The 1,4-benzodiazepine Ro5-4864 (4-chlorodiazepam) suppresses multiple pro-inflammatory mast cell effector functions
© Yousefi et al; licensee BioMed Central Ltd. 2013
Received: 10 September 2012
Accepted: 16 February 2013
Published: 20 February 2013
Activation of mast cells (MCs) can be achieved by the high-affinity receptor for IgE (FcεRI) as well as by additional receptors such as the lipopolysaccharide (LPS) receptor and the receptor tyrosine kinase Kit (stem cell factor [SCF] receptor). Thus, pharmacological interventions which stabilize MCs in response to different receptors would be preferable in diseases with pathological systemic MC activation such as systemic mastocytosis. 1,4-Benzodiazepines (BDZs) have been reported to suppress MC effector functions. In the present study, our aim was to analyze molecularly the effects of BDZs on MC activation by comparison of the effects of the two BDZs Ro5-4864 and clonazepam, which markedly differ in their affinities for the archetypical BDZ recognition sites, i.e., the GABAA receptor and TSPO (previously termed peripheral-type BDZ receptor). Ro5-4864 is a selective agonist at TSPO, whereas clonazepam is a selective agonist at the GABAA receptor. Ro5-4864 suppressed pro-inflammatory MC effector functions in response to antigen (Ag) (degranulation/cytokine production) and LPS and SCF (cytokine production), whereas clonazepam was inactive. Signaling pathway analyses revealed inhibitory effects of Ro5-4864 on Ag-triggered production of reactive oxygen species, calcium mobilization and activation of different downstream kinases. The initial activation of Src family kinases was attenuated by Ro5-4864 offering a molecular explanation for the observed impacts on various downstream signaling elements. In conclusion, BDZs structurally related to Ro5-4864 might serve as multifunctional MC stabilizers without the sedative effect of GABAA receptor-interacting BDZs.
1,4-Benzodiazepines (BDZs) are clinically used as anxiolytic, hypnotic, anti-convulsive, and muscle relaxing drugs [1–4]. BDZs are lipophilic and readily cross cell membranes. There are two major types of BDZ recognition sites. The first site is part of the GABAA receptor complex found in cells of the central nervous system  and, hence, is termed central-type BDZ receptor. The other one is an ubiquitously expressed transmembrane protein of the outer mitochondrial membrane (OMM) termed translocator protein (18 kDa) (TSPO)  (previously named peripheral-type BDZ receptor ). Interaction studies revealed that TSPO is associated with the OMM protein voltage-dependent anion channel (VDAC) and the inner mitochondrial membrane (IMM) protein adenine nucleotide transporter (ANT)  and the requirement for both TSPO and VDAC for BDZ binding has been suggested. Most BDZs clinically used possess nanomolar affinity for the central-type receptor, but only milli- to micromolar affinities for TSPO. However, there are also BDZs available with high affinity and selectivity for TSPO (e.g. Ro5-4864 = 4-chlorodiazepam) , thus, allowing the analysis of the potential involvement of TSPO function in biological processes.
Expression of TSPO has also been described in mast cells (MCs) [10–12]. MCs are hematopoietic, tissue-resident cells, which are involved in various physiological as well as pathophysiological scenarios. They are very important players in innate and adaptive immune responses, inflammation, and tissue changes [13, 14]. Important reactions during these processes are allergen-triggered degranulation of preformed mediators (e.g. histamine and proteases) and lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (e.g. IL-6 and TNF-α), respectively. In addition to the allergy-relevant high-affinity receptor for IgE (FcεRI) and the LPS receptor (TLR4), the receptor tyrosine kinase Kit represents another important signaling system, which regulates MC differentiation, proliferation, survival, chemotaxis, and production of pro-inflammatory cytokines .
BDZs have been reported to inhibit MC effector functions: Midazolam suppressed substance P-induced chemotaxis as well as degranulation  of canine MCs. Diazepam and midazolam inhibited proliferation of murine MCs as well as pro-inflammatory mediator release from these cells . With respect to systemic MC activation disease (MCAD) [17, 18], the clinical efficacy of the BDZs flunitrazepam, diazepam, bromazepam, and midazolam for the treatment of MC mediator-induced symptoms has been reported [18, 19]. The TSPO-selective BDZ Ro5-4864 was shown to inhibit concanavalin A-induced serotonin release from as well as 45Ca uptake into rat MCs, whereas the GABAA-receptor-selective BDZ clonazepam only had a slight impact on serotonin release and did not affect 45Ca uptake . In addition, diazepam, Ro5-4864, and flunitrazepam were demonstrated to reduce NECA-induced IL-8 production in human MC leukemia cells . Since on the one hand MCs did not express GABAA receptors in previous investigations and on the other hand most BDZs, such as flunitrazepam, diazepam, and midazolam possess considerable affinity for TSPO [7, 9, 12], it was conceivable that the inhibitory effects of BDZs on MCs may be due to their binding to TSPO in MCs. In this context the aim of the present study was to analyze the molecular processes underlying the inhibitory action of BDZs in MCs by using two selective BDZs: Ro5-4864 (4-chlorodiazepam) that possesses high affinity for TSPO but has only low affinity for GABAA receptors and clonazepam, a high-affinity ligand for GABAA receptors with only low affinity for TSPO .
We show here that Ro5-4864 but not clonazepam inhibited antigen (Ag)-triggered degranulation as well as Ag-, LPS- or SF-induced pro-inflammatory cytokine production. In addition, Ro5-4864 inhibited allergen-induced bronchoconstriction in precision-cut lung slices. Moreover, Ag-triggered Ca2+ mobilization and production of reactive oxygen species (ROS) were suppressed by Ro5-4864. By expressing a fluorescent TSPO fusion protein and using confocal microscopy, we were not able to detect a plasmalemnal localization of the TSPO-containing fusion protein in MCs which has been observed previously in some other cell types [21, 22]. Analysis of early Ag-triggered signaling events suggested Ro5-4864-dependent attenuation of Src family kinases (SFKs), which represent very early signaling molecules active in the chain of FcεRI signaling. Hence, attenuation of SFKs by direct inhibition and/or indirectly by targeting a so far unidentified upstream plasmalemnal recognition site could be the reason for the observed suppression of pro-inflammatory MC responses.
Ro5-4864 inhibits mast cell degranulation
Ro5-4864 suppresses Ag-triggered Ca2+ influx as well as ROS production
Ro5-4864 suppresses pro-inflammatory cytokine production in mast cells
Ro5-4864 attenuates activation of the PI3K pathway
Interestingly, a comparable concentration-dependent pattern was observed when analyzing the effect of Ro5-4864 on Ag- or LPS-triggered IL-6 production in SHIP1-deficient BMMCs (Figure 6D and Additional file 1: Figure S1). Pro-inflammatory cytokine production downstream of the FcεRI strongly depends on the NFκB as well as the p38 MAPK pathways . Whereas Ro5-4864 treatment resulted in suppressed activation of p38 (measured by bis-phosphorylation at T180/Y182) in response to Ag in wild-type cells, no such effect was observed in SHIP1-deficient BMMCs (Figure 6B). In addition, phosphorylation of IκBα at S32 indicating activation of the NFκB pathway was slightly attenuated by Ro5-4864 treatment in wild-type BMMCs, which was not the case in SHIP1-deficient cells (Figure 6B). These data suggest that Ro5-4864 suppresses MC activation and effector functions independently of its effect on the PI3K, p38, and/or NFκB pathways.
Ro5-4864 interferes with the tyrosine phosphorylation response in mast cells
TSPO is expressed in mitochondria of mast cells
In the present study, our aim was to analyze molecularly the inhibitory effect of BDZs on MC activity by comparison of the effects of the two BDZs Ro5-4864 and clonazepam. The two drugs differ markedly in their affinities for the archetypical BDZ recognition sites, i.e., the GABAA receptor and the TSPO, previously termed peripheral-type BDZ receptor. Ro5-4864 is an agonist at TSPO and has only low affinity to the GABAA receptor , whereas clonazepam is a high-affinity GABAA receptor agonist, but has only low affinity for TSPO . Ro5-4864 concentration-dependently inhibited Ag-triggered degranulation in BMMCs and PMCs, whereas clonazepam was essentially ineffective in this respect. In accordance with this observation, Ro5-4864 suppressed Ca2+ mobilization, production of ROS and activation of the PI3K pathway (as measured by phosphorylation of Akt at Ser473), which are all important signaling events in the positive regulation of the secretory response [26, 27]. These data suggest that Ro5-4864 and structurally related compounds might be applicable as versatile MC stabilizing drugs in MC-dependent diseases, e.g., hypersensitivity diseases, asthma, and systemic MCAD [18, 40]. This was also shown by the inhibition of allergen-induced bronchoconstriction in rat precision-cut lung slices. In this context it is interesting to note that Ro5-4864 did not change the basal activation of the MCs indicating a selective action of BDZs at (pathologically) activated MCs.
The question arises for the target sites at which Ro5-4864 and other 1,4-benzodiazepines mediate their inhibitory effects on MCs. Since the selective GABAA receptor agonist clonazepam did not mimic the effects of Ro5-4864, an action of Ro5-4864 at classical GABAA receptors is rather unlikely. One potential candidate structure is TSPO, a transmembrane protein located in the OMM and enriched in OMM-IMM contact sites. It is a central component of a multimeric protein complex, comprising amongst others TSPO, VDAC1, ANT, and PRAX-1, and is associated with the mitochondrial permeability transition pore (mPTP) . Therefore, functions of TSPO in regulating apoptotic processes have been discussed. Indeed, Ro5-4864 has been reported to induce apoptosis in some human and murine cancer cell lines and thymocytes, in particular by interfering with the mitochondrial membrane potential [41–45]. In these studies, cells were treated with Ro5-4864 for many hours to days, i.e. for a much longer time span compared to our experiments, which were performed within minutes to 3 h. The analysis of the effect of Ro5-4864 on BMMC survival showed only subtle apoptotic effects after 24 h (data not shown) excluding apoptotic effects within the significant shorter time windows of our MC activation experiments. Since we were able to reduce the pre-incubation time with Ro5-4864 to 1 min without loosing inhibitory efficiency, a mechanism via plasma membrane-located target sites instead of mitochondria-resident TSPO seems more likely. Interestingly, in certain cell types TSPO expression has also been detected in the plasma membrane [21, 22]. By expressing a fluorescent TSPO fusion protein and using confocal microscopy, however, we were not able to detect the TSPO-containing fusion protein in the plasma membrane of MCs. Though we consider the technique used sufficiently sensitive, we did not have the material to detect endogenous TSPO and, thus, cannot totally exclude expression of the endogenous protein in the plasma membrane.
The concentration-dependent inhibition of Ag-triggered degranulation by Ro5-4864 could be due to suppression of mitochondrial Ca2+ uptake. Ag-triggered degranulation is dependent on influx of extracellular Ca2+ ions through SOC channels [46, 47]. We found that Ro5-4864 suppressed SOC entry, whereas intracellular Ca2+ release from the ER appeared unaltered. Recently, Farsky and colleagues reported on a similar attenuation of fMLP-induced Ca2+ mobilization in neutrophils by Ro5-4864 . Optimal SOC entry requires efficient emptying of the ER. To prevent immediate re-uptake of Ca2+ into the ER via the SERCA, mitochondria are able to take up Ca2+ at the moment of release from the ER by the uniporter channel. Ro5-4864 treatment might suppress this mitochondrial Ca2+ uptake mechanism. If so, passive release of Ca2+ from the ER by means of treatment with the SERCA inhibitor thapsigargin should be independent of such a mitochondrial buffering mechanism. Indeed, thapsigargin-induced SOC influx was not suppressed by Ro5-4864 treatment, which would be in agreement with this idea of an interaction of Ro5-4864 with mitochondrial Ca2+ uptake.
However, the effects of Ro5-4864 on Ag-triggered signaling in BMMCs deficient for the negative regulator SHIP1 point to another target site of Ro5-4864. Intriguingly, whereas Ro5-4864 concentration-dependently suppressed degranulation in SHIP1-deficient BMMCs, Ag-triggered activation of Akt was not altered in these cells. Moreover, there was only a slight reduction of extracellular Ca2+ influx (Additional file 3: Figure S3). It is known that SHIP1-deficient MCs are less sensitive to drugs inhibiting PI3K compared to wild-type MCs . Since Akt phosphorylation and Ca2+ mobilization are PI3K-dependent [26, 49] suppression of PI3K activation by Ro5-4864 was not as effective in SHIP1-deficient BMMCs. These data suggested that Ro5-4864 very likely did not interfere with the mitochondrial Ca2+ buffering mechanism (such an effect should be observable in SHIP1-deficient BMMCs as well) and that a target site located more upstream should be involved in Ro5-4864-mediated regulation of the secretory response.
1,4-Benzodiazepines have been reported to inhibit SFKs , which are known to play multiple important roles in MC activation, in particular via the FcεRI . Amongst the first signaling events in MCs in response to FcεRI crosslinking are activation of the SFK Lyn, subsequent tyrosine phosphorylation of the FcεRI β-chain and γ-chain ITAMs by Lyn and activation of the tyrosine kinase Syk via interaction with the phosphorylated γ-chains and phosphorylation by Lyn . Moreover, immediate activation of the SFK Fyn leads to the activation of the PI3K pathway . Thus, pharmacological interference with SFK activation would have a negative impact on most FcεRI-mediated signaling pathways. Indeed, Ro5-4864 attenuated Ag-triggered tyrosine phosphorylation events in both wild-type and SHIP1-deficient MCs. Using a GST-SH2(Lyn) fusion protein to pull-down specific tyrosine-phosphorylated proteins, reduced phosphorylation of FcεRIβ and Syk was observed, indicating early interference of FcεRI signaling by Ro5-4864. Though both FcεRIβ and Syk are known targets of Lyn, involvement of other SFKs cannot be excluded, all the more since particularly Fyn seems involved in regulation of Ag-triggered degranulation and activation of the PI3K pathway . In this respect, enhanced Ag-induced phosphorylation of Akt in Lyn-deficient BMMCs was markedly suppressed by Ro5-4864, clearly indicating Lyn-independent effects of this BDZ and suggesting a Fyn-dependent effect (Additional file 4: Figure S4).
Whereas degranulation is a fast response after Ag triggering of MCs occurring within a few minutes, production of pro-inflammatory cytokines takes place with slower kinetics. Important signaling pathways for cytokine production downstream of the FcεRI include the PI3K, p38, and NFκB pathways . All of these pathways were attenuated by Ro5-4864 treatment in wild-type MCs underlining the role of central signaling elements, e.g. SFKs, being blocked by this drug. Intriguingly, Ro5-4864 also concentration-dependently suppressed cytokine production in response to stimulation of receptor systems such as Kit and TLR4. Also this effect can be explained by the inhibition of SFKs. Both Lyn and Fyn have been reported to play positive regulatory roles in the context of Kit signaling [52, 53]. In addition, a recent publication by Avila et al. has demonstrated the importance of Lyn for the production of TNF-α in response to LPS in MCs . Thus, all effects observed in the present study with the 1,4-benzodiazepine Ro5-4864 are explainable by attenuation of SFK activity.
In conclusion, the present data demonstrate that the 1,4-benzodiazepine Ro5-4864 significantly suppresses pro-inflammatory MC responses downstream of differential ligand/receptor systems, most likely by attenuating SFK activity by direct inhibition of the respective SFK and/or indirectly by acting at a so far unknown upstream plasmalemnal recognition site. Hence, Ro5-4864 and structurally related compounds might be applicable as effective MC stabilizing drugs in different MC-dependent diseases, such as allergies, asthma, and systemic MCAD. It is however mandatory to identify and characterize the direct molecular target(s) to exclude unwanted side effects on other immune and non-immune cells. For certain MC-dependent diseases, however, topical administration as cream, eye drops or nasal spray could be options for first applications.
Material and methods
Ro5-4864, clonazepam, DNP-HSA (containing 30–40 mol DNP per mole albumin), monoclonal IgE anti-DNP (SPE-7), ovalbumin, thapsigargin, and EDTA were purchased from Sigma-Aldrich, Munich, Germany. Fluo-3 AM, Fura Red AM, pluronic F-127, H2DCFDA, MitoTracker Red CMXRos, and recombinant mouse SCF were obtained from (Invitrogen, Karlsruhe, Germany). Monoclonal mouse anti-P-Akt (S473), monoclonal rabbit anti-P-Erk1/2 (T202/Y204), polyclonal rabbit anti-P-IκBαS32, and polyclonal rabbit anti-P-p38 (T180/Y182) antibodies were purchased from Cell Signaling Technology, Frankfurt, Germany, polyclonal rabbit anti-p85 from Millipore, Schwalbach, Germany, polyclonal anti-Syk antibody (N-19) from Santa Cruz Biotechnology, and DMSO from AppliChem, Darmstadt, Germany. Monoclonal anti-FcεRIβ antibody was kindly provided by Dr. R. Siraganian (Bethesda, MD). R-form LPS from S. Minnesota mutant R595 was extracted and purified as described [55–57] and was a gift from Dr. M. Freudenberg and Dr. C. Galanos (MPI for Immunobiology and Epigenetics, Freiburg, Germany).
Bone marrow-derived MCs (BMMCs): According to the technique established by Razin et al. , bone marrow cells (1×106/ml) from 6 to 8 week old mice (129/Sv × C57BL/6) were cultured (37°C, 5% CO2) as single cell suspensions in RPMI 1640 medium supplemented with 15% FCS, 1% X63Ag8-653-conditioned medium (source of IL-3 ), 2 mM L-glutamine, 10 μM β-mercaptoethanol, 50 units/ml penicillin, and 50 mg/ml streptomycin. At weekly intervals, the non-adherent cells were reseeded at 5×105 cells/ml in fresh medium. After 4–6 weeks in culture, greater than 99% of the cells were Kit and FcεRI positive as assessed by FACS using phycoerythrin-labeled anti-c-kit antibodies (Pharmingen, Mississauga, Canada) and FITC-labeled hamster anti-mouse FcεRIα antibodies (eBioscience, Frankfurt, Germany), respectively. SHIP1+/+ and −/− BMMCs as well as Lyn+/+ and Lyn−/− BMMCs were differentiated in vitro using the same protocol but starting from bone marrow cells of 6 to 8 week old SHIP1+/+ and −/− littermates (129/Sv × C57BL/6). Peritoneal MCs (PMCs) were cultivated according to Malbec et al. . RBL-2H3 cells were maintained (37°C; 5% CO2) in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 units/ml penicillin, 50 mg/ml streptomycin, and 50 μM β-mercaptoethanol.
Cellular stimulation and western blotting
IgE-loaded BMMCs were washed in PBS and resuspended in RPMI/0.1% BSA. Cells were adapted to 37°C for 15 min and pretreated and stimulated as indicated. After stimulation, cells were pelleted and solubilized with 0.5% NP-40 and 0.5% sodium deoxycholate in 4°C phosphorylation solubilisation buffer . After normalizing for protein content, the postnuclear supernatants (obtained after centrifugation at 4°C at 13,200 rpm in an Eppendorf 4515R centrifuge (F45-24-11 rotor) for 15 min) were subjected directly to SDS-PAGE and Western blot analysis . The GST-SH2(Lyn) construct was described previously  and production, pull-down and immunoblotting were performed as published .
For degranulation studies, MCs were preloaded with 0.15 μg/ml IgE anti-DNP overnight at 37°C. The cells were then washed and resuspended in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% bovine serum albumin (BSA) in 10 mM Hepes, pH 7.4). The cells were adapted to 37°C for 20 min and then treated at 37°C as mentioned. Vehicle (DMSO) and BDZ treatment was for 20 min prior to Ag (DNP-HSA) addition. The degree of degranulation was determined by measuring the release of β-hexosaminidase .
Preparation and use of precision-cut lung slices
Precision-cut lung slices (PCLS) were prepared from 8-week-old Wistar rats (220 ± 20 g) obtained from Charles River (Sulzfeld, Germany) and kept under controlled conditions (22°C, 55% humidity and 12-h day/night rhythm). Animal experiments were approved by the local ethics committee. Rat PCLS were prepared as previously described . Rats were sacrificed by an overdose of pentobarbital i.p. (60 mg/kg). Isolated lungs were filled with pre-warmed agarose solution (0.75%) via the trachea and subsequently chilled with ice. Then lobes were separated and cut into 5 to 10 mm thick tissue segments from which cores were made along the airways, and then cut into 250 ± 20 μm thick slices (Alabama Research and Development, Munford, AL). For studies with ovalbumin, the lung slices were incubated overnight with cell culture medium containing 1% serum from actively sensitized rats, as previously done . After overnight culturing, the airways in PCLS were imaged and digitized using a digital video camera. A control picture was taken before addition of DMSO or Ro5-4864 (10 μM, 30 μM, and 100 μM) and after addition of ovalbumin (10 μg/ml) frames were recorded every 30 s for 15 min. The images were analyzed by the image analysis program Optimas 6.5 (Optimas, Bothell, WA).
Mouse IL-6 and TNF-α ELISAs (BD Pharmingen, Heidelberg, Germany) were performed according to the manufacturer’s instructions. Absolute levels of cytokines in culture supernatants varied between experiments/BMMC cultures. Qualitative differences, however, were consistent throughout the study. Experiments were done in triplicates and performed at least three times.
Measurement of Ca2+ mobilization
IgE-preloaded BMMCs were washed with RPMI 1640 medium, resuspended at 5 × 106 cells/ml in RPMI 1640 containing 1% FCS, 1.3 μM Fluo-3 AM, 2.7 μM Fura Red AM, and 0.1% pluronic F-127, and incubated for 30 min at 37°C. Cells were then pelleted, resuspended in RPMI 1640 containing 1% FCS and analyzed in a FACSCalibur flow cytometer (BD Biosciences) after the indicated stimulation procedures. The FACS profiles were converted to line graph data using the FlowJo analysis software (Treestar, Ashland, OR, USA).
Flow cytometric analysis of ROS production
IgE-sensitized BMMCs were washed with PBS, resuspended in RPMI 1640/1% FCS (5 × 106 cells/ml), and stained with the free radical-sensitive dye H2DCFDA (final concentration: 10 μM) for 30 min at 37°C in the dark. Subsequently, stimulus (antigen) was added and flow cytometric analysis of cell samples was carried out using a FACSCalibur (Beckton Dickinson, San Jose, USA). Data were processed by FlowJo analysis software.
Molecular cloning and transfection
To obtain a fusion construct comprising murine TSPO and an enhanced green fluorescent protein (eGFP), TSPO cDNA at its 3′-end was fused to eGFP sequence. Murine TSPO full-length cDNA (FANTOM clone I830130P14) was obtained from imaGenes GmbH (Berlin, Germany) and plasmid pEGFP-N1 from Clontech Laboratories Inc (Mountain View, USA). The coding sequence of TSPO was inserted in-frame using EcoRI and BamHI restriction sites resulting in pEGFP-N1-TSPO. The final plasmid was controlled by DNA-sequencing. RBL-2H3 cells as well as BMMCs were transiently transfected with pEGFP-N1-TSPO via electroporation with the Neon Transfection System (Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer’s instructions.
RBL-2H3 cells were detached from the plate, reseeded on cover slips in a 12-well plate and incubated for another 24 h. BMMCs (24 h post transfection) were transferred to a 12-well plate containing cover slips pretreated with 0.1% poly-L-lysine in PBS and were also incubated for further 24 h. 48 h after transfection, mitochondria of RBL-2H3 cells and BMMCs were stained with MitoTracker Red CMXRos. Cells were incubated for 30 min at 37°C with 200 nM MitoTracker in stimulation medium. Cells were then washed twice with PBS containing 9 mM CaCl2 and 5 mM MgCl2 and fixed with methanol for 20 min in the dark at RT. Background fluorescence was then quenched for 5 min with 50 mM NH4Cl in PBS containing 9 mM CaCl2, 5 mM MgCl2, and 0.1% (v/v) Triton X 100 at RT. Finally, cells were washed in water and mounted on a glass slide with one drop of Immunomount. The prepared slides were analyzed with a Zeiss LSM 710 (Carl Zeiss AG, Jena, Germany) confocal laser scanning microscope. All images were taken with a 63x oil immersion objective. For the fluorophores, the following lasers, excitation wavelengths, and detected range of emission wavelengths were used: eGFP (Laser: Argon; Excitation Wavelength: 488 nm; detected wavelength range: 493–574 nm) and MitoTracker Red CMXRos (Laser: DPSS 561–10; Excitation Wavelength: 561 nm; Detected Wavelength Range: 568–691 nm). During all measurements, the pinhole was set to 1 AU. The obtained images were analyzed with the Zeiss ZEN 2009 software.
P values were calculated by the paired two-tailed Student’s t test. P values of * < 0.05, ** < 0.005, and *** < 0.0005 were considered statistically significant. ns, non-significant.
This work was supported by a grant from the Leonardis Stiftung to M. H. (001-2011). The expert technical assistance of T. Nöcker in early stages of this work is acknowledged.
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