C . Grothe et al. Brain Research 885 2000 172 –181
173
proliferation of astrocytes, oligodendrocytes, and Schwann receptor-immunoreactivity. Such cells were used for the
cells [7,8,37] and on survival, neurite outgrowth, transmit- experiments. Schwann cells were treated with IL-6 100
ter metabolism, and synapse formation of various neuron units ml; 10 ng ml for 1, 2, 6 and 24 h or with FGF-2
populations [18,42]. With regard to the functional role of 50 ng ml for 5, 10 and 24 h. Recombinant IL-6 and
FGF-2 after peripheral nerve injury, evidence from in vivo recombinant 18-kD FGF-2 were purchased from Preprotec.
studies suggests that the molecule could mediate neuro- Controls were cultured in the absence of cytokines in the
trophic effects on axotomized motor and sensory neurons. respective medium for 24 h. In a second series of
This notion is supported by the finding that nerve lesion experiments, transfected clonal Schwann cells over-ex-
results in an up-regulation of FGF-2 expression in pressing the 18-kD or 21 23-kD FGF-2 isoforms [20] were
motoneurons, dorsal root ganglia, and sympathetic ganglia analyzed for IL-6 and IL-6 receptor expression and com-
[19,24,26] and that exogenously applied FGF-2 can pre- pared with Schwann cells transfected with a control vector,
vent the lesion-induced neuron death [17,36]. At the lesion non-transfected
Schwann cells,
and non-transfected
site itself FGF-2 might be involved in the myelination Schwann cells treated with 18-kD FGF-2. Dislodging of
process [30]. The molecular signals which regulate FGF-2 cells for RNA or protein isolation was performed by
expression after lesion, as well as the events that are trypsinization 0.125 trypsin 20 mM EDTA. Pellets
triggered by the increased expression of FGF-2 are not were stored at 2808C.
known. In the present study we have investigated mutual effects
2.2. Sciatic nerve crush of IL-6 and FGF-2 on the expression of the respective
endogenous molecules in immortalized Schwann cells Hanover Wistar rats Charles River Wiga, Sulzfeld,
which were used as a model system. In immortalized Germany were anaesthetized using sodium pentobarbital
Schwann cells and immortalized Schwann cells over-ex- 50 mg kg, i.p.. After exposing the left sciatic nerve, the
pressing the 18-kD FGF-2 or the high-molecular-weight crush was performed with a fine forceps at the mid-thigh
FGF-2 isoforms, 18-kD FGF-2 stimulated the expression level. For quantitative studies, 5 h and 2 days after the
of the IL-6 and IL-6R mRNAs. Furthermore, physiological operation, rats were killed by decapitation and 8-mm nerve
secondary Schwann cells of the newborn rat also dis- segments proximal and distal to the crush site were
played an upregulation of IL-6 and IL-6R mRNAs in dissected; identical segments of the contralateral nerve
response to FGF-2. In addition, in situ hybridization of the were isolated in the same way. For each time point three
intact and injured sciatic nerve revealed Schwann cells as animals were used.
the main source of IL-6 and IL-6R. Finally, FGF-2 and the Experimental
protocols were
approved by
Re- IL-6 system are upregulated within the first hours after
¨ gierungsprasidium Freiburg AZ 37 9185.813 784 and
sciatic nerve injury. These data may reflect a functional Bezirksregierung Hannover AZ 509i-42502-98 47 and
coupling of Schwann cell-derived FGF-2 and IL-6 system meet the guidelines of the Tierschutzgesetz i.d.F.v.
in the injured peripheral nervous system. 17.02.93.
2.3. RNA preparation
2. Materials and methods
For total cellular RNA isolation frozen tissue samples were thoroughly homogenized in lysis buffer and RNA
2.1. Schwann cell cultures was isolated by acid guanidinium thiocyanate–phenol–
chloroform extraction [6] and quantified spectrophotomet- Immortalized Schwann cells 15–20 passages; [40]
rically by absorbance at 260 nm. The quality of RNA was were cultured in Dulbecco’s modified Eagle’s medium
checked by formaldehyde agarose gel electrophoresis. DMEM supplemented with 5 fetal calf serum FCS
2
and 10 horse serum. At a density of 25 000 cells cm medium was changed and cells received serum-containing
2.4. IL-6 and IL-6 receptor mRNA determination medium or serum-free medium supplemented with N1
additives [3] and 0.25 bovine serum albumin overnight. Transcripts of IL-1 and IL-6 receptor were analyzed by
Physiological Schwann cells were produced from spinal reverse transcription-polymerase chain reaction RT-PCR.
nerves of 1-day-old rats essentially as described by Total
RNA was
reverse-transcribed into
cDNA Brockes et al. [5]. After plating on poly-
L
-lysine 0.5 Superscript
E II, Gibco-BRL and then subjected to PCR mg ml cells were expanded in DMEM supplemented with
amplification using primers specific for IL-6 sense: 59- 10 FCS containing forskolin 2 mM and stored in
GACTGATGTTGTTGACAGCCACTG-39; antisense: 59- masterstocks. After removal of forskolin cells stopped
TAGCCACTCCTTCTGTGACTCTAA-39 and IL-6 re- proliferating and returned to their spindle-shaped pheno-
ceptor sense: 59-TGGTGCAGCCGGATCCACCTGCCA- type, expressing S100- and p75 nerve growth factor
39; anti:
59-CTGGGGCGAGGACACTCGTTGCTT-39.
174 C
The number of cycles used to amplify each cDNA was immunological detection was performed using a mono-
chosen to allow the PCR to proceed in a linear range clonal anti-FGF-2 antibody Transduction Laboratories,
according to the Elongase E enzyme mix-protocol Gibco-
USA and the ECL system Amersham, Braunschweig, BRL. PCR amplification of the constitutively expressed
Germany. ribosomal protein S12 cDNA was used as a measure of
input RNA S12: sense: 59-GGAAGGCATTGCTGCTGG- 2.7. In situ hybridization
39; anti: 59-CTTCAATGACATCCTTGG-39. Controls using RNA samples without reverse transcription or con-
At 5 h after sciatic nerve crush, ipsi- and contralateral trols without RNA were used to demonstrate absence of
nerves were fixed in 4 paraformaldehyde overnight and contaminating DNA. The amplification steps involved
embedded in paraffin. Sections of 8 mm thickness were denaturation at 948C for 1 min, annealing for 50 s at 558C
placed on coated slides aminoalkyl silane-prepared slides, with specific primers and extension for 1 min at 688C. The
Sigma. After proteinase K 2 mg ml treatment, prehy- PCR products 5 ml were analyzed by electrophoresis
bridization was performed in 53 standard sodium citrate using 1.5 agarose gels followed by alkaline blotting of
SSC; SSC3150.15 M NaCl and 0.015 M sodium citrate, the fragments onto nylon membranes and subsequent
pH 7.2 containing 50 formamide, 53 Dehnardt’s solu- hybridization
with specific
digoxigenin-labeled DNA
tion, tRNA 250 mg ml and salmon sperm DNA 500 probes.
Detection was
performed using
CDP-Star E
mg ml for 2 h at 558C. Hybridization was done in the Tropix as chemoluminescence substrate for alkaline
same solution containing digoxygenin-labelled cRNAs 2.5 phosphatase
conjugated to
anti-digoxigenin-antibodies mg 200 ml slide; labelling kit, Boehringer, Mannheim,
Boehringer Mannheim as indicated by the manufacturers. Germany for 24 h at 558C. Washing procedure was
Appropriate exposures of Kodak X-Omat films were performed in 53SSC for 5 min at 728C and in 0.23SSC
quantified using a video densitometer Model 620, Bio- for 1 h at 728C. Detection of the bound probes was
Rad. performed according to the supplier’s information Boeh-
ringer. Both, antisense and sense control sections were 2.5. FGF-2 and FGFR1 mRNA determination
used in all experiments. Five independent experiments were performed.
Transcripts of FGF-2 and FGFR1 were analyzed by ribonuclease protection assay performed as described
2.8. Immunocytochemistry previously [32]. Hybridization was performed overnight at
498C after total RNA was dissolved and heated to 958C for Consecutive sections were used for in situ hybridization
10 min in hybridization solution [80 formamide, 40 mM and S100 immunocytochemistry. Binding of S100 anti-
PIPES pH 6.4, 400 mM NaCl, and 1 mM EDTA]
33
body Dako, Hamburg, Germany was visualized using containing 100 000 cpm of
P-labeled cRNA probe. After peroxidase antiperoxidase system and diaminobenzidine.
addition of RNase digestion buffer [10 mM Tris–HCl pH For Ox-42 immunocytochemistry native sections were
7.4, 300 mM NaCl, and 5 mM EDTA] containing RNases fixed in ethanol or acetone, respectively. Binding of Ox-42
A and T1 incubated for 1 h at 308C, proteinase K and antibody Serotec, UK was visualized by Cy3-labelled
sodiumdodecyl sulfate were added. After an incubation for second antibodies.
25 min at 358C phenol–chloroform extraction and ethanol– glycogen precipitation were performed. Pellets were sus-
pended in loading buffer 0 formamide, 10 mM EDTA, and 0.1 bromophenol blue, heated for 5 min at 858C,
3. Results
