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Regular Articles |
From the Departments of Neuropharmacology *
and
Molecular Biology,
The Scripps Research
Institute, and the Department of Pathology,
University of California at San Diego, La Jolla, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the
pathogenesis of degenerative disorders of the central nervous system
(CNS), transgenic mice were developed in which expression of
murine TNF- was targeted to astrocytes using a glial fibrillary
acidic protein (GFAP)-TNF- fusion gene. In two independent
GFAP-TNF transgenic lines (termed GT-8 or GT-2) adult (>4 months of
age) animals developed a progressive ataxia (GT-8) or total paralysis
affecting the lower body (GT-2). Symptomatic mice had prominent
meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large
numbers of B cells and CD4+ and CD8+ T cells
accumulated at predominantly perivascular sites. The majority of these
lymphocytes displayed a memory cell phenotype
(CD44high, CD62Llow,
CD25-) and expressed an early activation marker (CD69).
Parenchymal lesions contained mostly CD45+ high,
MHC class II+, and Mac-1+ cells of the
macrophage microglial lineage with lower numbers of neutrophils and few
CD4+ and CD8+ T cells. Cerebral expression of
the cellular adhesion molecules ICAM-1, VCAM-1, and
MAdCAM as well as a number of - and ß-chemokines was induced or
up-regulated and preceded the development of inflammation,
suggesting an important signaling role for these molecules in the CNS
leukocyte migration. Degenerative changes in the CNS of the GFAP-TNF
mice paralleled the development of the inflammatory lesions and
included primary and secondary demyelination and neurodegeneration.
Disease exacerbation with more extensive inflammatory lesions that
contained activated cells of the macrophage/microglial lineage occurred
in GFAP-TNF mice with severe combined immune deficiency.
Thus, persistent astrocyte expression of murine TNF- in the
CNS induces a late-onset chronic inflammatory encephalopathy in which
macrophage/microglial cells but not lymphocytes play a central role in
mediating injury.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a
multifunctional pro-inflammatory cytokine pivotal in the regulation of
the host response during infection and inflammation and is also
implicated in the pathogenesis of many autoimmune
diseases.1-3
A convincing body of evidence implicates
TNF- in the pathogenesis of inflammation in the central nervous
system (CNS), including multiple sclerosis (MS),4,5
stroke,6
and infectious diseases ranging from cerebral
malaria7
and bacterial meningitis8
to HIV
encephalopathy.9
The sources for TNF- production in
these pathological states may be quite varied, and in addition to
infiltrating leukocytes such as macrophages and T cells, significant
local production of this cytokine may also come from
astrocytes,10,11
microglia,12,13
and possibly
neurons.14
Although it is clear that TNF- is present in the CNS during various
insults and that resident neural cells can express TNF- providing a
local source of production for this cytokine, divergent views (see, for
example, 15 and 16
) have evolved as to the role of TNF- in
these pathological states with experimental evidence supporting both
detrimental and protective functions. Various manipulations that reduce
TNF- levels, including administration of anti-TNF- neutralizing
antibodies or a soluble TNF type I receptor protein or treatment with
the TNF-suppressing drug Rolipram, ameliorate or even prevent
experimental autoimmune encephalomyelitis (EAE)17-20
or
cerebral malaria21,22
in mice. Intracisternal injection of
TNF- promotes a vigorous inflammatory response with infiltration of
the meninges and ventricles with large numbers of
leukocytes.23
Finally, numerous in vitro studies
also support the notion that TNF- plays a central role in the
evolution of neuroinflammation and as well may contribute directly to
degenerative CNS disease. In particular, TNF- is a potent inducer of
both cellular adhesion molecule expression by cerebrovascular
endothelial cells24
and astrocytes25
and
chemokine expression by microglia26,27
and
astrocytes27,28
and promotes demyelination and
oligodendrocyte injury.29,30
Against this body of evidence for pro-inflammatory and harmful effects
of TNF-, recent studies in gene knockout mice deficient for the TNF
genes or their corresponding TNF receptors have provided evidence for
an alternative, possible anti-inflammatory or protective function of
this cytokine in CNS disease. Mice deficient for TNF-31
or TNF- and TNF-ß32
were not only found to be
susceptible to the development of EAE but also invariably exhibited a
more severe and protracted form of the inflammatory demyelinating
disease. In a similar vein, the neuronal injury caused by cerebral
ischemia or excitotoxic amino acids was found to be exacerbated in mice
deficient for both the p55 and p75 TNF receptors.33
However, not all studies using knockout mice support a beneficial
action of TNF- in CNS disease; for example, mice deficient for both
TNF- and TNF-ß are resistant to cerebral malaria and show a marked
reduction in CNS inflammation.34
It should be noted, as has
recently been outlined,16
that experiments using the
current generation of gene knockout mice to understand the role of
cytokines such as TNF- in disease are confounded by a number of
issues that warrant caution in interpreting the results.
Transgenic mice with CNS-targeted expression of cytokines offer an
alternative approach to gene knockout animals for the study of cytokine
functions in the intact CNS.35
In the case of TNF-,
development of transgenic mice that express either murine TNF- from
its own promoter with expression apparently in neurons36
or
human TNF- from the GFAP promoter with expression in
astrocytes37
was reported by the same laboratory. In both
cases, transgenic animals appear to have had very high TNF-
expression in the brain and exhibited at an early age severe and
invariably lethal neurological deficits in association with extensive
CNS inflammation and degenerative pathology. In addition to the
possible adverse impact of TNF- on the normal development of the CNS
in these transgenic mice, the severe neurological disorder exhibited by
these animals necessitated intervening actions to suppress TNF-
levels to be able to breed transgenic mice for further study. In a
separate study, Taupin and colleagues reported that transgenic mice
with oligodendrocyte-targeted expression of TNF- driven by the
myelin basic protein promoter failed to develop spontaneous disease but
developed more severe disease when induced with EAE.38
Although in this latter report brain levels of transgene encoded
TNF- were shown to be similar to those found in mice with EAE, the
targeted expression of this cytokine to oligodendrocytes could be
problematic as these cells are not known to produce this cytokine. In
addition, oligodendrocytes may be susceptible to nonspecific injury
resulting from transgene expression per se.39
With the aim of addressing the issue of the role of TNF- in the
initiation and perpetuation of disease in the adult CNS, we set out
here to generate transgenic mice (termed GFAP-TNF) with expression
of this cytokine targeted to a cell (ie, the astrocyte) that is known
to produce TNF-10,11
at levels that did not compromise
the development and breeding viability of the animal. To this end we
describe two independent lines of transgenic mice that exhibit no
detectable spontaneous disease until greater than 4 months of age,
after which time a progressive and eventually fatal neurological
disorder ensues. On further characterization, florid
meningoencephalomyelitis and degenerative disease was found in the CNS
of symptomatic GFAP-TNF mice. Interestingly, disease exacerbation
with more extensive lesions that contained activated cells of the
macrophage/microglial lineage occurred in GFAP-TNF mice with severe
combined immune deficiency. Thus, persistent astrocyte expression of
murine TNF- in the CNS is sufficient to induce a chronic
inflammatory encephalopathy in which macrophage/microglial cells but
not lymphocytes play a central role in mediating injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J x SJL F2 mice used for the development of the
transgenic lines and Balb/cByJscid/scid (SCID) mice were
obtained from Jackson Laboratories (Bar Harbor, ME). For the breeding
of GFAP-TNFscid/scid mice, transgenic mice of the GT-2
line (see below) were mated with Balb/cByJscid/scid mice.
GFAP-TNF x Balb/cByJscid/+ F1 mice were backcrossed
with Balb/cByJscid/scid mice, and SCID offspring were
identified by analysis of plasma IgG levels using an immunodiffusion
assay kit (ICN, Costa Mesa, CA). All mice were maintained in
specific-pathogen-free conditions in the closed breeding colony of the
Scripps Research Institute.
Generation of Transgenic Mice
The CNS- and astrocyte-specific expression obtained for fusion
gene constructs under the control of the murine GFAP promoter is well
documented.35,40
To obtain astrocyte expression of murine
TNF-, a modified strategy was used to that previously used for the
generation of the GFAP-IL641
and GFAP-IL342
transgenic mice. Briefly, a 2.2-kb Sfi-1/Not-1
fragment containing the murine GFAP promoter and an SV-40 small intron
was excised from the GFAP-IL3 plasmid construct42
and
cloned into the vector pGEMEX-1 (Promega, Madison, WI) to generate the
construct pGF. Next, a 0.65-kb Sma-1/EcoRV
fragment containing the human growth hormone (hGH) polyadenylation
signal sequence was cut from the plasmid pIC-hGH (kindly provided by
Dr. Jan Allison, Walter and Eliza Hall Institute of Medical Research,
Melbourne, Australia) and ligated into the EcoRV site of pGF
to generate the construct pGF.GH. Finally, a 1.8-kb
EcoN1/EcoRI genomic DNA (a genomic clone
containing the entire murine TNF- and TNF-ß gene locus was kindly
provided by Dr. Beutler, Southwestern Medical Center, University of
Texas) fragment spanning the coding region of murine TNF- gene was
modified by adding Not-1 linkers and was then ligated into
the Not-1 site in pGF.GH. After screening for correct
orientation, the GFAP-TNF fusion gene fragment was excised with
Sfi-1/EcoRI and purified from plasmid DNA before
microinjection into fertilized eggs of C57BL/6J x SJL F2 hybrid
mice. Transgenic offspring were identified by slot blot analysis of
tail DNA using a 32P-labeled random primer hGH DNA fragment
as a probe.
RNA Preparation
Transgenic or control mice were killed, and the organs were then removed and immediately snap frozen in liquid nitrogen. Samples were stored at -70°C pending RNA preparation. Total RNA was extracted with TRIZOL reagent (Gibco-BRL, Grand Island, NY) used according to the manufacturer's instructions. The concentration of RNA was determined by ultraviolet spectroscopy at 260 nm.
RNase Protection Assay (RPA)
The production and characterization of the multiprobe RPA probe
sets used for the detection of cytokine or chemokine gene expression
have been described previously.43,44
To distinguish between
transgene-encoded TNF- (tgTNF) mRNA and the endogenously transcribed
TNF- (eTNF) mRNA, RPA probes were constructed that targeted the hGH
sequence (to detect tgTNF) (GenBank accession number M13438;
nucleotides (nt) 2453 to 2653) or the TNF- 3' untranslated region
(UTR; to detect eTNF) (GenBank accession number M11731; nt 1110 to
1357). In addition, to detect total TNF- mRNA, a previously
described43
RPA probe was used. In all cases, a fragment of
the RPL32-4A gene45
served as an internal loading control.
RPAs were performed as described previously.46
Routine Histology, in Situ Hybridization, and Immunohistochemistry
For routine histology, organs were removed, fixed in 4% buffered paraformaldehyde, and embedded in paraffin for hematoxylin and eosin or luxol fast blue staining, immunohistochemistry, and in situ hybridization. In addition, some fixed brain and spinal cord specimens were transferred to 20% sucrose in PBS and stored overnight at 4°C. After cryoprotection, the tissue was then snap frozen in liquid nitrogen and 10-µm cryostat sections prepared for oil red O staining to visualize neutral fat. Briefly, stock oil red O (Sigma Chemical Co., St. Louis, MO) was prepared at 3 mg/ml in 99% isopropanol. Immediately before use, stock was diluted 1:4 in distilled H2O to obtain a working solution. Sections were stained for 15 minutes in working solution, counterstained for 1 minute in Ehrlichs hematoxylin, rinsed in tap water, coverslipped using an aqueous mounting medium (Aquamount, Lerner Laboratories, Pittsburgh, PA), and immediately inspected by light microscopy.
For in situ hybridization, the TNF- probe used was
35S-labeled, single-stranded antisense or sense RNA to
murine TNF-. For probe synthesis, a cDNA for murine TNF- (kindly
provided by Genentech, South San Francisco, CA) was subcloned into
pBluescript-KS (Stratagene, La Jolla, CA) and antisense or sense RNAs
synthesized using T7 or T3 RNA polymerase, respectively. In
situ hybridization was performed as described by Simmons et
al47
with modifications. Briefly, paraffin sections were
deparaffinized and rehydrated in graded alcohols. After post-fixation
in 4% formaldehyde, proteinase K treatment (2.4 mg/100 ml of 5X TE
buffer at 37°C for 15 minutes) and acetylation (250 µl of acetic
anhydride in 100 ml of PBS for 10 minutes), the slides were dehydrated
in graded alcohol and dried. The 35S-labeled sense or
antisense probes were hybridized to the tissue overnight at 56°C.
After digestion with RNAse A (Promega), slides were washed in
decreasing concentrations of SSC buffer. After the last SSC step,
slides were blocked with serum (Vector Laboratories, Burlingame, CA)
and immunohistochemistry was performed where applicable. The primary
antibody against GFAP (Dako, Carpinteria, CA) was incubated overnight
at 4°C. Antibody-labeled cells were detected using a Vectastain kit
(Vector) according to the manufacturer's instructions. After
dehydration in graded alcohol, slides were air dried and exposed for 5
days to Cronex film (DuPont, Wilmington, DE). Slides were then dipped
in Kodak NTB-2 emulsion, dried, and stored in the dark for 2 weeks.
Subsequently, slides were developed, counterstained with Mayer's
hematoxylin, and examined by bright field microscopy.
Immunohistochemistry for the detection of TNF- protein was performed
on paraformaldehyde-fixed, paraffin-embedded sections. After dewaxing
and rehydration, slides were incubated in 1.5% normal goat serum
(Vector) in PBS containing 0.1% Saponin (Sigma) for 30 minutes.
After blocking, slides were incubated overnight at room temperature
with a polyclonal rabbit anti-murine TNF- antibody (Genzyme,
Cambridge, MA) diluted 1:1000 in the blocking buffer. All following
steps were performed using the Vectastain kit (Vector) according to the
manufacturer's instructions.
For immunophenotyping and cellular adhesion molecule immunostaining, mice were killed and organs were removed and immediately snap frozen in isopentane and stored at -70°C until sectioning. Sagittal cryomicrotome cut sections of 10 µm were air dried and either stored dehydrated at -70°C or directly processed. Immediately before staining, tissue sections were fixed in cold (-20°C) acetone/methanol (1:1) for 45 seconds and nonspecific binding was blocked by incubating sections for 30 minutes in PBS containing 3% rabbit and 3% goat serum. Sections were then incubated for 2 hours at room temperature in rat monoclonal antibodies to identify leukocytes (CD45 from Pharmingen, San Diego, CA), lymphocytes (CD4, CD8, and B220 from Pharmingen), NK cells (DX5; Pharmingen), neutrophils (MCA771; Serotec, Raleigh, NC), activation markers (MHC class II, clone M5/114 and Mac-1, and clone TIB 126 from American Type Culture Collection, Rockville, MD), and vascular or cellular adhesion molecules (MAdCAM, VCAM-1, and Endoglin from Pharmingen and ICAM-1 clone YN11.1 kindly provided by Dr. F. Takei, Toronto, Canada). All antibodies were used at a final concentration of 5 µg/ml diluted in the blocking buffer. Bound antibody was detected using a biotinylated anti-rat antibody (Southern Biotechnology Associates, Birmingham, AL) followed by avidin-labeled horseradish peroxidase (Sigma). Staining used 3',3'-diaminobenzidine (Sigma) as substrate. Before mounting, sections were counterstained with Mayer's hematoxylin and dehydrated in graded ethanols.
Quantitative Neuropathological Assessment
Briefly, as previously described,41,48 animals were perfused with cold 4% paraformaldehyde in pH 7.4 PBS. The right hemibrain was serially sectioned at 40 µm of thickness with the Vibratome 2000 (Leica) for subsequent immunocytochemical/computer-aided image analysis. The left hemibrain was embedded in paraffin and serially sectioned for studies of DNA fragmentation. To determine neuronal integrity, blind-coded vibratome sections were immunolabeled with antibodies against the dendritic marker MAP-2 (mouse monoclonal antibody, diluted 1:1000; Boehringer Mannheim, Indianapolis, IN) and against the marker for interneurons, parvalbumin (mouse monoclonal, diluted 1:1000; Sigma). For MAP-2, after overnight incubation, sections were incubated with the fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse IgG (Vector). The sections were then transferred to SuperFrost slides (Fisher Scientific, Tustin, CA), mounted under glass coverslips with anti-fading media (Vector), and analyzed with the MRC1024 LSCM (Bio-Rad, Richmond, CA).49 The percent area of the neuropil covered by MAP-2-immunoreactive dendrites was performed with the IMAGE software, as previously described.48 Anti-parvalbumin-immunostained cells were counted in 10 consecutive fields (0.1 mm; two each) along the side of the gyrus using a 40x objective and a gridded 10x eyepiece lens.
Cells undergoing DNA fragmentation were identified by using a commercially available kit (Apoptag, Oncor, Gaithersburg, MD) or a modified version of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling method of Gavriely.50 The main modification was the absence of tissue pretreatment with H2O2 (thus preventing nonspecific DNA fragmentation) and a slight difference in the ratio of TdT to biotinylated dUTP to reduce background. For this purpose, paraffin sections were dewaxed and rehydrated, washed in Tris-buffered saline, and treated with proteinase K in 5X TE buffer (at room temperature; 10 µg/ml for 10 minutes), followed by a 1-hour incubation at 37°C in a solution containing TdT (0.3 enzyme units/µl) and biotin-16-dUTP (0.02 nM/µl; Boehringer) in 1X TdT buffer with cobalt chloride. After this incubation, sections were washed in PBS and incubated for 30 minutes at room temperature in avidin-horseradish peroxidase complex (ABC Elite, Vector) followed by diaminobenzidine (0.05%). Paraffin sections from the adult rat mammary gland processed in parallel served as a positive control. Negative control experiments consisted of processing sections either in the absence of TdT or dUTP. Labeled sections were analyzed with an image analysis apparatus to determine the numbers of TdT-positive cells per unit area.
Ultrastructural Assessment
Unless stated otherwise, morphological examination of the brain
and spinal cord was performed on symptomatic GFAP-TNF mice or
age-matched normal littermates. Before killing, animals were
anesthetized with intraperitoneal injection (2 ml/kg) of a solution
consisting of pentobarbital (12.5 mg/ml) and diazepam (12.5 mg/ml) in
0.9% NaCl. The anesthetized animals were perfused intracardially with
a solution of 5% phosphate-buffered glutaraldehyde. The brain and
spinal cord were then removed, and after overnight fixation at 4°C,
tissues were post-fixed in 1% aqueous osmium tetroxide solution for 2
hours, dehydrated using graded alcohols and polyphylene oxide,
and infiltrated with resin. Overnight infiltration was followed by
embedding in fresh araldite resin. Thick sections (1 µm) were cut
with glass knives and stained with paraphenylenediamine (PPD) or
methylene blue azure II in preparation for light microscopy. Ultra-thin
sections from selected blocks were cut with a diamond knife and stained
with uranyl acetate and lead citrate before electron microscopic
examination.
Leukocyte Isolation
Leukocytes were isolated as described51,52 with minor modifications. Briefly, mice were killed by halothane inhalation and perfused with 20 ml of PBS unless stated otherwise. Brains were rapidly removed and mechanically dissociated by sequentially forcing the tissue through 210- and 70-µm Nitex meshes. The cell suspension was enzymatically digested with DNase I (28 U/ml) and collagenase (0.2 mg/ml) for 1 hour at 37°C in a shaking incubator in Hanks' balanced salt solution without serum. After quenching the digestion with the addition of 10% fetal bovine serum (final concentration), the cell suspension was separated on a discontinuous 1.033/1.088/1.122 Percoll gradient. Leukocytes were collected from the interfaces and the 1.033 Percoll fraction. Myelin and cell debris separated above the gradient.
Flow Cytometry
Biotin-conjugated, FITC-conjugated, and phycoerythrin-conjugated antibodies against mouse CD4, CD8, CD25, CD44, CD45, B220, CD62 (Mel-14), and CD69 (Pharmingen), and MHC class II (M5/114, ATCC) were reacted with the cells isolated from the Percoll gradient or lymph node cell suspensions. The cells were then analyzed with a FACScan by using Cell Quest acquisition software (Becton Dickinson, Mountain View, CA).
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Transgenic Mice
From the initial founder generation, five mice were identified as
positive for transgene integration. By postnatal day 14, two founder
animals appeared runted, developed severe motor impairment, and died
between days 21 and 32. Subsequent analysis of the brain from one of
these mice revealed high expression of TNF- in association with
extensive and severe encephalomyelitis (not shown). Of the remaining
founders, all were bred successfully, and analysis of transgene
expression confirmed TNF- expression in the CNS of offspring derived
from two of the three founders. For further study, stable
transgene-expressing lines termed GT-2 and GT-8 were then established
from the two transgene-expressing founders. Mice from the GT-2 line
developed paraparesis from 4 months of age or older and progressed to
total hind limb paralysis and premature death around 2 months after the
onset of paraparesis. Mice from the GT-8 line had a different physical
presentation and showed symptoms of ataxia from 6 months of age or
older, the severity of which increased progressively, leading to death
of the animal by approximately 12 months of age. After the onset of
motor impairment, mice from both lines displayed muscle spasms and
became progressively cachectic.
To determine the expression characteristics of the transgene-encoded
product versus the endogenous TNF- gene, an RPA was
developed that permitted distinction between the TNF- transcript,
the TNF- 3' UTR encoded by the endogenous TNF gene, and the 3' hGH
region encoded by the transgene. A representative assay is shown in
Figure 1A
. There was little detectable
expression of the TNF- gene in the CNS tissue of wild-type controls.
However, TNF- mRNA was found to be expressed at elevated levels in
the forebrain, cerebellum, spinal cord, and eye from mice of both
transgenic lines. In mice of the GT-2 line, the highest level of
TNF- mRNA expression was found in the spinal cord (Figure 1A)
,
whereas in the GT-8 line, the expression of TNF- mRNA was highest in
the cerebellum (data not shown). Consistent with the production of
transgene-encoded TNF-, in mice from both transgenic lines, a
similar pattern of expression was observed in these CNS regions for the
hGH transcript with the highest expression of the hGH mRNA detectable
in the spinal cord of symptomatic GT-2 animals (Figure 1A)
and
cerebellum of GT-8 mice (not shown). Although not as high as the
expression for the hGH mRNA, elevated expression of the TNF- 3' UTR
transcript was clearly detectable in these same CNS regions, indicating
there was also activation of the endogenous TNF- gene. A survey of a
number of peripheral organs revealed that in wild-type mice TNF- and
3' UTR but not hGH transcript was constitutively expressed in spleen,
lung, heart, and thymus (Figure 1A)
. In the transgenic mice, increased
expression of the TNF- and 3' UTR transcript was detectable in the
muscle, spleen, kidney, heart, lung, and thymus. A somewhat lower level
of hGH mRNA was observed in the lung, heart, and thymus, particularly
in symptomatic mice. Thus, there was some leakage of the transgene GFAP
promoter expression in the periphery of symptomatic mice, however, and
in contrast to the CNS, the elevated expression of TNF- mRNA seen in
these peripheral organs was due mostly to increased activation of the
endogenous gene.
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in the brain was demonstrated using a combination
of immunohistochemistry for GFAP and in situ hybridization
for TNF- RNA. Cells strongly positive for GFAP as well as for
TNF- RNA were scattered predominantly throughout the spinal cord and
to a lesser extent the cerebellum and thalamus of GT-2 mice or the
cerebellum, hippocampus, and cerebral cortex of GT-8 animals (Figure 1B)
. TNF- mRNA expression was not detectable by this method in CNS
tissue of wild-type mice. By immunohistochemical staining, cells
immunopositive for TNF- with the morphological appearance of
astrocytes were observed with a similar distribution as astrocytes
expressing TNF- RNA (as detected by in situ hybridization
above) in the CNS of mice from both the GT-2 and GT-8 lines (Figure 1C)
. No cells immunopositive for TNF- were observed in the CNS
tissue of wild-type controls (not shown).
Pathological Alterations in the CNS of Symptomatic but Not
Asymptomatic GFAP-TNF Mice
In mice of both the GT-2 (1 to 3 months of age) and GT-8 (1 to 6
months of age) lines, before the onset of physical signs, light
microscopic examination of the brain and spinal cord failed to reveal
any overt pathological alterations compared with wild-type controls.
However, in symptomatic transgenic mice, a most striking finding was
the presence of large areas of dense mononuclear cell infiltration at
meningeal, perivascular, and parenchymal locations (Figure 2)
. These infiltrates varied in intensity
throughout the brain, being minimal in cortical regions to particularly
florid in the spinal cord (Figure 2E)
or the cerebellum (Figure 2B)
of
mice from the GT-2 or GT-8 lines, respectively. The density of
mononuclear cell infiltration therefore coincided with the levels of
TNF- expressed in the various brain regions. Infiltrating cells
located at perivascular sites were, on the whole, quite homogeneous
with the morphological characteristics of lymphocytes and were often
seen to form well organized, follicular-like structures (Figure 2C)
whereas parenchymal lesions were less well organized and appeared to
contain a more heterogeneous cell population (Figure 2F)
. In more
advanced-stage lesions, areas of hemorrhage were present and indicated
vascular damage.
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mice was next
assessed. In specimens from symptomatic but not presymptomatic mice,
luxol fast blue staining revealed areas of marked white matter pallor
suggestive of demyelination overlapping with the inflammatory lesions
in the spinal cord of GT-2 mice or cerebellum of GT-8 mice (not shown).
Compared with wild type (Figure 3, A and C)
, active destruction of myelin was clearly evident in the GFAP-TNF
mice with an overt loss of CNPase immunostaining (Figure 3B)
and the
presence of numerous oil-red-O-positive (Figure 3D)
cells in
association with the white matter lesions. This was confirmed in
plastic sections from tissue prepared for electron microscopy (EM).
Both primary demyelination and Wallerian degeneration were identified
in areas of active disease (Figure 4)
. In
these regions, the normal compact appearance of the white matter
(Figure 4A)
was effaced by masses of infiltrating cells (Figure 4, B and C)
. Small groups of demyelinated axons and solitary axonal profiles
lacking their myelin sheaths appeared within the inflammatory
infiltrate (Figure 4, A–C)
. These groups of contiguous demyelinated
axons were more readily identified in electron micrographs from the
affected region (Figure 4, D and E)
. Clusters of macrophages were
scattered through the spinal cord parenchyma. Many of them were foamy
(Figure 4B)
, and ultra-thin sections from such clusters showed lipid
droplet accumulation in the vicinity of demyelinated axons (Figure 4E)
.
Mast cells were also observed in the inflamed meninges. Greatly swollen
axons undergoing Wallerian degeneration were found in regions of the
spinal cord compressed by infiltrating cells (Figure 4C)
and distal to
the site of inflammation. Inflammation and fibrosis of the spinal
meninges and Wallerian degeneration in nerve roots and peripheral
nerves was also identified. Axons undergoing Wallerian degeneration
were very swollen (Figure 4, B and F)
, and electron microscopy revealed
masses of darkly staining organelles, characteristic of that process
(Figure 4F)
.
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mice was further assessed using quantitative
morphometric analysis. The overall integrity of the dendritic
complexity was examined by labeling sections with antibodies against
MAP-2. Confocal analysis of the sections showed that in presymptomatic
transgenic mice there was a little or no detectable alterations in the
neocortex, hippocampus, or cerebellum. In contrast, in symptomatic
GFAP-TNF mice moderate to severe loss of dendritic structures was
evident both in pyramidal neurons in the hippocampus and neocortex as
well as in Purkinje cells in the cerebellum (Figure 5A)
. These neurodegenerative changes were
accompanied by significant astrogliosis as revealed by the GFAP
antibody (not shown). As neurons in these areas express high levels of
calcium-binding proteins and have been shown previously to be lost in
human49
and murine41
neuroinflammatory disease
states, we analyzed the integrity of these neuronal populations with
antibodies against parvalbumin. This analysis showed similar numbers of
parvalbumin-immunoreactive interneurons in the presymptomatic mice as
compared with wild-type controls (Figure 5B
, top panels). In contrast,
in the symptomatic mice there was widespread and severe loss of
parvalbumin-immunoreactive cells in the neocortex, hippocampus, and
cerebellum.
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is known to be a potent inducer of apoptosis, and based on
in vitro studies this pathway has been implicated in
TNF--mediated toxicity to oligodendrocytes.29,30
To
obtain evidence as to whether the astrocyte-targeted expression of
TNF- in vivo was associated with increased apoptosis we
analyzed for DNA fragmentation in situ, using TUNEL staining
methods. Little or no nuclear staining was evident in any brain region
analyzed from wild-type or presymptomatic GFAP-TNF mice (Figure 5, A and B
, lower panel). In contrast, in symptomatic GFAP-TNF mice,
numerous nuclei positive for TUNEL staining were observed in the CNS
inflammatory lesions of GT-8 mice (Figure 5B
, lower panel) or GT-2 mice
(not shown), suggesting these were leukocytes undergoing apoptosis. Immunophenotypic and Spatial Heterogeneity of the CNS-Infiltrating Cells
The phenotypic composition of the inflammatory infiltrates
observed in symptomatic mice from both transgenic lines was examined in
detail by immunohistochemical staining and revealed differences between
the perivascular and parenchymal compartments (Figure 6)
. Thus, the perivascular infiltrates
contained mostly lymphocytes of which B220-positive B lymphocytes were
abundant and formed dense accumulations that were likely responsible
for the follicular-like structure of these infiltrates noted above
(Figure 2)
. Both CD4- and CD8-positive T lymphocytes were also
prevalent and were scattered throughout the perivascular infiltrates
(Figure 6, E and I
, respectively). Cells strongly positive for Mac-1,
and presumed to be activated macrophage/microglia, circumscribed the
perivascular lymphocytic infiltrates (Figure 6G)
. In contrast to the
perivascular infiltrates, infiltrates in the parenchyma contained
mostly CD45-high, Mac-1-positive cells of the macrophage lineage with
fewer numbers of both CD4- and CD8-positive T lymphocytes and few B
lymphocytes (Figure 6, B, H, F, J, and D)
. Numerous neutrophils were
also identified scattered throughout the parenchymal lesions (Figure 6L)
. In addition to the perivascular and parenchymal infiltrates,
strong staining for the macrophage marker Mac-1 was observed on
ramified cells adjacent to lesion areas and were assumed to represent
activated microglia (Figure 6, G and H)
. A similar assessment of CNS
tissue from presymptomatic mice of each line failed to detect the
presence of increased numbers of leukocytes when compared with
wild-type controls (not shown).
|
. Lymph node T and B
cells were used as markers of CD44low,
CD62Lhigh cells. Although all B cells, naive and activated,
express relatively high levels of CD44 as compared with T cells, CD44
levels were still increased on B cells from the TNF mice.
Examination of two commonly used markers of acute activation showed an
unusual dissociation of CD25 (IL-2 receptor) and CD69 (very early
activation antigen) expression. CD25 was virtually absent in the
infiltrating leukocyte population (not shown), whereas CD69 expression
was elevated in both T and B cells, being the highest in
CD8+ cells (Figure 7A)
. Microglia activation was monitored
by analysis of levels of CD45 expression. In contrast to all other
CD45-expressing cells in adult mice, microglia express low levels in
their unactivated state and intermediate levels in their activated
state. All other nucleated cells of hemopoietic origin express high
levels of CD45.52,53
In all symptomatic mice (GT-2 and
GT-8), there is reduction or loss of the CD45low population
and the appearance of CD45intermediate and
CD45high populations, indicating both microglial activation
and leukocyte infiltration (Figure 7B)
. In early symptomatic mice, this
shift in CD45 levels was seen even in the absence of detectable T cell
infiltration, suggesting that large scale leukoctye infiltration was
secondary to microglia/macrophage activation (data not shown).
|
To begin to elucidate the mechanisms by which TNF- may promote
the recruitment of leukocytes to the brain, we examined the expression
of a number of cellular adhesion molecules (CAMs), cytokines, and
chemokines in CNS specimens from wild-type control and presymptomatic
and symptomatic GFAP-TNF mice from each line.
CAM expression was analyzed by immunohistochemical staining and
revealed low levels of ICAM-1 and VCAM-1 expression but no MAdCAM
expression by cerebrovascular endothelium in control mice (Figure 8)
. In contrast, in presymptomatic
GFAP-TNF mice, significant up-regulation of ICAM-1 and VCAM-1
expression was observed on large and small cerebrovascular endothelium
whereas induction of MAdCAM molecule expression was found to be
prominent on small vessel endothelium (Figure 8)
. Expression of these
CAMs was further increased in symptomatic GFAP-TNF mice with ICAM-1
and VCAM-1 also being expressed by some infiltrating leukocytes.
Endoglin expression was the same in wild-type and GFAP-TNF mice.
|
.
Consistent with our findings above, in presymptomatic and symptomatic
mice of each transgenic line, TNF- mRNA was readily detectable in
these brain regions. With the exception of the IL-1 and IL-1ß
mRNAs, which exhibited a modest increase in spinal cord or cerebellum
of symptomatic mice of the GT-2 or GT-8 mice, respectively, expression
of transcripts for other cytokines, including TNF-ß, IL-2, -3, -4,
-5, and -6, and IFN-
, were not detectable. Similar to the cytokines,
in control CNS tissue, no chemokine gene expression was detectable
(Figure 9B)
. In contrast to the cytokine genes, however, expression of
a number of and ß chemokine transcripts was induced in tissue
from presymptomatic transgenic mice and increased further in CNS
tissue from symptomatic GFAP-TNF mice and included C10, MIP-2,
MCP-3, MIP-1ß, MCP-1, crg-2, and RANTES (Figure 9B)
. The level of
chemokine mRNA expression in the different brain regions paralleled
that for the TNF- mRNA and was highest in the spinal cord or
cerebellum from GT-2 or GT-8 mice, respectively.
|
SCID/SCID Mice
To determine the relative contribution of the lymphocytes and
therefore the adaptive immune response to the development of the
neurological disorder observed in the GFAP-TNF mice, animals of the
GT-2 line were crossed with SCID mice that lack T and B
lymphocytes.54
Compared with GFAP-TNF+/+ or
GFAP-TNFSCID/+ animals, GFAP-TNFSCID/SCID
mice exhibited earlier physical deterioration by 2 months of age
characterized initially by the onset of severe ataxia, which then
progressed to hind limb paralysis and was followed soon after by the
death of the animals. Histological examination of the brain and spinal
cord revealed the presence of more widely disseminated lesions in the
spinal cord, cerebellum, and striatum of the
GFAP-TNFSCID/SCID mice. Luxol fast blue staining showed
that considerable demyelination was evident in these lesion areas (not
shown). Immunophenotypic analysis revealed the presence of large
numbers of CD45high (Figure 10A)
, Mac-1 (Figure 10B)
, and MHC class
II (Figure 10F)
positive macrophage/microglial cells within and
surrounding the lesions (Figure 10)
. However, and in contrast to the
GFAP-TNF+/+ or GFAP-TNFSCID/+ animals,
the inflammatory lesions in the GFAP-TNFSCID/SCID mice
did not contain T and B lymphocytes (Figure 10, C–E)
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is assumed to play a critical role in the pathogenesis of
many neuroinflammatory and neurodegenerative disorders of the CNS.
However, recent experimental results31-33
derived
particularly from studies in gene knockout mice deficient for either
TNF- itself or the TNF p55 and/or p75 receptors have brought into
question the previously widely held view that this cytokine was
pro-inflammatory and contributed to CNS injury. In the present study,
chronic expression of TNF- was targeted to astrocytes in the intact
CNS of transgenic mice to better understand the CNS pathobiology of
this cytokine. The results indicated that under these circumstances and
in the transgenic lines studied, TNF- expression was clearly
deleterious, resulting in the development of degenerative disease
affecting both white and gray matter. Significantly, this disorder
occurred with a late onset and was closely linked to the development of
a robust and rapidly progressing inflammatory process in the CNS.
Similar features exist in neurological disorders in adult humans where
dysregulated TNF- production is found, including
MS,55,56
HIV encephalopathy,57,58
and the
infectious meningoencephalitides8
or in the experimental
animal models of EAE59,60
and cerebral
malaria.61
Therefore, the GFAP-TNF mice represent a
relevant in vivo model to evaluate the contribution of
TNF- to the initiation and perpetuation of neuroinflammatory disease
in the adult CNS.
It was notable that in the GFAP-TNF mice described here, there was a
long preclinical phase in which little neurodegeneration or apoptosis
was discernible. Direct toxicity of TNF- to different neural cells
and structures has been documented using in vitro culture
systems. Thus, TNF- is cytotoxic to primary oligodendrocytes and
also mediates myelin injury29,30
whereas in primary
cultures of human neurons TNF- potentiates glutamate
neurotoxicity.62
In contrast to these findings, in
vitro63
and in vivo33
studies
provide evidence of a neuroprotective function of TNF-. In addition,
chronic intracerebral infusion of TNF- was found not to be
associated with significant neuronal or oligodendrocyte
toxicity.64
Clearly, there are major differences between
the different model systems; nevertheless, our findings suggest that
despite its chronic production in the CNS, at levels that eventually
provoke significant inflammatory disease, TNF- alone does not induce
apoptosis in neural cells such as oligodendrocytes and does not
directly cause CNS injury.
The preclinical phase in the GFAP-TNF mice was also marked by the
absence of detectable infiltrating leukocytes in the CNS. However, this
phase was later replaced by the sudden onset of an inflammatory
response with recruitment and infiltration of the CNS with large
numbers of leukocytes. Therefore, this transgenic model provides an
opportunity to study the role of TNF- in the initiation of
inflammation in the CNS and raises questions as to why there was such a
long delay before the activation of the inflammatory response and as to
what the nature of initiating signals may have been that were driving
this process. Evidence derived from the study of acute inflammatory
responses highlight an intrinsic resistance of the CNS to the
recruitment and activation of leukocytes.65
Such resistance
may emanate from structural elements such as the blood-brain
barrier66
as well as from suppressive factors produced
constitutively in the CNS, including cytokines such as
TGF-ß67
and certain classes of
gangliosides.68
Clearly, all of these may contribute to the
delayed initiation of the inflammatory response in the CNS of the
GFAP-TNF mice. In any case, the chronic production of TNF- by
astrocytes in the GFAP-TNF mice did eventually overcome these
barriers to leukocyte recruitment. Leukocyte trafficking is known to be
complex, involving multiple factors in which key participants include
the endothelial expressed cellular adhesion molecules (CAMs),
chemokines, and cytokines.69,70
A survey of the expression
of these molecules in the CNS of the GFAP-TNF mice documented marked
increases or induction in the expression of a number of endothelial
CAMs and of a variety of members of both the and ß chemokine gene
families. These alterations likely represent a direct action of
TNF-, which is known to be a potent regulator of CAM expression by
cerebrovascular endothelium,71
and of chemokine expression
by various neural cells types.70
The CNS expression of the
CAMs and the chemokine genes was found to be coordinately induced in
the presymptomatic stages in the GFAP-TNF mice and may therefore
indicate a central role of these factors in the subsequent initiation
of the inflammatory cascade.
The CNS inflammatory lesions in the GFAP-TNF mice displayed some
degree of heterogeneity between perivascular and parenchymal locations
with the former being composed of predominantly lymphocytes whereas the
latter contained predominantly macrophage/microglia, with neutrophils
and T lymphocytes and few B lymphocytes. The highly organized
appearance of the perivascular lesions was due mainly to the
accumulation of large numbers of B lymphocytes and was reminiscent of
follicular lymphoid tissue. The similarities of these lesions to
peripheral lymphoid tissue was further underscored by the phenotypic
properties of the cerebrovascular endothelium, which had the morphology
of high endothelial venules and expressed MAdCAM, a molecule usually
found on vessels in mesenteric lymph nodes and the expression of which
is known to be induced by TNF-.69
Despite the robust
nature and organization of the lymphocytic response, only a few of the
T lymphocytes (as judged by the IL-2R expression) and B lymphocytes (as
judged by the presence of plasma cells) were activated, with the vast
majority being memory-type cells. In addition, expression of a number
of cytokine genes associated with T lymphocyte activation, including
IFN- and IL-2, were not detectable in the CNS of the symptomatic
GFAP-TNF mice. In all, the cytoarchitectural characteristics and the
general lack of functional activation of the inflammatory response that
we have observed in the CNS of the GFAP-TNF mice are similar to
those reported for TNF- when expressed by pancreatic ß cells in
the islets of Langerhans72,73
and by pulmonary cells in the
lung74
of transgenic mice. Recent studies in mice show that
persistent exposure to TNF- in vivo or in
vitro suppresses Th1 and Th2 lymphocyte responses
markedly.75
Therefore, these and the transgenic studies
highlight a possible down-regulatory role at the level of the T
lymphocyte for TNF- in chronic inflammatory states.
The development of severe CNS degenerative features were observed to
parallel the onset and evolution of the inflammatory disease in the
GFAP-TNF mice. As noted above, in view of the absence of such
features in the presymptomatic mice, we concluded that the inflammatory
response is central to the pathogenesis of the CNS injury. The white
matter lesions, which were particularly prominent, shared many features
found in human inflammatory demyelinating disorders such as MS and
included primary demyelination, remyelination, and the presence of
foamy macrophages. However, unlike MS, extensive neurodegeneration
affecting hippocampus and frontal cortex as well as the cerebellum and
spinal cord was also seen and presumably reflected the more
disseminated nature of the inflammatory lesions found in the transgenic
mice. We suggest some of these neurodegenerative changes may have
arisen from ischemic injury and edema resulting from vascular damage
due to the massive and rapid accumulation of leukocytes within the
cerebrovascular compartment. In support of this, hemorrhaging was
evident in advanced lesions and resulted from injury to the vessel. In
addition, ischemic injury may help to explain the pronounced axonal
damage observed at sites distal to the inflammatory focus. It should be
noted that vascular damage consequent to TNF--mediated inflammation
appears to be a central mechanism in the pathogenesis of
neurodegeneration in a number of experimental disorders, including
cerebral malaria24
and bacterial meningitis.8
It is known from studies in EAE that although activated CD4-positive
lymphocytes constitute a small fraction of the lymphocyte population
that enters the CNS, these few cells are able to coordinate a
significant destructive autoimmune process that is targeted to the
white matter.76
It was therefore conceivable that although
there was little lymphocyte activation in the CNS of the symptomatic
GFAP-TNF mice, a possible autoimmune mechanism may still be involved
in contributing to the CNS injury. This notion was also supported by
the observation of induction of MHC class II expression in the
inflammatory lesions coupled with enhanced macrophage/microglial
activation and function. However, our data in the GFAP-TNF
SCID/SCID mice clearly suggest that if there is involvement
of a classical T-lymphocyte-regulated autoimmune response in these
transgenic mice it is not a significant direct determining factor in
the development of degenerative disease. On the contrary, the fact that
the clinical presentation was of earlier onset and the CNS inflammatory
lesions were more widespread in GFAP-TNF SCID/SCID mice
suggested that T and possibly B lymphocytes may have a role in
counteracting the recruitment and/or destructive potential of the
macrophage/microglia that formed the inflammatory lesions.
CD45 expression can be used to discriminate infiltrating macrophages
from resident microglia in the adult mouse CNS.51
In view
of their high expression of this marker, it is likely that the CNS
lesion-associated cells in the GFAP-TNF SCID/SCID mice
were derived from peripheral macrophages recruited to the CNS, although
it cannot be ruled out that these cells were also microglia with
up-regulated CD45 expression. In any case, the studies in the
GFAP-TNF SCID/SCID mice highlight a dramatic direct
response by cells of the macrophage microglial lineage to chronic
TNF- production in the CNS. This response, which included
recruitment, accumulation, and activation concomitant with significant
tissue destruction, therefore highlights a central role of the
macrophage/microglial cell as a mediator of inflammatory disease in the
CNS of the GFAP-TNF model. The CNS white matter appears to be a
major target in this regard in which macrophage/microglia were involved
in causing significant demyelination. We have previously documented the
spontaneous induction of macrophage/microglial-mediated demyelinating
disease after chronic astrocyte-targeted expression of the cytokine
IL-3.42
More recently, persistent disease with increased
macrophage/microglial reactivity was seen after induction of EAE in
MBP-TNF transgenic mice that do not develop spontaneous
disease.38
There is now accumulating evidence
implicating cells of the macrophage/microglial lineage as primary
effectors in the pathogenesis of inflammatory demyelinating
disorders77,78
and neurodegenerative
disorders79
in humans. Our findings here link directly
TNF-, a cytokine for which expression is increased in the CNS in
these same disorders, to the function of macrophage/microglia and
suggest a possible mechanism whereby the reactivity of these cells
could be perpetuated by both paracrine and autocrine pathways.
Therefore, approaches directed at breaking this link, for example,
suppressing macrophage/microglial reactivity, may provide potential
therapeutic strategies for CNS inflammatory disorders the efficacy of
which could be tested in the GFAP-TNF model.
|
Footnotes |
|---|
Supported by USPHS grant MH 50426 (I.L. Campbell). A.K. Stalder is a postdoctoral fellow of the National Multiple Sclerosis Society. A. Pagenstecher was a postdoctoral fellow of the Deutsche Forschungsgemeinschaft.
A. Pagenstecher's current address: Department of Neuropathology, University of Freiburg, Freiburg, Germany.
Accepted for publication June 25, 1998.
|
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