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The Journal of Immunology, 2004, 173: 4897-4907.
Copyright © 2004 by The American Association of Immunologists

Human Cytomegalovirus Induces Inhibition of Macrophage Differentiation by Binding to Human Aminopeptidase N/CD131

Sara Gredmark*, William B. Britt{ddagger}, Xun Xie{dagger}, Lennart Lindbom{dagger} and Cecilia Söderberg-Nauclér2,*

* Department of Medicine, Center for Molecular Medicine, and {dagger} Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; and {ddagger} Department of Pediatrics and Microbiology, University of Alabama, Birmingham, AL 35233


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human CMV (HCMV) infection in immunocompromised patients is frequently associated with impaired immunological functions. We have recently found that HCMV inhibits cytokine-induced differentiation of monocytes into macrophages. In this study, we demonstrate that HCMV-induced inhibition of macrophage differentiation was dependent on binding of virus particles to the cell surface molecule CD13/aminopeptidase N, which involved Ca2+-dependent intracellular signaling pathways. We found that treatment of cells with the CD13-specific mAbs My7 and WM15 inhibited macrophage differentiation, and that My7 and WM15 induced a rise in intracellular Ca2+ in similar ways as HCMV. In contrast, binding of the CD13-specific Ab clone SJ1D1 blocked the ability of HCMV to inhibit macrophage differentiation, and blocked the HCMV-induced intracellular Ca2+ response. In addition, the Ca2+ modulator thapsigargin partially blocked the ability of HCMV to inhibit cellular differentiation. Furthermore, we demonstrate that recombinant viral glycoprotein gB was able to inhibit macrophage differentiation in similar ways as the virus. Thus, these results suggest that binding of HCMV to monocytes induces an intracellular rise of Ca2+, of which one result is a block in the ability of the cells to differentiate into macrophages. These observations suggest an efficient viral strategy to interfere with cellular differentiation pathways, and may also in part explain the generalized immunosuppression that is often observed in HCMV-infected patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human CMV (HCMV)3 belongs to the herpesvirus family and between 60 and 100% of the population is persistently infected with and carries the virus. The interest of HCMV as an important human pathogen has increased over the past two decades, with the escalation in the number of patients undergoing immunosuppressive therapy following organ or bone marrow transplantation, as well as with the increasing amount of AIDS patients (1). HCMV infection is usually subclinical in immunocompetent individuals, but the virus can cause fatal disease in immunocompromised patients. Although 50–90% of bone marrow and organ transplant patients experience HCMV infections in the posttransplant period, the prevalence of HCMV approaches 100% in most HIV-infected groups of patients (1). Furthermore, with an incidence of 0.2–2.2% per live birth, HCMV infection is the most common congenital virus infection (1). The virus is also associated with the development of atherosclerosis, restenosis after coronary angioplasty, chronic rejection in organ transplant patients, and chronic graft vs host disease in bone marrow transplant patients (2, 3, 4). A primary HCMV infection is followed by a lifelong persistence of the virus in a latent state, and reactivation may occur later in life. The latency phase is characterized by viral genome persistence without production of infectious virus, that enables the virus to coexist with its host by reducing its visibility, thus leading to an avoidance of immune recognition. HCMV has developed a number of ways to avoid elimination by the immune system, such as inhibition of HLA class I and II molecule expression, inhibition of peptide presentation and T cell activation, inhibition of NK lysis, up-regulation of FcRs, and interference with cytokine signaling (as reviewed in Ref. 5).

Monocytes are major HCMV target cells in vivo, and are responsible for dissemination of the virus throughout the body during latent, acute, and late phases of infection. Monocytes are believed to be the predominant cell type that harbors HCMV in peripheral blood in seropositive individuals (6, 7). A number of studies have shown that HCMV infection of monocytes is nonpermissive and restricted to early events of gene expression (8, 9). In contrast, HCMV infection is permissive in differentiated macrophages, and a high number of HCMV-infected macrophages expressing late viral genes have been identified in tissue specimens obtained from HCMV-infected patients (10). A number of studies have shown that differentiation of monocytes into macrophages is a prerequisite for productive HCMV infection (11, 12, 13, 14). An absence of late viral gene expression and production of virus in monocytes supports the hypothesis that these cells are reservoirs for latent virus, and several studies have also demonstrated that latent virus is mainly present in monocytes in the peripheral blood (6, 8). We have previously shown that latent HCMV can be reactivated in differentiated macrophages by allogeneic stimulation of PBMC (15). Furthermore, we have shown an important role of the inflammatory cytokines IFN-{gamma} and TNF-{alpha} for reactivation of latent virus and growth of HCMV in differentiated macrophages (13, 16). Other investigators have also shown a positive role for TNF-{alpha} for direct activation of the HCMV immediate early (IE) promoter in myeloid cells (17, 18, 19, 20). These results suggest an important role of immune activation with concomitant production of inflammatory cytokines for virus reactivation and replication in HCMV-infected patients.

Because monocytes in the peripheral blood are cells with a short half-life, the ability to reactivate latent HCMV in macrophages suggests that HCMV is maintained in a precursor population of the myeloid cell lineage. Myeloid lineage cells would provide an ideal site for latency for a virus that is closely linked to the immune system for activation. In support of this hypothesis, HCMV has previously been reported to infect CD34+-positive pluripotent stem cells and CD33-positive myeloid-lineage-committed progenitor cells both in vitro and in vivo (21, 22, 23, 24, 25, 26, 27). However, because differentiation of all infected cells after virus entry would eliminate the virus during the infectious cycle, we speculated that the virus may interfere with cellular differentiation signals to establish latency in myeloid lineage cells. We have recently found that HCMV inhibits macrophage differentiation, resulting in an impaired ability of migration and phagocytosis (28). Here, we extended these studies to further examine the pathways by which HCMV inhibits macrophage differentiation by investigating the role of intracellular pathways and signaling molecules that are important for the inhibition of macrophage differentiation. Interestingly, HCMV has previously been reported to induce a number of physiological changes upon early interactions with its target cells. For example, HCMV has been demonstrated to activate intracellular signaling pathways during the early events of infection, such as hydrolysis of phosphatidylinositol (4,5)-bisphosphate and stimulation of arachidonic acid metabolism within minutes after binding, a virus-induced transient influx of calcium at 1–3 h postinfection (hpi), and a cellular and viral gene expression-dependent increase in cAMP and cGMP after 6–12 hpi (as reviewed in Ref. 29). Here, we found that binding of HCMV to monocytes induced a rapid increase in the levels of intracellular Ca2+ by a virus interaction with the cell surface molecule CD13, which has been shown to facilitate the entry of HCMV into target cells (30). A similar rise in intracellular Ca2+ levels was also observed using the CD13-specific mAb My7 and WM15, a finding that was accompanied by an induced inhibition of macrophage differentiation. Importantly, we found that the CD13-specific Ab clone SJ1D1 did not result in an intracellular Ca2+ response, and that this Ab could block the ability of HCMV to inhibit macrophage differentiation. Furthermore, we found that recombinant gB protein and microbeads coated with the viral glycoprotein gB, but not beads coated with gH, could inhibit macrophage differentiation. These results imply a role of gB in delivering inhibitory signals for macrophage differentiation, possibly by binding to CD13.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Establishment of macrophage cultures

PBMCs from healthy donors were isolated as previously described (31), and the cells were plated onto Primaria plates (Falcon; BD Biosciences, San Jose, CA) at a cell concentration of 10–18 x 106 cells/ml in Iscove’s modified medium with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Life Technologies, Grand Island, NY), 10% AB serum, and incubated at 37°C for 2 h. The nonadherent cells were removed, the cultures were extensively washed, and the monocyte-enriched cells were stimulated by addition of a "24 h allosupernatant" containing cytokines produced by the allogeneic reaction between T cells and monocytes that will be further referred to as allocytokines. The cells were infected with HCMV or mock-infected, at the same time point as allocytokines were added to the monocyte cultures. The allocytokine preparations were produced as follows: PBMCs from different donors were mixed and incubated for 24 h in Iscove’s complete medium. Thereafter, the allosupernatant was collected, cleared by centrifugation, and used to stimulate separate monocyte cultures. We have previously shown that treatment of monocytes with preparations of allocytokines induce differentiation of macrophages (13). At 24 h poststimulation, the cultures were washed with Iscove’s medium and thereafter cultured in 60% AIM-V medium, 30% Iscove’s modified medium, and 10% AB serum, with the addition of L-glutamine, penicillin, and streptomycin (complete 60/30 medium). The medium was changed to fresh complete 60/30 medium every 3–4 days.

Virus infections

At the time of allocytokine stimulation, the cells were challenged with HCMV at a multiplicity of infection (MOI) of 1 and the cell cultures were incubated for 24 h at 37°C. The following HCMV strains were used for infection of monocytes: the laboratory strains AD169 or Towne. Cell-free viral stocks were prepared from supernatants of infected HL cells, frozen, and stored until use at –70°C. Virus titers were determined by plaque assays as previously described (32).

Quantification of the number of macrophages in monocyte-derived macrophage (MDM) cultures and statistical analyses

Differentiated macrophages are readily distinguished from monocytic cells by the cell morphology, by their enlargement in size, and by the numerous vacuoles in the cytoplasm of these cells. To determine the number of macrophages in the cell cultures, cultures were fixed and stained at day 7 poststimulation with allocytokines in the presence or absence of virus infection, using an {alpha}-naphtyl acetate esterase staining kit with a fluoride inhibition procedure (Sigma-Aldrich, St. Louis, MO). The cell cultures were fixed and stained with the kit according to the manufacturer’s instructions. All cells of the monocytic lineage are negative for {alpha}-naphtyl acetate esterase enzyme activity with the exception of histiocytes and specialized macrophages, which are resistant to sodium fluoride. The enzyme activity appears as a black granulation in positive macrophages. Using this method, the number of macrophages was determined by counting the black granulated cells, which also exhibited a classical macrophage (histological phenotype) appearance in an inverted microscope.

At least 100 cells (either positive or negative) were counted in each well. The results were calculated as the percentage of monocytes in the cultures. In the monocyte-enriched cell cultures, up to ~20% of cells in the cultures (both infected and mock-infected cultures) are not of monocytic origin, but these numbers remain constant between different cultures obtained from the same donor. Hence, small cells can account for ~20% of the total cell number, and the numbers of these cells are included in the quantification of the monocytes (e.g., 20% monocytes in mock-infected cultures represent a complete differentiation of most monocytic cells in the cultures). All experiments were performed with cells from at least 2 (generally 6) independent donors and in duplicates. The data was either presented as mean ± SD or SEM of the indicated number of experiments, or Student’s (two-tailed, paired) t tests were performed.

Treatment of cells with aminopeptidase inhibitors

PBMC were isolated and plated as described above. PBMC were infected with AD169 at an MOI of 1.0 and simultaneously stimulated with allocytokines. Either bestatin (5 or 10 mM) or actinonin (1 mM), which are aminopeptidase N (APN) inhibitors (30), and arphamenine (1 mM) (inhibitor of aminopeptidase B activity), all from Sigma-Aldrich, were incubated with the cells for 1 h at 37°C before removal of drugs and subsequent infection or FACS analyses for CD13 or treatment with APN.

Cell surface aminopeptidase activity (APN)

APN activity of intact monocytes was measured using a spectrophotometric assay as previously described (33). Briefly 0.6 x 106 PBMC were incubated in a 96-well plate for 2 h. The nonadherent cells were removed, the cultures were extensively washed, and the monocyte-enriched cells were incubated with 5 or 10 mM bestatin for 1 h in 37°C. The cells were washed, and then incubated with alanine-p-nitroanilide (6 mM) (Sigma-Aldrich) in isotonic buffer at 37°C. At various times, the released free p-nitroanilide was measured spectrophotometrically at 410 nm. The APN activity was expressed as mean values of triplicate determinations. The cell viability was >95% as measured with trypan blue uptake.

Flow cytometric analysis of CD13 expression and binding

A fluorescence-activated cell sorter (FACSort; BD Biosciences) was used to analyze the bestatin-treated or untreated monocytes. The adherent monocytes were harvested by incubating the cells in versene (Invitrogen Life Technologies, Grand Island, NY) at room temperature for 60 min followed by scraping. The cells were stained with Abs recognizing CD13 (Dakopatts, Glostrup, Denmark) or isotype controls (IgG1; Dakopatts). The expression of CD13 on bestatin-treated and untreated monocytes was measured as the mean channel fluorescence value following treatment with the respective Ab compared with the isotype control. Binding of different CD13 Ab clones to fibroblasts and monocytes were analyzed by FACS. The cells were stained with the following CD13-specific Ab clones: WM15 (Serotec, Oslo, Norway), SJ1D1 (Immunotech, Marseille, France), My7 (Coulter-Immunotech, Hamburg, Germany), and the isotype control IgG1 (Immunotech) followed by appropriate FITC-conjugated secondary Abs (Dakopatts). The difference in the histogram mean channel value for uninfected and HCMV-infected cells was calculated on a linear scale, and a >10 channel difference between uninfected and HCMV-infected cells was considered as a positive or negative change as based on variations of controls.

Isolation of gB and gH

HCMV supernatants obtained from fibroblasts infected with AD169, or from mock-infected fibroblasts, were lysed by mixing supernatants with equal volumes of buffer B (4% Nonidet P40, 10 mM Tris-HCl, 0.5 M NaCl, 2 mM EDTA), and dialyzed overnight at 4°C in PBS. As a control for the effect of the micromagnetic beads, supernatants from mock-infected fibroblasts were used, treated in the same way as the HCMV-infected samples ("mock-beads"). The lysates were incubated with either gB (clone 27-78) or gH (clone 14-4b) specific Abs for 30 min on ice. Thereafter, 40 µl of rat anti-mouse IgG1 Mini MACS MicroBeads (Miltenyi Biotec, Auburn, CA) were added, and the mix was incubated for 30 min at 4°C. Magnetic separation was performed according to manufacturer’s protocol. Thirty microliters of Mini MACS complexes (all in 2.5 µg/µl concentration) were used for treatment of monocytes in a total volume of 100 µl of allosupernatant in 96-well plates.

Treatment of cells with recombinant soluble gB

PBMC were isolated and plated as described above. PBMC were infected with AD169 at a MOI of 1.0 and simultaneously stimulated with allocytokine supernatants. Recombinant soluble gB produced in Chinese hamster ovary (CHO) cells (34) was kindly provided by Aventis Pasteur (Marcy l’Etoile, France) and used in different concentrations (1–100 ng/ml) at the same time point as allostimulation. As controls to the recombinant gB, we used baculovirus-produced recombinant HIV-1 gp160 (Protein Sciences, Meriden, CT) in a concentration of 100 ng/ml, and crude CHO supernatant in a final dilution of 1/10; furthermore, HSV-1 glycoprotein D (recombinant human protein from Pichia pastoris) and HSV-2 glycoprotein G (recombinant protein from Saccharomyces cerevisiae) (both from Nordic Biosite, Täby, Sweden) were used (at a concentration of 100 ng/ml) at the same time point as allostimulation.

Inhibition of HCMV infection by CD13-specific Abs

Neutralization of HCMV infection of fibroblasts (permissive for HCMV infection) was performed by preincubation of fibroblasts with Abs to CD13 (WM15, SJ1D1, or My7) and the isotype control IgG1 (all in the concentration of 20 µg/ml) for 1 h at 37°C. Thereafter, the cells were HCMV infected and incubated for 4 h, washed three times with PBS, and subsequently the cultures were incubated for 20 h at 37°C and assayed for HCMV infection by detection of the expression of the IE Ag (IE-72-specific Ab; Argene, Biosoft, Parc Technologique Delta Sud, Varilhes, France) and thereafter conjugated with a FITC-labeled secondary Ab (DakoCytomation, Glostrup, Denmark). The cells were evaluated under fluorescence microscope and data are presented as the percent of control as compared with untreated HCMV-infected fibroblasts.

Inhibition of macrophage differentiation with anti-CD13 Abs

All experiments were performed by preincubation of cells, grown in 96-well Primaria plates, with the following Abs for 30 min on ice before removing the Ab by washing: the CD13-specific clone WM15 (Serotec Scandinavia, Oslo, Norway), the CD33-specific clone WM54, the isotype control mouse IgG1 (all Dakopatts), the isotype control mouse IgG1, CD14, the CD13-specific clone SJ1D1 (all Immunotech), the CD13 clone My7 (Coulter-Immunotech) in a concentration from 0.01 to 0.1 µg/µl. Thereafter, infection, treatment with recombinant soluble gB or micromagnetic beads, and allocytokine stimulation were performed as previously described.

Detection of intracellular calcium mobilization

Monocytes isolated from PBMC were allowed to adhere on chamber slides overnight. The adherent monocytes were loaded with fluo-4-AM (Molecular Probes, Eugene, OR), based on the manufacturer’s instruction. Changes in monocyte intracellular free Ca2+ following stimulation were measured as changes in fluorescent intensity detected by an inverted laser-scanning confocal microscope (Insight Plus; Meridian Instruments, Okemon, MI). The following stimuli were used: concentrated HCMV (pelleted at 10,000 rpm for 16 h, and solved in TNM: 50 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2), CD13 Ab clones My7 (in the concentration 2 µg/ml) and SJ1D1 (in the concentration of 20 µg/ml) and isotype control IgG1 (in the concentration of 20 µg/ml) (the parameters of the confocal microscope were set initially and applied to all experiments). Monocytes isolated from PBMC were also allowed to adhere in 96-well plates overnight. The adherent monocytes were loaded with fluo-4-AM (Molecular Probes), based on the manufacturer’s instruction. Changes in monocyte intracellular free Ca2+ following stimulation were also measured as changes in fluorescent intensity as detected with Fluoroscan II (Thermo Labsystems, Vantaa, Finland). The following stimuli were used: concentrated HCMV, CD13-specific Ab clones My7 (20 µg/ml), WM15 (20 µg/ml), SJ1D1 (20 µg/ml), or isotype control IgG1 (20 µg/ml) (the parameters of the fluoroscan were set initially and applied to all experiments).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HCMV-induced inhibition of macrophage differentiation involves Ca2+-dependent pathways

To examine whether HCMV could affect cellular differentiation, we mock infected or HCMV infected monocyte-enriched cultures with HCMV at a MOI of 1 in the presence of allocytokines that have previously been demonstrated to induce macrophage differentiation (13). We first tested whether HCMV inhibited macrophage differentiation at a MOI of 1 (Fig. 1A). Macrophages stimulated with allocytokines exhibited a classical macrophage morphology and were esterase positive (Fig. 1B and data not shown). In contrast, HCMV-infected monocytes were small and esterase negative (Fig. 1B and data not shown), and hence these results confirmed our previous findings (28). In our previous work, we found that the ability of HCMV to induce inhibition of macrophage differentiation was not dependent on viral replication (28). Instead, binding of virus particles to monocytes appeared to be sufficient to inhibit macrophage differentiation. Therefore, we hypothesized that binding of the virus to the cells may induce intracellular signaling pathways in the monocytes that would prevent cellular differentiation.



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  		FIGURE 1. HCMV inhibits differentiation of monocytes into macrophages. A, HCMV was tested for its ability to affect cellular differentiation by infecting monocyte-enriched cultures with the laboratory strain AD169 (MOI 1), or by mock infecting of monocyte-enriched cultures at the same time point as allocytokine stimulation. B, HCMV was tested for its ability to affect cellular differentiation by infecting monocytes. Panels represent a phase microscopy photo of (a) allostimulated MDMs and (b) HCMV-infected monocytes at an MOI of 1 after 7 days in culture.

 
HCMV and monocyte interaction involve Ca2+ signaling

Because virus binding inhibited macrophage differentiation, we speculated that binding of HCMV to its receptor induced the block in differentiation through intracellular Ca2+-dependent signaling pathways. We have previously demonstrated that human APN/CD13 functions as an important cell surface molecule for the entry of virus into susceptible cells (30). To examine the role of CD13 and intracellular Ca2+ signaling pathways in the ability of HCMV to inhibit macrophage differentiation, we examined the ability of HCMV to induce a rise in cytosolic-free Ca2+ by measuring Ca2+ levels in monocytes from PBMC during acute HCMV infection by confocal microscopy and by fluoroscan. We found that HCMV induced a rise in intracellular Ca2+ levels at 70 s (Fig. 2A). We further performed experiments with two CD13-specific Ab clones; two that inhibit APN activity (clone My7 and WM15 (35)); and one Ab which does not inhibit APN activity (clone SJ1D1 (35)). Interestingly, the CD13-specific Ab clones My7 and WM15 also induced a rise in intracellular Ca2+ in fresh monocytes, as demonstrated by confocal microscopy, fluoroscan, and FACS analysis (Fig. 2, B and C, and data not shown). In contrast, the CD13-specific Ab clone SJ1D1, as well as an isotype control (IgG1), did not induce a rise in intracellular Ca2+ levels (Fig. 2, B and C). When the monocytes were preincubated with SJ1D1 for 30 min, we observed an inhibition of intracellular Ca2+ mobilization upon HCMV infection (p < 0.001) (Fig. 2D). Furthermore, the drug thapsigargin induced a sustained rise in intracellular Ca2+ levels for at least 2 h, and at this time point, HCMV was not able to induce a rapid rise in intracellular Ca2+ (data not shown). Because HCMV has previously been reported to induce a number of physiological changes upon early interactions with its target cells, and we here found that HCMV upon binding to the monocyte induced a rapid rise in intracellular Ca2+, we further wanted to investigate the effect of increasing levels of intracellular Ca2+ in the HCMV-treated monocytes by treating monocytes with the Ca2+ modulator thapsigargin, at the same time point as HCMV infection. Interestingly, we found that when monocytes were treated with high concentrations of thapsigargin, which is an inhibitor of the sarcoplasmatic or endoplasmatic reticulum Ca2+-ATPase family (36), HCMV failed to block macrophage differentiation (Fig. 3). These results suggested that the HCMV-induced block in cellular differentiation involved calcium-dependent pathways.



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  		FIGURE 2. HCMV induces a rise in intracellular Ca2+ levels by binding to CD13. Intracellular calcium levels rise in HCMV-infected monocytes as measured by a change in fluorescence intensity of fluo-4, loaded in monocytes, in response to HCMV treatment. A, The images captured before (left) and at 70 s (right) after adding HCMV show resting and peak fluorescence intensity in the same group of cells. B, HCMV and the CD13-specific Ab clone My7 and clone SJ1D1 were used to test their ability to induce a rise in intracellular Ca2+ in monocytes, while a TNM buffer and an IgG1 isotype control Ab were used as negative controls by confocal microscopy (p < 0.001 when comparing HCMV with TNM, and My7 with SJ1D1 or IgG1, respectively). C, HCMV and the CD13-specific Ab clones My7 and WM15 and clone SJ1D1 were used to test their ability to induce a rise in intracellular Ca2+ in monocytes; an IgG1 isotype control Ab was used as negative control in fluoroscan. D, To test whether the CD13-specific Ab clone SJ1D1 abrogated the ability of HCMV to induce an increase in intracellular Ca2+ in monocytes, pretreatment with the Ab was performed for 30 min before the experiment was conducted as described in Materials and Methods (p ≤ 0.001 when comparing SJ1D1 pretreated cells with untreated cells).

 


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  		FIGURE 3. The ability of HCMV to block macrophage differentiation involves calcium-dependent pathways. To test whether the ability of HCMV to block cellular differentiation is a calcium-dependent process, monocytes were treated with thapsigargin at the same time point as HCMV or mock infection, and cells were quantified under light microscope after 7 days in culture (n = 7–9, mean value ± SEM; ***, p ≤ 0.001, thapsigargin-treated cultures as compared with HCMV-infected cultures).

 
HCMV-induced inhibition of macrophage differentiation requires an interaction with CD13

Because HCMV as well as the CD13-specific Ab clones My7 and WM15 induced an intracellular Ca2+ increase in the monocytes, we further investigated whether these Ab clones could influence macrophage differentiation. We found that treatment of cells with My7 as well as WM15 inhibited macrophage differentiation in a similar way as was observed following infection with HCMV (Fig. 4A). In addition, increasing concentrations of the SJ1D1 clone, but not CD14 and CD33 specific Abs or an isotype control Ab, prevented the ability of the virus to inhibit macrophage differentiation (Fig. 4, B and C).



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  		FIGURE 4. The inhibition of macrophage differentiation is directly mediated by binding to the HCMV cell surface receptor CD13. A, To examine whether ligation of CD13 with mAbs could inhibit macrophage differentiation, monocytes were pretreated for 30 min with either of the CD13-specific Ab clones WM15 and My7 before cultures were HCMV or mock-infected at the same time point as allocytokine stimulation. Treatment of cells with increasing concentrations of the CD13-specific Ab clone My7 (0.01–0.1 µg/µl) and WM15 (0.05 µg/µl) is shown in the presence or absence of HCMV. B, Treatment of cells with increasing concentrations of the CD13-specific Ab clone SJ1D1 (0.01–0.1 µg/µl) is shown in the presence or absence of HCMV. The Ab clone SJ1D1 was used to block the ability of HCMV to inhibit macrophage differentiation as described in Materials and Methods, and showed a highly significant ability to prevent the effects induced by HCMV. C, As negative controls, Abs specific for CD33, CD14, or an isotype control IgG1 Ab, were used (all at a concentration of 0.1 µg/µl) and did not affect the ability of HCMV to inhibit macrophage differentiation. (**, p ≤ 0.005; ***, p ≤ 0.001 represents results from treatment of cells compared with allostimulated cultures).

 
To further substantiate our findings that CD13 seems to be critically involved in HCMV-mediated inhibition of macrophage differentiation we studied the ability of the CD13 Ab clones My7, WM15 and SJ1D1 to bind to freshly isolated monocytes as well as to fibroblasts by flow cytometry. We found that all CD13 Ab clones reacted with both fibroblasts and monocytes (Fig. 5, A and B) at similar levels. We next examined the ability of the CD13 Ab clones My7, WM15, SJ1D1 to affect HCMV infection in fibroblasts, representing a cell type that is, in contrast to monocytes, highly permissive to HCMV infection. We found that all the HCMV CD13 Ab clones we tested reduced the HCMV infectibility by 70–85% (Fig. 5C).



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  		FIGURE 5. CD13-specific Abs react with monocytes and fibroblasts and neutralize HCMV infection of fibroblasts. Binding of different CD13-specific Ab clones to (A) monocytes and (B) fibroblasts was analyzed by flow cytometry. The cells were stained with CD13-specific Ab clones; WM15, SJ1D1, My7, and the isotype control IgG1. The data are presented as histogram overlays. C, Neutralization of HCMV infection of fibroblasts (permissive for HCMV infection) were performed by preincubation of fibroblasts with Abs to the CD13 clones WM15, SJ1D1, and My7 and the isotype control IgG1 (all at a concentration of 20 µg/ml) for 1 h at 37°C, thereafter the cells were HCMV infected at a MOI of 1. Data are presented as the percentage of IE-positive cells as compared with untreated HCMV-infected fibroblasts.

 
We have previously shown that pretreatment of cells with aminopeptidase inhibitors inhibits HCMV infection (30). Therefore, we further examined whether inhibition of the aminopeptidase activity influenced macrophage differentiation by testing three different aminopeptidase inhibitors for their ability to affect macrophage differentiation. Monocytes were pretreated with bestatin for 1 h before the cells were assayed for APN activity as previously described (30). As shown in Fig. 6A, pretreatment of monocytes with bestatin decreased the aminopeptidase activity by 50–70%. Next, we tested whether bestatin treatment of monocytes could affect the expression of CD13 on monocytes by analyzing the CD13 expression on monocytes by flow cytometry after 1 h of treatment with bestatin. We found that treatment of monocytes with bestatin did not influence the expression of CD13 on monocytes (Fig. 6B). Furthermore, bestatin and actinonin (APN inhibitors;30), and arphamenin B (inhibitor of aminopeptidase B activity), neither influenced the ability of HCMV to affect macrophage differentiation nor affected the ability of the cells to differentiate into macrophages in the absence of virus (Fig. 6C). These results indicate that even though CD13 is critically involved in the ability of HCMV to inhibit macrophage differentiation, the enzymatic activity of APN does not appear to be involved in this process.



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  		FIGURE 6. Treatment of cells with aminopeptidase inhibitors do not affect macrophage differentiation. To examine whether aminopeptidase activity was involved in the control of macrophage differentiation, we performed experiments using chemicals that inhibits aminopeptidase activity. A, To examine whether bestatin would alter the aminopeptidase activity on monocytes, we preincubated monocytes for 1 h with bestatin, the cells were then incubated with alanine-p-nitroanilide. At various time points, the released free p-nitroanilide was measured spectrophotometrically at 410 nm. The APN activity was expressed as mean values of triplicate determinations. B, To examine whether bestatin treatment of monocytes would alter the CD13 expression on monocytes, cells were incubated with bestatin for 1 h, whereafter the monocytes were examined for CD13 expression by flow cytometry. The figure represents a representative example of a histogram plot of the fluorescence values of CD13 of bestatin-treated monocytes (dotted line) and untreated monocytes (solid line) obtained by flow cytometry. C, Treatment of uninfected and HCMV-infected monocytes was performed with the aminopeptidase N inhibitors bestatin, actinonin, and the aminopeptidase B inhibitor arphamenine B. Cells were incubated for 1 h at 37°C before removal of drugs and subsequent infection. The figure shows the effect on macrophage differentiation in uninfected and HCMV-infected cells treated with the drugs bestatin, actinonin, and arphamenine B.

 
The viral glycoprotein gB, but not gH, appears to be involved in the ability of HCMV to inhibit macrophage differentiation

To examine which HCMV protein mediated binding to CD13, we performed experiments using blocking Abs against two of the main HCMV glycoproteins, gB and gH. Preincubation of the virus with these Abs reduced infectivity by 90 and 100%, respectively, but did not prevent the ability of HCMV to inhibit cellular differentiation (data not shown). However, because these mAbs only bind to specific epitopes on the viral glycoproteins, we isolated gB and gH from HCMV lysates by binding gB and gH, respectively, onto Ab-coated micromagnetic beads. The gB- and gH-coated beads were then tested for their ability to affect differentiation of monocytes into macrophages. We found that gB-coated beads could inhibit macrophage differentiation by 55–60%, whereas gH-coated beads inhibited macrophage differentiation by <20% (Fig. 7A), as compared with the effect observed by the virus particles. These results implied that HCMV gB rather than gH was involved in the ability of HCMV to inhibit macrophage differentiation. Therefore, we performed additional experiments using a recombinant gB protein and found that recombinant gB inhibited macrophage differentiation to a similar extent as HCMV, whereas control proteins (a recombinant glycoprotein HIV-1 gp 160, glycoproteins from HSV-1 and HSV-2, and a crude CHO supernatant) did not inhibit macrophage differentiation (Fig. 7, B and C).



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  		FIGURE 7. The HCMV glycoprotein gB, but not gH, plays a role in the ability of HCMV to inhibit macrophage differentiation. A, To evaluate the role of the HCMV glycoproteins gB and gH in the inhibition of macrophage differentiation, HCMV gB and gH proteins bound to micromagnetic beads were tested for their ability to inhibit macrophage differentiation. Cells were incubated with the micromagnetic beads at the same time point as when the allocytokine stimulation was performed; the cultures were quantified under light microscope after 7 days. The mean and SEM results shown are from six different donors performed in duplicates. Mock-gB 27-78 and mock-gH 14-4b refers to "mock-beads" treated with mock-infected supernatants, and HCMV gB 27-78 and HCMV gH 14-4b refers to the beads coated with the respective proteins by treatment of anti-gB and anti-gH coated beads with HCMV supernatants as described in Materials and Methods. B, Cells were incubated with recombinant gB (1–100 ng/ml) at the same time point as when the allocytokine stimulation was performed; the cultures were quantified under light microscope after 7 days. The mean and SEM results shown are from two different donors performed in duplicates. C, Cells were incubated with recombinant gB (100 ng/ml), recombinant HIV-1 gp160 (100 ng/ml), recombinant HSV-1 glycoprotein D (100 ng/ml), recombinant HSV-2 glycoprotein G (100 ng/ml), or crude CHO supernatant (in final dilution 1/10) at the same time point as when the allocytokine stimulation was performed, and the cultures were examined and cells were quantified under light microscope after 7 days. The mean and SEM results shown are obtained from four different donors performed in duplicates. D, Monocytes were pretreated for 30 min with either the CD13-specific Ab clone SJ1D1 or the isotype control (both in a concentration of 0.1 µg/µl) before the cultures were treated with the micromagnetic beads, at the same time point as when the allocytokine stimulation was performed; the cultures were examined and cells quantified under light microscope after 7 days. The mean and SEM results shown are from two different donors performed in duplicates. E, Monocytes were pretreated for 30 min with either the CD13-specific Ab clone SJ1D1 or the isotype control (both in a concentration of 0.1 µg/µl) before the cultures were treated with HCMV or recombinant glycoprotein B, at the same time point as when the allocytokine stimulation was performed; the cultures were examined and cells were quantified under light microscope after 7 days. The mean and SEM results shown are from two different donors performed in duplicates.

 
To further substantiate our findings that CD13 and HCMV glycoprotein B seem to be critically involved in HCMV-mediated inhibition of macrophage differentiation we performed blocking experiments of gB-coated beads as well as of recombinant gB using the CD13-specific Ab clone SJ1D1. We found that Abs directed at CD13 but not an isotype control Ab prevented the gB-coated beads as well as the ability of recombinant gB to inhibit macrophage differentiation (Fig. 7, D and E).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Monocytes/macrophages are key cell types in the immune system as well as in the pathogenesis of HCMV. We have recently found that HCMV-infected monocytes are unable to differentiate into macrophages upon cytokine stimulation (28). In the current study, we have extended our initial observation that HCMV inhibits macrophage differentiation and have found that virus binding to one of the cellular receptor molecules of HCMV, CD13, induced a Ca2+-dependent inhibition of macrophage differentiation. We have previously shown that HCMV interacts with the cell surface molecule CD13 during the early events of HCMV infection (30). CD13 is a membrane peptidase that belongs to a multifunctional group of ectoenzymes that interestingly have been implied in the control of differentiation and growth of many cell types (37). The aminopeptidases cleave peptides to activate or inactivate mediators, and function as molecules mediating adhesion and signal transduction pathways (37). In addition, a recent study has shown that inhibition of enzymatic activity of CD13 inhibited cellular proliferation, and CD13 has been suggested to play a role in the growth of DC/macrophage progenitor cells (38). Here, we interestingly found that binding of two CD13-specific Abs (My7 and WM15) to monocytes inhibited macrophage differentiation in the absence of HCMV. In contrast, pretreatment of cells with the CD13-specific Ab clone SJ1D1 blocked the ability of HCMV to inhibit macrophage differentiation. We also found that pretreatment of HCMV permissive cells with the CD13-specific Ab clones My7, WM15, and SJ1D1 reduced the number of infected cells by 70–85%, which further supports the hypothesis that CD13 is involved in binding and virus uptake by the cells. However, pretreatment of monocytes with aminopeptidase inhibitors that can inhibit HCMV infection (30) did not prevent the ability of HCMV to inhibit macrophage differentiation, and did not affect cell surface expression of CD13 on monocytes, although it significantly decreased aminopeptidase activity on these cells. These observations suggest that inhibition of macrophage differentiation involved binding of HCMV to CD13, but did not involve the enzymatic activity of APN for this effect. Our additional experiments suggested that the HCMV glycoprotein B was involved in the inhibition of cellular differentiation, because gB, but not gH, coated microbeads and recombinant gB inhibited macrophage differentiation, and because pretreatment of cells with the CD13-specific Ab clone SJ1D1 prevented the ability of gB-coated microbeads and recombinant gB to inhibit macrophage differentiation.

gB (UL55) is a major glycoprotein component of the HCMV envelope, and is essential for infectivity. HCMV gB appears to be involved in both attachment and penetration of the virus particles into the cell, whereas gH (UL75) and gL (UL115) are thought to be involved in fusion of the viral envelope with the host cell membrane (as reviewed in Ref. 39). DNA microarray analysis of human fibroblasts infected with HCMV recently revealed changes in the expression of >200 host genes, and a major finding of this analysis was that HCMV induced known IFN-stimulated genes (40). Boyle et al. (41) have found that gB is the viral structural component responsible for intracellular signaling and gene induction by interference with the IFN-responsive pathways. Recent evidence also suggest that gB, most likely through an interaction with its cellular binding partner, leads to an altered cellular transcription profile early after HCMV infection (42). Using DNA microarrays, Simmen et al. (42) showed that fibroblasts treated with gB exhibited the same transcriptional profile as HCMV-infected cells, which supports the well-known concept that ligand-receptor interactions trigger signal-transduction cascades. Furthermore, Yurochko et al. (43) have found that the binding of HCMV to monocytes induced a number of immunoregulatory genes such as IL-1{beta}, A20, NF-{kappa}B p105/p50, and I{kappa}B{alpha} in unactivated monocytes, and treatment of virus with neutralizing Abs to gB and gH inhibited the induction of these genes. Furthermore, HCMV has been suggested to activate different intracellular signaling pathways, since HCMV transiently activates the PI3K pathway in fibroblasts already at 15–30 min postinfection (44). Treatment of the cells with the potent PI3K inhibitor LY294002 prevented viral replication, but not virus entry (44). The MAPK p38 has also been shown to be strongly induced following viral infection in fibroblasts, and the MAPK has been shown to activate numerous transcription factors (45).

A rise in cytosolic calcium levels is one of the first events occurring after stimulation of a variety of membrane receptors. Here, we found that the drug thapsigargin, which interferes with Ca2+ release from intracellular stores by preventing uptake into both inositol 1,4,5-triphosphate and GTP-sensitive intracellular stores, prevented the ability of HCMV to block macrophage differentiation. These results imply that the inhibition of macrophage differentiation caused by HCMV involves Ca2+-dependent pathways. In support of this hypothesis, a previous study has demonstrated that HCMV infection of cells leads to an increase in intracellular calcium levels (46). Interestingly, aminopeptidases have also been shown to mediate activation of signal transduction pathways. Previous findings also suggest that CD13-specific Abs transiently increased the intracellular Ca2+ concentration, which was accompanied by a decrease in proliferation of U937 cells (47). These observations suggest that CD13, by inducing changes in the intracellular Ca2+ concentrations (47), may serve as a regulatory protein in global cell processes such as proliferation, that could lead to an altered control of many intracellular signaling events. Interesting, evidence also suggest that CD13 is linked to phosphorylation of MAPKs and to an increase in intracellular Ca2+ levels in monocytes (48). Ligation of the CD13-specific Ab clones WM15 and My7 to cells of Mono Mac-6 and U937 cell lines have also been shown to increase the intracellular Ca2+ levels (48). Here, we found that the Ab clone My7 and WM15 induced a rise intracellular Ca2+ levels in monocytes, and that both the Ab clones My7 and WM15 inhibited macrophage differentiation. In contrast, treatment of cells with the CD13-specific Ab clone SJ1D1 did not result in an increase in intracellular Ca2+ levels in monocytes, that was in accordance with previous observations by Santos et al. (48). However, we interestingly found that the SJ1D1 Ab was able to prevent the ability of HCMV to induce a rise in intracellular Ca2+, and that the Ab prevented the inhibitory viral effect on cellular differentiation. Although not yet confirmed, it is possible that the change in intracellular Ca2+ levels in monocytes by HCMV, or by the CD13-specific mAbs My7 and WM15, changes the activation of one or more transcription factors that regulate macrophage differentiation that would negatively influence cellular differentiation. In support of this hypothesis, a number of different transcription factors have been described that regulate the control of macrophage differentiation (49).

A number of previous publications have clearly shown the importance of cellular differentiation for productive HCMV infection in certain cell types including macrophages (11, 12, 13, 50). Interestingly, while viral adsorption and penetration seem to occur in cells of different differentiation stages (11), a clear correlation exists between cellular differentiation and the permissiveness for HCMV replication in monocytes/macrophages (11, 12, 13, 14). Here, we found that HCMV has a unique ability to transiently shut down further cellular differentiation of monocytes into macrophages. Therefore, one could speculate whether the virus may be silent in monocytes early in the infectious process due to an inactivation of the infected cells. The inability of the HCMV-infected monocytes to differentiate into macrophages may also further facilitate the establishment of latency in myeloid cell populations. In contrast to our findings, Smith et al. (51) recently published data which suggest that HCMV instead could induce differentiation of monocytes into macrophages. Collagen-coated plates were used in their experimental system, which may explain the different results obtained, and importantly, these differences may also reflect differences in the in vivo situation when monocytes in the peripheral blood or monocytes entering tissues as macrophages will become HCMV infected.

The viral and cellular mechanisms for maintaining HCMV latency are today mainly unknown. HCMV latency associated transcripts have been identified in HCMV in vitro-infected fetal liver-derived CD33-positive granulocyte-macrophage progenitor cells and in HCMV-seropositive individuals, and may play a critical role in the control of latency (27). The HCMV latency associated transcripts are represented by sense (ORF94) and antisense transcripts (ORF152, 154), which are expressed from a region of the genome (UL122/UL123) normally involved in the expression of the transcriptional activators (IE1 and IE2) that are involved in lytic replication (52). However, recent observations also suggest that inactivation of ORF94 does not affect reactivation of latent HCMV in the fetal liver-derived CD33-positive granulocyte-macrophage progenitor cells (53). The ability of HCMV to inhibit macrophage differentiation may also have important clinical implications in the acute phase of disease in infected patients because an inhibition of these cells would decrease the ability for the host to clear the virus infection as well as secondary bacterial and fungal infections. In support of this hypothesis, HCMV has been reported to predispose for other opportunistic infections and to be associated with bacterial and fungal infections (54, 55, 56). In concordance with these results, we have recently found that HCMV inhibits differentiation of monocytes into dendritic cells with the consequence of depressed immunological functions (57). Because monocytes/macrophages play a key role in the immune defense against microbes by functioning as phagocytic and APCs, and thereby orchestrating the activation of different subsets of lymphocytes, it is interesting to note that HCMV-infected patients are known to suffer from a generalized immunosuppression during the active phase of an acute HCMV infection.

In summary, we here found that HCMV was able to directly inhibit the development of one of the most important cell types in the immune system, the functionally active macrophage, through a viral interaction with monocytes that did not involve viral replication. The virus inactivated cellular differentiation pathways through binding to a cellular molecule, CD13, that plays an important role in the early events of HCMV infections, and that also has been implied to regulate cellular activation and differentiation events. Our findings provide new tools to examine cellular differentiation response pathways and may help in the development of new strategies to improve immunological functions in HCMV-infected patients.


   Acknowledgments
 
We thank Dr. Erna Möller for helpful discussions, Dr. Mats Ohlin for human gB Abs, and Dr. Kjell-Olof Hedlund for help with electron microscopy. We also thank Aventis Pasteur for providing us with the recombinant gB.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Swedish Medical Research Council (K98-06X-12615-01A, K2001-16X-12615-04A (to C.S.-N.), 14X-4342 (to L.L.), the Tobias Foundation (1313/98, 20/01, 33/02), the Swedish Children’s Cancer Foundation (1998/065, 01/046), the Heart-Lung Foundation (199941305, 200241138), Swedish Society for Medicine (1999-02-0347), and the Emil and Wera Cornells Foundation. C.S.-N. is a fellow of the Wenner-Gren Foundation, Sweden. Back

2 Address correspondence and reprint requests to Dr. Cecilia Söderberg-Nauclér, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden. E-mail address: cecilia.soderberg.naucler{at}cmm.ki.se Back

3 Abbreviations used in this paper: HCMV, human CMV; IE, immediate early; hpi, hours postinfection; MOI, multiplicity of infection; MDM, monocyte-derived macrophage; APN, aminopeptidase N; CHO, Chinese hamster ovary. Back

Received for publication November 21, 2002. Accepted for publication August 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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