INFECTION AND IMMUNITY, Aug. 1979, p. 565-568 0019-9567/79/08-0565/04$02.00/0
Vol. 25, No. 2
Depression of Bovine Monocyte Chemotactic Responses by Bovine Viral Diarrhea Virus ARLEN T. KETELSEN, DONALD W. JOHNSON, AND CHARLES C. MUSCOPLAT* Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108
Received for publication 2 May 1979
Incubation of bovine peripheral blood monocytes with bovine viral diarrhea virus (either Singer or NY-1 strain) caused a consistent, statistically significant decrease in their random locomotion (no chemoattractant) and chemotaxis towards a chemotactic lymphokine. Chemotaxis was determined by a modification of the Boyden method. Incubation of bovine viral diarrhea virus with mononuclear cells depressed chemotaxis by a mean of 56% (P < 0.0005). Heat-killed virus had no effect on monocyte motility. Data suggest that bovine viral diarrhea virus can rapidly suppress monocyte functions in vitro, but by unknown mechanisms, not by killing cells.
Mononuclear leukocytes are important cells involved in host immunity. The accumulation of macrophages at local sites of inflammation is of particular importance in host defense against infectious agents (5). The ability of some viruses to alter the functions of an infected host's immune system is also well recognized (8, 14, 19, 20). _ The bovine viral diarrhea (BVD) virus has an apparent affinity for cells of the immune system. In vivo, BVD virus has been shown to cause necrotic lesions and destruction of cells and tissues within lymphoid elements (11, 12), and to produce immunological abnormalities in BVD virus-infected calves (6, 12, 13). It has also been demonstrated in vitro that lymphocytes and macrophages prepared from peripheral blood supported the replication of BVD virus (16, 18). Cattle chronically infected with BVD virus generally exhibit signs of immune dysfunction, such as lymphocyte unresponsiveness (6), lack of specific neutralizing antibody (12), and recurring severe secondary bacterial infections. Any abnormality in a host's ability to localize monocytes and/or macrophages at antigenically stimulated sites would be expected to have profound effects on the rest of the immune system as well as on the immune status of the host in general. Consequently, since many tissue macrophages are derived from the circulating pool of peripheral blood monocytes (4), any abnormal chemotactic response by the peripheral monocytes could result in significantly increased morbidity or mortality due to infectious organisms. The purpose of the present study was to see whether inhibition of the chemotactic responsiveness of circulating peripheral monocytes is
one possible mechanism by which BVD virus can depress cellular immune responses. MATERIALS AND METHODS Animals. Ten healthy, normal cattle, 9 to 15 months of age, consisting of various breeds and of both sexes, were used in this study. Preparation of mononuclear cells. Blood was obtained by jugular venipuncture using heparinized Vacutainer tubes. Mononuclear cells were fractionated out by the standard Ficoll-Hypaque gradient technique (2) and subsequently washed two times with Hanks balanced salt solution. Cells were then counted and diluted to a concentration of 5 x 106/nml in RPMI1640 media, supplemented with 25 mM HEPES (N-2hydroxyethyl piperazine-N'-2-ethanesulfonic acid) buffer, 0.3% bovine serum albumin, and 25 Ig of gentamicin per ml. Viruses. In this study, two strains of BVD virus were tested for chemotactic inhibitory activity. The cytopathic Singer strain and the noncytopathic NY-1 strain of BVD virus were grown on monolayers of bovine embryonic turbinate cells (BT cells) in roller bottles. When the cytopathic effect due to the Singer strain reached 60%, the culture fluid was removed, pooled, and centrifuged at 2,000 x g for 15 min to remove cell debris. The supernatant was decanted from the cell debris pellet and ultracentrifuged at 82,500 x g for 35 min. The supernatant was discarded, and the pellet containing virus was resuspended in 10 ml of RPMI1640 containing 25 mM HEPES, 0.3% bovine serum albumin, and 25 ,ug of gentamicin per ml and frozen in 1-ml aliquots at -70'C. Subsequent titration on BT cells gave a titer of 5 x 107 50% tissue culture infective doses per ml. For use in this chemotaxis assay, this higher-titered stock was diluted in the same medium as above, to 2.5 x 105 50% tissue culture infective doses per ml. The NY-I strain of BVD virus was grown for 5 days,
565
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KETELSEN, JOHNSON, AND MUSCOPLAT
after which the culture fluid was pooled and centrifuged at 2,000 x g for 15 min to remove cell debris. The cell debris pellet was discarded, and the supernatant was frozen in 1-ml aliquots at -70'C. Subsequent titration by the interference method yielded a result of 5 x 105 50% tissue culture infective doses per ml, and the culture fluid was used at this titer throughout the rest of the experiment. Culture fluid from uninfected monolayers of BT cells was handled in the same manner as the NY-I strain and later used as control fluid for the incubation period. Chemotactic factor. The chemotactic factor used in this experiment was obtained by incubating purified mononuclear leukocytes in serum-free RPMI-1640 medium containing 6 /ig of concanavalin A and 25 jig of gentamicin per ml. After 72 h of incubation at 370C, the supernatant was harvested by centrifugation at 2,000 x g for 10 min and frozen in 1-ml aliquots at -70'C. Control chemotactic factor was obtained in an identical manner, except no concanavalin A was added to the media. Prior to this experiment, testing of this lymphocyte-derived chemotactic fluid (LDCF) determined the optimal concentration for chemotaxis to be at a 50% concentration. Concanavalin A by itself had no affect on monocyte chemotactic responses. Incubation of mononuclear cells with virus. Either Singer strain or NY-I strain of BVD was added to the purified mononuclear cells at a ratio of 1 ml of virus to 4 ml of cells. This mixture was then placed on a slow rotator for 30 min at 370C. After this absorption period, the cells were washed in Hanks balanced salt solution to get rid of any unabsorbed viral particles and then suspended in RPMI-1640 supplemented with 25 mM HEPES, 0.3% bovine serum albumin, and 25 Mg of gentamicin per ml to the original concentration of 5 x 106 cells per ml. As a control experiment, to see if only absorption of the viral particles is necessary to depress chemotaxis, heat-killed (60°C for 2 h) virus of both strains of BVD was incubated with cells in an identical manner as above. Also, as a second control experiment, control fluid supernatant from uninfected tissue cultures was incubated with cells in an identical manner as above. Chemotactic assay. Chemotactic assays were performed by modifying the double-filter membrane method of Keller et al. (7) for mononuclear leukocytes. In comparison to the conventional leukotaxis assay based on the original work of Boyden (1), employing one filter membrane, this assay, utilizing two filter membranes, offers increased reliability, reproducibility, and sensitivity (3). Briefly, the procedures involve using Blind-Well chemotactic chambers (Neuroprobe, Inc., Bethesda, Md.); 0.2 ml of the 50% LDCF or control chemotactic medium, prewarmed to 37°C, is placed in the lower well of the chamber. A lower cellimpermeable 0.45-,um pore size Millipore membrane filter is inserted, followed by a second, upper Millipore membrane filter with cell-permeable 8.0-nm pore size. The filter retainer is inserted into the upper well, followed by 0.5 ml of the incubated cell suspension of 5 x 10' cells per ml. The assembled chamber is then incubated for 3 h at 37°C in a 5% CO2-95% air atmosphere. After the incubation period, the filters are removed from the chamber, fixed in methanol, stained, dehy-
INFECT. IMMUN.
drated, and cleared in xylene before being mounted on glass slides. Chemotactic responses are scored by counting at least 20 random high-powered fields (HPF) (X450) of measured size and averaging the numbers of cells that have migrated completely through the cell-permeable filter. The random migratory activity of monocytes was assessed by determining the number of cells that had migrated completely through the cell-permeable filter in response to the control chemotactic medium alone (8-10).
RESULTS The monocyte chemotactic response to LDCF was determined after the cells were exposed to BVD virus in vitro. Figure 1, which is the graph of monocytes exposed to Singer strain of BVD virus, clearly shows a significant depression (P < 0.0005) of the chemotactic response of cells exposed to the virus before being assayed with LDCF. This is in marked contrast to the response of cells incubated with tissue culture control fluid only before assaying with LDCF. The mean response of the cells incubated with tissue culture control fluid was 35.9 cells per HPF, whereas the cells that had been first exposed to the virus had a mean of 14.97 cells per HPF. Figure 2, which shows the results of cells exposed to the NY-i strain of BVD virus, shows 60 60
5050
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CL
4'
O
0.
k 8a
4)
0
430
C.)
0m
E * 200
O
10-
O
10
00I
r
_
LDCF+CF
LDCF+BVD
CF only
BVD only
FIG. 1. Depression of bovine monocyte chemotactic responses by Singer strain of B VD virus. Suppression of migration occurred in cultures incubated with LDCF as well as in control cultures showing only random migration.
VOL. 25, 1979
BVD DEPRESSION OF BOVINE MONOCYTES
chemotactic response. When the normal response to LDCF was tested for cells that were not exposed to virus, the mean response of 48 test replicates was 35.6 cells per HPF. When the cells were assayed for normal random background activity, 46 test replicates gave a mean response of 14.85 cells per HPF. But if the cells were exposed to NY-1 or Singer BVD virus before assaying with LDCF, the response was only 16 or 15.97 cells per HPF, respectively. If we consider cells incubated without virus as having 100% of the normal migratory response, we see (Table 2) that cells exposed to NY-1 BVD virus before assaying with LDCF had a mean inhibition of the normal chemotactic response of 55.1%. Likewise, cells exposed to Singer BVD virus before testing with LDCF were inhibited by 57.9%. The random migration can also be seen to be inhibited by both strains of virus as compared to the nonexposed cells.
0
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600
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j 50U
S
8
a:0 0.
CM 40a a
.20 Lu
301
S E
8
0 0
U
OD
20 00
0
8 0
am
10-I
LDCF+CF LDCF+BVD
CF only
BVD only
FIG. 2. Depression of bovine monocyte chemotactic by NY-I strain of B VD virus. Suppression of migration occurred in cultures incubated with LDCF as well as in control cultures showing only random migration. responses
567
DISCUSSION The results of this study show that there is a marked depression of the normal response of bovine monocytes to a chemotactic stimulus, if these cells are exposed to BVD virus. Other investigators have found a similar response in monocytes exposed to other viruses. It has been shown that patients with acute influenza have a dramatic and transient depression of their peripheral blood monocyte chemotactic responsiveness (10, 15). When normal human periphTABLE 1. Monocyte chemotactic responsiveness of normal mononuclear leukocytes after incubation with either LDCF or control fluid and B VD virus Virus strain
N'
LDCF Cells per HPFh
N
Control fluid Cells per HPF
48 35.6 ± 1.76 47 14.85 ± 0.3 No virus 26 16.0 ± 0.73 18 12.88 ± 0.45 NY-1 33 14.97 ± 0.48 29 13.79 ± 0.28 Singer aN, Number of determinations. b Number of cells per HPF ± 1 standard error of the mean.
pattern of depression similar to that seen in Fig. 1. Cells incubated with NY-1 virus before assaying with LDCF had a mean of 16 cells per HPF, which is significantly lower (P < 0.0005) than the mean response (35.1 cells per HPF) of cells that were not exposed to the virus, yet were TABLE 2. Inhibition of bovine monocyte assayed in an identical manner. chemotactic responsiveness of normal mononuclear If cells were exposed to heat-killed BVD virus leukocytes after incubation with LDCF or control fluid and BVD virus before assaying with LDCF or control chemotactic medium, the results of both test conditions LDCF Control fluid were the same as those for cells not exposed to Virus strain % Sup% Suppa p any virus at all. To see whether heat-killed BVD pression pression virus could cause cell death, viability of the cells 0 0 No virus was tested by the trypan blue exclusion method 13.3
Vol. 25, No. 2
Depression of Bovine Monocyte Chemotactic Responses by Bovine Viral Diarrhea Virus ARLEN T. KETELSEN, DONALD W. JOHNSON, AND CHARLES C. MUSCOPLAT* Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108
Received for publication 2 May 1979
Incubation of bovine peripheral blood monocytes with bovine viral diarrhea virus (either Singer or NY-1 strain) caused a consistent, statistically significant decrease in their random locomotion (no chemoattractant) and chemotaxis towards a chemotactic lymphokine. Chemotaxis was determined by a modification of the Boyden method. Incubation of bovine viral diarrhea virus with mononuclear cells depressed chemotaxis by a mean of 56% (P < 0.0005). Heat-killed virus had no effect on monocyte motility. Data suggest that bovine viral diarrhea virus can rapidly suppress monocyte functions in vitro, but by unknown mechanisms, not by killing cells.
Mononuclear leukocytes are important cells involved in host immunity. The accumulation of macrophages at local sites of inflammation is of particular importance in host defense against infectious agents (5). The ability of some viruses to alter the functions of an infected host's immune system is also well recognized (8, 14, 19, 20). _ The bovine viral diarrhea (BVD) virus has an apparent affinity for cells of the immune system. In vivo, BVD virus has been shown to cause necrotic lesions and destruction of cells and tissues within lymphoid elements (11, 12), and to produce immunological abnormalities in BVD virus-infected calves (6, 12, 13). It has also been demonstrated in vitro that lymphocytes and macrophages prepared from peripheral blood supported the replication of BVD virus (16, 18). Cattle chronically infected with BVD virus generally exhibit signs of immune dysfunction, such as lymphocyte unresponsiveness (6), lack of specific neutralizing antibody (12), and recurring severe secondary bacterial infections. Any abnormality in a host's ability to localize monocytes and/or macrophages at antigenically stimulated sites would be expected to have profound effects on the rest of the immune system as well as on the immune status of the host in general. Consequently, since many tissue macrophages are derived from the circulating pool of peripheral blood monocytes (4), any abnormal chemotactic response by the peripheral monocytes could result in significantly increased morbidity or mortality due to infectious organisms. The purpose of the present study was to see whether inhibition of the chemotactic responsiveness of circulating peripheral monocytes is
one possible mechanism by which BVD virus can depress cellular immune responses. MATERIALS AND METHODS Animals. Ten healthy, normal cattle, 9 to 15 months of age, consisting of various breeds and of both sexes, were used in this study. Preparation of mononuclear cells. Blood was obtained by jugular venipuncture using heparinized Vacutainer tubes. Mononuclear cells were fractionated out by the standard Ficoll-Hypaque gradient technique (2) and subsequently washed two times with Hanks balanced salt solution. Cells were then counted and diluted to a concentration of 5 x 106/nml in RPMI1640 media, supplemented with 25 mM HEPES (N-2hydroxyethyl piperazine-N'-2-ethanesulfonic acid) buffer, 0.3% bovine serum albumin, and 25 Ig of gentamicin per ml. Viruses. In this study, two strains of BVD virus were tested for chemotactic inhibitory activity. The cytopathic Singer strain and the noncytopathic NY-1 strain of BVD virus were grown on monolayers of bovine embryonic turbinate cells (BT cells) in roller bottles. When the cytopathic effect due to the Singer strain reached 60%, the culture fluid was removed, pooled, and centrifuged at 2,000 x g for 15 min to remove cell debris. The supernatant was decanted from the cell debris pellet and ultracentrifuged at 82,500 x g for 35 min. The supernatant was discarded, and the pellet containing virus was resuspended in 10 ml of RPMI1640 containing 25 mM HEPES, 0.3% bovine serum albumin, and 25 ,ug of gentamicin per ml and frozen in 1-ml aliquots at -70'C. Subsequent titration on BT cells gave a titer of 5 x 107 50% tissue culture infective doses per ml. For use in this chemotaxis assay, this higher-titered stock was diluted in the same medium as above, to 2.5 x 105 50% tissue culture infective doses per ml. The NY-I strain of BVD virus was grown for 5 days,
565
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KETELSEN, JOHNSON, AND MUSCOPLAT
after which the culture fluid was pooled and centrifuged at 2,000 x g for 15 min to remove cell debris. The cell debris pellet was discarded, and the supernatant was frozen in 1-ml aliquots at -70'C. Subsequent titration by the interference method yielded a result of 5 x 105 50% tissue culture infective doses per ml, and the culture fluid was used at this titer throughout the rest of the experiment. Culture fluid from uninfected monolayers of BT cells was handled in the same manner as the NY-I strain and later used as control fluid for the incubation period. Chemotactic factor. The chemotactic factor used in this experiment was obtained by incubating purified mononuclear leukocytes in serum-free RPMI-1640 medium containing 6 /ig of concanavalin A and 25 jig of gentamicin per ml. After 72 h of incubation at 370C, the supernatant was harvested by centrifugation at 2,000 x g for 10 min and frozen in 1-ml aliquots at -70'C. Control chemotactic factor was obtained in an identical manner, except no concanavalin A was added to the media. Prior to this experiment, testing of this lymphocyte-derived chemotactic fluid (LDCF) determined the optimal concentration for chemotaxis to be at a 50% concentration. Concanavalin A by itself had no affect on monocyte chemotactic responses. Incubation of mononuclear cells with virus. Either Singer strain or NY-I strain of BVD was added to the purified mononuclear cells at a ratio of 1 ml of virus to 4 ml of cells. This mixture was then placed on a slow rotator for 30 min at 370C. After this absorption period, the cells were washed in Hanks balanced salt solution to get rid of any unabsorbed viral particles and then suspended in RPMI-1640 supplemented with 25 mM HEPES, 0.3% bovine serum albumin, and 25 Mg of gentamicin per ml to the original concentration of 5 x 106 cells per ml. As a control experiment, to see if only absorption of the viral particles is necessary to depress chemotaxis, heat-killed (60°C for 2 h) virus of both strains of BVD was incubated with cells in an identical manner as above. Also, as a second control experiment, control fluid supernatant from uninfected tissue cultures was incubated with cells in an identical manner as above. Chemotactic assay. Chemotactic assays were performed by modifying the double-filter membrane method of Keller et al. (7) for mononuclear leukocytes. In comparison to the conventional leukotaxis assay based on the original work of Boyden (1), employing one filter membrane, this assay, utilizing two filter membranes, offers increased reliability, reproducibility, and sensitivity (3). Briefly, the procedures involve using Blind-Well chemotactic chambers (Neuroprobe, Inc., Bethesda, Md.); 0.2 ml of the 50% LDCF or control chemotactic medium, prewarmed to 37°C, is placed in the lower well of the chamber. A lower cellimpermeable 0.45-,um pore size Millipore membrane filter is inserted, followed by a second, upper Millipore membrane filter with cell-permeable 8.0-nm pore size. The filter retainer is inserted into the upper well, followed by 0.5 ml of the incubated cell suspension of 5 x 10' cells per ml. The assembled chamber is then incubated for 3 h at 37°C in a 5% CO2-95% air atmosphere. After the incubation period, the filters are removed from the chamber, fixed in methanol, stained, dehy-
INFECT. IMMUN.
drated, and cleared in xylene before being mounted on glass slides. Chemotactic responses are scored by counting at least 20 random high-powered fields (HPF) (X450) of measured size and averaging the numbers of cells that have migrated completely through the cell-permeable filter. The random migratory activity of monocytes was assessed by determining the number of cells that had migrated completely through the cell-permeable filter in response to the control chemotactic medium alone (8-10).
RESULTS The monocyte chemotactic response to LDCF was determined after the cells were exposed to BVD virus in vitro. Figure 1, which is the graph of monocytes exposed to Singer strain of BVD virus, clearly shows a significant depression (P < 0.0005) of the chemotactic response of cells exposed to the virus before being assayed with LDCF. This is in marked contrast to the response of cells incubated with tissue culture control fluid only before assaying with LDCF. The mean response of the cells incubated with tissue culture control fluid was 35.9 cells per HPF, whereas the cells that had been first exposed to the virus had a mean of 14.97 cells per HPF. Figure 2, which shows the results of cells exposed to the NY-i strain of BVD virus, shows 60 60
5050
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CL
4'
O
0.
k 8a
4)
0
430
C.)
0m
E * 200
O
10-
O
10
00I
r
_
LDCF+CF
LDCF+BVD
CF only
BVD only
FIG. 1. Depression of bovine monocyte chemotactic responses by Singer strain of B VD virus. Suppression of migration occurred in cultures incubated with LDCF as well as in control cultures showing only random migration.
VOL. 25, 1979
BVD DEPRESSION OF BOVINE MONOCYTES
chemotactic response. When the normal response to LDCF was tested for cells that were not exposed to virus, the mean response of 48 test replicates was 35.6 cells per HPF. When the cells were assayed for normal random background activity, 46 test replicates gave a mean response of 14.85 cells per HPF. But if the cells were exposed to NY-1 or Singer BVD virus before assaying with LDCF, the response was only 16 or 15.97 cells per HPF, respectively. If we consider cells incubated without virus as having 100% of the normal migratory response, we see (Table 2) that cells exposed to NY-1 BVD virus before assaying with LDCF had a mean inhibition of the normal chemotactic response of 55.1%. Likewise, cells exposed to Singer BVD virus before testing with LDCF were inhibited by 57.9%. The random migration can also be seen to be inhibited by both strains of virus as compared to the nonexposed cells.
0
700o
600
0. IN
j 50U
S
8
a:0 0.
CM 40a a
.20 Lu
301
S E
8
0 0
U
OD
20 00
0
8 0
am
10-I
LDCF+CF LDCF+BVD
CF only
BVD only
FIG. 2. Depression of bovine monocyte chemotactic by NY-I strain of B VD virus. Suppression of migration occurred in cultures incubated with LDCF as well as in control cultures showing only random migration. responses
567
DISCUSSION The results of this study show that there is a marked depression of the normal response of bovine monocytes to a chemotactic stimulus, if these cells are exposed to BVD virus. Other investigators have found a similar response in monocytes exposed to other viruses. It has been shown that patients with acute influenza have a dramatic and transient depression of their peripheral blood monocyte chemotactic responsiveness (10, 15). When normal human periphTABLE 1. Monocyte chemotactic responsiveness of normal mononuclear leukocytes after incubation with either LDCF or control fluid and B VD virus Virus strain
N'
LDCF Cells per HPFh
N
Control fluid Cells per HPF
48 35.6 ± 1.76 47 14.85 ± 0.3 No virus 26 16.0 ± 0.73 18 12.88 ± 0.45 NY-1 33 14.97 ± 0.48 29 13.79 ± 0.28 Singer aN, Number of determinations. b Number of cells per HPF ± 1 standard error of the mean.
pattern of depression similar to that seen in Fig. 1. Cells incubated with NY-1 virus before assaying with LDCF had a mean of 16 cells per HPF, which is significantly lower (P < 0.0005) than the mean response (35.1 cells per HPF) of cells that were not exposed to the virus, yet were TABLE 2. Inhibition of bovine monocyte assayed in an identical manner. chemotactic responsiveness of normal mononuclear If cells were exposed to heat-killed BVD virus leukocytes after incubation with LDCF or control fluid and BVD virus before assaying with LDCF or control chemotactic medium, the results of both test conditions LDCF Control fluid were the same as those for cells not exposed to Virus strain % Sup% Suppa p any virus at all. To see whether heat-killed BVD pression pression virus could cause cell death, viability of the cells 0 0 No virus was tested by the trypan blue exclusion method 13.3
