Adrenaline-induced immunological changes are altered in patients with rheumatoid arthritis

J. M. Kittner, R. Jacobs, C. R. Pawlak1, C. J. Heijnen2, M. Schedlowski3 and R. E. Schmidt1

Department of Clinical Immunology, Hannover Medical School
1 Department of Medical Psychology, Hannover Medical School, Germany,
2 Laboratory for Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands and
3 Department of Medical Psychology, University of Essen, Germany


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
 References
 
Objective. To investigate whether in rheumatoid arthritis (RA) patients the immunological changes induced by adrenaline are different from healthy controls (HC).

Methods. Fifteen female RA patients and 14 HC were infused with 1 µg/kg adrenaline over 20 min. Blood was drawn before, immediately after, and 1 h after the end of infusion. Lymphocyte subpopulations, cytokine production and natural killer cell cytotoxicity were determined.

Results. Subjects exhibited mild cardiovascular changes with no differences between patients and controls. CD16+CD56+CD3- NK cells increased by a factor of 5.7, CD3+ T cells by 1.5, monocytes by 1.6 and PMN by 1.2 in both groups. The numbers of IL-8- and IL-10-producing monocytes were higher in patients and presented a larger increase after infusion. NK cytotoxic activity was higher in RA patients and increased after infusion in both groups. Activated monocytes and T cells were preferentially recruited in patients and controls. Values returned to baseline 1 h later.

Conclusion. We describe an altered response to adrenaline in patients with RA with both pro- and anti-inflammatory effects. Additionally, activated T cells and monocytes recruited to the peripheral blood may influence disease activity.

KEY WORDS: Rheumatoid arthritis, Adrenaline infusion, Cytotoxicity, Cytokines, Chemokines, Activated T cells, CD14+CD16+ monocytes, Perforin.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
 References
 
The sympathetic nervous system and the immune system are closely linked which is apparent in the expression of adrenergic receptors on leucocytes and the dense innervation of lymphoid tissue [1]. Adrenaline is one of the key hormones released during acute stress and contributes decisively to the ‘fight or flight’ reaction [2] in the cardiovascular, pulmonal, metabolic and immunological system. In situations of acute psychological stress such as a first-time parachute jump adrenaline increases to a level up to 10-fold from baseline [3]. Acute psychological stress and physical exercise have been shown to affect immune functions including significant increases in NK, T cell and monocyte numbers in the peripheral blood [46]. These changes are most likely due to recruitment phenomena and are mediated by catecholamines via ß2-adrenergic receptors as they can be blocked by selective ß2-receptor antagonists. Consequently, analogous immunological changes can be induced by intravenous application of adrenaline [7, 8]. Clinical observations suggest an association between stressful life events and disease activity in patients with autoimmune disease [9, 10]. The common autoimmune disease rheumatoid arthritis (RA) leads to pain and immobility by joint and bone destruction. Genetic factors, sex hormones and one or several unknown antigens may lead to stimulation and oligoclonal expansion of T cells, production of pro-inflammatory cytokines, chemokines and autoantibody production (reviewed in [11]). The sympathetic nervous system was reported to be dysregulated in patients with RA [12]. A down-regulation of G-protein coupled receptor kinases in cells from RA patients and an increased sensitivity to ß2-adrenergic agonists in vitro have been described [13]. On the other hand, Baerwald et al. [14] demonstrated a decreased number of ß-adrenoceptors on peripheral blood mononuclear cells (PBMC) of RA patients and reduced response of their lymphocytes to adrenaline when stimulated with mitogens [14]. In this study we tested the hypothesis that adrenaline-induced immunological changes in patients with RA differ from adrenaline effects in healthy controls (HC). Immunological parameters such as circulating leucocytes, lymphocyte subpopulations, cytotoxic activity, cytokine and chemokine patterns, killer cell immunoglobulin-like receptors (KIRs), adhesion molecule expression and perforin production were assessed before, immediately after, and 1 h after the intravenous application of adrenaline.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
 References
 
Patients and subjects
Fifteen female RA patients (mean age 51.5 yr, S.D. 12.32, range 22–65) and 14 healthy female volunteers (mean age 43.6 yr, S.D. 11.32, range 27–58) participated in the study after written consent. RA patients were recruited from the rheumatology out-patient clinic at Hannover Medical School. Patients had been diagnosed according to the criteria of the American Rheumatism Association [15]. The study was approved by the ethical review board of the Hannover Medical School. Subjects with significant cardiovascular diseases, hypertension, or concurrent infections were excluded. Patients displayed no exacerbation of disease activity at least 4 weeks prior to onset of the experiment. All patients were free of steroids, methotrexate, azathioprine, leflunomide or gold therapy. One patient took hydroxychloroquine (250 mg/day), and one patient was treated with penicillamine (300 mg/day). Seven patients were on non-steroidal anti-inflammatory drugs regularly or as needed. In addition, all subjects were free of medication such as anti-depressants, adrenoceptor antagonists and benzodiazepines, suspected of altering cardiovascular or immunologic responsiveness.

Assessment of disease activity
All patients filled out the RADAR score, a self-rating scale for disease activity in RA [16]. C-reactive protein (CRP) was highly sensitive measured using a Dade–Behring nephelometer (Dade Behring, Marburg, Germany) with polystyrene particles coated with monoclonal antibodies (mAb) against human CRP.

Experimental design and procedure
Experimental sessions were conducted between 08:00 and 12:00 h. Subjects were requested to avoid caffeine in the morning of the study. After giving informed consent a medical history and an ECG were taken. Subjects were seated in a chair and remained so throughout the experiment. An intravenous cannula was inserted into a cubital vein and serum potassium was determined. Fifteen minutes after insertion of the cannula the first blood sample (baseline) was drawn. Adrenaline (Hoechst Pharma, Germany) was freshly diluted in 50 ml NaCl (0.9%) to the desired concentration of 0.05 µg/kg/min, corresponding to 60 µg for a 60-kg person. The solution was applied continuously over 20 min via an intravenous pump. Blood pressure and heart rate were supervised every 5 min. Immediately after infusion, a second blood sample (infusion) was drawn by the cannula. One hour after the end of the infusion a last blood sample was drawn.

White blood cells
Blood was drawn using sodium-heparin-MonovettenTM (Sarstedt, Nümbrecht, Germany) or in heparinized syringes for immunological analyses. White blood cells were counted automatically with a STKS (Coulter Electronics Inc., Hialeah, USA).

Monoclonal antibodies
Labelled mAb directed against the respective human leucocyte antigens CD2 FITC, CD4 FITC, CD8 PE, CD16 FITC (DAKO, Hamburg, Germany), CD3 FITC, CD11a FITC, CD158 a and b PE (Immunotech, Hamburg, Germany), CD3 PE, CD3 APC, CD20 FITC and CD56 PE, CD56 APC (Becton Dickinson, Heidelberg, Germany), CCR2 PE, CCR5 FITC (R&D Systems) and CD14 (Pharmingen, Hamburg, Germany) were used. The following mAb for the detection of intracellular cytokines and perforin were purchased from Pharmingen (Hamburg, Germany): IFN{gamma} FITC, IL-2 PE, IL-4 PE, IL-10 PE, TNF{alpha} FITC, RANTES PE, MCP1 FITC, perforin FITC. Monoclonal antibodies against MIP1{alpha} FITC and IL-8 PE were purchased from R&D Systems (Minneapolis, USA). For all cytofluorimetric experiments appropriate isotype control antibodies (PE/FITC/APC) were utilized.

Phenotypic analyses
Mononuclear cells were separated using Ficoll-Hypaque centrifugation. Phenotypic analyses were performed using directly labelled mAb in two- or three-colour immunofluorescence. Briefly, 1–3x105 cells/well were incubated with murine mAb against the appropriate antigen at an optimal dilution for 30 min at 4°C. Non-specific binding was eliminated by mixing the samples with a 1:5 solution of a commercial human IgG (Endoglobin, ImmunoGmbH, Heidelberg, Germany). Samples were washed three times in PBS/BSA, and at least 104 cells per lymphocyte or monocyte gate were analysed using a FACS Calibur (Beckon Dickinson, Heidelberg, Germany). The gates were set according to the forward scatter and sideward scatter properties of the cells.

Cell stimulation and staining of intracellular cytokines
Cytokine and chemokine production is frequently determined from supernatants of stimulated PBMC by ELISA. These results are hampered by consumption of cytokines during culture; therefore, we decided to determine cytokine- and chemokine-producing cells by intracellular staining: after Ficoll separation 2x106 PBMC were resuspended in 1 ml culture medium (RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 1% penicillin–streptomycin solution). Cells were then stimulated by addition of 10 ng phorbol 12-myristate 13-acetate (PMA) and 1 mM ionomycin. One hour later the transport inhibitor brefeldin A (2.5 mM) was added to prevent the secretion of the induced cytokines into the supernatant. After 16 h of culture at 37°C and 5% CO2 the cells were harvested. Cells were washed with PBS/BSA and stained for surface molecules for 30 min. After washing, cells were fixed for 10 min at room temperature in PBS containing 4% paraformaldehyde. Cells were washed again and resuspended in saponin buffer (PBS supplemented with 5 mM HEPES and 0.1% saponin). Subsequently, aliquots were stained with mAb against intracellular cytokines. Unspecific binding of the mAb via Fc-receptors was reduced by adding human IgG solution. After 30 min incubation at 4°C, cells were washed once with saponin buffer and twice with PBS/BSA. After resuspension cells were ready for FACS analysis.

Perforin staining
Cells were surface-stained with mAb against CD3 and CD56, fixed with paraformaldehyde, and permeabilized with saponin without prior stimulation. Cells were then stained with mAb against perforin for 20 min. After two washes and resuspension in PBC they were analysed by three-colour FACS analysis.

NK assay
Standard 4 h 51chromium (51Cr) release cytotoxicity assays were performed using cryopreserved PBMC. Frozen samples were rapidly thawed by three washes in RPMI 1640 and finally resuspended in medium supplemented with 10% FCS. One hour later, cells were counted and added in triplicate at four effector to target (E/T) ratios (60:1, 30:1, 15:1 and 7.5:1) in V-bottom microtitre plates with 5x103 51Cr-labelled K562 target cells per well. The medium used for cytotoxicity assays was RPMI 1640 supplemented with 5% FCS and 1% penicillin–streptomycin. After 4 h of incubation, plates were centrifuged, supernatants were harvested and released 51Cr was measured in a gamma counter. Background values were determined by incubating target cells without effector cells. Maximal values were obtained by lysing target cells with Triton X-100 (Merck, Darmstadt, Germany). Specific lysis was calculated by:


For a more precise analysis of cytotoxic capacity lytic units (LU) were calculated according to the method of Bryant et al. [17]. Using this mathematical transformation the sigmoid dose response relation between number of employed effectors and measured cytotoxicity can be linearized. LU calculated this way allow comparison of cytotoxicity assays performed at different points of time.

Statistical analyses
Statistical analyses of the data were performed by using ANOVAs with repeated measures. The group x time interaction effect is given for the differences between RA patients and HC. SPSS 10.0.7 was used.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
 References
 
Disease activity
The pain/tenderness score of the RADAR comprising number and intensity of affected joints revealed 8.13±6.54 points (scale 0–60). The level of function score evaluating the impairment of normal life activities revealed 1.33±0.45 points (scale 0–4). ESR was not determined. A CRP of 5.14±4.30 mg/l (normal range <5 mg/l) was measured in RA patients.

Cardiovascular system
Heart rate increased by 20% directly after adrenaline infusion (P<0.001, time effect). A significant increase of 5% (P=0.008, time effect) of the systolic blood pressure was observed, whereas the diastolic blood pressure decreased by 15% after the infusion (P<0.001, time effect) (data not shown). However, no significant differences in values and time kinetics between patients and healthy subjects were observed.

Leucocytes
Blood samples drawn before, immediately after and 1 h after the end of the infusion were analysed using a Coulter counter. Neutrophil granulocyte numbers increased significantly after infusion and slightly further 1 h later (P<0.001, time effect). Both monocyte and lymphocyte numbers also increased after infusion, and were back at baseline levels at the follow-up time point (P<0.001, time effect). However, RA patients and HC did not differ in any of these cell numbers at any time point (Table 1Go).


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TABLE 1. Leucocytes: average cell numbers per microlitre are shown, as determined by Coulter counter

 

Lymphocyte subsets
CD3+ T-cell numbers increased by a factor of 1.5 (P<0.001) after infusion and were back to baseline 1 h later. The increase in CD3+ numbers was predominantly due to CD3+CD8+ cells which doubled (P<0.001, time effect). CD4+ T-helper cells presented only a moderate increase (P=0.020, time effect) (Table 2Go). CD20+ B cells, in contrast, decreased by 20% after infusion (P=0.009, time effect) and remained on this level 1 h later (Table 2Go). The most distinct changes after infusion were observed in CD3-CD16+CD56+ NK cell numbers which increased by almost 6-fold (P<0.001, time effect) and decreased below baseline levels 1 h later in both groups (Fig. 1Go). Except for a tendency to elevated numbers of CD4+ T cells in RA patients (P=0.071, group effect) no significant differences at any time point were observed between patients and controls for all lymphocyte subsets.


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TABLE 2. Lymphocyte and monocyte subsets as determined by FACS analysis

 


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FIG. 1. NK cells (CD16+CD56+CD3-) increased after infusion (P<0.001). No differences were observed between patients ({triangleup}, n=15) and controls ({blacksquare}, n=14). Data are presented as mean ± S.E.

 
In addition, the numbers of CD56+CD3- NK and CD3+ CD56- T cells expressing CD158a or b (KIRs) were determined. Although no differences were observed at baseline the CD158a+ NK cells increased significantly less in RA patients after infusion compared with HC (interaction, P=0.033) (Fig. 2Go).



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FIG. 2. After infusion, in RA patients ({triangleup}, n=13) a significantly lower increase of CD158a+CD3-CD56+ cells was observed (interaction, P=0.033) compared with HC ({blacksquare}, n=8). Data are presented as mean ± S.E.

 

Monocytes
CD14++ monocytes increased by a factor of 1.4 after application of adrenaline in patients and controls. The subset of CD14+CD16+ monocytes, which comprises about 10% of the monocytes, demonstrated similar cell numbers in both patients and controls (P=0.772, group effect). Interestingly, this subset of cells increased by a factor of 2.6 in both groups after the infusion. One hour later, baseline levels were achieved (Table 2Go).

Cytotoxic activity
Cytotoxic activity was determined by standard cytotoxicity assay using 51Cr-labelled K562 target cells. Cytotoxicity increased significantly in both groups (P<0.001). Cells from RA patients exhibited a higher lytic activity over all three time points (P=0.041, group effect). One hour after the end of the infusion, cytotoxicity was slightly below baseline levels in both groups (Fig. 3Go).



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FIG. 3. NK cytotoxicity assays revealed higher lytic activity in RA patients ({triangleup}, n=13) compared with HC ({blacksquare}, n=13) (group effect, P=0.041). A difference in reaction pattern was not observed (interaction, P=0.78). Data are presented as mean ± S.E.

 

Cytokine and chemokine production
In order to determine cytokine and chemokine production, lymphocytes were stimulated with ionomycin and PMA, monocytes with lipopolysaccharide (LPS) prior to permeabilization and intracellular staining with specific antibodies. Without prior stimulation, no production of cytokines or chemokines could be observed in any sample of any donor. Interleukin-2 (IL-2)-, interferon {gamma} (IFN{gamma})-, interleukin-4 (IL-4)- and tumour necrosis factor {alpha} (TNF{alpha})-producing lymphocytes increased after infusion with no differences between patients and controls. IL-2+ lymphocytes in tendency were more frequent in patients with RA than in HC (not reaching significance, P=0.055, group effect) (Table 3Go). Differences between RA patients and HC were observed in the number and time kinetics of monocytes producing IL-8, a potent chemokine, and IL-10, a cytokine promoting Th2 differentiation: RA patients exhibited significantly higher numbers of IL-8+ monocytes at all three time points (P=0.009, group effect) and after the infusion of adrenaline in RA patients a significantly stronger increase of these cells was observed (P=0.043, interaction). At the follow-up time point, cell numbers returned to baseline values (Fig. 4Go). The number of IL-10-producing monocytes was also higher at all three time points (P=0.048, group effect). After the application of adrenaline RA patients presented a larger increase of IL-10-producing monocytes compared with HC (P=0.022, interaction) (Fig. 5Go).


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TABLE 3. Number of cytokine-producing lymphocytes per microlitre after stimulation with PMA/ionomycin as determined by FACS analysis

 


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FIG. 4. In RA patients ({triangleup}, n=15) IL-8+ monocytes displayed higher numbers compared with HC ({blacksquare}, n=13) (group effect, P=0.009). The increase of IL-8+ monocytes was significantly stronger in RA patients (interaction, P=0.043). Data are presented as mean ± S.E.

 


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FIG. 5. IL-10+ monocytes were higher in RA patients ({triangleup}, n=15) compared with HC ({blacksquare}, n=13) (group effect, P=0.048) and increased significantly stronger after infusion (interaction, P=0.022). Data are presented as mean ± S.E.

 

Recruitment of activated T cells
Perforin is responsible for the lysis of target cells by NK and T cells. Eighty-five percent of CD56+CD3- NK cells displayed an expression of perforin, but only 12% of CD3+ T cells in patients (n=13) and controls (n=8) were perforin+ at baseline. After application of adrenaline the percentage of perforin+ T cells significantly increased to 29.4% (P<0.001) in both groups. One hour later, the level returned to baseline. The percentage of perforin+ NK cells did not change significantly. No differences between patients and controls were observed (Fig. 6Go).



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FIG. 6. At baseline, 12% of CD3+CD56- T cells and 85% of CD3-CD56+ NK cells were positive for perforin. After infusion, the percentage of perforin+ T cells increased to 29% (P<0.001), whereas the percentage of perforin+ NK cells did not change significantly. No differences were observed between patients ({triangleup}, n=13) and controls ({blacksquare}, n=8). Data are presented as mean ± S.E.

 
CD11a is an adhesion molecule important for activation and transmigration of lymphocytes expressed on all lymphocytes. CD8+ T cells in patients (n=12) and controls (n=7) presented two populations of either low (+) or high (++) CD11a expression, whereas all CD56+CD3- NK cells showed a high density of CD11a. Adrenaline caused a selective increase of CD11a++CD8+ T cells by a factor of 4 (P=0.012) in peripheral blood in both patients and controls (Fig. 7Go shows a representative histogram).



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FIG. 7. Two distinct populations, CD11a low and high positive CD8+ T cells were detected. After infusion, the CD11a++ cells increased by a factor of 4 without differences between patients and controls. A representative histogram is shown.

 


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
 References
 
In several clinical studies an association between psychological stress and the course of RA was observed [9, 10]. It is well established that catecholamines via ß2-receptor stimulation increase intracellular cAMP levels in lymphocytes, thereby blocking the production of pro-inflammatory cytokines and enhancing the secretion of IL-10 [18, 19]. Thus, a shift towards a Th2-response is induced which may lead to less cellular infiltration and less tissue destruction. However, local interactions can be different, and also pro-inflammatory effects of catecholamines have been reported [20, 21]. The role of catecholamines in RA is not definite yet. In animal models of RA, studies with either adrenergic agonists or antagonists yielded inconsistent results [2224].

In this study, RA patients and HC were infused intravenously with adrenaline in order to analyse whether patients with RA would present differences in leucocyte and lymphocyte distribution, in NK cell activity, cytokine and chemokine production before, immediately after and 1 h after the end of the infusion. The observed cardiovascular changes were mild, and did not differ between both groups. Lymphocyte numbers doubled after infusion, mainly due to increases of CD16+CD56+CD3- NK- and CD3+ T cells which is in accordance with previous findings in normal controls [5]. In contrast to the literature, cytotoxic activity was higher over all three time points in RA patients compared with HC. A reduced activity of NK cells in patients with RA has been described, presumably due to chronic activation [25]. As our patients presented only a low inflammatory activity, the NK cells might not yet be exhausted but, on the contrary, activated.

The number of KIR-expressing T and NK cells revealed a weaker increase of CD158a+ NK cells in RA patients after the infusion compared with HC. KIRs play a pivotal role in regulating cytotoxic activity, inhibiting lysis of cells that express ‘self’ MHC molecules, thereby contributing to tolerance. Recently, Kogure et al. [26] reported a decreased density of CD158a on CD8+ T cells of RA patients. Our results demonstrate that a lower number of tolerance-providing cells is present after adrenaline infusion in RA patients compared with HC which might contribute to the autoimmune process [27].

Monocyte numbers are known to be increased by psychological stress [28]. Steppich et al. [6] described an increase of monocytes after physical exercise and a selective enhancement of the subset of CD14+CD16+ monocytes. These cells are known to be efficient producers of TNF, but not IL-10 and were thus addressed as pro-inflammatory monocytes [29]. The CD14+CD16+ monocytes were shown to be elevated in inflammatory conditions [30], but up to now, no study in patients with RA was performed. However, in our experiments we could not detect differences in CD14+CD16+ monocyte numbers or time kinetics between both groups. We confirmed the assumption of Steppich et al. that the selective increase of this subset is caused by adrenaline.

The number of monocytes producing IL-8 and IL-10 after LPS stimulation was higher at baseline in RA patients, and also the increase of both IL-8+ and IL-10+ monocytes after infusion of adrenaline was significantly higher in RA patients. Both cytokines play an important role in RA, although their function is contradictory. IL-8 acts as a chemokine, attracts PMN and monocytes to the area of inflammation [31] and activates them. In an animal model the infusion of simply IL-8 into joints induced arthritis [32] and a positive correlation of IL-8 serum levels with disease activity in patients with juvenile RA was found [33]. IL-10 levels, on the other hand, are elevated in RA patients in both serum and synovial fluid [34]. In animal models, IL-10 presented a clear anti-inflammatory activity by blocking the production of pro-inflammatory cytokines and shifting towards a favourable Th2-response [35]. In humans, the production of IL-10 in peripheral PBMC was negatively correlated to disease activity and progression of joint destruction [36]. Interestingly, LPS-induced IL-8 and IL-10 production in human monocytes was potentiated by ß2-adrenergic agonists in vitro [37]. In summary, adrenaline led to higher numbers of both pro- and anti-inflammtory cells in RA patients. It needs to be elucidated whether and to what extent these cells influence systemic and articular inflammatory activity. However, we cannot exclude that the observed changes are due to a chronic inflammatory state rather than specifically to RA. In earlier work of our group specific differences in the reaction pattern to an acute stress situation between RA and SLE patients were found [38]. Therefore, further studies in patients with other chronic inflammatory diseases should be performed.

Perforin is involved in the inflammatory process of RA, apparent in an elevated number of perforin-expressing cells in synovial fluids of RA patients [39]. In our study, perforin-expressing T cells were preferably recruited into peripheral blood after application of adrenaline. To our knowledge, up to now a selective increase of perforin-expressing T cells after exercise or application of catecholamines has not been described. However, differences in cell numbers or kinetics between both groups were not observed. Also, adhesion molecules may play a role in RA for transmigration of lymphocytes into the joints, since a higher surface expression on cells from patients with reactive arthritis had been described [40].

CD11a (LFA-1) belongs to the integrin family and is decisive for cell adhesion and activation. CD11a+ cells are considered to be naive, CD11a++ to be primed T cells [41]. An increase of CD11a density on CD8+ lymphocytes after infusion of ß-adrenergic agonists was demonstrated [42]. We confirmed these findings, but we did not identify differences in CD11a++ cell numbers or kinetics between patients and controls.


    Conclusion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
 References
 
The infusion of adrenaline proved to be a safe and valid method to induce immunological changes in RA patients. Patients differed in their response to adrenaline in presenting a higher increase of both IL-8- and IL-10-producing monocytes after infusion. Surprisingly, this suggests that adrenaline has both pro- and anti-inflammatory effects in patients with chronic inflammatory disease. Due to the short time, we assume that these changes are caused by recruitment phenomena, but also an induction of cytokine expression is conceivable. Activated monocytes displayed similar increases as during physical activity without differences between RA patients and HCs. A selective increase of perforin-expressing T cells after infusion of adrenaline was demonstrated for the first time. Elevated levels of activated cells in the peripheral blood may facilitate a recruitment into the joints and consecutively enhance the inflammatory process.


    Acknowledgments
 
The authors would like to thank all participants in the study, Mrs Verhey from the Rheumaliga Hannover, Dr J. Hülsemann, Dr L. Köhler and Dr J. Kuipers for their help in recruiting patients. This work was supported by the Volkswagen Foundation Germany, Grant I/72 032/031.


    Notes
 
Correspondence to: J. Kittner, Division of Clinical Immunology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. The first two authors contributed equally to this article. Back


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Conclusion
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Submitted 24 January 2002; Accepted 25 March 2002





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