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Altered cytokine production in whole blood cultures of chronic fatigue syndrome patients and a disturbed regulation by glucocorticoids
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 (1998) 1 3 - 1 0 5
239 Association of A u t o n o m i c Nervous Hyperreflexia a n d Systemic I n f l a m m a t i o n in Patients with Systemic L u p u s E r y t h e m a t o s u s ILH. Straub. M. Zeuner, J. Seholm erich, B. Lung, UniversityMedical Center, Regensburg,Germany
This study aimed at finding autonomic nervous function parameters in patients with systemic lupus erythematosus (SLE) which are suitable to demonstrate the inflammation-induced activation of the autonomic nervous system (AN$). 34 patients with SLE (age: 35.3:1:1.9 yr) were investigated by standardized AN8 function tests (Straub el al., J. Rheumatol 1996;23:87-92). The SLEDAI and laboratory parameters of systemic inflammation were assessed by standard techniques. PupiUary latency time hyperreflexia was found in 29.4% whereas maximal pupillary area was hyperresponsive in only 2.9%. 12% had overall cardiovascular autonomic nervous hyperreflexia. Patients with latency time hyperreflexia had more severe systemic inflammation (erythrocyte sedimentation rate (ESR): p
45
242 Altered Cytokine Production in Whole Blood Cultures of Chronic Fatigue S y n d r o m e Patients a n d a Disturbed Regulation by Glueoeorticoids J. Visser+B. Blauw, TNOPreventionandHealth, TheNetherlands, W. Graffelman, Facultyof GeneralPractician,Leiden, TheNetherlands, T. Bnmt, TNOPreventionand Health, TheNetherlands, R+de Kloet, MedicalPharmaeologyLACDR,Leiden, The Netherlands, L. Nagelkerken,/NO PreventionandHealth, The Netherlands Recently, we have shown that CD4 + T cells from patients with the chronic fatigue syndrome (CFS) produce less IFN+v and display an increased sensitivity to glucoeorticoids (GC) with regard to proliferation and IL-4 production (J Infect Dis 1998;177:451-4). Further studies with cells from healthy donors revealed that IL-12 was very sensitive to GC suppression, while 1L-IO was relatively resistant (Blood 1998, in press). In view of the importance of these cytokines in the regulation of IFN-y, we studied the regulation of IL- 10 and IL12 by endogenous and exogenous GC in whole blood cultures of 30 CFS patients and controls. CFS patients showed an increased production of LPS induced IL-tO (p<0.05), but no changes in IL-12. No difference in sensitivity to dexamethasone was detected, but the patients showed an increased sensitivity to hydrocortisone (HC). Addition of 10 M HC resulted in an increase of IL- 10 and a decrease of IL-12(p40) production, whereas the cytokine production of the controls was not affected. This might imply an altered functioning of GCreceptors (GR), also because IL-IO production showed an inverse correlation with serum levels of cortisol in the controls, but not in the patients. Further studies regarding the nuraber and affinity of GR in lymphocytes from CFS patients are heing performed to get more insight in the altered regulation of cytokines by GC.
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243
N e u r o n a l Regulation of Interleukin IL-6 Secretion by Substance P in the Intestinal Wall of Balb/C l ~ e e ILH. Straub. M. Kubitza, H. Heffarth, W. FaIL J. Scholm erich, UniversityMedical Center,Regensburg,Germany
Brain Neuropeptide Y (NPY) Y1 and Y2 Receptors Both Produce Anxiolysis b u t Differentially Affect Cortieosterone Levels and I n n a t e I m m u n e Functions S, van Htrsten, H. Nave, J. Ballof, D. Meyer, R.E. Sehmidt, HannoverMedieal ,~chool,Germany
Substance P (SP) is a neuropeptide which is associated with neurogenic inflammation in arthritic joints and inflamed mucosa of the gastrointestinal tract. -->To study SP effects in the intestinal mucosa. Exogenously added SP stimulated IL-6 secretion of intestinal wall strips at a concentration of 10"t° M (p=0.0023), but inhibited IL-6 secretion at 10"tt M (p=0.034). Thus, the concentration - response curve was bimodal. Electrical field stimulation (ES) of intestinal wall strips, which releases endogenous neurotransmitters from presynaptic nerve terminals, inhibited IL-6 secretion (p<0.0OOl). The neurekinin-1 (SP) receptor antagonist L668,169 further increased the electrically induced inhibition of IL-6 secretion at 10 .7 M (p=0.08). However, 1.668,'169 at a concentration of 10"B M significantly attenuated the electrically induced inhibition of IL-6 secretion (p=0.004). In conclusion, SP is involved in the neuromodulation of intestinal IL-6 secretion in Balb/C mice. The neuronal regulation of IL-6 by $P is bimodal with inhibition at lower and stimulation at higher concentration of SP. This indicates a differential influence of SP on immune function in the intestinal wall depending on the local SP concentration.
The involvement of brain NI~ Yt and Yz receptor subtypes in the regulation of activity and anxiety (home-cage, open-field, and plus-maze behavior), HPA-axis activation (serum cortieosterone levels), and innate immune functions (blood and splenic NKcell mediatedlysis), was studied in rats. Intracerebroventheularly (i.c.v.) applied combinations of pepfides and antagonists with various selectivity for Y~, and Yz, subtypes were used. At lh after application, the Y~3.s,+,-agonist [LeuJt,Pro~+]-NPY induced behavioral activation, exeerted anxiolytic effects on the plus-maze, and furthermore stimulated blood and splenic NKcell function. These effects were blocked by pre-trearment with the selective Yt+-antagonist BIBP-3226. However, the Y,,antagonist itself was anxiogenie, increased corticosterone levels and suppressed splenic NK function. The Y2.s,,,-agonist PYY(3-36) also produced anxiolysis, but in contrast to [Leu~t.Pro~4]-Nl~, i.c.v, application of this peptide increased corticosterone levels, and suppressed NK cell function. These PYY(3-36)-mediated effects were antagonized by pre-treatment with the competitive Y2,-antagonist T4-[NPY(33-36)]+. Thus, both Y=- and Y2,-mediate anxiolysis at lh after i.c.v. application. However, HPA-activily and innate immune functions are differentially affected. While Y~, stimulation increases innate immune functions, the Yz, activates the HPA-axisand suppresses NKcell function.
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Effect of P e r i p h e r a l A d m i n i s t r a t i o n of Bacterial Endotoxin on Neurosteroid Binding Sites of the G A B A - A Receptor Complex: A n A u t o r a d i o g r a p h i e Study in Mouse Brain J. Remigereau, S. Behar, E Boudou, M. Vineens, HopitalLariboisiere, Univer~ite Paris VII,France
Evaluation of Antioxidative Features of Melatonin in a L y m p h o c y t e Model A. Woclfler~P.M. Liebmann, H_C. Calnba, G. Debt, K. SGhaucnstcin, Universityof Graz,Austria
We have studied in mice brain the effect of inflammatory factors on neurosteroid function. Usin~ quantitative autoradiography , we have lnve.utigated the effect of the acute administration of b a c t e r i a l endotoxin (lipopoiysaecharide, LPS, 120 lag .I.P.) on neurostemid bindit,~ sites of the GABA-A receptor c,,mplex labelled with [3"~l-tb u t y l b l c y c l o p h o s p h o c o t h l o n a t e ([35S]-TBPS) in v a r i o u s brain gtruclurea. Results; 1 / t h e apparent n u m b e r of [35S]-TBPS binding sites w a s decrea,+ed in LPS treated animals as Compared to control group in regions studied (corteX, CA b CA3, Dentate Cyrus). 21 In ¢onh~ol Stoup, IC.'i0 values of the neuroateroid, allopregnanolone for the Inhibition of [~SS]-TBP9 binding were 3x10"6 and 5.4 x 10"6M respectively in cortex IV and V, and IA1 and 3.8x 10-'~M in CAI and CA3. in LPS treated group, ICS0 v a l u e s of allopregnanolone In inhibiting [35S]-TBPS binding w e r e decreased by a factor 5 to 10. Conclusion: these data gtrol~gly ~ul?.Be~.l. that LPS treatmen! Induced a modulation of tile interaction of neuroateroid with the GABA-A receptor complex.
Oxidative stress is known to impair various lymphocyte functions. One major target of oxygen radicals is the intracelinlar calcium response triggering lymphocyte activation. Since melatonin (MEL), the main hormone secreted by the pineal gland, has been reported to have strong antioxidative properties, we investigated whether MEL exerts beneficial effects on the impaired mitogen-induced calcium response of Jurkat T cells exposed to oxidative stress. Thus, we incubated Jurkat cells for 40 minutes with the hydroxyl radical producing system glucose oxidase + glucose, and measured the mitogeninduced intracellular calcium signal immediately thereafter using the calcium sensitive dye indo-I and FACS analysis. Jurkat cells exposed to hydroxyl radicals showed a delayed and impaired calcium response. While addition of glutathione during exposition to hydroxyl radicals readily restored the mitogen-induced calcium response, MEL was without any effect. Since even millimolar concentrations of MEL did not counteract the impairment of the calcium signal, we conclude that MEL does not exert protective features against oxidative stress in our cellular model. (Supported by the Austrian Science Foundation P-9925 Mad)
239 Association of A u t o n o m i c Nervous Hyperreflexia a n d Systemic I n f l a m m a t i o n in Patients with Systemic L u p u s E r y t h e m a t o s u s ILH. Straub. M. Zeuner, J. Seholm erich, B. Lung, UniversityMedical Center, Regensburg,Germany
This study aimed at finding autonomic nervous function parameters in patients with systemic lupus erythematosus (SLE) which are suitable to demonstrate the inflammation-induced activation of the autonomic nervous system (AN$). 34 patients with SLE (age: 35.3:1:1.9 yr) were investigated by standardized AN8 function tests (Straub el al., J. Rheumatol 1996;23:87-92). The SLEDAI and laboratory parameters of systemic inflammation were assessed by standard techniques. PupiUary latency time hyperreflexia was found in 29.4% whereas maximal pupillary area was hyperresponsive in only 2.9%. 12% had overall cardiovascular autonomic nervous hyperreflexia. Patients with latency time hyperreflexia had more severe systemic inflammation (erythrocyte sedimentation rate (ESR): p
45
242 Altered Cytokine Production in Whole Blood Cultures of Chronic Fatigue S y n d r o m e Patients a n d a Disturbed Regulation by Glueoeorticoids J. Visser+B. Blauw, TNOPreventionandHealth, TheNetherlands, W. Graffelman, Facultyof GeneralPractician,Leiden, TheNetherlands, T. Bnmt, TNOPreventionand Health, TheNetherlands, R+de Kloet, MedicalPharmaeologyLACDR,Leiden, The Netherlands, L. Nagelkerken,/NO PreventionandHealth, The Netherlands Recently, we have shown that CD4 + T cells from patients with the chronic fatigue syndrome (CFS) produce less IFN+v and display an increased sensitivity to glucoeorticoids (GC) with regard to proliferation and IL-4 production (J Infect Dis 1998;177:451-4). Further studies with cells from healthy donors revealed that IL-12 was very sensitive to GC suppression, while 1L-IO was relatively resistant (Blood 1998, in press). In view of the importance of these cytokines in the regulation of IFN-y, we studied the regulation of IL- 10 and IL12 by endogenous and exogenous GC in whole blood cultures of 30 CFS patients and controls. CFS patients showed an increased production of LPS induced IL-tO (p<0.05), but no changes in IL-12. No difference in sensitivity to dexamethasone was detected, but the patients showed an increased sensitivity to hydrocortisone (HC). Addition of 10 M HC resulted in an increase of IL- 10 and a decrease of IL-12(p40) production, whereas the cytokine production of the controls was not affected. This might imply an altered functioning of GCreceptors (GR), also because IL-IO production showed an inverse correlation with serum levels of cortisol in the controls, but not in the patients. Further studies regarding the nuraber and affinity of GR in lymphocytes from CFS patients are heing performed to get more insight in the altered regulation of cytokines by GC.
240
243
N e u r o n a l Regulation of Interleukin IL-6 Secretion by Substance P in the Intestinal Wall of Balb/C l ~ e e ILH. Straub. M. Kubitza, H. Heffarth, W. FaIL J. Scholm erich, UniversityMedical Center,Regensburg,Germany
Brain Neuropeptide Y (NPY) Y1 and Y2 Receptors Both Produce Anxiolysis b u t Differentially Affect Cortieosterone Levels and I n n a t e I m m u n e Functions S, van Htrsten, H. Nave, J. Ballof, D. Meyer, R.E. Sehmidt, HannoverMedieal ,~chool,Germany
Substance P (SP) is a neuropeptide which is associated with neurogenic inflammation in arthritic joints and inflamed mucosa of the gastrointestinal tract. -->To study SP effects in the intestinal mucosa. Exogenously added SP stimulated IL-6 secretion of intestinal wall strips at a concentration of 10"t° M (p=0.0023), but inhibited IL-6 secretion at 10"tt M (p=0.034). Thus, the concentration - response curve was bimodal. Electrical field stimulation (ES) of intestinal wall strips, which releases endogenous neurotransmitters from presynaptic nerve terminals, inhibited IL-6 secretion (p<0.0OOl). The neurekinin-1 (SP) receptor antagonist L668,169 further increased the electrically induced inhibition of IL-6 secretion at 10 .7 M (p=0.08). However, 1.668,'169 at a concentration of 10"B M significantly attenuated the electrically induced inhibition of IL-6 secretion (p=0.004). In conclusion, SP is involved in the neuromodulation of intestinal IL-6 secretion in Balb/C mice. The neuronal regulation of IL-6 by $P is bimodal with inhibition at lower and stimulation at higher concentration of SP. This indicates a differential influence of SP on immune function in the intestinal wall depending on the local SP concentration.
The involvement of brain NI~ Yt and Yz receptor subtypes in the regulation of activity and anxiety (home-cage, open-field, and plus-maze behavior), HPA-axis activation (serum cortieosterone levels), and innate immune functions (blood and splenic NKcell mediatedlysis), was studied in rats. Intracerebroventheularly (i.c.v.) applied combinations of pepfides and antagonists with various selectivity for Y~, and Yz, subtypes were used. At lh after application, the Y~3.s,+,-agonist [LeuJt,Pro~+]-NPY induced behavioral activation, exeerted anxiolytic effects on the plus-maze, and furthermore stimulated blood and splenic NKcell function. These effects were blocked by pre-trearment with the selective Yt+-antagonist BIBP-3226. However, the Y,,antagonist itself was anxiogenie, increased corticosterone levels and suppressed splenic NK function. The Y2.s,,,-agonist PYY(3-36) also produced anxiolysis, but in contrast to [Leu~t.Pro~4]-Nl~, i.c.v, application of this peptide increased corticosterone levels, and suppressed NK cell function. These PYY(3-36)-mediated effects were antagonized by pre-treatment with the competitive Y2,-antagonist T4-[NPY(33-36)]+. Thus, both Y=- and Y2,-mediate anxiolysis at lh after i.c.v. application. However, HPA-activily and innate immune functions are differentially affected. While Y~, stimulation increases innate immune functions, the Yz, activates the HPA-axisand suppresses NKcell function.
241
244
Effect of P e r i p h e r a l A d m i n i s t r a t i o n of Bacterial Endotoxin on Neurosteroid Binding Sites of the G A B A - A Receptor Complex: A n A u t o r a d i o g r a p h i e Study in Mouse Brain J. Remigereau, S. Behar, E Boudou, M. Vineens, HopitalLariboisiere, Univer~ite Paris VII,France
Evaluation of Antioxidative Features of Melatonin in a L y m p h o c y t e Model A. Woclfler~P.M. Liebmann, H_C. Calnba, G. Debt, K. SGhaucnstcin, Universityof Graz,Austria
We have studied in mice brain the effect of inflammatory factors on neurosteroid function. Usin~ quantitative autoradiography , we have lnve.utigated the effect of the acute administration of b a c t e r i a l endotoxin (lipopoiysaecharide, LPS, 120 lag .I.P.) on neurostemid bindit,~ sites of the GABA-A receptor c,,mplex labelled with [3"~l-tb u t y l b l c y c l o p h o s p h o c o t h l o n a t e ([35S]-TBPS) in v a r i o u s brain gtruclurea. Results; 1 / t h e apparent n u m b e r of [35S]-TBPS binding sites w a s decrea,+ed in LPS treated animals as Compared to control group in regions studied (corteX, CA b CA3, Dentate Cyrus). 21 In ¢onh~ol Stoup, IC.'i0 values of the neuroateroid, allopregnanolone for the Inhibition of [~SS]-TBP9 binding were 3x10"6 and 5.4 x 10"6M respectively in cortex IV and V, and IA1 and 3.8x 10-'~M in CAI and CA3. in LPS treated group, ICS0 v a l u e s of allopregnanolone In inhibiting [35S]-TBPS binding w e r e decreased by a factor 5 to 10. Conclusion: these data gtrol~gly ~ul?.Be~.l. that LPS treatmen! Induced a modulation of tile interaction of neuroateroid with the GABA-A receptor complex.
Oxidative stress is known to impair various lymphocyte functions. One major target of oxygen radicals is the intracelinlar calcium response triggering lymphocyte activation. Since melatonin (MEL), the main hormone secreted by the pineal gland, has been reported to have strong antioxidative properties, we investigated whether MEL exerts beneficial effects on the impaired mitogen-induced calcium response of Jurkat T cells exposed to oxidative stress. Thus, we incubated Jurkat cells for 40 minutes with the hydroxyl radical producing system glucose oxidase + glucose, and measured the mitogeninduced intracellular calcium signal immediately thereafter using the calcium sensitive dye indo-I and FACS analysis. Jurkat cells exposed to hydroxyl radicals showed a delayed and impaired calcium response. While addition of glutathione during exposition to hydroxyl radicals readily restored the mitogen-induced calcium response, MEL was without any effect. Since even millimolar concentrations of MEL did not counteract the impairment of the calcium signal, we conclude that MEL does not exert protective features against oxidative stress in our cellular model. (Supported by the Austrian Science Foundation P-9925 Mad)