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Expression of interleukin-6 and its receptor in the sciatic nerve and
cultured Schwann cells: relation to 18-kD fibroblast growth factor-2
a ,
*
b a a bClaudia Grothe
, Klaus Heese , Christof Meisinger , Konstantin Wewetzer , Dieter Kunz ,
c b
Peter Cattini , Uwe Otten
a
Hannover Medical School, Center of Anatomy, OE 4140, D-30623 Hannover, Germany
b
Department of Physiology, Vesalianum, Vesalgasse 1, CH-4051 Basel, Switzerland
c
Department of Physiology, University of Manitoba, 730 William Ave., Winnipeg, Manitoba R3E 3J7, Canada Accepted 22 August 2000
Abstract
Expression of interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2) in Schwann cells is modulated by external stimuli. To study possible interactions of both factors we have analyzed mutual effects of exogenous IL-6 and FGF-2 on the expression of each other and the corresponding receptor (R) molecules IL-6R and FGFR1 after peripheral nerve lesion in vivo and in vitro using cultured Schwann cells. Using rat Schwann cells we found that IL-6 did not exert any effects on the expression of FGF-2 and FGF receptor type 1 (R1) whereas exogenously applied 18-kD FGF-2 strongly increased the expression of the mRNAs of IL-6 and its receptor. In addition, immortalized Schwann cells over-expressing the 18-kD FGF-2 isoform showed elevated levels of IL-6 and IL-6R whereas immortalized Schwann cells over-expressing the high-molecular-weight isoforms (21 kD and 23 kD) displayed unaltered IL-6 and IL-6R expression levels. According to in situ hybridization studies of intact and crushed sciatic nerves in vivo, Schwann cells seems to be the main source of IL-6 and IL-6R. Following sciatic nerve crush, the FGF-2 and the IL-6 system are upregulated after the first hours. Furthermore, we showed that the early increase of the FGF-2 protein is mainly confined to the 18-kD isoform. These results are consistent with the idea of a functional coupling of FGF-2 and the IL-6 system in the early reaction of Schwann cells to nerve injury. 2000 Elsevier Science B.V. All rights reserved.
Keywords: IL-6; Schwann cell; Sciatic nerve; FGF-2; Rat
1. Introduction physiological role of IL-6 in the nervous system [12,13]. Central nervous system-derived IL-6 might be involved in The expression of interleukin (IL)-6 and fibroblast neuronal survival, differentiation, and transmitter metabo-growth factor (FGF)-2 in cultured Schwann cells and lism [15]. Sympathetic and sensory ganglia also show a Schwann cell lines is modulated by external stimuli [2,19]. development-dependent expression [14]. IL-6 appears to Since both molecules are upregulated after peripheral mediate effects on survival and neuropeptide expression of nerve injury [2,19,27], the question arises whether the sensory and sympathetic neurons [15]. Other cytokines, expression of both molecules is causally linked to each such as IL-1b and tumor necrosis factor (TNF)-a are other. IL-6 belongs to a family of neuropoietic cytokines known to modulate IL-6 expression in fibroblasts and which also includes ciliary neurotrophic factor (CNTF) astrocytes [15,43]. However, nothing is known about the leukemia inhibitory factor (LIF), oncostatin M, signals triggering the up-regulation of IL-6 in the injured cardiotrophin-1, growth promoting activity, and IL-11 nerve.
[23,25]. The fact that the IL-6 and IL-6 receptor mRNAs FGF-2 is a member of the FGF family which comprises are developmentally regulated in the brain is evidence for a heparin-binding proteins that promote mitogenesis of mesoderm- and neuroectoderm-derived cells [4]. FGF-2 expression has been reported in distinct areas of the
*Corresponding author. Tel.:149-511-532-2896; fax: 1
49-511-532-developing and mature nervous system [1,21,41]. In vitro
2880.
E-mail address: [email protected] (C. Grothe). studies have revealed stimulating effects of FGF-2 on
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 at2808C.
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
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 ElongaseE 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-StarE
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 and separated on a 4% polyacrylaminde–urea sequencing
gel. Following fixation and drying, gels were exposed to
3.1. Effects of IL-6 on immortalized Schwann cells Kodak bioMax film. tRNA was used as negative control.
It has been previously demonstrated that immortalized
2.6. Western blot analysis Schwann cells and physiological (secondary) Schwann
level 1, 2, 6, and 24 h after administration of IL-6 (not whether FGF-2 can influence IL-6 and IL-6 receptor shown). In addition, the level of FGFR1 transcript was not expression, immortalized Schwann cells were treated with altered after IL-6 treatment for 2, 6 and 24 h (not shown). FGF-2 for 5, 10 and 24 h under serum-containing and serum-free culture conditions. Both IL-6 and IL-6 receptor transcript levels were elevated after FGF-2 treatment under 3.2. Effects of FGF-2 on immortalized and physiological both culture conditions. For example, IL-6 mRNA level
Schwann cells showed a 2.5-fold increase 5 and 10 h after addition of FGF-2 which had returned to control values by 24 h (Fig. Recently, it was shown that IL-6 expression in Schwann 1). Levels for the IL-6 receptor mRNA displayed a 2.2-cells is modulated by various cytokines [2]. To determine fold increase 5 h after FGF-2 treatment and were slightly
reduced after 10 h; after 24 h the transcript level was still sion peak of the IL-6 transcript was found after a 10 1.5-fold elevated compared to untreated cultures (Fig. 1). h-treatment with FGF-2, the IL-6 receptor mRNA
maxi-In a second set of experiments, transfected clonal mum was reached after 5 h (not shown). immortalized Schwann cells over-expressing the 18-kD or
the 21 / 23-kD FGF-2 isoforms [20] were analyzed for IL-6 3.3. Cellular localization of IL-6 and IL-6 receptor and IL-6 receptor expression and compared with immortal- mRNAs in the sciatic nerve
ized Schwann cells transfected with a control vector,
non-transfected immortalized Schwann cells, and non-transfect- Although immortalized physiological Schwann cells ed immortalized Schwann cells treated with 18-kD FGF-2. express IL-6 and IL-6 receptor mRNAs and up-regulate The IL-6 and IL-6 receptor mRNA levels were found to be these transcripts in response to 18-kD FGF-2, the cellular significantly increased only in those Schwann cell clones source of these factors in the peripheral nerve is not yet that were either over-expressing or receiving the 18-kD known. Therefore, in situ hybridization was performed FGF-2 isoform (Fig. 2). Exogenous administration as well using sciatic nerve preparations 5 h after nerve crush in as endogenous over-expression of the 18-kD FGF-2 but comparison to contralateral intact nerves. In the intact not of the 21 / 23-kD isoforms led to an increased IL-6 and nerve, prominent staining for both transcripts (IL-6, IL-6
IL-6 receptor expression. receptor) was found in vascular endothelial cells and in
Physiological Schwann cells of the newborn rat dis- Schwann cells as revealed by S100 immunostaining (Fig. played a 2.5- and 2.8-fold increase of IL-6 and IL-6 3). Staining pattern refers to the cellular distribution which receptor mRNA in response to FGF-2 treatment as well, was unaltered 5 h after crush lesion (Fig. 4). Ox-42 however, with a slightly different time pattern. The expres- immunoreactive cells were sporadically found 5 h after
Fig. 3. Cellular localization of IL-6 and IL-6 receptor mRNAs in Schwann cells and vascular endothelial cells. (A) Immunocytochemical staining for S100 localized in Schwann cells (short arrows); (B) non-radioactive in situ hybridization with digoxygenin-labelled IL-6 receptor (IL6R) riboprobes localized in Schwann cells (short arrows) and vascular endothelial cells (long arrow). Magnification3390.
sciatic nerve crush. However, in order to verify Ox-42 FGF-2 is known to be up-regulated after peripheral immunostaining sciatic nerves 24 h after injury were used. nerve injury [19,30]. However, mRNA and protein values Positive cells were exclusively localized around the lesion for early time points after lesion have not been published site (Fig. 5). The temporal and spatial pattern of the Ox-42 so far. Therefore, FGF-2 mRNA was analyzed 5 h and immunostaining was completely different to the IL-6 and FGF-2 protein was studied 5 h and 2 days after crush IL-6 receptor mRNA staining. These results clearly dem- lesion. Ribonuclease protection analysis revealed elevated onstrate that Schwann cells and vascular endothelial cells FGF-2 mRNA levels in the proximal and distal nerve but not macrophages represent the main sources for IL-6 stump compared to the contralateral side 5 h after injury and IL-6R, also after sciatic nerve crush with increased (Fig. 7). In addition, the FGF-2 protein displayed a
IL-6 and IL-6R in RNA levels. significant increase after 5 h, at which the 18-kD FGF-2
isoform showed the strongest up-regulation compared to
3.4. Sciatic nerve crush the 21- and 24-kD isoforms (Fig. 8). Two days after
peripheral nerve crush the FGF-2 protein level was only From previous studies it is known that peripheral nerve slightly elevated (not shown).
injury results in an elevated IL-6 mRNA expression level in the nerve stumps as early as 2 and 3 h after operation [2,38]. To investigate whether the IL-6 receptor transcript
level is also increased after lesioning, the mRNA was 4. Discussion analyzed 5 h after sciatic nerve crush. Nerve segments
in vitro interaction of FGF-2 and IL-6, the early increase of both molecules after nerve injury suggests a functional coupling of FGF-2 and IL-6.
Previously, it was shown that in addition to various cytokines, co-culturing of Schwann cells with phaeoch-romocytoma (PC)12 cells or NIH 3T3 fibroblasts stimulate IL-6 synthesis in Schwann cells [2]. Since it is known that both cell types express FGF-2 constitutively [33,35], the present data suggest that this up-regulation of IL-6 might be mediated by FGF-2 released from PC12 cells and 3T3 fibroblasts.
Different FGF-2 isoforms between 18 and 24 kD have been described representing alternative translation products from a single mRNA which occur in a tissue- and species-specific manner [10,16,31]. Initiation at the AUG codon results in the 18-kD FGF-2, the higher-molecular-weight forms (21 kD, 23 / 24 kD) arise after initiation at CUG codons [9]. Our data indicate that it is the 18-kD FGF-2 isoform which is mainly responsible for the induction of IL-6 and IL-6 receptor. This is based on the following observations: (1) Exogenously applied 18-kD FGF-2 in-duced an up-regulation of both transcripts. (2) Over-ex-pression of 18-kD but not of 21 / 24-kD FGF-2 resulted in increased IL-6 and IL-6 receptor transcript levels. Stable over-expression of the FGF-2 isoforms in transfected Schwann cells was documented previously [20]. (3) Peripheral nerve crush showed specific induction of the 18-kD FGF-2 isoform in the proximal and distal nerve stumps after 5 h. Further experiments, aiming to selective-ly eliminate the endogenous 18-kD FGF-2, e.g. by using specific antisense probes, should verify this hypothesis.
The present in situ hybridization data are the first demonstration of IL-6 and IL-6 receptor synthesis in Schwann cells and vascular endothelial cells. Macrophages did not seem to be a major source of IL-6 and IL-6 receptor mRNAs. This result correlates with studies in IL-6-deficient mice where the injury-induced invasion of macrophages was not altered compared to the wild-type animals [44]. These data suggest that the IL-6 and its
Fig. 4. Expression of IL-6 and IL-6 receptor (IL6R) in the sciatic nerve 5
receptor are synthesized in Schwann cells and possibly
h after crush lesion. In situ hybridization was performed on consecutive
involved in the Schwann cell reaction to injury whereas the
sections using A, digoxygenin-labelled IL-6 antisense (as) riboprobes, B,
macrophage reaction takes place independently of IL-6.
digoxygenin-labelled IL-6 receptor antisense riboprobes, and C,
digox-ygenin-labelled IL-6 receptor sense (s) riboprobes. Long arrows, crush We have shown previously that the FGF-2 transcript site; short arrows, stained vascular endothelial cells. Magnification x85. level is up-regulated over a period of 3 weeks after
peripheral nerve lesion [19]. This, together with the observation that FGF-2 is capable of suppressing the cells. IL-6 in turn, however, was not able to alter the forskolin-induced myelin protein Po [34] and stimulates expression of FGF-2 in the same cell type. The immortal- the proliferation of Schwann cells in vitro [7] suggests that ized Schwann cells which were used as a model system do FGF-2 is involved in the initial steps of regeneration, e.g. express Schwann cell markers like S100, P0, and galac- by preventing myelination and inducing Schwann cell
¨
Fig. 5. Immunolocalization of Ox-42 in macrophages 24 h after sciatic nerve crush. Macrophages accumulate at the lesion site. Magnification3273.
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