Previous Article | Next Article 
The Journal of Neuroscience, April 15, 2002, 22(8):3052-3060
The Cytokine Network of Wallerian Degeneration: Tumor Necrosis
Factor-
, Interleukin-1, and Interleukin-1
Shlomit
Shamash,
Fanny
Reichert, and
Shlomo
Rotshenker
Department of Anatomy and Cell Biology, Hebrew University-Hadassah
Medical School, and The Eric Roland Center for Neurodegenerative
Diseases, Jerusalem 91120, Israel
 |
ABSTRACT |
Wallerian degeneration (WD) is the inflammatory response of the
nervous system to axonal injury, primarily attributable to the
production of cytokines, the mediator molecules of inflammation. We
presently document the involvement of the inflammatory cytokines TNF
, interleukin (IL)-1, and IL-1
in peripheral nerve (PNS) injury in C57/BL/6NHSD (C57/BL) mice that display the normal rapid progression of WD (rapid-WD) and C57/BL/6-WLD/OLA/NHSD mice that display abnormal slow progression of WD (slow-WD). TNF and IL-1 mRNAs were expressed, whereas TNF but not IL-1 protein was
synthesized in intact PNS of C57/BL mice. TNF and IL-1 protein
synthesis and secretion were rapidly upregulated during rapid-WD in
Schwann cells. IL-1 mRNA expression and protein synthesis and
secretion were induced sequentially in Schwann cells with a delay after injury. Thereafter, recruited macrophages contributed to the production of TNF, IL-1, and IL-1, which in turn augmented myelin
phagocytosis by macrophages. Observations suggest that TNF and
IL-1 are the first cytokines with protein production that is
upregulated during rapid-WD. TNF and IL-1 may initiate,
therefore, molecular and cellular events in rapid-WD (e.g., the
production of additional cytokines and NGF). TNF, IL-1, and
IL-1 may further regulate, indirectly, macrophage recruitment,
myelin removal, regeneration, and neuropathic pain. In contrast to
rapid-WD, the production of TNF, IL-1, and IL-1 protein was
deficient in slow-WD, although their mRNAs were expressed. mRNA
expression and protein production of TNF, IL-1, and IL-1 were
differentially regulated during rapid-WD and slow-WD, suggesting that
mRNA expression, by itself, is no indication of the functional
involvement of cytokines in WD.
Key words:
TNF; IL-1; IL-1; Wallerian degeneration; regeneration; pain; Schwann cell; fibroblast; macrophage; myelin; phagocytosis
| |
INTRODUCTION |
PNS injury is followed by Wallerian
degeneration (WD) (Ramon y Cajal, 1928
). WD is significant to future
regeneration and to the development of neuropathic pain. This is
evident from comparing WD in normal C57/BL/6NHSD (C57/BL) and mutant
C57/BL/6-WLD/OLA/NHSD (Wld) mice (for review, see Stoll and
Muller, 1999). Cellular and molecular events are efficient and
rapid (rapid-WD) in C57/BL mice but deficient and slow (slow-WD) in Wld
mice. Myelin, which inhibits regeneration (McKerracher et al., 1994;
Mukhopadhyay et al., 1994; Schafer et al., 1996; Bandtlow and Schwab,
2000), is removed rapidly during rapid-WD but slowly during slow-WD
(Brown et al., 1991; Reichert et al., 1994; Be'eri et al., 1998). NGF, which promotes growth and survival of some PNS neurons (Bibel and
Barde, 2000), is injury induced in PNS of C57/BL but not Wld mice
(Brown et al., 1991). Delayed myelin removal and deficient NGF
production therefore delay regeneration in Wld mice (Brown et al.,
1991; Chen and Bisby, 1993; Schafer et al., 1996). NGF and interleukin
(IL)-6 regulate neuropathic pain (Safieh-Garabedian et al., 1995; Woolf
et al., 1997; Mendell et al., 1999; Murphy et al., 1999). Deficient
production of NGF and IL-6 (Reichert et al., 1996) reduces
neuropathic pain in Wld mice (Myers et al., 1996).
We view WD as an injury-induced inflammatory response attributable to
the production of cytokines, the mediator molecules of inflammation.
The classical view of inflammation suggests a network of cytokine
production (Oppenhheim and Feldman, 2001) in which inflammatory
cytokines TNF, IL-1, and IL-1 first upregulate the production
of additional inflammatory cytokines and thereafter the production of
anti-inflammatory cytokines. Anti-inflammatory cytokines
downregulate production altogether. Consequently, inflammation is
turned off. We termed the orchestrated production of cytokines during
WD "the cytokine network of Wallerian degeneration" (Rotshenker, 1997; Be'eri et al., 1998).
TNF, IL-1, and IL-1 are of particular significance to WD.
First, they could initiate the cytokine network of WD as they do in
other networks of inflammation (Oppenhheim and Feldman, 2001). Second,
they contribute to macrophage recruitment to inflammatory sites through
endothelial cell activation and chemokine production (Oppenhheim and
Feldman, 2001). Likewise, they could contribute to macrophage
recruitment in WD and consequently to macrophage-dependent functions
(e.g., myelin removal by phagocytosis). Third, they could indirectly
regulate survival and growth of PNS neurons and neuropathic pain
through the regulation of NGF production in PNS resident fibroblasts
[see above and Lindholm et al. (1987) and Hattori et al. (1993,
1994)].
Our present study was aimed at studying the production of TNF,
IL-1, and IL-1 in intact and injured PNS of C57/BL and Wld mice.
Transcription, translation, and secretion of cytokines can be
differentially regulated such that mRNA expression does not necessarily
indicate protein synthesis and synthesis does not necessarily indicate
secretion (Dinarello, 2001b). Therefore, we studied mRNA expression and
protein synthesis and secretion of TNF, IL-1, and IL-1 and
further identified the cellular sources of their production, as we did
previously with respect to granulocyte macrophage colony stimulating
factor (GM-CSF), IL-6, and IL-10 (Reichert et al., 1996; Saada et al.,
1996; Rotshenker, 1997; Be'eri et al., 1998). The use of the same
animal models in all our studies provides the essential common grounds
for elucidating the cytokine network of WD, the orchestrated production
of cytokines (TNF, IL-1, IL-1, GM-CSF, IL-6, and IL-10). It
further enables unraveling of the intricate relations between the
cytokine network and additional events in WD (e.g., NGF production,
macrophage recruitment, myelin removal, regeneration, and neuropathic
pain) that were also studied in the same animal models.
| |
MATERIALS AND METHODS |
Animals and surgical procedures. The experimental
protocol was approved by the authority for research and development of
the Hebrew University of Jerusalem. Two strains of mice, 2-4 months old, were used. The normal strain, C57/BL/6NHSD (Harlan Sprague Dawley,
Jerusalem, Israel), which displays rapid-WD, and the mutant strain, C57/BL/6-WLD/OLA/NHSD (Harlan Olac, Bicester, UK), which displays abnormal slow-WD. We refer to them as C57/BL and Wld, respectively. All surgical procedures were performed under
anesthesia. The sciatic nerve was transected after leaving the pelvis.
Freeze-damaged PNS segments were obtained by removing 15 mm PNS
segments from intact animals and subjecting them to three cycles of
freeze-thaw in distilled water to extinguish all resident cells.
Freeze-damaged PNS segments were returned in situ to donor
animals. Macrophages, and to a lesser extent fibroblasts, are recruited
to the freeze-damaged nerve. Freeze-damaged nerve segments serve as
Schwann cell free sources from which recruited macrophages are isolated
for cell culture studies.
Cell dissociation and culture. Detailed protocols for
obtaining nerve-derived non-neuronal cell cultures are given in
previous studies (Reichert et al., 1994, 1996; Be'eri et al., 1998).
Schwann cells, fibroblast, and macrophages were distinguished from each other by their distinct morphology and immunocytochemistry. Macrophages are positive to F4/80, complement receptor 3 (CR3)/MAC-1, and Galectin-3/MAC-2, Schwann cells are positive for S-100 and
Galectin-3/MAC-2 but negative for F4/80, and fibroblasts are negative
to all (for review, see Reichert et al., 1994, 1996; Be'eri et al.,
1998).
TNF, IL-1, and IL-1
immunocytochemistry in tissues. PNS segments were fixed in 4%
buffered formalin (3 hr), washed in PBS, cryoprotected in 30% sucrose
(overnight at 4°C), and sectioned in a cryostat. Sections were fixed
in 2% neutral formalin, washed in PBS, incubated in 0.05% Triton
X-100 in PBS (10 min), blocked in 10% FCS in PBS (3 hr), incubated
(overnight at 4°C) in goat anti-mouse TNF (4 µg/ml), rabbit
anti-human IL-1 (2 µg/ml), or goat anti-mouse IL-1 (4 µg/ml),
washed in PBS, incubated in biotinylated donkey anti-goat or
anti-rabbit IgG (1 hr), washed in PBS, incubated in FITC-conjugated
streptavidin (2.5 µg/ml for 45 min), and washed in PBS. TNF,
IL-1, and IL-1 immunocytochemistry in isolated non-neuronal cells
was performed using the same steps with the following exceptions.
Cultures were first fixed in 4% buffered formalin at 37°C, incubated
or not in 0.05% Triton X-100, and thereafter treated as tissue
sections after Triton X-100. Triton X-100 permeabilizes cells, enabling
antibodies (Abs) access to the cytoplasm and thereby detection
of cytoplasmic molecules. In the absence of Triton X-100, Abs cannot
gain access to the cytoplasm, and thus only cell surface molecules are
visualized. Normal rabbit or goat IgG was used as negative control.
Because there were variations between batches of antibodies, we have
always performed "positive controls" simultaneously and side by
side with all experimental samples. Positive controls were 3 d
injured PNS for tissue samples and macrophages for other non-neuronal cells. The two positive controls displayed consistent positive immunoreactivity to all three cytokines.
Detailed protocols for F4/80, CR3/MAC-1, Galectin-3/MAC-2, and S-100
immunocytochemistry of non-neuronal cells are given and have been shown
in previous studies (Reichert et al., 1994, 1996; Be'eri et al.,
1998).
TNF, IL-1, and IL-1
mRNA detection by RT-PCR. PNS segments were removed from
non-operated and operated mice at various time points after injury and
rapidly frozen in liquid nitrogen. Total RNA was isolated from the
tissues using an RNA isolation kit according to manufacturer's
instructions (Biological Industries, Beit-Haemek, Israel). Equal
amounts of total RNA were used to synthesize cDNA.
Primer sequences for the PCR reaction were as follows: IL-1:
5'-CTCTAGAGCACCATGCTACA-3' and 5'-TGGAATCCAGGGGAAACACTG-5'; IL-1: 5'-GCAACTGTTCCTGAACTCA-3' and 5'-CTCGGAGCCTGTAGTGCAG-3'; TNF: 5'-GGCAGGTCTACTTTGGAGTC-3' and
5'-ACATTCGAGGCTCCAGTGAATTCGG-3'; -actin: 5'-CAGCTTCTTTGCAGCTCCTT-3'
and 5'-TCACCCACATAGGAGTCCTT-3'. The number of cycles were 30 for
TNF and IL-1 and 25 for IL-1. -actin reaction was used as a
positive control and performed together with each of the cytokines at
their specific conditions. PCR amplification without Moloney murine
leukemia virus ensured that RNA preparations were not contaminated with
genomic DNA. To increase reaction specificity, the EX-Taq
polymerase was added to each tube just before insertion into the PCR
cycler, which was preheated to 94°C. PCR amplification products were
separated on ethidium bromide-stained 1.5% agarose gel, visualized by
ultraviolet light, and photographed. The photographs were scanned, and
densitometric analysis was performed using the Bio-Rad Multi Analyst/PC
version 1.1. The amplified bands showed their predicted sizes: IL-1, 312 bp; IL1- 364 bp; TNF 301 bp; and -actin, 219 bp.
Conditioned medium. Intact and injured PNS segments were cut
into 5 mm pieces and washed for 10 min in DMEM/F-12. PNS tissues were
incubated in DMEM/F-12 (0.15-0.25 ml per nerve) supplemented by 10%
heat-inactivated FCS in a humidified incubator saturated by 5%
CO2 at 37° C for 5 hr. Conditioned media were
separated from PNS tissues by aspiration, followed by 10 min of
centrifugation. Each sample of conditioned medium was produced by four
distinct nerves. Media and supplements were obtained from Biological Industries.
PNS extract. Ten 10 mm PNS segments were frozen immediately
in liquid nitrogen after removal from mice. Thereafter, the frozen PNS
segments were extracted in 1 ml PBS containing a mixture of protease
inhibitors (Sigma, Rehovot, Israel) and centrifuged for 10 min.
Different dilutions of the supernatant were tested for cytokine content
by ELISA.
Identification and quantification of TNF and
IL-1 by ELISA. We used two-site sandwich ELISA to
identify and quantify TNF and IL-1 in conditioned media and PNS
extract, according to manufacturer's instructions (Duo-Set; R & D
Systems, Minneapolis, MN). Levels of detection of the two cytokines
were between 8 and 16 pg/ml.
ELISA assay to study myelin basic protein-myelin
phagocytosis. Detailed protocols are given in previous studies
(Reichert et al., 2001; Slobodov et al., 2001). In principle,
thioglycollate-elicited macrophages were seeded (50 × 103 cells per well) in 96-well tissue
culture plates in quadruplicates. Isolated myelin was added for 1 hr to
allow phagocytosis, and excess myelin was washed out. Then, levels of
phagocytosed myelin were determined by ELISA. The ELISA method is based
on the ability to detect myelin basic protein (MBP) in lysate of
macrophages that phagocytosed myelin. Because MBP is unique to the
structure of myelin, levels of MBP are proportional to levels of
myelin. We have determined that >90% of the MBP associated with
macrophage lysate is cytoplasmic, resulting from myelin phagocytosis
(Slobodov et al., 2001).
Myelin isolation. Myelin was isolated from brains of
C57/BL/6NHSD mice (Norton and Poduslo, 1973; Reichert et al., 2001;
Slobodov et al., 2001). The resulting isolated myelin preparation is
made of fragments of myelin membrane that form small vesicles. Isolated myelin vesicles retain the fundamental ultrastructural and biochemical characteristics of intact membrane myelin. Therefore, levels of MBP are
proportional to the amount of myelin vesicles.
ELISA assay to study cell surface CR3/MAC-1. In each
experiment, an individual macrophage population that was obtained from a single mouse was seeded in quadruplicate (50 × 103 per well). Macrophages were fixed (4%
formalin at 37°C for 30 min), washed, blocked in 3% BSA (overnight
at 4°C), washed, incubated in monoclonal antibody (mAb) M1/70 rat
anti-mouse CR3/MAC-1 (2 hr at 37°C), washed, incubated in alkaline
phosphatase-conjugated donkey anti-rat IgG, washed, and incubated in
100 µl per well substrate (1 mg/ml p-nitrophenyl phosphate
sodium in 10% diethanolamine, pH 9.8; Sigma), and the reaction product
was read in a Dynatec ELISA reader at 405 nm wavelength.
Antibody sources. Antibody sources are as follows: hybridoma
cell lines producing mAb M1/70 rat anti-mouse CR3/MAC-1 (Developmental Studies Hybridoma Bank, Iowa City, IA) and rat anti-mouse F4/80 and
Galectin-3/MAC-2 (American Type Culture Collection, Rockville, MD); mAb
rat anti-mouse MBP and matched isotype control mAbs (Serotec, Oxford,
UK); antibodies anti-S-100 and FITC-conjugated rabbit anti-rat and goat
anti-rabbit IgG (Sigma); alkaline phosphatase-conjugated goat anti-rat,
FITC-conjugated streptavidin, and goat and rabbit IgG (Jackson
ImmunoResearch, West Grove, PS); and goat anti-mouse TNF and IL-1
and rabbit anti-human IL-1 (Santa Cruz Biotechnology, Santa Cruz, CA).
Statistical analysis. One-way ANOVA and the Bonferroni's
multiple comparison test were used. All p values of
significance are two tailed. Values given are average ± SEM.
| |
RESULTS |
TNF, IL-1, and IL-1 mRNAs expression in intact and injured
PNS of C57/BL mice undergoing rapid-WD
The expression of TNF, IL-1, and IL-1 mRNAs was studied
in intact and rapid-WD PNS of C57/BL mice by RT-PCR (Fig.
1). Tissues were frozen immediately after
removal from animals and used thereafter as sources for the detection
of all three mRNAs. The detection of mRNA thus indicates in
vivo expression. TNF and IL-1 mRNAs were detected in intact
PNS, whereas IL-1 mRNA was not. TNF and IL-1 mRNAs were
further detected in the distal PNS segments undergoing rapid-WD 5 hr
and 1, 3, and 6 d after injury. IL-1 mRNA was detected 5 hr and
1 and 3 d but barely at 6 d after injury. Thus TNF and
IL-1 mRNAs were constitutively expressed in intact PNS of C57/BL
mice and during the first 6 d of rapid-WD tested. In contrast,
IL-1 mRNA expression was injury induced and most probably transient.
Furthermore, TNF and IL-1 mRNAs display a similar pattern of
expression, which differs from that of IL-1 mRNA. It is most
probable, therefore, that the expression of TNF and IL-1 mRNAs,
on one hand, and IL-1 mRNA, on the other, is differentially
regulated in intact PNS of C57/BL mice and during rapid-WD.
View larger version (80K):
[in this window]
[in a new window]
|
Figure 1.
TNF and IL-1 mRNAs but not IL-1 mRNA are
detected in intact PNS of C57/BL mice (N). TNF
and IL-1 mRNAs are further detected during the first 6 d of
rapid-WD studied. IL-1 mRNA is detected 5 hr and 1 and 3 d but
barely at 6 d after injury during rapid-WD. Intact
(N) and rapid-WD PNS segments were removed 5 hr
(5h) and 1, 3, and 6 d (D-1,
D-3, and D-6, respectively) after injury.
The same tissues were the source for the detection of TNF, IL-1,
IL-1, and -actin mRNAs by RT-PCR. RT-PCR amplification products
were separated on ethidium bromide-stained 1.5% agarose gel,
visualized by ultraviolet light, and photographed (top
panel). The photographs were scanned, and densitometric
analysis was performed. Levels of mRNA of each cytokine at each time
point were further calculated as percentage of levels of -actin mRNA
in the same tissue sample (bottom panel).
|
|
TNF, IL-1, and IL-1 protein production in intact and
injured PNS of C57/BL mice undergoing rapid-WD
TNF (Aggawal et al., 2001) and IL-1 (Dinarello, 2001a)
protein exert their biological activity as soluble and cell
membrane-associated molecules, whereas IL-1 (Dinarello, 2001b)
exerts its biological activity as a soluble molecule only. We tested
for synthesis and secretion of TNF, IL-1, and IL-1 protein by
immunocytochemistry, ELISA, and bioassay. Immunocytochemistry in tissue
sections visualizes cytoplasmic TNF, IL-1, and IL-1 and
membrane-associated TNF and IL-1. Because immunocytochemistry was
performed in PNS segments that were fixed immediately after removal
from animals, positive immunoreactivity indicates the in
vivo synthesis and association of cytokines with the producing
cells. This was further verified by detecting cytokines, by ELISA, in
extracts of PNS segments that were frozen immediately after removal
from mice. We further tested for the presence of soluble TNF,
IL-1, and IL-1 in medium conditioned by PNS segments using ELISA
and bioassay. The presence of these cytokines in conditioned medium
indicates the secretion of synthesized cytokines. Immunocytochemistry,
ELISA, and bioassay use different mAbs and Abs to detect each cytokine
protein. Therefore, these are two independent assays that we used to
test for the synthesis of each cytokine protein. The resulting positive
identifications are thus specific and significant.
TNF in PNS tissue was studied by immunocytochemistry (Fig.
2). Low levels of TNF immunoreactivity
were detected occasionally in intact PNS in a small number of Schwann
cells. Higher levels of immunoreactivity to TNF were detected in
rapid-WD PNS segments 5 and 12 hr and 1 and 3 d after injury. The
in vivo presence of TNF was further verified in 1 d
injured PNS segments that were frozen immediately after removal from
mice and then extracted. TNF content in the extract, determined by
ELISA, was 6.53 pg in 5 mg wet weight tissue. Secreted TNF was
studied further. Intact and rapid-WD PNS segments that were removed at
various time points after injury were incubated immediately in medium for 5 hr. Conditioned medium thus obtained was tested for the presence
of soluble TNF by ELISA (Fig.
3A). Soluble TNF was detected in medium conditioned at all time points tested. Thus the
onset of secretion was rapid, within 5 hr after injury, and continuous,
peaking at 1 d after injury.
View larger version (145K):
[in this window]
[in a new window]
|
Figure 2.
TNF protein but not IL-1 or IL-1 protein
is detected in intact PNS of C57/BL mice (N).
TNF protein is further detected during the first 3 d of
rapid-WD studied. IL-1 protein is first detected 5 hr after injury
and thereafter during the first 3 d of rapid-WD studied. IL-1
protein is detected 1 and 3 d after injury but not 5 and 12 hr
after injury. Cryostat sections of intact (N) and
rapid-WD PNS segments that were removed 5 and 12 hr (5h
and 12h, respectively) and 1 and 3 d
(D-1 and D-3, respectively) after injury
were used to detect TNF, IL-1, and IL-1 protein by
immunofluorescence microscopy. Magnification: 250×.
|
|
View larger version (10K):
[in this window]
[in a new window]
|
Figure 3.
TNF and IL-1 are synthesized and
secreted during rapid-WD. The onset of TNF synthesis and secretion
is rapid, within the first 5 hr after PNS injury. The onset of IL-1
synthesis and secretion is delayed, between 5 and 10 hr after PNS
injury. PNS segments were removed from non-operated C57/BL mice and
used to condition medium for 5 hr (0-5h). Similarly,
PNS segments situated distal to transection sites were removed from
C57/BL mice 5 hr and 1, 3, 6, and 9 d after injury and used to
condition medium for 5 hr (5-10h,
D-1, D-3, D-6, and
D-9, respectively). The same conditioned media were
assayed for TNF and IL-1 content by ELISA, and production levels
were calculated (picograms per milligram wet weight of tissue in 5 hr).
Bars are the average of three experiments except for time point
D-3, where six experiments were performed. In each
experiment, four different PNS segments were used. Error bars indicate
±1 SEM.
|
|
The PNS segments that were incubated in medium for 5 hr were further
incubated for an additional 19 hr in fresh medium (5 
24 hr time
period) and conditioned medium assayed for TNF content by ELISA.
Secretion of TNF was calculated, picograms per milligram wet weight
of tissue in 5 hr, to enable comparison with secretion levels during
the first 5 hr of incubation (Fig. 3A). Secretion of TNF
by PNS segments removed 1 d after injury averaged 0.18 ± 0.01 pg/mg per 5 hr during the 5 24 hr time period compared with
0.49 ± 0.04 pg/mg per 5 hr during the first 5 hr. TNF was not
detected in medium conditioned during the 5 24 hr time period by
PNS segments removed 0, 3, 6, and 9 d after injury, although it
was detected in medium conditioned by the same PNS segments during the
first 5 hr of incubation (Fig. 3A). In vitro
conditions thus impose progressive reduction in secreted TNF.
In vitro imposed progressive reduction, not augmentation,
suggests that TNF secretion during the first 5 hr after removal of
injured PNS from animals is most likely a continuation and reflection
of cytokine secretion in vivo. This suggestion is supported
by the observations that TNF mRNA and protein are expressed and
synthesized in vivo in injured PNS (Figs. 1, 2) (and tissue
content by ELISA). Combined, immunocytochemistry and ELISA thus suggest
constitutive low levels of TNF synthesis and transient
injury-induced upregulation of TNF synthesis and secretion.
IL-1 in PNS tissue was studied by immunocytochemistry (Fig. 2).
Immunoreactivity to IL-1 was not detected in intact PNS of C57/BL
mice. It was detected in rapid-WD PNS segments 5 and 12 hr and 1 and
3 d after injury. We have previously documented the biological
activity of soluble IL-1 in medium conditioned within the first 5 hr
of rapid-WD and by 7 d rapid-WD PNS segments (Rotshenker et al.,
1992). Combined, immunocytochemistry and bioassay suggest no
constitutive production but injury-induced rapid onset of synthesis and
secretion of soluble IL-1 within 5 hr after injury that continued
thereafter. The injury-induced synthesis and secretion of IL-1
protein is further supported by the observations of injury-induced
upregulation of IL-1 mRNA expression (Fig. 1). Note, however, that
in intact PNS, IL-1 mRNA was detected whereas IL-1 protein was
not (see Discussion).
IL-1 in PNS tissue was studied by immunocytochemistry (Fig. 2).
Immunoreactivity to IL-1 was not detected in intact PNS of C57/BL
mice or in rapid-WD PNS segments 5 and 12 hr after injury. Immunoreactivity to IL-1 was detected in rapid-WD PNS segments 1 and
3 d after injury. The in vivo presence of IL-1 was
further verified in 1 d injured PNS segments that were frozen
immediately after removal from mice and then extracted. IL-1 content
in the PNS extract, determined by ELISA, was 37.69 pg in 5 mg wet
weight tissue. We further studied by ELISA the secretion of IL-1 in medium conditioned for 5 hr by PNS segments removed from non-operated mice and at various time points after PNS injury (Fig. 3B).
Soluble IL-1 was first detected in medium conditioned 5-10 hr after
injury and thereafter, peaking at 1 d after injury.
PNS segments that were incubated in medium for 5 hr were further
incubated for an additional 19 hr in fresh medium (5 24 hr time
period), and conditioned medium was assayed for IL-1 content by
ELISA. Secretion of IL-1 was calculated, picograms per milligram wet
weight of tissue in 5 hr, to enable comparison with secretion levels
during the first 5 hr of incubation (Fig. 3B). Secretion of
IL-1 during the 5 24 hr time period by PNS segments removed 1 and 3 d after injury averaged, respectively, 0.08 ± 0.007 and 0.28 ± 0.02 pg/mg per 5 hr compared with 0.54 ± 0.11 and 1.72 ± 0.29 pg/mg per 5 hr during the first 5 hr (Fig. 3).
IL-1 was not detected in medium conditioned by PNS segments removed
from non-operated animals either during the first 5 hr of incubation
(Fig. 3) or during the 5 24 hr time period. In vitro
conditions thus impose progressive reduction in secreted IL-1, as
was the case for TNF (see above). In vitro imposed progressive reduction, not augmentation, suggests that IL-1
secretion during the first 5 hr after removal of injured PNS from
animals is most likely a continuation and reflection of cytokine
secretion in vivo. Combined, immunocytochemistry and ELISA
of conditioned medium suggest no constitutive production of IL-1 and
relatively late onset, between 5 and 10 hr after injury, of IL-1
synthesis and secretion that continued thereafter. Injury-induced
synthesis and secretion of IL-1 protein is further supported by the
detection of injury-induced IL-1 mRNA expression (Fig. 1) and
IL-1 protein in injured PNS extract.
Note that low levels of secreted soluble IL-1 were detected by ELISA
in medium conditioned during the period of 5-10 hr after injury,
whereas IL-1 immunoreactivity was not detected by
immunocytochemistry in PNS tissue 12 hr after injury. This discrepancy
may be explained by the fact that positive immunoreactivity depends on
the accumulation of IL-1 in the cytoplasm to levels detectable by
immunocytochemistry. If onset of synthesis is accompanied by rapid
secretion, then newly synthesized IL-1 could first reach detectable
levels by ELISA in its secreted form in conditioned medium. In
contrast, newly synthesized TNF is first incorporated into the
plasma membrane and then released by proteolytic cleavage, and a
proportion of newly synthesized IL-1 precursor, which is
biologically active, becomes cell membrane associated. It is likely,
therefore, that cytoplasmic and membrane-associated TNF and IL-1
reach detectable levels by immunocytochemistry as fast as the secreted
forms reach detectable levels by ELISA and bioassay in conditioned medium.
TNF, IL-1, and IL-1 production by Schwann cells,
fibroblasts, and macrophages
Schwann cells and fibroblasts compose most of the non-neuronal
cell population in intact PNS, whereas macrophages, which are scarce in
intact PNS, are recruited in large numbers as of the third day after
injury (Perry et al., 1987; Reichert et al., 1994). We established
mixed and single-cell type cultures from intact PNS, 3 and 5 d
rapid-WD PNS, and 5 d freeze-damaged PNS. Freezing extinguishes
all resident non-neuronal cells. Numerous macrophages are recruited
into the Schwann cell-free freeze-damaged PNS, which then serve as a
source for nerve-derived macrophages. Macrophage cultures were also
established from thioglycollate-elicited peritoneal macrophages.
The identification of the non-neuronal cells was based on morphology
and immunological markers [reviewed and shown in the same model system
in Reichert et al. (1994) and Reichert and Rotshenker (1996, 1999)]:
F4/80 is a murine-specific monocyte, macrophage, and microglia marker;
Galectin-3/MAC-2 marks in the nervous system-activated myelin
phagocytosing macrophages, Schwann cells, and microglia; complement
receptor-3 (CR3/MAC-1) is expressed by monocytes, macrophages, and
microglia; and S-100 is specific to Schwann cells in peripheral nerves.
Macrophages were F4/80, CR3/MAC-1, and Galectin-3/MAC-2 positive.
Schwann cells were Galectin-3/MAC-2 and S-100 positive but F4/80
CR3/MAC-1 negative. Schwann cells featured heterogeneous morphology.
Some had the "classical" spindle shape, bipolar morphology. Many
assumed other shapes that could lead to false identification as
macrophages if not for the fact that they were F4/80 and CR3/MAC-1 negative. Fibroblasts were F4/80, CR3/MAC-1, Galectin-3/MAC-2, and
S-100 negative.
The cultured non-neuronal cells were studied for TNF,
IL-1, and IL-1 protein production
by immunocytochemistry (Fig. 4) and mRNA expression by RT-PCR (Fig.
5). Positive immunoreactivity to TNF,
IL-1, and IL-1 was detected in Schwann cells that were derived
from intact and rapid-WD PNS and in macrophages that were derived from
rapid-WD and freeze-damaged PNS. Low levels of immunoreactivity to
TNF, but not to IL-1 or IL-1, were detected in fibroblasts derived from intact and rapid-WD PNS. Positive immunoreactivity to all
cytokines (Fig. 4) was detected after cells were permeabilized by
Triton X-100, thereby enabling access of antibodies to the cytoplasm.
Immunoreactivity was not detected if Triton X-100 was omitted (data not
shown). Immunocytochemistry thus localized cytokines to the cytoplasm
and not to the cell surface of cells, as expected from the synthesis of
cytokines by these cells. Schwann cells further expressed TNF,
IL-1, and IL-1 mRNAs (Fig. 5). Thioglycollate-elicited peritoneal
macrophages, which express TNF, IL-1, and IL-1, were used as
positive controls. They displayed positive immunoreactivity to TNF,
IL-1, and IL-1, which were localized to the cytoplasm, and
further expressed TNF, IL-1, and IL-1 mRNA.
View larger version (107K):
[in this window]
[in a new window]
|
Figure 4.
Schwann cells and recruited macrophages produce
TNF, IL-1, and IL-1 protein. Nerve-derived fibroblasts produce
low levels of TNF but not IL-1 or IL-1 protein. Single-cell
type cultures of resident Schwann cells and fibroblasts and recruited
macrophages were obtained from intact, rapid-WD, and freeze-damaged PNS
of C57/BL mice. The non-neuronal cells were studied for the presence of
TNF, IL-1, and IL-1 protein by immunofluorescence microscopy.
High levels of immunoreactivity for all cytokines were detected in the
cytoplasm of Schwann cells and macrophages. In fibroblasts, levels of
immunoreactivity to TNF were slightly above control levels, whereas
levels of immunoreactivity to IL-1 and IL-1 were the same as for
control. Immunoreactivity of cytokines was localized to the
cytoplasm and not cell surfaces because immunoreactivity was detected
in cells permeabilized by Triton X-100 but not in non-permeabilized
cells (data not shown). Magnification: 400×.
|
|
View larger version (37K):
[in this window]
[in a new window]
|
Figure 5.
TNF and IL-1 and IL-1 mRNA are detected
in Schwann cells and macrophages. Schwann cell cultures were obtained
from rapid-WD PNS of C57/BL mice, as in Figure 4, and macrophages were
thioglycollate elicited. Schwann cells and macrophages were used as
source for the detection of TNF, IL-1, IL-1, and -actin
mRNAs by RT-PCR. RT-PCR amplification products were separated on
ethidium bromide-stained 1.5% agarose gel, visualized by ultraviolet
light, and photographed. The photographs were scanned, and
densitometric analysis was performed. Levels of mRNA of each cytokine
were further calculated as percentage of levels of -actin mRNA in
the same cell sample.
|
|
TNF, IL-1, and IL-1 mRNA expression and protein production
in intact and injured PNS of Wld mice undergoing slow-WD
In mutant Wld mice, the PNS segment situated distal to an injury
site can be divided into two domains: the injury domain, which is <1
mm in length and situated immediately after the injury site, and the
remainder more distally located domain that makes most of the distal
PNS segment. The injury domain undergoes rapid-WD, whereas the
remainder more distally located domain undergoes slow-WD (Reichert et
al., 1994).
The expression of TNF, IL-1, and IL-1 mRNAs was studied by
RT-PCR in intact and slow-WD Wld mice PNS 3 d after injury. TNF
and IL-1 mRNAs were detected in intact PNS, whereas IL-1 mRNA was
not. The mRNAs of all three cytokines were detected in the slow-WD
domains 3 d after injury (Fig.
6).
View larger version (44K):
[in this window]
[in a new window]
|
Figure 6.
TNF and IL-1 mRNAs but not IL-1 mRNA are
detected in intact PNS of Wld mice (N). TNF,
IL-1, and IL-1 mRNAs are detected in slow-WD domains of Wld mice
3 d after injury (D-3). Intact PNS segments and
slow-WD PNS domains, 3 d after injury, were removed from Wld mice.
The same tissues were the source for the detection of TNF, IL-1,
IL-1, and -actin mRNAs by RT-PCR. RT-PCR amplification products
were separated on ethidium bromide-stained 1.5% agarose gel,
visualized by ultraviolet light, and photographed. The photographs were
scanned, and densitometric analysis was performed. Levels of mRNA of
each cytokine at each time point were further calculated as percentage
of levels of -actin mRNA in the same tissue sample.
|
|
The production of TNF, IL-1, and IL-1 protein was studied by
immunocytochemistry in intact and the two domains of injured Wld mice
PNS, the injury domains, and the slow-WD domains 3 d after injury.
Immunoreactivity to TNF, IL-1, and IL-1 was not detected in
intact PNS (data not shown). Immunoreactivity to the three cytokines
was detected 3 d after injury in the injury domains but not in the
slow-WD domains (Fig. 7). It should be
noted that each injury domain and mate slow-WD domain, taken from the
same injured PNS, were processed simultaneously side by side for
immunocytochemistry. The discrepancy between injury domains and slow-WD
domains of the same PNS nerve segments is thus significant, suggesting
extremely low levels or no production of TNF, IL-1, and IL-1
proteins in slow-WD domains.
View larger version (48K):
[in this window]
[in a new window]
|
Figure 7.
TNF, IL-1, and IL-1 protein are detected
in injury domains but not slow-WD domains of Wld mice 3 d after
injury. The PNS segment, which is situated distal to the injury site,
was removed 3 d after injury, and cryostat sections were taken
from the injury domain and its mate slow-WD domain. These were then
processed simultaneously side by side for the detection of TNF,
IL-1, and IL-1 protein by immunofluorescence microscopy.
Magnification: 250×.
|
|
TNF and IL-1 augment myelin phagocytosis by macrophages
One of the hallmarks of Wallerian degeneration is the removal, by
phagocytosis, of myelin that otherwise may inhibit regeneration. We
have previously documented the rapid and efficient removal of myelin
during rapid-WD by resident Schwann cells and recruited macrophages
(Reichert et al., 1994; Be'eri et al., 1998). Complement receptor-3
(CR3/MAC-1) plays a major role in mediating myelin phagocytosis by
macrophages (Bruck and Friede, 1990; Mosley and Cuzner, 1996; van der
Laan et al., 1996; Reichert et al., 2001; Slobodov et al., 2001). There
are conflicting reports regarding the effect of TNF on myelin
phagocytosis by macrophages. In one (Bruck et al., 1992), TNF
inhibited myelin phagocytosis by downregulating cell surface expression
of CR3/MAC-1. In others (Smith et al., 1998; Liefner et al., 2000),
TNF had no effect on myelin phagocytosis by macrophages but
augmented myelin phagocytosis by microglia.
We studied the effect of TNF and IL-1 on myelin phagocytosis and
cell surface expression of CR3/MAC-1 in macrophages.
Thioglycollate-elicited peritoneal macrophages were incubated in medium
in the absence or presence of either recombinant mouse TNF (1 ng/ml)
or IL-1 (0.6 ng/ml) for 36 hr. Cultures were then studied for myelin
phagocytosis, cell surface levels of CR3/MAC-1, and cell number (Fig.
8). In each experiment, an individual
macrophage population that was obtained from a single mouse was tested
for myelin phagocytosis in quadruplicate. Myelin phagocytosis was
quantified by determining levels of MBP in lysate of macrophages by
ELISA and further calculated as percentage of phagocytosis by
macrophages that were not treated by the cytokines. Myelin phagocytosis
was augmented significantly by TNF (120.85 ± 5.38% of normal;
p < 0.01) and by IL-1 (129.17 ± 6.18% of
normal; p < 0.001). Duplicate cultures were used to study cell surface levels of CR3/MAC-1 by ELISA. The same cultures were
further used to quantify cell number by counting cells in a sample area
in the center of wells. Cell surface levels of CR3/MAC-1 and cell
number were further calculated as percentage of their values in
experiments in which macrophages were not treated by the cytokines.
Cell surface levels of CR3/MAC-1 and cell number did not differ from
normal. Thus the augmentation of myelin phagocytosis was not the result
of either TNF- or IL-1-induced upregulation of cell surface
levels of CR3/MAC-1 or macrophage proliferation.
View larger version (46K):
[in this window]
[in a new window]
|
Figure 8.
TNF and IL-1 augment myelin phagocytosis by
macrophages. Thioglycollate-elicited peritoneal macrophages were
incubated in the absence or presence of TNF (1 ng/ml) or IL-1
(0.6 ng/ml) for 36 hr. In each experiment, a macrophage population from
a different mouse was studied. Thereafter, myelin phagocytosis, cell
surface levels of CR3/MAC-1, and cell number (#) were quantified and
further calculated as percentage of normal. Values obtained in the
absence of cytokines were defined 100% normal. Bars are the average of
six experiments, each performed in quadruplicate. Error bars indicate
±1 SEM.
|
|
DICUSSION
We presently document the involvement of TNF, IL-1, and
IL-1 during normal WD. TNF and IL-1 are likely to be the first cytokines the protein production of which is upregulated in Schwann cells after PNS injury. The production of IL-1 protein is induced in
Schwann cells with a delay after injury. TNF and IL-1 therefore may play a role in the initiation of the cytokine network of WD. TNF, IL-1, and IL-1 can further regulate additional molecular and cellular events in WD. In agreement with and support of this notion
is the deficient production of the three cytokines during slow-WD.
Transcription, translation, and secretion of cytokines can be
differentially regulated such that mRNA expression does not necessarily
indicate protein synthesis and synthesis does not necessarily indicate
secretion (Dinarello, 2001b). Therefore, we examined TNF, IL-1,
and IL-1 mRNA expression (RT-PCR), protein synthesis
(immunocytochemistry and ELISA), and protein synthesis and secretion
(ELISA and bioassay of conditioned medium) and further identified the
cellular sources of their production. The functional involvement of any of the cytokines is indicated by combining positive independent observations on mRNA expression, protein synthesis, and protein secretion and not by just one observation. Each
observation complements and verifies the others.
TNF mRNA and protein are constitutively expressed and synthesized at
low levels in Schwann cells that reside in intact PNS of C57/BL mice.
Injury induces the rapid upregulation of TNF mRNA expression and
protein synthesis and secretion during rapid-WD. This notion stems from
integrating observations on the detection of TNF mRNA and protein in
intact PNS and during rapid-WD, the detection of secreted TNF within
the first 5 hr after PNS injury and thereafter, and the detection of
TNF mRNA and protein in Schwann cells. These findings, along with
the fact that Schwann cells are in intimate contact with axons that
they ensheathe, suggest that Schwann cells may be the first among the
non-neuronal cells to "sense" and respond to axonal injury by
secreting already existing constitutively synthesized TNF protein.
Then, Schwann cells further synthesize and secrete TNF protein using
constitutively and newly injury-induced expressed mRNA. Our
observations and conclusions agree with and further extend previous
findings: injury-induced mRNA expression of TNF in PNS of mice (La
Fleur et al., 1996), constitutive and injury-induced upregulation of
TNF mRNA in rat PNS (Taskinen et al., 2000), and constitutive and
injury-induced upregulation of TNF protein content in rat PNS and
Schwann cells (Wagner and Myers, 1996b; George et al., 1999); however,
see injury-induced but not constitutive TNF mRNA expression in rat
PNS (Wagner and Myers, 1996b) and injury-induced but not constitutive
TNF protein content in rat PNS (Stoll et al., 1993).
IL-1 mRNA is constitutively expressed but IL-1 protein is not
synthesized in intact PNS of C57/BL mice. Injury induces the rapid
upregulation of IL-1 mRNA expression and IL-1 protein synthesis
and secretion in Schwann cells during rapid-WD. This conclusion is
based on integrating the detection of IL-1 mRNA but not protein in
intact PNS, the detection of IL-1 mRNA during rapid-WD, the
detection of synthesized and secreted IL-1 within the first 5 hr
after PNS injury and thereafter (Rotshenker et al., 1992), and the
detection of IL-1 mRNA and protein in Schwann cells. The
observations in intact PNS that TNF and IL-1 mRNAs are expressed
and TNF but not IL-1 protein is synthesized suggest that
injury-induced IL-1 protein production may lag after TNF protein
production. Our observations agree with and further extend previous
findings on constitutive expression of IL-1 mRNA in intact rat PNS
(Perry et al., 1987) and injury-induced IL-1 mRNA expression in
injured mouse PNS (La Fleur et al., 1996).
IL-1 mRNA is not expressed and IL-1 protein is not synthesized in
intact PNS of C57/BL mice. Injury induces the rapid expression of
IL-1 mRNA but the delayed synthesis and secretion of IL-1 protein
in Schwann cells. This notion arises from integrating the inability to
detect IL-1 mRNA and protein in intact PNS, the detection of IL-1
mRNA 5 hr and 1 and 3 d after PNS injury, and the detection of
synthesized and secreted IL-1 protein between 5 and 10 hr but not
within the first 5 hr of rapid-WD. Constitutive and injury-induced
upregulation of IL-1 mRNA expression was reported in rat PNS (Gillen
et al., 1998).
Macrophages, which are recruited to the injured nerves as of the third
day of WD, may further contribute to TNF, IL-1, and IL-1 production.
In contrast to rapid-WD, mRNA expression but not protein production of
TNF, IL-1, and IL-1 is upregulated during slow-WD in Wld mice.
This contrast between efficient versus deficient protein production of
TNF, IL-1, and IL-1 during rapid-WD and slow-WD, respectively,
follows a similar pattern of efficient versus deficient production of
GM-CSF, IL-6, and IL-10 that we reported previously in these PNS
tissues (Saada et al., 1996; Reichert et al., 1996; Be'eri et al.,
1998). This overall efficient versus deficient injury-induced cytokine
production points to the inflammatory nature of WD and further suggests
that cytokines play significant roles in regulating molecular and
cellular events in WD. This notion is supported by a number of
observations. GM-CSF, IL-6, and IL-10 are produced efficiently in
rapid-WD but not slow-WD, which is in accord with TNF, IL-1, and
IL-1 inducting GM-CSF, IL-6, and IL-10 production (Aggawal et al.,
2001; Dinarello, 2001a,b). Schwann cells are activated during rapid-WD
but not slow-WD (Reichert et al., 1994), which is in agreement with the
efficient production of GM-CSF that activates Schwann cells during
rapid-WD but not slow-WD (Saada et al., 1996). Macrophages are
recruited and activated during rapid-WD but not slow-WD (Perry et al.,
1987; Brown et al., 1991; Reichert et al., 1994), which is in accord
with the role of TNF, IL-1, and IL-1 in the recruitment of
macrophages to sites of inflammation (Liefner et al., 2000;
Oppenhheim and Feldman, 2001; Ousman and David, 2001; Subang and
Richardson, 2001). Deficient macrophage recruitment and deficient
Schwann cell activation result, in turn, in delayed myelin removal by phagocytosis during slow-WD (Brown et al., 1991; Reichert et al., 1994;
Be'eri et al., 1998). NGF production is upregulated during rapid-WD
but not slow-WD (Brown et al., 1991), which is in accord with TNF,
IL-1, and IL-1 inducing NGF production in fibroblasts (Lindholm
et al., 1987; Hattori et al., 1993; Hattori et al., 1994). In turn,
deficient NGF production combined with delayed myelin removal
contributes to delayed regeneration in Wld mice in which slow-WD occurs
(Brown et al., 1991; Chen and Bisby, 1993; Schafer et al., 1996).
Furthermore, deficient production of NGF and IL-6 during slow-WD
contributes to the reduction of neuropathic pain after PNS injury in
Wld mice (Safieh-Garabedian et al., 1995; Myers et al., 1996; Wagner
and Myers, 1996a; Woolf et al., 1997; Mendell et al., 1999; Murphy et
al., 1999).
TNF, IL-1, and IL-1 mRNA expression and protein production may
be differentially regulated during WD. IL-1 mRNA was detected in
intact PNS of C57/BL mice, whereas IL-1 protein was not. IL-1 mRNA was detected within the first 5 hr of rapid-WD, whereas synthesis and secretion of IL-1 protein were not. TNF, IL-1, and IL-1 mRNAs were detected during slow-WD in injured PNS of Wld mice, whereas
TNF, IL-1, and IL-1 protein were not. The inability to detect
cytokine protein by immunocytochemistry and ELISA can reflect either no
production or extremely low levels of production that are likely to be
functionally insignificant. This differential regulation of mRNA
expression and protein production suggests that mRNA expression, by
itself, is no indication of the functional involvement of TNF,
IL-1, or IL-1 in WD. Similar observations have been made
previously in other cells and tissues (for review, see Dinarello,
2001b).
We have previously studied mRNA expression, the time course of protein
synthesis and secretion, and the identity of the non-neuronal cells
producing IL-6, GM-CSF, and IL-10 during rapid-WD and slow-WD (Reichert
et al., 1996; Saada et al., 1996; Be'eri et al., 1998) as we have
presently done for TNF, IL-1, and IL-1. We further studied
macrophage recruitment and activation, Schwann cell activation, and
myelin removal by phagocytosis. These studies were all performed using
the same animal and injury models, which provide the required common
grounds for comparison and integration of data. These observations suggest a network of cytokine production that is orchestrated in time
and magnitude: the cytokine network of Wallerian degeneration (Fig.
9). Timing and magnitude of cytokine
production are determined to a large extent by the non-neuronal cell
types producing them, the spatial distribution of non-neuronal cells in
the PNS tissue, and the timing of macrophage recruitment. In intact
PNS, Schwann cells form close contact with axons that they ensheathe,
whereas fibroblasts are scattered between nerve fibers. Macrophages,
which are scarce in intact PNS, are recruited as of the third day after injury but reach large numbers within the next few days. TNF and
IL-1 are most likely the first inflammatory cytokines to be produced
in rapid-WD by Schwann cells, which are the first to sense and respond
to axotomy because of their intimate contact with axons (see above).
Then follows the production of IL-6 by fibroblasts, within 2 hr after
injury, and GM-CSF, within 4 hr after injury (Reichert et al., 1996;
Saada et al., 1996). Fibroblasts do not express or produce IL-6 and
GM-CSF mRNA or protein in intact PNS. IL-6 and GM-CSF production
can be induced by diffusible TNF and IL-1 produced by Schwann
cells (Saada et al., 1996). The onset of IL-1 production by Schwann
cells is 5-10 hr after injury. IL-1 can further contribute to IL-6
and GM-CSF production. IL-6, which inhibits TNF production (Aggawal
et al., 2001), may be responsible for the reduction in TNF
production after the first day of rapid-WD. TNF, IL-1, and
IL-1 produced by Schwann cells can further induce recruited
macrophages to produce mostly IL-6 (Reichert et al., 1996) but also
TNF, IL-1, and IL-1. TNF, IL-1, and IL-1 induce IL-10
production (de Waal Malefyt, 2001). Indeed, the onset of IL-10
production by fibroblasts is rapid (Be'eri et al., 1998), but levels
of production are low and insignificant because fibroblasts are poor
producers of IL-10. The timing of high levels of IL-10 production,
after the fourth day of rapid-WD, is determined by the timing of
macrophage recruitment, which produces large amounts of IL-10. IL-10
then downregulates the production of the inflammatory cytokines and
itself (Be'eri et al., 1998; de Waal Malefyt, 2001), thereby
downregulating WD.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 9.
The cytokine network of Wallerian degeneration.
The cellular elements depicted are a resident Schwann cell ensheathing
an axon, a resident fibroblast, and a recruited macrophage.
Solid lines represent induction and upregulation, and
dotted lines represent downregulation of the production
of cytokine protein. Axotomy induces the production of TNF |
|
TOP
ABSTRACT
INTRODUCTION
