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Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions
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Immunobiology 209 (2004) 11–20 www.elsevier.de/imbio
Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions Petronela Ancutaa, Ashlee Mosesb, Dana Gabuzdaa,* a
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, JFB 816, Boston, MA 02115, USA b Vaccine and Gene Therapy Institute, Oregon Health & Sciences University, 505 NW, 185th Avenue, Beaverton, OR 97006, USA Received 20 March 2004; accepted 13 April 2004
Abstract CD16+ monocytes represent 5–10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-g (IFN-g) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16 and CD16+ monocyte subsets adhered to TNF/IFN-g-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16 monocytes across TNF/IFN-gstimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-g triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, proinflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules. r 2004 Elsevier GmbH. All rights reserved. Keywords: Monocytes, dendritic cells, siglecs
Abbreviations: BMVEC, brain microvascular endothelial cells; CD62E, E-selectin; CNS, central nervous system; FKN/CX3CL1, fractalkine; HUVEC, human umbilical vascular endothelial cells; ICAM-1, intercellular adhesion molecule-1; IFN-g, interferon-g; JAM, junctional adhesion molecule; MCP-1, monocyte chemoattractant protein-1; MIP-1a, macrophage inflammatory protein-1a; PECAM-1/CD31, platelet endothelial cell adhesion molecule-1; SDF-1a, stromal derived factor-1a; TEM, transendothelial migration; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1 *Corresponding author. Tel.: +1-617-632-2154; fax: +1-617-632-3113. E-mail address: dana [email protected] (D. Gabuzda). 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.04.001
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Introduction FcgRIII (CD16) expression distinguishes two subsets of human peripheral blood monocytes (Ziegler-Heitbrock, 1996). CD16+ monocytes represent 5–10% of peripheral blood monocytes in healthy individuals and are dramatically expanded in several pathological conditions including sepsis (Fingerle et al., 1993), HIV-1 infection (Pulliam et al., 1997; Thieblemont et al., 1995), metastatic cancer (Saleh et al., 1995), and asthma (Rivier et al., 1995). CD16+ monocytes exhibit a macrophage-like morphology ex vivo (Ziegler-Heitbrock et al., 1993), produce high levels of pro-inflammatory cytokines (e.g. tumor necrosis factor (TNF) and IL-1) (Belge et al., 2002; Thieblemont et al., 1995) and neurotoxic factors (Pulliam et al., 1997), and may represent dendritic cell precursors in vivo (Randolph et al., 2002). CD16 and CD16+ monocyte subsets exhibit distinct patterns of cell surface molecule expression and migratory properties. Consistent with the differential expression of CCR1, CCR2, and CX3CR1 on these monocyte subsets, CD16 monocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) (Weber et al., 2000), whereas fractalkine (FKN/CX3CL1) preferentially triggers migration and arrest of CD16+ monocytes (Ancuta et al., 2003). Thus, CD16 and CD16+ monocytes may be recruited into distinct anatomic sites in response to locally produced chemokines. FKN is a unique membrane-bound chemokine with a CX3C motif atop a mucin like stalk (Bazan et al., 1997), first identified by screening of a murine choroid plexus complementary DNA library (Pan et al., 1997). Human FKN messenger RNA is expressed under constitutive conditions in several tissues including heart, brain, lung, kidney, and pancreas (Bazan et al., 1997). Although the cellular distribution of FKN under constitutive conditions in vivo is still controversial (Lucas et al., 2001), its expression on cytokine-activated endothelial cells is well established (Bazan et al., 1997) and suggests a role of this chemokine in regulating leukocyte trafficking (Schall, 1997). Expression of the FKN receptor, CX3CR1, is detected on subsets of leukocytes including CD4+ T, CD8+ T, NK cells, and monocytes (Imai et al., 1997). The soluble form of FKN is released from the cell surface by proteolytic cleavage mediated by disintegrinlike metalloproteinases (Garton et al., 2001; Hundhausen et al., 2003) and is a chemoattractant for CX3CR1+ leukocytes (Ancuta et al., 2003; Fong et al., 1998; Imai et al., 1997). The membrane-bound form of FKN mediates adhesion of leukocytes under static and flow conditions in a selectin- and integrin-independent manner (Ancuta et al., 2003; Bazan et al., 1997; Fong et al., 1998; Imai et al., 1997). The ability of FKN to mediate firm arrest of leukocytes onto endothelial cells is
not due to its presentation atop a rigid stalk, but rather to the CX3C domain itself, which has a slow off-rate from its receptor (Haskell et al., 2000). FKN–CX3CR1 interaction has been implicated in the pathogenesis of several inflammatory diseases through the accumulation and/or activation of CX3CR1+ leukocytes on vascular beds and their deleterious infiltration into peripheral tissues (Umehara et al., 2001). For example, increased levels of FKN are expressed in the brain of patients with HIV-1 associated dementia (HAD) (Pereira et al., 2001; Tong et al., 2000) and may trigger monocyte infiltration into the central nervous system (CNS), which is a critical event in the pathogenesis of HAD (Gartner, 2000; Gartner and Liu, 2002). Given the dramatic expansion of CD16+ monocytes in patients with HAD (Crowe et al., 2003; Gartner, 2000; Gartner and Liu, 2002; Pulliam et al., 1997) and other inflammatory conditions (Grage-Griebenow et al., 2001; Ziegler-Heitbrock, 1996), and their ability to produce high levels of pro-inflammatory cytokines (Belge et al., 2002; Thieblemont et al., 1995) and neurotoxic factors (Pulliam et al., 1997), we investigated the migration of these cells across endothelial monolayers under constitutive and inflammatory conditions. Here we demonstrate that soluble FKN preferentially triggers migration of CD16+ monocytes across unactivated brain microvascular (BMVEC) and human umbilical vascular endothelial (HUVEC) cell monolayers. Stimulation of endothelial cells with TNF and IFN-g induces expression of membrane-bound FKN together with other activation-induced molecules and results in strong attachment of CD16+ monocytes, which fail to undergo subsequent diapedesis in response to a chemotactic gradient. These results suggest that CD16+ monocytes, which are dramatically expanded in the peripheral blood of HIV-1-infected patients and produce pro-inflammatory cytokines and neurotoxic factors, may contribute to the pathogenesis of HAD and possibly other inflammatory CNS diseases by accumulating onto blood-brain barrier endothelial cells that express FKN and other adhesion molecules under inflammatory conditions.
Material and methods Reagents and antibodies Recombinant human MCP-1, MIP-1a, stromal derived factor-1a (SDF-1a), FKN, TNF, and IFN-g were purchased from R&D Systems. Versene was purchased from Life Technologies. The following antibodies were used: FITC anti-CD14, -CD16b, PE anti-CD33, -CD56, and -intercellular adhesion molecule-1 (ICAM)-1, and PC5 anti-CD3 and -CD16 mAbs (Beckman
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Coulter); FITC anti-CD62E and PE anti-CCR2 and -FKN mAbs (R&D Systems); FITC anti-CX3CR1 mAb (MBL International); PE anti-vascular cell adhesion molecule (VCAM)-1 mAbs (BD Pharmingen).
Fluorospheres (Beckman Coulter). The index of migration (IM) was calculated as the ratio between the numbers of cells migrated in response to chemokine and medium alone.
Cell isolation
Adhesion assay under static conditions
PBMC were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation (Pharmacia Biotech). The study protocol and informed consent forms were approved by the Dana-Farber Cancer Institute Institutional Review Board. Monocytes were negatively isolated from PBMC using the Monocyte Isolation Kit II, according to the manufacturer’s protocol (Miltenyi Biotec). The purity of the monocyte fraction was 95–98% as determined by flow cytometry analysis after staining with fluorescence conjugated antiCD14, -CD33, -CD16b, -CD3, and -CD56 mAbs. CD16+ monocytes were not depleted by this approach as determined by staining with CD16 mAbs before and after monocyte isolation.
HUVEC were cultured at 80% confluence and incubated in the presence or absence of TNF (40 ng/ ml) and IFN-g (50 ng/ml), for 18 h at 37 C. Stimulated (ST) or unstimulated (NS) HUVEC were washed 2 times with media and co-cultured with PE anti-CD33 and PC5 anti-CD16 mAbs-labeled monocytes for 30 min at 37 C in DMEM/10% FBS. Non-adherent monocytes were removed by extensive washing with PBS. Monocytes attached to HUVEC were harvested together with endothelial cells using Trypsin. The number of adherent monocytes was determined by FACSs using FlowCount Fluorospheres. The percentage of adherent CD16 and CD16+ monocytes was calculated as the ratio between the number of monocytes in the adherent fraction and the number of monocytes in the input fraction multiplied by 100.
Endothelial cell culture Primary HUVEC were purchased from Clonetics/ BioWhittaker and immortalized with the recombinant retrovirus LXSN16 E6/E7 as previously described (Moses et al., 1999) to expand their lifespan in vitro. Primary brain endothelial microvascular endothelial cells (BMVEC) were isolated from temporal resections as previously described (Moses et al., 1993). Immortalized HUVEC and primary BMVEC were cultured on collagen-coated (50 mg/ml, SIGMA) flasks using endothelial cell medium EGM-MV (BioWhittaker) containing 200 mg/ml G418 (Cellgro).
Statistical analysis Data are presented as the mean7SD. Student’s t test was used for analysis of statistical significance. Values of po0:05 were considered statistically significant.
Results CX3CR1 and CCR2 expression on purified monocytes
Transwell and transendothelial migration assays Chemotaxis assays were performed as previously described (Kishimoto et al., 1990; Luscinskas et al., 1996). Briefly, HUVEC and BMVEC were cultured on collagen-coated transwells (6.5 mm diameter and 5 mm pore size, Corning Costar) at 2 105 cells/well in EGMMV medium for 2 days. The confluence of the monolayers was assessed by measure of electrical resistance using a Millicells–ERS electrode (Millipore). Monocytes were isolated as described above and stained with PE anti-CD33 and PC5 anti-CD16 mAb prior to migration assays. Serial dilutions of chemokines in DMEM (Invitrogen) containing 0.5% BSA were placed in the bottom chamber of the transwell system. Monocytes (106) were placed in the upper chamber of the transwell system and incubated for 2.5–4 h at 37 C. The number of migrated CD16 and CD16+ monocytes was determined by FACS analysis using Flow-Count
Monocytes were isolated from PBMC by negative selection using magnetic beads and analyzed for the cell surface expression of CD14, CD16, CCR2, and CX3CR1. CD16 expression distinguished two subsets: CD16 and CD16+ monocytes (Fig. 1, left panel). Among CD16+ monocytes, two subsets were further identified by distinct levels of CD14 expression: CD14highCD16+ and CD14lowCD16+ (Fig. 1, left panel). CD16 monocytes expressed CCR2 (MCP-1 receptor) but not CX3CR1 (FKN receptor), whereas CD16+ monocytes expressed CX3CR1 but low to undetectable levels of CCR2 (Fig. 1, middle and right panel). Thus, CCR2 and CX3CR1 are differentially expressed on CD16 and CD16+ monocytes, suggesting distinct migratory properties of these monocyte subsets in response to MCP-1 and FKN. The pattern of chemokine receptor expression on monocytes isolated by negative selection was similar to that previously
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Fig. 1. CCR2 and CX3CR1 expression on CD16 and CD16+ monocytes. Monocytes were purified from peripheral blood of healthy individuals by negative selection using magnetic beads. Monocytes were triple stained with FITC anti-CD14, PE anti-CD33, PC5 anti-CD16, PE anti-CCR2, and FITC anti-CX3CR1 mAbs, and analyzed by flow cytometry. Values in each quadrant represent the percentage of cells. Results are representative of experiments performed with cells from ten different donors.
described for monocytes from whole blood or PBMC (Ancuta et al., 2003).
TNF and IFN-c induce FKN expression on both BMVEC and HUVEC To investigate the interaction of CD16 and CD16+ monocytes with endothelial cells under constitutive and inflammatory conditions, we used immortalized HUVEC, which exhibit morphologic and phenotypic characteristics similar to primary HUVEC (Ancuta et al., 2003), and primary brain microvascular endothelial cells (BMVEC). BMVEC cultured at confluence on collagen-coated plates exhibited a fibroblast-like morphology, whereas HUVEC exhibited a rounded morphology (data not shown). Under constitutive conditions, HUVEC expressed moderate levels of ICAM-1 but not the activation-induced adhesion molecules VCAM-1, CD62E, and FKN, whereas BMVEC expressed both VCAM-1 and ICAM-1 but not CD62E and FKN (Fig. 2). Expression of VCAM-1, ICAM-1, CD62E, and FKN was then assessed on HUVEC and BMVEC after stimulation with TNF and IFN-g, a combination of cytokines known to induce high levels of FKN expression on HUVEC (Ancuta et al., 2003) and other cell types (Ludwig et al., 2002; Yoshida et al., 2001). Results presented in Fig. 2, left panels, demonstrate that HUVEC stimulation with TNF and IFN-g induces high levels of FKN, VCAM-1, and CD62E expression together with dramatic up-regulation of ICAM-1 expression. Stimulation of BMVEC with the same combination of cytokines results in up-regulation of VCAM-1 and ICAM-1 expression, moderate expression of FKN molecules, but no detectable surface expression of CD62E (Fig. 2, right panels). Stimulation of BMVEC with TNF and IL-1b, alone or in combination, failed to induce detectable CD62E expression (data not shown). These results demonstrate that BMVEC can
be distinguished from HUVEC by constitutive VCAM-1 expression and that TNF and IFN-g induce FKN expression on both HUVEC and BMVEC, but detectable CD62E expression is induced only on HUVEC.
FKN triggers CD16+ monocyte migration across a confluent BMVEC monolayer We previously reported that CD16 and CD16+ monocytes undergo efficient chemotactic migration across confluent HUVEC monolayers (Ancuta et al., 2003). Given the constitutive expression of VCAM-1 on unactivated BMVEC, we investigated the ability of BMVEC monolayers to support migration of these monocyte subsets in response to MCP-1 and soluble FKN. The spontaneous migration of CD16 monocytes was higher compared to that of CD16+ monocytes (data not shown). Both monocyte subsets underwent efficient chemotactic migration across BMVEC (Fig. 3A and data not shown): CD16 monocytes migrated with a high index of migration in response to MCP-1 and MIP-1a, whereas FKN triggered efficient transendothelial migration (TEM) of CD16+ monocytes (Fig. 3B). Of note, TEM of CD16+ monocytes across unactivated BMVEC was triggered by low (1–10 ng/ml) but not high (50–100 ng/ml) doses of FKN, whereas higher concentrations of MCP-1 and MIP-1a (50–100 ng/ml) were required for maximal migration of CD16 monocytes (Fig. 3B and data not shown). These results indicate that BMVEC, similar to HUVEC, support TEM of CD16+ monocytes in response to soluble FKN.
Transendothelial migration of CD16+ monocytes across TNF/IFN-c-stimulated HUVEC We previously demonstrated that CD16+ monocytes express high levels of CX3CR1 and arrest onto cell surface-expressed FKN with higher frequency compared
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Fig. 2. Phenotypic characterization of HUVEC and BMVEC. Immortalized HUVEC and primary BMVEC were cultured at 80% confluence on collagen-coated plates and incubated in the presence or absence of TNF (40 ng/ml) and IFN-g (50 ng/ml). After 18 h, cells were harvested using Versene, stained with PE anti-VCAM, FITC anti-CD62E, PE anti-ICAM-1, and PE anti-FKN mAbs, and analyzed by flow cytometry. Results are representative of three independent experiments.
to CD16 monocytes under flow conditions in vitro (Ancuta et al., 2003). It remains unknown whether CD16+ monocytes attached to inflamed endothelial cells via the interaction of CX3CR1 with FKN undergo subsequent diapedesis. We first investigated adhesion of monocyte subsets onto TNF/IFN-g-activated HUVEC monolayers that express high levels of membrane-bound FKN. CD16+ monocytes adhered onto resting HUVEC under static conditions with significantly higher frequency compared to CD16 monocytes (16.572.1% compared to 8.270.2%, mean7SD, n ¼ 3; po0:05; Student’s t test). When HUVEC were stimulated with TNF and IFN-g, a dramatic increase in the frequency of adherent monocytes was observed, and CD16 and CD16+ monocytes attached to TNF/IFN-g-stimulated HUVEC with similar frequencies (59.2712.2% and 47.7719.2%, respectively, mean7SD, n ¼ 3) (Fig. 4). We further investigated CD16 and CD16+ monocyte migration in the transwell system as compared to TEM across unactivated or TNF/IFN-g-activated HUVEC in response to the appropriate chemokines. In the transwell system, CD16 and CD16+ monocytes
migrated with similar efficiency in response to MCP-1 and FKN, respectively (230 147752 563 and 24 368711 061, respectively, mean7SD, n ¼ 3) (note that CD16+ monocytes represented o10% of total monocytes) (Fig. 5, left panels). In the transendothelial system, the number of CD16 monocytes that migrated in response to MCP-1 was only slightly lower compared to that observed in the transwell system (230 147752 563), regardless of whether HUVEC were stimulated (174 601793 438) or not (208 525769 247) with TNF and IFN-g (Fig. 5, upper panels). In contrast, the yield of CD16+ monocyte migration was dramatically decreased when migration was assessed across a resting HUVEC monolayer (11127734) compared to their migration in the transwell system (24 368711 061) (Fig. 5, lower panels). Moreover, stimulation of HUVEC with TNF and IFN-g dramatically reduced the number of transmigrated CD16+ monocytes (2907270) (Fig. 5, lower right panel). These results demonstrate that CD16 and CD16+ monocytes adhere to TNF/IFN-g-activated HUVEC with similar frequency, but only CD16 monocytes are able to detach
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Fig. 3. Migration of CD16 and CD16+ monocytes across BMVEC. (A) Primary BMVEC were cultured at confluence on collagencoated transwell filters. Monocytes were pre-stained with PE anti-CD33 and PC5 anti-CD16 mAbs and allowed to migrate across a BMVEC monolayer in response to optimal concentrations of MCP-1 and FKN. After 4 h, CD33+ monocytes that migrated in the bottom chamber of the transwell system were recovered and the % of CD16+ monocytes in each migrated fraction was determined by flow cytometry. (B) The numbers of CD16 and CD16+ monocytes that migrated in response to medium alone, MCP-1, and FKN were counted using fluorescent beads. The index of migration of monocyte subsets was calculated at the chemokine concentrations indicated in the figure. Results are representative of three independent experiments performed with monocytes from different donors.
from inflamed endothelium and migrate in response to MCP-1. In contrast, CD16+ monocytes remain strongly attached to inflamed endothelial cells and fail to undergo diapedesis in response to soluble FKN.
Discussion Massive infiltration of activated and/or HIV-1 infected monocytes is observed postmortem in the brain of patients with AIDS and represents a critical event in
HIV-1 neuropathogenesis (Gartner, 2000; Persidsky & Gendelman, 2003). CD16+ monocytes are dramatically expanded (up to 50%) in the peripheral blood of patients with HAD, produce high levels of proinflammatory cytokines and neurotoxic factors and may, if recruited into the brain, contribute to alteration of blood-brain barrier integrity and neuronal loss (Gartner, 2000; Gartner and Liu, 2002; Nottet, 1999; Pulliam et al., 1997; Thieblemont et al., 1995). We previously identified FKN as the major chemokine and adhesion molecule mediating arrest and migration of CD16+ monocytes (Ancuta et al., 2003). Similarly,
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Fig. 4. Adhesion of CD16 and CD16+ monocytes to HUVEC under static conditions. Immortalized HUVEC were cultured at 80% confluence and incubated in the presence or absence of TNF (40 ng/ml) and IFN-g (50 ng/ml), for 18 h at 37 C. Stimulated (ST) or unstimulated (NS) HUVEC were washed 2 times with media and co-cultured with PE anti-CD33 and PC5 anti-CD16 mAbs-labeled monocytes for 30 min at 37 C. Non-adherent monocytes were removed by extensive washing, whereas monocytes attached to HUVEC were harvested together with endothelial cells using trypsin. CD16 and CD16+ monocytes in the adherent fraction were counted by FACSs using fluorescent beads and the percentage of adherent cells was calculated taking into account the number of CD16 and CD16+ monocytes in the input fraction (mean7SD, n ¼ 2). p ¼ 0:02 Student’s t test (CD16 versus CD16+ monocytes); po0:05; Student’s t test (NS HUVEC versus ST HUVEC).
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mouse CX3CR1high monocytes, which resemble human CD16+ monocytes, are constitutively recruited into the brain and other peripheral tissues via CX3CR1 interaction with FKN (Geissmann et al., 2003). FKN is constitutively expressed in different peripheral tissues (Bazan et al., 1997) and is dramatically up-regulated in the CNS of patients with HAD (Pereira et al., 2001; Tong et al., 2000). Together, these findings suggest that CD16+ monocytes may migrate into the brain under constitutive and inflammatory conditions in vivo and contribute to the pathogenesis of HAD and other pathological conditions. Monocyte recruitment into tissues involves interaction with endothelial cells and locally expressed chemokines (Muller, 2001) and is a multistep process regulated by a large number of adhesion molecules with overlapping functions (Kubes, 2002; Luscinskas et al., 2001; Schenkel et al., 2004). We previously demonstrated that stimulation of HUVEC with TNF and IFN-g results in high expression of VCAM-1, and CD62E, together with membrane-bound FKN and promotes firm arrest of both CD16+ and CD16 monocytes under flow conditions (Ancuta et al., 2003). However, the TEM of monocytes attached to TNF/IFN-g-activated HUVEC
Fig. 5. Migration of CD16 and CD16+ monocytes across unactivated and TNF/IFN-g-activated HUVEC. Monocytes were assessed for their ability to undergo transwell migration and cross unstimulated (NS) and TNF/IFN-g-stimulated (ST) immortalized HUVEC monolayers in response to optimal concentrations of MCP-1 (50 ng/ml), FKN (10 ng/ml) and SDF-1a (50 ng/ml). CD16 and CD16+ monocytes, which migrated in the bottom chamber of the transwell system, were counted by FACSs using fluorescent beads (mean7SEM, n ¼ 3). po0:05 Student’s t test (medium versus MCP-1 or FKN); po0:05; Student’s t test (NS HUVEC versus ST HUVEC).
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was not determined. Here we investigated TEM of CD16 and CD16+ monocytes across unactivated and TNF/IFN-g-activated endothelial cell monolayers under static conditions. We used two types of endothelial cells as models of TEM in vitro: brain microvascular (BMVEC) and human umbilical vascular (HUVEC) endothelial cells. BMVEC were distinguished from HUVEC by constitutive expression of VCAM-1. Unactivated BMVEC and HUVEC supported efficient TEM of CD16+ and CD16 monocytes in response to soluble FKN and MCP-1, respectively, consistent with our previous studies (Ancuta et al., 2003). Activation with TNF and IFN-g induced high levels of VCAM-1, CD62E, and FKN expression on HUVEC, whereas, under the same experimental conditions, BMVEC expressed moderate and undetectable levels of FKN and CD62E expression, respectively. Nottet et al. (1996) reported that BMVEC express high levels of CD62E and VCAM-1 after interaction with HIV-1-infected monocytes in vitro, which may induce massive infiltration of monocytes into the brain of HIV-1 infected patients. The relative resistance of BMVEC to cytokine-induced activation may be a unique property of endothelial cells bordering an immune privileged tissue. However, induction of moderate levels of FKN expression on BMVEC under inflammatory conditions in vitro suggests that FKN may mediate accumulation of CD16+ monocytes onto blood-brain barrier endothelial cells in vivo. Phenotypic differences between BMVEC and HUVEC revealed in this study are consistent with the immense heterogeneity of human vascular endothelium and the existence of molecular zip codes on endothelial cells bordering each tissue (Trepel et al., 2002). Further investigation to identify other BMVEC-specific molecules will help to characterize molecular mechanisms that underlie the specific recruitment of leukocyte subsets into the brain. Consistent with our previous studies performed under flow conditions (Ancuta et al., 2003), here we show that similar high percentages of CD16 and CD16+ monocytes attach to TNF/IFN-g-stimulated HUVEC under static conditions. However, only CD16 monocytes have the ability to undergo TEM in response to MCP-1. In contrast, CD16+ monocytes fail to migrate across activated HUVEC. Whether CD16+ monocytes remained attached at the apical or basal surface of TNF/IFN-g-stimulated HUVEC monolayers and whether CD16+ monocytes require shear stress to detach from the apical pole of endothelial cells and cross intercellular junctions (Luscinskas et al., 2001, 2002) remain to be determined. The block in the TEM of CD16+ monocytes observed under static conditions in vitro is not explained by the previously reported down-regulation and/or redistribution of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and junctional adhesion molecule (JAM) at the surface of endothelial cells stimulated with TNF and IFN-g
(Ozaki et al., 1999; Rival et al., 1996), since CD16 monocytes successfully crossed these monolayers. Rather, the inability to undergo TEM across TNF/IFN-g-stimulated HUVEC may be due to high expression of CX3CR1 on CD16+ monocytes (Ancuta et al., 2003), which may lead to more efficient interaction of these cells with membrane-bound FKN as compared to CD16 monocytes. Previous reports demonstrated that FKN has an unusually high affinity for its receptor and this interaction has a very slow offrate (Haskell et al., 1999, 2000). Thus, after capture on membrane-bound FKN, CX3CR1+ cells may remain firmly and irreversibly arrested. The importance of the deattachment of arrested monocytes in TEM is better understood in the light of a recent report that identified a new essential step, called locomotion, in which monocytes move from a site of firm adhesion to the nearest junction in order to begin diapedesis (Schenkel et al., 2004). Whether or not the interaction of CD16+ monocytes with membrane-bound FKN prevents this locomotion step or any other step involved in TEM remains to be investigated. The ability of attached monocytes to detach and undergo subsequent diapedesis may also depend on the level of FKN expression on inflamed endothelial cells. The possibility that CD16+ monocytes can cross endothelial cell monolayers expressing low levels of FKN under inflammatory conditions (e.g., TNF/IFN-g-stimulated BMVEC) merits investigation and may provide new insights into the regulation of monocyte trafficking by membrane-bound FKN. Massive accumulation of perivascular CD14+/CD16+ macrophages has been detected in the CNS of patients with HIV-1 encephalitis and HAD (Fischer-Smith et al., 2001). CD16+ monocytes that migrate across the bloodbrain barrier may be the origin of perivascular CD16+ macrophages detected in brain. However, CD16 monocytes differentiate into CD16+ macrophages in vitro (Ancuta et al., 2000) and may, therefore, acquire CD16 expression during differentiation in vivo. Whether CD16+ monocytes/macrophages in brain are derived from CD16 or CD16+ monocytes that cross the bloodbrain barrier in vivo remains to be determined. Taken together, our data demonstrate that CD16+ monocytes fail to undergo TEM across TNF/IFN-gactivated HUVEC, which express high levels of FKN, whereas CD16 monocytes efficiently migrate across activated endothelial cell monolayers. Thus, CD16 monocytes may migrate across inflamed endothelia and infiltrate subjacent tissues in response to a chemotactic gradient in vivo. In contrast, CD16+ monocytes may arrest onto cytokine-activated endothelial cells in an irreversible fashion, at least in part via CX3CR1–FKN interaction, and may thereby contribute to vascular injury during pathological conditions where this subset is expanded. CD16+ monocytes may contribute to the pathogenesis of HAD by altering blood-brain barrier
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integrity as a consequence of their massive and irreversible accumulation onto inflamed brain vascular endothelial cells expressing FKN and other activationinduced adhesion molecules. Understanding molecular mechanisms of CD16+ monocyte trafficking under inflammatory conditions will help the development of new therapeutic strategies to prevent deleterious consequences of monocyte infiltration at sites of chronic inflammation in vivo.
Acknowledgements We thank Mr. Lee Jae Morse for excellent technical assistance in blood collection. This work was supported by NIH grant DA016549 to DG. Core facilities were supported by a Center for AIDS Research grant (AI2869) and the DFCI/Harvard Center for Cancer Research grant.
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Immunobiology 209 (2004) 11–20 www.elsevier.de/imbio
Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions Petronela Ancutaa, Ashlee Mosesb, Dana Gabuzdaa,* a
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, JFB 816, Boston, MA 02115, USA b Vaccine and Gene Therapy Institute, Oregon Health & Sciences University, 505 NW, 185th Avenue, Beaverton, OR 97006, USA Received 20 March 2004; accepted 13 April 2004
Abstract CD16+ monocytes represent 5–10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-g (IFN-g) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16 and CD16+ monocyte subsets adhered to TNF/IFN-g-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16 monocytes across TNF/IFN-gstimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-g triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, proinflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules. r 2004 Elsevier GmbH. All rights reserved. Keywords: Monocytes, dendritic cells, siglecs
Abbreviations: BMVEC, brain microvascular endothelial cells; CD62E, E-selectin; CNS, central nervous system; FKN/CX3CL1, fractalkine; HUVEC, human umbilical vascular endothelial cells; ICAM-1, intercellular adhesion molecule-1; IFN-g, interferon-g; JAM, junctional adhesion molecule; MCP-1, monocyte chemoattractant protein-1; MIP-1a, macrophage inflammatory protein-1a; PECAM-1/CD31, platelet endothelial cell adhesion molecule-1; SDF-1a, stromal derived factor-1a; TEM, transendothelial migration; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1 *Corresponding author. Tel.: +1-617-632-2154; fax: +1-617-632-3113. E-mail address: dana [email protected] (D. Gabuzda). 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.04.001
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Introduction FcgRIII (CD16) expression distinguishes two subsets of human peripheral blood monocytes (Ziegler-Heitbrock, 1996). CD16+ monocytes represent 5–10% of peripheral blood monocytes in healthy individuals and are dramatically expanded in several pathological conditions including sepsis (Fingerle et al., 1993), HIV-1 infection (Pulliam et al., 1997; Thieblemont et al., 1995), metastatic cancer (Saleh et al., 1995), and asthma (Rivier et al., 1995). CD16+ monocytes exhibit a macrophage-like morphology ex vivo (Ziegler-Heitbrock et al., 1993), produce high levels of pro-inflammatory cytokines (e.g. tumor necrosis factor (TNF) and IL-1) (Belge et al., 2002; Thieblemont et al., 1995) and neurotoxic factors (Pulliam et al., 1997), and may represent dendritic cell precursors in vivo (Randolph et al., 2002). CD16 and CD16+ monocyte subsets exhibit distinct patterns of cell surface molecule expression and migratory properties. Consistent with the differential expression of CCR1, CCR2, and CX3CR1 on these monocyte subsets, CD16 monocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) (Weber et al., 2000), whereas fractalkine (FKN/CX3CL1) preferentially triggers migration and arrest of CD16+ monocytes (Ancuta et al., 2003). Thus, CD16 and CD16+ monocytes may be recruited into distinct anatomic sites in response to locally produced chemokines. FKN is a unique membrane-bound chemokine with a CX3C motif atop a mucin like stalk (Bazan et al., 1997), first identified by screening of a murine choroid plexus complementary DNA library (Pan et al., 1997). Human FKN messenger RNA is expressed under constitutive conditions in several tissues including heart, brain, lung, kidney, and pancreas (Bazan et al., 1997). Although the cellular distribution of FKN under constitutive conditions in vivo is still controversial (Lucas et al., 2001), its expression on cytokine-activated endothelial cells is well established (Bazan et al., 1997) and suggests a role of this chemokine in regulating leukocyte trafficking (Schall, 1997). Expression of the FKN receptor, CX3CR1, is detected on subsets of leukocytes including CD4+ T, CD8+ T, NK cells, and monocytes (Imai et al., 1997). The soluble form of FKN is released from the cell surface by proteolytic cleavage mediated by disintegrinlike metalloproteinases (Garton et al., 2001; Hundhausen et al., 2003) and is a chemoattractant for CX3CR1+ leukocytes (Ancuta et al., 2003; Fong et al., 1998; Imai et al., 1997). The membrane-bound form of FKN mediates adhesion of leukocytes under static and flow conditions in a selectin- and integrin-independent manner (Ancuta et al., 2003; Bazan et al., 1997; Fong et al., 1998; Imai et al., 1997). The ability of FKN to mediate firm arrest of leukocytes onto endothelial cells is
not due to its presentation atop a rigid stalk, but rather to the CX3C domain itself, which has a slow off-rate from its receptor (Haskell et al., 2000). FKN–CX3CR1 interaction has been implicated in the pathogenesis of several inflammatory diseases through the accumulation and/or activation of CX3CR1+ leukocytes on vascular beds and their deleterious infiltration into peripheral tissues (Umehara et al., 2001). For example, increased levels of FKN are expressed in the brain of patients with HIV-1 associated dementia (HAD) (Pereira et al., 2001; Tong et al., 2000) and may trigger monocyte infiltration into the central nervous system (CNS), which is a critical event in the pathogenesis of HAD (Gartner, 2000; Gartner and Liu, 2002). Given the dramatic expansion of CD16+ monocytes in patients with HAD (Crowe et al., 2003; Gartner, 2000; Gartner and Liu, 2002; Pulliam et al., 1997) and other inflammatory conditions (Grage-Griebenow et al., 2001; Ziegler-Heitbrock, 1996), and their ability to produce high levels of pro-inflammatory cytokines (Belge et al., 2002; Thieblemont et al., 1995) and neurotoxic factors (Pulliam et al., 1997), we investigated the migration of these cells across endothelial monolayers under constitutive and inflammatory conditions. Here we demonstrate that soluble FKN preferentially triggers migration of CD16+ monocytes across unactivated brain microvascular (BMVEC) and human umbilical vascular endothelial (HUVEC) cell monolayers. Stimulation of endothelial cells with TNF and IFN-g induces expression of membrane-bound FKN together with other activation-induced molecules and results in strong attachment of CD16+ monocytes, which fail to undergo subsequent diapedesis in response to a chemotactic gradient. These results suggest that CD16+ monocytes, which are dramatically expanded in the peripheral blood of HIV-1-infected patients and produce pro-inflammatory cytokines and neurotoxic factors, may contribute to the pathogenesis of HAD and possibly other inflammatory CNS diseases by accumulating onto blood-brain barrier endothelial cells that express FKN and other adhesion molecules under inflammatory conditions.
Material and methods Reagents and antibodies Recombinant human MCP-1, MIP-1a, stromal derived factor-1a (SDF-1a), FKN, TNF, and IFN-g were purchased from R&D Systems. Versene was purchased from Life Technologies. The following antibodies were used: FITC anti-CD14, -CD16b, PE anti-CD33, -CD56, and -intercellular adhesion molecule-1 (ICAM)-1, and PC5 anti-CD3 and -CD16 mAbs (Beckman
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Coulter); FITC anti-CD62E and PE anti-CCR2 and -FKN mAbs (R&D Systems); FITC anti-CX3CR1 mAb (MBL International); PE anti-vascular cell adhesion molecule (VCAM)-1 mAbs (BD Pharmingen).
Fluorospheres (Beckman Coulter). The index of migration (IM) was calculated as the ratio between the numbers of cells migrated in response to chemokine and medium alone.
Cell isolation
Adhesion assay under static conditions
PBMC were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation (Pharmacia Biotech). The study protocol and informed consent forms were approved by the Dana-Farber Cancer Institute Institutional Review Board. Monocytes were negatively isolated from PBMC using the Monocyte Isolation Kit II, according to the manufacturer’s protocol (Miltenyi Biotec). The purity of the monocyte fraction was 95–98% as determined by flow cytometry analysis after staining with fluorescence conjugated antiCD14, -CD33, -CD16b, -CD3, and -CD56 mAbs. CD16+ monocytes were not depleted by this approach as determined by staining with CD16 mAbs before and after monocyte isolation.
HUVEC were cultured at 80% confluence and incubated in the presence or absence of TNF (40 ng/ ml) and IFN-g (50 ng/ml), for 18 h at 37 C. Stimulated (ST) or unstimulated (NS) HUVEC were washed 2 times with media and co-cultured with PE anti-CD33 and PC5 anti-CD16 mAbs-labeled monocytes for 30 min at 37 C in DMEM/10% FBS. Non-adherent monocytes were removed by extensive washing with PBS. Monocytes attached to HUVEC were harvested together with endothelial cells using Trypsin. The number of adherent monocytes was determined by FACSs using FlowCount Fluorospheres. The percentage of adherent CD16 and CD16+ monocytes was calculated as the ratio between the number of monocytes in the adherent fraction and the number of monocytes in the input fraction multiplied by 100.
Endothelial cell culture Primary HUVEC were purchased from Clonetics/ BioWhittaker and immortalized with the recombinant retrovirus LXSN16 E6/E7 as previously described (Moses et al., 1999) to expand their lifespan in vitro. Primary brain endothelial microvascular endothelial cells (BMVEC) were isolated from temporal resections as previously described (Moses et al., 1993). Immortalized HUVEC and primary BMVEC were cultured on collagen-coated (50 mg/ml, SIGMA) flasks using endothelial cell medium EGM-MV (BioWhittaker) containing 200 mg/ml G418 (Cellgro).
Statistical analysis Data are presented as the mean7SD. Student’s t test was used for analysis of statistical significance. Values of po0:05 were considered statistically significant.
Results CX3CR1 and CCR2 expression on purified monocytes
Transwell and transendothelial migration assays Chemotaxis assays were performed as previously described (Kishimoto et al., 1990; Luscinskas et al., 1996). Briefly, HUVEC and BMVEC were cultured on collagen-coated transwells (6.5 mm diameter and 5 mm pore size, Corning Costar) at 2 105 cells/well in EGMMV medium for 2 days. The confluence of the monolayers was assessed by measure of electrical resistance using a Millicells–ERS electrode (Millipore). Monocytes were isolated as described above and stained with PE anti-CD33 and PC5 anti-CD16 mAb prior to migration assays. Serial dilutions of chemokines in DMEM (Invitrogen) containing 0.5% BSA were placed in the bottom chamber of the transwell system. Monocytes (106) were placed in the upper chamber of the transwell system and incubated for 2.5–4 h at 37 C. The number of migrated CD16 and CD16+ monocytes was determined by FACS analysis using Flow-Count
Monocytes were isolated from PBMC by negative selection using magnetic beads and analyzed for the cell surface expression of CD14, CD16, CCR2, and CX3CR1. CD16 expression distinguished two subsets: CD16 and CD16+ monocytes (Fig. 1, left panel). Among CD16+ monocytes, two subsets were further identified by distinct levels of CD14 expression: CD14highCD16+ and CD14lowCD16+ (Fig. 1, left panel). CD16 monocytes expressed CCR2 (MCP-1 receptor) but not CX3CR1 (FKN receptor), whereas CD16+ monocytes expressed CX3CR1 but low to undetectable levels of CCR2 (Fig. 1, middle and right panel). Thus, CCR2 and CX3CR1 are differentially expressed on CD16 and CD16+ monocytes, suggesting distinct migratory properties of these monocyte subsets in response to MCP-1 and FKN. The pattern of chemokine receptor expression on monocytes isolated by negative selection was similar to that previously
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Fig. 1. CCR2 and CX3CR1 expression on CD16 and CD16+ monocytes. Monocytes were purified from peripheral blood of healthy individuals by negative selection using magnetic beads. Monocytes were triple stained with FITC anti-CD14, PE anti-CD33, PC5 anti-CD16, PE anti-CCR2, and FITC anti-CX3CR1 mAbs, and analyzed by flow cytometry. Values in each quadrant represent the percentage of cells. Results are representative of experiments performed with cells from ten different donors.
described for monocytes from whole blood or PBMC (Ancuta et al., 2003).
TNF and IFN-c induce FKN expression on both BMVEC and HUVEC To investigate the interaction of CD16 and CD16+ monocytes with endothelial cells under constitutive and inflammatory conditions, we used immortalized HUVEC, which exhibit morphologic and phenotypic characteristics similar to primary HUVEC (Ancuta et al., 2003), and primary brain microvascular endothelial cells (BMVEC). BMVEC cultured at confluence on collagen-coated plates exhibited a fibroblast-like morphology, whereas HUVEC exhibited a rounded morphology (data not shown). Under constitutive conditions, HUVEC expressed moderate levels of ICAM-1 but not the activation-induced adhesion molecules VCAM-1, CD62E, and FKN, whereas BMVEC expressed both VCAM-1 and ICAM-1 but not CD62E and FKN (Fig. 2). Expression of VCAM-1, ICAM-1, CD62E, and FKN was then assessed on HUVEC and BMVEC after stimulation with TNF and IFN-g, a combination of cytokines known to induce high levels of FKN expression on HUVEC (Ancuta et al., 2003) and other cell types (Ludwig et al., 2002; Yoshida et al., 2001). Results presented in Fig. 2, left panels, demonstrate that HUVEC stimulation with TNF and IFN-g induces high levels of FKN, VCAM-1, and CD62E expression together with dramatic up-regulation of ICAM-1 expression. Stimulation of BMVEC with the same combination of cytokines results in up-regulation of VCAM-1 and ICAM-1 expression, moderate expression of FKN molecules, but no detectable surface expression of CD62E (Fig. 2, right panels). Stimulation of BMVEC with TNF and IL-1b, alone or in combination, failed to induce detectable CD62E expression (data not shown). These results demonstrate that BMVEC can
be distinguished from HUVEC by constitutive VCAM-1 expression and that TNF and IFN-g induce FKN expression on both HUVEC and BMVEC, but detectable CD62E expression is induced only on HUVEC.
FKN triggers CD16+ monocyte migration across a confluent BMVEC monolayer We previously reported that CD16 and CD16+ monocytes undergo efficient chemotactic migration across confluent HUVEC monolayers (Ancuta et al., 2003). Given the constitutive expression of VCAM-1 on unactivated BMVEC, we investigated the ability of BMVEC monolayers to support migration of these monocyte subsets in response to MCP-1 and soluble FKN. The spontaneous migration of CD16 monocytes was higher compared to that of CD16+ monocytes (data not shown). Both monocyte subsets underwent efficient chemotactic migration across BMVEC (Fig. 3A and data not shown): CD16 monocytes migrated with a high index of migration in response to MCP-1 and MIP-1a, whereas FKN triggered efficient transendothelial migration (TEM) of CD16+ monocytes (Fig. 3B). Of note, TEM of CD16+ monocytes across unactivated BMVEC was triggered by low (1–10 ng/ml) but not high (50–100 ng/ml) doses of FKN, whereas higher concentrations of MCP-1 and MIP-1a (50–100 ng/ml) were required for maximal migration of CD16 monocytes (Fig. 3B and data not shown). These results indicate that BMVEC, similar to HUVEC, support TEM of CD16+ monocytes in response to soluble FKN.
Transendothelial migration of CD16+ monocytes across TNF/IFN-c-stimulated HUVEC We previously demonstrated that CD16+ monocytes express high levels of CX3CR1 and arrest onto cell surface-expressed FKN with higher frequency compared
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Fig. 2. Phenotypic characterization of HUVEC and BMVEC. Immortalized HUVEC and primary BMVEC were cultured at 80% confluence on collagen-coated plates and incubated in the presence or absence of TNF (40 ng/ml) and IFN-g (50 ng/ml). After 18 h, cells were harvested using Versene, stained with PE anti-VCAM, FITC anti-CD62E, PE anti-ICAM-1, and PE anti-FKN mAbs, and analyzed by flow cytometry. Results are representative of three independent experiments.
to CD16 monocytes under flow conditions in vitro (Ancuta et al., 2003). It remains unknown whether CD16+ monocytes attached to inflamed endothelial cells via the interaction of CX3CR1 with FKN undergo subsequent diapedesis. We first investigated adhesion of monocyte subsets onto TNF/IFN-g-activated HUVEC monolayers that express high levels of membrane-bound FKN. CD16+ monocytes adhered onto resting HUVEC under static conditions with significantly higher frequency compared to CD16 monocytes (16.572.1% compared to 8.270.2%, mean7SD, n ¼ 3; po0:05; Student’s t test). When HUVEC were stimulated with TNF and IFN-g, a dramatic increase in the frequency of adherent monocytes was observed, and CD16 and CD16+ monocytes attached to TNF/IFN-g-stimulated HUVEC with similar frequencies (59.2712.2% and 47.7719.2%, respectively, mean7SD, n ¼ 3) (Fig. 4). We further investigated CD16 and CD16+ monocyte migration in the transwell system as compared to TEM across unactivated or TNF/IFN-g-activated HUVEC in response to the appropriate chemokines. In the transwell system, CD16 and CD16+ monocytes
migrated with similar efficiency in response to MCP-1 and FKN, respectively (230 147752 563 and 24 368711 061, respectively, mean7SD, n ¼ 3) (note that CD16+ monocytes represented o10% of total monocytes) (Fig. 5, left panels). In the transendothelial system, the number of CD16 monocytes that migrated in response to MCP-1 was only slightly lower compared to that observed in the transwell system (230 147752 563), regardless of whether HUVEC were stimulated (174 601793 438) or not (208 525769 247) with TNF and IFN-g (Fig. 5, upper panels). In contrast, the yield of CD16+ monocyte migration was dramatically decreased when migration was assessed across a resting HUVEC monolayer (11127734) compared to their migration in the transwell system (24 368711 061) (Fig. 5, lower panels). Moreover, stimulation of HUVEC with TNF and IFN-g dramatically reduced the number of transmigrated CD16+ monocytes (2907270) (Fig. 5, lower right panel). These results demonstrate that CD16 and CD16+ monocytes adhere to TNF/IFN-g-activated HUVEC with similar frequency, but only CD16 monocytes are able to detach
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Fig. 3. Migration of CD16 and CD16+ monocytes across BMVEC. (A) Primary BMVEC were cultured at confluence on collagencoated transwell filters. Monocytes were pre-stained with PE anti-CD33 and PC5 anti-CD16 mAbs and allowed to migrate across a BMVEC monolayer in response to optimal concentrations of MCP-1 and FKN. After 4 h, CD33+ monocytes that migrated in the bottom chamber of the transwell system were recovered and the % of CD16+ monocytes in each migrated fraction was determined by flow cytometry. (B) The numbers of CD16 and CD16+ monocytes that migrated in response to medium alone, MCP-1, and FKN were counted using fluorescent beads. The index of migration of monocyte subsets was calculated at the chemokine concentrations indicated in the figure. Results are representative of three independent experiments performed with monocytes from different donors.
from inflamed endothelium and migrate in response to MCP-1. In contrast, CD16+ monocytes remain strongly attached to inflamed endothelial cells and fail to undergo diapedesis in response to soluble FKN.
Discussion Massive infiltration of activated and/or HIV-1 infected monocytes is observed postmortem in the brain of patients with AIDS and represents a critical event in
HIV-1 neuropathogenesis (Gartner, 2000; Persidsky & Gendelman, 2003). CD16+ monocytes are dramatically expanded (up to 50%) in the peripheral blood of patients with HAD, produce high levels of proinflammatory cytokines and neurotoxic factors and may, if recruited into the brain, contribute to alteration of blood-brain barrier integrity and neuronal loss (Gartner, 2000; Gartner and Liu, 2002; Nottet, 1999; Pulliam et al., 1997; Thieblemont et al., 1995). We previously identified FKN as the major chemokine and adhesion molecule mediating arrest and migration of CD16+ monocytes (Ancuta et al., 2003). Similarly,
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Fig. 4. Adhesion of CD16 and CD16+ monocytes to HUVEC under static conditions. Immortalized HUVEC were cultured at 80% confluence and incubated in the presence or absence of TNF (40 ng/ml) and IFN-g (50 ng/ml), for 18 h at 37 C. Stimulated (ST) or unstimulated (NS) HUVEC were washed 2 times with media and co-cultured with PE anti-CD33 and PC5 anti-CD16 mAbs-labeled monocytes for 30 min at 37 C. Non-adherent monocytes were removed by extensive washing, whereas monocytes attached to HUVEC were harvested together with endothelial cells using trypsin. CD16 and CD16+ monocytes in the adherent fraction were counted by FACSs using fluorescent beads and the percentage of adherent cells was calculated taking into account the number of CD16 and CD16+ monocytes in the input fraction (mean7SD, n ¼ 2). p ¼ 0:02 Student’s t test (CD16 versus CD16+ monocytes); po0:05; Student’s t test (NS HUVEC versus ST HUVEC).
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mouse CX3CR1high monocytes, which resemble human CD16+ monocytes, are constitutively recruited into the brain and other peripheral tissues via CX3CR1 interaction with FKN (Geissmann et al., 2003). FKN is constitutively expressed in different peripheral tissues (Bazan et al., 1997) and is dramatically up-regulated in the CNS of patients with HAD (Pereira et al., 2001; Tong et al., 2000). Together, these findings suggest that CD16+ monocytes may migrate into the brain under constitutive and inflammatory conditions in vivo and contribute to the pathogenesis of HAD and other pathological conditions. Monocyte recruitment into tissues involves interaction with endothelial cells and locally expressed chemokines (Muller, 2001) and is a multistep process regulated by a large number of adhesion molecules with overlapping functions (Kubes, 2002; Luscinskas et al., 2001; Schenkel et al., 2004). We previously demonstrated that stimulation of HUVEC with TNF and IFN-g results in high expression of VCAM-1, and CD62E, together with membrane-bound FKN and promotes firm arrest of both CD16+ and CD16 monocytes under flow conditions (Ancuta et al., 2003). However, the TEM of monocytes attached to TNF/IFN-g-activated HUVEC
Fig. 5. Migration of CD16 and CD16+ monocytes across unactivated and TNF/IFN-g-activated HUVEC. Monocytes were assessed for their ability to undergo transwell migration and cross unstimulated (NS) and TNF/IFN-g-stimulated (ST) immortalized HUVEC monolayers in response to optimal concentrations of MCP-1 (50 ng/ml), FKN (10 ng/ml) and SDF-1a (50 ng/ml). CD16 and CD16+ monocytes, which migrated in the bottom chamber of the transwell system, were counted by FACSs using fluorescent beads (mean7SEM, n ¼ 3). po0:05 Student’s t test (medium versus MCP-1 or FKN); po0:05; Student’s t test (NS HUVEC versus ST HUVEC).
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was not determined. Here we investigated TEM of CD16 and CD16+ monocytes across unactivated and TNF/IFN-g-activated endothelial cell monolayers under static conditions. We used two types of endothelial cells as models of TEM in vitro: brain microvascular (BMVEC) and human umbilical vascular (HUVEC) endothelial cells. BMVEC were distinguished from HUVEC by constitutive expression of VCAM-1. Unactivated BMVEC and HUVEC supported efficient TEM of CD16+ and CD16 monocytes in response to soluble FKN and MCP-1, respectively, consistent with our previous studies (Ancuta et al., 2003). Activation with TNF and IFN-g induced high levels of VCAM-1, CD62E, and FKN expression on HUVEC, whereas, under the same experimental conditions, BMVEC expressed moderate and undetectable levels of FKN and CD62E expression, respectively. Nottet et al. (1996) reported that BMVEC express high levels of CD62E and VCAM-1 after interaction with HIV-1-infected monocytes in vitro, which may induce massive infiltration of monocytes into the brain of HIV-1 infected patients. The relative resistance of BMVEC to cytokine-induced activation may be a unique property of endothelial cells bordering an immune privileged tissue. However, induction of moderate levels of FKN expression on BMVEC under inflammatory conditions in vitro suggests that FKN may mediate accumulation of CD16+ monocytes onto blood-brain barrier endothelial cells in vivo. Phenotypic differences between BMVEC and HUVEC revealed in this study are consistent with the immense heterogeneity of human vascular endothelium and the existence of molecular zip codes on endothelial cells bordering each tissue (Trepel et al., 2002). Further investigation to identify other BMVEC-specific molecules will help to characterize molecular mechanisms that underlie the specific recruitment of leukocyte subsets into the brain. Consistent with our previous studies performed under flow conditions (Ancuta et al., 2003), here we show that similar high percentages of CD16 and CD16+ monocytes attach to TNF/IFN-g-stimulated HUVEC under static conditions. However, only CD16 monocytes have the ability to undergo TEM in response to MCP-1. In contrast, CD16+ monocytes fail to migrate across activated HUVEC. Whether CD16+ monocytes remained attached at the apical or basal surface of TNF/IFN-g-stimulated HUVEC monolayers and whether CD16+ monocytes require shear stress to detach from the apical pole of endothelial cells and cross intercellular junctions (Luscinskas et al., 2001, 2002) remain to be determined. The block in the TEM of CD16+ monocytes observed under static conditions in vitro is not explained by the previously reported down-regulation and/or redistribution of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and junctional adhesion molecule (JAM) at the surface of endothelial cells stimulated with TNF and IFN-g
(Ozaki et al., 1999; Rival et al., 1996), since CD16 monocytes successfully crossed these monolayers. Rather, the inability to undergo TEM across TNF/IFN-g-stimulated HUVEC may be due to high expression of CX3CR1 on CD16+ monocytes (Ancuta et al., 2003), which may lead to more efficient interaction of these cells with membrane-bound FKN as compared to CD16 monocytes. Previous reports demonstrated that FKN has an unusually high affinity for its receptor and this interaction has a very slow offrate (Haskell et al., 1999, 2000). Thus, after capture on membrane-bound FKN, CX3CR1+ cells may remain firmly and irreversibly arrested. The importance of the deattachment of arrested monocytes in TEM is better understood in the light of a recent report that identified a new essential step, called locomotion, in which monocytes move from a site of firm adhesion to the nearest junction in order to begin diapedesis (Schenkel et al., 2004). Whether or not the interaction of CD16+ monocytes with membrane-bound FKN prevents this locomotion step or any other step involved in TEM remains to be investigated. The ability of attached monocytes to detach and undergo subsequent diapedesis may also depend on the level of FKN expression on inflamed endothelial cells. The possibility that CD16+ monocytes can cross endothelial cell monolayers expressing low levels of FKN under inflammatory conditions (e.g., TNF/IFN-g-stimulated BMVEC) merits investigation and may provide new insights into the regulation of monocyte trafficking by membrane-bound FKN. Massive accumulation of perivascular CD14+/CD16+ macrophages has been detected in the CNS of patients with HIV-1 encephalitis and HAD (Fischer-Smith et al., 2001). CD16+ monocytes that migrate across the bloodbrain barrier may be the origin of perivascular CD16+ macrophages detected in brain. However, CD16 monocytes differentiate into CD16+ macrophages in vitro (Ancuta et al., 2000) and may, therefore, acquire CD16 expression during differentiation in vivo. Whether CD16+ monocytes/macrophages in brain are derived from CD16 or CD16+ monocytes that cross the bloodbrain barrier in vivo remains to be determined. Taken together, our data demonstrate that CD16+ monocytes fail to undergo TEM across TNF/IFN-gactivated HUVEC, which express high levels of FKN, whereas CD16 monocytes efficiently migrate across activated endothelial cell monolayers. Thus, CD16 monocytes may migrate across inflamed endothelia and infiltrate subjacent tissues in response to a chemotactic gradient in vivo. In contrast, CD16+ monocytes may arrest onto cytokine-activated endothelial cells in an irreversible fashion, at least in part via CX3CR1–FKN interaction, and may thereby contribute to vascular injury during pathological conditions where this subset is expanded. CD16+ monocytes may contribute to the pathogenesis of HAD by altering blood-brain barrier
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integrity as a consequence of their massive and irreversible accumulation onto inflamed brain vascular endothelial cells expressing FKN and other activationinduced adhesion molecules. Understanding molecular mechanisms of CD16+ monocyte trafficking under inflammatory conditions will help the development of new therapeutic strategies to prevent deleterious consequences of monocyte infiltration at sites of chronic inflammation in vivo.
Acknowledgements We thank Mr. Lee Jae Morse for excellent technical assistance in blood collection. This work was supported by NIH grant DA016549 to DG. Core facilities were supported by a Center for AIDS Research grant (AI2869) and the DFCI/Harvard Center for Cancer Research grant.
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