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(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


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ABSTRACT
 
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-{alpha}) 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
 
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-{alpha}). 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.


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MATERIALS AND METHODS
 
Human astrocyte cultures
Human fetal CNS tissue (18–23 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 Earl’s 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 [Dulbecco’s modified Eagle’s 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 (3–5 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 85–90% 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 manufacturer’s 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-{alpha} (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 Student’s 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 Dunnet’s procedure or least significant difference (LSD) test.


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RESULTS
 
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.

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.

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).

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).

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).

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 stimulation—monocytes 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).

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-{alpha} 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-{alpha} in modulating MCP-1 production in monocyte:astrocyte co-cultures.



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  		Figure 9. Effect of immunoneutralization of IL-1ß and TNF-{alpha} on MCP-1 production in monocyte:astrocyte co-cultures. Astrocytes were pretreated for 3 h with polyclonal anti-IL-1ß antibody (10 µg/ml) and/or polyclonal anti-TNF-{alpha} (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 48 h of co-culture. Data are expressed as mean ± SE of three experiments (n=6). *p<0.001 (Dunnet’s procedure) when compared with untreated monocyte:astrocyte co-cultures receiving no antibodies; #p<0.05 (LSD test) when compared with co-cultures receiving anti-IL1ß or anti-TNF-{alpha} antibody alone.

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-{alpha} 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 (Dunnet’s 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
 
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-{alpha} 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 type—not the monocyte—was 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{alpha}) 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{alpha}, 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-{alpha} 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-{alpha} mediate the stimulation of MCP-1 expression in monocyte:astrocyte co-cultures. IL-1{alpha}, 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-{alpha} 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.


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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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