1 Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106; 2 Institute of Physiology, Philipps University, 35033 Marburg; and 3 Department of Pediatric Pneumology and Immunology and 4 Neuroscience Research Center, Charité, Humboldt University, 10117 Berlin, Germany
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ABSTRACT |
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The transport mechanism
mediating brain uptake of tumor necrosis factor (TNF)- has been
studied. When 125I-labeled rat TNF-
was used in internal
carotid artery perfusions in rats, the cytokine showed transcytosis
through the blood-brain barrier in intact form (permeability-surface
area product 0.34 ± 0.13 µl · min
1 · g
1).
Uptake was inhibited by low nanomolar concentrations of unlabeled rat
TNF-
. Human TNF-
, which does not interact with the p80 TNF receptor in rodents, showed no brain uptake. mRNA expression of both
p60 and p80 receptors could be demonstrated in native brain microvessel
preparations. These transcripts increased to 149% (p60) and 127%
(p80) of control 4 h after a systemic immune stimulation (2 mg/kg
bacterial endotoxin ip). Lipopolysaccharide treatment did not alter the
rate of brain uptake of TNF-
measured between 4 and 24 h later.
In conclusion, a receptor-mediated mechanism is responsible for the
transcytosis of TNF-
. Saturable transport, requiring the p80
receptor, occurs at concentrations encountered under pathophysiological
conditions and therefore constitutes a relevant mechanism of
communication between the immune system and the brain.
brain perfusion; Northern blot; lipopolysaccharide
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INTRODUCTION |
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SYSTEMICALLY RELEASED
PROINFLAMMATORY CYTOKINES, like tumor necrosis factor-
(TNF-
), act as signals in the complex network of
immune-neuro-endocrine interactions. They cause unequivocal effects in
the central nervous system (CNS), such as fever and stimulation of the
hypothalamo-pituitary-adrenal axis (5, 9, 25). However,
the mechanisms of transmittal from the circulation to the brain, in
particular the role of the blood-brain barrier (BBB), are still not
fully understood. Studies utilizing diverse experimental approaches
suggest at least four distinct pathways of communication (9,
17). Cytokines may act on cognate receptors on brain cells after
physically entering the organ, either by 1) transport across
the BBB or 2) by gaining access at circumventricular organs,
i.e., at sites where the BBB is absent. Alternatively, 3)
cytokines may trigger the release of second messengers (prostaglandins, nitric oxide) by brain microvascular endothelial cells and perivascular microglia. Finally, 4) cytokines may stimulate afferent
nerve fibers in the periphery to send signals to the brain parenchyma.
Regarding the first mechanism, the intact mammalian BBB does not allow
nonspecific uptake of proteins by diffusional transport. However, as
initially demonstrated for insulin (16) and transferrin (18), brain capillary endothelial cells express
receptor-mediated transport systems for peptides and proteins. These
enable transcytosis of their respective ligands from blood to brain
extracellular space. An analogous receptor-mediated transport of
proinflammatory cytokines through the BBB could contribute to their
centrally mediated effects. In this regard, TNF- is of particular
interest, as it is the first cytokine released into the circulation
after stimulation of the immune system with endotoxin (6),
and it triggers the inflammatory cascade in concert with interleukin (IL)-1 (14). In contrast to IL-1, TNF-
concentrations
in plasma rise into the high picomolar range under pathophysiological
conditions in humans and experimental animals. Brain uptake of TNF-
from the systemic circulation has been described (3, 11, 21, 30), and a saturable, specific transport mechanism at the BBB has been postulated (21). Available evidence suggests that
the two known TNF receptors, designated TNFR1 (receptor type-1, also known as p55/p60 receptor or CD120a) and TNFR2 (receptor type-2, also
known as p75/p80 receptor or CD120b) (4, 42), are involved in the transport. These receptors are expressed on many cell types, and
endothelial cells are among the primary targets of TNF-
(28). The detection of TNF receptor transcripts within the
rat brain has been reported by use of in situ hybridization
(29). In the basal state, a low-expression level of TNFR1
over blood vessels was described, whereas the TNFR2 signal was
negative. After systemic injection of either lipopolysaccharide (LPS)
or recombinant TNF-
, the message for the TNFR1 was upregulated, and
TNFR2 became detectable. Recent experiments in receptor knockout mice
indicated that both TNFR1 and TNFR2 are required for brain uptake of
TNF-
from the circulation (32), with no uptake seen in
double-knockout animals.
A local immune response or tissue damage in the CNS enhances BBB
transport of TNF- from the systemic circulation, as shown in
experimental autoimmune encephalomyelitis (30) and spinal cord injury (33). It is not known whether a systemic
immune stimulation would also modulate brain uptake of TNF-
. In the case of insulin, systemic endotoxin treatment apparently increased the
transport rate of the peptide hormone at the BBB (47).
In the present studies, we addressed three questions: 1)
whether brain uptake of TNF- is reflected in binding of the cytokine by freshly isolated brain microvessels, 2) whether native
brain microvessels express both TNF receptor types, and 3)
how systemic immune stimulation with endotoxin would affect receptor
expression and brain uptake of TNF-
.
The transport of TNF- through the BBB in rats was measured with or
without prior intraperitoneal administration of LPS. These transport
studies were performed using the internal carotid artery perfusion
technique. This method is particularly suitable for brain uptake
studies of labile substances, because it eliminates systemic
metabolism. The latter point is relevant with respect to TNF-
: in
rats, a distribution half-life of only 5 min and a mean residence time
of 24 min have been reported (45). In the present
experiments, uptake into brain parenchyma was differentiated from
vascular sequestration by the capillary depletion method. Binding and
cellular uptake of TNF-
were also studied in receptor-binding assays
with use of freshly isolated brain cortical microvessels and cell
membrane preparations from these microvessels. Northern blot analysis
of brain microvessel-derived RNA was applied to determine the TNF
receptor gene expression in untreated rats and after systemic LPS administration.
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MATERIALS AND METHODS |
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Materials.
Male Sprague-Dawley rats (Harlan-Winkelmann, Borchem, Germany) weighing
200-300 g were used in the animal experiments. D. Gemsa (Institute
for Immunology, Philipps University, Marburg, Germany) kindly provided
L929 mouse fibroblasts. Recombinant rat TNF- was purchased from
PeproTech (Rocky Hill, NJ) and from the National Institute for
Biological Standards and Control (Potters Bar, UK). Recombinant human
TNF-
was also obtained from PeproTech. LPS from Escherichia
coli 0111:B4 [lethal dose of 50% (LD50) = 12.3 mg/kg in mice] was obtained from Difco Laboratories (Detroit, MI). The rat TNF-
ELISA was obtained from Endogen (Woburn, MA), and
the EZ4Y cytotoxicity assay was purchased from Biomedica (Vienna, Austria). Na125I was purchased from Amersham Pharmacia
(Braunschweig, Germany) and iodogen from Pierce (Rockford, IL). If not
specifically mentioned, all other chemicals were of analytical grade
and were purchased from Sigma (Deisenhofen, Germany).
Tracer.
TNF- was radioiodinated with the iodogen method (19) to
specific activities between 600 and 3,000 µCi/nmol (referring to trimeric TNF-
). Briefly, 2 µg of TNF-
in 30 µl of 0.1 M Na
phosphate (pH 7.4) were allowed to react for 3 min with 0.5 mCi
Na125I in an iodogen-coated microfuge tube. The reaction
mixture was transferred to a second tube with 100 µl of 1%
K127I. The labeled protein was purified from free
125I by Sephadex G25 gel chromatography. An aliquot of the
peak fractions was tested for TCA precipitability. Only a tracer of
>98% precipitability was used within 48 h of labeling. Rat serum
albumin (RSA) was labeled with
[3H]N-hydroxysuccinimidyl propionate, as
described (7).
Cytotoxicity assay.
Bioactivity of labeled and unlabeled rat TNF- was determined using
the L929 mouse fibroblast assay. Briefly, L929 fibroblasts were seeded
in 96-well plates at 25,000 cells/well and grown in RPMI 1640 with 10%
fetal calf serum. After 24 h, the medium was replaced with 50 µl
of fresh medium containing Actinomycin D (2 µg/ml), and 1 h
later the TNF-
dilutions in 50 µl of medium were added. After
18 h, incubation at 37°C cell viability was quantified by the
EZ4Y assay according to the manufacturer's instructions. To obtain the
exact TNF-
protein concentration, 125I-labeled TNF-
was quantified with the ELISA specific for rat TNF-
.
Internal carotid artery perfusion technique.
Unilateral vascular brain perfusions were performed in anesthetized
rats (100 mg/kg ketamine and 4 mg/kg xylazine im) via retrograde
cannulation of the external carotid artery after cauterization of the
occipital artery, superior thyroid artery, and pterygopalatine artery.
The common carotid artery was ligated just before initiation of the
perfusion. Krebs-Henseleit buffer containing 1% bovine serum albumin
(BSA) equilibrated with 95% O2-5% CO2 was
perfused at a flow rate of 1.25 ml/min by use of a peristaltic pump
(Spetec, Munich, Germany). The perfusate contained 1 µCi/ml of
125I-labeled rat TNF- (0.33-1.67 nM, referring to
trimeric TNF-
) or human TNF-
and 10 µCi/ml 3H-RSA.
Inhibition experiments were performed by adding unlabeled rat TNF-
(0.83-16.7 nM) to 125I-labeled rat TNF-
tracer in
the perfusate. Perfusion times of 1, 5, or 10 min were chosen. To keep
intravascular volume constant during the 5-min and 10-min perfusions,
blood was withdrawn at the same rate via a catheter in the femoral
artery. Perfusions were terminated by decapitation. The brain was
quickly removed and cleaned from meninges, and the ipsilateral
hemisphere (forebrain without hypothalamus and olfactory bulb) was
weighed. The tissue was gently homogenized on ice in physiological
buffer, and "capillary depletion" analysis was performed, as
described (43) with the following modifications: dextran
(molecular mass 60-90 kDa) was added to a final
concentration of 20%, and the centrifugation was performed at 3,200 g for 15 min at 4°C in a table top centrifuge with
swinging bucket rotor. Efficiency of separation into the vascular
pellet and the postvascular supernatant was tested in a pilot series by
measurement of the capillary-enriched marker enzymes alkaline
phosphatase and
-glutamyl transpeptidase. The results were
comparable with those of the original protocol, with 93.5 ± 2%
(alkaline phosphatase) and 93.2 ± 1.6% (
-glutamyl
transpeptidase) of enzyme activities accumulated in the pellet fraction
(means ± SD, n = 4, based on enzyme activities
per mg protein). The pellet and aliquots of homogenate and postvascular
supernatant were digested with tissue solubilizer (Soluene 350, Canberra Packard, Dreieich, Germany) and measured in a Wallac 1210 liquid scintillation counter with a dual-isotope program (Wallac, Ohu,
Finland). The results are expressed as the apparent volume of
distribution, VD, which is calculated as
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TCA precipitation of brain postvascular supernatant.
Two milliliters of ice-cold 20% TCA in water were added to 0.5 ml of
postvascular supernatant, briefly vortexed, incubated on ice for 10 min, and centrifuged at 4,300 g for 5 min at 4°C. The
TCA-precipitable pellet and the TCA supernatant were measured in a
-counter. The TCA-precipitable fraction of radioactivity was
expressed as
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SDS-PAGE.
After 5-min internal carotid artery perfusion with
125I-TNF-, the perfused hemisphere was homogenized for
10 s in 3 ml of ice-cold 0.9% NaCl with an Ultra Turrax
(Kinematica, Lucerne, Switzerland). A 300-µl aliquot was
loaded on a 3-mm discontinuous 5%/15% SDS-PAGE gel. The gel was dried
and exposed to autoradiographic film (Kodak X-OMAT) for 2 wk.
Isolation of rat brain capillaries. Animals were killed under halothane anesthesia, and the brains were removed. Meninges, choroid plexus, and white matter were carefully removed from the cortical shell. The tissue was mechanically homogenized in 20% dextran and centrifuged at 3,200 g for 15 min at 4°C. Microvessels were purified by sieving through an 80-µm nylon mesh and pouring onto a glass bead column held by a 40-µm nylon mesh as described (35). The microvessels were harvested from the glass beads and the 40-µm nylon mesh and were spun down by gentle centrifugation (300 g, 5 min, 4°C). The capillary pellet was resuspended in Ringer-HEPES buffer (RHB) with 0.1% BSA and visually inspected by light microscopy. An aliquot was removed for protein measurement by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL).
Binding of 125I-TNF- to rat brain capillaries.
Freshly isolated capillaries (50 µg of protein per vial) were used
for binding studies and incubated in a final volume of 0.45 ml
RHB-0.1% BSA with 125I-TNF-
at a concentration of
0.167-0.27 nM (100,000 counts · min
1 · vial
1)
at 4°C or 37°C for up to 120 min. Unlabeled TNF-
(33 nM) was added in competition studies. At the end of the incubation times, the
samples were centrifuged and the supernatant was removed. In some assay
series, the pellet was subjected to a brief acid wash to remove cell
surface-bound tracer (6 min at 4°C in 0.12 M NaCl, 0.028 M Na
acetate, and 0.02 M Na barbital, pH 3) (35). Radioactivity
of the pellet was measured in a
-counter. Protein was subsequently
determined by solubilization (500 µl of 1 M NaOH, 60°C for 30 min)
and BCA assay (Pierce).
Binding of 125I-TNF- to isolated rat brain
capillary membranes.
The membrane fraction was prepared from freshly isolated rat brain
capillaries according to Lidinsky et al. (26). After cell
lysis in 0.01 M Tris · HCl (pH 7.4), the basement
membrane was separated from cell membranes by sonication and spun out
(25,000 g, 30 min). The supernatant with cell membranes was
used immediately or stored in 0.05 M Tris · HCl
(pH 7.4) at
70°C.
Total RNA and poly(A)-RNA preparation.
Total RNA from cortical microvessels of 12 rats was extracted with the
single-step phenol-chloroform extraction method (13). The
isolated microvessels were solubilized in 1 ml of lysis buffer containing 4 M guanidine thiocyanate, 25 mM Na citrate, 10% (wt/vol) N-lauroylsarcosine, and 0.7% -mercaptoethanol. The
lysates were vortexed and incubated on ice for 10 min. A centrifugation
step was performed at >10,000 g for 15 min at 4°C. The
upper aqueous phase contained the isolated total RNA. After addition of
1/10 volume of 2 M Na acetate (pH 4.0), the RNA was extracted with an
equal volume of phenol-chloroform-isoamylalcohol (25:24:1). Isopropanol
was used for precipitation of the RNA pellet, and 70% ethanol was used
for washing the RNA. The total RNA (yield ~20 µg, measured by UV
detection at 260 nm) was used for RT-PCR. Poly(A)-RNA for Northern
blots was sampled from brain capillaries, choroid plexus, pineal
glands, and anterior and posterior pituitaries from two groups of rats,
either untreated control rats or rats after injection of 2 mg/kg ip
LPS, 4 h before decapitation (n = 12/group). The
tissues of each group were pooled and homogenized with a Teflon-glass
homogenizer in 5 ml of 0.1 M Tris · HCl
with 1% SDS, 0.5 M LiCl, 10 mM EDTA, and 5 mM dithiothreitol (pH 8.0). poly(A)-RNA was isolated using the oligo(dT)25 Dynabeads single-step method according to the manufacturer's instructions (Dynal Biotech, Hamburg, Germany).
RT-PCR. Primers for amplification of p60 (TNFR1) were selected from the rat sequence (EMBL accession code m63122) for amplification of bp 813 to 1167 of the cDNA sequence by using the HUSAR software. Primers for cDNA of rat p80 (TNFR2) were taken from the partial sequence of 256 bp (EMBL accession code u55849). Four additional primer pairs for overlapping regions spanning the coding region were selected using the mouse p80 cDNA sequence (EMBL accession code m60469).
One microgram of total RNA was reverse transcribed with Superscript II-reverse transcriptase (GIBCO, Karlsruhe, Germany) and pd(N)6 random primer hexamers (Amersham Pharmacia Biotech, Freiburg, Germany) for 90 min at 42°C, and 2 µl of reverse transcription product (cDNA) were used for PCR without further purification. Hot start PCR with addition of 2.5 U AmpliTaq polymerase (Applied Biosystems, Weiterstadt, Germany) at 94°C was performed in a total volume of 50 µl for 35 amplification cycles with annealing temperatures of 55°C and annealing times of 40 s. PCR products (10 µl) were resolved by electrophoresis on 1.5% agarose gels.Northern blot hybridization.
Enriched poly(A) RNA (2.5-5 µg) was separated by electrophoresis
on denaturating agarose gels (1.5% agarose, 1× MOPS, 6 M formaldehyde
37%), transferred to nylon membranes (Nytran NY 12N, Schleicher & Schuell, Dassel, Germany), and cross-linked by UV irradiation. To
obtain p60 and p80 cDNA probes, RT-PCR products were T/A cloned into
the pGEM-T vector (Promega, Mannheim, Germany) by following the
manufacturer's protocol. A 0.5-kb cDNA fragment for GLUT1 was cut from
the Bluescript KS plasmid containing the bovine GLUT1 cDNA clone pGT51
(10). A 1.2-kb cDNA probe pRB15 for mouse C1q was excised
from pCR 1000 by EcoRI digest (39). As a
standard, a commercial human GAPDH cDNA fragment was used (Clontech,
Heidelberg, Germany). All cDNA probes were labeled to high specific
activity (>109 cpm/µg) using [-32P]dCTP
(Hartmann Analytic, Braunschweig, Germany) and Prime-it RmT random
primer labeling kit (Stratagene, Austin, TX).
Statistical analysis. Statistical analysis was performed with the InStat program (GraphPad, San Diego, CA). Comparison of the means of two groups was performed by Student's t-test; ANOVA was used for multiple group comparisons. The significance level was set at P < 0.05. Linear regression analysis was performed with GraphPad Prism.
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RESULTS |
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The brain uptake of radiolabeled rat TNF- was measured in rats
by use of the internal carotid artery perfusion method. We confirmed
that the labeling procedure did not compromise bioactivity of TNF-
by measuring the EC50 of unlabeled and
125I-labeled TNF-
in a cytotoxicity assay with L929
cells. EC50 for tracer was not significantly different from
unlabeled TNF-
, with values of 9.5 ± 1 and 11.4 ± 3.7 pg/ml, respectively (unpaired t-test, n = 4, P > 0.1). Brain perfusions over 1, 5, and 10 min with
rat 125I-TNF-
provided evidence of time-dependent organ
uptake. Consistent with a relatively slow transport, the VD
of TNF-
after a 1-min perfusion was not yet significantly different
from the vascular volume, with VD values of 10.77 ± 1.38 and 12.27 ± 0.75 µl/g for 125I-TNF-
and
3H-RSA, respectively. However, after 5- and 10-min
perfusions, the VD of rat 125I-TNF-
was
significantly higher than that of the vascular marker 3H-RSA. This is shown in Fig.
1, A and B.
Separation of brain tissue into the vascular component and postvascular
supernatant by capillary depletion revealed that, after correction for
intravascular content, the uptake could largely be attributed to
transcytosis through the BBB. The vascular pellet contains only a minor
fraction of total brain radioactivity. For example, the pellet
VD of 0.99 ± 0.29 µl/g after 5 min corresponds to
only 6% of the total tracer in brain. The radioactivity in
postvascular supernatant represented intact tracer. This can be
concluded from the TCA-precipitable fraction in these samples of
91.3 ± 1.0 (mean ± SE, n = 43; pooled data
from all 5- and 10-min perfusions), which approached the precipitability of the freshly labeled tracer. The integrity of the
tracer in brain was further demonstrated by SDS-PAGE and
autoradiography. A single band at the expected molecular mass of the
TNF-
monomer (17 kDa) was detected (Fig.
2).
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The permeability-surface area product (PS) at the BBB for TNF- was
estimated from 1-, 5-, and 10-min perfusions by linear regression
analysis, as shown in Fig. 1B. Uptake into whole brain and
into postvascular supernatant occurred at rates of 0.81 ± 0.13 and 0.34 ± 0.13 µl · min
1 · g
1, respectively.
Figure 1C provides evidence that a TNF--specific
mechanism is involved in that transport. Co-perfusions of rat
125I-TNF-
with unlabeled rat TNF-
at concentrations
between 0.83 and 16.7 nM led to a decrease in brain VD
compared with tracer perfusion alone (concentration = 1.25 nM), down to VD values as seen for RSA. Further
evidence of a specific and TNF receptor-related mechanism can be
derived from the perfusion experiment with human 125I-TNF-
, depicted in Fig. 1A. Human TNF-
has no affinity to the rodent p80 receptor (24). There was
no difference between the VD of human
125I-TNF-
and 3H-RSA after a 5-min brain perfusion.
We studied next whether the in vivo brain uptake of TNF- is
reflected in binding and uptake by isolated rat brain microvessels. The
result is shown in Fig. 3A.
There was a time-dependent increase in the amount of rat
125I-TNF-
bound by a brain microvessel preparation. Part
of the tracer binding was displaced either by competition with
unlabeled rat TNF-
or by a mild acid wash. As apparent from Fig.
3B, similar results were obtained with a membrane
preparation derived from the isolated brain microvessels. A 100-fold
molar excess of rat TNF-
displaced ~30% of the binding, whereas
no competition was observed by a 900-fold molar excess of the unrelated
polypeptide leptin (negative control). The relatively high level of
nonspecific binding of TNF-
with both the intact microvessels (Fig.
3A) and the membranes (Fig. 3B) did not allow the
performance of nonlinear binding analysis or Scatchard transformation.
Modifications of the binding buffer or of the separation method
(filtration or centrifugation) did not reduce nonspecific binding (data
not shown).
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Analysis of gene expression at the mRNA level was conducted to further
characterize the binding sites. As shown in Fig.
4, RT-PCR with RNA from rat microvessels
resulted in the generation of amplicons of the predicted lengths for
both TNF receptor types. Furthermore, sequencing of the overlapping
amplified segments of the rat type 2 receptor (p80) covered 1,300 nucleotides in the coding region (deposited in GenBank under accession
no. AF420214). The sequence homology with the published murine type 2 receptor was 90% at the mRNA level and 85.5% for the deduced amino
acid sequence. No additional PCR products of different lengths that could indicate p80 isoforms were observed. Northern blots with 32P-labeled cDNA probes encompassing the transmembrane
domain (in the case of the p60 transcript) or part of the cytosolic
domain (in the case of the p80 transcript) showed distinct bands for both messages in a brain capillary polyA-RNA preparation (Fig. 5). The signals for both receptors were
clearly detectable in native capillaries, albeit weak. Consistent with
the high enrichment of endothelial cells in the capillary preparation,
a strong signal for the BBB endothelium-enriched message GLUT1 was
detected. In contrast, the absence of a signal for C1q argues against
the contribution of perivascular microglia as a mRNA source in our
microvascular samples. For comparison, Fig. 5 also shows blots of other
CNS-derived tissues that do not possess an endothelial barrier system.
The same pattern of expression of TNFR1 and TNFR2 was seen in all tissues. No evidence of additional transcripts could be detected in the
capillary preparation for either receptor. Systemic treatment with LPS
as immune stimulant resulted in moderate upregulation of the message
for both TNF receptors in brain capillaries to 149% of control level
(p60) and 127% of control level (p80), as given in Table
1. The corresponding upregulation of p60
mRNA in the other tissues ranged from 136% (choroid plexus) to 225% (pineal gland), and from 190% (choroid plexus) to 348% (posterior pituitary) for p80 mRNA.
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The question of whether an increase in mRNA for the two TNF receptors
at the BBB results in an increased brain uptake of TNF- was
addressed by comparing the BBB transport in vehicle (0.9% NaCl)-injected animals and that in rats which had received
intraperitoneal injections of LPS between 4 and 24 h earlier. As
shown in Fig. 6, the preceding systemic
immune challenge with LPS did not cause significant changes in brain
uptake of rat 125I-TNF-
as measured by 5-min brain
perfusion. This was true for brain homogenate (Fig. 6) as well as for
the vascular pellet and postvascular supernatant (not shown). The
VD of the vascular marker 3H-RSA remained
unchanged from untreated controls, also, confirming our earlier
observations that LPS administration does not open the BBB to proteins
nonspecifically (7).
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DISCUSSION |
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The results of the study are compatible with the following
conclusions. 1) A saturable transport system for TNF- is
present at the BBB, which recognizes rat TNF-
but not human TNF-
.
The p80 receptor appears to be required for the BBB transport.
2) The TNF receptors p60 and p80 are constitutively
expressed by brain microvascular endothelial cells of untreated rats.
3) The rat p80 receptor shows 90% sequence homology with
the corresponding mouse cDNA. No isoforms were detected at the mRNA
level. 4) Systemic treatment with LPS moderately upregulated
the mRNA levels of both receptors in brain microvasculature and in
brain tissues without endothelial barrier function. 5) The
LPS treatment did not change the rate of uptake of TNF-
at the BBB
in vivo, suggesting that the receptor protein level is not increased on
the endothelial cell surface.
The rate of BBB transport of TNF- was measured using the internal
carotid artery perfusion method. Although the PS product for TNF-
was low, it was clearly much higher than would be expected for a
macromolecule of its size (molecular mass of the homotrimer = 51 kDa). For comparison, a PS value of ~0.01
µl · min
1 · g
1
for native albumin has been reported in intravenous studies
(36). Albumin does not show measurable brain uptake in
short-term perfusion experiments, as used here, and is a suitable
intravascular marker. When the fraction of TNF-
tracer truly
penetrating the BBB is considered (represented by the postvascular
supernatant after capillary depletion), the PS product amounted to
0.34 ± 0.13 µl · min
1 · g
1.
This value is similar to the PS product of 0.2 µl · min
1 · g
1
for murine TNF-
in mice, as measured by the intravenous bolus technique (21, 32). It is also in the same range as the PS product of 0.32-0.39
µl · min
1 · g
1
reported for the low molecular mass substance sucrose (342 kDa) (37, 41). The latter comparison with a substance often
used as vascular marker highlights the importance of excluding
metabolic artifacts. This requirement was met in the present perfusion
studies, as shown by the integrity of the tracer in brain tissue, which was independently determined with TCA precipitation and SDS-PAGE.
The saturability of TNF- transport is consistent with specific,
receptor-mediated transcytosis as the underlying mechanism. Although
exceptions exist, as postulated for epidermal growth factor
(31), in which the cell surface receptor mediating the classical physiological effect of a peptide hormone is distinct from
its transport protein at the BBB, such may not apply to TNF-
. Evidence from the present studies strongly argues for the involvement of at least the known p80 receptor in brain uptake of the cytokine. Uptake in the rat model was seen only with homologous rat TNF-
, whereas human TNF-
was not transported (Fig. 1A). It is
known that p80 receptors are species specific, and rodent p80 receptors do not recognize human TNF-
(24). The absence of brain
uptake of human TNF-
thus indicates critical involvement of the p80 receptor in the BBB transport process. In this regard, the present result is in good agreement with recently reported data in knockout mice (32). These studies showed a decreased rate of
transport of murine TNF-
at the mouse BBB, when either one of the
TNF receptor types was absent, and no brain uptake in double knockout animals.
The range of concentrations over which the saturation of BBB transport
occurs (between 1 and 17 nM, Fig. 1) is compatible with the p80
receptor. The dissociation constant (Kd) of p80
for TNF- has been reported as 0.42 nM (20). The
affinity of the p60 receptor, on the other hand, seems to be higher,
with a Kd of 19 pM determined under
physiological conditions on human cell lines (20).
The characterization of the receptors in binding and internalization
studies with isolated brain microvessels and membrane preparations
proved difficult. We were able to demonstrate specific binding sites
for 125I-labeled rat TNF- on these microvessels by the
partial competition with a molar excess of TNF-
. However, a
calculation of maximal binding (Bmax) and
Kd was not feasible. Presumably the endothelial expression of these binding sites at the protein level is relatively low compared with the degree of nonspecific binding of TNF-
to microvessels. The latter could be due to adsorption to cytoskeletal elements, which are partially exposed in microvessel preparations such
as those used here (29).
However, we could readily identify both the p60 and the p80 receptor on Northern blots of native rat brain capillary mRNA. These transcripts were of endothelial origin, as demonstrated by the presence of a strong GLUT1 signal, which is known to be selectively expressed at the BBB (34). Absence of the message for C1q, which represents an exclusive marker of microglia and perivascular macrophages in brain tissue (38), served as a negative control in support of the pureness of the microvessel preparation. Microglia are known not only as a source of cytokines but also to constitutively express both types of TNFR (15). Therefore, the lack of a signal for C1q in the capillary mRNA indicates that these cells were not present in the microvessel preparation.
Our results for p60 and p80 mRNA expression in brain microvessels after
systemic LPS administration extend the observations with in situ
hybridizations performed by Nadeau and Rivest (29). These
authors found low constitutive expression of the p60 transcript over
brain parenchymal blood vessels, whereas they did not detect basal p80
expression by BBB endothelium. The discrepancy between the negative in
situ hybridization data and the present findings with respect to the
basal p80 signal on brain microvessels may be explained by the
different methods. We achieved high sensitivity in our Northern
hybridizations due to the use of poly(A)-RNA from brain microvessels of
six pooled brains. After provocation of a systemic immune response with
intraperitoneal injection of LPS, the mRNA levels of p60 and p80 in
brain capillaries were moderately elevated in the present study, and
these Northern blot data 4 h after LPS are consistent with peak
stimulations by LPS or TNF- found between 3 and 6 h in the in
situ hybridization study over brain parenchymal blood vessels and over
choroid plexus (29). The signals seen on our Northern
blots did not reveal additional transcripts in brain capillary
endothelial cells compared with non-BBB tissues. Similar transcript
sizes for the p60 and p80 receptor have been reported in rat bronchial
epithelium (2) and rat glia (15). In
addition, all of the PCR products for TNFR1 and TNFR2 described here
were of the expected size, as confirmed by sequencing. In this regard,
it is interesting to note that a novel isoform of the human TNFR2,
icp75TNFR, which is intracellularly expressed at a low abundance, was
recently isolated (40) and found in nonbrain endothelial
cells (HUVEC) by these authors.
Concerning the expression of TNFR1 and TNFR2 in pituitary and pineal
gland, TNF- binding was detected autoradiographically in the
anterior pituitary (46), and the transcripts for the two
receptors have been measured in the corticotroph-derived cell line
AtT-20 (23). Functional evidence for pineal TNF receptors is derived from a decrease in pinealocyte serotonin secretion and
stimulation of pineal microglia of explant cultures under TNF-
treatment (44). Receptor expression at the mRNA level has
not been previously reported in the pineal gland.
No significant changes occurred in the rate of brain uptake of rat
125I-TNF- between 4 and 24 h after intraperitoneal
administration of LPS. We have previously shown that LPS administration
at low (50 µg/kg) and high doses (2 mg/kg) did not result in a
measurable breakdown of the BBB in rats for vascular markers of small
and high molecular weight (sucrose and serum albumin, respectively) (7). The present data with RSA confirmed that finding.
Therefore, no increase in the rate of brain uptake of TNF-
due to a
damaged BBB had to be expected in our study. As outlined, the present results (Fig. 1) clearly argue in favor of TNF receptor-mediated transcytosis of TNF-
through the BBB. This is supported by the expression of both TNF receptor types on native brain microvessel endothelial cells and by the saturability of transport at low nanomolar
concentrations. TNF receptor-mediated transport could theoretically
change in either direction during the course of an immune challenge by
LPS with its associated release of cytokines. 1) The
receptor expression may be upregulated. Our Northern blot data support
this option at the level of gene transcription, and this is also in
agreement with the published in situ hybridization data
(29). In primary endothelial cell culture from mouse
brain, TNF-
caused a peak increase in mRNA for TNFR1 and TNFR2
levels at 6 h, whereas the corresponding increase in protein
expression was significant after 12-24 h (27).
However, at the protein level, in vivo effects of LPS on TNF receptor
regulation in brain endothelial cells have not been reported.
2) Ligand-stimulated receptor shedding may actually decrease
the number of surface-expressed TNF receptors. Systemic TNF-
levels
are increased after LPS. Receptor shedding is a physiological response
that is considered protective under septic conditions (1)
and could protect the BBB from damage in vivo. Shedding of both types
of TNF receptors by endothelial cells has been observed within the
first 2 h after ligand stimulation (27).
3) Even without shedding, the number of TNF receptors on the
cell surface may be downregulated by ligand-induced internalization, which has also been demonstrated on endothelial cells
(12). Finally, a combination of two or all of the
mechanisms listed above could result in a condition in which no net
increase or decrease of cell surface receptor expression would occur.
From a physiological point of view, a substantial increase of the
transport rate of TNF- into brain under inflammatory conditions may
be undesirable, considering the potent cytotoxic effect of the
cytokine. However, the amount of the cytokine transported would
increase proportionately with plasma concentrations, as long as the
concentrations stay within a range seen in physiological conditions.
These concentrations are insufficient to saturate the BBB transport
system for TNF-
(Fig. 1). Here, the rate of BBB transport after an
LPS stimulus (Fig. 6) was measured at perfusate TNF-
tracer
concentrations of ~1 nM, which already correspond to high levels seen
in conditions like sepsis (48).
In conclusion, our data strengthen the concept that receptor-mediated
transport of TNF- at the BBB occurs under native conditions and
after systemic immune stimulation. This could form part of a feedback
regulation between the immune system and the CNS, analogous to a
feedback loop regulating food intake, which seems to be mediated by the
BBB transport of leptin and may be disturbed in obesity (22). The transport of the cytokine across the BBB would
complement the well described direct effect of circulating TNF-
on
the endothelial cells (29), which releases soluble
mediators such as prostaglandins and nitric oxide and activates the
hypothalamo-pituitary-adrenal axis.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the expert technical assistance of G. Hohorst and S. Schaefer-Dewald. The plasmid containing the GLUT1 cDNA was kindly provided by Dr. R. Boado, and the C1q cDNA probe was a gift from Dr. W. Schwaeble. We are indebted to Drs. K. Bauer and J. Weidanz for stimulating discussions.
![]() |
FOOTNOTES |
---|
This research was supported in part by the Deutsche Forschungsgemeinschaft (SFB 297). B. Osburg is the recipient of a stipend from the Daimler-Benz Foundation, Germany.
Address for reprint requests and other correspondence: U. Bickel, Dept. of Pharmaceutical Sciences, Texas Tech Univ. Health Sciences Center, School of Pharmacy, 1300 Coulter Dr., Amarillo, Texas 79106 (E-mail: ubickel{at}ama.ttuhsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 31, 2002;10.1152/ajpendo.00436.2001
Received 27 September 2001; accepted in final form 11 July 2002.
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