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Tumor necrosis factor-α and interleukin-6 regulate secretion of brain-derived neurotrophic factor in human monocytes
Journal of Neuroimmunology 160 (2005) 204 – 209 www.elsevier.com/locate/jneuroim
Tumor necrosis factor-a and interleukin-6 regulate secretion of brain-derived neurotrophic factor in human monocytes Olaf Schulte-Herbrqggena,1, Christina Nassensteinb,1, Marek Lommatzschc, David Quarcood, Harald Renze, Armin Braunb,* b
a Department of Neurology, Charite´ , Humboldt University, Berlin, Germany Immunology and Allergology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs Strasse 1, 30625 Hannover, Germany c Department of Pneumology, University Rostock, Schillingallee 35, 18057 Rostock, Germany d Department of Pediatrics, Charite´, Humboldt University, 13353 Berlin, Germany e Department of Clinical Chemistry and Molecular Diagnostics, Philipps-University Marburg, Baldingerstrasse, 35033 Marburg, Germany
Received 23 July 2004; received in revised form 25 October 2004; accepted 25 October 2004
Abstract Activated macrophages have been shown to produce brain-derived neurotrophic factor (BDNF) in diseases such as multiple sclerosis (MS) or allergic bronchial asthma (BA). However, there is little data on BDNF regulation in these cells. We demonstrate that unstimulated human peripheral blood monocytes, but not lymphocytes, constitutively secrete BDNF. IL-6 and TNF-a specifically enhanced BDNF secretion in monocytes, whereas typical Th1- and Th2-cytokines did not show any effect. None of the cytokines induced BDNF secretion in T- or B-cells. Thus, our data provide evidence that IL-6 and TNF-a represent a specific link between monocyte infiltration and neuronal changes in inflammatory diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: BDNF; Neurotrophins; Neuroimmunology; IL-6; TNF-a; Inflammation; Monocytes
1. Introduction Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is an essential mediator of neuronal activity and synaptic plasticity in the central and peripheral nervous system (Lewin and Barde, 1996; Kerr et al., 1999; Lommatzsch et al., 1999; McAllister et al., 1999). Initially, BDNF was thought to be predominantly derived from neuronal tissues. This concept has recently been revised by the finding that BDNF is produced in inflamed tissues of diseases such as allergic bronchial asthma (BA) and multiple sclerosis (MS). BDNF levels are specifically enhanced in the late phase reaction following segmental * Corresponding author. Tel.: +49 511 5350 263; fax: +49 511 5350 155. E-mail address: [email protected] (A. Braun). 1 Both authors contributed equally. 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.026
allergen provocation in patients with BA (Virchow et al., 1998). In a mouse model of BA, we have identified macrophages as predominant cellular sources of enhanced BDNF production in the inflamed lung. This BDNF production was specifically induced by allergic inflammation, since macrophages from control animals did not show any detectable BDNF expression (Braun et al., 1999). Furthermore, infiltrating mononuclear cells, predominantly representing activated macrophages, were shown to be BDNF immunoreactive in inflammatory brain lesions of subjects with MS (Kerschensteiner et al., 1999). These data suggest that activated macrophages represent a major source of BDNF in inflamed tissues. However, to date, there is no data on inflammatory mediators regulating BDNF secretion in macrophages. In addition, it is unknown whether unstimulated human monocytes of the peripheral blood constitutively secrete BDNF. Therefore, the aim of this study was to investigate the cellular sources of BDNF in
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unstimulated peripheral blood mononuclear cells (PBMC) and to test the impact of cytokines typically involved in BA and MS on BDNF secretion. We demonstrate here that peripheral blood monocytes constitutively secrete BDNF, representing the major source of this neurotrophin among PBMC. In addition, we show that TNF-a and IL-6 specifically enhance BDNF secretion in human monocytes. These findings may help to further elucidate the mechanisms of neuroimmune interactions in chronic inflammatory diseases.
2. Materials and methods 2.1. Cell separation PBMC were isolated from healthy individuals (aged 23– 35 years) using density centrifugation (Lymphoprep, Nycoprep Pharma, Oslo, Norway) as previously described (Nasert et al., 1996). T-cell, B-cell and monocyte separation from PBMC was performed by negative selection using commercial magnet-activated cell separation (MACS) extraction kits (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity was determined using flow cytometry. B-cells had a purity of N80%, T-cells N95% and monocytes N90% (data not shown). 2.2. Cell culture Cells (2105 PBMC, 4105 CD3+ cells, 4105 CD19+ and 4105 CD14+ per well) were cultured in serum-free AIM-V (Gibco BRL, Paisley, Scotland) with 1% lglutamine (Biochrom, Berlin, Germany) and 1.25 Ag/ml fungizone (Gibco BRL) at 37 8C in a 95% air/5% CO2 humidified atmosphere for 24 h. Cytokine stimulation experiments were performed with 10, 50 and 100 ng/ml of each cytokine for 24, 48 and 96 h (R&D Systems, Minneapolis, USA). Optimal conditions for both IL-6 and TNF-a were 50 ng/ml after 24 h. Cytokine blocking experiments were performed with 50 ng/ml of cytokine and 10 Ag/ml of monoclonal anti-human-cytokine antibody. Viability of cells was quantified by a Trypan blue assay (Biochrom, Berlin, Germany). Only cell cultures with a viability of N95% were chosen for BDNF determination. 2.3. Determination of BDNF by ELISA BDNF was measured in cell-free cell-culture supernatants with commercial ELISA kits according to the manufacturer’s instructions (Promega, Madison, WI) as previously described (Lommatzsch et al., 1999). Briefly, flat-bottom 96-well plates (Nunc, Wiesbaden, Germany) were coated with monoclonal mouse anti-BDNF antibodies. Captured neurotrophins were detected with secondary chicken polyclonal anti-BDNF and tertiary antibodies conjugated to horseradish peroxidase. Neurotrophin content was quantified against a standard curve
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generated with known amounts of protein. Detection limits were 4 pg/ml. To exclude eventual cross reactivity with other neurotrophins, the BDNF ELISA was tested with rhNGF, rhNT3 and rhNT4 (R&D Systems). There was no cross reactivity detectable. 2.4. BDNF immunoreactivity Immunoreactivity against BDNF protein was studied on acetone-fixed cytospins of freshly isolated cells following the manufacturer’s instruction (EnVisionk System Alkaline Phosphatase, DAKO, Glostrup, Denmark). Monoclonal mouse IgG1 anti-human BDNF antibodies (10 Ag/ml) were used as primary antibody and incubated overnight at 4 8C (R&D Systems). Incubation of cytospins with purified mouse IgG1 antibodies served as negative control (R&D Systems). 2.5. Determination of BDNF mRNA by real-time PCR Total cellular RNA was extracted from monocytes 2 h after incubation with culture medium (control), 50 ng/ml TNF or 50 ng/ml IL-6, respectively, using a commercial available RNA-isolation kit (RNeasy Mini Kit, Qiagen, Hilden, Germany). After digestion of genomic DNA by DNase treatment (RNase Free DNase Set, Qiagen), reverse transcription was performed with 0.1 Ag of total RNA from each sample in a 20 Al reaction for 60 min at 37 8C followed by 5 min at 95 8C according to the manufacturer’s recommendation (Omniscript RT-Kit, Qiagen). As control, 0.1 Ag RNA of each sample was treated according to the same protocol with addition of water instead of the reverse transcriptase. Real-time PCR was performed on the Light Cycler Instrument (Roche, Mannheim, Germany) using DNA binding dye SYBR green (Light Cycler-Fast Start DNA Master SYBR Green I, Roche). PCR reaction mix contained 2 Al PCR master mix supplement with 2.5 mM MgCl2, custom synthesized primers (MWG Biotech, Ebersberg, Germany) and 2 Al of either external standard, cDNA or RNA filled up with water to a final volume of 20 Al. As specific primers, we used porphobilinogen deaminase (PBGD, sense: 5V-ACA CAG CCT ACT TTC CAA GCG GAG-3V, antisense: 5V-TCT TGT CCC CTG TGG TGG ACA TAG CAA-3V) and BDNF (sense: 5V-CCA AGG CAG GTT CAA GAG G-3V, antisense: 5V-TCC AGC AGA AAG AGA AGA GGA-3V). After an initial denaturation step at 95 8C for 10 min, the PCR reaction was initiated with an annealing temperature of 60 8C for 10 s, followed by an extension phase at 72 8C for 9 s and a denaturation cycle at 95 8C for 1 s. At the end of each extension phase, fluorescence was observed at 72 8C and the PCR was completed after 45 cycles. The melting point analysis was carried out by heating the amplicon from 65 to 958 C and revealed the characteristic melting point for each product. After cooling down to 408 C, the product was extracted from the capillary. A total of 10 Al of each reaction was run onto a 1.5% agarose gel and visualized by staining with
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and cultured them for 24 h (12 individuals). We observed a constitutive secretion of BDNF by unstimulated PBMC (Fig. 1A). In order to identify the cell subsets responsible for this BDNF-secretion, we examined supernatants of purified T-cell, B-cell and monocyte cultures. Unstimulated monocytes constitutively secreted BDNF, whereas BDNF was not detectable in B- and Tcell supernatants (Fig. 1A). These data were confirmed by immunocytochemistry. A subpopulation of PBMC morphologically identified as monocytes as well as purified monocytes revealed BDNF immunoreactivity. In contrast, purified T-cells and B-cells remained BDNF-negative (Fig. 1B). 3.2. Stimulation of PBMC with cytokines involved in BA and MS In order to identify the mediators which regulate BDNF secretion in PBMC, we tested the impact of cytokines typically involved in BA or MS on BDNF secretion (Begolka and Miller, 1998). The proinflammatory cytokines TNF-a and IL-6 significantly enhanced BDNF levels in PBMC cultures after 24 h in a dose-dependent manner, whereas the typical Th1- and Th2-cytokines IL-1h, IFN-g, IL-2, IL-4, IL-5, IL-13 and IL-15 did not have any effect (Table 1). The specificity was demonstrated by neutralizing antibodies against IL-6 or TNF-a, which completely abolished cytokine-induced BDNF elevations in PBMC supernatants (n=10).
Fig. 1. Constitutive BDNF synthesis by mononuclear cells from the peripheral blood. (A) The BDNF protein level in cell free supernatants of PBMC, CD3+ cells, CD19+ cells and CD14+ cells from healthy donors was measured by ELISA after 24 h of culture. Columns include 12 individuals, 2 experiments were performed per person. In every experiment, three to four supernatants from separate wells were taken. Columns represent mean valuesFS.D. *Under detection limit (4 pg/ml). (B) Cell subsets were centrifuged and stained using a mouse anti-human BDNF mAb. Shown are representative photographs of the cell subsets of one individual out of four independent experiments: 1=BDNF staining in PBMC, 2=BDNF staining in CD19+ cells, 3=BDNF staining in CD3+ cells, 4=BDNF staining in CD14+ cells, 5=isotype control (IgG1 in CD14+ cells).
ethidium bromide. After sequencing the PCR product (GATC Biotech, Konstanz, Germany) its homology to human BDNF was confirmed by BLAST search.
3. Results 3.1. Human peripheral blood monocytes constitutively secrete BDNF To evaluate whether unstimulated human peripheral blood mononuclear cells (PBMC) are permanent sources of BDNF, we separated PBMC from healthy individuals
3.3. TNF-a and IL-6 directly enhance BDNF secretion by monocytes In order to reveal whether TNF-a and IL-6 directly induce BDNF secretion in monocytes or whether this pathway requires additional cell–cell interactions, we stimulated MACS-separated monocytes with TNF-a and IL-6. Both cytokines significantly enhanced BDNF levels in
Table 1 BDNF secretion in cytokine-stimulated PBMC cultures Cytokine
Mean (pg/ml)
SD (pg/ml)
Medium control IL-1h IL-2 IL-4 IL-5 IL-13 IL-15 IFN-g
59.8 46.8 65.3 44.6 54.8 54.0 63.7 50.0
19.3 19.0 12.8 15.0 9.0 13.2 17.3 14.9
The BDNF protein level in cell free supernatants of cytokine-stimulated PBMC from healthy donors was measured by ELISA after 24 h of culture. Shown are means including three individuals, two experiments were performed per person. In every experiment, three to four supernatants from separate wells were taken. None of these cytokines had a significant effect on BDNF secretion. There was no significant difference between stimulated and medium control PBMC using Student’s t-test, pb0.05.
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supernatants of monocyte cultures (Fig. 2). These effects were neutralized by specific monoclonal antibodies against TNF-a and IL-6 (Fig. 2) (n=6). Stimulation of isolated T-cell and B-cell cultures with different concentrations of IL-6 and TNF-a did not induce any detectable amounts of BDNF protein (data not shown). 3.4. BDNF-mRNA expression in monocytes after TNF-a and IL-6 stimulation In order to distinguish whether TNF-a or IL-6 cause a secretion of preformed BDNF from intracellular vesicles or whether these cytokines also directly specifically induce BDNF mRNA, quantitative RT-PCR for BDNF was performed in freshly isolated monocytes and in cells which were stimulated for 2 h. No difference was observed between the ratio of BDNF mRNA and PBGD mRNA in freshly isolated monocytes and unstimulated control cells (7.79E 03 vs. 7.95E 03 calculated units). After stimulation of monocytes with TNF-a, or IL-6,
Fig. 3. BDNF mRNA expression in monocytes stimulated with IL-6 and TNF-a 3106 MACS-separated monocytes were stimulated with either TNF-a (50 ng/ml) or IL-6 (50 ng/ml). To assess BDNF expression, mRNA for BDNF and PBGD was quantified after 2 h of stimulation in six independent experiments by real-time PCR. The ratio of BDNF mRNA and the mRNA of the housekeeping gene PDGB was calculated. The results are expressed in % of the appropriate medium control; Student T-test revealed no statistically significant difference of BDNF mRNA expression in cytokine stimulated monocytes compared to the medium control (=100% level).
respectively, no change in BDNF mRNA levels were detectable (Fig. 3).
4. Discussion
Fig. 2. BDNF synthesis in monocytes stimulated with IL-6 and TNF-a BDNF was measured in supernatants of 4105 monocytes after 24 h of culture. Stimulations were performed using TNF-a (50 ng/ml) (A), IL-6 (50 ng/ml) (B) and cytokine plus specific monoclonal antibodies (10 Ag/ml). Shown are means of 12 wells with standard deviation from one representative experiment. *pb0.05 significant difference (Student’s t-test).
Apart from its essential role as a target-derived neurotrophic factor for developing neurons, BDNF is currently discussed as a key regulator of neuronal and synaptic plasticity in the adult (Kang and Schuman, 1995; Winter, 1998; McAllister et al., 1999). The concept that BDNF serves as an exclusive interneuronal mediator has recently been revised by the finding that biologically active BDNF can also be produced by immune cells in several inflammatory processes (Braun et al., 1999; Kerschensteiner et al., 1999; Hammarberg et al., 2000). In the present study, we demonstrate for the first time that unstimulated monocytes constitutively secrete BDNF. Thus, monocytes could be the major cellular source for BDNF, which can be measured in the plasma of healthy adults (Radka et al., 1996). Notably, unstimulated lymphocytes of the peripheral blood did not contain or secrete BDNF protein. There is recent data describing BDNF protein in supernatants of human B- and T-cells, which were stimulated with irradiated PBMC and PHA (T-cells) or Staphylococcus aureus extracts (B-cells) (Kerschensteiner et al., 1999). The hypothesis that BDNF synthesis can be induced in human Tand B-cells is supported by data of animal models of BA and MS demonstrating BDNF mRNA synthesis in activated Tcells infiltrating the lung or brain (Braun et al., 1999; Hammarberg et al., 2000). Whereas we have many data dealing with BDNF-regulation in neurons, depending on various stimuli like, e.g., CREB-dependent depolarization (Zha, 2001) and cytokines (Murphy et al., 2000), the specific lymphocyte activation pathways are still unknown and
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probably do not involve the cytokines examined in this study. We focused on the question, how BDNF is regulated in monocytes. Our data suggest that BDNF secretion by monocytes can be specifically enhanced by IL-6 and TNFa. Since there is only a slight up regulation of BDNFmRNA in these cells after TNF-a or IL-6, we presume that the increase of BDNF in supernatants is based also on a release of preformed BDNF in monocytes. Interestingly, specific Th1- or Th2-cytokines did not show any effect on BDNF secretion by monocytes. This is in line with previous data showing BDNF up regulation in very different types of inflammation, with no apparent Th1- or Th2-restriction. Furthermore, our findings in monocytes constitute a parallel mechanism to BDNF regulation in rat and mouse sensory neurons. Here, Murphy et al. (2000) found a reciprocal induction of BDNF and IL-6. Thus, BDNF up regulation seems to be a general inflammatory phenomenon. However, the significance of enhanced BDNF levels in different inflammatory diseases does not seem to be unequivocal. On one hand, a neuroprotective role of immune-cell-derived BDNF has been postulated, especially in MS (Kerschensteiner et al., 1999; Hammarberg et al., 2000). It is known that injection of stimulated macrophages in artificially injured spinal cord enhances nerve recovery (Rapalino et al., 1998). In addition, supernatants of CD14+activated monocytes enhance survival of BDNF-dependent neurons of the nodose ganglia (Kerschensteiner et al., 1999). Therefore, BDNF is currently thought to protect neurons against cytotoxic damage in inflammatory brain lesions (Hohlfeld et al., 2000). Since steroids are known to down regulate BDNF synthesis, at least in neurons, the discussion on negative effects of non-selective immunosuppression has recently been re-opened (Schaaf et al., 1998; Hansson et al., 2000). On the other hand, BDNF is a candidate for mediating hyperreactivity of sensory and motor neurons in BA (Braun et al., 2000). BA is characterized by an infiltration of peripheral blood cells into the airways, a specific cytokine pattern and by bronchoconstriction (Barnes et al., 1998). Neuronal hyperreactivity is a pathogenetic hallmark of bronchial hyperresponsiveness and late phase bronchoconstriction following allergen challenge (late asthmatic response, LAR) (Undem et al., 1999, 2000). However, the link between cell infiltration and neuronal hyperreactivity is poorly understood. IL-6 and TNF-a levels are specifically enhanced in bronchoalveolar lavage fluid of allergic asthmatic patients with LAR (Virchow et al., 1995). Only alveolar macrophages of patients with LAR display significant IL-6 and TNF-a production following allergen or IgE stimulation (Gosset et al., 1991). Therefore, both cytokines are thought to be essential mediators of LAR bronchoconstriction (Gosset et al., 1992). Furthermore, TNF-a is known to induce bronchial hyperresponsiveness (Kips et al., 1992; Thomas, 2001). However, there is to date no pathogenetic concept describing the mechanism of
IL-6 and TNF-a induced neuronal hyperreactivity in the lung. Our data suggest that BDNF-secreting monocytes from the peripheral blood play a role in this neuroimmune cross talk. In conclusion our data demonstrate that BDNF, a factor that very effectively regulates neuronal function, can be produced by human blood monocytes and that this production is up regulated by inflammatory mediators as TNF-a and IL-6. This may have implication for the understanding of the development of neuroimmune interactions in diseases like MS and BA.
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Tumor necrosis factor-a and interleukin-6 regulate secretion of brain-derived neurotrophic factor in human monocytes Olaf Schulte-Herbrqggena,1, Christina Nassensteinb,1, Marek Lommatzschc, David Quarcood, Harald Renze, Armin Braunb,* b
a Department of Neurology, Charite´ , Humboldt University, Berlin, Germany Immunology and Allergology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs Strasse 1, 30625 Hannover, Germany c Department of Pneumology, University Rostock, Schillingallee 35, 18057 Rostock, Germany d Department of Pediatrics, Charite´, Humboldt University, 13353 Berlin, Germany e Department of Clinical Chemistry and Molecular Diagnostics, Philipps-University Marburg, Baldingerstrasse, 35033 Marburg, Germany
Received 23 July 2004; received in revised form 25 October 2004; accepted 25 October 2004
Abstract Activated macrophages have been shown to produce brain-derived neurotrophic factor (BDNF) in diseases such as multiple sclerosis (MS) or allergic bronchial asthma (BA). However, there is little data on BDNF regulation in these cells. We demonstrate that unstimulated human peripheral blood monocytes, but not lymphocytes, constitutively secrete BDNF. IL-6 and TNF-a specifically enhanced BDNF secretion in monocytes, whereas typical Th1- and Th2-cytokines did not show any effect. None of the cytokines induced BDNF secretion in T- or B-cells. Thus, our data provide evidence that IL-6 and TNF-a represent a specific link between monocyte infiltration and neuronal changes in inflammatory diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: BDNF; Neurotrophins; Neuroimmunology; IL-6; TNF-a; Inflammation; Monocytes
1. Introduction Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is an essential mediator of neuronal activity and synaptic plasticity in the central and peripheral nervous system (Lewin and Barde, 1996; Kerr et al., 1999; Lommatzsch et al., 1999; McAllister et al., 1999). Initially, BDNF was thought to be predominantly derived from neuronal tissues. This concept has recently been revised by the finding that BDNF is produced in inflamed tissues of diseases such as allergic bronchial asthma (BA) and multiple sclerosis (MS). BDNF levels are specifically enhanced in the late phase reaction following segmental * Corresponding author. Tel.: +49 511 5350 263; fax: +49 511 5350 155. E-mail address: [email protected] (A. Braun). 1 Both authors contributed equally. 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.026
allergen provocation in patients with BA (Virchow et al., 1998). In a mouse model of BA, we have identified macrophages as predominant cellular sources of enhanced BDNF production in the inflamed lung. This BDNF production was specifically induced by allergic inflammation, since macrophages from control animals did not show any detectable BDNF expression (Braun et al., 1999). Furthermore, infiltrating mononuclear cells, predominantly representing activated macrophages, were shown to be BDNF immunoreactive in inflammatory brain lesions of subjects with MS (Kerschensteiner et al., 1999). These data suggest that activated macrophages represent a major source of BDNF in inflamed tissues. However, to date, there is no data on inflammatory mediators regulating BDNF secretion in macrophages. In addition, it is unknown whether unstimulated human monocytes of the peripheral blood constitutively secrete BDNF. Therefore, the aim of this study was to investigate the cellular sources of BDNF in
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unstimulated peripheral blood mononuclear cells (PBMC) and to test the impact of cytokines typically involved in BA and MS on BDNF secretion. We demonstrate here that peripheral blood monocytes constitutively secrete BDNF, representing the major source of this neurotrophin among PBMC. In addition, we show that TNF-a and IL-6 specifically enhance BDNF secretion in human monocytes. These findings may help to further elucidate the mechanisms of neuroimmune interactions in chronic inflammatory diseases.
2. Materials and methods 2.1. Cell separation PBMC were isolated from healthy individuals (aged 23– 35 years) using density centrifugation (Lymphoprep, Nycoprep Pharma, Oslo, Norway) as previously described (Nasert et al., 1996). T-cell, B-cell and monocyte separation from PBMC was performed by negative selection using commercial magnet-activated cell separation (MACS) extraction kits (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity was determined using flow cytometry. B-cells had a purity of N80%, T-cells N95% and monocytes N90% (data not shown). 2.2. Cell culture Cells (2105 PBMC, 4105 CD3+ cells, 4105 CD19+ and 4105 CD14+ per well) were cultured in serum-free AIM-V (Gibco BRL, Paisley, Scotland) with 1% lglutamine (Biochrom, Berlin, Germany) and 1.25 Ag/ml fungizone (Gibco BRL) at 37 8C in a 95% air/5% CO2 humidified atmosphere for 24 h. Cytokine stimulation experiments were performed with 10, 50 and 100 ng/ml of each cytokine for 24, 48 and 96 h (R&D Systems, Minneapolis, USA). Optimal conditions for both IL-6 and TNF-a were 50 ng/ml after 24 h. Cytokine blocking experiments were performed with 50 ng/ml of cytokine and 10 Ag/ml of monoclonal anti-human-cytokine antibody. Viability of cells was quantified by a Trypan blue assay (Biochrom, Berlin, Germany). Only cell cultures with a viability of N95% were chosen for BDNF determination. 2.3. Determination of BDNF by ELISA BDNF was measured in cell-free cell-culture supernatants with commercial ELISA kits according to the manufacturer’s instructions (Promega, Madison, WI) as previously described (Lommatzsch et al., 1999). Briefly, flat-bottom 96-well plates (Nunc, Wiesbaden, Germany) were coated with monoclonal mouse anti-BDNF antibodies. Captured neurotrophins were detected with secondary chicken polyclonal anti-BDNF and tertiary antibodies conjugated to horseradish peroxidase. Neurotrophin content was quantified against a standard curve
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generated with known amounts of protein. Detection limits were 4 pg/ml. To exclude eventual cross reactivity with other neurotrophins, the BDNF ELISA was tested with rhNGF, rhNT3 and rhNT4 (R&D Systems). There was no cross reactivity detectable. 2.4. BDNF immunoreactivity Immunoreactivity against BDNF protein was studied on acetone-fixed cytospins of freshly isolated cells following the manufacturer’s instruction (EnVisionk System Alkaline Phosphatase, DAKO, Glostrup, Denmark). Monoclonal mouse IgG1 anti-human BDNF antibodies (10 Ag/ml) were used as primary antibody and incubated overnight at 4 8C (R&D Systems). Incubation of cytospins with purified mouse IgG1 antibodies served as negative control (R&D Systems). 2.5. Determination of BDNF mRNA by real-time PCR Total cellular RNA was extracted from monocytes 2 h after incubation with culture medium (control), 50 ng/ml TNF or 50 ng/ml IL-6, respectively, using a commercial available RNA-isolation kit (RNeasy Mini Kit, Qiagen, Hilden, Germany). After digestion of genomic DNA by DNase treatment (RNase Free DNase Set, Qiagen), reverse transcription was performed with 0.1 Ag of total RNA from each sample in a 20 Al reaction for 60 min at 37 8C followed by 5 min at 95 8C according to the manufacturer’s recommendation (Omniscript RT-Kit, Qiagen). As control, 0.1 Ag RNA of each sample was treated according to the same protocol with addition of water instead of the reverse transcriptase. Real-time PCR was performed on the Light Cycler Instrument (Roche, Mannheim, Germany) using DNA binding dye SYBR green (Light Cycler-Fast Start DNA Master SYBR Green I, Roche). PCR reaction mix contained 2 Al PCR master mix supplement with 2.5 mM MgCl2, custom synthesized primers (MWG Biotech, Ebersberg, Germany) and 2 Al of either external standard, cDNA or RNA filled up with water to a final volume of 20 Al. As specific primers, we used porphobilinogen deaminase (PBGD, sense: 5V-ACA CAG CCT ACT TTC CAA GCG GAG-3V, antisense: 5V-TCT TGT CCC CTG TGG TGG ACA TAG CAA-3V) and BDNF (sense: 5V-CCA AGG CAG GTT CAA GAG G-3V, antisense: 5V-TCC AGC AGA AAG AGA AGA GGA-3V). After an initial denaturation step at 95 8C for 10 min, the PCR reaction was initiated with an annealing temperature of 60 8C for 10 s, followed by an extension phase at 72 8C for 9 s and a denaturation cycle at 95 8C for 1 s. At the end of each extension phase, fluorescence was observed at 72 8C and the PCR was completed after 45 cycles. The melting point analysis was carried out by heating the amplicon from 65 to 958 C and revealed the characteristic melting point for each product. After cooling down to 408 C, the product was extracted from the capillary. A total of 10 Al of each reaction was run onto a 1.5% agarose gel and visualized by staining with
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and cultured them for 24 h (12 individuals). We observed a constitutive secretion of BDNF by unstimulated PBMC (Fig. 1A). In order to identify the cell subsets responsible for this BDNF-secretion, we examined supernatants of purified T-cell, B-cell and monocyte cultures. Unstimulated monocytes constitutively secreted BDNF, whereas BDNF was not detectable in B- and Tcell supernatants (Fig. 1A). These data were confirmed by immunocytochemistry. A subpopulation of PBMC morphologically identified as monocytes as well as purified monocytes revealed BDNF immunoreactivity. In contrast, purified T-cells and B-cells remained BDNF-negative (Fig. 1B). 3.2. Stimulation of PBMC with cytokines involved in BA and MS In order to identify the mediators which regulate BDNF secretion in PBMC, we tested the impact of cytokines typically involved in BA or MS on BDNF secretion (Begolka and Miller, 1998). The proinflammatory cytokines TNF-a and IL-6 significantly enhanced BDNF levels in PBMC cultures after 24 h in a dose-dependent manner, whereas the typical Th1- and Th2-cytokines IL-1h, IFN-g, IL-2, IL-4, IL-5, IL-13 and IL-15 did not have any effect (Table 1). The specificity was demonstrated by neutralizing antibodies against IL-6 or TNF-a, which completely abolished cytokine-induced BDNF elevations in PBMC supernatants (n=10).
Fig. 1. Constitutive BDNF synthesis by mononuclear cells from the peripheral blood. (A) The BDNF protein level in cell free supernatants of PBMC, CD3+ cells, CD19+ cells and CD14+ cells from healthy donors was measured by ELISA after 24 h of culture. Columns include 12 individuals, 2 experiments were performed per person. In every experiment, three to four supernatants from separate wells were taken. Columns represent mean valuesFS.D. *Under detection limit (4 pg/ml). (B) Cell subsets were centrifuged and stained using a mouse anti-human BDNF mAb. Shown are representative photographs of the cell subsets of one individual out of four independent experiments: 1=BDNF staining in PBMC, 2=BDNF staining in CD19+ cells, 3=BDNF staining in CD3+ cells, 4=BDNF staining in CD14+ cells, 5=isotype control (IgG1 in CD14+ cells).
ethidium bromide. After sequencing the PCR product (GATC Biotech, Konstanz, Germany) its homology to human BDNF was confirmed by BLAST search.
3. Results 3.1. Human peripheral blood monocytes constitutively secrete BDNF To evaluate whether unstimulated human peripheral blood mononuclear cells (PBMC) are permanent sources of BDNF, we separated PBMC from healthy individuals
3.3. TNF-a and IL-6 directly enhance BDNF secretion by monocytes In order to reveal whether TNF-a and IL-6 directly induce BDNF secretion in monocytes or whether this pathway requires additional cell–cell interactions, we stimulated MACS-separated monocytes with TNF-a and IL-6. Both cytokines significantly enhanced BDNF levels in
Table 1 BDNF secretion in cytokine-stimulated PBMC cultures Cytokine
Mean (pg/ml)
SD (pg/ml)
Medium control IL-1h IL-2 IL-4 IL-5 IL-13 IL-15 IFN-g
59.8 46.8 65.3 44.6 54.8 54.0 63.7 50.0
19.3 19.0 12.8 15.0 9.0 13.2 17.3 14.9
The BDNF protein level in cell free supernatants of cytokine-stimulated PBMC from healthy donors was measured by ELISA after 24 h of culture. Shown are means including three individuals, two experiments were performed per person. In every experiment, three to four supernatants from separate wells were taken. None of these cytokines had a significant effect on BDNF secretion. There was no significant difference between stimulated and medium control PBMC using Student’s t-test, pb0.05.
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supernatants of monocyte cultures (Fig. 2). These effects were neutralized by specific monoclonal antibodies against TNF-a and IL-6 (Fig. 2) (n=6). Stimulation of isolated T-cell and B-cell cultures with different concentrations of IL-6 and TNF-a did not induce any detectable amounts of BDNF protein (data not shown). 3.4. BDNF-mRNA expression in monocytes after TNF-a and IL-6 stimulation In order to distinguish whether TNF-a or IL-6 cause a secretion of preformed BDNF from intracellular vesicles or whether these cytokines also directly specifically induce BDNF mRNA, quantitative RT-PCR for BDNF was performed in freshly isolated monocytes and in cells which were stimulated for 2 h. No difference was observed between the ratio of BDNF mRNA and PBGD mRNA in freshly isolated monocytes and unstimulated control cells (7.79E 03 vs. 7.95E 03 calculated units). After stimulation of monocytes with TNF-a, or IL-6,
Fig. 3. BDNF mRNA expression in monocytes stimulated with IL-6 and TNF-a 3106 MACS-separated monocytes were stimulated with either TNF-a (50 ng/ml) or IL-6 (50 ng/ml). To assess BDNF expression, mRNA for BDNF and PBGD was quantified after 2 h of stimulation in six independent experiments by real-time PCR. The ratio of BDNF mRNA and the mRNA of the housekeeping gene PDGB was calculated. The results are expressed in % of the appropriate medium control; Student T-test revealed no statistically significant difference of BDNF mRNA expression in cytokine stimulated monocytes compared to the medium control (=100% level).
respectively, no change in BDNF mRNA levels were detectable (Fig. 3).
4. Discussion
Fig. 2. BDNF synthesis in monocytes stimulated with IL-6 and TNF-a BDNF was measured in supernatants of 4105 monocytes after 24 h of culture. Stimulations were performed using TNF-a (50 ng/ml) (A), IL-6 (50 ng/ml) (B) and cytokine plus specific monoclonal antibodies (10 Ag/ml). Shown are means of 12 wells with standard deviation from one representative experiment. *pb0.05 significant difference (Student’s t-test).
Apart from its essential role as a target-derived neurotrophic factor for developing neurons, BDNF is currently discussed as a key regulator of neuronal and synaptic plasticity in the adult (Kang and Schuman, 1995; Winter, 1998; McAllister et al., 1999). The concept that BDNF serves as an exclusive interneuronal mediator has recently been revised by the finding that biologically active BDNF can also be produced by immune cells in several inflammatory processes (Braun et al., 1999; Kerschensteiner et al., 1999; Hammarberg et al., 2000). In the present study, we demonstrate for the first time that unstimulated monocytes constitutively secrete BDNF. Thus, monocytes could be the major cellular source for BDNF, which can be measured in the plasma of healthy adults (Radka et al., 1996). Notably, unstimulated lymphocytes of the peripheral blood did not contain or secrete BDNF protein. There is recent data describing BDNF protein in supernatants of human B- and T-cells, which were stimulated with irradiated PBMC and PHA (T-cells) or Staphylococcus aureus extracts (B-cells) (Kerschensteiner et al., 1999). The hypothesis that BDNF synthesis can be induced in human Tand B-cells is supported by data of animal models of BA and MS demonstrating BDNF mRNA synthesis in activated Tcells infiltrating the lung or brain (Braun et al., 1999; Hammarberg et al., 2000). Whereas we have many data dealing with BDNF-regulation in neurons, depending on various stimuli like, e.g., CREB-dependent depolarization (Zha, 2001) and cytokines (Murphy et al., 2000), the specific lymphocyte activation pathways are still unknown and
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probably do not involve the cytokines examined in this study. We focused on the question, how BDNF is regulated in monocytes. Our data suggest that BDNF secretion by monocytes can be specifically enhanced by IL-6 and TNFa. Since there is only a slight up regulation of BDNFmRNA in these cells after TNF-a or IL-6, we presume that the increase of BDNF in supernatants is based also on a release of preformed BDNF in monocytes. Interestingly, specific Th1- or Th2-cytokines did not show any effect on BDNF secretion by monocytes. This is in line with previous data showing BDNF up regulation in very different types of inflammation, with no apparent Th1- or Th2-restriction. Furthermore, our findings in monocytes constitute a parallel mechanism to BDNF regulation in rat and mouse sensory neurons. Here, Murphy et al. (2000) found a reciprocal induction of BDNF and IL-6. Thus, BDNF up regulation seems to be a general inflammatory phenomenon. However, the significance of enhanced BDNF levels in different inflammatory diseases does not seem to be unequivocal. On one hand, a neuroprotective role of immune-cell-derived BDNF has been postulated, especially in MS (Kerschensteiner et al., 1999; Hammarberg et al., 2000). It is known that injection of stimulated macrophages in artificially injured spinal cord enhances nerve recovery (Rapalino et al., 1998). In addition, supernatants of CD14+activated monocytes enhance survival of BDNF-dependent neurons of the nodose ganglia (Kerschensteiner et al., 1999). Therefore, BDNF is currently thought to protect neurons against cytotoxic damage in inflammatory brain lesions (Hohlfeld et al., 2000). Since steroids are known to down regulate BDNF synthesis, at least in neurons, the discussion on negative effects of non-selective immunosuppression has recently been re-opened (Schaaf et al., 1998; Hansson et al., 2000). On the other hand, BDNF is a candidate for mediating hyperreactivity of sensory and motor neurons in BA (Braun et al., 2000). BA is characterized by an infiltration of peripheral blood cells into the airways, a specific cytokine pattern and by bronchoconstriction (Barnes et al., 1998). Neuronal hyperreactivity is a pathogenetic hallmark of bronchial hyperresponsiveness and late phase bronchoconstriction following allergen challenge (late asthmatic response, LAR) (Undem et al., 1999, 2000). However, the link between cell infiltration and neuronal hyperreactivity is poorly understood. IL-6 and TNF-a levels are specifically enhanced in bronchoalveolar lavage fluid of allergic asthmatic patients with LAR (Virchow et al., 1995). Only alveolar macrophages of patients with LAR display significant IL-6 and TNF-a production following allergen or IgE stimulation (Gosset et al., 1991). Therefore, both cytokines are thought to be essential mediators of LAR bronchoconstriction (Gosset et al., 1992). Furthermore, TNF-a is known to induce bronchial hyperresponsiveness (Kips et al., 1992; Thomas, 2001). However, there is to date no pathogenetic concept describing the mechanism of
IL-6 and TNF-a induced neuronal hyperreactivity in the lung. Our data suggest that BDNF-secreting monocytes from the peripheral blood play a role in this neuroimmune cross talk. In conclusion our data demonstrate that BDNF, a factor that very effectively regulates neuronal function, can be produced by human blood monocytes and that this production is up regulated by inflammatory mediators as TNF-a and IL-6. This may have implication for the understanding of the development of neuroimmune interactions in diseases like MS and BA.
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