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(Journal of Leukocyte Biology. 2006;80:1156-1164.)
© 2006 by Society for Leukocyte Biology

CD16+ monocytes produce IL-6, CCL2, and matrix metalloproteinase-9 upon interaction with CX3CL1-expressing endothelial cells

Petronela Ancuta*,{dagger}, Jianbin Wang*,{dagger} and Dana Gabuzda*,{ddagger},1

* Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Departments of
{dagger} Pathology and
{ddagger} Neurology, Harvard Medical School, Boston, Massachusetts, USA

1 Correspondence: Dana-Farber Cancer Institute, 44 Binney St., JFB 816, Boston, MA 02115. E-mail: dana_gabuzda{at}dfci.harvard.edu

ABSTRACT

The CD16+ subset of peripheral blood monocytes (Mo) is expanded dramatically during inflammatory conditions including sepsis, HIV-1 infection, and cancer. CD16+ express high levels of CX3CR1, which mediates arrest onto CX3CL1-expressing endothelial cells (EC) under flow conditions. In contrast, attachment of CD16– Mo onto cytokine-activated EC is independent of CX3CL1. Here, we investigate the ability of CD16+ and CD16– Mo to produce proinflammatory cytokines upon interaction with CX3CL1-expressing HUVEC. We demonstrate that CD16+ but not CD16– Mo produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9 when cocultured with TNF/IFN-{gamma}-activated HUVEC or nonactivated HUVEC expressing CX3CL1. Furthermore, supernatants from Mo cocultured with cytokine-activated HUVEC induce neuronal death in vitro. These results suggest that membrane-bound CX3CL1 stimulates production of IL-6, CCL2, and MMP-9 by CD16+ Mo, likely via engagement of CX3CR1. Thus, expansion of CD16+ Mo and their accumulation onto CX3CL1-expressing EC may result in recruitment of Mo and T cell subsets at sites of inflammation in response to CCL2, IL-6-induced cell activation and/or differentiation, and MMP-9-mediated vascular and tissue injury.

Key Words: vascular injury • chemokines • CX3CR1 • neurotoxicity • neurogeneration

INTRODUCTION

The expression of CD16 (Fc{gamma}RIII) identifies a subset of peripheral blood monocytes (Mo) with high levels of HLA-DR and low levels of CD14, CD64 (Fc{gamma}RI), and CD62L (L-selectin) expression [1 2 3 4 ]. The CD16+ Mo subset represents 5–15% of total Mo (tMo) in healthy individuals [1 , 4 , 5 ] but is expanded dramatically in several pathological conditions including sepsis [6 ], HIV-1 (HIV) infection [7 , 8 ], tuberculosis, metastatic cancer, and asthma [1 ]. CD16+ Mo share phenotypic and functional characteristics with macrophages (M{Phi}) and dendritic cells (DC) [1 , 3 , 5 , 9 ] and preferentially differentiate into DC after transendothelial migration (TEM) [10 ] or GM-CSF treatment [11 , 12 ]. In contrast to CD16– Mo, CD16+ Mo produce high levels of proinflammatory cytokines upon TLR engagement [9 , 13 ] and are a source of TNF, IL-1, and neurotoxic factors in HIV-infected patients [7 , 8 ]. Furthermore, CD16+ Mo-derived M{Phi} activate resting CD4+ T cells for susceptibility to HIV infection by producing the CCR3 and CCR4 ligands CCL2, CCL5, CCL17, CCL22, and CCL24 [14 ]. These findings suggest that CD16+ Mo may represent DC precursors and an important source of proinflammatory cytokines and chemokines in vivo.

Consistent with tissue-specific recruitment of leukocyte subsets [15 , 16 ], the interaction of CD16+ and CD16– Mo with endothelial cells (EC) and subsequent TEM is mediated by distinct combinations of adhesion molecules and chemokine receptors [4 , 17 , 18 ]. We demonstrated previously that CD16+ Mo express high levels of CX3CR1 [4 ], the receptor for CX3CL1 [19 ], and undergo TEM in response to soluble CX3CL1 [4 ]. In contrast, CD16– Mo express low levels of CX3CR1 but high levels of CCR2 and migrate efficiently in response to CCL2/MCP-1 but not CX3CL1 [4 , 17 ]. The heterogeneity of Mo subsets is conserved in humans and mice [18 , 20 ]. Geissmann et al. [18 ] identified Gr1+CCR2+ and Gr1–CX3CR1+ Mo subsets in mice as the homologs of human CD16– and CD16+ Mo subsets, respectively. The Gr1–CX3CR1+ Mo subset is constitutively recruited into tissues such as lymph nodes, intestine, and brain, whereas Gr1+CCR2+ Mo migrate into inflamed peritoneum [18 ], likely in response to CCL2 [21 ]. Thus, CX3CL1 and CCL2 differentially regulate recruitment of CD16+ and CD16– Mo into peripheral tissues. Expression of CX3CL1 [22 , 23 ] and CCL2 [24 ] is up-regulated dramatically in the brain of patients with HIV-associated dementia (HAD), suggesting that CD16+ and CD16– Mo may be recruited into the brain of HIV-infected patients and contribute to neuropathogenesis.

CX3CL1 is expressed as soluble and membrane-bound forms with the chemokine domain positioned atop an extracellular stalk attached to the cell membrane via a transmembrane domain [25 ]. A disintegrin and metalloprotease (ADAM)10 and ADAM17 cleave the ectodomain of CX3CL1, resulting in release of the chemokine domain [26 , 27 ]. In addition to their role in orchestrating leukocyte trafficking, chemokines induce cell activation and differentiation [28 , 29 ]. The membrane-bound form of CX3CL1 mediates firm arrest of leukocytes under flow conditions, independent of signal transduction via CX3CR1 [30 , 31 ]. However, CX3CR1 ligation by CX3CL1 induces G-protein [31 ] and subsequent MAPK pathway activation [32 , 33 ] and triggers production of MIP-1ß and IL-8 in M{Phi} [34 ]. Thus, preferential accumulation and stable attachment of CD16+ Mo onto CX3CL1-expressing EC [4 , 35 ] may trigger signal transduction pathways downstream of CX3CR1 and thereby activate the transcriptional program of genes that regulate cell activation and/or differentiation.

The up-regulation of CX3CL1 expression in lymph nodes, GALT, and brain [22 , 23 ], together with the increased frequency of CD16+ Mo in HIV-infected individuals, prompted us to investigate the production of proinflammatory cytokines by CD16+ Mo upon interaction with CX3CL1-expressing EC. Here, we show that CD16+ Mo cocultured with CX3CL1-expressing EC produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9. Furthermore, supernatants from Mo cocultured with cytokine-activated EC induce neuronal death in vitro. These results support a model in which expansion of CD16+ Mo and their accumulation onto CX3CL1-expressing EC in vascular beds create an environment favorable for recruitment of CD16– Mo and T cell subsets into the brain and other inflamed tissues in response to CCL2; Mo differentiation and T cell activation in response to IL-6; and destruction of vascular endothelium and/or blood-brain-barrier (BBB) integrity through production of high levels of MMP-9.

MATERIALS AND METHODS

Reagents and antibodies
The following mAb were used for FACS analysis: FITC anti-CD14, -CD16b, and -CD66b, PE anti-CD33, -CD19, and -CD56, PC5 anti-CD16, -CD3, and -CD54 (Beckman Coulter, Fullerton, CA); PE anti-CD1a, -CD4, -CD8, -LFA-1/CD11a, -CD31, -CD44, -VLA4, -VCAM-1, -P-selectin glycoprotein ligand-1, and -HLA-DR (BD PharMingen, San Diego, CA); FITC anti-CD1c (Miltenyi Biotec, Auburn, CA); FITC anti-CD62E and PE anti-fractalkine (FKN; R&D Systems, Minneapolis, MN); and FITC or PE anti-CX3CR1 (MBL International, Woburn, MA). Matched isotype controls purchased from the same company were used as negative controls.

Mo sorting
PBMC were isolated from fresh peripheral blood of healthy individuals. The Dana-Farber Cancer Institute Institutional Review Board (Boston, MA) approved the study protocol and informed consent forms. Highly pure tMo and CD16+ and CD16– Mo subsets were isolated from PBMC using magnetic beads (Miltenyi Biotec), as described previously [4 , 14 , 35 ]. Mo subsets were stained on the cell surface with fluorochrome-conjugates antibodies and analyzed by FACS.

EC generation and culture
Primary HUVEC were purchased from Clonetics/BioWhittaker (San Diego, CA) and immortalized with the recombinant retrovirus LXSN16 E6/E7 as described previously [36 ] to expand their lifespan in vitro. Immortalized HUVEC were cultured on collagen-coated (50 µg/ml; Sigma Chemical Co., St. Louis, MO) flasks (Costar, Corning, NY) using EC basal medium supplemented with bovine brain extract (12 µg/ml), human epidermal growth factor (12 µg/ml), hydrocortisone (1 µg/ml), FBS (5%), gentamycin/amphotericin-B (GA-1000; 0.1%, Clonetics/BioWhittaker), and G418 (200 µg/ml, Cellgro, Herndon, VA; EGM-MV). The CX3CL1-expressing HUVEC line (CX3CL1-HUVEC) was generated as described previously [4 , 35 ].

Mo-EC cocultures
HUVEC and FKN-HUVEC were cultured at 80% confluence on collagen-coated 24-well plates (Costar) and incubated in the presence (ST-HUVEC) or absence of TNF (40 ng/mL) and IFN-{gamma} (100 ng/mL; NS-HUVEC; R&D Systems) for 5 h. ST-HUVEC monolayers were washed extensively with medium to remove exogenous cytokines prior to establishing Mo-HUVEC cocultures. Mo subsets isolated as described above were cocultured with HUVEC monolayers at 5 x 105 cells/well in 0.5 ml RPMI with 10% FBS final volume. Mo:HUVEC coculture supernatants were recovered 48 h later. In parallel experiments, HUVEC cultured at 80% confluence were stimulated with TNF and IFN-{gamma}, washed extensively, and then harvested using versene, stained on the surface with fluorochrome-conjugates antibodies, and analyzed by FACS.

Neurotoxicity assay
Mixed primary human brain cell cultures consisting of neurons, astrocytes, and microglia were prepared from human fetal brain tissue (16–22 weeks gestation) procured in accordance with institutional and federal regulations, dissociated with trypsin, and then cultured on poly-lysine-coated 48-well plates at 2 x105 cells/well in high glucose DMEM with 10% heat-inactivated calf serum for 1 week. Cultures were then incubated with RPMI 10% FBS or supernatants derived from tMo:HUVEC cocultures (prepared as described above) diluted to 50% in DMEM. After 2 weeks in culture, cells were harvested using 0.25% trypsin without EDTA, washed, and incubated with FITC-conjugated annexin-V (BD PharMingen), PE-conjugated anti-CD44 and PE-Cy5-conjugated anti-CD45 antibodies (BD PharMingen), and/or 7-amino actinomycin D (7-AAD; BD PharMingen) at 4°C for 20 min. Cells were then analyzed using an EPICS XL flow cytometer (Beckman Coulter) and counted using Flow-Count fluorospheres (Beckman Coulter). The percentage of neuronal loss was calculated as [(NRPMI–NMo;HUVEC)/NRPMIx100]. NRPMI and NMO:HUVEC are the numbers of viable neurons in the presence of RPMI control medium and Mo:HUVEC supernatants, respectively. Viable neurons were identified as cells with small size [forward-scatter (FSC)] and granularity [side-scatter (SSC)] lacking the expression of CD44 and CD45 and staining negative for annexin-V and 7-AAD. In some experiments, intracellular TUNEL staining was performed (BD PharMingen kit), and cells were analyzed by flow cytometry.

Cytokine antibody array
A cytokine antibody array was used to detect expression of 60 cytokines in Mo:HUVEC coculture supernatants (antibody array C1000 VI, RayBiotech Inc., Norcross, GA). The expression of each cytokine was evaluated by measuring the OD of duplicate spots using Eagle Sight software (Stratagene, La Jolla, CA). The ratio between each cytokine spot and the positive control were calculated.

MMP-9, IL-6, and CCL2 quantification
ELISA kits for MMP-9 (Amersham Pharmacia Biotech Inc., UK), IL-6 (RayBiotech Inc.), and CCL2 (R&D Systems) were used to quantify cytokine levels in Mo:HUVEC coculture supernatants.

RESULTS

Membrane-bound CX3CL1 triggers IL-6 and CCL2 production in CD16+ Mo
We demonstrated previously that CD16+ but not CD16– Mo adhere strongly to CX3CL1-expressing EC [4 ] and fail to undergo subsequent TEM across cytokine-activated EC [35 ], but the consequences of CD16+ Mo accumulation onto inflamed EC are unknown. To investigate the ability of CD16+ and CD16– Mo to produce proinflammatory cytokines upon interaction with EC under constitutive and inflammatory conditions, tMo and the CD16+ and CD16– Mo subsets were isolated from PBMC of healthy individuals using a protocol we described previously [14 , 37 ]. Both Mo subsets express the adhesion molecules CD11a, CD31, CD44, and VLA-4, but the levels of expression are higher on CD16+ than on CD16– Mo [4 , 35 ]. In addition, CD16+ compared with CD16– Mo express higher levels of CX3CR1 [4 , 35 ], the receptor for CX3CL1 [19 ].

To study Mo interaction with EC, we used an immortalized HUVEC cell line, which we characterized in previous studies [4 , 35 ]. The HUVEC model has been used extensively as an in vitro model to dissect molecular mechanisms of leukocyte adhesion and TEM [10 , 38 ]. Furthermore, we previously used the HUVEC cell line to demonstrate TEM of CD16+ Mo in response to soluble CX3CL1 and firm arrest of these Mo onto EC-expressed CX3CL1 under physiological flow [4 ] or static conditions [35 ]. EC from different blood vessels (e.g., arteries and veins) and microvascular/capillary EC from different tissues have distinct gene-expression profiles [39 ]. Thus, functional differences are likely to be associated with each type of EC. However, compared with other types of primary human EC such as microvascular EC, HUVEC offer several important advantages for in vitro studies. In particular, HUVEC are easier to harvest and maintain in culture and have a greater inherent, useful passage number in vitro. Therefore, the HUVEC model is valuable for investigating functions of human EC and their interactions with other cell types. HUVEC incubated in the presence (ST-HUVEC) or absence (NS-HUVEC) of TNF and IFN-{gamma} for 5 h express high levels of CD54, CD31, and CD44 (data not shown). In contrast, the expression of VCAM-1 and CX3CL1 is up-regulated dramatically on ST-HUVEC compared with NS-HUVEC [4 , 35 ]. Thus, CD16+ and CD16– Mo subsets have the potential to attach to EC via CD11a-CD54, CD31-CD31, CD44-CD44, and VLA-4-VCAM-1 interactions under constitutive and/or inflammatory conditions, whereas CX3CR1-CX3CL1 interaction mediates preferential arrest of CD16+ Mo onto activated EC [4 , 35 ].

To investigate the consequences of CD16+ Mo interaction with CX3CL1-expressing EC, we used a cytokine/chemokine antibody array to screen for 60 soluble factors in supernatants from Mo-EC cocultures. Compared with Mo cocultured with NS-HUVEC, tMo cocultured with ST-HUVEC for 48 h produced higher levels of IL-6, CCL-2, CCL-7, and CCL-8 (Fig. 1A and 1B ). Similarly, high levels of IL-6, CCL-2, CCL-7, and CCL-8 expression were observed when ST-HUVEC were cocultured with CD16+ but not CD16– Mo (Fig. 1C and 1D) . Higher levels of IL-6 and CCL2 were also detected in supernatants from cocultures of CX3CL1-HUVEC with CD16+ compared with CD16– Mo (Fig. 1E and 1F) . Quantification of IL-6 and CCL2 expression by ELISA confirmed differences in expression detected by cytokine antibody array (Fig. 1G and 1J) . IL-6 production was undetectable in Mo cultured alone (Fig. 1G) , whereas low levels of CCL2 were produced constitutively by CD16+ Mo (data not shown) [14 ]. Coculture of Mo subsets with NS-HUVEC and ST-HUVEC resulted in low and high levels of IL-6 and CCL2 expression, respectively, and there was significantly higher expression of these cytokines/chemokines upon HUVEC coculture with CD16+ compared with CD16– Mo (Fig. 1G and 1I) .


Figure 1
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  		Figure 1. EC-expressed CX3CL1 triggers production of IL-6, CCL2, CCL7, and CCL8 in CD16+ Mo. tMo, or CD16– or CD16+ Mo subsets were cultured alone or cocultured with NS-HUVEC, ST-HUVEC, or CX3CL1-HUVEC for 48 h. (A, C, E) Supernatants were blotted against a membrane spotted with 60 anticytokine antibodies in duplicate (antibody array C1000 VI), as described in Materials and Methods. (B, D, F) The OD of each spot was determined using Eagle Sight software (Stratagene), and the ratio between the OD of cytokine and positive control spots was calculated. Results are representative of experiments performed with cells from two different donors (mean±SD of duplicate spots). (G–J) IL-6 and CCL2 levels in supernatants were quantified by ELISA. (G) Results are representative of experiments performed with cells from two different donors (mean±SD of duplicate wells). (H) Shown are IL-6 levels in cocultures of Mo subsets with CX3CL1-HUVEC (mean±SD, n=3). (I and J) Shown are CCL2 levels produced in cocultures of Mo with (I) NS-HUVEC (mean±SD of duplicate wells) or ST-HUVEC (mean±SD, n=3) and (J) CX3CL1-HUVEC (mean±SD, n=2). *, P < 0.05, Student’s t-test.

 
To demonstrate the ability of CX3CL1 to promote cytokine production in Mo, a HUVEC cell line stably expressing CX3CL1 (CX3CL1-HUVEC) in the absence of other activation-induced adhesion molecules such as VCAM-1 and CD62E was generated as described previously [4 , 35 ] and cocultured with CD16+CX3CR1high and CD16–CX3CR1low Mo. Moderate levels of IL-6 and CCL2 were detected in supernatants of CD16+ Mo cocultured with CX3CL1-HUVEC (Fig. 1H and 1J) . In contrast, IL-6 and CCL2 levels were low-to-undetectable when CD16–CX3CR1low/– Mo were cocultured with CX3CL1-HUVEC (Fig. 1H and 1J) or when CD16+ Mo were cocultured with HUVEC transfected with an empty vector (data not shown). Cytokine production by CD16+ Mo was higher when Mo were cocultured with ST-HUVEC compared with CX3CL1-HUVEC (Fig. 1G and 1I) . Thus, other activation-induced molecules expressed on cytokine-activated EC are also involved in CD16+ Mo activation. NS-HUVEC and CX3CL1-HUVEC cultured alone produced low levels of IL-6, and HUVEC stimulation by TNF and IFN-{gamma} up-regulated IL-6 production (data not shown). However, IL-6 levels in supernatants from ST-HUVEC cultured alone were 20- to 160-fold lower compared with those in CD16+ Mo-ST-HUVEC cocultures (data not shown). In contrast, CCL2 was not detected in supernatants from NS-HUVEC, CX3CL1-HUVEC, or ST-HUVEC cultured in the absence of Mo (data not shown). Other cytokines such as CCL24/eotaxin-2 and CXCL6/CGP-2 were also detected in supernatants from Mo cocultured with ST-HUVEC (Fig. 1C 1D 1E 1F) , consistent with our previous studies reporting constitutive production of CCL24 by CD16+ Mo [14 ]. Donor-to-donor variation may explain the differential expression of CCL24/eotaxin-2 and CGP2 in blots shown in Figure 1A 1C and 1E . Together, these results demonstrate that coculture of CD16+ Mo with cytokine-activated HUVEC leads to high levels of IL-6, CCL2, CCL7, and CCL8 expression and that the membrane-bound form of CX3CL1 triggers production of IL-6 and CCL2 in CD16+ Mo, likely by engagement of CX3CR1.

CD16+ Mo are a major source of MMP-9
High levels of MMP-2 and MMP-9 are detected in cerebrospinal fluid and brain tissue of patients with HAD [40 41 42 43 ]. Increased CX3CL1 expression may contribute to preferential recruitment of CD16+ Mo into the brain of HIV-infected patients [4 , 18 , 23 ]. Therefore, we assessed the production of MMP-2 and MMP-9 by CD16+ and CD16– Mo after interaction with CX3CL1-expressing EC. The results shown in Figure 2 demonstrate that CD16+ Mo are a major source of MMP-9 but not MMP-2. CD16+ but not CD16– Mo secreted low levels of MMP-9 when cultured alone or in the presence of NS-HUVEC for 48 h (Fig. 2A) . Coculture of CD16+ Mo with ST-HUVEC resulted in a dramatic increase in MMP-9 production, whereas MMP-9 levels remained low in CD16– Mo-ST-HUVEC cocultures (Fig. 2A) . When Mo were stimulated directly with TNF-{alpha} (10–100 ng/ml), CD16+ and CD16– Mo subsets produced high levels of MMP-9 (data not shown). However, CD16– Mo did not produce MMP-9 when incubated with ST-HUVEC. These findings indicate that residual cytokines used to stimulate EC were removed efficiently by extensive washing before coculturing ST-HUVEC with Mo. Similar to HUVEC, brain microvascular EC (BMVEC) express CX3CL1 upon TNF-{alpha}/IFN-{gamma} stimulation [35 ], and high levels of MMP-9 were observed when CD16+ Mo were incubated with ST-BMVEC (data not shown). Thus, cytokine-activated HUVEC and BMVEC have a similar ability to trigger CD16+ Mo activation, at least in part via CX3CL1-CX3CR1 interaction. Given the expression of CX3CL1 on ST-HUVEC [4 , 35 ], we sought to determine whether CX3CL1 expressed on EC in the absence of other activation-induced molecules triggers MMP-9 production in CD16+ Mo. When Mo subsets were cocultured with CX3CL1-HUVEC [4 , 35 ], moderate levels of MMP-9 were detected in supernatants of CD16+ but not CD16– Mo-CX3CL1-HUVEC cocultures (Fig. 2B) . MMP-9 production was also detected in the supernatants of tMo cocultured with CX3CL1-HUVEC or ST-HUVEC, indicating that the anti-CD16 beads used for CD16+ Mo isolation did not interfere with MMP-9 production (Fig. 2C) . MMP-9 was not detected in the supernatants of NS-HUVEC, ST-HUVEC, or CX3CL1-HUVEC cultured alone (data not shown). In contrast to the selective production of MMP-9 in cocultures of EC with CD16+ Mo, MMP-2 levels were similarly high when NS-HUVEC and ST-HUVEC were cocultured with CD16+ or CD16– Mo (Fig. 2D) . These results demonstrate that CD16+ Mo are a major source of MMP-9 and that EC-expressed CX3CL1 triggers MMP-9 but not MMP-2 production in this Mo subset.


Figure 2
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  		Figure 2. EC-expressed CX3CL1 triggers MMP-9 release by CD16+ Mo. HUVEC were incubated in the presence (ST-HUVEC) or absence (NS-HUVEC) of TNF and IFN-{gamma} (ST-HUVEC) for 5 h or stably transfected with CX3CL1 (CX3CL1-HUVEC). CD16+ and CD16– Mo subsets were isolated and then cultured in 24-well plates at 5 x 105 Mo/well with 80% confluent HUVEC. Supernatants were recovered 48 h postcoculture, and levels of MMP-9 and MMP-2 were quantified by ELISA. (A–C) Shown are MMP-9 levels in Mo-HUVEC cocultures. (A) Shown are MMP-9 levels (mean±SEM, n=4). (B and C) Results are representative of experiments performed with cells from two different donors (mean±SD of duplicate wells). (D) Shown are MMP-2 levels (mean±SEM, n=4). * and **, P < 0.05, Student’s t-test.

 
Mo interaction with inflamed EC leads to release of neurotoxic factors
Previous studies demonstrated that CD16+Mo isolated from peripheral blood of patients with AIDS release soluble factors with neurotoxic activity [8 ]. We therefore investigated whether soluble factors produced upon interaction of CD16+ Mo with ST-HUVEC induce neuronal death. Cultures containing a mixture of neurons, astrocytes, and microglia isolated from human fetal brain tissue were cultured for 2 weeks in the presence of medium alone or conditioned media from tMo cocultured with NS-HUVEC or ST-HUVEC. Neuronal cell bodies were identified on the basis of their lack of CD44 (astrocyte marker) and CD45 (microglia marker) expression and small size (FSC) and granularity (SSC) compared with CD44+CD45– astrocytes and CD44–CD45+ microglia (J. Wang and D. Gabuzda, unpublished results). Viable neurons excluded 7-AAD and were negative for annexin-V staining. Significantly higher levels of neuronal cell death were observed in the presence of supernatants from Mo-ST-HUVEC compared with Mo-NS-HUVEC cocultures, whereas neuronal viability was close to 100% in the presence of medium alone (Fig. 3 ). In other experiments, Mo were cocultured with HUVEC in the upper chamber of a transwell system with primary human brain cells cultured in the bottom chamber. Under these experimental conditions, the appearance of TUNEL+ and/or 7-AAD+ neurons was detected as early as Day 5, when cocultures contained ST-HUVEC but not NS-HUVEC (data not shown). The deleterious effect on neuronal viability was dependent on Mo interaction with ST-HUVEC, as neuronal death was not induced in the presence of Mo or ST-HUVEC cultured alone (data not shown). These results demonstrate that interaction of Mo with cytokine-activated HUVEC results in production of as-yet unidentified, soluble factors, which are detrimental for neuronal survival.


Figure 3
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  		Figure 3. Neuronal toxicity induced by soluble factors produced in Mo-HUVEC cocultures. Mixed primary human brain cell cultures consisting of neurons, astrocytes, and microglia were maintained in the presence or absence of supernatants from Mo cocultured with NS-HUVEC or ST-HUVEC for 2 weeks. The percentage of neuronal loss was determined by counting the number of viable neurons relative to the control, as described in Materials and Methods. Viable neurons were identified as cell lacking the expression of the astrocyte and microglia markers CD44 and CD45, respectively, and stained negative for annexin-V and 7-AAD. Results are representative of experiments performed with cells from two different donors (mean±SD of duplicate wells). *, P < 0.05, Student’s t-test.

 
DISCUSSION

In this study, we demonstrate that CX3CL1-expressing EC trigger production of IL-6, CCL2, and MMP-9 in CD16+ Mo, likely by engagement of CX3CR1 expressed on this Mo subset [4 ]. Moreover, we demonstrate that supernatants from Mo cocultured with cytokine-activated EC induce neuronal death in vitro. These findings elucidate functional differences between CD16+ and CD16– Mo under inflammatory conditions and provide evidence that accumulation of CD16+ Mo onto EC expressing CX3CL1 may create a proinflammatory microenvironment deleterious for the integrity of vascular beds and subjacent tissues such as brain.

Recruitment of Mo subsets into tissues requires firm arrest and attachment onto vascular endothelia under shear stress conditions [44 ] and is mediated by specific combinations of adhesion molecules and chemokine receptors [4 , 16 17 18 ]. In addition to their role in regulating cell trafficking [28 , 29 , 45 ], adhesion molecules and chemokines may control leukocyte activation, differentiation, and function in tissues where these cells are recruited preferentially. We reported previously that CD16+ and CD16– Mo subsets attach with similar frequency onto cytokine-activated EC under flow conditions, but membrane-bound CX3CL1 triggers preferential arrest of CD16+ Mo [4 ]. Moreover, CD16+ Mo attach strongly to cytokine-activated EC expressing CX3CL1 and fail to undergo TEM, in contrast to CD16– Mo, which efficiently cross inflamed endothelia under static conditions in vitro [35 ]. However, experiments performed under physiological shear stress demonstrated that CD16+ Mo migrate underneath a confluent EC monolayer (unpublished observations), consistent with previous studies demonstrating the critical role of shear flow stress in triggering lymphocyte diapedesis [46 ]. Therefore, CD16+ Mo attached to inflamed EC monolayers may migrate into tissues in response to soluble CX3CL1, stromal cell-derived factor (SDF) [4 ], or other chemokines under flow conditions in vivo. The adhesion molecules CD11a, CD31, CD44, and VLA-4 are expressed at high levels on CD16+ and CD16– Mo, suggesting that CD11a-CD54, CD31-CD31, CD44-CD44, and VLA-4-VCAM-1 interactions may be involved in mediating adherence of these Mo subsets to EC under constitutive and/or inflammatory conditions. However, CD16+ Mo express higher CX3CR1 levels and have a superior ability to interact with CX3CL1-expressing EC compared with CD16– Mo [4 ]. Here, we demonstrate that CD16+ Mo produce high levels of IL-6, CCL2, and MMP-9 as a consequence of the CX3CR1-CX3CL1 interaction. In contrast, low levels of these cytokines are produced by CD16– Mo, likely as a result of low levels of CX3CR1 expression. Cytokine production is higher when CD16+ Mo are cocultured with EC expressing CX3CL1 together with other activation-induced molecules compared with CX3CL1 alone. Thus, other molecular interactions in addition to CX3CL1-CX3CR1 may contribute to CD16+ Mo activation upon arrest onto inflamed vascular beds in vivo. These studies provide the first evidence that engagement of CX3CR1 by CX3CL1 not only regulates differential trafficking of CD16+ and CD16– Mo subsets but also selectively induces production of IL-6, CCL2, and MMP-9 in CD16+ Mo. Thus, by inducing IL-6, CCL2, and MMP-9, engagement of CX3CR1 may influence the activation and/or differentiation of CD16+ Mo and their functions in innate and/or adaptive immunity.

IL-6 is produced by a variety of cell types including Mo/M{Phi} [47 ], DC [48 ], and EC [49 ], and its receptor is expressed in lymphoid and nonlymphoid tissues [50 ]. IL-6 regulates biological processes involved in innate and adaptive immunity [51 ] and inflammation [52 ] including T cell activation [53 ] and Mo differentiation into M{Phi} [54 ]. In addition, up-regulation of IL-6 production contributes to the pathogenesis of inflammatory conditions such as rheumatoid arthritis [55 ], atherosclerosis [56 , 57 ], and CNS inflammation during HAD [58 , 59 ]. IL-6 also stimulates HIV replication by M{Phi} in vitro [60 ]. The finding that IL-6 is produced preferentially by CD16+ Mo upon interaction with CX3CL1-expressing EC suggests that expansion of CD16+ Mo may contribute to increased levels of IL-6 production in inflamed tissues, such as the brain of HAD patients, where these cells may be preferentially recruited [4 , 18 , 22 , 23 ].

In a recent study, we demonstrated that CD16+ Mo-derived M{Phi} constitutively produce CCL2 and that significantly higher levels are produced by CD16+ Mo from HIV-infected patients compared with uninfected individuals [14 ]. High levels of CCL2 in brain or CSF are associated with HAD [24 ], and a CCR2 polymorphism and mutant CCL2 promoter allele linked to increased CCL2 levels are associated with accelerated progression to AIDS and increased risk of HAD [24 , 61 ]. Here, we show that EC-expressed CX3CL1 dramatically up-regulates expression of CCL2 in CD16+ Mo. CCL2 plays a critical role in regulation of leukocyte trafficking to sites of inflammation [28 ]. Thus, accumulation of CD16+ Mo onto CX3CL1-expressing EC may result in further recruitment and possibly activation of Mo and T cell subsets expressing the CCL2 receptors CCR2 and CCR4. Together, these findings suggest that recruitment of CD16+ Mo into the brain in response to CX3CL1 may contribute to HIV pathogenesis and development of HAD, at least in part, by producing CCL2.

MMP-2 (gelatinase A) and MMP-9 (gelatinase B) digest specific extracellular matrix (ECM) components during normal processes and contribute to vascular and tissue injury when present at high levels during pathological conditions. MMP-2 and MMP-9 also modulate the activity of several chemokines and cytokines including TNF-{alpha}, IL-1ß, CXCL8, CCL2, CCL7, and CCL8 by proteolytic cleavage, thereby providing positive and negative regulation during inflammatory cell recruitment (reviewed in ref. [62 ]). MMP-2 and MMP-9 are expressed in many different cell types including activated M{Phi} and DC [43 , 63 , 64 ]. MMP-9 production by Mo increases as a function of differentiation into M{Phi} [65 ] or upon infection with HIV [43 ]. High levels of MMP-2 and MMP-9 are detected in cerebrospinal fluid and brain tissue of patients with HAD [40 41 42 43 ]. Here, we identify CD16+ Mo as a major source of MMP-9 following interaction with CX3CL1-expressing EC, and similar levels of MMP-2 are detected upon coculture of CD16+ and CD16– Mo with cytokine-activated or nonactivated EC. The capacity of CD16+ Mo to produce high levels of MMP-9 upon interaction with CX3CL1-expressing EC may increase their capacity to transmigrate but may also have deleterious consequences for vascular and tissue integrity during certain pathological conditions. For example, massive accumulation of CD16+ Mo onto brain EC during HAD [4 , 18 , 23 ] may alter the integrity of BBB and contribute to neurodegeneration [66 ]. Consistent with this hypothesis, our results demonstrate that supernatants from Mo cocultured with cytokine-activated EC, rich in MMP-2 and MMP-9, induced neuronal death in vitro. Whether engagement of CX3CR1 on Mo via CX3CL1 leads to production of neurotoxic factors remains to be determined.

Pulliam et al. [8 ] demonstrated previously that soluble factors produced by Mo from AIDS patients with an increased frequency of CD16+ Mo induce neuronal toxicity in vitro, but whether CD16+ Mo were the source of neurotoxic factors remains unclear. CD16+ and CD16– Mo have the potential to be recruited into the brain of HIV-infected patients expressing high levels of CX3CL1 and CCL2 [24 , 67 ] and may contribute to neuropathogenesis. CD16+ Mo may contribute to HIV neuropathogenesis by carrying virus into the brain, promoting productive infection in T cells or possibly other bystander cells [14 ], and/or producing proinflammatory cytokines such as IL-6, CCL2, and MMP-9. CD16– Mo may also carry virus into the brain or after trafficking into the brain, may differentiate into M{Phi}, which can serve as targets for productive HIV infection and/or possibly produce proinflammatory cytokines and neurotoxic factors. Further studies are needed to identify neurotoxic factors produced by specific Mo subsets and determine their roles in HIV neuropathogenesis.

Studies using CX3CR1–/– mice suggest a role for CX3CR1-CX3CL1 interaction in atherogenesis [68 , 69 ], cerebral ischemia-reperfusion injury [70 ], and clearance of entero-invasive pathogens by DC [71 ]. Genetic polymorphism in CX3CR1 is likely to be associated with increased risk for coronary artery disease [72 , 73 ] and more rapid HIV disease progression [74 , 75 ]. A model has been proposed in which CX3CL1 mediates vascular injury through accumulation of effector leukocytes onto inflamed endothelia [76 ]. The consequences of CD16+ Mo accumulation onto vascular beds at sites of inflammation in vivo are unknown. Here, we provide evidence that attachment of CD16+ Mo onto CX3CL1-expressing endothelia may exacerbate local inflammation by producing IL-6 and CCL2 and may affect the integrity of vascular beds and subjacent tissues by producing high levels of MMP-9. Further studies are needed to determine whether under physiological flow conditions, CD16+ Mo remain irreversibly attached to inflamed EC expressing CX3CR1 or migrate into subjacent tissues in response to FKN, SDF-1, or other chemokines. Further studies are also required to address the question of whether CD16+ Mo activation occurs via membrane-bound and/or proteolytically cleaved forms of CX3CL1.

In summary, we have demonstrated that EC-expressed CX3CL1 preferentially triggers production of IL-6, CCL2, and MMP-9 in CD16+ Mo, likely by engagement of CX3CR1. These findings support a model in which accumulation of CD16+ Mo onto inflamed endothelial beds expressing CX3CL1 contributes to vascular and tissue injury during HIV infection and other pathological conditions in which this Mo subset is expanded [1 , 7 , 8 ]. Engagement of CX3CR1 on CD16+ Mo may lead to a second wave of Mo and T cell subset recruitment into the brain and other inflamed tissues in response to CCL2, followed by IL-6-triggered activation and differentiation of Mo and T cells and EC damage in response to high levels of MMP-9 production. Thus, our findings suggest that new therapeutic strategies to block CX3CR1-CX3CL1 interactions may reduce pathogenic consequences of CD16+ Mo infiltration into sites of inflammation.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DA016549 and NS35734. Core facilities were supported by the Harvard Medical School Center for AIDS Research and DFCI/Harvard Center for Cancer Research grants. We thank A. Moses for providing HUVEC cell lines and Ashley Moses and Francis W. Luscinskas for valuable discussions.

Received February 28, 2006; revised May 15, 2006; accepted May 17, 2006.

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