(Journal of Leukocyte Biology. 2000;68:545-552.)
© 2000
by Society for Leukocyte Biology
Monocyte:astrocyte interactions regulate MCP-1 expression in both cell types
Anuska V. Andjelkovic*,
Danielle Kerkovich and
Joel S. Pachter*
* Blood-Brain Barrier Laboratory, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut
Correspondence: J. S. Pachter, Blood-Brain Barrier
Laboratory, Department of Pharmacology, University of Connecticut
Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail: PACHTER{at}SUN.UCHC.EDU
 |
ABSTRACT
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As astrocytes are a source of monocyte chemoattractant protein-1
(MCP-1) and lie in close apposition to brain microvessels, interactions
between astrocytes and infiltrating monocytes might regulate production
of this chemokine. To investigate this possibility, a
monocyte:astrocyte co-culture model was utilized to assess the
respective roles of these two cell types in regulating MCP-1
production. Results indicate that, while neither monocytes nor
astrocytes alone produce detectable levels of MCP-1, co-culture of
these two cell types results in time-dependent production of this
chemokine. Such production requires de novo protein
synthesis and is dependent on physical contact between monocytes
and astrocytes, involving engagement of the cell-adhesion molecules
ICAM-1 and VCAM-1. Additionally, interleukin 1-beta (IL-1ß) and tumor
necrosis factor-alpha (TNF-
) are soluble mediators of this response.
These findings imply that monocyte extravasation into the CNS may be
critically regulated at the blood-brain barrier by specialized
monocyte:astrocyte interactions.
Key Words: blood-brain barrier neuroinflammation chemokines
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INTRODUCTION
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Considerable attention has focused recently on chemokines as
critical effectors of inflammatory processes in the central nervous
system (CNS) [1
2
3
4
]. In particular, the ß chemokine
monocyte chemoattractant protein-1 (MCP-1) has been evidenced
consistently to be associated with animal models of CNS inflammatory
disease [5
] and the human neuroinflammatory condition
multiple sclerosis (MS) [6
, 7
]. MCP-1
expression was initially described to be transiently upregulated in the
CNS in a rat model of acute experimental autoimmune encephalomyelitis
(EAE) in a manner that temporally correlated with symptomatology
[8
]. CNS levels of MCP-1 have been determined
subsequently to be dramatically increased during the relapsing phase of
chronic relapsing EAE and highly localized to astrocytes in this
disease model as well as in MS lesions [6
,
7
, 9
]. Administration of antibody to MCP-1
has been shown, furthermore, to effectively mitigate the severity of
relapsing EAE [10
], underscoring the prominence of this
particular chemokine in mediating clinical inflammatory disease of the
CNS. Lastly, the central role of astrocyte-derived MCP-1 in stimulating
leukocyte transendothelial migration has been highlighted recently in a
tissue culture model [11
].
Despite the extreme importance attributed to MCP-1 in regulating
leukocyte extravasation and CNS inflammation, the signal(s) inciting
astrocyte production of this chemokine remain unclear. A possible clue
to this might be provided from the anatomical arrangement of astrocytes
around cerebromicrovessels. In this regard, microvessels in the brain
are subtended by the glial limitans, which is composed of a
nearly continuous array of astrocyte foot processes that project onto
the subendothelial basement membrane [12
,
13
]. By virtue of being positioned at the
endothelial:brain interface, astrocytes are thus likely to come into
contact with invading leukocytes that have newly penetrated the
blood-brain barrier (BBB). Such contact could result in at least
temporary interaction between the two cell types. Indeed, results from
this and another laboratory have indicated specific, adhesion
molecule/receptor-mediated attachment between astrocytes and monocytic
cells [14
, 15
]. Adhesive events, in turn,
could provide the necessary signal(s) to stimulate MCP-1 production in
monocytes, astrocytes, or both cell types. A precedent for cell
adhesion-mediated induction of MCP-1 expression has, in fact, already
been established. For example, adherence of monocytes to cultured human
umbilical vein endothelial cells (HUVECs) has been shown to stimulate
MCP-1 production in the latter cell type [16
].
Additionally, de novo synthesis of MCP-1 has been shown to
be induced in monocytes during transendothelial migration in
vitro [17
], presumably as a result of a specialized
interaction with endothelial cells during diapedesis. Co-cultures of
monocytes and fibroblasts have also been shown to exhibit augmented
MCP-1 production, further implying that cell:cell interaction can drive
MCP-1 production [16
].
In light of these considerations, the objective of this study was to
determine whether monocyte:astrocyte interaction(s) governed expression
of MCP-1 in either of these two cells. Results indicate that monocyte
attachment to astrocytes can signal expression of MCP-1 in both cell
types and that this induction in chemokine expression is mediated, in
part, by proinflammatory cytokines, interleukin-1 beta (IL-1ß) and
tumor necrosis factor-alpha (TNF-
). Moreover, this attachment-driven
stimulation of MCP-1 expression appears to require engagement of the
adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and
vascular cell adhesion molecule 1 (VCAM-1). These findings point toward
monocyte:astrocyte adhesive interaction(s) at the BBB as playing a
critical role in the generation and maintenance of CNS inflammatory
lesions.
 |
MATERIALS AND METHODS
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Human astrocyte cultures
Human fetal CNS tissue (1823 weeks) was obtained from the
elective termination of intrauterine pregnancies from otherwise normal,
healthy females. The astrocyte isolation procedure was done essentially
as described by Liu et al. [18
] with minor
modifications. Briefly, human fetal CNS tissue was separated from the
meninges, dissociated, and digested for 20 min at 37°C in a solution
containing 0.025% trypsin (Sigma, St. Louis, MO), 1 x
HEPES-buffered Earls balanced salt solution (He-EBSS, Gibco-BRL,
Paisley, UK), and DNAse I (Sigma). The resulting cell suspension was
then passed sequentially through 250 µm and 150 µm nylon filters
(Becton Dickinson, Rutherford, NJ), and the filtrate was centrifuged at
800 g for 8 min. The cell pellet was resuspended in complete
astrocyte media [Dulbeccos modified Eagles medium (DMEM),
Gibco-BRL], 1 x antibiotic/antimycotic (Gibco-BRL), 10%
heat-inactivated fetal calf serum (FCS; Gemini, Bio-Products,
Calabasos, CA), 2 mM glutamine (Sigma), counted, and seeded at
a density of 2.5 x 107 per 75 cm2 tissue
culture flask. Two weeks after the initial plating, flasks were shaken
at 220 RPM for 2 h at 4°C on a horizontal rotary shaker, after
which the supernatant containing mainly microglia was removed. Cultures
were then shaken for an additional 18 h at 37°C, and the
resulting supernatant, containing predominantly neurons, was again
removed. Cell-culture purity was determined by immunocytochemistry
using a monoclonal anti-human glial fibrillary acidic protein (GFAP)
antibody (Boehringer Mannheim, Mannheim, Germany). Astrocyte cultures
(35 passages) were
99% positive for GFAP, and all experiments
were observed in this time period.
Isolation of human monocytes
Peripheral blood monocytes were isolated from blood samples
taken from healthy volunteers at the University of Connecticut Health
Center, by a modification of the Percoll density gradient method of
Denholm and Wolber [19
], as previously described
[20
, 21
]. Monocyte preparations isolated in
this manner typically contained 8590% monocytes as determined by
dual Giemsa staining and the expression of CD14 by immunofluorescence.
Monocyte:astrocyte cell co-cultures
Astrocytes (third passage) were plated into eight-well
chamber slides (Becton Dickinson) at a density of 2.5 x
105/well. After
18 h, astrocytes were washed and
incubated with monocytes (5x105/well in 400 µl) in assay
media (DMEM, 5% FCS, 1x antibiotic/antimycotic, 2 mM glutamine) for
1, 3, 6, 9, 12, 18, 24, and 48 h at 37°C in 5% CO2.
To determine whether the monocyte:astrocyte co-cultures required
cell-to-cell contact for stimulation of MCP-1 production, Transwell
culture inserts (Costar, Cambridge, MA; 0.45 µm porosity) were
utilized, which physically separated the cells into upper and lower
compartments. Astrocytes were cultured in the lower compartment, grown
to a density of 2.5 x 105, washed twice, and
incubated in assay media. Monocyte suspensions containing 5 x
105 cells in 400 µl of assay media were added to the
upper compartment. After the desired time of coincubation at 37°C,
the media from upper and lower compartments were collected and stored
at -20°C until assayed.
Quantification of MCP-1 production by enzyme-linked immunosorbent
assay (ELISA)
Cell supernatants were quick-thawed for analysis by ELISA, and
the level of MCP-1 protein detected using the sandwich-type immunoassay
kit (R&D Systems, Minneapolis, MN) was according the manufacturers
instructions. The lower level of detection for MCP-1 was 32 pg/ml.
Immunocytochemistry
Monocyte:astrocyte co-cultures were fixed in 4%
paraformaldehyde for 30 min at 20°C. To minimize nonspecific binding,
cells were treated for 1 h at 20°C with blocking buffer of the
following composition: 5% normal goat serum, 0.05% Tween in 0.02 M
phosphate-buffered saline (PBS), pH 7.4. For detection of MCP-1 alone,
samples were incubated with mouse anti-human MCP-1 antibody (R&D
Systems) overnight at 4°C. Following incubation with anti-MCP-1, the
samples were washed in PBS, after which they were exposed to
fluorescein-conjugated goat anti-mouse antibody (Sigma) for 2 h at
20°C and then rinsed again in PBS. In the case of
double-immunolabeling, cells were next reacted with rabbit anti-cow
GFAP antibody (DAKO, Carpinteria, CA) to label astrocytes, washed in
PBS, then exposed to Texas Red-conjugated goat anti-rabbit antibody
(Vector Labs, Burlingame, CA). Control slides were processed in a
similar manner, except for the exclusion of primary antibodies. All
samples were viewed with a Zeiss LSM 410 confocal microscope.
In situ hybridization
Cytoplasmic detection of MCP-1 mRNA was performed using a
combination of standard methodologies. In brief, co-cultures of
astrocytes and monocytes were initially fixed in 4% paraformaldehyde
for 30 min at 20°C, rinsed in PBS, and stored overnight in 70%
ethanol at 4°C. Subsequently, co-cultures were treated with
proteinase K (1 µg/ml; Boehringer Mannheim) in 0.05 M Tris/EDTA
buffer (pH 7.6) for 1 h at 37°C, rinsed with Tris/EDTA buffer,
and post-fixed in 4% paraformaldehyde for 20 min at 20°C.
Co-cultures were then washed with Tris/EDTA buffer and prehybridized
for 1 h at 37°C in a solution (prehybridization buffer)
consisting of 50% (v/v) deionized formamide (Sigma), 4 x SSC
(1x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 500
µg/ml heat-denatured herring sperm DNA (Boehringer Mannheim), 250
µg/ml yeast tRNA (Boehringer Mannheim), and 10% (w/v) dextran
sulfate (Sigma). Next, prehybridization buffer was decanted, and cells
overlaid with fresh buffer containing a digoxigenin 3', 5' end-labeled,
30 nucleotide-probe cocktail complimentary to human MCP-1 (290 ng/ml;
R&D Systems). After overnight hybridization at 37°C, co-cultures were
consecutively rinsed three times for 10 min at 37°C in each of the
following buffers: 4 x SSC, 2 x SSC, and 0.2 x SSC,
all containing 30% (w/v) deionized formamide. The hybridization
reaction was visualized with a rhodamine-conjugated antidigoxigenin
antibody (1:100 dilution; Roche, Basel, Switzerland).
Antibody-blocking experiments
To determine the possible role of adhesion molecules and
cytokines in MCP-1 production resulting from monocyte:astrocyte
interaction, purified neutralizing monoclonal antibodies against ICAM-1
and VCAM-1 (R&D Systems) and polyclonal rabbit antibodies against
IL-1ß and TNF-
(Endogen, Woburn, MA) were used. Astrocytes and
monocytes were incubated separately with assay media containing 10%
mouse serum or 10% rabbit serum for 6 h. Monocytes were then
added to astrocytes in assay media along with purified antibody (10
µg/ml for single antibody treatment; 20 µg/ml for combined
treatment). After 24 h of incubation at 37°C, the media was
collected and stored at -20°C until assayed.
Statistical analysis
Data were analyzed using InStat (Graphpad Software, San Diego,
CA) and SPSS (SSPS Inc., Chicago, IL) software. Differences
between means were assessed directly by paired Students
t-test. For the analysis of multiple groups, overall
differences were assessed by analysis of variance (ANOVA), and
individual group differences were determined post hoc using
Dunnets procedure or least significant difference (LSD) test.
 |
RESULTS
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MCP-1 production in separate monocyte and astrocyte cultures and
monocyte:astrocyte co-cultures
Figure 1
shows the experimental model used to assess regulation of MCP-1
production in monocyte:astrocyte co-cultures. Association between the
two cell types is readily observed and could play a role in signaling
MCP-1 expression. Immunofluorescence analysis indicated that virtually
all mononuclear cells attached to the astrocyte substratum were
CD14-positive (unpublished results), thus revealing their monocyte
identity. As seen in Figure 2
, neither isolated monocytes nor astrocytes produces detectable
levels of MCP-1, as judged by the amount of this chemokine secreted
into the culture supernatant. In response to stimulation with IL-1ß,
however, both cell types significantly augment their production of
MCP-1. This stimulated production of MCP-1 is achieved also when
monocytes and astrocytes are co-cultured in the absence of any
exogenous IL-1ß. Figure 3
further reveals cytoplasmic expression of MCP-1 in these two
cells. As can be seen clearly, there is heightened cytoplasmic
expression of MCP-1 in monocytes and astrocytes as a consequence of
IL-1ß treatment and co-culture of the two cell types, paralleling the
increase in secreted chemokine detected under these conditions.
Notably, cytoplasmic expression of MCP-1 appeared more intense in
monocytes than in astrocytes under conditions of IL-1ß treatment or
co-culture. It is important to bear in mind, however, that this
discrepancy might reflect simply significant differences in the size
and shape of these two cell types and, thus, unequal dissolution of the
MCP-1 signal. Alternatively, a dissimilar responsiveness in the
magnitude of MCP-1 induction might exist.

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Figure 1. Co-culture of human monocytes and astrocytes. Astrocytes
(2.5x105/well), plated in eight-well chamber slides for
18 h, were then incubated with freshly isolated monocytes
(5x105) in assay medium for 48 h. Arrows demarcate
monocytes, and arrowheads indicate astrocytes. Note the extensive
association of monocytes with astrocyte processes.
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Figure 2. MCP-1 production by separate monocyte and astrocyte cultures and by
monocyte:astrocyte co-cultures. MCP-1 levels in culture supernatants
were evaluated by ELISA, as described in Materials and Methods.
Separate cultures of untreated monocytes or astrocytes do not produce
detectable levels of MCP-1 except when stimulated with IL-1ß (10
ng/ml, 48 h). On the contrary, untreated monocyte:astrocyte
co-cultures produced significant levels of MCP-1. Data are expressed as
mean ± SE of three experiments (n=6).
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Figure 3. Immunocytochemical detection of MCP-1 expression in monocyte:astrocyte
co-cultures. Monocytes and astrocytes were cultured alone or together
for 24 h in the presence or absence of IL-1ß (10 ng/ml).
Afterward, cells were fixed in paraformaldehyde and then processed for
immunocytochemistry using a monoclonal anti-human MCP-1 antibody,
followed by fluorescein-conjugated goat anti-mouse IgG. Micrograph
shows MCP-1 expression under the following conditions: (A) untreated
monocytes; (B) monocytes treated with IL-1ß; (C) untreated
astrocytes; (D) astrocytes treated with IL-1ß; and (E)
monocyte:astrocyte co-culture. Original scale bar = 80 µ.
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To exclude the possibility that MCP-1 released from astrocytes was
merely adhering to the surface of monocytes, and thus giving the false
impression that both cell types were expressing MCP-1 in co-culture,
confocal microscopy and in situ hybridization were
additionally performed (Fig. 4
). Optical sectioning through the entirety of the cytoplasm of
individual monocytes revealed MCP-1 immunoreactivity to be dispersed
throughout the interior of these cells, strongly suggesting that they
exhibit a de facto induction of MCP-1 expression. This
interpretation was corroborated by detection of MCP-1 messenger RNA in
monocytes and astrocytes.

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Figure 4. MCP-1 is produced by monocytes and astrocytes in co-cultures.
Monocyte:astrocyte co-cultures were established for 24 h, fixed,
and then subject to (A) immunocytochemical detection of MCP-1 and GFAP
[the latter to label astrocytes (red)], followed by confocal
microscopy or (B) in situ hybridization of MCP-1 mRNA.
Confocal optical sections through three different planes (e.g., bottom,
middle, and top) of individual monocytes (green) reveal MCP-1
immunoreactivity throughout the cytoplasm of these cells, indicating
that MCP-1 is not adsorbed merely to the monocyte surface. In
situ hybridization analysis is consistent with this depiction,
indicating heightened MCP-1 mRNA expression in monocytes (arrowheads)
and astrocytes (arrows) in co-cultures, compared with that in single
cultures of either cell type. Original scale bar = 20 µ.
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Additional confirmation of the argument that monocytes and astrocytes
were sources of MCP-1 in co-cultures was provided by varying the number
of one cell type and keeping the other constant. Figure 5
reveals that an increase of monocytes or astrocytes produced
gradually higher levels of chemokine, reflecting the propensity of each
of these cell types to produce MCP-1 after being cultured together.
Presumably, such heightened MCP-1 production was the result of
increased frequency of direct or indirect monocyte:astrocyte
interactions at higher plating densities. It is further noteworthy to
point out here that, in contrast to their apparently less-intense
cytoplasmic staining of MCP-1 (Fig. 3)
, astrocytes were observed to
secrete MCP-1 at a level that approximated or was greater than that
achieved by monocytes, following initiation of co-culture (compare the
relative rates of rise of detectable MCP-1 in co-culture supernatants
when astrocyte number was varied vs. when the number of monocytes was
altered). A priori, this may point to distinct physiological
roles for astrocyte-derived vs. monocyte-derived MCP-1 within the CNS.

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Figure 5. Effect of varying numbers of monocytes or astrocytes on MCP-1
production in monocyte:astrocyte co-cultures. In one set of experiments
(solid bars), varying numbers of monocytes were layered atop a constant
number of astrocytes (2x105). In the other set (shaded
bars), a constant number of monocytes (5x105) were layered
atop varying numbers of astrocytes. MCP-1 levels were evaluated by
ELISA in culture supernatants after 48 h of co-culture. Data are
expressed as mean ± SE of three experiments
(n=6).
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MCP-1 production shows a time-dependent increase in
monocyte:astrocyte co-cultures
To gain a better appreciation of the kinetics of MCP-1 induction
in monocyte:astrocyte co-cultures, a time course analysis was
performed, and the results are depicted in Figure 6
. Detectable levels of MCP-1 in culture supernatant were first
observed at 6 h post co-culture and rose steadily thereafter until
at least 48 h (the last time point determined). That MCP-1 was not
detected until at least 6 h after co-culture commenced, nor
observed in isolated monocyte or astrocyte cultures even as much as
48 h after plating (Figs. 2
and 3)
, may be taken as indication
that neither of these two cell types was significantly activated by the
isolation/culture process. Instead, stimulated MCP-1 production in
co-cultures may be argued to have resulted solely from interaction
between monocytes and astrocytes. Confirmation of this argument was
provided by observations that immunocytochemical detection of MCP-1
expression, by monocytes or astrocytes, was not apparent until
monocytes had migrated near or onto the astrocyte surface (unpublished
results).

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Figure 6. Time course of MCP-1 production in monocyte:astrocyte co-cultures.
Co-cultures were established and allowed to remain for the indicated
periods of time, after which supernatants were retrieved and assayed
for MCP-1 levels. MCP-1 was first detected 6 h post initiating
co-culture and, thereafter, increased steadily. Data are expressed as
mean ± SE of three experiments (n=6).
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The delay in detection of secreted MCP-1 further suggested that the
induction of MCP-1 expression might be dependent on de novo
protein synthesis. This implication was affirmed using the translation
inhibitor cycloheximide, as shown in Figure 7
. Cycloheximide treatment completely inhibited the induction of
MCP-1 expression seen up to 9 h after establishing
monocyte:astrocyte co-cultures. This inhibition was not because of
toxicity, as indicated by the continued ability of cells in co-culture
to exclude trypan blue (unpublished results). Cessation of protein
synthesis could not be extended beyond this time, however, because this
did lead to loss of cell viability. These findings, thus, highlight
that protein synthesis is required to initiate the induction of
chemokine expression in monocyte:astrocyte co-cultures.

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Figure 7. MCP-1 production in monocyte:astrocyte co-cultures requires de
novo protein synthesis. Prior to establishing co-cultures,
monocytes and astrocytes were separately treated for 1 h with
cycloheximide (10 µg/ml). Co-cultures were subsequently exposed to
the same level of cycloheximide for the indicated periods of time, and
then supernatants were retrieved for quantification of MCP-1 levels.
Data are expressed as mean ± SE of three experiments
(n=6).
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Monocyte:astrocyte contact regulates MCP-1 production
After establishing that MCP-1 production is clearly upregulated in
monocyte:astrocyte co-cultures, we next turned to the question of
whether physical contact between monocytes and astrocytes is a
requirement for this occurrence. To investigate this issue, a
dual-chamber configuration was employed. Specifically, the two cell
types were separated by a semipermeable filter that allowed for the
free diffusion of soluble substances between upper and lower chambers.
When monocyte:astrocyte co-cultures were established under this
arrangement, MCP-1 production was undetectable (Fig. 8
), thus underscoring an obligatory role for cell-cell association
in initiating the heightened release of this chemokine. That such
results were not solely a result of inefficient diffusion of soluble
factors in this configuration was realized by experiments in which
monocytes or astrocytes were prestimulated with IL-1ß, washed free of
exogenous cytokine, and then co-cultured with the other cell type in
the same dual-chamber arrangement. In this case, prior cytokine
stimulation of monocytes or astrocytes resulted in augmented
cytoplasmic detection of MCP-1 in the other physically separated cell
type, as determined by immunofluorescence (unpublished results). Thus,
under the appropriate signal(s)e.g., physical contact or IL-1ß
stimulationmonocytes and astrocytes release soluble factors that can
direct the other cell type to increase MCP-1 expression.

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Figure 8. The role of cell:cell contact in the stimulation of MCP-1 production in
monocyte:astrocyte co-cultures. Co-cultures were established in
microwell chambers that allowed for physical contact between the two
cell types (left side), or in Transwell chambers that precluded
physical interaction (right side). In the latter configuration,
monocytes (5x105) were placed in the upper chamber, and
astrocytes (2.5x105) were seeded in the lower chamber.
After 48 h, culture supernatants were retrieved and assayed for
MCP-1 by ELISA. Data are expressed as mean ± SE of
three experiments (n=6).
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Cytokine regulation of MCP-1 expression in monocyte and astrocyte
cultures
Next, the factors mediating augmentation of MCP-1 expression
following monocyte:astrocyte contact were explored. Because IL-1ß and
TNF-
have been shown to induce MCP-1 expression when exogenously
applied to a wide variety of cell types
[22
23
24
25
26
27
], it was reasoned that these cytokines
may be released in response to initial monocyte:astrocyte association
and relay the signal to stimulate production of this chemokine.
Figure 9
reveals that neutralizing antibodies to each of these cytokines,
when added individually to monocyte:astrocyte co-cultures, attenuated
MCP-1 production significantly. Furthermore, when both types of
antibodies were added simultaneously, an even more pronounced reduction
in stimulated MCP-1 production occurred. Immunoglobulin (Ig) from
normal rabbit sera, however, was completely ineffective in blocking
this stimulation of MCP-1 production, thus attesting to the specificity
of IL-1ß and TNF-
in modulating MCP-1 production in
monocyte:astrocyte co-cultures.
The role of adhesion molecules in regulating MCP-1 production in
monocyte:astrocyte co-cultures
Lastly, it was determined whether specific adhesion molecules were
necessary for mediating the physical contact-dependent increase in
MCP-1 production in monocyte:astrocyte co-cultures. Results presented
in Figure 10
show that antibodies to ICAM-1 or VCAM-1 reduced significantly the
stimulated production of MCP-1 in monocyte:astrocyte co-cultures. The
combined use of both antibodies further inhibited MCP-1 production to
an extent greater than that achieved by either antibody alone. As was
the case in confirming the specificity of action of anticytokine
antibodies, isotype control antibodies did not diminish
co-culture-stimulated MCP-1 production. Engagement of ICAM-1 and/or
VCAM-1, therefore, might be an obligatory step in signaling stimulated
expression of MCP-1 when monocytes come into contact with astrocytes.
It is further conceivable that such engagement might be fostered by
IL-1ß and/or TNF-
released subsequent to incipient
monocyte:astrocyte association, because cytokines are known to
upregulate ICAM-1 and VCAM-1 expression in astrocytes
[28
].

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Figure 10. Effect of cell surface neutralization of ICAM-1 and VACM-1 on MCP-1
production in monocyte:astrocyte co-cultures. Astrocytes were
pretreated for 3 h with monoclonal anti-ICAM-1 antibody (10
µg/ml) and/or monoclonal anti-VCAM-1 (10 µg/ml) antibody.
Monocytes, diluted in media containing the respective antibodies, were
then layered on top of the pretreated astrocyte monolayers. MCP-1
concentration was evaluated by ELISA after 24 h of co-culture.
MCP-1 concentration was evaluated by ELISA after 48 h of
co-culture. Data are expressed as mean ± SE of three
experiments (n=6). *p < 0.001 (Dunnets
procedure) when compared with untreated monocyte:astrocyte co-cultures
receiving no antibodies; #p < 0.05 (LSD test) when compared
with co-cultures receiving anti-ICAM-1 or anti-VCAM-1 alone.
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DISCUSSION
|
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In this study, monocyte:astrocyte co-cultures were utilized to
present evidence that interaction between these two cell types leads to
heightened production of MCP-1. Specifically, the following
observations were made. 1) Augmentation of MCP-1 production in these
co-cultures, as judged by accumulation of this chemokine in the culture
supernatant, occurred in a time-dependent manner and required de
novo protein synthesis. 2) Monocytes and astrocytes clearly
exhibited induction of MCP-1 expression, as revealed by combined
immunocytochemistry/confocal microscopy and in situ
hybridization. 3) Physical contact between the two cell types was
required for increased chemokine production. 4) Antibody blockade of
IL-1ß and TNF-
significantly attenuated co-culture-induced
stimulation of MCP-1 production. 5) Functionally blocking antibodies to
ICAM-1 and VCAM-1 mitigated stimulation of MCP-1 production in
co-cultures also. Collectively, these observations point toward a
scenario in which adhesive events between astrocytes and monocytes
generate proinflammatory cytokines, which, in turn, stimulate MCP-1
production in both cell types.
That MCP-1 production was undetectable in isolated cultures of
monocytes or astrocytes but in monocyte:astrocyte co-cultures was
time-dependent and proportional to the number of allowable
monocyte:astrocyte interactions (achieved by varying the number of
monocytes or astrocytes) initially highlighted that induction of MCP-1
was unlikely to stem merely from isolation and/or culture-induced
cellular activation. This view was corroborated by additional
experiments, which revealed that physically separating the two cell
types by a semipermeable membrane precluded MCP-1 induction.
In these experiments, monocytes were clearly a significant source of
MCP-1 production in monocyte:astrocyte co-cultures. This is in contrast
to other studies, which described heightened MCP-1 production in
analogous co-culture systems. For example, in co-cultures of monocytes
with HUVECs, fibroblasts, or a glioblastoma cell line, the other cell
typenot the monocytewas observed to be the chief producer of MCP-1
[16
, 24
]. The distinction in our results
may underlie the fact that co-culture-induced MCP-1 expression is
dictated by highly specialized interactions between unique cell pairs
that can selectively alter chemokine expression in one cell type over
the other. In this regard, the fact, that in co-cultures of monocytes
with tumor-derived glioblastoma cells chemokine induction was
determined to be restricted mostly to the latter [24
],
may reflect differences in glial subtype and/or transformation state
that are crucial in regulating interactions with monocytes. Further
support for this conceptualization is provided by studies indicating
that although monocytes can produce MCP-1 and the functionally related
CC chemokine macrophage inflammatory peptide 1-alpha (MIP-1
)
in response to a particular stimulus, e.g., IL-1ß stimulation
[29
, 30
], in monocyte:HUVEC and
monocyte:fibroblast co-cultures, monocytes selectively augment their
production of MIP-1
, and HUVECs and fibroblasts dominate in MCP-1
production [16
].
Methodological issues may have contributed additionally to the
discrepancy between our observations regarding co-culture-induced
expression of MCP-1 in monocytes and those described by other
laboratories. For example, in the study by Kasahara et al.
[24
], paraformaldehyde-fixed monocytes or glioblastoma
cells were co-cultured with unfixed glioblastoma cells or monocytes,
respectively, to determine which cell type was responsible for the
observed increase in MCP-1 secretion. In this case, the combination of
fixed monocytes/unfixed glioblastoma cells resulted in MCP-1
production, and that of fixed glioblastoma cells/unfixed monocytes did
not, prompting the authors to argue that glioblastoma cells but not
monocytes manifest augmented MCP-1 expression in their co-culture
model. It remains plausible, however, that paraformaldehyde fixation
variably affects signaling molecules on different cell types and that
such treatment adversely impacts monocyte responsiveness, relatively
sparing that of glioblastoma cells.
The specialized intercellular reactions that determine which chemokine
is secreted by which cell type probably involve adhesive events of one
sort or another. Underscoring this argument, MCP-1 production in
monocyte:astrocyte co-cultures was shown to require physical contact
between monocytes and astrocytes. That MCP-1 production in
monocyte:astrocyte co-cultures could be inhibited by anti-ICAM-1
antibody is consistent with the previous finding that adhesion between
these two cell types is dependent on ICAM-1 and the ß2 integrin Mac-1
(CR3), the latter a ligand for ICAM-1 [14
]. The close
physical proximity of astrocyte foot processes to endothelial cells
in situ, and thus to sites of monocyte extravasation, would
further argue that such contact/adhesion could easily occur following
diapedesis of monocytes across microvessels in the CNS. Reasoning in
this manner, infiltrating monocytes may be envisioned to stimulate
MCP-1 production as a consequence of penetrating the BBB, resulting in
further mononuclear recruitment to a defined CNS locale. Such a
feed-forward cycle may be the basis for the development of
site-specific inflammatory lesions in neuroinflammatory conditions.
What molecular signals for MCP-1 production could be derived from
contact/adhesion between monocytes and astrocytes? The ability of
antibodies to IL-1ß and TNF-
to significantly reduce production of
MCP-1 in monocyte:astrocyte co-cultures implies a strategic role for
these cytokines in regulating MCP-1 expression. This interpretation of
IL-1ß action is consistent with that recently described by Kasahara
et al. [24
], who also showed an efficacy of
anti-IL1ß antibody in suppressing MCP-1 induction in co-cultures of
monocytes with a glioblastoma cell line. That the combination of
antibodies to both these cytokines did not completely abrogate MCP-1
induction may indicate that soluble effectors other than IL-1ß and
TNF-
mediate the stimulation of MCP-1 expression in
monocyte:astrocyte co-cultures. IL-1
, in particular, might function
in this capacity [24
]. Additionally, residual MCP-1
expression in the face of combined antibody treatment might also be
derived from Fc receptor-mediated monocyte stimulation
[31
]. Lastly, the presence of IL-1ß and TNF-
in
inflammatory lesions in EAE at the sites of MCP-1 production
[32
, 33
] is consistent with the notion that
these cytokines are physiological effectors of MCP-1 expression.
Lastly, it is of significance to note that IL-1ß treatment of
astrocytes and co-culture of astrocytes with monocytes were observed to
induce consistently profound and similar alterations in astrocyte
morphology (see Figs. 1
and 3
). Specifically, each of these
manipulations causes marked glial arborization, affecting a phenotype
more closely resembling the "stellate-shape" of astrocytes observed
in situ. Potentially, such an observation could hint at
there being cytokine-mediated control of astrocyte morphology in
vivo, possibly stemming from astrocyte interactions with
monocyte-derived perivascular macrophages or resident microglia. The
similarity in astrocyte morphological effects caused by IL-1ß and
monocyte co-culture treatments is also consistent with the
interpretation that co-culturing of astrocytes with monocytes results
in IL-1ß production, which, in turn, contributes to stimulating MCP-1
expression.
Further comprehension of the scenario of cellular and molecular events
responsible for stimulating MCP-1 production in monocyte:astrocyte
co-cultures is likely to provide key information about the initiation
and propagation of the inflammatory process at the BBB. In turn, such
information may well define susceptible targets for therapeutic
intervention of neuroinflammatory disease.
 |
ACKNOWLEDGEMENTS
|
|---|
This work was supported by grants to J. S. P. from the
National Institutes of Health and the National Multiple Sclerosis
Society. The authors thank Dr. Karen Waldenheim (Albert Einstein
College of Medicine) for supplying brain tissue and Mr. Kirk Dzenko for
assisting in the preparation of monocytes.
Received November 22, 1999;
revised May 3, 2000;
accepted May 5, 2000.
 |
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