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The Journal of Neuroscience, 2000, 20:RC114:1-6
RAPID COMMUNICATION
Reciprocal Regulation of the Junctional Proteins Claudin-1 and
Connexin43 by Interleukin-1 in Primary Human Fetal Astrocytes
Heather S.
Duffy1,
Gareth R.
John2,
Sunhee C.
Lee2,
Celia F.
Brosnan1, 2, and
David C.
Spray1
Departments of 1 Neuroscience and
2 Pathology, Albert Einstein College of Medicine, Bronx,
New York 10461
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ABSTRACT |
Vertebrate tissues use multiple junctional types to establish and
maintain tissue architecture, including gap junctions for cytoplasmic
connectivity and tight junctions (TJs) for paracellular and/or cell
polarity barriers. The integral membrane proteins of gap junctions are
connexins, whereas TJs are a complex between occludin and members of a
recently characterized multigene family, the claudins. In normal brain,
astrocytes are coupled by gap junctions composed primarily of
connexin43 (Cx43), whereas TJs have not been detected in these cells.
We now show that treatment of primary human astrocytes with the
cytokine interleukin-1 (IL-1) causes rapid induction of
claudin-1, with an expression pattern reciprocal to loss of Cx43.
Treatment also led to protracted downregulation of occludin but no
change in expression of zonula occludens proteins ZO-1 and -2. Immunofluorescence staining localized claudin-1 to cell membranes in
IL-1-treated astrocytes, whereas freeze-fracture replicas showed
strand-like arrays of intramembranous particles in treated cells
resembling rudimentary TJ assemblies. We conclude that in human
astrocytes, IL-1 regulates expression of the claudin multigene
family and that gap and tight junction proteins are inversely regulated
by this proinflammatory cytokine. We suggest that in pathological
conditions of the human CNS, elevated IL-1 expression fundamentally
alters astrocyte-to-astrocyte connectivity.
Key words:
gap junction; tight junction; cytokine; astrocyte; CNS; human
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INTRODUCTION |
The
cytokine interleukin-1 (IL-1) is found at low levels in the
CNS, where its expression is restricted to specific neuronal tracts (Breder et al., 1988 ). However, levels of IL-1
increase dramatically in a number of different inflammatory and
degenerative conditions, with expression localized to activated
microglia and macrophages. Experiments both in vivo and
in vitro have strongly implicated a role for this cytokine
in pathogenesis of CNS disease (for review, see Rothwell et al., 1997).
IL-1 is a key activator for human astrocytes in vitro
(Lee et al., 1993) and has been implicated in the induction of reactive
astrogliosis, a common response to brain injury (Giulian et al., 1988).
Studies using in vivo models have suggested that reactive
astrogliosis is associated with the development of a CNS environment
less permissive for the movements of solutes (Nicholson and Sykova,
1998), but the mechanisms underlying this are unknown. One possibility
is that changes in astrocyte-to-astrocyte connectivity play a role in this response.
In the CNS, distinct specialized junctional complexes mediate
cell-cell connectivity. Intercellular communication is mediated by gap
junctions, which allow the direct exchange of small molecules between
cells (Spray et al., 1999). Gap junctions may also form a
macromolecular signaling complex, or nexus, at appositional membranes
via interaction of the cytoplasmic domain with the scaffolding protein
zonula occludens-1 (ZO-1) (Giepmans and Moolenaar, 1998; Toyofuku et
al., 1998). In contrast, tight junctions (TJs) are complexes of
proteins that mediate paracellular sealing, thereby restricting
diffusion of fluid and small molecules in the extracellular space
("barrier function") and also function to establish and maintain
cell polarity ("fence function") (Tsukita and Furuse, 1999).
Although the first intramembrane TJ-associated protein identified was
the 65 kDa protein occludin (Furuse et al., 1993), recent studies have
also strongly supported a role for members of a novel family of
integral membrane proteins, the claudins (Furuse et al., 1998a,b) such
that TJ formation is now thought to involve an interaction between
occludin and one or more of the claudins. The carboxyl terminus of
claudin, in turn, associates with the second PDZ domain of ZO-1,
linking the complex to a cellular scaffold (Tsukita et al., 1999).
In the normal CNS, gap junctions composed primarily of connexin43
(Cx43), with a minor contribution by other connexins such as Cx30,
Cx40, Cx45, and Cx46 (Dermietzel, 1996; Spray et al., 1998), abundantly
interconnect human cortical astrocytes, linking these cells into a
functional syncytium. In contrast, TJs have not been noted among these
cells (Massa and Mugnaini, 1985; Rash et al., 1996). However, we now
show that IL-1 causes a rapid and robust induction of the
TJ-associated protein, claudin-1 (Cln-1) with a temporal pattern
reciprocal to the previously reported IL-1-induced loss of Cx43 in
the same cells (John et al., 1999). Striking morphological changes at
the cell membrane accompanied this response. Thus, a switch from gap
junction to TJ-associated proteins and morphology occurs under
conditions in which IL-1 is elevated, which may alter intercellular
and extracellular diffusion and thereby contribute to the dysfunctional
state associated with inflammatory or degenerative conditions of the CNS.
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MATERIALS AND METHODS |
Astrocyte cultures and cytokines. Human fetal
cortical astrocyte cultures were established, and purity (>98%) was
determined as described previously (Lee et al., 1992). Tissue
collection was approved by the Albert Einstein College of
Medicine Institutional Clinical Review Committee (CCI number
94-32). Recombinant human IL-1 was a gift from National Cancer
Institute Biological Response Modifiers Program (Bethesda,
MD). Recombinant human IL-1Ra, the endogenous IL-1 receptor
antagonist, was from R & D Systems (Minneapolis, MN).
Immunostaining. For primary antibodies (except occludin),
cells were fixed 5 min in 1% paraformaldehyde, rinsed, then blocked 30 min with PBS + 10% normal goat serum + 0.4% Triton X-100. For occludin, cells were fixed in 2% paraformaldehyde incubated in ethanol
and acetic acid (95:5 v:v) 20 min at  20°C and blocked with 10%
goat serum in PBS 30 min at RT. Cells were stained overnight at 4°C
with primary antibodies against junctional proteins (Cln-1, ZO-1, ZO-2,
ZO-3, and occludin; Zymed, South San Francisco, CA; Cx43 and ZO-3,
Chemicon, Temecula, CA) diluted 1:1000 in blocking solution (see
above). After rinsing in PBS with 0.4% Triton X-100 (except for
occludin staining, in which no Triton was used), cells were incubated 1 hr at RT with secondary antibodies conjugated to Alexa 488 or 546 (Molecular Probes, Eugene, OR), examined on a Nikon Eclipse TE300
microscope and photographed using a SPOT-RT digital camera (Diagnostic
Imaging) with phase-contrast and epifluorescence optics.
SDS-PAGE and immunoblotting. Confluent astrocyte cultures
(treated as described in Results) were lysed in boiling 1×
SDS-PAGE loading buffer to obtain whole-cell extracts, which were then centrifuged, and pellets were discarded. For cell membrane extracts, astrocytes were lysed in 10 mM Tris-HCl buffer (4°C, pH
7.5), centrifuged, and pellets were resuspended in boiling 2× SDS-PAGE loading buffer. Proteins were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membrane as described (John et al., 1999). Parallel blots were probed with polyclonal antisera for
Cln-1, occludin, ZO-1, ZO-2, or ZO-3, or with monoclonal anti-Cx43 or
anti-ZO-3.
RT-PCR for human Cln-1. Total RNA was isolated from
confluent control or IL-1-treated cultures and subjected to RT-PCR
as previously described (John et al., 1999). Primers were based on reported sequence of human Cln-1: forward, 5'-AAC GCG GGG CTG CAG CTG
TTG-3'; and reverse, 5'-GGA TAG GGC CTT GGT GTT GGG T-3' (GenBank
accession numbers AF115546, AF134160, and AF101051). Human -actin
primers were obtained commercially (Clontech, Palo Alto, CA).
Conditions applied for PCR were: 95° for 5 min, 31 cycles of 95°C
for 1 min, 71.5°C for 1 min, 72°C for 1 min, and 72°C for 7 min.
Samples were separated by electrophoresis in ethidium bromide-impregnated 1.6% agarose gels. PCR product was identified by
cloning and sequencing of five independent clones using methods previously reported (John et al., 1999).
Thin section electron microscopy. Human astrocyte monolayers
cultured in 5% fetal calf serum were treated with 0 or 10 ng/ml IL-1 for 24 hr. Cells were fixed 1 hr at RT in 2.5% glutaraldehyde, 0.1 M cacodylate, and 0.1% tannic acid, post-fixed in
osmium tetroxide, dehydrated stepwise in graded alcohols to 100%, and
embedded in Epon. Ultrathin sections counterstained with lead citrate
and uranyl acetate were then examined on a JEOL 100CX transmission electron microscope.
Freeze-fracture electron microscopy. Human astrocytes grown
on 100 mm dishes were treated with 10 ng/ml IL-1 for 0 or 24 hr,
fixed in 2.5% glutaraldehyde in 0.1% cacodylate buffer 60 min at RT,
and rinsed in 0.1% cacodylate buffer (3× for 10 min). Cells were
brought stepwise to 20% glycerol in 0.1% cacodylate buffer, rapidly
frozen by immersion in ultracold Freon-22, and fractured in a
Cressington CFE-50 Freeze Etch unit. Samples were platinum shadowed at
a 45° angle, rotary shadowed with carbon, and examined on a JEOL
100CX transmission electron microscope, and stereo pairs were taken at
8° angles.
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RESULTS |
Immunolocalization of Cln-1 to cell membranes in IL-1 treated
astrocyte cultures
Localization of Cx43, Cln-1, occludin, and ZO-1, -2, and -3 were
examined in control and IL-1-treated primary human astrocyte cultures using immunofluorescence staining. In control cultures there
was robust expression of Cx43, particularly at cell membranes in which
punctate staining was observed at sites of cell-cell contact (Fig.
1A). Conversely, in the
same cultures Cln-1 staining was totally absent (Fig. 1C).
After treatment of cultures with 10 ng/ml IL-1 for 24 hr, Cx43 was
lost (Fig. 1B), which contrasted with the robust
expression of Cln-1, particularly at cell membranes (Fig.
1D). The striking change from Cx43 to Cln-1
expression after 24 hr of 10 ng/ml IL-1 treatment was particularly
evident when images obtained from double-labeled cells were
superimposed (Fig. 1E,F).
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Figure 1.
Immunofluorescence labeling of junctional
proteins in control (Ctrl) and Interleukin-1
(IL-1)-treated human astrocytes. Connexin43
(Cx43) labeling (red) is seen within the
cytoplasm and at cell-cell junctions (arrows) in
control cells (A), with cell nuclei
counterstained with DAPI (blue). Examination of the
phase image shows that the punctate staining is localized to cell-cell
contacts in control cells (G). IL-1 treatment
caused a complete loss of Cx43 (B). Double
labeling of cells for Cln-1 (green) showed no
staining in control cells (C), whereas treatment
with IL-1 caused Cln-1 staining to be evident at cell membranes of
treated astrocytes (D), although the phase image
(H) shows that Cln-1 is not uniquely
localized to sites of cell-cell contact. Note the striking
change in cell morphology from flat to stellate, which is a
characteristic effect of treatment with IL-1. Overlay of the
red and green channels is shown in
control (E) and IL-1-treated cells
(F). Scale bar, 25 µm.
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Expression of occludin and ZO-1 and -2 was also observed at cell
membranes in control cultures, and no substantive change was seen in
their localization after 10 ng/ml IL-1 treatment, although over a
period of ~30-55 hr of treatment the number of cells with occludin
localized to cell membranes gradually decreased. In cells that
maintained occludin expression, no change in intensity or membrane
localization was seen (data not shown). Expression of ZO-3 was not
detected in either control or cytokine-treated cultures using two
commercially available antibodies (data not shown).
IL-1 treatment of primary human astrocytes causes rapid and
robust induction of Cln-1
The patterns of expression of Cx43, Cln-1, occludin and ZO-1, -2, and -3 were examined in control and IL-1-treated primary human
astrocyte cultures using immunoblotting of whole-cell extracts (Fig.
2A). No signal for
Cln-1 was detected in control cultures; however, robust expression of a
single 22 kDa band corresponding to Cln-1 was observed within 6 hr
after addition of IL-1 (10 ng/ml) to cultures, reaching a plateau at
15 hr after treatment. The expression pattern of Cln-1 was reciprocal
to the dramatic downregulation of Cx43 that was observed within 6 hr
after treatment with IL-1, and Cx43 was not detected by
immunoblotting from 15 hr onward, in agreement with previous findings
(John et al., 1999). After IL-1 treatment there was progressive
downregulation of occludin over a more protracted time course (Fig.
2A). Expression of both ZO-1 and ZO-2 was detected in
control cultures and did not alter after IL-1 treatment, but as with
the immunolocalization studies, no expression of ZO-3 was detected
(negative data not shown).
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Figure 2.
IL-1 induces claudin-1 expression in human
fetal astrocytes. A, Western blot analysis of the
expression of Cx43 and Cln-1 after treatment with IL-1. Whole-cell
extracts of control or IL-1 (10 ng/ml)-treated astrocytes were
probed for Cln-1, Cx43, occludin and ZO-1, -2, and -3. No signal for
Cln-1 was detected in control cultures; in contrast, expression of a
single 22 kDa band corresponding to Cln-1 was observed within 6 hr
after addition of IL-1 to cultures, with expression reaching a
plateau at 15 hr. Levels of occludin were high in untreated cells but
declined commencing 15-30 hr after IL-1 treatment. Treatment with
IL-1 had no effect on expression of either ZO-1 or ZO-2.
B, Western blot analysis of membrane fractions in
control and IL-1 (10 ng/ml at 16 and 24 hr)-treated cells probed for
Cx43 and Cln-1. No signal for Cx43 was detected at 16 hr or 24 hr after
addition of IL-1 to cultures. In contrast, strong Cln-1 expression
was observed at both 16 and 24 hr after IL-1 treatment
(bottom panel). This signal for Cln-1 was
enriched in the membrane fraction (mb) compared with
whole-cell extract (wc). C,
Concentration-dependent effects of IL-1 on Cx43 and Cln-1 expression
as determined by Western blot analysis. Effects of IL-1 on Cln-1 and
Cx43 expression was detected at a dose as low as 10 pg/ml and were
significantly inhibited by cotreatment with the naturally occurring
IL-1 receptor antagonist (IL-1Ra, 500 ng/ml). Analysis by RT-PCR of
mRNA expression for Cln-1 in astrocytes exposed to IL-1 is shown in
D. Total RNA from untreated (control labeled
C) and IL-1-treated (16 hr) human astrocyte cultures
was subjected to first-strand reverse transcription followed by PCR for
human Cln-1 and -actin as described in Materials and Methods (lanes
labeled RT). As a control, the procedure was also
performed in the absence of reverse transcriptase (lanes labeled
N). No signal for Cln-1 was detected in untreated
cultures (top panel) although -actin
expression (bottom panel) was readily detected.
In contrast, in astrocyte cultures treated with 10 ng/ml IL-1 for 16 hr robust expression of a single 650 bp band was detected using primers
specific for human Cln-1.
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Immunoblotting of whole-cell extracts versus cell membrane fractions
from control and IL-1-treated cultures was used to determine whether
Cln-1 expressed after IL-1 treatment was associated with cell
membranes. As a control, these blots were also probed for Cx43. In
agreement with previous work from our laboratories, levels of the 46 and 44 kDa (P2 and P1) isoforms of Cx43 were enriched in membrane
fraction compared with whole-cell extract, in which the 42 kDa, or NP,
isoform of Cx43 predominated (Fig. 2B). Neither membrane fraction nor whole-cell extract contained detectable Cx43
signal at 16 and 24 hr after treatment of cultures with IL-1. In
contrast, whereas no signal for Cln-1 was detectable by immunoblotting either in whole-cell extracts or membrane fractions from control cultures, strong Cln-1 expression was observed at both 16 and 24 hr
after IL-1 treatment, and the signal was enriched in the membrane fractions.
Immunoblots of whole-cell extracts from astrocyte cultures demonstrated
that the effect of IL-1 on Cx43 and Cln-1 expression was
concentration-dependent and could be detected at doses as low as
10-100 pg/ml (Fig. 2C). In addition, the effects of the highest concentrations of IL-1 (10 ng/ml) were significantly inhibited by cotreatment with the naturally occurring selective IL-1
receptor antagonist (IL-1Ra, 500 ng/ml; Fig. 2C).
Induction of Cln-1 mRNA after IL-1 treatment of
human astrocytes
RT-PCR using specific primers demonstrated induction of Cln-1 mRNA
in human astrocyte cultures after 16 hr treatment with IL-1 (Fig.
2D). To confirm the identity of the PCR product,
bands were excised from the gel, cloned, and sequenced. Data obtained from five clones gave sequences >97% identical to GenBank sequence of
Cln-1/SEMP-1 cloned from Caco-2 and mammary epithelial cells (GenBank
accession numbers AF115546, AF134160, and AF101051). No band was
detected in PCR samples in the absence of reverse transcriptase. This
induction of Cln-1 mRNA is directly reciprocal to our previously
reported loss of Cx43 (John et al., 1999).
Freeze-fracture and thin-section electron microscopy
Control and IL-1-treated cell membranes were examined using
both transmission (TEM) and freeze-fracture (FF) electron microscopy. Membranes were well preserved, and on FF images multiple square arrays
(Fig. 3A,C), a classic
identifying marker for astrocytes (Massa and Mugnaini, 1985; Rash et
al., 1996), were seen in both control and treated cells. Examination of
both FF and TEM images showed a marked difference in the morphology of
cell membranes between control and IL-1-treated cells. In control
cells, FF images showed multiple E- and P-face gap junctional plaques
of varying sizes (Fig. 3A). Gap junctions were also found
between astrocytes using TEM (Fig. 3B). In contrast,
examination of cell membranes after treatment of cells with IL-1
showed no gap junctions, suggesting loss of all gap junction proteins.
Instead, FF images of membranes of IL-1-treated cells showed the
presence of strand-like arrays of ~10 nm particles at multiple sites
on P-face membranes (Fig. 3C). While these strands were
clearly not classical TJs, their morphology was reminiscent of
rudimentary tight junction strands. In agreement with these findings,
multiple sites of close membrane apposition were observed in
IL-1-treated astrocytes using TEM, which again did not resemble
classical TJs (Fig. 3D). These ultrastructural data indicate
that treatment with IL-1 induces morphological changes in primary
human astrocyte junctional membranes that parallel the switch from
expression of Cx43 to Cln-1 in these cells.
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Figure 3.
Ultrastructural examination of control and
IL-1-treated primary human astrocytes. Control (A, B)
and IL-1 treated (C, D) astrocytes were examined by
FF (A, C) and TEM (B, D). Astrocytes were
identified by the presence of well preserved square particle arrays on
their P-faces (A, C, bottom right insets). Extensive gap
junctions were found between control cells by both FF
(A, gap junction shown at higher magnification in
top left inset) and TEM (B). After
IL-1 treatment no gap junctions were seen. Instead, FF images showed
strands of 10 nm intramembranous particles reminiscent of rudimentary
tight junctions (C, higher magnification in top
left inset) and TEM revealed areas of close membrane apposition
(D). A lower power view of a membrane from an
IL-1-treated cell is seen in E, demonstrating the
large numbers of particle strands found on these cells. Scale bars:
A and C insets, 0.05 µm.
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DISCUSSION |
This study demonstrates that treatment of primary human
astrocytes with the proinflammatory cytokine IL-1 results in the specific induction of an integral TJ protein, claudin-1, over a time
course similar to the reciprocal loss of the gap junction protein Cx43.
Furthermore, this change in expression of junctional proteins is
associated with morphological changes in the appositional astrocyte
membranes, such that the gap junctions present in control cultures are
apparently replaced, after IL-1 treatment, by loose particle strands
reminiscent of rudimentary TJs. Although structural features of gap and
TJs and expression of several protein components of these junctions are
known to be susceptible to environmental stimuli (Citi, 1993;
Spray et al., 1999), this is the first report of such an effect on
claudin expression in CNS.
There is increasing evidence that both gap junctions and TJs are
macromolecular assemblies consisting of integral membrane proteins and
other proteins that bind to them. In the case of TJs, occludin, members
of the claudin family, and junction adhesion molecule (JAM) appear to
be the membrane-embedded proteins (Fanning et al., 1999), with zonula
occludens family members (ZO-1, -2, and -3), cingulin, p130, 7H6, and
possibly symplekin and ZA-1TJ being cytosolic binding partners (for
review, see Mitic and Anderson, 1998). In the case of Cx43, binding by
v-src and by ZO-1 have been demonstrated, suggesting that gap junctions
may also form a macromolecular structure (Giepsman and Moolenar, 1998;
Toyofuku et al., 1998; Loo et al., 1999). In this regard, it is
interesting to note that connexin and claudin multigene families share
analogous structural and physical properties. They both exist as four
transmembrane domain (tetraspan) proteins with intracellular amino and
carboxyl termini, and both bind the second PDZ domain of the
membrane-associated guanylate kinase protein ZO-1 within the
cell (Giepmans and Moolenaar, 1998; Itoh et al., 1999). Connexins are
known to form hexamers surrounding a hydrophilic pore through which
ions and small molecules pass from cell to cell; claudins have been
proposed to form hexamers that may create paracellular pores for ion
and molecular diffusion in the spaces between cells (Tsukita and
Furuse, 1999).
In our experiments, the patterns of expression of Cln-1 and Cx43
protein in control and IL-1-treated cultures showed minimal overlap,
with Cx43 rapidly being replaced by Cln-1 after IL-1 treatment. The
observation that the expression pattern of Cln-1 was reciprocal to that
of Cx43 suggests that Cln-1 may substitute for Cx43 at the scaffolding
complex localized to astrocyte cell membranes. Together with our
findings that levels of ZO-1 do not change in IL-1-treated
astrocytes, this suggests the interesting possibility that exposure to
IL-1 may result in the replacement of one tetraspan hexamer composed
of connexons with another tetraspan hexamer composed of claudins and/or
occludin at the same location in the scaffold localized to astrocyte
cell membranes.
The switch in expression from one type of junctional protein to another
is expected to result in profound effects on intercellular communication and extracellular diffusion in the CNS under inflammatory conditions. Under normal conditions, astrocytes are highly coupled by
gap junctions formed primarily of Cx43, allowing for passage of
information directly between cells. However, under inflammatory conditions this pathway could also allow the passage of apoptotic signals leading to widespread cell death (Lin et al., 1998). Thus, IL-1 induced loss of Cx43 could provide a protective mechanism by
which apoptosis is minimized in the damaged CNS. Alternatively, the
signaling domain associated with Cx43 in normal astrocytes may be
damaging to the cell under pathological conditions.
Although astrocytes are not normally connected by TJs, our results
indicate that they express the tight junction-associated proteins ZO-1,
ZO-2, and occludin, but lack expression of Cln-1. The electron
micrographs of astrocyte appositional membranes after Cln-1 induction
by IL-1 treatment showed small regions of close membrane apposition,
and in FF we observed short linear particle arrays in P face membrane,
but we did not observe membrane fusions or the elaborate tight
junctional strands reported in Cln-1 transfected fibroblasts (Furuse et
al., 1998b). The fact that induction of Cln-1 in IL-1-treated
astrocytes did not form complete tight junctional strands suggests that
some component of the tight junction strands may have been missing or
nonfunctional in these cells. This possibility is further suggested by
the decline in occludin expression after IL-1 treatment, which began
to occur at about the same time that Cln-1 expression was induced.
Because it has been suggested that the tight junction complex may
contain occludin in addition to claudin, it is possible that the mature
tight junctional strands require this or another integral membrane protein.
We speculate that the rudimentary tight junction strands detected in
human astrocytes after IL-1 treatment may perform a function of
paracellular sealing, thereby contributing to the reduced effective
volume of the extracellular space and the increased tortuosity observed
after inflammatory lesions (Nicholson and Sykova, 1998). Alternatively,
or in addition, the junctional assemblies seen in astrocytes after
IL-1 treatment may represent specialized microdomains that
constitute a signalsome with novel roles in intercellular or
extracellular signaling or adhesion (Denker et al., 1996; Saha et al.,
1998).
We conclude that the junctional complexes in human astrocytes are
dynamic structures that can be modified by environmental stimuli,
including exposure to inflammatory cytokines and that in pathological
conditions of the human CNS, elevated IL-1 expression significantly
alters glial connectivity. The combined action of IL-1 on Cx43 and
Cln-1 would be expected to reduce intercellular coupling and to
decrease bulk fluid movement in the extracellular space. Both actions
could contribute to local confinement of inflammatory responses,
thereby limiting spread to surrounding uninvolved tissue.
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FOOTNOTES |
Received July 7, 2000; revised Sept. 5, 2000; accepted Sept. 11, 2000.
This work was supported by United States Public Health Service Grants
MH55477 (S.C.L.), NS11920 (C.F.B.), and NS07512 and NS34931 (D.C.S.),
CA09475 and NS07098 (H.S.D.), and National Multiple Sclerosis Society
Fellowship FG1355 (G.R.J.). We thank Dr. Karen Weidenheim (Director of
the Human Fetal Tissue Repository), Dr. Meng-Liang Zhao and Wa Shen for
culture preparations, Dr. Frank Macaluso and the Albert Einstein
College of Medicine Analytical Imaging Facility, and Yvonne
Kress of the Pathology Department EM Facility for their assistance with
sample preparation.
H.D. and G.J. contributed equally to this work.
Correspondence should be addressed to Dr. David C. Spray, Department of
Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, Bronx, NY 10461. E-mail: spray{at}aecom.yu.edu.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2000, 20:RC114 (1-6). The
publication date is the date of posting online at
www.jneurosci.org.
| |
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Copyright © 2000 Society for Neuroscience 0270-6474/00/$05.00/0
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