA. References 01 Medawar, P.B. (1948) Br. J. Exp. Pathol. 29, 58–69 02 Cserr, H.F., Harling-Berg, C.J. and Knopf, P.M. 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A. 91, 12912–12916 13 Bergquist, J., Josefsson, E., Tarkowski, A., Ekman, R. and Ewing, A. (1997) Electrophoresis 18, 1760–1766 14 Josefsson, E., Bergquist, J., Ekman, R. and Tarkowski, A. (1996) Immunology Concluding remarks 88, 140–146 The small volume capabilities of capillary electrophoresis, along with the sensitivity of electrochemical detection, make it an invaluable tool in the study of the neuroimmunological network at the single cell level. None of the currently available alternative technical approaches could be applied to resolve some of the properties reported here for single lymphocytes. Sample handling for lymphocytes with 10215 litre volume is relatively straightforward and detection limits for catecholamines at levels of 10219–10220 mol have been achieved. Key areas of future work need to include laser-induced fluorescence, mass spectrometry, and perhaps immunoassay based detection schemes for determining neuroimmune interactions on a molecular level. 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