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Departments of
*
Pathology and
Microbiology/Immunology, Albert Einstein College of Medicine, Bronx, NY 10461;
Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595; and
§
Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI 48201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
, or IL-1ß
treatment of cocultures also induced significant migration of monocytes
that correlates with the induction of MCP-1 protein. TGF-ß,
previously shown to induce MCP-1 expression in astrocytes, but not in
EC, caused migration of monocytes across cocultures, but not across EC
grown alone. Monocytes and lymphocytes transmigrated across
cytokine-treated cocultures in greater numbers than across EC alone.
Astrocytes were the main source of cytokine-induced MCP-1, supporting a
role for astrocytes in facilitating leukocyte transmigration. A
blocking Ab to MCP-1 inhibited MCP-1- and cytokine-induced
transmigration of monocytes by 85–90%. Cytokine treatment of
cocultures also resulted in the transmigration of activated,
CD69-positive lymphocytes. The MCP-1-mediated transmigration of
monocytes across cocultures was blocked using an Ab to ICAM-1 and
inhibited by 55% using an Ab to E-selectin. These data suggest a
central role for astrocyte-derived MCP-1 in directing the migration of
monocytes and lymphocytes across the BBB. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are small, secreted proteins that mediate the recruitment and activation of leukocytes via binding to specific cell surface receptors (reviewed in 8 . Families of chemokines are categorized by the presence of conserved cysteine residues in their primary sequence. Monocyte-chemoattractant protein (MCP-1) belongs to the C-C family of chemokines and acts as a chemoattractant for monocytes, activated T cells, basophils, and NK cells (9, 10). MCP-1 expression is prominent within the atherosclerotic lesion (11), inflamed synovium (12), and glomerulus (13). Of relevance to the CNS and leukocyte trafficking across the BBB, MCP-1 expression has been detected in the brains and cerebrospinal fluid of patients with HIV-1-associated dementia (14). HIV-1 Tat protein was further found to induce MCP-1 expression in cultured astrocytes. The expression of MCP-1 has also been demonstrated in MS (15) and in its animal model, experimental autoimmune encephalomyelitis (EAE) (16, 17, 18, 19). In these studies, the expression of MCP-1 was localized to astrocytes, EC, and infiltrating leukocytes. Fuentes et al. confirmed a direct role for MCP-1 in mediating leukocyte recruitment in the CNS through the transgenic overexpression of MCP-1 in the brain, which resulted in a marked mononuclear infiltrate (20). The kinetics of chemokine expression may thus determine the nature of the inflammatory process. These findings suggest an important role for MCP-1 in the recruitment of monocytes and lymphocytes into the CNS.
The BBB is formed, in part, by the interactions between microvascular endothelial cells with astrocyte foot processes (21). The BBB functions to restrict the passage of cells and macromolecules into the CNS. During pathologic conditions, the BBB may become leaky and permit or even facilitate the passage of leukocytes into the CNS, a process believed to be an early event in the pathogenesis of MS and AIDS encephalitis (reviewed in 22 . Additionally, the production of cytokines (6, 7) and chemokines (23, 24, 25) by EC and astrocytes is likely to participate in the recruitment and activation of leukocytes.
Previous tissue culture models examined chemokine-mediated leukocyte
transmigration across EC monolayers (26). To model more closely the
human BBB, we characterized a tissue culture system in which HUVEC were
cocultured with autologous astrocytes on opposite sides of a porous
insert (27). Astrocyte processes contact the EC through the insert
pores and induce the expression of BBB markers, including glucose
transport protein-1 and glutamyl-transpeptidase (21, 27). These
markers are not expressed on umbilical vein EC grown in the absence of
astrocytes or in the absence of direct astrocyte-EC contact
through the insert pores. The same coculture system was utilized by
Hayashi et al., who also demonstrated the induction of barrier
properties, as evidenced by an increased resistance to the passage of
radiolabeled inulin (28). Using our coculture system, we demonstrated a
role for MCP-1 in facilitating leukocyte transmigration and determined
the effects of proinflammatory cytokines upon EC and astrocyte-derived
chemokines that may mediate the transmigration process. We demonstrate
an important role for astrocyte-derived MCP-1 in mediating the
migration of monocytes and activated lymphocytes across
cocultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study is part of an ongoing research protocol that has been approved by the Albert Einstein College of Medicine Committee on Clinical Investigation (Bronx, NY), and the City of New York Health and Hospitals Corporation. Informed consent was obtained from all participants. Fetal tissues were obtained at the time of elective termination of intrauterine pregnancy from healthy females (29).
Cell culture
Astrocyte cultures were prepared according to a modified protocol of McCarthy and de Vellis (30). Briefly, human fetal CNS tissue was separated from the meninges, minced, and digested in 0.25% trypsin (Life Technologies, Grand Island, NY)/0.1% collagenase (Sigma, St. Louis, MO). The resulting cell suspension was serially filtered through sterile 100- and 80-µm nylon mesh filters (Tetko, Elmsford, NY) and pelleted at 900 rpm. Cultures were established in RPMI media supplemented with 10% FCS and 1% penicillin/streptomycin (Life Technologies). After 12 days, microglial cells were removed from the mixed culture by shaking 30 min at 4°C. Cultures were passaged after trypsinization and were examined by immunofluorescence microscopy for glial fibrillary acidic protein (IgG1; 1:50; Boehringer Mannheim, Indianapolis, IN), anti-macrophage (HAM56; IgM; 1:50; Enzo Diagnostics, Farmingdale, NY), as a marker for cells of the monocyte/microglia lineage, and isotype-matched control Abs (IgG1 mouse myeloma; 1:50; Organon Teknika, Durham, NC; and antimedullary and subcapsular cortical thymic epithelial cells; IgM; 1:50; Sigma, respectively). Astrocyte cultures (3–5 passages) were >99% glial fibrillary acidic protein positive and unreactive for either HAM56 or control Abs. Thus, cells of passages 3–5 were used to ensure the absence of contaminating microglial cells.
HUVEC were obtained by digesting umbilical cords with type II collagenase (2 mg/ml; Worthington Biochemical, Freehold, NJ) and were grown on 0.2% gelatin (Fisher Scientific, Pittsburgh, PA)-coated tissue culture plates in M199 media (Life Technologies) supplemented with 20% newborn calf serum (Life Technologies), 5% heat-inactivated human serum (Biocell Laboratories, Rancho Dominguez, CA), 1% penicillin/streptomycin, and 12 ng/ml endothelial cell growth factor (Sigma). Human brain microvascular endothelial cells were purchased from Cell Systems (Kirkland, WA). EC cultures were >99% factor VIII positive, as demonstrated by immunofluorescence using an anti-factor VIII-related Ag Ab (IgG1; 1:100; Dako, Carpenteria, CA) and unreactive for an isotype-matched negative control (IgG1 mouse myeloma protein; 1:50; Organon Teknika).
Establishment of EC and astrocyte cocultures
Cocultures of umbilical vein EC and astrocytes were established on opposite sides of a gelatin-coated, 3-µm pore-size tissue culture insert (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ). Astrocytes (1 x 105) were first seeded onto the underside of the insert. Cells were allowed to attach for 4–5 h, at which time the insert was placed into a 24-well tissue culture plate containing complete M199 media. EC (1.6 x 104) were applied to the gelatin-coated opposite side of the insert. Cocultures were maintained until 16 h before the transmigration assay, at which time inserts were transferred to new wells containing cytokines using M199 media supplemented with only 10% newborn calf serum and no human serum, so as to minimize the effect of serum on MCP-1 expression (31). These media were used throughout the transmigration assay. The transmigration assay was always conducted 3 days following establishment of cocultures.
Reagents
EC and astrocytes on the insert were treated with either 1 ng/ml
TGF-ß1 (endotoxin <0.1 ng/µg; R&D Systems, Minneapolis, MN), 100
IU/ml TNF- (endotoxin <0.1 ng/µg; R&D), 200 IU/ml IFN-
(endotoxin <0.2 ng/mg; Genzyme, Cambridge, MA), or 2 IU/ml IL-1ß
(endotoxin = 2.5 EU/mg; National Cancer Institute, Frederick, MD)
for 16 h. Human rMCP-1 was purchased from Genzyme. For cytokine
treatments, 400 µl media containing cytokine were placed in a 24-well
plate, and the insert tops were gently washed with warmed media. For
MCP-1-blocking experiments, cultures were washed and preincubated with
10 µg/ml mAb 10F7 to MCP-1 (a generous gift of Dr. Charles Mackay,
formerly of Leukosite, Cambridge, MA) or IgG1 mouse myeloma protein
(Organon Teknika) as a negative control for 1 h.
Isolation of PBMC and PBMC transmigration
Human PBMC were isolated from freshly drawn human blood by Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ) centrifugation, as previously described (32). Briefly, whole blood was diluted 1/3 with PBS, layered over Ficoll (1:2.5), and centrifuged at 1400 rpm for 40 min at 18°C. The layer containing PBMC was removed and washed with cold PBS containing 1% FCS. Cells were spun at 1400 rpm for 20 min at 4°C and resuspended with cold media. Cells were brought to a final concentration of 1.5 x 106 PBMC/ml with room temperature media. The tops of the inserts were washed again with warmed media. PBMC (200 µl or 3 x 105 cells) were added to the top of the insert, and inserts were verified for integrity. For wells receiving MCP-1, 400 µl MCP-1 at different concentrations were added below the culture insert. The transmigration assay was conducted for 2.5 h at 37°C. To minimize variability, one blood donor was used for the majority of experiments.
Monocyte immunocytochemistry and quantification
Monocytes that had transmigrated and adhered to the bottom of the well were fixed briefly in 5% Formalin. After washing and blocking in 2% BSA/PBS, cells were incubated with a monocyte-specific mAb (Mac 387; Dako) overnight at 4°C. Cells were washed and incubated with peroxidase-conjugated secondary Abs for 1 h. For visualization, cells were washed and stained with diaminobenzidine (Sigma). The number of transmigrated cells was calculated by counting the number of stained cells under x40 magnification. Five nonoverlapping fields were counted, and the average of five fields was termed the number of cells/field. The total number of cells transmigrated is equivalent to the number of cells/field x 20.
Flow-cytometric analysis
For the majority of experiments, the cells that had transmigrated were collected from the culture supernatant below the insert. Cells adherent to the well bottoms were detached with 0.5 mM EDTA/PBS and pooled with the previously collected cells. Cells were centrifuged 5 min at 300 x g and resuspended in a final volume of 400 µl media. A total of 10 µl Leukogate Ab mixture (Becton Dickinson, San Jose, CA) was added for a 30-min incubation at 4°C. This mixture contains FITC-conjugated anti-CD45, a pan-leukocyte marker, and PE-conjugated anti-CD14, a monocyte marker. For some experiments, a FITC-conjugated CD14 Ab was used in combination with a PE-conjugated CD69 Ab (Becton Dickinson). Cells were centrifuged, fixed in 2% paraformaldehyde, and analyzed within 24 h by FACS.
Leukocyte transmigration was analyzed with a FACScan flow cytometer (Becton Dickinson). The numbers of monocytes and lymphocytes per sample were determined by acquisition and analysis of List Mode Data files using Lysis II software and the Consort 32 computer system. The instrument was calibrated with CALIBRITE beads and autocomp software BDIS using a 4-parameter, 4-decade logarithmic fluorescence amplifier. Gated acquisition was determined by a combination of gates based on forward scatter, side scatter, and CD45-FITC reactivity. The number of cells in each sample and the starting leukocyte sample was determined by analyzing the number of events recorded in a 3-min interval.
Adhesion molecule-blocking studies
Cocultures were stimulated with TNF- (100 U/ml) on the EC
side to induce the expression of adhesion molecules. For ICAM-1, the
contribution of basal expression (untreated) was also examined. The
length of TNF- treatment was 4 h for E-selectin and IG9, and
7 h for VCAM-1 studies. Cocultures were washed and incubated with
50 µg/ml of one of the following Abs: anti-ICAM-1 (IgG1; Dako),
anti-E-selectin (IgG2a; Becton Dickinson), anti-VCAM-1 (IgG1;
Becton Dickinson), anti-IG9 (IgG3; purified in our laboratory
(33)), or isotype-matched myeloma proteins as negative controls
(Cappel, West Chester, PA). After 1 h, the upper and lower
sides of cocultures were washed extensively with media. Monocyte
transmigration was assayed as above by the addition of 100 ng/ml MCP-1
to the lower chamber supernatants.
ELISA
Culture supernatants were collected from both sides of the
insert after the transmigration assay and stored at -20°C until use.
MCP-1 protein was determined using a sandwich-type immunoassay using
capture and biotinylated detection Abs, both purchased from PharMingen
(San Diego, CA). MIP-1, MIP-1ß, RANTES, and IP-10 protein were
determined using Abs purchased from R&D. The minimum detection limit
for these assays is <15 pg/ml.
Statistical analyses
Student’s one-tailed, paired t test was used to compare the numbers of transmigrated leukocytes and MCP-1 protein in cytokine-treated cultures with that of untreated cultures. For MCP-1-blocking experiments, significance between anti-MCP-1- and negative control-treated supernatants was similarly compared. Results were considered to be significant for p < 0.05 values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first determined whether exogenously added MCP-1 induces the
migration of monocytes across cocultures. A range of MCP-1 doses was
added to the lower chamber of coculture inserts, and 3 x
105 PBMC were added to the upper chamber. After 2.5 h,
the number of transmigrated monocytes was quantified by staining cells
that had passed through the filter and adhered to the bottom of the
plate with an Ab for monocytes, Mac 387. As shown in Fig. 1
, MCP-1 induced the dose-dependent
transmigration of monocytes, with 100 ng/ml MCP-1 resulting in maximal
migration (p < 0.001; n = 3).
Significant transmigration was observed using a dose of 20 ng/ml MCP-1
as well (p < 0.03). Transmigration could be
detected by 1 h and was maximal after 2.5 h (data not shown).
In the absence of exogenously added MCP-1, there was a low level of
monocyte transmigration due to minimal baseline expression of MCP-1 by
astrocytes (Fig. 1). The addition of 100 ng/ml MCP-1 to both the upper
and lower chambers, so as to abolish the chemokine gradient, inhibited
monocyte transmigration to levels below this basal level of migration
(Fig. 1).
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A, the addition of 100 ng/ml
MCP-1 to the lower chamber resulted in the significant transmigration
of monocytes across a coculture of EC and astrocytes
(p < 0.0003; n = 15). With
this method, we also demonstrated monocyte migration using as little as
5 or 10 ng/ml MCP-1 (data not shown). Lymphocytes migrated to a lesser,
yet significant, degree (p < 0.002 as compared
with untreated cocultures). The migrated sample reflected a dramatic
shift in the ratio of monocytes to lymphocytes. Whereas the starting
CD45-positive leukocyte population consisted of approximately 90%
lymphocytes and 10% monocytes (Fig. 2B), the migrated
sample was typically >90% monocytes (Fig. 2C). The few
lymphocytes that migrated are likely to be activated, given that MCP-1
is chemotactic for activated T cells (34). In the absence of MCP-1,
there was minimal migration of either monocytes or lymphocytes (Fig. 2A). As before, the addition of MCP-1 to the upper and
bottom chambers abolished the chemotactic gradient and completely
inhibited migration (Fig. 2A).
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To determine whether proinflammatory cytokines activate EC and
astrocytes to express functional MCP-1, cocultures or EC cultured alone
were treated for 16 h with TNF-, IFN-, or IL-1ß on the
underside (astrocyte side) of the insert. We previously determined that
the timing and dose of these cytokine treatments induce MCP-1
expression in both EC and astrocytes (35, 55). After 16 h, the top
chamber was washed once with warm media, and PBMC were added for the
transmigration experiment. The supernatant from the bottom chamber was
untouched. A total of 3 x 105 PBMC was added to the
top chamber, and cultures were incubated for 2.5 h. As shown in
Fig. 3, significant monocyte migration
across cocultures was detected with TNF-, IFN-, or IL-1ß
treatments (p < 0.003; n = 11
each), as compared with untreated cocultures. TNF-, IFN-, or
IL-1ß also induced significant monocyte migration across EC cultures
(p < 0.04; n = 6). When
cocultures, but not EC alone, were treated with TGF-ß, significant
monocyte migration was also detected (p <
0.0005). This is consistent with our earlier findings that TGF-ß
induces MCP-1 expression in human fetal astrocytes (25). Greater
monocyte migration was detected across cocultures, as compared with EC
alone. These differences were significant for TGF-ß and TNF-
(p < 0.04 each). We also examined leukocyte
transmigration across cocultures and EC monolayers using human brain
microvascular endothelial cells. Identical transmigration profiles of
monocytes and lymphocytes across cytokine-treated cultures were
detected as with cultures established using HUVEC (data not
shown).
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). Cytokine-treated cocultures released
significant levels of MCP-1 protein into the lower chamber, as compared
with untreated cocultures (p < 0.00001). By
comparing MCP-1 expression in cocultures with that of EC or astrocytes
grown on the inserts separately, 75–80% of the MCP-1 expression in
cocultures was attributed to the astrocytes (Fig. 4). With TGF-ß
treatment, all of the MCP-1 expression was astrocyte derived.
Cytokine-treated EC released significant MCP-1 protein in amounts
sufficient to promote leukocyte migration (p <
0.004; n = 8), albeit to a lesser extent than
cocultures or astrocytes alone.
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- or
IL-1ß-treated EC and detected approximately 150 and 110 ng/ml MCP-1,
respectively, as compared with 30 ng/ml MCP-1 in the lower chamber
supernatants for each cytokine. Thus, EC production of MCP-1 was
predominantly into the upper chamber. The upper chamber supernatants of
cytokine-treated astrocytes contained equivalent MCP-1 protein as the
lower chamber (70–100 ng/ml). In our protocol, we removed the upper
chamber supernatant before the addition of PBMC, so as to mimic what
might be occurring in vivo as a result of the blood flow removing any
unbound chemokine.
Exogenous addition of 100 ng/ml MCP-1 to the lower chamber of EC or
astrocyte cultures resulted in an approximately 50% increase (150
± 12 and 160 ± 37 ng/ml, respectively) in MCP-1 expression by
both cell types. We are currently investigating this apparent autocrine
regulation of chemokine expression in EC and astrocytes. In EC or
astrocyte cultures, as well as in EC/astrocyte cocultures, we did not
detect other monocyte chemoattractants, such as MIP-1, MIP-1ß, or
RANTES (data not shown). By ELISA, IFN- treatment of cocultures
inconsistently induced 10–20 ng/ml IP-10 in the lower chamber (data
not shown).
Cytokine treatments induce the transmigration of activated lymphocytes
As shown in Fig. 5, significant
migration of lymphocytes was observed following TGF-ß, TNF-,
IFN-, or IL-1ß treatment of cocultures (p
< 0.004; n = 10), as compared with untreated
conditions. The order of potency was IL-1ß or TNF- > IFN-
>> TGF-ß. With EC grown in the absence of astrocytes, the
baseline trafficking of lymphocytes was greater, and only IFN-
induced significant lymphocyte migration above that of untreated
cultures (p < 0.04). We determined whether
these lymphocytes were activated, because MCP-1 is chemotactic for
activated, but not resting T cells (34). Expression of CD69 was used to
assess the state of activation of the lymphocytes. As compared with the
starting population of PBMC, which consisted of 95% CD69-negative
lymphocytes, the transmigrated lymphocytes were >50% positive for
CD69 expression, and thus were in an activated state (36) (Fig. 6). Even in the case of untreated
cultures, the few lymphocytes that did transmigrate were CD69 positive
(data not shown). Monocytes were negative for CD69 expression, except
for a minimal increase with IFN- treatment (data not shown),
consistent with previous findings that IFN- induces CD69 expression
in monocytes (36).
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or
IL-1ß for 2.5 h, since this is the maximum length of time that
these cells would encounter the cytokines in our transmigration assay.
No difference in CD69 expression was detected on either TNF-- or
IL-1ß-treated lymphocytes, as compared with untreated lymphocytes
(data not shown). MCP-1 directly mediates leukocyte migration across EC and astrocyte cocultures
To determine whether the transmigration of monocytes in our system
was specifically due to MCP-1, the supernatant from the lower chamber
was incubated with a neutralizing Ab to MCP-1 for 1 h before
addition of PBMC. As shown in Fig. 7,
preincubation with mAb 10F7 (10 µg/ml) resulted in the marked
inhibition of monocyte transmigration across cocultures. The percentage
of inhibition was 90% for treatments with MCP-1 or IFN-, and 85%
for treatments with TGF-ß, TNF-, or IL-1ß
(p < 0.05; n = 4). An
isotype-matched, mouse myeloma protein was used at 10 µg/ml as a
negative control, and this did not have any effect on leukocyte
migration for any treatment condition. Anti-MCP-1 pretreatment
inhibited the TNF-- or IL-1ß-induced lymphocyte migration across
cocultures by 55 and 45%, respectively (p <
0.04; data not shown). Although IP-10 was detected inconsistently by
ELISA in IFN--treated cocultures, experiments using an
IP-10-blocking Ab (R&D; 10 µg/ml) had no effect on monocyte migration
(data not shown). No IP-10 was detected in TNF-- or IL-1ß-treated
cocultures, suggesting the presence of an additional chemokine(s) that
is not MIP-1, MIP-1ß, or RANTES, responsible in part for mediating
T cell migration.
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We determined the roles of adhesion molecules in the MCP-1-induced
transmigration of monocytes across cocultures. Cocultures were treated
with TNF- on the EC side (as described in Materials and
Methods). Cocultures were washed and preincubated with mAbs to
either ICAM-1, E-selectin, VCAM-1, IG9, or isotype-matched myeloma
proteins as negative controls. As shown in Fig. 8A, anti-ICAM-1 completely
blocked MCP-1-induced monocyte transmigration across cocultures
(p < 0.03; n = 3). Results
were similar, irrespective of whether cocultures were left untreated or
stimulated with TNF-. As shown in Fig. 8B,
anti-E-selectin treatment reduced monocyte transmigration across
TNF--stimulated cocultures by 55% (p <
0.05; n = 4). Pretreatment with either anti-VCAM-1
or anti-IG9 had no significant effect upon MCP-1-induced monocyte
transmigration (n = 2 each; data not shown). In all
cases, the expression of the respective adhesion molecules by EC in the
cocultures was confirmed by immunocytochemistry (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
MIP-1ß, and RANTES (37, 38), that contribute to this ongoing process. Numerous in vivo studies have suggested a role for the expression of MCP-1 in the pathogenesis of CNS inflammatory disease. MCP-1 is expressed in the brain and cerebrospinal fluid of patients with AIDS dementia (14). This was the only chemokine detected in the CSF of these individuals. During the course of EAE, the marked infiltration of monocytes and reactive T lymphocytes is preceded by MCP-1 expression by astrocytes, EC, and additional leukocytes (17, 18, 19). In addition, MCP-1 was found to play a role in relapsing disease (16). In a mouse transgenic model in which MCP-1 expression was targeted to the brain using a myelin basic protein promoter, the overexpression of MCP-1 in the brain resulted in a significant mononuclear infiltrate (20). In humans, reactive astrocytes within and surrounding the MS lesion also expressed MCP-1, implicating this chemokine in the development of MS lesions (15).
Our findings are in agreement with others who have demonstrated an
important role for proinflammatory cytokines in promoting the
transmigration of activated leukocytes. In a study on the migration of
uninfected and HIV-infected monocytes, Persidsky et al. found that the
activation state, not infection, of macrophages correlated with their
migratory potential (39). Furthermore, Birdsall et al. showed that
TNF- primes leukocytes for enhanced transendothelial migration (40).
Our data indicate that the expression of proinflammatory cytokines in
the CNS facilitates the recruitment of activated leukocytes via
astrocyte-derived MCP-1.
We found that TGF-ß potently induced MCP-1 expression by astrocytes and facilitated the subsequent transmigration of monocytes across cocultures. A role for TGF-ß in the recruitment of mononuclear cells into the CNS was also found by Wyss-Coray et al., who showed that transgenic mice whose astrocytes produce high levels of TGF-ß had increased inflammation and disease severity in EAE (41). We believe that this may be attributed, in part, to TGF-ß acting on the astrocytes themselves to induce MCP-1 expression. It would be important to examine chemokine expression in these transgenic mice to characterize this possible mechanism. This proinflammatory role for TGF-ß contrasts with previous findings that TGF-ß treatment ameliorates EAE (42, 43). This may indicate important distinctions between murine and human studies, and also underscores the pleiotropic nature of TGF-ß, whose effects depend upon the cytokine and cellular environment (44).
EC are a major source of MCP-1 (23, 24), and a role for EC-derived MCP-1 in facilitating leukocyte transmigration across cocultures cannot be excluded. We show in this study that cytokine treatment induced MCP-1 expression to levels sufficient to promote a small, but significant leukocyte transmigration across EC monolayers. MCP-1 bound to extracellular matrix components may also mediate the initial attraction of the leukocytes for the endothelium, leading to firm adherence. Additionally, when we performed ELISA analysis on the upper chamber supernatants of cytokine-treated EC cultures, we detected significant MCP-1 protein. We removed the upper chamber supernatants and washed the insert tops before adding PBMC for the assay, since this is what may occur in vivo as a result of blood flow. Thus, EC-derived MCP-1 that is not matrix bound would not play a significant role in our system.
In our studies, Abs to MCP-1 blocked 85–90% of the monocyte and
45–55% of the lymphocyte migration across cocultures. Although
astrocyte expression of IP-10 has been noted in EAE (18), we detected
little or no IP-10 and found no significant role for this chemokine in
mediating either monocyte or lymphocyte migration across cocultures. It
is possible, however, that the induction of IP-10 is more complex,
involving the interactions of multiple cytokines with cell types. We
also did not detect MIP-1, MIP-1ß, or RANTES in the
cytokine-treated cocultures, suggesting that, for lymphocytes, other
chemokines contribute to their transmigration across the cocultures.
We found that the MCP-1-induced transmigration of monocytes was dependent upon ICAM-1 and partially dependent upon E-selectin. No significant role for either VCAM-1 or IG9 in this process was found. The enhanced expression of ICAM-1, VCAM-1, and E-selectin has been demonstrated in several CNS inflammatory diseases, including MS (45, 46) and HIV-1 encephalitis (47, 48). In those studies, the increased expression of adhesion molecules by EC and/or astrocytes was generally correlated with the extent of leukocyte infiltration. VLA-4/VCAM-1 interactions have been shown to play an important role in mediating leukocyte adhesion in both EAE (49, 50) and SIV encephalitis (51). Our findings demonstrate that pathways other than VCAM-1 may be utilized in the transmigration of leukocytes across the BBB in response to MCP-1. Furthermore, our data are consistent with those of several groups who showed that, using EC only, the transendothelial transmigration of monocytes is dependent upon LFA-1 and Mac1, which each bind ICAM-1 (52, 53). In these studies, the two processes of leukocyte adherence and transmigration could be distinguished. VLA-4 was more important than LFA-1 in mediating the initial binding of monocytes to cytokine-activated EC. Once the monocytes had adhered to the EC, the addition of anti-VLA-4 Ab had no significant effect on their subsequent transmigration. In contrast, the migration of monocytes could be blocked using an Ab to CD18, the integrin chain common to both LFA-1 and Mac1. Thus, in our coculture system: 1) LFA-1 or Mac-1 binding to ICAM-1 may be sufficient for monocyte binding, as well as transmigration, or 2) VLA-4 on monocytes may bind ligands other than VCAM-1, such as fibronectin (54). Our mAb to VCAM-1 binds to domains 6 and 7. Thus, it is also possible that other domains of VCAM-1 may mediate monocyte adhesion that would not have been detected with our reagent. Future studies using activated and/or HIV-infected leukocytes may suggest additional mechanisms whereby these cells cross the BBB and enter the CNS.
MCP-1 appears to play a critical role in both the initiation and/or amplification stages of CNS inflammatory diseases. The transmigrated leukocytes and other CNS elements, such as microglia, may be sources of additional MCP-1, as well as other chemokines and cytokines (38), that contribute further to this process.
This is the first report of cytokine-induced MCP-1 causing the migration of leukocytes across a coculture of human EC and astrocytes and the role that astrocyte-derived factors play in this process. Strategies aimed at regulating astrocyte-derived MCP-1 expression may therefore yield useful approaches to limit the progression of CNS inflammatory diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jonathan M. Weiss, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address:
3 Abbreviations used in this paper: CNS, central nervous system; BBB, blood-brain barrier; EAE, experimental autoimmune encephalomyelitis; EC, endothelial cells; IP, inflammatory protein; MCP-1, monocyte-chemoattractant protein-1; MIP, macrophage-inflammatory protein; MS, multiple sclerosis; PE, phycoerythrin.
Received for publication April 6, 1998. Accepted for publication August 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  S. Do Carmo, H. Jacomy, P. J. Talbot, and E. Rassart Neuroprotective Effect of Apolipoprotein D against Human Coronavirus OC43-Induced Encephalitis in Mice J. Neurosci., October 8, 2008; 28(41): 10330 - 10338. [Abstract] [Full Text] [PDF]
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 M. Malik, Y.-Y. Chen, M. F. Kienzle, B. E. Tomkowicz, R. G. Collman, and A. Ptasznik Monocyte Migration and LFA-1-Mediated Attachment to Brain Microvascular Endothelia Is Regulated by SDF-1{alpha} through Lyn Kinase J. Immunol., October 1, 2008; 181(7): 4632 - 4637. [Abstract] [Full Text] [PDF]
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 J. Sun, J. H. Zheng, M. Zhao, S. Lee, and H. Goldstein Increased In Vivo Activation of Microglia and Astrocytes in the Brains of Mice Transgenic for an Infectious R5 Human Immunodeficiency Virus Type 1 Provirus and for CD4-Specific Expression of Human Cyclin T1 in Response to Stimulation by Lipopolysaccharides J. Virol., June 1, 2008; 82(11): 5562 - 5572. [Abstract] [Full Text] [PDF]
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C. C. Clay, D. S. Rodrigues, Y. S. Ho, B. A. Fallert, K. Janatpour, T. A. Reinhart, and U. Esser Neuroinvasion of Fluorescein-Positive Monocytes in Acute Simian Immunodeficiency Virus Infection J. Virol., November 1, 2007; 81(21): 12040 - 12048. [Abstract] [Full Text] [PDF]
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 M. H. Lehmann, S. Masanetz, S. Kramer, and V. Erfle HIV-1 Nef upregulates CCL2/MCP-1 expression in astrocytes in a myristoylation- and calmodulin-dependent manner J. Cell Sci., November 1, 2006; 119(21): 4520 - 4530. [Abstract] [Full Text] [PDF]
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 D. G. Walker, J. Link, L.-F. Lue, J. E. Dalsing-Hernandez, and B. E. Boyes Gene expression changes by amyloid {beta} peptide-stimulated human postmortem brain microglia identify activation of multiple inflammatory processes J. Leukoc. Biol., March 1, 2006; 79(3): 596 - 610. [Abstract] [Full Text] [PDF]
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J. Sun, T. Soos, V. N. KewalRamani, K. Osiecki, J. H. Zheng, L. Falkin, L. Santambrogio, D. R. Littman, and H. Goldstein CD4-Specific Transgenic Expression of Human Cyclin T1 Markedly Increases Human Immunodeficiency Virus Type 1 (HIV-1) Production by CD4+ T Lymphocytes and Myeloid Cells in Mice Transgenic for a Provirus Encoding a Monocyte-Tropic HIV-1 Isolate J. Virol., February 15, 2006; 80(4): 1850 - 1862. [Abstract] [Full Text] [PDF]
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E. A. Eugenin, K. Osiecki, L. Lopez, H. Goldstein, T. M. Calderon, and J. W. Berman CCL2/Monocyte Chemoattractant Protein-1 Mediates Enhanced Transmigration of Human Immunodeficiency Virus (HIV)-Infected Leukocytes across the Blood-Brain Barrier: A Potential Mechanism of HIV-CNS Invasion and NeuroAIDS J. Neurosci., January 25, 2006; 26(4): 1098 - 1106. [Abstract] [Full Text] [PDF]
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 A. Chahlavi, P. Rayman, A. L. Richmond, K. Biswas, R. Zhang, M. Vogelbaum, C. Tannenbaum, G. Barnett, and J. H. Finke Glioblastomas Induce T-Lymphocyte Death by Two Distinct Pathways Involving Gangliosides and CD70 Cancer Res., June 15, 2005; 65(12): 5428 - 5438. [Abstract] [Full Text] [PDF]
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T. S. Kim and S. Perlman Viral Expression of CCL2 Is Sufficient To Induce Demyelination in RAG1-/- Mice Infected with a Neurotropic Coronavirus J. Virol., June 1, 2005; 79(11): 7113 - 7120. [Abstract] [Full Text] [PDF]
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 G. P.A. Rice, H.-P. Hartung, and P. A. Calabresi Anti-{alpha}4 integrin therapy for multiple sclerosis: Mechanisms and rationale Neurology, April 26, 2005; 64(8): 1336 - 1342. [Abstract] [Full Text] [PDF]
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C.-F. Lin, S.-C. Chiu, Y.-L. Hsiao, S.-W. Wan, H.-Y. Lei, A.-L. Shiau, H.-S. Liu, T.-M. Yeh, S.-H. Chen, C.-C. Liu, et al. Expression of Cytokine, Chemokine, and Adhesion Molecules during Endothelial Cell Activation Induced by Antibodies against Dengue Virus Nonstructural Protein 1 J. Immunol., January 1, 2005; 174(1): 395 - 403. [Abstract] [Full Text] [PDF]
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J. Falsig, P. Porzgen, J. Lotharius, and M. Leist Specific Modulation of Astrocyte Inflammation by Inhibition of Mixed Lineage Kinases with CEP-1347 J. Immunol., August 15, 2004; 173(4): 2762 - 2770. [Abstract] [Full Text] [PDF]
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 G. R. John, S. C. Lee, and C. F. Brosnan Cytokines: Powerful Regulators of Glial Cell Activation Neuroscientist, February 1, 2003; 9(1): 10 - 22. [Abstract] [PDF]
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E. A. Eugenin, M. C. Branes, J. W. Berman, and J. C. Saez TNF-{alpha} Plus IFN-{gamma} Induce Connexin43 Expression and Formation of Gap Junctions Between Human Monocytes/Macrophages That Enhance Physiological Responses J. Immunol., February 1, 2003; 170(3): 1320 - 1328. [Abstract] [Full Text] [PDF]
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 A. Prat, K. Biernacki, J.-F. Lavoie, J. Poirier, P. Duquette, and J. P. Antel Migration of Multiple Sclerosis Lymphocytes Through Brain Endothelium Arch Neurol, March 1, 2002; 59(3): 391 - 397. [Abstract] [Full Text] [PDF]
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S. Delaire, C. Billard, R. Tordjman, A. Chedotal, A. Elhabazi, A. Bensussan, and L. Boumsell Biological Activity of Soluble CD100. II. Soluble CD100, Similarly to H-SemaIII, Inhibits Immune Cell Migration J. Immunol., April 1, 2001; 166(7): 4348 - 4354. [Abstract] [Full Text] [PDF]
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 D. Goldman, X. Song, R. Kitai, A. Casadevall, M.-L. Zhao, and S. C. Lee Cryptococcus neoformans Induces Macrophage Inflammatory Protein 1{alpha} (MIP-1{alpha}) and MIP-1{beta} in Human Microglia: Role of Specific Antibody and Soluble Capsular Polysaccharide Infect. Immun., March 1, 2001; 69(3): 1808 - 1815. [Abstract] [Full Text] [PDF]
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M. A. Wetzel, A. D. Steele, T. K. Eisenstein, M. W. Adler, E. E. Henderson, and T. J. Rogers {micro}-Opioid Induction of Monocyte Chemoattractant Protein-1, RANTES, and IFN-{gamma}-Inducible Protein-10 Expression in Human Peripheral Blood Mononuclear Cells J. Immunol., December 1, 2000; 165(11): 6519 - 6524. [Abstract] [Full Text] [PDF]
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A. V. Andjelkovic, D. Kerkovich, and J. S. Pachter Monocyte:astrocyte interactions regulate MCP-1 expression in both cell types J. Leukoc. Biol., October 1, 2000; 68(4): 545 - 552. [Abstract] [Full Text]
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Y. Persidsky, J. Zheng, D. Miller, and H. E. Gendelman Mononuclear phagocytes mediate blood-brain barrier compromise and neuronal injury during HIV-1-associated dementia J. Leukoc. Biol., September 1, 2000; 68(3): 413 - 422. [Abstract] [Full Text] [PDF]
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N. Mozaffarian, A. Casadevall, and J. W. Berman Inhibition of Human Endothelial Cell Chemokine Production by the Opportunistic Fungal Pathogen Cryptococcus neoformans J. Immunol., August 1, 2000; 165(3): 1541 - 1547. [Abstract] [Full Text] [PDF]
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R. Szymocha, H. Akaoka, M. Dutuit, C. Malcus, M. Didier-Bazes, M.-F. Belin, and P. Giraudon Human T-Cell Lymphotropic Virus Type 1-Infected T Lymphocytes Impair Catabolism and Uptake of Glutamate by Astrocytes via Tax-1 and Tumor Necrosis Factor Alpha J. Virol., July 15, 2000; 74(14): 6433 - 6441. [Abstract] [Full Text]
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J. M. Weiss, A. Nath, E. O. Major, and J. W. Berman HIV-1 Tat Induces Monocyte Chemoattractant Protein-1-Mediated Monocyte Transmigration Across a Model of the Human Blood-Brain Barrier and Up-Regulates CCR5 Expression on Human Monocytes J. Immunol., September 1, 1999; 163(5): 2953 - 2959. [Abstract] [Full Text] [PDF]
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