Published online before print April 23, 2004
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,
,1
* Department of Microbiology and Immunology,
Center for Molecular and Tumor Virology,
Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport
1Correspondence: Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932. E-mail: ayuroc{at}lsuhsc.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cytomegalovirus • lifecycle • endothelial cells • inflammation • atherosclerosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of our laboratory’s interest in HCMV pathogenesis, we began an investigation into the potential mechanisms by which HCMV infection of monocytes promotes chronic inflammation. We have evidence demonstrating that HCMV infection of monocytes results in their aberrant activation and the induction of an inflammatory phenotype that is consistent with HCMV pathogenesis. We showed that HCMV infection of monocytes induces nuclear factor-
B activation, which in turn promotes inflammatory cytokine production [14
]. Furthermore, we demonstrated that this viral induction of inflammatory mediators occurs through viral binding alone [14
], suggesting that HCMV binding to target cells mimics cytokine signaling by activating cellular signaling pathways through a receptor ligand-mediated process. These findings raise the following question: What is the benefit of HCMV-induced monocyte activation and inflammatory cytokine induction for the HCMV lifecycle in the host?
We hypothesized that HCMV uses an inflammatory response to activate newly infected monocytes in the peripheral blood and drive their transendothelial migration as a mechanism of viral dissemination to host tissue, an essential element for the viral lifecycle in the infected host and in a broader sense, in the human population [15 ]. Such a monocyte-driven mechanism of HCMV spread fits with observations that infected macrophages are found in the tissue of seropositive hosts following viremia [15 16 17 18 19 ]. Furthermore, monocytes are the primary cell type infected during viremia [15 , 20 , 21 ] and the predominant infiltrating cell type found in infected organs [22 ]. Monocytes have also been shown to be a source of viral latency [21 , 23 24 25 26 27 ].
To test our hypothesis, we examined if HCMV infection activates monocytes to promote monocyte-to-macrophage differentiation, viral replication, and migration. We demonstrated that HCMV infection of monocytes, which are nonpermissive for viral gene expression and replication, promotes their differentiation into macrophages permissive for viral replication, thus providing a mechanism of viral replication and spread in tissue [28 ]. In addition, we documented that HCMV infection of monocytes promotes monocyte extravasation as a potential mechanism of viral spread from the peripheral blood to host tissue during viremia [28 ], a finding that is consistent with recent studies in animal models of cytomegalovirus (CMV) pathogenesis [29 30 31 32 ]. However, in this initial study, we did not address the viral mechanism for the induction of monocyte differentiation and migration. We propose that the activation of monocytes by HCMV through cellular signaling pathways is necessary to drive their migration out of host tissue, to allow for viral replication, and to promote viral spread and the establishment of life-long persistence. A consequence of this aberrant monocyte activation and migration would be the migration of activated monocytes into the subendothelial space of vessels and peripheral tissues, a biological event that could promote chronic inflammatory disease such as atherosclerosis.
Atherosclerosis is a chronic inflammatory disease, which at the cellular level, involves the migration of activated monocytes from the peripheral blood into the subendothelial space and their production of inflammatory cytokines and chemokines [33 , 34 ]. The aberrant activation and migration of monocytes are critical to the initiation and progression of atherosclerotic disease and represent the first recognizable events detected during atherosclerotic plaque development [35 36 37 38 ]. Migrating activated monocytes contribute to atherogenesis through their production of proinflammatory cytokines and chemokines, which promote additional leukocyte recruitment into plaques [39 40 41 ], endothelial and smooth muscle cell proliferation [42 43 44 ], and atherosclerotic plaque rupture and thrombus formation [45 46 47 ]. Together, these findings point to a central role monocytes play in atherosclerotic disease. As the effects of HCMV infection of monocytes appear to mimic the biology of monocytes in atherosclerotic plaques, our findings may provide a biological link between the lifecycle of HCMV and diseases associated with infection.
We hypothesize that viral-mediated, cellular activation is the driving force behind the HCMV-induced changes in monocyte function that we observed. We suggest that HCMV usurps cellular signal-transduction pathways in monocytes to allow viral spread to host tissues and in so doing, acts as a promoter of inflammation in the host. As HCMV-induced monocyte migration is critical for viral spread [28 ] and would provide a biological mechanism to account for the role of HCMV in atherogenesis [12 ], we initiated a study to focus on the cellular pathway(s) activated during HCMV infection that promotes monocyte migration. Here, we report that HCMV up-regulates phosphatidylinositol 3,4,5 triphosphate kinase [PI(3)K] activity in monocytes and provide the first evidence that this viral-induced PI(3)K activity is required for monocyte-transendothelial migration following HCMV infection. Furthermore, we showed that the increased PI(3)K activity induces monocyte motility and regulates the expression of inflammatory chemokines and chemokine receptors related to monocyte migration and implicated in atherogenesis. Together, our data describe a novel viral mechanism that makes biological "sense" for HCMV; aberrant PI(3)K activation would allow newly infected peripheral blood monocytes to infiltrate host tissue, differentiate into macrophages, and subsequently, establish the persistent infection seen in the tissue of seropositive hosts [15 ]. This abnormal, HCMV-induced monocyte migration could also promote chronic inflammatory diseases by increasing monocyte infiltration into sites such as atherosclerotic plaques, which with the subsequent release of inflammatory mediators and virus, would exacerbate the disease.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monocyte isolation, treatment, and culture
Human peripheral blood monocytes were purified by double-density gradient centrifugation [14
, 14
, 50
]. Whole blood was taken by venipuncture from HCMV-seronegative donors and centrifuged through a Ficoll Histopaque 1077 (Sigma Chemical Co., St. Louis, MO) gradient. The collected mononuclear cells were washed six times to remove platelets. The monocytes were then isolated by centrifugation through a Percoll (Pharmacia, Piscataway, NJ) gradient. This technique yields 3–5 x 107 monocytes per donor at a purity of 
90%. Monocytes were pretreated nonadherently with the PI(3)K inhibitor LY294002 (25 or 50 µM, Promega, Madison, WI), cytochalasin D (4 µM, Promega), or the control drug solvent dimethyl sulfoxide (DMSO) for 1 h before HCMV infection, mock infection, or phorbol 12-myristate 13-acetate (PMA; 10 ng/ml, Sigma Chemical Co.) treatment as described above. Monocytes were then plated at the cell densities described in each experimental section and cultured in endotoxin-free RPMI supplemented with 10% human serum (Sigma Chemical Co.) at 37°C with 5% CO2. A dose response of LY294002 treatment revealed that 25 and 50 µM LY294002 did not affect monocyte viability at 48 h post-treatment (data not shown). University Institutional Review Board and Health Insurance Portability and Accountability Act guidelines were followed for all experimental protocols.
Western blot analysis
Monocytes were isolated, treated, and plated on fibronectin (Calbiochem, San Diego, CA)-coated 100 mm tissue-culture plates (prepared according to the manufacturer’s protocol) at a density of 2 x 106 cells per plate [28
]. Monocytes were harvested in Laemmli sample buffer (Bio-Rad, Hercules, CA) at the indicated time-points and boiled for 10 min. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose (Immobilon-P, Millipore, Billerica, MA). Blots were blocked in a Tris-buffered saline with 0.1% Tween 20 and 0.1% gelatin and then incubated with an anti-Pan-Akt polyclonal antibody (Cell Signaling Technology, Beverly, MA), an anti-phospho-Akt (anti-p-Akt; Ser473) polyclonal antibody (Cell Signaling Technology), or an anti-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Blots were washed, incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology) or a HRP-conjugated anti-goat secondary antibody (Santa Cruz Biotechnology), and detected using the ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ) protocol.
Transendothelial migration assay
Transendothelial migration assays were performed using human dermal microvascular endothelial cells (HMECs) cultured on cell-culture inserts (BD Falcon, Bedford, MA) with an 8-µm pore size in 24-well plates (Corning Costar, Cambridge, MA). HMECs were incubated and grown to confluence in endothelial growth medium-1 (Clonetics, Walkersville, MD) supplemented with 10% heat-inactivated FBS (Clonetics), hydrocortisone (1 µg/ml, Clonetics), human epidermal growth factor (10 ng/ml, Clonetics), bovine brain extract (12 µg/ml, Clonetics), and gentamicin sulfate amphotericin-B (Clonetics) at 37°C with 5% CO2. Monocytes were isolated and treated as described above. Treated monocytes were labeled with CellTracker Green 5-chloromethylfluorescein diacetate (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol. Monocytes were then washed and resuspended in RPMI supplemented with 10% human serum. The media within the transwells and the wells of the plate were removed and replaced with RPMI supplemented with 10% human serum. Monocytes (2.5x104) were then plated per transwell and incubated at 37°C with 5% CO2. The cell-tracker dye alone had no effect on monocyte viability and cell migration nor did it interfere with HCMV infection (data not shown). Furthermore, the endothelial cells were viable for the entire course of our experiment and maintained their junctional integrity (data not shown). The ratios of cells undergoing diapedesis versus those cells that were stationary on the surface of the endothelial monolayer were determined by inverted fluorescence microscopy at 24 h post-addition of monocytes. Results are plotted as mean ± SD of 15 random fields of view (200x magnification) from three separate experiments using different human blood donors. The number of monocytes that had migrated completely through the endothelial monolayer and the cell culture insert was determined by inverted fluorescence microscopy at 96 h post-addition of monocytes. Results are plotted as mean ± SEM of 30 random fields of view (200x magnification) from three separate experiments using different human blood donors. The percentage of monocytes that had undergone total migration at 96 h out of the total number of monocytes added per transwell (input cells) was determined as follows. Transwells were removed from the 24-well plates, and monocytes adhered to the bottom of each well were incubated with versene (0.2 mg/ml EDTA, BioWhittaker, Wakersville, MD) for 1 h at 37°C. Monocytes were then removed from the wells by gentle scraping, washed, and counted with a hemocytometer to determine the total number of monocytes per well. Results are plotted as mean ± SD of three counted wells per experimental arm. Statistical significance between experimental means (P value) was determined using Student’s t-test.
F-actin staining
To stain for F-actin, monocytes were isolated and treated as described above, and 2.5 x 104 monocytes were plated on 15 mm fibronectin-coated glass coverslips in 24-well plates and incubated for 24 h. As a negative control, monocytes were treated with cytochalasin D (4 µM, Promega) for 30 min before cell fixation and staining. Following 24 h of incubation, monocytes were fixed with 1% paraformaldehyde/2% sucrose for 15 min and subsequently stained with Alexa Fluor 594 phalloidin (Molecular Probes) and 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes) according to the manufacturer’s protocol. Images of monocytes were captured using a fluorescent microscope (1000x magnification).
Phagokinetic track motility assay
Phagokinetic track motility assays were performed using a modified protocol of Scott et al. [51
]. To prepare colloidal gold-coated coverslips, 15 mm glass coverslips were heated in a 300-bloom gelatin solution (0.5 g in 300 ml, Sigma Chemical Co.) at 90°C for 10 min and dried at 70°C for 45 min. Colloidal gold suspensions were prepared by mixing 11 ml endotoxin-free water (Sigma Chemical Co.), 6 ml Na2CO3 (36.5 mM), and 1.8 ml AuHCl4 (14.5 mM, Fisher Scientific, Pittsburgh, PA). The solution was brought to a rapid boil, and 1.8 ml 0.1% formaldehyde was added immediately to the solution. The suspension (4 ml) was added to each coverslip while still hot and incubated at 37°C for 1 h. Coverslips were washed in calcium- and magnesium-free saline and transferred to 24-well plates containing RPMI supplemented with 10% human serum. Monocytes were isolated and treated as described above, and 500 monocytes were added to each well. After 6 h of incubation, monocytes were fixed for 15 min in calcium- and magnesium-free phosphate-buffered saline containing 1% paraformaldehyde and mounted on slides. Track images of individual cells were video-captured at 400x magnification. The average area cleared per cell out of 50 cells per sample was determined in square arbitrary units using Scion Image software (Scion Corporation, Frederick, MD). Results are plotted as mean ± SEM of 50 cells. Statistical significance between experimental means (P value) was determined using Student’s t-test.
Gene-chip analysis
Monocytes were isolated and treated as described above, and 2 x 106 monocytes were plated per fibronectin-coated 100 mm dish. After 48 h of incubation, total RNA was harvested from 1 x 107 monocytes (five plates) per experimental arm, purified using the Stat-60 RNA isolation kit (Tel-Test, Inc., Friendswood, TX), and converted into cRNA according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA). Affymetrix Human Genome U95Au2 arrays were hybridized, washed, and scanned according to the manufacturer’s instructions (Affymetrix). Data were analyzed using Affymetrix Microarray Suite 4.0 software (Affymetrix) to determine signal intensity for each probe set. Table 1
represents gene products related to inflammation and migration, which were up-regulated twofold or greater in DMSO-pretreated, HCMV-infected monocytes compared with DMSO-pretreated, mock-infected monocytes and also down-regulated twofold or greater in LY294002-pretreated, HCMV-infected monocytes compared with DMSO-pretreated, HCMV-infected monocytes. Only those gene products that met these criteria in two separate experiments using monocytes isolated from two blood donors were included in Table 1
.
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Table 1. Cellular Genes Related to Migration and Inflammation Induced by HCMV Infection and Regulated by PI(3)Kab
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32P]uridine 5'-triphosphate (UTP)-labeled human cytokine multiprobe (hCM-8) template set (BD Biosciences, San Diego, CA) overnight according to the manufacturer’s protocol. RNase protection assays were then performed with labeled RNA using the Multi-Probe RNase protection assay system (BD Biosciences) according to the manufacturer’s protocol. Briefly, samples were treated with RNase, and purified, protected probes were resolved on a denaturing polyacrylamide gel. The gel was dried, and radioactivity was quantified by phosphorimaging. Band intensities (see representation in Fig. 6B
) were determined using Quantity One image analysis software (Bio-Rad) and normalized to the respective L32 band intensities.
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Figure 6. HCMV infection induces chemokine expression in a PI(3)K-dependent manner. DMSO-pretreated mock-infected, LY294002-pretreated mock-infected, DMSO-pretreated HCMV-infected, and LY294002-pretreated HCMV-infected monocyte RNA from two independent experiments using different human blood donors was hybridized with an [32P]UTP-labeled hCM-8 template set (BD Biosciences) and subjected to RNase protection assays using the Multi-Probe RNase protection assay system (BD Biosciences) according to the manufacturer’s protocol (A). Band intensities were determined using Quantity One image analysis software (Bio-Rad), normalized to the respective L32 band intensities, and plotted in arbitrary units (B). HCC-1, Hemofiltrate CC chemokine 1.
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Figure 1. HCMV infection of monocytes up-regulates PI(3)K activity. Monocytes were pretreated with LY294002 (50 µM) or the solvent control DMSO for 1 h, infected (Inf) with HCMV (M.O.I. of 15) or mock-infected, and plated on fibronectin-coated dishes. After the incubation times indicated, protein was harvested. (a) Western blot analyses of Pan-Akt and Ser473 p-Akt were performed with equal protein loading of each sample normalized to actin band intensity. LY+ indicates pretreatment with LY294002, while LY– indicates pretreatment with DMSO. (b) Bands were analyzed by densitometry to determine relative levels of Pan-Akt and p-Akt and are expressed in arbitrary units as a ratio of x/actin.
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Figure 2. HCMV-induced monocyte diapedesis is dependent on PI(3)K activity. Monocytes were pretreated with LY294002 (25 µM and 50 µM) or the solvent control DMSO for 1 h, infected with HCMV (M.O.I. of 15), mock-infected, or treated with PMA (10 ng/ml), and subsequently, were labeled with CellTracker Green. Labeled monocytes were added to cell-culture inserts containing confluent monolayers of HMECs. The ratios of cells undergoing diapedesis versus those cells that were stationary at the monolayer surface were determined by fluorescence microscopy after a 24-h incubation. Results are plotted as mean ± SD of 15 random fields of view (200x original magnification) from three separate experiments using different human blood donors. The HCMV-infected and PMA-treated groups are significantly different from the mock-infected, LY294002-pretreated mock-infected, LY294002-pretreated HCMV-infected, and LY294002-pretreated PMA-treated groups (P<0.01).
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Figure 3. HCMV-induced monocyte total migration is dependent on PI(3)K activity. Monocytes were pretreated with LY294002 (25 µM or 50 µM) or the solvent control DMSO for 1 h, infected with HCMV (M.O.I. of 15), treated with PMA, or mock-infected, and subsequently, labeled with CellTracker Green. Labeled monocytes were added to cell-culture inserts containing confluent monolayers of HMECs. (A) After 96 h of incubation, the number of monocytes that had migrated through the HMEC monolayer and the pores of the cell-culture insert was determined by fluorescence microscopy. Results are plotted as mean ± SEM of 30 random fields of view (200x original magnification) from three separate experiments using different human blood donors. The HCMV-infected and PMA-treated groups are significantly different from the mock-infected, LY294002-pretreated mock-infected, LY294002-pretreated HCMV-infected, and LY294002-pretreated PMA-treated groups (P<0.01). (B) The percentage of input monocytes that had undergone total migration for each experimental arm was determined after 96 h of incubation by removing monocytes adhered to the bottom of the wells with versene and counting the total number of cells present using a hemocytometer. Results are plotted as mean ± SD of three wells per experimental arm. Results are representative of three independent experiments from different human blood donors. The HCMV-infected and PMA-treated groups are significantly different from the mock-infected, LY294002-pretreated mock-infected, LY294002-pretreated HCMV-infected, and LY294002-pretreated PMA-treated groups (P<0.01).
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95% of mock-infected monocytes exhibited a nonmotile monocyte morphology in these assays (data not shown). In contrast, HCMV-infected monocytes pretreated with LY294002 (50 µM) only exhibited a rounded, nonmotile phenotype (Fig. 4C)
similar to mock-infected monocytes and LY294002-pretreated (50 µM), mock-infected monocytes (Fig. 4G)
, suggesting that PI(3)K activity is required for HCMV induction of actin reorganization and lamellipodium formation. Like HCMV-infected monocytes, PMA-treated monocytes also lost the motile cell morphology following LY294002 (50 µM) pretreatment (Fig. 4K)
. As a negative control for nonmotile monocyte morphology, HCMV-infected (Fig. 4D)
, mock-infected (Fig. 4H)
, and PMA-treated (Fig. 4L)
monocytes were treated with cytochalasin D, an inhibitor of actin polymerization, for 30 min before cell fixation. Although LY294002-pretreated mock-infected and LY294002-pretreated HCMV-infected monocytes exhibited a significant decrease in cell size and spreading, LY294002 treatment did not affect cell viability at 24 and 48 h post-treatment (data not shown).
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Figure 4. PI(3)K is required for lamellipodium formation in infected monocytes. Monocytes were pretreated with LY294002 or the DMSO solvent control for 1 h, HCMV-infected (M.O.I of 15), mock-infected, or PMA-treated (10 ng/ml), and subsequently, cultured on fibronectin-coated glass coverslips for 24 h. As a negative control, mock-infected, HCMV-infected, and PMA-treated monocytes were treated with cytochalasin D for 30 min before cell fixation. DMSO-pretreated HCMV-infected (A and B), LY294002-pretreated HCMV-infected (C), cytochalasin D-treated HCMV-infected (D), DMSO-pretreated mock-infected (E and F), LY294002-pretreated, mock-infected (G), cytochalasin D-treated, mock-infected (H), PMA-treated (I and J), LY294002-pretreated, PMA-treated (K), and cytochalasin D-treated PMA-treated (L) monocytes were then fixed and stained with Alexa Fluor 594 phalloidin and DAPI (original magnification, 1000x). For each experimental arm, representative panels of single cells are shown. Results are representative of experiments from three experiments using different human blood donors.
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Figure 5. PI(3)K is required for HCMV-induced monocyte motility. Monocytes were pretreated with LY294002 or the DMSO solvent control for 1 h and were HCMV-infected (M.O.I of 15), mock-infected, or treated with PMA (10 ng/ml). DMSO-pretreated HCMV-infected (A), LY294002-pretreated HCMV-infected (B), cytochalasin D-pretreated HCMV-infected (C), DMSO-pretreated mock-infected (D), LY294002-pretreated mock-infected (E), cytochalasin D-pretreated, mock-infected (F), PMA-treated (G), LY294002-pretreated PMA-treated (H), and cytochalasin D-pretreated PMA-treated (I) monocytes were plated on colloidal gold-coated glass coverslips and incubated for 6 h. Monocytes were fixed, and individual cell track images were captured (400x original magnification). Panels are representative of individual cell tracks from each experimental arm. The average area (arbitrary units) of colloidal gold cleared per monocyte was determined for each experimental arm from captured images. Results are plotted as mean ± SEM of 50 cells per experimental arm (J). The HCMV-infected and PMA-treated groups are significantly different from the mock-infected, LY294002-pretreated, mock-infected, cytochalasin D-pretreated, mock-infected, LY294002-pretreated, HCMV-infected, cytochalasin D-pretreated, HCMV-infected, LY294002-pretreated, PMA-treated, and cytochalasin D-pretreated, PMA-treated groups (P<0.01). Results are representative of three independent experiments from separate blood donors.
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Table 1 lists gene products with known relevance to migration and inflammation that were up-regulated more than twofold in expression by DMSO-pretreated HCMV-infected monocytes compared with DMSO-pretreated mock-infected monocytes and were down-regulated twofold or greater in LY294002-pretreated HCMV-infected monocytes compared with DMSO-pretreated HCMV-infected monocytes. Only those gene products that met these criteria in two independent experiments using monocytes isolated from different human blood donors were included in this table. Although two adhesion molecules were induced by HCMV infection in a PI(3)K-dependent manner, these analyses revealed that the majority of genes related to migration and inflammation, which were up-regulated by HCMV-induced PI(3)K activation, were chemokines and chemokine receptors (Table 1) .
The up-regulation of chemokines by HCMV infection could have proatherosclerotic effects by activating and recruiting additional monocytes and T cells into the vessel lumen. In fact, MCP-1, MCP-2, MCP-3, GRO-, and ELC have been implicated in atherogenesis [59
60
61
62
63
64
65
66
]. These chemokines could also assist in viral dissemination by recruiting additional leukocytes to sites of infection, where they then could be infected, or by amplifying the migration of HCMV-infected monocytes. Proinflammatory cytokines produced by activated monocytes and macrophages in plaques also play a pivotal role in promoting atherogenesis as discussed above [33
, 34
, 39
40
41
42
43
44
, 46
, 47
, 59
]. Although a number of proatherosclerotic cytokines were induced greater than twofold by HCMV infection, including interleukin (IL)-1ß, tumor necrosis factor , IL-6, IL-12, and IL-15, their expression was not consistently altered by LY294002 pretreatment (data not shown). These data suggest that although HCMV-induced PI(3)K activation is pivotal in mediating many aspects of HCMV-induced migration and inflammation, other cellular signaling pathways are involved.
To confirm the up-regulation of chemokine expression by HCMV-induced PI(3)K activation, RNase protection assays were preformed on RNA harvested from DMSO-pretreated mock-infected, LY294002-pretreated mock-infected, DMSO-pretreated HCMV-infected, and LY294002-pretreated HCMV-infected monocytes using the hCM-8 human cytokine multiprobe template set (BD Biosciences; Fig. 6A
). Band intensities were analyzed using Quantity One image analysis software (Bio-Rad), normalized to the respective L32 band intensities, and plotted in arbitrary units (Fig. 6B)
. Consistent with our gene-chip array data (Table 1)
, these analyses revealed that MCP-1, MCP-2, MCP-3, HCC-1, and MIP-1 were dramatically up-regulated by HCMV infection in two experiments using monocytes purified from different human blood donors (Fig. 6B)
. Of these chemokines, MCP-1, MCP-2, MCP-3, and HCC-1 exhibited a twofold or greater decrease in HCMV-infected monocytes that were pretreated with LY294002 (50 µM) prior to infection (Fig. 6B)
. MIP-1 was reduced twofold or greater in LY294002-pretreated HCMV-infected monocytes from Donor 2 but exhibited a less-than-twofold reduction in LY294002-pretreated HCMV-infected monocytes from Donor 1 (Fig. 6B)
. This finding is consistent with our gene-chip array experiments in which we found that MIP-1 expression was up-regulated greater than twofold in HCMV-infected monocytes in two independent experiments using monocytes isolated from different human blood donors and was reduced in expression by twofold or greater in LY294002-pretreated HCMV-infected monocytes in only one of these experiments (data not shown). These findings suggest that like many of the inflammatory cytokines induced by HCMV infection, additional signaling pathways regulate MIP-1 expression. Together, our analyses show a role for HCMV-induced PI(3)K activation in promoting chemokine and chemokine receptor expression and are consistent with our proposed roles of HCMV-infected monocytes as vectors for viral dissemination and promoters of inflammatory disease. This study sets the stage for future studies on the regulation of additional inflammatory mediators induced by HCMV infection of monocytes.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There have been several obstacles, however, in determining the role monocytes play in dissemination during HCMV infection. First, HCMV infection is limited to human cells, and there are no animal models for directly studying HCMV pathogenesis, although animal models studying the related murine and rat CMVs do exist [29 30 31 32 ]. Second, monocytes are nonpermissive for viral gene expression and replication following a primary infection in vitro [28 , 68 69 70 71 ]. Studies have shown, however, that macrophages, which have been differentiated in vitro from monocytes using certain stimuli, become productive for viral replication only after differentiation [68 69 70 71 ].
To address this seeming paradox, we have developed a novel system to examine changes induced by HCMV infection in human primary peripheral blood monocytes cultured in vitro [28 ]. We recently showed that HCMV infection of monocytes drives monocyte-transendothelial migration and their differentiation into macrophages and that these HCMV-differentiated macrophages begin to replicate the original HCMV following this differentiation event [28 ]. Furthermore, we showed through the use of UV-inactivated HCMV that HCMV-induced differentiation and migration occurred in the absence of viral gene expression [28 ], suggesting that HCMV exerts these events through activation of cellular signal-transduction pathways. This makes biological sense, as HCMV cannot express viral gene products or replicate until the infected monocytes differentiate into macrophages. We proposed, therefore, that HCMV upon infection initiates a cellular signal-transduction event and subsequently induces cellular changes in monocytes conducive to replication and spread [28 ]. Widespread viral dissemination to host organs through monocytes correlates with the multiple organ system involvement and the severe disease associated with HCMV infection of the immunocompromised [15 , 72 ]. In immunocompetent hosts, the aberrant induction of monocyte migration following infection could exacerbate inflammatory diseases such as atherosclerosis. Further supporting this concept, we reported that HCMV-differentiated macrophages have an inflammatory macrophage phenotype at 2 weeks postinfection [28 ]. Together, our initial study suggests that HCMV-induced monocyte activation is critical for the viral lifecycle and that as a consequence of this viral strategy of dissemination, this activation event also plays a role in the pathogenesis of HCMV-mediated diseases.
To understand this critical step of monocyte activation, our laboratory has been investigating the cellular signaling-transduction pathways responsible for HCMV-induced monocyte activation. In this study, we first showed that HCMV infection of monocytes resulted in increased PI(3)K activity. This increase in PI(3)K activity in monocytes is the first evidence of this pathway being altered in cells relevant to in vivo HCMV infection. The finding is consistent with a study by Johnson et al. [73 ], showing that HCMV up-regulates PI(3)K activity in HEL fibroblasts, although they did not examine the functional consequences of increased PI(3)K activity at the cellular level. However, they did show that PI(3)K activity was required for viral replication in HEL fibroblasts [73 ]. As HCMV does not replicate in monocytes, we proposed that the HCMV-induced PI(3)K activation in monocytes was likely a critical mediator in other aspects of monocyte infection.
We next addressed the potential biological significance of the induced PI(3)K activity and showed that HCMV promotes monocyte diapedesis at 24 h postinfection and total migration through the endothelial monolayer and the transwell insert at 96 h in a PI(3)K-dependent manner. When PI(3)K was blocked before HCMV infection with the PI(3)K-specific drug inhibitor LY294002, levels of diapedesis and total migration returned to levels comparable with mock-infected monocytes. These data indicate that the up-regulation of PI(3)K activity by HCMV is central to the viral induction of monocyte migration. Therefore, we next examined if HCMV-induced PI(3)K activation promotes migration through the up-regulation of monocyte motility. HCMV-infected monocytes exhibited lamellipodium and retractile tail formation, characteristic of a motile cell phenotype. In contrast, infected monocytes pretreated with LY294002 before infection exhibited a nonmotile cell phenotype similar to mock-infected monocytes, which was characterized by cortical actin ring-staining and an absence of pseudopodium and lamellipodium. Quantification of cell motility using the phagokinetic track assay confirmed that PI(3)K activity was required for HCMV-induced monocyte motility. Consistent with our findings, previous studies have shown that chemokines promote monocyte migration in a PI(3)K-dependent manner through the up-regulation of cell motility [54 , 55 ], although in our studies, we did not add chemokines to the monocytes. Monocytes were treated with only HCMV, and at early times post-infection, PI(3)K activity was regulated by the effect of HCMV alone. Based on our gene chip-array data, it is possible that at later times postinfection, the release of chemokines by infected monocytes could, in an autoregulatory manner, enhance PI(3)K activity and thus amplify monocyte migration.
To elucidate additional roles that HCMV-induced PI(3)K activation might play in monocyte migration and inflammatory mediator production, we performed gene-chip array analyses on mock-infected, HCMV-infected, and LY294002-pretreated HCMV-infected monocytes at 48 h postadhesion. Because of their role in migration, we expected to see the up-regulation of a multitude of adhesion molecules by HCMV in a PI(3)K-dependent manner in our gene-array analyses. However, only two adhesion molecules were up-regulated by HCMV in a PI(3)K-dependent manner, suggesting that changes in adhesion molecule expression occur primarily at early times postinfection or that PI(3)K does not regulate the process of adhesion per se but rather regulates the process of motility. Nevertheless, an early induction of adhesion molecules would be consistent with our previous reports of HCMV-induced ß1 integrin and occludin expression, which begins to occur at 4 h postinfection and plateaus by 24 h postinfection [28 ]. We are currently addressing the regulation of HCMV-induced adhesion molecule expression by PI(3)K activation in monocytes at early times postinfection. The gene-chip array study and the RNase protection assays did reveal a PI(3)K-dependent induction of multiple chemokines by HCMV infection. As discussed in Results, these chemokines are known to promote atherogenesis through activation of the cellular components of the plaques or through the recruitment of additional monocytes and T cells into the plaque [59 60 61 62 63 64 65 66 ]. Such a chemokine-induced monocyte recruitment event could also play a role in recruiting additional infected monocytes into host tissue or promoting the recruitment of more monocytes to serve as targets for viral infection in the tissue. This initial project sets the stage for additional studies in which we will investigate HCMV-induced changes in monocytes that are known to promote inflammation and atherogenesis. Currently, we are investigating whether HCMV-induced chemokine expression promotes leukocyte and lymphocyte migration directly.
Altogether, these results support our hypothesis that viral-mediated cellular activation is the driving force behind HCMV-induced monocyte migration. This study demonstrated that HCMV infection activated PI(3)K in monocytes and that PI(3)K was essential for HCMV-induced monocyte-transendothelial migration. Additionally, we provided evidence that the viral induction of PI(3)K results in inflammatory mediator expression, which could have additional pathological consequences for the host. By deciphering the cellular pathways usurped during HCMV infection, we hope to provide an understanding of the steps necessary for viral infection and thus, the identification of new target sites for therapeutic intervention.
Received December 8, 2003; revised March 15, 2004; accepted March 26, 2004.
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and tumor necrosis factor- specifically induce formation of cytomegalovirus-permissive monocyte-derived macrophages that are refractory to the antiviral activity of these cytokines J. Clin. Invest. 100,3154-3163[Medline]This article has been cited by other articles:
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