Technical Reference #1640

1640.

Immunoglobulin-Free Light Chains Mediate Antigen-Specific Responses of Murine Dorsal Root Gangloin Neurons Anneke Rijnierse; Alfons Kroese; Frank Redegeld; Bart Blokhuis; Maurice van der Heijden; Andries Koster; Jean-Pierre Timmermans; Frans Kijkamp; Aletta Kraneveld, Utrecht University, Journal of Neuroimmunology, 208(1640), (2009)

Abstract:
Immunoglobulin-free light chains (IgLC) secreted by B lymphocytes have been shown to mediatehypersensitivity by inducing antigen-specific mast cell activation. Although both mast cells and sensoryneurons contribute to the hypersensitivity response t

Keywords:
DRG neurons; immunoglobulin-free light chain; intracellular calcium

Materials & Methods:
2.1. Animals Male BALB/c mice were obtained from Charles River (Maastricht The Netherlands). Mice were 6–8 weeks of age and weighed 20–25 g at the time of use. The animals were housed in groups not exceeding 8 mice per cage. Tapwater and chow food were allowed ad libitum and a 12/12 h day–night cycle was maintained. All experiments were conducted in accordance with The Animal Care Committee of Utrecht University (Utrecht The Netherlands). 2.2. DRG neuron cultures Single neuronal cells originating from DRG from the cervical to the lumbal segments were cultured as described previously (De Jonge et al. 2004) with small modifications. Animals were sacrificed by i.p. injection of overdoses of sodium pentobarbital and pinned down dorsally. All organs were removed and the spinal cord was revealed. DRGwere dissected one-by-one by carefully picking up the spinal cord exposing the DRG. Spinal roots were cut as close to the ganglion as possible. DRG were enzymatically digested with 10 mg/ml collagenase in L-15 medium at 37 °C 5% CO2 for 30 min. The reaction was stopped by adding an equal volume of FCS for 2 min. The ganglia were suspended in DRG culture medium (DMEM high glucose without glutamine and DMEM F-12 mediumat a 1:1 ratio enriched with 1% N2 human serum supplement 1% glutamine 50 mg/ml gentamicin 2 μM cytosine arabinoside) by mechanically dissociating with pipettes with decreasing diameters. The cell suspension was placed on serumcoated petri dishes to allow non-neuronal cells to adhere for 90 min at 37 °C 5% CO2. After separation neuronal cells were collected from the dishes and placed onto poly-L-lysine-coated petri dishes with glass bottoms. Cultures were maintained at 37 °C 5% CO2 and studied at days 2–5 of the culture; medium was refreshed at day 3. 2.3. Isolation of antigen-specific IgLC and IgE Mouse TNP-IgG1 was purified from culture supernatant of mouse hybridoma (1B7-11; ATCC) with affinity chromatography with protein G sepharose. The IgG fraction was dialyzed against 150 mM NaCl 50mMTris pH 8.0 and concentrated to the desired concentration using a Vivaspin 20 (MWCO 10.000) spin filter. Subsequently TNP-IgG1 was reduced in 200mM2-mercaptoethanol for 60 min at 37 °C followed by alkylation with 300 mM iodoacetamide for 30 min on ice. Heavy and light chainswere separated with size exclusion chromatography in 6M guanidine HCl pH 6.5 on a HiLoad Superdex200 column (Amersham). Fractions with immunoglobulin light chains were immediately dialyzed against PBS for several days with multiple buffer exchange. For antigen-specific immunoglobulin-free light chains (TNP)-specific IgG (1B7-11) was reduced alkylated and purified as described previously (Redegeld et al. 2002). Mouse TNP-specific IgE (clone H1DNP ε26.82 kindly provided by J. Rivera NIH USA) was isolated with a happen-affinity column containing TNP-BSA coupled to sepharosebeads. The column was washed with PBS and subsequently eluted with 0.1Mglycine pH 2.5. The eluate was dialyzed against PBS. Both immunoglobulin free light chains and IgEwere concentrated with a spin filter and checked for purity with (non-reducing) SDS-PAGE/ Coomassie staining. 2.4. Immunohistochemistry Cultured primary DRG neurons were fixed in 4% paraformaldehyde for 24 h at 4 °C on the glass bottom of poly-L-lysine-coated petri dishes. Subsequently they were washed with and kept in PBS until further use. To visualize binding affinity of IgLC and IgE to DRG neurons the following stainingwas performed. Cells were incubated with 25 μg/ml FITC-labeled mouse IgLC and FITC-labeled mouse IgE diluted in TBS with 1% BSA and 0.1% Triton X-100 for 90 min. The binding of IgLC and IgE to DRG neurons was dose-dependent and 25 μg/ml per DRG neuronal culture was found to be the optimal concentration. Aspecific binding of the secondary antibody was blocked with 10% normal goat serum (NGS) in TBS with 0.1% Triton X-100. Subsequently rabbit-anti- FITC (1/1000) was incubated for 1 h. The third antibody HRPconjugated goat-anti-rabbit was incubated for 1 h. The staining was developed with DAB enriched with 0.025% nickel ammonium sulphate to expose an intense and sharply outlined dark-brown/black color at the surface of the neurons. To expose the morphological structure of the cells an eosin background staining of the cytoplasm was performed subsequently. All incubations were performed at room temperature in a darkened incubation chamber. The secondary and tertiary antibodies were diluted in TBS containing 1% NGS and 0.1% Triton X-100. After each incubation step the cells were washed with TBS with 0.1% Triton X-100. DRG neurons with a crisp dark brown to black color sharply outlining the surface were considered to be positive for either IgLC or IgE DRG neurons with diffuse eosin background staining without distinct borders were regarded as negative. The number of positive and negative stained DRG neurons were in a blinded manner manually counted in a randomly selected region of each culture dish (view field 575 μm by 425 μm) containing on average 67 neurons. Criteria to distinguish DRG neurons from fibroblasts (the two cell types in culture) used were shape diameter and thickness of the soma presence of neurites and neurite size. Fibroblasts appear as thin long-stretched cells with no distinct neuritis and were excluded from further analysis. To investigate specific binding affinity of IgLC and IgE competition binding studies were performed with cold (i.e. unlabeled) ligand. In these studies the same staining protocol as described above was carried out in the presence of 250 μg/ml unlabeled IgLC or unlabeled IgE diluted in TBS with 1% BSA and 0.1% Triton X-100 for 15 min. Without washing the cells 25 μg/ml of FITC-labeled IgLC or IgE was added. The staining was further performed as described above. 2.5. Sensitization with IgLC and IgE Primary cultures of DRG neurons were used at days 2–5 of the culture. Cells were washed twice with DRG medium and incubated with 25 μg/ml IgLC IgE or IgG at 37 °C 5% CO2 for 90 min. After this incubation cells were washed twice with DRG medium and used for optical imaging of cytosolic [Ca2+]i. 2.6. Solutions The buffer used in the optical Ca2+ recordings was artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 122 NaCl 2.1 KCl 25 NaHCO3 2.5CaCl2 1.3 MgSO4 1.2 KH2PO4 and 11 glucose (pH 7.4; oxygenated with 95%CO2–5%O2). In the ACSF used to evoke brief (10 s) membrane depolarization the concentration of KCl was increased to 70 mM while the NaCl concentrationwas reduced to maintain osmolarity. To induce antigen-specific activation dinitrophenyl coupled to human serum albumin (DNP-HSA)was used (10 ng/ ml in ACSF). A solution of 10 ng/ml BSA in ACSF was used as a control solution. 2.7. Optical recording of cytosolic [Ca2+]i Intracellular calcium mobilization was assessed by confocal laser scanning microscopy (CLSM) and used as an index of neural activation. Dishes with DRG neurons were incubated with the Ca2+ indicator dye Fluo-4 AM (1 μM) and 0.005% of the detergent pluronic F-127 in ACSF buffer for 40 min at RT. Approximately 20–30 min after stopping dye loading by refreshing the buffer the dishes were transferred to an inverted microscope (Axiovert 100 M; Zeiss) equipped with a ×16 water immersion objective (Zeiss; Plan-neofluar; numerical aperture 0.8) in a CLSM system (Zeiss; LSM-510). Digital images (size 368.5× 368.5 μm) were recorded (usually for a period of 40 min) at RT with a spatial resolution of 256×256 pixels and a temporal resolution of one image per 6 s. The 488-nm argon laser line (200 mW) was used to excite Fluo-4 fluorescence in the cells which was measured using a long-pass 505-nm filter. Laser illumination intensity was kept to a minimum (max 1% of laser output) to avoid phototoxicity and photobleaching. The calcium signals of the cultured DRG neurons were recorded in the culture dishes. All fluorescence measurements were made from subconfluent areas of the dishes enabling identification of individual cells. To study intracellular calcium changes in response to application of KCl DNP-HSA and BSA these compounds were dissolved in ACSF buffer and perfused through the bath in a constant flow rate (4 ml/min) by using a 4-channel perfusion setup. Because the culture dishes with DRG neurons also contained some fibroblasts all neurons within the image were identified by visual inspection. Criteria used were shape diameter and thickness of the soma presence of neurites and neurite size. Fibroblasts appear as thin long-stretched cells with no distinct neurites and were excluded from analysis. Image data were analyzed off-line using the Zeiss LSM510 analyzing software V2.53. A selected image in each image set was used as a template for designating each neuronal soma as a region of interest. The Ca2+ response to depolarization with 70 mM KCl was used to localize a subconfluent area with vital neurons. At the end of each experiment depolarization with KCl was again performed to check cell viability. Neurons lacking a Ca2+ response of the same order of magnitude as the response to the first KCl depolarization were excluded from further analysis. Because Fluo-4 is a single-wavelength indicator it was not possible to apply the ratiometric method for quantitative determination of [Ca2+]i. Therefore datawere normalized with respect to the mean fluorescence intensity (F0) during approximately the first 3 min of the recording. The temporal fluorescence intensity of the dye during the 40-min recording was divided by F0. The relative fluorescence (RF) obtained in this way represents integrated [Ca2+]i. The RF values of each region of interestwere plotted as a function of time. The amplitude of the responses of neurons to application of DNP-HSA was quantified as the RF level reached at the end of the 40 min measuring period using the last 15 images of the recording. Neurons were identified as being responsive on the basis of the following criteria: 1) an initial and transient response to application of DNP-HSA (see Results) followed by 2) a clear and gradual rise in RF values as a function of time and 3) a RF value at the end of the DNPHSA application exceeding 1.2. The percentage of responsive neurons was determined by dividing their number by the total amount of vital neurons in that particular recording. The lag time of the neural response to application of specific compounds was determined by measuring the time between compound application (as recorded by computer) and the first noticeable rise in RF. 2.8. Western blotting Cultured DRG neurons of 3 days old were lysed in RIPA buffer and lysates were frozen (−70 °C) until use. An amount of 200 μg protein of DRG neuron lysate and 40 μg protein of bone marrow-derived mast cell lysate (control) of boiled non-reduced samples were separated electrophoretically (SDS-PAGE 12%) and transferred onto polyvinylidene difluoride membranes (BIO-RAD Laboratories). The membrane was blocked with Tween-PBS containing 2% milk proteins. FcεRI α-chain was detected with hamster anti-mouse FcεRIα (1/500) by incubation for 4 h at room temperature. HRP-labeled goat antihamster (1/2000) was applied for 1 h at room temperature as secondary antibody. Blots were washed in Tween-PBS three times for 10 min incubated in commercial ECL reagent and exposed to photographic film. Films were scanned on a GS710 Calibrated Imaging Densitometer (Bio-Rad Veenendaal The Netherlands). 2.9. Materials L-Glutamine cytosine β-D-arabinofuranoside (ara-C) diaminobenzidine (DAB) DNP-HSA pluronic F-127 and BSA were all purchased from Sigma Chemical Co. St. Louis MO USA. Collagenase A was purchased from Roche Diagnostics Almere The Netherlands. The following materials were obtained from Invitrogen Breda The Netherlands: L15 medium DMEM F12 DMEM high glucose without glutamine gentamicine and N2 supplement. FCS came from Perbio Ettenleur The Netherlands. Glass bottom culture dishes were bought at MatTek Cultureware AslandMA USA. The Cy-3-conjugated donkey anti-rabbit antibody was from Jackson ImmunoResearch Europe Soham UK. Hamster anti-mouse FcεRIα was purchased from eBioscience. Rabbit anti-FITC HRP-conjugated goat anti-rabbit IgG and HRP-labeled goat anti-hamsterwere fromDakoCytomationHeverlee Belgium.Molecular Probes Eugene OR USA supplied the Fluo-4 AM. 2.10. Statistics The responses to stimulation of IgLC- and IgE-sensitized DRG neurons with DNP-HSA were subjected to a two-way analysis of variance (ANOVA) to test whether the responses on the different days of the culture could be pooled for further analysis. These statistical tests revealed no difference between the different days of culture within experimental conditions. Consequently results obtained at different days were pooled which resulted in mean values for the response amplitude for every experimental group. Responses in control groups and non-responsive neurons from experimental groups were compared in a one-way ANOVA followed by a Dunn's multiple comparison test. In experimental groups (IgLC- and IgE-sensitized) responsive neurons were distinguished from non-responsive neurons (see criteria above) and comparisonswere made by an unpaired t-test. The results are expressed as mean±sd. The results in Fig. 4 are presented in a box-and-whisker plot indicating the median (horizontal line in the box) distribution of the data by 25th and 75th percentiles (boxes) and lower and upper extremes (whiskers). The dot indicates an outlier of the dataset. Pb0.05 was considered to be statistically significant.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
Neurons