© 1991 S. Karger AG, Basel 0300-5526/91/0323-0137S2.75 /0
Intervirology 1991;32:137-148
Effects of Poliovirus Replication on Undifferentiated and Differentiated Monocytic U937 Cells: Comparative Studies with Human Macrophages José A. López-Guerrero3, Carlos Cabañas*, Carmelo Bernabeub , Manuel Fresnoa , Miguel A. Alonso3 “Centro de Biología Molecular, Universidad Autónoma, CSIC, Madrid; bCentro de Investigaciones Biológicas, CSIC, Madrid, Spain
Key Words. Surface antigens • Superoxide anions • Macrophages • Poliovirus
Introduction Macrophages play a unique role in the tissue response to microbial invasion, crit ically positioned at most portals of entry and also surrounding epithelial and mesenchy mal cells. Macrophages interact with T and B cells controlling the immune response, and
Address inquiries to: Miguel A. Alonso, Centro de Biología Molecular, Universidad Autónoma, Cantoblanco, E-28049 Madrid (Spain) Received: December 18,1989 Accepted: April 10,1990
they are very active in the secretion of im munological modulators [1], Given this cen tral role of macrophages in the immune re sponse, many pathogens have developed mechanisms to neutralize their activity. Hu man immunodeficiency virus is an example of a virus that uses cells of the monocyte/ macrophage lineage as host cells for replica tion [2, 3]. Studies on visna virus infection have established that both susceptibility to infection and virus gene expression increase during monocyte maturation to macro phages [4]. Herpes simplex virus type 1 repli cation is also severalfold enhanced in macro phages compared to that in monocytes [5],
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Summary. Poliovirus infection of either undifferentiated or differentiated U937 cells produced a decrease in the percentage of cells positive for the surface expression of the CD4, CDlic, CD14, or 8E11 antigens. The number of 4F2 surface molecules per cell increased in infected normal U937 cells, but was unaffected in differentiated cells. The level of Oi production in infected differentiated U937 cells was approximately 50% of that found when not infected. Finally, poliovirus RNA levels and infectious particle production were similar in either cell type.
Poliovirus, the étiologie agent of paralytic poliomyelitis, infects humans via the mouth. Primary replication occurs in epithelial cells lining the intestine and in lymphoid tissue within tonsils, lymph nodes, and Peyer’s patches [6,7]. Northern blot analysis of RNA from different human tissues has recently demonstrated expression of poliovirus re ceptor mRNA in cells of the immune system, including B ant T cell lines and freshly iso lated macrophages [8]. In spite of the import ance of lymphoid cells in the progression of the infection, the cell types which are infec ted have not been unambiguously identified. As mononuclear phagocytes are present at most portals of entry and also in lymphoid tissue, we investigated the susceptibility of the monocytic U937 cell line to poliovirus infection. Our results show that a restriction at the poliovirus mRNA translation level oc curs in infected U937 cells [9]. U937 cells can be differentiated into macrophage-like cells by stimulation with a variety of biological and chemical agents [10]. This capability has made U937 cells widely used as a model to study in vitro the maturation of monocytes to macrophages. Poliovirus infection on U937 cells undergoing differentiation to macro phages interferes with the gene activation process triggered by phorbol-12-myristate13-acetate (PMA) addition [11]. To further understand the effect of poliovirus infection on macrophages, we now have analyzed pol iovirus RNA replication, infectious particle production, oxidative metabolism, and anti gen surface expression in undifferentiated and differentiated U937 cells infected with poliovirus. In addition, we have carried out experiments using human adherent mononu clear cells to reinforce the idea of the possible role of macrophages as host cells in poliovi rus infection.
López-Guerrgro/Cabañas/Bernabeu/Fresno/Alonso
Material and Methods Cells. Viruses, and Monoclonal Antibodies U937 cells [12] were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, and the experiments were carried out in DMEM containing 1%fetal calf serum. Wild-type poliovirus type I (Mahoney strain) was grown on HeLa cell cultures in DMEM with 1%calf serum. Monoclonal antibodies (mAbs) were used as hybridoma culture supernatants. mAbs FG 1/8, Bear 1, Bear 2, HC1/1, and HP2/6 recognizing the 4F2, CDllb, CDI4, CDIlc, and CD4 antigens, respec tively, have been previously described [13-16], Human adherent mononuclear cells were purified from peri pheral blood samples of healthy donors by FicollHypaque gradient sedimentation followed by adher ence to plastic flasks for 3 h at 37°. After an additional incubation period of 24 h at 37° the cells were used for experiments. RNA Blot Analysis Total cytoplasmic RNA was extracted by the Nonidet P-40 lysis method [17]. For RNA dot blot analysis, 10 ug of RNA was denatured in 50% formamide and 2.2 M formaldehyde at 65° for 5 min, and twofold serial dilutions were made in 20 x SSC (lxS S C = 0.15 M NaCl/0.015 M sodium citrate. pH 7.0) and spotted onto nitrocellulose membranes using a microfiltration apparatus (Bio-Rad). RNA samples immobilized on nitrocellulose were hybridized under standard conditions to nick-translated [18] poliovirus cDNA [19], Final blot washing conditions were 0.1 xSSC/0.1%SDS at 50°. Immunofluorescence Flow Cytometry Cells at 5 x lOVml were incubated with approxi mately 10gg/ml of monoclonal antibody for 30 min at 4° washed twice with phosphate-buffered saline, and incubated for 30 min at 4° in the same volume of fluorescein-isothiocyanate-conjugated goat anti mouse IgG previously diluted at 1:50 in phosphatebuffered saline. Cells were washed three times and analyzed in an EP1CS-C cytofluorometer as de scribed. P3X63 myeloma culture supernatant was used as a negative control. Measurement o f Superoxide Anion Generation The method for assaying reactive oxygen species production has been previously described [20].
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Briefly, U937 cells incubated in the absence or in the presence of 25 ng/ml of PMA for 2 days were mock infected or infected with poliovirus at 20 PFU/cell. After 24 h, the cells were washed twice with a bal anced salt solution (10 mA/Hepes, 150 mM NaCl, 1.2 mM MgCK, 1.3 mM CaCl, 5.5 mM glucose, pH 7.5) and resuspended in this buffered solution. I x 10“ U937 cells were incubated with 0.90 mg of cytochro me and 5.0 pg of cytochalasin B either in the presence or in the absence of 0.4 pg of superoxide dismutase in a total reaction volume of 1.0 ml for 10 min at 37°. Subsequently, 100 ng of PMA was added and the reaction proceeded for an additional period of 30 min at 37°. At the end of this period, the samples were centrifuged to remove cells, and the supernatants were collected. The extent of cytochrome c reduction was measured in a Bausch-Lomb (Spectronic 710) spectrophotometer at 550 nm. The difference in ab sorbance between samples treated or untreated with superoxide dismutase was a measure of the amount of reduced cytochrome c. Protein Labeling and PAGE In vivo labeling of newly synthesized protein was carried out by giving a I-hour pulse with 5 pCi of [35S]-methionine (1,450 Ci/mmol; Amersham) to cell cultures in methionine-free DMEM. At the end of the labeling period, the medium was removed, and the cells washed with phosphate-buffered saline and dis solved in 100 pi 0.02 M NaOH/1% SDS and 200 pi of sample buffer: 62.5 m A/Tris (pH 6.8)/2% SDS/0.1 M dithiothreitol/17% glyceroi/0.024% bromophenol blue. Volumes of 5 pi were applied to a 15%polyacryl amide gel and electrophoresed overnight at 100 V.
Results Phenotypic Changes in Poliovirus-Infected U937 Cells U937 cells were induced to differentiate by treatment with 25 ng/ml PMA for 2 days. After extensive washing, PMA-treated and normal U937 cell cultures were infected with poliovirus at 10 PFU/cell. At different times after infection, the cells were analyzed for antigen expression in a flow fluorescence cytometer using a panel of six different mAbs
139
against distinct membrane antigens. The re sult of typical experiment is presented in figure I. The response of the expression of these antigens to poliovirus infection al lowed their classification in three groups. The antigens represented in the first group include CD4 CDllc, CD14, and 8E1I which were partially downmodulated by poliovirus infection in both undifferentiated or PMAdifferentiated U937 cells. Figure 1 shows a time course of the surface expression of CD4 that was chosen as a prototype of the antigens included in the first group. Interestingly, PMA also exerted an inhibitory effect on the surface expression of this group of antigens, and an additive inhibitory effect of poliovi rus and PMA was observed in infected PM Adifferentiated U937 cells. The second group includes the 4F2 antigen whose number of surface molecules per cell was increased in infected differentiated cells. The CD llb anti gen, which was not affected by poliovirus infection in either normal or differentiated U937 cells, is included in the third group (fig. 1). The dependence of the surface antigen reactivity with the MOI of poliovirus used is shown in figure 2. No variation with the MOI was observed in the expression of CD llb and 4F2 in the infected undifferentiated or the differentiated U937 cells. In contrast, although CD4 surface expression was not affected by increasing the MOI in undiffer entiated cells, in cells differentiated with PMA, both the number of reactive molecules per cell and the number of cells positive for CD4 greatly diminished. Effect o f Poliovirus Infection on the Production o f Superoxide Anion The interference that poliovirus produces on the host cell metabolism in permissive
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Replication of Poliovirus in Macrophages
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140
-P M A h p.i.
CD4
+ PMA
CDU b
4F2
CD4
C D llb
4F2
a. LU CE
Z 3 _l
LU
O
FLUORESCENCE
INTENSITY
Fig. 1. Time course analysis of the surface expres sion of different antigens in poliovirus-infected un differentiated or differentiated U937 cells. Cultures of U937 cells incubated in the presence or absence of 25 ng/ml PMA for 48 h were mock infected or infec ted with 10 PFU/cell of poliovirus. At the indicated times after infection (h p.i.), the cells were subjected
to fluorescence flow cytometry analysis with different monoclonal antibodies. The position of the fluores cence threshold for discriminating positive from ne gative cells is indicated on the x-axis. Numbers above histograms represent the percentage of positive cells. The experiment shown represents three different experiments.
cells [21] is well characterized. Since the pro duction of oxidative metabolites has been associated with the microbicidal action of macrophages [22], we have examined the effect of poliovirus on the capacity of U937 cells to produce superoxide anion (Oj). U937 cells were differentiated to macro phages with 25 ng/m l PMA for 48 h and infected with poliovirus. The production of 0 2 was determined 24 h after infection, mea-
suring the reduction of cytochrome c by U937 cell supernatants in the presence or absence of superoxide dismutase. Figure 3 shows that unstimulated U937 cells pro duced undetectable levels of 0 2, but incuba tion with PMA for 72 h increased the levels of superoxide anion. Infection of PMAstimulated cells with poliovirus reduced the level of 0 2 in noninfected cells to approxi mately 50%.
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LOG
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Replication of Poliovirus in Macrophages
m.O.i.
____________- P M A ____________
___________ + P M A ___________
CD4
CD4
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4F2
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98 99
9 5 59
9 9 19
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9 9 .6 4
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9 8 .4 4
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9 8 .2 4
9 8 .6 9
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FLUORESCENCE
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Fig. 2. Effect of the multiplicity of infection on the surface antigen expression in poliovirus-infected un differentiated or differentiated U937 cells. Parallel cultures of U937 cells incubated in the presence or absence of 25 ng/ml PMA for 48 h were mock infec ted or infected with the indicated MOI of poliovirus. After 24 h of incubation at 37°, the cells were sub
jected to fluorescence flow cytometry analysis with different monoclonal antibodies. The position of the fluorescence threshold for discriminating positive from negative cells is indicated on the x-axis. Num bers above histograms represent the percentage of positive cells. The experiment represents three differ ent experiments.
Morphologic Alterations and Infectious Particle Production in Infected Cultures Figure 4 shows the morphology of U937 cells 24 h after infection with poliovirus at two different multiplicities of infection. Pol iovirus infection at 10 PFU/cell produced no evident CPE in normal or differentiated U937 cells. In contrast, cells infected at 100 PFU/cell presented a cytopathology with ex tensive vésiculation and clear features of cel-
lular damage that resemble that found in permissive cells. Phorbol ester treatment induces the inter nalization of a number of surface molecules, including receptors for hormones and growth factors [23,24], Prolonged PMA treat ment also causes the loss of the receptors for human immunodeficiency virus [25] and pol iovirus [26]. To check if (under our conditions of infection) most of the cells in the culture
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/V
became infected, we carried out a limiting dilution assay using HeLa or U937 cells preincubated (or not) with 25 ng/ml PM A for 48 h and infected with 10 PFU/cell of poliov irus. Figure 5 shows that, according to the Poisson distribution, more than 99% of the cells in the cultures were readily infected by poliovirus. In contrast to fully permissive HeLa cells, U937 cells present a low permissiveness for poliovirus replication [27]. Table I shows a comparative analysis of the PFU yield per cell in HeLa as well as in undifferentiated or differentiated U937 cells. Production of PFU in undifferentiated U937 cells was 16-fold, reduced as compared with HeLa cells, which is in agreement with our previous Findings [9]. PM A treatment of both HeLa or U937 cells exerted little effect on the production of infectious particles. This result contrasts with those found in other viral systems such as visna virus and herpesvirus in which a marked enhancement of PFU formation was observed with U937 cell differention [4, 5]. Table 1also shows that cell viability 24 h after infection was greater than 90% in undifferen tiated and differentiated U937 cells infected with poliovirus, whereas only approximately 10% of poliovirus-infected HeLa cells re mained viable 8 h after infection. Infection with poliovirus of cells differentiated with PM A did not affect the PM A-induced adher ence to plastic [10], and only at times after infection >48 h did cells lose their adherence as a consequence of cell killing. Comparison o f Poliovirus RNA Replication in Undifferentiated and Differentiated U937 Cells and in Fresh Human Macrophages The low yield of infectious particles in differentiated U937 cells could be due to a
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Poliovirus
-
+
-
+
Fig. 3. Generation of superoxide anion in poliovirus-infecied U937 cells. Cultures of U937 cells differ entiated (or not) with 25 ng/ml PM A for 48 h were mock infected or infected with 25 PFU/ml of poliovi rus. The production of O2 was determined 24 h after infection as described under Materials and Methods. The values shown are the mean of four independent experiments.
lower rate of RNA replication as compared with that in undifferentiated U937 cells. To addressthis point, we extracted total RNA at two different times after infection from nor mal and PMA-treated U937 cells infected with poliovirus, and viral RNA levels were compared by hybridization to a poliovirus cDNA probe. Figure 6A shows that PMAinduced differention of U937 cells did not significantly change the level of poliovirus RNA in U937 cells. In addition we have carried out comparative studies of poliovirus infection to test whether infection of U937 cells reflects the behavior of freshly isolated human macrophages. Figure 6A shows that poliovirus RNA replication readily took place in infected adherent mononuclear
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Replication of Poliovirus in Macrophages
-PM A
+ PMA
CONTROL
Poliovirus (m.o.¡.=10)
Fig. 4. Cytopathology of poliov irus infected U937 cells. Cultures of U937 cells undifferentiated (B, D, F) or not (A, C, E) with 25 ng/ml PMA for 48 h were mock infected (A, B) or infected with poliovirus at 10(C, D)or 100 (E, F) PFU/cell. The cell morphology was examined 24 h after virus addition, x 160.
Discussion Macrophages are often one of the first cell types to take up inoculated virus at the site of entry. A number of viruses can replicate in macrophages, and certain viruses replicate almost exclusively in this cell type. It has been suggested that differentiation of mono nuclear phagocytes plays an important role in persistence of latency of cytomegalovirus [28] and Friend leukemia virus [29]. This is also particularly evident in persistently visna virus infected sheep where bone marrow precursors of macrophages carry only a few copies of viral RNA in contrast to thousands of copies of viral genome in tissue macro phages [4,30]. A reduction in the permissive-
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cells, although the poliovirus RNA levels in these cells were reduced compared to those in undifferentiated or differentiated U937 cells and HeLa cells. When the proteins (synthesized 24 h after infection) were ana lyzed by PAGE, we detected no differences between uninfected and infected macro phage cultures, and no viral proteins were evident in the infected cells as was also the case in infected U937 cells differentiated (or not) with PMA (fig. 6B). However, as we described previously [9], both the existence of viral RNA replication and the results of immunofluorescence analysis with antisera to nonstructural proteins of poliovirus sug gest that some viral RNA translation takes place in U937 cells.
Poliovirus (moi =100)
Lopez-Guerrepo/Cabanas/Bernabeu/Fresno/Alonso
Cells/well
Table t. Production of infectious virus, viability, and adherence of U937 and HeLa cell cultures infec ted with poliovirus PFU/cell"
HeLa 200 ± 15 HeLa + PMA 145 ± 12 U937 12±3 U937 + PMA 10±3
Fig. 5. Estimation of the percentage of poliovirusinfected cells by a limiting dilution assay. Cultures of either U937 or HeLa cells treated or not with 25 ng/ml PMA for 48 h were incubated with 10 PFU/ml polio virus for 1 h to allow virus absorption. The cells were then washed six times with phosphate-buffered sa line, counted, and diluted in normal medium. Ali quots of each cell dilution were used so that 0.5,1,1.6, or 5 cells/well were added to HeLa cell monolayers grown on 96-well plates. After 72 h at 37°, crystal violet was added to determine the number of wells where the cell monolayer remained intact. B = HeLa cells; □ = HeLa cells treated with PMA: • = U937 cells: O = U937 cells treated with PMA.
ness to human immunodeficiency virus in fection has been observed in PMA-treated U937 cells compared to normal U937 cells due to downmodulation of the surface ex pression of the human immunodeficiency vi rus receptor, the CD4 antigen [25]. Monocyte differentiation of macrophages involves regulated changes of cell surface antigen expression and gene transcription concomitant to the development of specific
Viability °/ob
Adherence %c
< 10 < 10 >90 >90
10-20 10-20 95
Cell cultures of either HeLa or U937 cells pretrea ted or not with 25 ng/ml PMA for 48 h were infected with poliovirus at 10 PFU/cell. a Total PFU production was assayed 24 h after infection. The values shown are the mean of three independent experiments. b Viability was determined by the trypan blue ex clusion techniques h (HeLa cells)or24 h (U937 cells) after infection. c Adherence to plastic was determined by examina tion with a phase-contrast microscope 8 h (HeLa cells) or 24 h (U937 cells) after infection.
macrophage functions such as the oxidative microbicidal metabolism [31, 32]. We have shown that poliovirus infection interfered with the surface expression of the antigens CD4, CDllc, CD14, and 8E11 both in undif ferentiated and differentiated cells. The number of 4F2 surface molecules increased in undifferentiated cells and slightly de creased in differentiated cells, although the number of positive cells remained constant. The fact that the surface expression of CD1 lb did not change in poliovirus-infected cells makes it very unlikely that the changes in the expression observed in other surface markers were due to a generalized proteolytic degrad ation. Although we cannot rule out the possi bility that the surface antigen reactivity could
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Replication o f Poliovirus in Macrophages
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U-937 -PMA
Poliovirus (h pi.)
Hela
U-937 -P M A
U-937 + PMA
0
0 12 2A 0
•• ■ I Î! 9 •• 6
U-937 +PMA
M* s
S i
M0s
12 2A 0 12 2A •
•
•
•
•
• !
•
•
•
•
•
•
•
•
•
•
•
•
•
A
tI
Fig. 6. Poliovirus RNA replication and protein synthesis in different cells infected with poliovirus. A Cultures of HeLa cells, undifferentiated or differ entiated U937 cells, or human macrophages (MOs) were incubated with 10 PFU/cell poliovirus for 1 h to allow virus adsorption. The cells were then washed to remove nonadsorbed viral particles (zero time) and incubated at 37° for the indicated times. Total RNA was extracted, and starting with 10 pg of RNA, two-
fold erial dilutions were made. RNA samples immo bilized on nitrocellulose were subjected to hybridiza tion to a poliovirus cDNA probe. B Cultures of undif ferentiated or differentiated U937 cells, human mac rophages, or HeLa cells were mock infected or infec ted with 10 PFU/cell poliovirus. After 24 h at 37°, the cells were incubated with 5 pCi [35S]-methionine for 1 h and subjected to PAGE analysis.
be differentially affected by proteolysis, we believe this is not the case, since the varia tions in the expression of surface molecules occurred in cells infected by 10 PFU/cell of poliovirus as early as 5 h after infection, when no CPE was observed. In addition, supporting this view, we have found that, in general, a higher MOl of poliovirus did not
result in a more extensive loss of surface antigen reactivity. The way by which poliovi rus infection interferes with the surface anti gen expression is presently unknown, al though the kinetics of disappearance of some of the markers analyzed suggest that pro cesses involved in membrane protein turn over might be affected.
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B
The capacity to generate oxidative metab olites is a characteristic of phagocytic cells [33]. Mouse macrophages activated by Calmette-Guérin bacillus or lipopolysaccharide produced elevated levels of 0 2 and H20 2and showed enhanced microbicidal capacity against trypanosomes, toxoplasma, and Can dida [34, 35]. Production of 0 2 anions in dif ferentiated U937 cells infected with poliovi rus was reduced to approximately 50% of the level in noninfected cultures, suggesting that poliovirus replication affects either the syn thesis of oxidative metabolites or the devel opment of the enzymatic machinery to pro duce 0 2 and H20 2. The role of oxygen metab olites in mediating virucidal activity in mac rophages is not completely elucidated [33]. In murine macrophage cell lines, the intracellu lar resistance to vesicular stomatitis virus in fection seems to be mediated through nonoxidative mechanisms, although there was some extracellular killing of vesicular stoma titis virus by oxidation [22]. Vaccinia virus and poliovirus are susceptible to cell-free systems able to generate 0 2 and H20 2 [36], However, we found no differences in the rate of poliovirus RNA replication between un differentiated and differentiated U937 cells, and the PFU yield per cell was also similar in both cell types. These observations suggest that 0 2anions do not seem to play any impor tant role in reducing the production of in fectious poliovirus particles in differentiated U937 cells. It has been widely accepted that U937 cells differentiated with PMA represent a bona fide model of human macrophages. However, due to the leukemic origin of U937 cells [12], we decided to carry out compara tive studies of poliovirus replication on hu man macrophages. A lower level of poliovi rus RNA replication was found in these cells
López-Guerrero/Cabañas/Bernabeu/Fresno/Alonso
compared to that in undifferentiated or dif ferentiated U937 cells. A restriction in the translation of poliovirus RNA was evidenced in infected macrophages by PAGE analysis of newly synthesized proteins, as shown in U937 cells [9], Studies on the role of macrophages in poliovirus infection are still lacking. In fectious virus can be recovered from lym phoid tissue in autopsy samples of individu als affected with polyomyelitis [7], Replica tion of poliovirus in lymphoid tissue appears to be important in the establishment of viremia which facilitates the infection of the central nervous system, leading to paralysis. As macrophages are present in lymphoid tis sue, our findings could be useful to under stand how poliovirus and macrophages in teract during the initial steps of poliovirus replication in infected humans, both at the portal of entry and during primary replica tion. They also may help to elucidate the mechanism by which poliovirus evades de struction by the phagocytic cells.
Acknowledgments We acknowledge Dr. Jean de Vries and Dr. F. Sánchez-Madrid for their generous gift of mAbs. J.A.L. istheholderofa FPI fellowship. This work was supported by an institutional grant from the Funda ción Ramón Areces and grants from DG1CYT and F.C. de la A.E.C.C.
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Görden P, Orci L: Role of intracellular calcium and protein kinase C in the endocytosis of trans ferrin and insulin by HL60 cells. J Cell Biol 1986:103:851-856. Clapham PR, Weiss RA, Dalgleish AG, Exley M, Whitby D, Hogg N: Human immunodeficiency virus infection of monocytic and T-lymphocytic cells. Receptor modulation and differentiation in duced by phorbol ester. Virology 1987;158:44-51. Madshus IH, Sandvig K., Olsnes S: Tumor-pro moting phorbol esters and vanadate alter the sen sitivity of HeLa S} cells to poliovirus type I. Exp Cell Res 1985;160:240-244. Okada Y, Toda G, Oka H, Nomoto A, Yoshikura H: Poliovirus infection of established human blood cell lines: Relationship between differen tiation stage and susceptibility or cell killing. Vi rology 1987;156:238-245. Bräutigam AR, Dutko FJ, Olding LB, Oldstone MBA: Pathogenesis of murine cytomegalovirus infection: The macrophage as a permissive cell for cytomegalovirus infection, replication and lat ency. J Gen Virol 1979;44:349-359. Marcelletti J, Furmanski P: Infection of macro phages with Friend virus: Relationship to the spontaneous regression of viral erythroleukemia. Cell 1979;16:649-659. Peluso R, Haase A, Stowring L, Edwards M, Ven
31
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33 34
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tura P: A Trojan horse mechanism for the spread of visna virus in monocytes. Virology 1985:147: 231-236. Minta JO, Pambrun L: In vitro induction of cyto logic and functional differentiation of the imma ture human monocytelike cell line U937 with phorbol myristate acetate. Am J Pathol 1985; 119:111-126. Collins SJ: The HL-60 promyelocytic leukemia cell line: Proliferation, differentiation, and cel lular oncogene expression. Blood 1987:70: 1233-1244. Babior BM: Oxygen-dependent microbial killing by phagocytes. N Engl J Med 1978;298:659-668. Nathan C, Nogueira N, Juangbhanich C, Ellis J, Cohn Z: Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide re lease and killing of Trypanosoma cruzi. J Exp Med 1979;149:1056-1068. Sasada M, Johnston RB Jr: Macrophage microbi cidal activity. Correlation between phagocytosisassociated oxidative metabolism and the killing of Candida by macrophages. J Exp Med 1980; 152:85-98. Belding ME, Klebanoff SJ, Ray CG: Peroxidasemediated virucidal systems. Science 1970:167: 195-196.
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Löpez-Guerrero/Cabanas/Bernabeu/Fresno/Alonso
Intervirology 1991;32:137-148
Effects of Poliovirus Replication on Undifferentiated and Differentiated Monocytic U937 Cells: Comparative Studies with Human Macrophages José A. López-Guerrero3, Carlos Cabañas*, Carmelo Bernabeub , Manuel Fresnoa , Miguel A. Alonso3 “Centro de Biología Molecular, Universidad Autónoma, CSIC, Madrid; bCentro de Investigaciones Biológicas, CSIC, Madrid, Spain
Key Words. Surface antigens • Superoxide anions • Macrophages • Poliovirus
Introduction Macrophages play a unique role in the tissue response to microbial invasion, crit ically positioned at most portals of entry and also surrounding epithelial and mesenchy mal cells. Macrophages interact with T and B cells controlling the immune response, and
Address inquiries to: Miguel A. Alonso, Centro de Biología Molecular, Universidad Autónoma, Cantoblanco, E-28049 Madrid (Spain) Received: December 18,1989 Accepted: April 10,1990
they are very active in the secretion of im munological modulators [1], Given this cen tral role of macrophages in the immune re sponse, many pathogens have developed mechanisms to neutralize their activity. Hu man immunodeficiency virus is an example of a virus that uses cells of the monocyte/ macrophage lineage as host cells for replica tion [2, 3]. Studies on visna virus infection have established that both susceptibility to infection and virus gene expression increase during monocyte maturation to macro phages [4]. Herpes simplex virus type 1 repli cation is also severalfold enhanced in macro phages compared to that in monocytes [5],
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Summary. Poliovirus infection of either undifferentiated or differentiated U937 cells produced a decrease in the percentage of cells positive for the surface expression of the CD4, CDlic, CD14, or 8E11 antigens. The number of 4F2 surface molecules per cell increased in infected normal U937 cells, but was unaffected in differentiated cells. The level of Oi production in infected differentiated U937 cells was approximately 50% of that found when not infected. Finally, poliovirus RNA levels and infectious particle production were similar in either cell type.
Poliovirus, the étiologie agent of paralytic poliomyelitis, infects humans via the mouth. Primary replication occurs in epithelial cells lining the intestine and in lymphoid tissue within tonsils, lymph nodes, and Peyer’s patches [6,7]. Northern blot analysis of RNA from different human tissues has recently demonstrated expression of poliovirus re ceptor mRNA in cells of the immune system, including B ant T cell lines and freshly iso lated macrophages [8]. In spite of the import ance of lymphoid cells in the progression of the infection, the cell types which are infec ted have not been unambiguously identified. As mononuclear phagocytes are present at most portals of entry and also in lymphoid tissue, we investigated the susceptibility of the monocytic U937 cell line to poliovirus infection. Our results show that a restriction at the poliovirus mRNA translation level oc curs in infected U937 cells [9]. U937 cells can be differentiated into macrophage-like cells by stimulation with a variety of biological and chemical agents [10]. This capability has made U937 cells widely used as a model to study in vitro the maturation of monocytes to macrophages. Poliovirus infection on U937 cells undergoing differentiation to macro phages interferes with the gene activation process triggered by phorbol-12-myristate13-acetate (PMA) addition [11]. To further understand the effect of poliovirus infection on macrophages, we now have analyzed pol iovirus RNA replication, infectious particle production, oxidative metabolism, and anti gen surface expression in undifferentiated and differentiated U937 cells infected with poliovirus. In addition, we have carried out experiments using human adherent mononu clear cells to reinforce the idea of the possible role of macrophages as host cells in poliovi rus infection.
López-Guerrgro/Cabañas/Bernabeu/Fresno/Alonso
Material and Methods Cells. Viruses, and Monoclonal Antibodies U937 cells [12] were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, and the experiments were carried out in DMEM containing 1%fetal calf serum. Wild-type poliovirus type I (Mahoney strain) was grown on HeLa cell cultures in DMEM with 1%calf serum. Monoclonal antibodies (mAbs) were used as hybridoma culture supernatants. mAbs FG 1/8, Bear 1, Bear 2, HC1/1, and HP2/6 recognizing the 4F2, CDllb, CDI4, CDIlc, and CD4 antigens, respec tively, have been previously described [13-16], Human adherent mononuclear cells were purified from peri pheral blood samples of healthy donors by FicollHypaque gradient sedimentation followed by adher ence to plastic flasks for 3 h at 37°. After an additional incubation period of 24 h at 37° the cells were used for experiments. RNA Blot Analysis Total cytoplasmic RNA was extracted by the Nonidet P-40 lysis method [17]. For RNA dot blot analysis, 10 ug of RNA was denatured in 50% formamide and 2.2 M formaldehyde at 65° for 5 min, and twofold serial dilutions were made in 20 x SSC (lxS S C = 0.15 M NaCl/0.015 M sodium citrate. pH 7.0) and spotted onto nitrocellulose membranes using a microfiltration apparatus (Bio-Rad). RNA samples immobilized on nitrocellulose were hybridized under standard conditions to nick-translated [18] poliovirus cDNA [19], Final blot washing conditions were 0.1 xSSC/0.1%SDS at 50°. Immunofluorescence Flow Cytometry Cells at 5 x lOVml were incubated with approxi mately 10gg/ml of monoclonal antibody for 30 min at 4° washed twice with phosphate-buffered saline, and incubated for 30 min at 4° in the same volume of fluorescein-isothiocyanate-conjugated goat anti mouse IgG previously diluted at 1:50 in phosphatebuffered saline. Cells were washed three times and analyzed in an EP1CS-C cytofluorometer as de scribed. P3X63 myeloma culture supernatant was used as a negative control. Measurement o f Superoxide Anion Generation The method for assaying reactive oxygen species production has been previously described [20].
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Briefly, U937 cells incubated in the absence or in the presence of 25 ng/ml of PMA for 2 days were mock infected or infected with poliovirus at 20 PFU/cell. After 24 h, the cells were washed twice with a bal anced salt solution (10 mA/Hepes, 150 mM NaCl, 1.2 mM MgCK, 1.3 mM CaCl, 5.5 mM glucose, pH 7.5) and resuspended in this buffered solution. I x 10“ U937 cells were incubated with 0.90 mg of cytochro me and 5.0 pg of cytochalasin B either in the presence or in the absence of 0.4 pg of superoxide dismutase in a total reaction volume of 1.0 ml for 10 min at 37°. Subsequently, 100 ng of PMA was added and the reaction proceeded for an additional period of 30 min at 37°. At the end of this period, the samples were centrifuged to remove cells, and the supernatants were collected. The extent of cytochrome c reduction was measured in a Bausch-Lomb (Spectronic 710) spectrophotometer at 550 nm. The difference in ab sorbance between samples treated or untreated with superoxide dismutase was a measure of the amount of reduced cytochrome c. Protein Labeling and PAGE In vivo labeling of newly synthesized protein was carried out by giving a I-hour pulse with 5 pCi of [35S]-methionine (1,450 Ci/mmol; Amersham) to cell cultures in methionine-free DMEM. At the end of the labeling period, the medium was removed, and the cells washed with phosphate-buffered saline and dis solved in 100 pi 0.02 M NaOH/1% SDS and 200 pi of sample buffer: 62.5 m A/Tris (pH 6.8)/2% SDS/0.1 M dithiothreitol/17% glyceroi/0.024% bromophenol blue. Volumes of 5 pi were applied to a 15%polyacryl amide gel and electrophoresed overnight at 100 V.
Results Phenotypic Changes in Poliovirus-Infected U937 Cells U937 cells were induced to differentiate by treatment with 25 ng/ml PMA for 2 days. After extensive washing, PMA-treated and normal U937 cell cultures were infected with poliovirus at 10 PFU/cell. At different times after infection, the cells were analyzed for antigen expression in a flow fluorescence cytometer using a panel of six different mAbs
139
against distinct membrane antigens. The re sult of typical experiment is presented in figure I. The response of the expression of these antigens to poliovirus infection al lowed their classification in three groups. The antigens represented in the first group include CD4 CDllc, CD14, and 8E1I which were partially downmodulated by poliovirus infection in both undifferentiated or PMAdifferentiated U937 cells. Figure 1 shows a time course of the surface expression of CD4 that was chosen as a prototype of the antigens included in the first group. Interestingly, PMA also exerted an inhibitory effect on the surface expression of this group of antigens, and an additive inhibitory effect of poliovi rus and PMA was observed in infected PM Adifferentiated U937 cells. The second group includes the 4F2 antigen whose number of surface molecules per cell was increased in infected differentiated cells. The CD llb anti gen, which was not affected by poliovirus infection in either normal or differentiated U937 cells, is included in the third group (fig. 1). The dependence of the surface antigen reactivity with the MOI of poliovirus used is shown in figure 2. No variation with the MOI was observed in the expression of CD llb and 4F2 in the infected undifferentiated or the differentiated U937 cells. In contrast, although CD4 surface expression was not affected by increasing the MOI in undiffer entiated cells, in cells differentiated with PMA, both the number of reactive molecules per cell and the number of cells positive for CD4 greatly diminished. Effect o f Poliovirus Infection on the Production o f Superoxide Anion The interference that poliovirus produces on the host cell metabolism in permissive
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Replication of Poliovirus in Macrophages
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-P M A h p.i.
CD4
+ PMA
CDU b
4F2
CD4
C D llb
4F2
a. LU CE
Z 3 _l
LU
O
FLUORESCENCE
INTENSITY
Fig. 1. Time course analysis of the surface expres sion of different antigens in poliovirus-infected un differentiated or differentiated U937 cells. Cultures of U937 cells incubated in the presence or absence of 25 ng/ml PMA for 48 h were mock infected or infec ted with 10 PFU/cell of poliovirus. At the indicated times after infection (h p.i.), the cells were subjected
to fluorescence flow cytometry analysis with different monoclonal antibodies. The position of the fluores cence threshold for discriminating positive from ne gative cells is indicated on the x-axis. Numbers above histograms represent the percentage of positive cells. The experiment shown represents three different experiments.
cells [21] is well characterized. Since the pro duction of oxidative metabolites has been associated with the microbicidal action of macrophages [22], we have examined the effect of poliovirus on the capacity of U937 cells to produce superoxide anion (Oj). U937 cells were differentiated to macro phages with 25 ng/m l PMA for 48 h and infected with poliovirus. The production of 0 2 was determined 24 h after infection, mea-
suring the reduction of cytochrome c by U937 cell supernatants in the presence or absence of superoxide dismutase. Figure 3 shows that unstimulated U937 cells pro duced undetectable levels of 0 2, but incuba tion with PMA for 72 h increased the levels of superoxide anion. Infection of PMAstimulated cells with poliovirus reduced the level of 0 2 in noninfected cells to approxi mately 50%.
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LOG
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Replication of Poliovirus in Macrophages
m.O.i.
____________- P M A ____________
___________ + P M A ___________
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Fig. 2. Effect of the multiplicity of infection on the surface antigen expression in poliovirus-infected un differentiated or differentiated U937 cells. Parallel cultures of U937 cells incubated in the presence or absence of 25 ng/ml PMA for 48 h were mock infec ted or infected with the indicated MOI of poliovirus. After 24 h of incubation at 37°, the cells were sub
jected to fluorescence flow cytometry analysis with different monoclonal antibodies. The position of the fluorescence threshold for discriminating positive from negative cells is indicated on the x-axis. Num bers above histograms represent the percentage of positive cells. The experiment represents three differ ent experiments.
Morphologic Alterations and Infectious Particle Production in Infected Cultures Figure 4 shows the morphology of U937 cells 24 h after infection with poliovirus at two different multiplicities of infection. Pol iovirus infection at 10 PFU/cell produced no evident CPE in normal or differentiated U937 cells. In contrast, cells infected at 100 PFU/cell presented a cytopathology with ex tensive vésiculation and clear features of cel-
lular damage that resemble that found in permissive cells. Phorbol ester treatment induces the inter nalization of a number of surface molecules, including receptors for hormones and growth factors [23,24], Prolonged PMA treat ment also causes the loss of the receptors for human immunodeficiency virus [25] and pol iovirus [26]. To check if (under our conditions of infection) most of the cells in the culture
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/V
became infected, we carried out a limiting dilution assay using HeLa or U937 cells preincubated (or not) with 25 ng/ml PM A for 48 h and infected with 10 PFU/cell of poliov irus. Figure 5 shows that, according to the Poisson distribution, more than 99% of the cells in the cultures were readily infected by poliovirus. In contrast to fully permissive HeLa cells, U937 cells present a low permissiveness for poliovirus replication [27]. Table I shows a comparative analysis of the PFU yield per cell in HeLa as well as in undifferentiated or differentiated U937 cells. Production of PFU in undifferentiated U937 cells was 16-fold, reduced as compared with HeLa cells, which is in agreement with our previous Findings [9]. PM A treatment of both HeLa or U937 cells exerted little effect on the production of infectious particles. This result contrasts with those found in other viral systems such as visna virus and herpesvirus in which a marked enhancement of PFU formation was observed with U937 cell differention [4, 5]. Table 1also shows that cell viability 24 h after infection was greater than 90% in undifferen tiated and differentiated U937 cells infected with poliovirus, whereas only approximately 10% of poliovirus-infected HeLa cells re mained viable 8 h after infection. Infection with poliovirus of cells differentiated with PM A did not affect the PM A-induced adher ence to plastic [10], and only at times after infection >48 h did cells lose their adherence as a consequence of cell killing. Comparison o f Poliovirus RNA Replication in Undifferentiated and Differentiated U937 Cells and in Fresh Human Macrophages The low yield of infectious particles in differentiated U937 cells could be due to a
López-Guerrero/Cabañas/Bernabeu/Fresno/Alonso
Poliovirus
-
+
-
+
Fig. 3. Generation of superoxide anion in poliovirus-infecied U937 cells. Cultures of U937 cells differ entiated (or not) with 25 ng/ml PM A for 48 h were mock infected or infected with 25 PFU/ml of poliovi rus. The production of O2 was determined 24 h after infection as described under Materials and Methods. The values shown are the mean of four independent experiments.
lower rate of RNA replication as compared with that in undifferentiated U937 cells. To addressthis point, we extracted total RNA at two different times after infection from nor mal and PMA-treated U937 cells infected with poliovirus, and viral RNA levels were compared by hybridization to a poliovirus cDNA probe. Figure 6A shows that PMAinduced differention of U937 cells did not significantly change the level of poliovirus RNA in U937 cells. In addition we have carried out comparative studies of poliovirus infection to test whether infection of U937 cells reflects the behavior of freshly isolated human macrophages. Figure 6A shows that poliovirus RNA replication readily took place in infected adherent mononuclear
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Replication of Poliovirus in Macrophages
-PM A
+ PMA
CONTROL
Poliovirus (m.o.¡.=10)
Fig. 4. Cytopathology of poliov irus infected U937 cells. Cultures of U937 cells undifferentiated (B, D, F) or not (A, C, E) with 25 ng/ml PMA for 48 h were mock infected (A, B) or infected with poliovirus at 10(C, D)or 100 (E, F) PFU/cell. The cell morphology was examined 24 h after virus addition, x 160.
Discussion Macrophages are often one of the first cell types to take up inoculated virus at the site of entry. A number of viruses can replicate in macrophages, and certain viruses replicate almost exclusively in this cell type. It has been suggested that differentiation of mono nuclear phagocytes plays an important role in persistence of latency of cytomegalovirus [28] and Friend leukemia virus [29]. This is also particularly evident in persistently visna virus infected sheep where bone marrow precursors of macrophages carry only a few copies of viral RNA in contrast to thousands of copies of viral genome in tissue macro phages [4,30]. A reduction in the permissive-
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cells, although the poliovirus RNA levels in these cells were reduced compared to those in undifferentiated or differentiated U937 cells and HeLa cells. When the proteins (synthesized 24 h after infection) were ana lyzed by PAGE, we detected no differences between uninfected and infected macro phage cultures, and no viral proteins were evident in the infected cells as was also the case in infected U937 cells differentiated (or not) with PMA (fig. 6B). However, as we described previously [9], both the existence of viral RNA replication and the results of immunofluorescence analysis with antisera to nonstructural proteins of poliovirus sug gest that some viral RNA translation takes place in U937 cells.
Poliovirus (moi =100)
Lopez-Guerrepo/Cabanas/Bernabeu/Fresno/Alonso
Cells/well
Table t. Production of infectious virus, viability, and adherence of U937 and HeLa cell cultures infec ted with poliovirus PFU/cell"
HeLa 200 ± 15 HeLa + PMA 145 ± 12 U937 12±3 U937 + PMA 10±3
Fig. 5. Estimation of the percentage of poliovirusinfected cells by a limiting dilution assay. Cultures of either U937 or HeLa cells treated or not with 25 ng/ml PMA for 48 h were incubated with 10 PFU/ml polio virus for 1 h to allow virus absorption. The cells were then washed six times with phosphate-buffered sa line, counted, and diluted in normal medium. Ali quots of each cell dilution were used so that 0.5,1,1.6, or 5 cells/well were added to HeLa cell monolayers grown on 96-well plates. After 72 h at 37°, crystal violet was added to determine the number of wells where the cell monolayer remained intact. B = HeLa cells; □ = HeLa cells treated with PMA: • = U937 cells: O = U937 cells treated with PMA.
ness to human immunodeficiency virus in fection has been observed in PMA-treated U937 cells compared to normal U937 cells due to downmodulation of the surface ex pression of the human immunodeficiency vi rus receptor, the CD4 antigen [25]. Monocyte differentiation of macrophages involves regulated changes of cell surface antigen expression and gene transcription concomitant to the development of specific
Viability °/ob
Adherence %c
< 10 < 10 >90 >90
10-20 10-20 95
Cell cultures of either HeLa or U937 cells pretrea ted or not with 25 ng/ml PMA for 48 h were infected with poliovirus at 10 PFU/cell. a Total PFU production was assayed 24 h after infection. The values shown are the mean of three independent experiments. b Viability was determined by the trypan blue ex clusion techniques h (HeLa cells)or24 h (U937 cells) after infection. c Adherence to plastic was determined by examina tion with a phase-contrast microscope 8 h (HeLa cells) or 24 h (U937 cells) after infection.
macrophage functions such as the oxidative microbicidal metabolism [31, 32]. We have shown that poliovirus infection interfered with the surface expression of the antigens CD4, CDllc, CD14, and 8E11 both in undif ferentiated and differentiated cells. The number of 4F2 surface molecules increased in undifferentiated cells and slightly de creased in differentiated cells, although the number of positive cells remained constant. The fact that the surface expression of CD1 lb did not change in poliovirus-infected cells makes it very unlikely that the changes in the expression observed in other surface markers were due to a generalized proteolytic degrad ation. Although we cannot rule out the possi bility that the surface antigen reactivity could
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Replication o f Poliovirus in Macrophages
145
U-937 -PMA
Poliovirus (h pi.)
Hela
U-937 -P M A
U-937 + PMA
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•
•
•
•
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•
•
•
•
•
•
•
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•
•
•
•
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Fig. 6. Poliovirus RNA replication and protein synthesis in different cells infected with poliovirus. A Cultures of HeLa cells, undifferentiated or differ entiated U937 cells, or human macrophages (MOs) were incubated with 10 PFU/cell poliovirus for 1 h to allow virus adsorption. The cells were then washed to remove nonadsorbed viral particles (zero time) and incubated at 37° for the indicated times. Total RNA was extracted, and starting with 10 pg of RNA, two-
fold erial dilutions were made. RNA samples immo bilized on nitrocellulose were subjected to hybridiza tion to a poliovirus cDNA probe. B Cultures of undif ferentiated or differentiated U937 cells, human mac rophages, or HeLa cells were mock infected or infec ted with 10 PFU/cell poliovirus. After 24 h at 37°, the cells were incubated with 5 pCi [35S]-methionine for 1 h and subjected to PAGE analysis.
be differentially affected by proteolysis, we believe this is not the case, since the varia tions in the expression of surface molecules occurred in cells infected by 10 PFU/cell of poliovirus as early as 5 h after infection, when no CPE was observed. In addition, supporting this view, we have found that, in general, a higher MOl of poliovirus did not
result in a more extensive loss of surface antigen reactivity. The way by which poliovi rus infection interferes with the surface anti gen expression is presently unknown, al though the kinetics of disappearance of some of the markers analyzed suggest that pro cesses involved in membrane protein turn over might be affected.
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B
The capacity to generate oxidative metab olites is a characteristic of phagocytic cells [33]. Mouse macrophages activated by Calmette-Guérin bacillus or lipopolysaccharide produced elevated levels of 0 2 and H20 2and showed enhanced microbicidal capacity against trypanosomes, toxoplasma, and Can dida [34, 35]. Production of 0 2 anions in dif ferentiated U937 cells infected with poliovi rus was reduced to approximately 50% of the level in noninfected cultures, suggesting that poliovirus replication affects either the syn thesis of oxidative metabolites or the devel opment of the enzymatic machinery to pro duce 0 2 and H20 2. The role of oxygen metab olites in mediating virucidal activity in mac rophages is not completely elucidated [33]. In murine macrophage cell lines, the intracellu lar resistance to vesicular stomatitis virus in fection seems to be mediated through nonoxidative mechanisms, although there was some extracellular killing of vesicular stoma titis virus by oxidation [22]. Vaccinia virus and poliovirus are susceptible to cell-free systems able to generate 0 2 and H20 2 [36], However, we found no differences in the rate of poliovirus RNA replication between un differentiated and differentiated U937 cells, and the PFU yield per cell was also similar in both cell types. These observations suggest that 0 2anions do not seem to play any impor tant role in reducing the production of in fectious poliovirus particles in differentiated U937 cells. It has been widely accepted that U937 cells differentiated with PMA represent a bona fide model of human macrophages. However, due to the leukemic origin of U937 cells [12], we decided to carry out compara tive studies of poliovirus replication on hu man macrophages. A lower level of poliovi rus RNA replication was found in these cells
López-Guerrero/Cabañas/Bernabeu/Fresno/Alonso
compared to that in undifferentiated or dif ferentiated U937 cells. A restriction in the translation of poliovirus RNA was evidenced in infected macrophages by PAGE analysis of newly synthesized proteins, as shown in U937 cells [9], Studies on the role of macrophages in poliovirus infection are still lacking. In fectious virus can be recovered from lym phoid tissue in autopsy samples of individu als affected with polyomyelitis [7], Replica tion of poliovirus in lymphoid tissue appears to be important in the establishment of viremia which facilitates the infection of the central nervous system, leading to paralysis. As macrophages are present in lymphoid tis sue, our findings could be useful to under stand how poliovirus and macrophages in teract during the initial steps of poliovirus replication in infected humans, both at the portal of entry and during primary replica tion. They also may help to elucidate the mechanism by which poliovirus evades de struction by the phagocytic cells.
Acknowledgments We acknowledge Dr. Jean de Vries and Dr. F. Sánchez-Madrid for their generous gift of mAbs. J.A.L. istheholderofa FPI fellowship. This work was supported by an institutional grant from the Funda ción Ramón Areces and grants from DG1CYT and F.C. de la A.E.C.C.
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![Docosahexaenoic acid inhibits PAF and LTD4 stimulated [Ca2+]i-increase in differentiated monocytic U937 cells.](/assets/img/hold.png)
