INFETON AND IMMUNITY, Mar. 1975, p. 424-428 Copyright i 1975 American Society for Microbiology

Vol. 11, No. 3 Printed in U.S.A.

Virus Susceptibility of Mouse Hemopoietic Cells In Vitro: Inhibition of Granulocyte-Macrophage Precursor Cells by Newcastle Disease Virus T. A. McNEILL,* M. HAVREDAKI,I E. A. GOULD, AND L. COSBY Department of Microbiology and Immunobiology, The Queen's University of Belfast, Belfast, BT12 6BN, Northern Ireland

Received for publication 11 September 1974

Normal mouse bone marrow cells were exposed to encephalomyocarditis virus (EMC), reovirus type 3 (RE03), influenza virus (FLU), and Newcastle disease virus (NDV) and then assayed for granulocyte-macrophage precursor cells by the technique of colony formation in agar. Exposure to EMC, RE03, and FLU caused a slight but variable loss of colony-forming potential, whereas exposure to NDV caused a very marked loss. NDV acted directly on the cells, not indirectly through release of colony-inhibiting factors or destruction of colony-stimulating factor. Experiments with NDV inactivated by heat, ether, or ultraviolet irradiation indicated that colony inhibition was associated with fully infective virus, even though some of the inactivated preparations had retained full hemagglutinin, neuraminidase, or hemolytic activity.

The technique of colony formation in soft agar culture by granulocyte-macrophage precursor cells has provided a quantitative experimental system by which some aspects of hemopoietic regulation niay be studied in vivo and in vitro (reviewed in 13). The precursor cells which initiate agar colonies are termed colony-forming cells (CFC) and are progeny of the pluripotential hemopoietic stem cells (4, 21). Colony formation in vitro by mouse bone marrow cells is dependent upon the presence of a glycoprotein colony-stimulating (CS) factor which has a concentration-dependent effect upon the number and growth rate of colonies (12). Previous studies on the response and fate of CFC during ectromelia infection indicated that increased numbers could exist in the presence of a high level of infection within the marrow (9, 10). Bro-Jorgensen and Volkert (2), using the in vivo spleen colony assay for pluripotential stem cells, found that mice acutely infected with lymphocytic choriomeningitis virus were incapable of supporting the growth of these cells. However, since colony formation was not inhibited in viremic persistently infected mice the effect was not due to a direct action of virus on the stem cells. The present report extends these observations to the effect of several other viruses on the colony-forming potential of normal mouse bone marrow cells in vitro.

MATERIALS AND METHODS Viruses. Encephalomyocarditis virus (EMC), reovirus type 3 (REO3), influenza Mel strain (FLU), and the Ulster strain of Newcastle disease virus (NDV) were used. EMC and REO3 were grown in monolayer cultures of mouse L cells (L-929, Flow Laboratories, Ltd.). Cells were infected by approximately 1 plaqueforming unit (PFU) per cell, and virus was harvested after the cytopathic effect became pronounced by collecting the culture medium (EMC) or preparing a lysate of infected cells by three cycles of freezing and thawing (REO3). Cell debris was removed by centrifugation, and virus was stored at -70 C. These viruses were titrated by plaque assay on monolayers of L cells in 50-mm plastic dishes under agar. FLU and NDV were grown in the allantoic cavity of 10-day-old embryonated eggs and titrated by infectivity or hemagglutination as described below. Partially purified viruses were obtained by sedimentation in a Spinco L ultracentrifuge with resuspension in Eagle medium (BHK Eagle, Wellcome Reagents Ltd.). NDV inactivation. A preparation of partially purified virus (2 x 10' hemagglutination units [HAUyml) was inactivated by (i) heat (56 C for 60 min), (ii) ether (45 min exposure at 37 C with shaking in a sealed tube followed by ether evaporation with nitrogen), or (iii) ultraviolet irradiation (12 cm from a 30-W source for 60 s; 9 x 103 ergs/s per cm2). Control and inactivated preparations were assayed for infectivity, hemagglutination, neuraminidase, and hemolysin as follows. (i) Infectivity. Ten-day-old fertile hen eggs were inoculated with 0.2 ml of virus dilutions in saline and incubated for 48 h at 37 C. Allantoic fluid from each egg was tested for hemagglutination, and the infectivI Present address: Nuclear Research Center "Democritos," ity titer was estimated by a 50% end point (14). (ii) Hemagglutinin. Serial twofold dilutions of Aghia Paraskevi, Attiki, Greece. 424

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ANTIMARROW EFFECTS OF NDV

virus were mixed in tubes with an equal volume (0.25 ml) of 2% chick erythrocytes in saline and incubated at 37 C for 40 min. Titers were recorded as the reciprocal of the highest dilution showing visible agglutination. (iii) Hemolysin. After estimation of hemagglutinin the tubes were centrifuged at 200 x g for 5 min, supernatant fluids were replaced with 0.5 ml of saline, and erythrocytes were resuspended by shaking and incubated for 2 h at 37 C. Titers were recorded as the reciprocal of the highest dilution showing visible lysis (this represented release of 10% of total hemoglobin). (iv) Neuraminidase. Serial 10-fold dilutions of virus in phosphate-buffered saline (0.25 ml) were mixed with 0.25 ml of 2.4% fetuin substrate, 0.25 ml of 0.4 M sodium phosphate (pH 5.9) and incubated for 18 h at 37 C. Neuraminidase was estimated by a thiobarbituric acid assay (17, 18). Titers were recorded as the dilution which gave an optical density of 0.4 at 549 nm. Cells. Bone marrow cells were obtained by flushing femoral shafts of 6 to 8-week-old mice into collecting medium (BHK Eagle, Wellcome Reagents Ltd.), supplemented by 10% fetal bovine serum (Flow Laboratories Ltd.) and 10% Trypticase soy broth (Difco), and buffered by 16 mM N-2-hydroxyethyl-piperazineN'-2-ethanesulfonic acid. A single-cell suspension was made by gentle pipetting. Exposure of cells to virus. Cells and virus (concentrations given in Results) were mixed in 2 ml of collecting medium in glass tubes and incubated for 2 h at 37 C in a water bath. Cells were maintained in suspension during this period. After this, in most experiments, the cells were washed to remove excess virus using light centrifugation (160 x g). Each experiment included tubes containing cells without virus. Preliminary experiments had confirmed that no loss of CFC occurred under these conditions of preincubation in the absence of virus. CFC culture. Colony cultures were performed in 30-mm Nunclon plastic dishes using the underlayer technique previously described (5). In brief, a 2-ml underlayer of modified Eagle medium containing 1.2% (wt/vol) agar and 5% (vol/vol) mouse embryo conditioned medium (1) to provide an excess of CS factor was placed in each dish. Aliquots of modified Eagle medium containing 0.3% agar were held at 37 C, and virus-treated cells were diluted in this to a concentration of 5 x 104/ml. One-milliliter amounts of each cell suspension were placed on each of four prepared base layers, and the cultures were incubated in sealed boxes containing 10% CO. in humidified air at 37 C for 7 days, when colonies were counted using an Olympus dissecting microscope. RESULTS

Table 1 summarizes the results of all experiments in which cells were exposed to viruses at concentrations of 5 x 106 cells/ml and 5 x 107

PFU (EMC and REO3) or 100 HAU of virus (FLU and NDV). Colony counts were calculated to give the colony-forming potential of virustreated cell suspensions as a percentage of the

TABLE 1. Colony-forming potential of bone marrow cells preincubated with virusesa Virus

No. of expt

EMC REO 3 FLU NDV

12 10 9 6

Colonies per culture (Virus/control x 100) Mean + SD Range

69 s 22 66 15 65 15 1 1

26-94 41-88 50-100 0-2

a Bone marrow cells (5 x 106/ml) were pretreated with 5 x 107 PFU (EMC and REO3) or 100 HAU (FLU and NDV) of virus for 2 h at 37 C. Cells were then cultured in agar at 5 x 104/ml. SD, Standard deviation.

non-virus-treated control in each experiment. The mean plus or minus standard deviation for each group of experiments is shown. Partial purification of the virus preparations or washing of cells after exposure to virus made no difference to the results. It is evident that preincubation with NDV caused an almost complete loss of colony-forming potential and that the other viruses caused a slight but variable (from experiment to experiment) loss of CFC, which, nevertheless, could be significant in some individual experiments when colony counts in control and virus-treated cultures were compared. Other experiments with EMC and FLU showed that a similar variable reduction could be obtained with heat-inactivated virus, and the effect was also seen occasionally when marrow cells were preincubated with lysates of uninfected L cells. When the same marrow and EMC virus suspensions were incubated in a series of different tubes, there was considerable variation in CFC reduction from tube to tube (Table 2), suggesting that this effect depends upon unknown but apparently critical conditions within each tube. Experiments with NDV. Further experiments were performed to study the characteristics of the very pronounced CFC inactivation caused by NDV. Table 3 shows the results of five separate experiments in which bone marrow cells at 5 x 106/ml were preincubated with dirt:erent concentrations of either unpurified or ,partially purified NDV, washed, and cultured at 5 x 104 cells/ml of agar culture. Over the midrange of the virus concentrations used the number of surviving CFC was indirectly proportional to the virus concentration in the preincubation mixture. Unpurified virus had the same colonyinhibiting activity relative to hemagglutinin concentration as the partially purified preparation, suggesting that the effect was associated

426

McNEILL ET AL.

INFECT. IMMUN.

TABLE 2. Colony-forming potential of bone marrow cells preincubated with EMC virusa Cell suspension

Control 1 2 3 4 5

Coloniesofper SD) (Avg 4 ±iculture

72 9 72±5 74±6 75 8 75±7

es

CL

10

z

_

Virus 1 2 3 4 5

69±8 56±3 64±3 50±6 45±5

aResults are for five replicate control and virustreated suspensions of the same bone marrow. Bone marrow cells (5 x 106/ml) were pretreated with 5 x 107 PFU of EMC virus for 2 h at 37 C. Cells were then cultured in agar at 5 x 10'/ml. SD, Standard deviation.

MINUTES

FIG. 1. Kinetics of CFC inactivation by NDV; colonies per culture (average of 4) with untreated cells and with cells exposed to 12.5 (A), 25 (0), or 50 (0) HAU of virus for different times.

bone marrow colony growth (3, 6-8). Bone marrow cells at 5 x lOf/ml were incubated with or without 100 HAU of partially purified NDV for 2 h at 37 C, washed, and Colonies per culture (avg of 4 + SD) Virus concn cultured at 5 x 10' cells/ml for 1, 4, or 7 days. At (HAU) Expt 1 Expt 2 Expt 3 Expt 4 Expt 5 each of these times the top cell-containing layer was removed from some cultures, and the unNil (control) 52 + 2 70 5 67 5 55 + 3 52 t 3 derlayers were reused for fresh marrow cultures. 0 0 200 NT NT NT 1 0 0 0 0 100 The colony counts in these secondary cultures 0 50 2 1 2 1 1 2 1 after 7 days of incubation were expressed as a 4±1 5±1 6±2 2 1 5±1 25 percentage of those for unconditioned underlay10±2 10±2 12±3 7±2 11 4 12.5 ers incubated for the same period as the condi18±3 19±2 24±1 18±3 23±6 6.25 34 ± 3 41 2 27 2 34 3 3.125 NT tioned underlayers. Table 4 shows the results of 1.6 NT 51 3 NT NT 42 + 4 such an experiment, where it is clear that the a Bone marrow cells (5 x 106/ml) were pretreated with the virus-treated cells did not release detectable given virus concentrations for 2 h at 37 C. Cells were then colony inhibitors into the culture underlayers. It cultured in agar at 5 x 10'/ml. SD, Standard deviation; NT, should be noted that, in other experiments not tested. where the cell concentration in primary cultures was 106/ml instead of 5 x 10', underlayers with the virus particles. Figure 1 shows the conditioned by NDV-treated cells developed a kinetics of CFC inactivation by partially puri- very marked inhibitory effect compared with fied virus in an experiment where three virus concentrations were used. Amounts (0.05 ml) of virus-cell mixtures were withdrawn at 5-min TABLE 4. Conditioning effect of 5 x 104 normal and NDV-treated bone marrow cells on culture intervals after mixing and immediately diluted underlayers after 1, 4, or 7 days of incubation 1:100 in Eagle-agar medium for colony culture. These results again indicate the proportionality Primary culturesa conditioned for Bone marrow between CFC inhibition and virus concentracells 1 Day 4 Days 7 Days tion with single-hit inactivation kinetics. These findings suggested that CFC inactivaNormal 99 + 13 100 + 11 92 ± 6 tion by NDV was due to a direct action of virus NDV 91 ± 10 treated 94 + 3 92 ± 10 on the cells. However, several indirect mecha anisms were also considered since, for example, Colonies per culture in secondary cultures are NDV is well known to be a potent inducer of expressed as a percentage of those of unconditioned interferon and interferon causes inhibition of primary cultures incubated for the same periods. TABLE 3. Effect of NDV concentration on the colony-forming potential of bone marrow cellsa

ANTIMARROW EFFECTS OF NDV

VOL. 11, 1975

untreated cells (unpublished data), which was possibly due to release of interferon. The results given in Table 4 show that colony-inhibiting factors are unlikely to be an important cause of CFC inhibition by NDV under the experimental conditions used for the present observations. CS factor is sensitive to some neuraminidase preparations (15), and since NDV contains this enzyme an indirect effect of virus on colony growth could be caused by inactivation of CS factor. However, experiments in which conditioned medium was incubated with 100 HAU (per ml) of virus, centrifuged, and tested in parallel with untreated material did not show any fall in CS activity. All experiments indicated, therefore, that CFC inactivation by NDV was due to a direct effect of the virus particles. The fact that FLU did not have the same effect (Table 1) suggested that virus hemagglutinin or neuraminidase was not responsible. This conclusion was supported by failure to demonstrate any competitive effect of sialic acid (Sigma Ltd.; synthetic crystalline, 1 mg/ml) included in the NDV-cell preincubation mixture. NDV inactivated by heat, ether, or ultraviolet light gave preparations which varied in infectivity and hemagglutinating, neuraminidase, and hemolytic activities. Table 5 shows the effect of these preparations on colony formation and indicates that only the fully infective virus gave clear inhibition. The ultraviolet-inactivated preparation gave a slight colony-inhibiting effect when tested at high concentration (200 HAU/ml) in the bone marrow preincubation mixture. DISCUSSION These experiments show that exposure of mouse

bone

marrow

cells to EMC, RE03, FLU,

TABLE 5. Marrow colony-inhibiting activity of NDV preparations relative to other viral activities Infec NDV prepn

Untreated Ultraviolet treated Ether treated Heat treated a

Colony tivity Hemag- Neura- Hemolinhibi- (log1o glumini- ysidn tion EID5/ tinin° dasec Y

+++ 4 -

-

10.5

Virus susceptibility of mouse hemopoietic cells in vitro: inhibition of granulocyte-macrophage precursor cells by Newcastle disease virus.

INFETON AND IMMUNITY, Mar. 1975, p. 424-428 Copyright i 1975 American Society for Microbiology Vol. 11, No. 3 Printed in U.S.A. Virus Susceptibility...
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