growth of human cytomegalovirus in primary macrophages

Growth of Human Cytomegalovirusin Primary Macrophages

Cecilia Soderberg-Naucler,*,† Kenneth N. Fish,* and Jay A. Nelson*,1

*Department of Molecular Microbiology and Immunology, Oregon Health Sciences University,Portland, Oregon 97201; and †Department of Biosciences at Novum,Karolinska Institute, Novum, 141 57 Huddinge, Sweden

Human cytomegalovirus (HCMV) is a major human patho-gen that causes considerable disease among immunocom-promised individuals. A primary infection results in life-longpersistence of the virus in a latent form. HCMV is known to betransferred by blood products, bone marrow, and solid or-gans, but the cell type that carries the latent infection hasbeen difficult to identify. We have recently demonstratedreactivation of latent HCMV in allogeneically stimulatedmonocyte-derived macrophages (Allo-MDM). Reactivation oc-curred only in macrophages produced by allogeneic but notmitogenic stimulation. The presence of dendritic cell mark-ers on some Allo-MDM cells suggested that these macro-phages were related to dendritic cells. However, dendriticcells obtained by stimulation of monocytes with interleukin-4(IL-4) and granulocyte-macrophage colony stimulating factor(GM-CSF) were not permissive for HCMV infect ion.The cellular and cytokine components which are essentialfor HCMV replication and reactivation of virus were alsoexamined in Allo-MDM. The importance of both CD4- or CD8-positive T cells in the generation of HCMV permissive Allo-MDM was demonstrated by negative selection or blockingexperiments using antibodies directed against both HLAclass I and HLA class II molecules. Examination of the cy-tokines essential for the generation of HCMV permissiveAllo-MDM identified g-interferon (IFN-g, but not IL-1, IL-2,tumor necrosis factor a, or GM-CSF as critical componentsin the generation of these macrophages. However, additionof IFN-g to unstimulated macrophage cultures was insuffi-cient to reactivate virus. These results indicate the impor-tance of a specific moncyte stimulus in the generation ofa unique HCMV permissive macrophage phenotype as

well as why virus is commonly reactivated in transplant pa-tients. © 1998 Academic Press

Human cytomegalovirus (HCMV) is an opportunis-tic virus that has evolved to coexist with immunolog-ically healthy individuals. The importance and theinterest of HCMV as a pathogen has increased overthe past two decades as a result of the escalation inthe number of patients undergoing immunosuppres-sive therapy following organ or bone marrow trans-plantation, as well as with the increasing amount ofAIDS patients. HCMV is a member of the humanherpesvirus family, which includes herpes simplexvirus type 1 (HSV-1), herpes simplex virus type 2(HSV-2), varicella zoster virus (VZV), Epstein–Barrvirus (EBV), human herpesvirus 6 (HHV-6), and thenew members: human herpesvirus 7 (HHV-7) andthe Kaposi’s associated herpesvirus, human herpes-virus 8 (HHV-8). The prevalence of HCMV, similar toother herpesviruses, is ubiquitous worldwide with anestimated infected population of 70 –100% (1). Gen-erally, primary HCMV infection occurs during child-hood and results in lifelong persistence of the virusin a latent state. Latent infection is defined as thepersistence of viral genome without the ability todetect the virus in tissue cultures or secretions byconventional virus isolation techniques. Only a mi-nority of genes, if any, are expressed in cells thatharbor latent virus. Although the cellular latent res-ervoir of several of the herpesviruses has beenknown (i.e., neuronal cells for HSV-1, HSV-2, andVZV, and B cells for EBV), until recently the latentsite of HCMV remained elusive.

1 To whom correspondence should be addressed. Fax: (503) 494-2441. E-mail: [email protected].

METHODS: A Companion to Methods in Enzymology 16, 126–138 (1998)

Article No. ME980650

126 1046-2023/98 $25.00Copyright © 1998 by Academic Press

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REPLICATION OF HCMV

The herpesviruses have been grouped together basedon similar virion architecture. Mature HCMV virionsare composed of a core containing a linear viral doublestranded DNA genome in the form of a “toras” (2),which is enclosed within a 100-nm icosahedral capsid(20-sided body), consisting of 162 capsomers (penta-mers or hexamers) (3). The capsid is coated with ategument/matrix structure, which is surrounded by alipid bilayer envelope containing a number of viralglycoproteins as well as host cell derived proteins. Ma-ture virions range in size from 150 to 200 nm. TheHCMV genome is the largest of the known herpesvi-ruses with a DNA molecule equivalent to 230 kilobasepairs or a molecular weight of 150–155 3 106 (4–6).The HCMV genome is composed of two unique do-mains, the unique long (UL) and unique short (US),which are flanked by internal (IRS and IRL) and termi-nal (TRS and TRL) repetitive sequences. By convention,open reading frames (ORF) are designated with the UL,US, TR, or IR prefixes, depending on their location inthe genome. During HCMV DNA replication the USand UL components are capable of inversion (6). There-fore, four isomers of DNA molecules exist as a result ofthe orientations of the US and UL components (7, 8).The HCMV genome encodes over 200 ORFs of whichonly a small number have been characterized (9).

The replication cycle of HCMV is slow in comparisonto HSV, requiring 48–72 h to yield detectable levels ofprogeny virus (10). Infection of a permissive cell withHCMV leads to an ordered expression of viral genes.According to the appearance of viral mRNA or proteinsin the infected cell, the HCMV genes have been dividedinto three different categories: immediately early (IE),early (E), and late (L) genes (11, 12). Studies of thethree kinetic classes of genes suggest that HCMV rep-lication proceeds along a regulated transcription pro-gram in which each phase controls the progression tothe next phase. Transcription of IE genes proceeds inthe presence of protein synthesis inhibitors such ascycloheximide or anisomycin. Generally, the IE pro-teins are nonstructural nuclear proteins, which tran-scriptionally regulate both homologous and heterolo-gous promoters (10). A diagram of the life cycle ofHCMV is shown in Fig. 1.

The early phase of HCMV infection initiates approx-imately 4–8 h postinfection and proceeds until theonset of viral DNA synthesis, at approximately 24 hpostinfection (13). Generally, the HCMV E genes aredefined as those genes that are transcribed in the pres-ence of HCMV DNA polymerase inhibitors such asphosphonoacetic acid, gancyclovir, or foscarnet. Ap-proximately 75% of the HCMV genome is transcribedduring the early phase of infection and results in theproduction of mainly nonstructural proteins involved

in viral DNA replication such as the HCMV DNA poly-merase (10). The L HCMV genes which encode mainlystructural viral proteins such as proteins involved incapsid formation, tegument proteins, and envelope gly-coproteins are expressed 24–48 h postinfection (10).Some of these genes are transcribed at low levels priorto viral DNA replication, but most of the late proteinsrequire prior viral DNA synthesis for transcription.Posttranscriptional mechanisms have been suggestedto be responsible for the delay in the appearance of lateproteins (14, 15). Infectious virus particles are pro-duced in the cell approximately 48 h postinfection.

IN VITRO SYSTEMS TO EXAMINE REPLICATIONOF HCMV

Immunohistochemical analyses of HCMV infectedtissues have identified a variety of cell types such asepithelial cells, endothelial cells, macrophages, fibro-blasts, neuronal cells, smooth muscle cells, and hepa-tocytes as targets of viral infection (16–39). Only a fewcell lines are permissive for HCMV replication. There-fore, primary cells established from some of the cell

FIG. 1. The life cycle of HCMV.

127HUMAN CYTOMEGALOVIRUS IN PRIMARY MACROPHAGES

lineages described above have been utilized to examineHCMV replication in vitro. The prototype cell forgrowth of virus is the fibroblast, which produces hightiters of infectious virus after in vitro infection. Otherprimary cells such as macrophages, endothelial cells,and epithelial cells have been shown to be HCMV per-missive. However, their rate of viral production is con-siderably less than that of fibroblasts (40, 41). Recentadvances in the growth of these cell types have re-vealed that HCMV grows efficiently in macrophagesand endothelial cells depending on the state of differ-entiation or, as in the case of endothelial cells, theorgan of cellular origin (21, 41–46). Cellular differen-tiation has also been shown to be of importance forHCMV replication in a few cell lines. For example, themonocytic cell line THP-1 is permissive for HCMVreplication only after differentiation into monocyte/macrophages by stimulation with 12-O-tetradecanoy-phorbol 13-acetate (47). Similar results of restrictedreplication of HCMV in undifferentiated cells was ob-served in the teratocarcinoma cell line N-Tera-2 (48).These data strongly suggest a mechanism of persis-tence of HCMV in the peripheral blood that is indepen-dent of HCMV lytic gene expression and that the initialphases of lytic gene expression in monocytes can beinduced by differentiation of these cells to monocyte-derived macrophages (49). Since HCMV has beenshown to infect but not replicate in undifferentiatedcells, these observations also indicate that cellular dif-ferentiation events are linked to HCMV activation.

IMPORTANCE OF MACROPHAGES IN BIOLOGYOF HCMV

Although transmission of latent HCMV early wasshown to occur through transfusion of blood products,bone marrow grafts, and solid organs (1, 50–52), thelatent cellular reservoir was difficult to identify. Anumber of animal models have been established tounderstand mechanisms involved in latency and reac-tivation of CMV (53–57). In murine organ transplantmodels, reactivation of murine cytomegalovirus (MCMV)is influenced by the state of immunosuppression andhistoincompatibility between the donor and the recip-ient (53–57). In MCMV latently infected mice, thespleen, kidneys, and bone marrow are importantsources of virus (58–60). In these animals, activationof virus replication in latently infected animals occursthrough either intraperitoneal injection of thioglycol-late (61) or allogeneic stimulation (60, 62). In addition,the peripheral blood of latently infected animals hasbeen demonstrated to be a reservoir of virus sinceallogeneic stimulation resulted in activation of MCMVreplication (60, 62). Thus, while studies of human cellshave implicated monocyte/macrophages as reservoirs

for latent virus, these animal studies have suggestedthat T cells, B cells, or macrophages may be potentialsites for viral latency (58–60).

A prominent source of HCMV during acute diseaseare cells in the peripheral blood (17). HCMV is knownto be transmitted from healthy carriers to patientsthrough transfusion of the leukocyte fraction of theperipheral blood (51, 63–71). Although HCMV can beisolated from both the mononuclear and polymorpho-nuclear cell fractions obtained from HCMV infectedindividuals (30, 72), only a low percentage of virusinfected blood cells are detected in these individuals.These observations contrast the high frequency ofHCMV positive leukocytes in biopsies of transplantedkidneys and liver tissues during viral disease (73).These results suggested that cells in the peripheralblood may be a latent reservoir of HCMV and thatstimulation of these cells to respond to inflammatoryand/or allogeneic events may trigger activation of thevirus. Examination of separated cell populations fromthe peripheral blood mononuclear cells obtained fromHCMV seropositive individuals has identified mono-cytes as the predominant infected cell type (38). Theseobservations correlate with the identification of themacrophage as the predominant HCMV infected infil-trating cell in tissues (33) and suggest that themonocyte-derived macrophage (MDM) would be anideal vector for trafficking virus to different tissues.Although a number of viruses in addition to HCMVsurvive and persist in macrophages, the mechanismsinvolved in intracellular survival are unknown (74,75). The optimal situation for both virus and host cellsurvival would be for viral accumulation to occur with-out killing the host cell. Therefore, after HCMV infectsthe permissive MDM, HCMV persistence would re-quire a strategy for interacting with the macrophagethat did not expose progeny to the immune system orcompletely debilitate the macrophage. The questionbecomes, can HCMV use macrophages as a TrojanHorse similar to lentiviruses (74, 75)?

A primary mechanism through which HCMV avoidsdetection by the immune system is the ability of the virusto infect monocytes or monocyte precursor cells and re-main for the most part transcriptionally inactive. Second,in differentiated macrophages the virus has developed amechanism to remain compartmentalized in the cellwithout release of infectious virus. Immunofluorescenceanalysis has demonstrated accumulation of viral proteinsin discrete cytoplasmic vacuoles which were not associ-ated with plasma membrane. These intracytoplasmicvacuoles have been proposed to contain sequesteredHCMV. In support of this hypothesis, human immuno-deficiency virus (HIV) also appears to accumulate inMDM vacuoles (76). Ultrastructural studies of HIV-infected MDM indicate that these vacuoles are derivedfrom the Golgi complex (76). Similarly, we have found

128 SODERBERG-NAUCLER, FISH, AND NELSON

that the Golgi marker mannosidase II is associated withHCMV infected MDM vacuoles (77). However, neithermarkers for early endosomes such as rab 5 and trans-ferrin receptor nor late endosomes or lysosomes such aslamp 1 and lamp 2 colocalized with these viral structures(77). The latter observation suggests that HCMV evadeshost cell-mediated degradation induced by fusion ofHCMV containing vacuoles with lysosomes, which coulddegrade infectious virus.

During the latter stages of HCMV replication in MDM(13–15 days postinfection) the normal structure of theGolgi is disrupted without obvious cellular cytopathiceffect (77). Examination of infected MDM structural ele-ments revealed the progressive loss of an ordered micro-tubule network, which correlated with disruption of theGolgi apparatus (77). Since the microtubule networkmaintains placement of the Golgi and regulates intracel-lular vesicle movement, one would expect a cause andeffect relationship between the disaggregation of the mi-crotubules and disruption of the Golgi apparatus. How-ever, when the microtubule network is stabilized withtaxol disruption of the Golgi network still occurs. Theability of HCMV to disrupt microtubule structure or for-mation may serve two purposes. First, the loss of micro-tubules would block the release of virus by preventing thetrafficking of intracytoplasmic vacuoles containingHCMV to the plasma membrane. The fusing of vacuolescontaining HCMV with the PM may result in cytopathiceffect. Thus, HCMV has developed mechanisms to repli-cate in MDM that allows cellular survival as the cellsmigrate through tissues as well as protection from theimmune system.

PRIMARY MONOCYTE/MACROPHAGE SYSTEMSTO EXAMINE HCMV REPLICATION

Macrophages are a heterogenous population of ter-minally differentiated myeloid lineage cells. Macro-phage precursors include monocytes, monoblasts andpromonocytes. A number of different macrophage phe-notypes have previously been described that are func-tionally different depending on the method of isolationand stimulation (78). Generally monocyte cultures areobtained by plating PBMC on plastic culture dishes for1–20 h. The monocytes will adhere to the plastic sur-face and nonadherent cells are thereafter removedfrom the culture. The process of adherence activatesthe monocytes and by definition these cells are consid-ered macrophages. The purity of these cultures gener-ally varies from 50 to 80% and provide an easy methodof monocyte enrichment. Elutriation of PBMC by cen-trifugation has also been utilized to obtain monocytepopulations but usually these techniques result in1–10% contamination with other blood cell types. How-ever, positive selection for monocytes or negative selec-

tion of other populations of cells produces the mosthomogeneous cultures of macrophages. A number ofmethods have been utilized to stimulate adhered mac-rophages into other differentiated forms of these cells.The method of choice depends on what phenotype ofmacrophage will be studied. For example, macro-phages can be activated by a number of different cyto-kines such as IL-1, IL-2, IL-4, IL-6, GM-CSF, TNF-a,IFN-g, or other molecules such as LPS, PAF, PGE2. Inregards to HCMV, several primary monocyte/macro-phage systems have been established to examinemechanisms of HCMV activation and replication inmacrophages (24, 43, 72, 79–81). These studies havedemonstrated that the ability of the virus to replicatein macrophages is dependent on the state of cellulardifferentiation. Infection of unstimulated monocytesresulted in either the lack of viral gene expression orreplication restricted to immediate early gene products(43). The block in HCMV expression in unstimulatedmonocytes was not at the level of virus entry and fusionwith the cell, but rather at the level of transcriptionalor posttranscriptional events (41, 43).

HCMV INFECTION OF MACROPHAGESGENERATED BY CONCANAVALIN ASTIMULATION OF PERIPHERALBLOOD MONONUCLEAR CELLS

The best characterized in vitro primary monocyte/macrophage system in which differentiated macrophagesare fully permissive for HCMV is based on cocultivationof monocytes with concanavalin A (Con A) stimulatedautologous nonadherent cells for a defined period of timeto allow for monocyte stimulation (40, 43). Stimulatedmonocytes differentiate into morphologically distinctphenotypes of MDM (Con A-MDM), which include multi-nucleated giant cells (MNGC) (Fig. 2). The MDM at thisstage can be maintained for long periods of time withoutthe addition of exogenous cytokines, and these macro-phages are permissive for HCMV infection. An exampleof an infected Con A-MDM is demonstrated in Fig. 3. Wehave found that HCMV infection of MDM is nonlytic (40,43). Virus produced by HCMV infected Con A-MDM isexclusively cell associated (40, 43, 77). Classically, HCMVinfection of HF cells causes cellular fusion and lysis,resulting in the release of virus into culture supernatant.In contrast HCMV is not found in supernatants of in-fected Con A-MDM cultures (40, 43, 77). Furthermore,the HCMV replication cycle is significantly delayed inMDM relative to HF cells (40, 43). In Con A-MDM IEantigen expression initiates at 12 h postinfection, peak-ing at 3 days postinfection, while gB antigen expressionoccurred at 5 days postinfection, peaking at 7–13 dayspostinfection (40). In contrast, expression of these HCMV


antigens in human fibroblasts initiated at 1–5 and 48 hpostinfection for IE and gB, respectively. Viral productionpeaked at 12 days postinfection in Con A-MDM culturesand 4–5 days postinfection in HF cells. The delay in viralexpression may be a unique adaptation of HCMV to per-sist in MDM, since the retarded accumulation of HCMVgene products would prevent the rapid increase in viralfactors which may be toxic to the cell.

CELLULAR AND CYTOKINE FACTORSNECESSARY FOR GROWTH OF HCMVIN CON A-MDM

In the HCMV permissive Con A-MDM, the formationof multinucleated giant cells (MNGC) correlated with theability of virus to infect macrophage cultures. Investiga-tion of the cellular components of the nonadherent PBMCpopulation necessary for the generation of MDM by neg-ative selection revealed that HCMV replication in mac-rophages was dependent on CD8-positive T lymphocytes(45). A schematic diagram is shown in Fig. 4, whichdemonstrates the technical procedure of the negative se-lection experiments. Analysis of the cytokines necessaryfor generation of HCMV permissive Con A-MDM re-vealed that neutralization of IFN-g and TNF-a, but notIL-1, IL-2, TGF-b, or GM-CSF, resulted in a dramaticreduction of HCMV replication. These observations sug-gested that production of IFN-g and TNF-a by activatedCD8-positive T lymphocytes stimulate the generation of

HCMV permissive Con A-MDM. Therefore, recombinantIFN-g and TNF-a were added independently to monocytecultures to examine the effects of the individual cytokineson the formation of HCMV permissive MDM. While theeffect of these cytokines on the formation of MNGC andthe number of infected cells varied between the differentdonors, addition of TNF-a and IFN-g independently tomonocyte cultures consistently resulted in the significantproduction of viral progeny (45). These observations in-dicate the importance of TNF-a and IFN-g on the forma-tion of HCMV permissive macrophages.

The macrophage activation pathway induced by IFN-gand TNF-a was specific, since IL-1, IL-2, TGF-b, or GM-CSF were not critical components in the production ofHCMV permissive Con A stimulated MDM. Previousstudies have demonstrated that the secretion of IFN-g byCon A stimulated CD81 T lymphocytes is mediated bythe binding of Con A to at least three cell surface mole-cules (T200, LFA-1, and Lyt-2), even in the absence ofantigen (82). Con A receptors are believed to be inti-mately involved in antigen recognition and effector func-tions of CD81 T lymphocytes (83), since these cells ap-pear to produce IFN-g in response to specific interactionswith their target cells (82–86). Similarly, we demon-strated that direct interactions between CD81 T lympho-cytes and macrophages were crucial for macrophage dif-ferentiation in the Con A-MDM system, since antibodiesto HLA class I blocked HCMV replication in infectedMDM. However, cell free supernatants from Con A stim-ulated PBMC or recombinant IFN-g and TNF-a weresufficient to produce the HCMV permissive MDM. These

FIG. 2. A phase microscopy photograph of Con A-MDM at 3 days (A) and 8 days (B) poststimulation (original magnification, 3200).


observations suggest that direct contact between mono-cytes and T lymphocytes resulted in the production ofIFN-g and TNF-a in the Con A mediated production ofHCMV permissive MDM. While IFN-g and TNF-a pos-sess antiviral properties, addition of these cytokines topermissive MDM cultures did not affect the production ofHCMV. Thus, rather than inhibiting replication ofHCMV, IFN-g, and TNF-a specifically induce differenti-ation of monocytes into HCMV permissive MDM, whichwere resistant to the antiviral effects of these cytokines.

HCMV INFECTION OF MACROPHAGESGENERATED BY ALLOGENEICSTIMULATION OF PBMC

Since HCMV frequently reactivates in recipients ofallogeneic blood transfusions or organ and bone mar-row transplant patients, differences in HCMV replica-tion were examined in macrophages generated throughallogeneic stimulation of PBMC (Allo-MDM) in com-parison to Con A-MDM. HCMV infection of Allo-MDMat an m.o.i. of 10 resulted in a rapid lytic infection ofcells (Fig. 5). Cytopathic effects (CPE) were first detect-able at 3 days postinfection and complete lysis of thecells were observed by 7–10 days postinfection. In con-trast, HCMV infection of Con A-MDM was nonlytic at

FIG. 3. HCMV in vitro infection of Con A-MDM. Con A-MDM wereinfected at 7 days poststimulation with HCMV and cells were fixed andstained for the presence of IE (red) and pp65 tegument protein (green)at 8 days postinfection. Virus accumulates in cytoplasmic vacuoles ininfected cells (original magnification, 3480). (Reproduced here in blackand white. See special color plate section for reproduction in color.)FIG. 6. Reactivation of latent HCMV in Allo-MDM. Allo-MDMwere established as described under Methods from two healthy blooddonors. Reactivation was detected at 20 days after allogeneic stim-ulation of cells. Cells were fixed and stained for the presence of IE(red) and pp65 tegument protein (green) (original magnification,3200). (Reproduced here in black and white. See special color platesection for reproduction in color.)

FIG. 4. The method for negative selection of specific cell popula-tions from PBMC. As described under Methods, freshly isolatedmononuclear cells are stained with monoclonal antibody directedagainst specific PBMC populations to be removed from the mix. Cellswhich react with antibody are removed prior to plating with micro-magnetic MACS beads coated with rat antibody directed against themouse antibody.


intervals up to 90 days postinfection at an m.o.i. . 10.These observations suggested a functional difference inthe capacity of Allo-MDM to support HCMV replicationin comparison to the Con A-MDM.

The kinetics of HCMV replication in Allo-MDM wasrapid and large quantities of virus were found in boththe cellular and extracellular fractions. In addition,greater than 50% of the Allo-MDM expressed both IEand gB antigens at 12 days postinfection, in contrast to,10% of cells expressing viral antigens in the ConA-MDM cultures. Furthermore, the kinetics of expres-sion of the early–late structural protein gB correlatedwith the rapid production of virus within Allo-MDM.These characteristics are similar to viral replication in

fibroblasts and suggest that the allogeneically drivendifferentiation process, which results in the differenti-ation of a specific macrophage phenotype, ensures amore efficient replication cycle of HCMV in comparisonto mitogenically differentiated MDM.

CELLULAR AND CYTOKINE FACTORSNECESSARY FOR GROWTH OF HCMVIN ALLO-MDM

Activation of HCMV in vivo frequently occurs follow-ing allogeneic organ and bone marrow transplantation

FIG. 5. Phase microscopy of an HCMV infected Con A-MDM and Allo-MDM. HCMV in vitro infection of Allo-MDM at an m.o.i. of 10 resultsin the lysis of cells by 8 days postinfection (D), while Con A-MDM are unaffected by viral infection (B). Uninfected control Con A-MDM (A)and Allo-MDM (C) are shown (magnification, 3160).


as well as bacterial infections. In addition, elevatedlevels of both IFN-g and TNF-a have been found in thesera of patients with HCMV disease (87–91). Duringan allogeneic immune-mediated process that involvesthe activation of T cells and the production of thesecytokines, latently infected monocytes, which havebeen recruited to tissue sites and exposed to T cells,may differentiate into HCMV permissive macro-phages. To examine the cytokines involved in the acti-vation pathway of allo-MDM, these cells were infectedwith HCMV in vitro. Depletion of either CD41 or CD81

T cells from the PBMC before challenge of virus re-sulted in a 90% reduction in the number of Allo-MDMexpressing IE proteins as well as a 3–4 log decrease inthe production of virus (92). The addition of antibodiesdirected against HLA class I or HLA class II to PBMCprior to the allogeneic reaction also resulted in a sig-nificant decrease in virus production and suggests thatactivation of both T cell subsets and the sequentialproduction of cytokines are necessary for the specificdifferentiation of these cells. In order to determine if aspecific cytokine mediated the formation of HCMV per-missive Allo-MDM, polyclonal antibodies with neutral-izing activity to IL-1, IL-2, TNF-a, TGF-b, and IFN-g,were added separately to Allo-MDM cultures. Neutral-ization of IL-2 and IFN-g, but not IL-1, TNF-a, orTGF-b, within HCMV in vitro infected Allo-MDM cul-tures resulted in an 85–90% reduction in cells express-ing IE antigen and a 3 log reduction in the productionof infectious virus. The requirement of IL-2 for thedevelopment of HCMV permissive Allo-MDM corre-lates with the necessity of CD41 T cells in this system.Therefore, these experiments suggest that while CD81

T cells and the production of IFN-g were critical ele-ments in both the Con A-MDM as well as the Allo-MDM system, the activation of CD81 T cells by Con Aor by IL-2 produced by CD41 T cells was different.

REACTIVATION OF LATENT HCMVIN ALLO-MDM

The unusual growth of HCMV in the Allo-MDM sug-gested that this mechanism of macrophage activationmay induce reactivation of HCMV. Therefore, HCMVreactivation in vitro was examined in macrophagesobtained from seven consecutive seropositive (or PCRpositive) healthy donors through allogeneic stimula-tion of PBMC (44). Allo-MDM cultures were estab-lished by mixing PBMC from two healthy blood donorsas described below. HCMV replication was detected inallo-MDM cultures at 17 days poststimulation and vi-rus was recovered after long-term culture. Failure todetect HCMV transcripts and protein in the Allo-MDMprior to day 17 indicates that the virus is latent and notpersistent in this cell population. The frequency of

Allo-MDM containing HCMV proteins was 1/1000 to12/1000 (0.1–1.2%) when viral antigens were first de-tected in each culture. By day 60 poststimulation, be-tween 2 and 25% of the cells expressed HCMV proteinsin the different Allo-MDM cultures (Fig. 6). The highnumber of infected cells in some of the cultures at thisinterval most likely reflects reinfection of Allo-MDM invitro with reactivated virus.

To identify the cell type that reactivated HCMV,Allo-MDM were analyzed by flow cytometry for theexpression of specific cell surface markers expressed onT and B cells, NK cells, myeloid cells, and dendriticcells. Control analyses were performed with freshmonocytes and with dendritic cells generated by stim-ulation of monocytes with recombinant IL-4 and GM-CSF (93). Cells staining for T, B, and NK cell markerswere absent from the adherent cell population. TheAllo-MDM demonstrated a remarkably uniform sur-face expression of the monocyte/macrophage markersCD14 and CD64. Surprisingly, expression of the den-dritic cell markers CD1a and CD83 were also observed.In contrast, control dendritic cells expressed CD1a andCD83, but were negative for CD14, as previously re-ported (93). High levels of HLA class II, HLA class I,CD13, VCAM-1, ICAM-1, and CD40 were observed onthe Allo-MDM as well as on monocytes and dendriticcells. The presence of dendritic cells markers on someAllo-MDM suggested that these macrophages were re-lated to dendritic cells. Therefore, dendritic cells gen-erated by treatment of monocyte enriched cultureswith IL-4 and GM-CSF were examined for their capac-ity to support HCMV production. These cells expressedthe dendritic cell markers CD1a and CD83, but not themacrophage markers CD14 and CD64. HCMV infec-tion of these cultures was nonproductive. In addition,stimulation of dendritic cells with TNF-a and IFN-gdid not induce cellular permissiveness to HCMV infec-tion. These observations indicate that the Allo-MDM isa distinct macrophage phenotype which is completelydifferent from dendritic cells. Thus, these observationsdemonstrate that PBMC harbor latent HCMV thatreactivates in a myeloid lineage cell upon allogeneicstimulation. Further characterization of the cytokinefactors necessary for reactivation of latent HCMV inAllo-MDM revealed that neutralization of IFN-g butnot IL-1, IL-2, TNF-a, or GM-CSF in the cultures earlyduring the differentiation process prevented reactiva-tion of latent HCMV. These results imply that a spe-cific macrophage differentiation pathway, which maybe dependent on the production of IFN-g, is a keyelement in the reactivation of HCMV. A model forreactivation of HCMV in Allo-MDM is shown in Fig. 7.In summary, reactivation of latent HCMV exclusivelyoccurs in specific macrophages, which suggests theimportance of a specific monocyte stimulus in the re-activation of latent HCMV. These observations have


important implications for the understanding of mech-anisms for viral latency and persistence in the humanhost.

CONCLUSIONS

The development of an in vitro system that can beused to examine different aspects of HCMV latencyand reactivation in the human host represents signif-icant progress in the field of HCMV. The initial studiesin this system that have been performed in our labo-ratory address different aspects of immune activationas a triggering factor for reactivation of latent virus.Experimental evidence for immune activation ofHCMV from the peripheral blood of healthy donors hasimportant clinical implications. While control ofHCMV replication is generally well managed by theimmune system in immunocompetent individuals,HCMV reactivation within the blood or donor organ inimmunocompromised patients frequently results inprofound disease. Since blood transfusion is commonlyperformed over histoincompatibility barriers, reactiva-tion of HCMV through allogeneic stimulation of PBMCwould be expected if either the donor or the recipientwere virus positive. Even more important is the clinical

relevance of the allogeneic reactivation of HCMV fororgan and bone marrow transplant patients. HCMVinfection has been implicated in acute rejection in or-gan transplant patients and acute graft versus hostdisease (GVHD) in bone marrow transplant patients(94). The allogeneic activation of cells in solid organs orbone marrow grafts may provide a microenvironmentconducive to cellular differentiation and reactivation ofHCMV. Immune activation of myeloid-lineage cellsthrough allogeneic stimulation by T cells provides animmunologic stimulus that facilitates reactivation ofHCMV. Thus, the allogeneic activation of residual in-fected leukocytes in the solid organ or bone marrowrecipient tissue may be the primary mechanism of viralactivation in transplant patients. Furthermore, iatro-genic immunosuppression in the transplant settingwould exacerbate the severity of HCMV disease. Inter-estingly, transplant patients commonly reactivateHCMV between 1 and 4 months posttransplantation(95), which correlates with the time interval requiredto reactivate infectious virus in vitro by allogeneicstimulation of PBMC from healthy individuals. In ad-dition, initiation of an immune response by otherpathogens may also result in reactivation of latentvirus. This situation is exemplified by the increasedincidence of HCMV disease following bacterial infec-tions in different groups of patients (96) as well as inHIV infected individuals who experience other oppor-tunistic infections. The hypothesis of viral activationhas also been implied for other viruses such as HIV,HHV-8, and HHV-6. For example, allogeneic stimula-tion of HIV infected macrophages induces high levels ofviral expression (80) and conjugates of dendritic cellsand T cells have been suggested to be important sitesfor the replication of HIV (97).

Whether HCMV latency can be established exclu-sively in monocytes or also in immature hematopoieticprogenitor cells remains unknown. HCMV infectsmonocytes, and viral latency is established in thesecells with the capacity of viral reactivation upon im-munologic stimulation and differentiation of the mono-cyte into a specific phenotype of macrophage. As dis-cussed above, macrophages derived from monocytes bydifferent methods demonstrate a remarkable differ-ence in their ability to replicate HCMV. Furthermore,stimulation of monocytes into monocyte derived den-dritic cells does not result in reactivation of virus, andonly a restricted replication can be demonstrated in invitro infected dendritic cells derived from monocytes.Thus, the difference in virus replication on in vitroinfection of different cell phenotypes derived frommonocytes is reflected in the ability of latent virus toreactivate in a specific macrophage phenotype. Al-though there is not a specific marker known today thatcan be used to characterize specific macrophage phe-notypes, the difference in the ability of the virus to

FIG. 7. A model for the reactivation of HCMV in PBMC. In thismodel for the reactivation of latent HCMV from PBMC, latentlyinfected monocytes in the peripheral blood are activated throughimmune response by an allogeneic reaction or by an immune re-sponse against other pathogens. In this response CD41 and CD81 Tcell contact with the monocyte through the HLA class I and HLAclass II pathways is required for the generation of the HCMV per-missive Allo-MDM. Production of IL-2 by the CD41 T cells stimu-lates the CD81 T cells to produce IFN-g, which induces the mono-cytes with latent HCMV to differentiate into Allo-MDM, whichactivates the production of infectious virus.


grow in different macrophages clearly demonstratesthe functional differences between these cells. Thus,HCMV replication may be used as a marker for differ-entiation of monocytes. Most likely the virus is depen-dent on the presence of a cellular factor that is onlypresent in certain differentiated myeloid cells. How-ever, whether the restriction in virus replication de-pends on cellular or viral factors remains to be proven.Elucidation of the cellular activation pathway of T cellrecognition that results in viral reactivation will leadus closer to the understanding of the basis for latencyand reactivation of HCMV. The different primary cellsystems described above may be useful as a tool toidentify the crucial components required for successfulvirus replication and may lead us to new targets forantiviral therapies.

METHODS

Isolation and Culture of Con A-MDM and Allo-MDMPeripheral blood mononuclear cells (PBMC) were iso-

lated from blood samples of healthy HCMV-seronegativeindividuals. PBMC were isolated by density gradient cen-trifugation on Histopaque (Sigma Chemical Co., St.Louis, MO) at 800g for 25 min. The PBMC band wascollected, washed twice in sterile phosphate-buffered sa-line (PBS) and once with serum-free medium, and resus-pended at 1.8 3 107 cells per ml in Iscove’s medium(GIBCO Laboratories, Grand Island, NY) containing pen-icillin (100 IU/ml), streptomycin (100 mg/ml, both fromGIBCO), L-glutamine (2 mM) and 10% human AB serum(Sigma). Cells were plated onto Lab-Tek 2 chamber slides(Nunc, Naperville, IL), Primaria 96-well plates, or Pri-maria 60-mm dishes (Becton Dickinson, Lincoln Park,NJ) at 37°C with 5% CO2. The cell cultures were stimu-lated with Con A (5 mg/ml, Sigma) for 16–20 h, whereaf-ter nonadherent cells were removed by three washes inserum free medium. The adherent MDMs were culturedin complete 60/30 medium (60% AIM-V medium (GIBCO)and 30% Iscove’s medium supplemented with 10% hu-man AB serum, penicillin, and streptomycin andL-glutamine in the same concentrations as describedabove). MDM cultures were fed every 3 days with 50%fresh medium and 50% conditioned medium clarified bycentrifugation.

For Allo-MDM cultures, equal numbers of cells fromtwo different blood donors at a cell concentration of1.8 3 107 cells per ml were mixed before plating onPrimaria dishes (Becton Dickinson). After 48 h of cul-ture at 37°C in 5% CO2, nonadherent cells were re-moved. The cultures were washed and fed with 50%spent media/50% fresh media every 3–4 days and keptin culture for up to 90 days poststimulation. Controlcell cultures were established by stimulation of PBMCfrom individual donors with Con A as previously de-

scribed (77). Day 1 of differentiation is defined as theday after the initial PBMC isolation and stimulationwith Con A or allogeneic stimulation.

Virus Infection of MDM Cultures

Recent patients isolates of HCMV or laboratorystrains were used to infect primary cultures of MDM.The clinical isolates were isolated from transplant pa-tients with HCMV disease and passaged through hu-man fibroblasts (HF). Cell free viral stocks were pre-pared from supernatants of HF cultures, frozen, andstored until use at 270°C. The clinical viral strainsused for infections of MDM cultures should be keptbelow passage 15 in HF cells. MDM cultures are gen-erally infected with HF supernatants at a m.o.i. of 1–10at 7 to 10 days after allogeneic stimulation or stimula-tion with Con A. For mock infection, cells should beexposed to media from uninfected HF cultures. Thecultures should be fed every third day, and collected forviral titer assays and immunocytochemistry at differ-ent time points after infection.

Immunocytochemistry

HCMV infected and mock infected MDM or dendriticcell cultures grown in 8-well chamber slides or in Pri-maria 96-well plates were collected at different timepoints after infection. The cells were washed in PBSand fixed in phosphate buffered 1% paraformaldehyde(PFA) or methanol/acetone (1:1) for 10 min at roomtemperature and permeabilized with 0.3% TritonX-100 in PBS. Cells were blocked with 10% normalgoat serum or 10% human AB serum in PBS for 30 minat room temperature and thereafter with antibodiesagainst different HCMV gene products in a 1:100 dilu-tion for 1–6 h at room temperature: antibodies againstthe major immediately early protein (rabbit anti-MIE(98), or gB (mouse anti-gB (UL55)). Both murine mono-clonal antibodies were a kind gift from Dr. William(University of Alabama, Birmingham) (99). Cells werewashed three times in PBS and binding of the primaryantibody was detected with a fluorescein isothiocyanate(FITC) conjugated goat anti-mouse or goat anti-rabbitantibody for 1–2 h at room temperature. Double immu-nocytochemistry for cell surface markers was performedon live cells before fixation and staining for the HCMV IEantigen was performed. Stained cells were washed inPBS and mounted in Slow Fade antifade kit (MolecularProbes Inc., Eugene, OR) to ensure minimal fluorescencefading. Fluorescence positive cells were visualized on anupright or inverted Leitz fluorescent microscope and thenumber of infected cells were counted.

PCR Analysis

For PCR analysis, cell samples were collected atdifferent time points postinfection by scraping andDNA and RNA were prepared with QIAGEN blood and


cell culture DNA kit and RNeasy kit, respectively, ac-cording to the manufacturer’s instructions. HCMV spe-cific primer pairs were used in nested RT-PCR reac-tions detecting IE and pp150 RNA (100). As a positivecontrol for the presence of DNA or cDNA in each sam-ple primers detecting glucose-6-phosphatase dehydro-genase (G6PD) were used for each sample (100). Theprimers used for PCR analysis for HCMV IE (exon 1and exon 2 of the IE gene) and G6PD are spanning anintron and yield different band sizes for DNA andRNA, whereas the pp150 gene does not contain anintron and yield DNA and RNA PCR products of thesame size. The PCR products were visualized by directgel analysis on a 1% agarose gel.

Virus Titer Assays

At different days postinfection, supernatants fromMDM and dendritic cell cultures were collected, andcells were harvested by scraping adherent cells intoDulbecco’s modified Eagle’s medium (DMEM) contain-ing 2% fetal bovine serum (FBS), 2 mM L-glutamine,100 IU/ml penicillin, 100 mg/ml streptomycin. Super-natants or sonicated MDM cells were plated ontomonolayers of HF cells at subconfluency. After an ini-tial 24 h of viral adherence at 37°C, cells were washedtwice in medium and overlaid with DMEM mediumcontaining 10% FBS, 2 mM L-glutamine, 100 IU/ml ofpenicillin, 100 mg/ml of streptomycin, and 0.5% auto-claved SeaKem agarose (Sigma). The cultures wereincubated for 14 days, with feeding every fourth day.The cells were fixed with 25% formaldehyde in PBS for15 min, stained with a 0.05% solution of methyleneblue, and plaques counted (101). To obtain viral strainsfrom the individual donors, HF cells infected as de-scribed above were scraped into DMEM medium every7–10 days, sonicated, and plated onto new monolayersof HF cells at subconfluency. Plaques were generallyobserved in the different cultures at 10–16 weeks afterthe initial infection.

Flow Cytometry

A fluorescence-activated cell analyzer (FACScalibur;Becton Dickinson, San Jose, CA) producing 15 mW oflight at 488 nm was used for all analyses. The fluores-cence signal from 104 cells from samples before andafter negative selection was obtained. Data were han-dled with logarithmic amplification and fluorescenceintensity was displayed on a 1024-channel, 4-decadelog scale delineated in arbitrary log units. Histogramsdisplaying the log fluorescence of FITC (FL1) of thesamples before and after negative selection were gen-erated, and the percentage of positive cells was esti-mated by setting the level for positive cells not toinclude the background staining of uninfected cells inthe negative control.

Negative Selection of Blood Cells Priorto Concanavalin A Stimulation

In order to obtain CD41 or CD81 T lymphocyte or Bcell- or NK cell-depleted MDM cultures, the Mini MACSsystem (Miltenyi Biotec, Bergish Gladbach, Germany)was used for negative selection of the respective cell type.Freshly isolated PBMC were stained with monoclonalantibodies directed against the following cell-type specificmolecules: anti-human Leu-3a (CD4, T lymphocytes),anti-human Leu-2a (CD8, T lymphocytes), both from Bec-ton Dickinson, anti-human CD19 (DAKO-CD19, B Lym-phocytes), anti-human CD56 (DAKO-CD56, NK cells),and for negative controls anti-human CD31 (DAKO-CD31, endothelial cells), all from Dakopatts (Glostrup,Denmark) or mouse IgG1 (Fc, R&D Systems, Minneapo-lis, MN). In 500 ml serum free Iscove’s medium 1 3 108

cells were incubated with a titered excess of the respec-tive antibody at 4°C for 45 min. The cells were washedtwice in cold PBS and resuspended in 250 ml of MACSbuffer (PBS containing 5 mM EDTA and 0.5% BSA) andincubated with 160 ml MACS beads conjugated with ratanti-mouse IgG1 antibodies or rat anti-mouse IgG2a andIgG2b antibodies for 20 min at 4°C. Each MACS columnwas washed with 15 ml of MACS buffer before the addi-tion of the respective sample. PBMC coupled to MACSbeads were eliminated from the samples by flow throughthe column in a magnetic field under flow resistance.Each column was washed with 4 ml MACS buffer, andthe collected cells were washed twice in serum free me-dium, and resuspended in Iscove’s complete medium withthe addition of Con A (5 mg/ml) as described above. Smallaliquots of each sample before and after negative selec-tion were analyzed by flow cytometry to ensure satisfac-tory purity of each sample before the establishment ofeach MDM culture.

Blocking of HLA Class I and HLA Class II MoleculesTo block the interaction between T lymphocytes and

monocytes, monoclonal antibodies directed against con-stant regions of HLA A B C or HLA-DR (both from Im-munotech, Westbrook, ME) or isotype controls, mouseIgG2a or mouse IgG2b (both from R&D Systems), at aconcentration of 35 mg/ml were preincubated with 7 3107 cells in Iscove’s complete medium for 1 h at 4°C beforethe addition of Con A. The cells were washed after 16–20h of incubation to remove nonadherent followed by theaddition of complete 60/30 medium as described above.

Generation of Condition Medium from Con AStimulated PBMC

Supernatants from Con A stimulated PBMC collectedat 4 and 20 h poststimulation were filtered through a45-mm filter, and cell-free supernatants and transferredto fresh monocyte enriched cell cultures. At 7–9 days thecultures were infected with HCMV at an m.o.i. of 10.Cells were collected for virus titer assays by scraping at14 days postinfection as described for viral titer assays.


Neutralization of Lymphokines in MDM CulturesFor neutralization experiments, polyclonal neutral-

izing goat antibodies against human TNF-a, IL-1a,IL-2, TGF-b, or IFN-g (R&D Systems) were used toblock the respective lymphokine produced in MDMcultures. Antibodies were added to the cultures at thesame time as Con A, and were present in the culturesfor 16–20 h poststimulation. Thereafter, nonadherentcells and antibodies in the cultures were removed bythree washes in serum-free medium, and the MDMcultures were cultured in complete 60/30 medium forup to 20 days.

Stimulation of Monocyte Enriched Cultureswith Recombinant Cytokines

Fresh PBMC at a concentration of 1.8 3 107 cells/mlwere enriched for monocytes by plastic adherence in Pri-maria dishes for 2 h at 37°C. Nonadherent cells wereremoved and the adherent monocyte cultures were stim-ulated with recombinant IFN-g (500 U/ml), IL-1 (2 ng/ml), or TNF-a (10 ng/ml) (all from R&D Systems) incomplete 60/30 medium. Parallel dishes were stimulatedwith Con A as described above. All the cultures wereinfected with HCMV at 9 days poststimulation at anm.o.i. of 10. Cells were collected for virus titer assays asdescribed elsewhere or fixed in 1% PFA, or methanol/acetone (1:1) for immunocytochemistry.

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