JOURNAL OF VIROLOGY, June 1994, p. 4017-4021 Vol. 68, No. 60022-538X/94/$04.00+0Copyright ©D 1994, American Society for Microbiology

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Human Cytomegalovirus Infection of the Monocyte/MacrophageLineage in Bone Marrow

E. J. MINTON,t C. TYSOE, J. H. SINCLAIR, AND J. G. P. SISSONS*Department of Medicine, University of Cambridge Clinical School, Cambridge CB2 2QQ, Unzited Kingdom

Received 7 September 1993/Accepted 21 February 1994

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthycarriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect withHCMV, it has been suggested that they may acquire HCMV during development in the bone marrow.Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers containendogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infectedby clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells becomedifferentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strainAD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry ofAD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMVand transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave thecirculation and undergo tissue-specific differentiation to macrophages.

Human cytomegalovirus (HCMV) is a ubiquitous pathogenand persistently infects over 50% of the normal population(12), but primary infection in normal individuals is usuallyasymptomatic, even though virus may be shed in body fluidssuch as urine and saliva (23). In contrast, primary infection orreactivation of endogenous virus in immunosuppressed indi-viduals, such as transplant recipients (25) or patients infectedwith the human immunodeficiency virus (7), may be severe andis sometimes fatal.HCMV may be transmitted by blood transfusion (15, 20);

and this may be prevented by leukocyte depletion, suggestingthat the virus is present in one or more types of bloodleukocyte. In general, it is not possible to culture HCMV fromnormal seropositive blood, although the virus can readily becultured from peripheral blood of patients with symptoma-tic HCMV infection (14). Consequently, normal peripheralblood leukocytes are unlikely to be productively infected withHCMV. Recently, we have shown that HCMV DNA is detect-able predominantly in the peripheral blood monocytes (PBM)of normal seropositive individuals (30, 31) but with little or noaccompanying lytic-gene expression (32). However, becausemonocytes are only transiently present in the circulation (34)and there is no evidence of free virus in the peripheral blood ofnormal subjects, it has been suggested that monocytes mayacquire HCMV earlier in their development, in the bonemarrow.

Previous work has suggested that bone marrow cells requiredifferentiation to express viral genes upon infection withHCMV (9, 21, 29). Similarly, only a very small number offreshly isolated PBM express immediate-early (IE) genesfollowing in vitro infection with HCMV (24, 26), and PBM

* Corresponding author. Mailing address: Department of Medicine,University of Cambridge Clinical School, Hills Rd., Cambridge CB22QQ, United Kingdom. Phone: 223-336844. Fax: 223-336846.

t Present address: Brigham and Women's Hospital, Boston, MA02115.

become fully permissive for productive infection only if stim-ulated to differentiate (13, 16). Also, in monocytic cell lines,repression of HCMV IE gene expression by cell-specific fac-tors also appears to be related to cellular differentiation (28,33). Thus it seems likely that cell differentiation may influencethe expression of HCMV genes in cells of the monocyte/macrophage lineage infected in vivo.

In the experiments described here, we demonstrate thatmonocytic cells at different developmental stages are efficientlyinfected with clinical isolates of HCMV but that viral geneexpression is absolutely dependent on the differentiation ofthese cells. In contrast, the AD 169 laboratory isolate ofHCMV inefficiently infected even differentiated monocytes,and this was, at least in part, due to the inability of AD169 toenter these cells. Finally, analysis of bone marrow progenitorscollected from seropositive individuals also shows that endog-enous HCMV DNA was present in colonies derived from thesecells, arguing that the HCMV genome is present in bonemarrow progenitors in vivo.

Results. (i) HCMV gene expression in cells of the monocytelineage requires differentiation. There have been a number ofreports showing that long-term-cultured bone marrow cells canbe productively infected with laboratory isolates of HCMV (9,19, 21) and that the cell types infected include stromal cells (1)as well as bone marrow cells of the monocyte/macrophagelineage (9, 19, 21). However, long-term bone marrow cultures(LTBMCs) contain a mixture of monocytes at different devel-opmental stages. Thus, the exact stage of development atwhich monocytes/macrophages in LTBMC infected withHCMV become permissive for viral gene expression is unclear.To address this, we obtained monocytic cells at different stagesof development by separation of CD34+ progenitor cells frombone marrow mononuclear cells with anti-CD34 immunomag-netic beads and by aspiration of defined colonies of immaturemonocytic cells grown from CD34+ progenitor cells in semi-solid medium in standard bone marrow progenitor assays (18).We then analyzed their permissiveness for expression of the

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TABLE 1. IEl expression in the monocyte lineage at differentdevelopmental stages 16 h after infection with HCMV

strains AD169 and M

% of cells positive for IEI

Developmental stage fluorescence" after inifection with:AD169 M

CD34' progenitor U U7-day monocyte colony U U14-day monocyte colony U <1PBM U <lDifferentiated monocyte U 50Pulmonary macrophage ND 15

' A minimum of 21111 cells were counted. U, undetectaible; ND, not donc.

HCMV major IEI protein by indirect immunofluorescencewith an IEI-specific monoclonal antibody (32), after infectionwith cell-free virus. In repeated experiments, no IEI expres-sion could be detected when CD34+ bone marrow progenitorcells were infected for 16 h with a clinical isolate of HCMVthat we have termed strain M (Table 1). Similarly, no evidenceof IEI expression was detected when 7-day-old monocytecolonies (monoblasts) were infected with isolate M of HCMVand fewer than 1% of cells showed evidence of IEI expressionwhen freshly isolated PBM were infected with this or otherclinical isolates (Table 1). In contrast, up to 50% of monocyte-derived macrophages expressed IEI 16 h after infection withclinical isolates of HCMV. These cells were induced to differ-entiate to monocyte-derived macrophages by being culturedfor 7 days in LTBMC medium (10) containing 5 x 10-7 Mhydrocortisone sodium succinate and 15% donor horse serum(which appeared essential for monocyte survival). Viral gHexpression was also detectable by indirect immunofluorescencewith a monoclonal antibody to gH (5) 72 h postinfection (datanot shown). If the same cells were harvested 7 days later,nearly 100% of them expressed IEI (data not shown). Thisdifferentiation protocol for PBM resulted in increased fre-quencies of IEI expression after shorter periods of culturecompared with other protocols (13, 16) and did not requireT-cell activation as previously described (13). As with previousreports (8), up to 15% of in vivo differentiated pulmonaryalveolar macrophages readily expressed IEI (Table 1) and gH(data not shown) antigens without preculture after infectionwith these clinical isolates of HCMV. In contrast, no IEIexpression was seen in cells at any stage of differentiation wheninfected with AD169 (Table 1). Consistent with this, infectiousvirus could also be detected by routine plaque assays withprimary human fibroblast cells, in the supernatants of culturedmonocyte-derived macrophages infected with isolate M butnot AD169 (Fig. 1). Our data demonstrate that HCMV geneexpression occurs only in differentiated monocytes/macro-phages and that recent clinical isolates may infect monocyticcells more efficiently than laboratory-adapted strains do, inagreement with other workers (9, 13, 16, 19, 21). We did notobserve appreciable IE (or late) gene expression in CD34-'bone marrow progenitors or immature monocytes culturedfrom these progenitors, consistent with these studies but incontrast to one recent report (17). This latter study used arecombinant HCMV carrying the lacZ gene under the controlof a major early promoter to analyze HCMV gene expressionin freshly isolated monocytes. However, identical experimentsin our laboratory with a similar lacZ recombinant virus (3)routinely showed P-galactosidase expression in monocyteseven after viral inactivation with UV; this was due to the

PFU/nil

0 4 8 12 16 20

Days post-infection

FIG. 1. Plaque assay of supernatants obtained from monocytesprecultured for 7 days after infection with AD169 and isolate M.Supernatants from infected mycloid cells were titrated onto F2002fibroblasts. Each point represents the arithmetic mean of three esti-mations. Symbols: A, supernatant derived from cells infected withAD169; 0, supernatant derived from cells infected with isolate M; *,supernatant derived from mock-infected cells.

inability to remove free 3-galactosidase from viral preparations(18a).

(ii) HCMV clinical isolates can enter cells of the monocyte/macrophage lineage more efficiently than laboratory strainsdo. Previous workers have shown that both peripheral bloodleukocytes and bone marrow cells are more likely to beinfected by low-passage clinical isolates of HCMV than bylaboratory strains, such as AD169, that have been extensivelypassaged in fibroblasts (9, 26, 27, 29). To exclude the possibilitythat the lack of IEl expression seen in differentiated mono-cytes after incubation with AD169 is due to the inability ofthese cells to permit virus entry, we used nuclear pp65 expres-sion, as previously reported (22), to determine whether HCMVvirions were able to enter monocyte precursor cells. As ex-pected, the lower matrix structural protein, pp65, could bedetected in the nuclei of fibroblasts within 3 h of inoculation ofAD169 (Fig. 2A). However, we were unable to detect pp65after incubation of in vitro differentiated monocytes withAD169 even at multiplicity of infection of 100 (Fig. 2C),although pp65 could be detected when these cells were incu-bated with the M clinical isolate (Fig. 2B). In addition, no pp65was detected in freshly isolated monocytes incubated withAD169 (data not shown). This relative inability of the AD169strain of HCMV to enter monocytes compared with that of anHCMV clinical isolate suggests that there are differencesbetween AD169 and the clinical isolate which affect themechanism of HCMV entry into cells of the monocyte lineage.We used the same approach to determine whether lack of

IEI expression in immature monocytic cells could also beexplained by the inability of clinical isolates to enter these cells.Both CD34+ cells and cells from 7-day-old monocyte colonies(monoblasts) showed nuclear pp65 staining after incubationwith isolate M (Fig. 2D and E), as did freshly isolated PBM

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FIG. 2. HCMV pp65 expression in cells incubated with HCMV for 3 h. Cells were counterstained with Evans Blue. (A) F2002 fibroblastsincubated with AD169; (B) monocytes cultured for 7 days and incubated with M; (C) monocytes cultured for 7 days and incubated with AD169(multiplicity of infection, 100); (D) CD34 cells incubated with isolate M (arrowhead indicates nuclear staining in a cell surrounded by residualCD34+ dynabeads); (E) cells derived from 7-day-old monocyte colonies (monoblasts) (arrowheads indicate nuclear staining); (F) freshly isolatedPBM incubated with isolate M. Magnification, X400.

(Fig. 2F), suggesting that virions of this strain were indeed ableto enter such cells despite the lack of de novo gene expression.

(iii) Monocyte progenitors infected with an HCMV clinicalisolate express IEl and gH on subsequent differentiation.Because HCMV virions were able to enter monocytic cellsearly in development, we wished to confirm reports that bonemarrow progenitors infected with HCMV could subsequentlybe induced to express HCMV IEl and gH after differentiation(17, 29). Monocyte colonies derived from bone marrow cellsafter 7 days in culture were infected with the M strain ofHCMV and showed no IEl expression after a further 7 days inculture, but their subsequent differentiation in LTBMC me-dium resulted in high frequencies of cells expressing TEl andgH (Table 2). Infectious virus could also be detected in culturesupernatants from isolated monocytic colonies by passage onfibroblast monolayers. In contrast, a similar analysis withAD169 virus failed to show detectable levels of IEl or gHexpression (results not shown). Analysis of monocytes pooledin groups of five (the survival of single isolated colonies was

poor) suggested that between 1 in 20 and 1 in 30 colonies couldbe induced to express HCMV antigens. These results areconsistent with previous observations (17, 29) that, afterHCMV infection, monocyte progenitors can maintain theHCMV genome with little or no viral gene expression until the

TABLE 2. HCMV gene expression and virus production inmonocyte colonies grown from bone marrow

progenitors infected with isolate M

HCMV gene expressiona at day following infection:Subject

7 14 28

1 U U U2 U U U3 U U IE1, gH4 U IEl IE1, gH5 U lE1 IE1, gH6 U U U7 U U IE1, gH8 U U IE1, virus9 U U IEl, gH, virus10 U U IEl11 U U U

a IEl expression at 14 and 28 days was 5 to 10% and 50 to 60% of the total cellscounted, respectively. gH expression at 28 days was 10 to 20% of total cellscounted. U, undetectable.

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*. .;....

*MNM - 4 t..w ;st

1 2 3 4 5 6 7 8 9 m

44

m 1 2 3 4 5 6 7 8 9

FIG. 3. Detection of HCMV DNA in colonies derived from bonemarrow progenitors collected from seropositive subjects. (A) Ethidiumbromide-stained 1.5% agarose gel of PCR for HCMV DNA. Thearrowhead indicates the gB PCR product of 244 bp. (B) Ethidiumbromide-stained 2% agarose gel of PCR for histidyl-tRNA synthetaseDNA on the same samples. The arrowhead indicates the PCR productof 360 bp. Lanes: 1, subject A without GM-CSF; 2, subject A withGM-CSF; 3, subject B without GM-CSF; 4, subject B with GM-CSF; 5,subject C without GM-CSF; 6, subject C with GM-CSF; 7, subject Dwith GM-CSF; 8, subject E with GM-CSF; 9, peripheral bloodmononuclear cells from a normal seropositive subject. Subjects A to Dwere HCMV seropositive, and subject E was an HCMV-seronegativecontrol. Lanes m, molecular weight markers, a 100-bp ladder in panelA and HindIlI-digested X DNA in panel B.

cells reach a certain stage of differentiation, after which IE1and subsequently gH are expressed.

(iv) Detection of endogenous HCMV DNA in bone marrow

progenitors derived from seropositive individuals. Becauseour data suggested that undifferentiated monocytes could beinfected with HCMV in vitro, we asked whether endogenousHCMV DNA could be detected in bone marrow progenitorcells of healthy seropositive individuals. Figure 3 shows that a

PCR amplification of DNA from 105 cells which targeted thegB-encoding region of HCMV (30) detected viral genomes inmonocyte colonies grown, in semisolid media plus methylcel-lulose, from granulocyte-macrophage colony-stimulating factor(GM-CSF)-stimulated progenitors (18) obtained from threeof four seropositive subjects. Subject B was undergoing hipreplacement for osteoarthritis and had no hematological or

immunological disease. The other samples were obtained fromharvests for autologous bone marrow transplantation in sub-jects who were well and in remission. There was no evidence ofIEI expression as detected by indirect immunofluorescence inthe cultures from these individuals (data not shown), ruling outactive infection. A sample of DNA derived from the peripheralblood mononuclear cells of a normal seropositive volunteerwas included as a positive control, and a control PCR to detecthistidyl-tRNA synthetase DNA (4) confirmed that all samplesanalyzed contained amplifiable DNA.

This observation implies that endogenous HCMV DNA was

present in progenitor cells when the cultures were initiated.We were unable to detect HCMV DNA in cultures grownwithout GM-CSF stimulation (which might in theory containcontaminating PBM), suggesting that small numbers of PBMcould not account for the positive results obtained in thepresence of GM-CSF (which does not favor PBM prolifera-tion). Under GM-CSF stimulation, myeloid progenitors un-

dergo intense proliferation (6), probably leading to replicationof viral DNA, which is more readily detectable by PCR.These results suggest that, in normal individuals persistently

infected with HCMV, the bone marrow may act as a reservoirfor the virus. Following primary infection, HCMV may infectprimitive bone marrow stem cells with the capacity for self-renewal. If replication and partition of the HCMV genomeoccur as these cells divide, the viral genome could be main-tained and amplified without entry into the productive cycle,avoiding expression of proteins against which the cell-medi-ated immune response is directed (2). Upon differentiation ofthese cells to tissue macrophages, however, virus reactivationcould occur. One analogy for this is visna virus, which has beenshown to infect monocyte progenitors in bone marrow withintegration of proviral DNA without viral gene expression,thus avoiding the host immune response (11).

It is usually inferred that HCMV can reactivate fromleukocytes in vivo following blood transfusion. In other work,we have found that in vitro differentiation of monocytes fromnormal virus carriers in the presence of hydrocortisone andphorbol myristate acetate can result in IE gene expressionfrom endogenous virus, detectable by reverse transcription andPCR (32). However, so far we have not been able to demon-strate full reactivation of endogenous HCMV from monocytesby cocultivation. Therefore, as well as reaching a stage of celldifferentiation which is permissive for HCMV, it is possiblethat PBM also require further stimuli to allow full reactivationof the latent genome from monocytes/macrophages.

This work was supported by the Medical Research Council (MRC)and by an MRC Training Fellowship to E.J.M.

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