Veterinary Immunology and Immunopathoiogy, 29 ( 1991 ) 251-265
251
Elsevier Science Publishers B.V., Amsterdam
Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin- 1 activity of bovine monocytes* J. Jensen ~ and R.D. Schultz Department of Pathobiological Sciences, School of VeterinaryMedicine, Universityof WisconsinMadison, Madison, W153 706, USA (Accepted 12 November 1990)
ABSTRACT Jensen, J. and Schultz, R.D., 1991. Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin-1 activity of bovine monocytes. Vet. Immunol. Immunopathol., 29:251-265. The effect of bovine viral diarrhea virus (BVDV) infection in vitro on the interleukin-I (IL-I) activity of bovine monocytes was studied. Supernatants from BVDV-infected monocytes suppressed IL-l-stimulated proliferation of mouse thymocytes and masked lipopolysaccharide-stimulated IL-I activity of bovine monocytes in the mouse comitogen thymocyte assay. Suppression of mouse thymocyte proliferation was restored by the addition of IL-1. IL-1 inhibitory activity was induced both by the prototype variants BVDV/NADi. cytopathic and BVDV/NY-1 noncytopathic and by BVDV variants isolated from persistently infected cattle. Suppressed IL-I a ~ ; u v l t y w i a ~ a l a o found in supernatants from monocytes from persistently infected cattle following infection with BVDV in vitro. No differences in levels of IL-l mRNA synthesis were detected between BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. These results suggest that infection of bovine monocytes with BVDV results in the production and/or activation of a soluble inhibitor of IL-l activity. ABBREVIATIONS BVDV, bovine viral diarrhea virus; CMV, cytomegalovirus; Con A, concanavalin A; CP, cytopathic; FBS, fetal bovine serum; HIV, human immunodeficiency virus; IL-l, interleukin-1; LBT, lymphocyte blastogenesis test; LPS, lipopolysaccharide; NCP, non-cytopathic; PBML, peripheral blood mononuclear leukocyte; PHA, phytohemagglutinin; PI, persistently infected; RSV, respiratory syncytial virus; VSV, vesticular stomatitis virus. ~Present address: Department of Surgery, University of Wisconsin Medical School, 4679 Medical Science Center, 1300 University Avenue, Madison, WI 53706, U.S.A. *This work was supported in part by the large Animal Research Fund, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706 and by USDA-CSRA Grant 89-341164528.
0165-2427/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.
252
J. JENSEN AND R.D. SCHULTZ
INTRODUCTION
Infection of cattle by bovine viral diarrhea virus (BVDV) can cause generalized or specific immunosuppression. Postnatal infection with BVDV resuits in increased susceptibility of the infected host to secondary infection by bacteria or other viruses (Reggiardo and Kaebede, 1981; Potgeiter et al., 1984a,b). Infection with BVDV in utero can result in specific immunotolerance to the infecting strain of BVDV, depending on the age of the fetus at the time of infection and the biotype of the infecting BVDV (Brownlie et al., 1984; McClurkin et al., 1985; reviewed by Radostits and Littlejohns, 1988). In the infected host, BVDV replicates in lymphoid tissues, including B cells, T cells, monocytes and macrophages (Truitt and Schechmeister, 1973; Bolin et at., 1985). BVDV-induced suppression of several cellular immune functions, including T cell proliferation (Muscoplat et al., 1973; Pospisil et al., 1975), neutrophil phagocytic activity (Roth and Kaebefle, 1983; Roth et al., 1986) and monocyte chemotactic response (Akkina et al., 1979) has been observed in vitro and in vivo. Interleuidn-1 (IL-1 ) is an immunoregulatory molecule with a wide range of biological activities, including T cell maturation, B cell activation and stimulation of fibroblast and epidermal cell proliferation (Dinarello, 1985; Durum et al., 1985; reviewed by Oppenheim et al., 1986). BVDV infection of monocytes and macrophages and consequent perturbation of IL- 1 activity by BVDV could result in suppression of the host's immune response. Perturbation ofIL-1 activity following viral infection of monocytes or macrophages has been noted with several human viruses, including cytomegalovirus (CMV) (Kapasi and Rice, 1988 ), respiratory syncytial virus (RSV) and influenza virus [ Koberts et ai., i 986; McCarthy et ai., 1989) and human immunodeficiency virus (HIV) (Berman et al., 1987). Infection of macrophages with these viruses causes botih enhanced production of IL-I activity and induction of humoral inhibitors of IL-I activity. In some cases, virusinduced humoral inhibitors of IL-1 activity have been associated with antiproliferative effects of mitogen-stimulated T cell proliferation (McCarthy et al., 1989) and suppressed B cell maturation (Berman et al., 1986). Considering the immunosuppressive effect of BVDV infection on the host's immune response and the central immunomodulatory role of IL-1, the present study was designed to look at the effect of in vitro BVDV infection of bovine monocytes on IL- 1 activity. MATERIALS AND METHODS
Animals Adult cattle not infected with BVDV and three persistently infected (PI) cattle (Jensen et al., 1990) were used.
253
EFFECT OF BVDVON BOVINE MONOCYTES
Viruses and cell cultures BVDV/NADL was obtained from the stock virus collection at the James A. Baker Institute for Animal Health, Cornell University. BVDV/NY-I was obtained from Dr. S.R. Bolin, National Animal Disease Center, Ames, IA. Virus was propagated in mycop!asma-free primary bovine testicle cells in minimal essential media (MEM) with 10% fetal bovine serum (FBS) (low endotoxin lot 127F-0279, Sigma, St. Louis, MO) and 1% penicillin-streptomycin. The FBS used was free of noncytopathie (NCP) BVDV and did not have detectable antibody to BVDV. Cytopathic (CP) BVDV/NADL was titrated in bovine testicle cells by limiting dilution. NCP BVDV/NY-1 and the three NCP field isolates of BVDV from PI cattle were titrated in bovine testicle cells by limiting dilution, using the indirect fluorescent antibody technique as described previously (Jensen et al., 1990). For preparation of inactivated BVDV, virus was ~,-irradiated (3000 rads) or heat-inactivated at 60°C for 2 h (Atluru et al., 1979).
Preparation of monocyte-derived supernatants Bovine peripheral blood mononuclear leukocytes (PBML) were isolated by centrifugation over Ficoll-hypaque (Schultz, 1982) and resuspended in RPMI- 1640 media with 1% penicillin-streptomycin (RPMI) and 10% FBS (Jensen et al., 1988). PBML were plated in 24-well tissue culture plates (Coming Cell Wells, Coming, NY) at a cell density of 1 X 106 cells/cm 2 (2.6× 106 cells per well), incubated for 2 h at 37°C and nonadherent cells were removed by washing with RPMI. To prepare BVDV-infected monocyte cultures, monocytes were infected with 1 × 10s TCIDso BVDV (multiplicity of infection, two to five) in RPMI, the plates incubated for 1 h at 37°C and l~ g,.nL Aa~ ,rg . l~ ,c~ V,l ~ r hK,, .~l ~ ttJ Jt. .•, A n l c r,~mrLv,~tl hL , IvJ u~a©hlnt~ uV Vl l.e.t• h JLA ~ e , ~ m. ~ g A A ~ J • ~l~. vv V~AAAAA~
R P~.4I
I Indar
thane
~anditian.~
more than 90% of adherent cells are infected with BVDV as determined by indirect fluorescent antibody staining (Jensen et al., 1990). Uninfected monocytes were incubated with RPMI for 1 h and then washed. RPMI with 10% FBS or RPMI with 10% FBS and i 0 gg/ml LPS (Escherichia coli lipopolysaccharide (LPS) 0.55:B5; Difco Laboratories, Detroit, MI) was then added to both infected and uninfected monocytes, the cultures incubated for 18 h at 37°C and the supernatants harvested by centrifugation (Lederer and Czuprynski, 1989). The 18 h incubation was chosen based on data from initial experiments showing optimal IL-l inhibitory activity in supernatants harvested at this time. Supernatants were stored frozen at - 2 0 ° C until assayed for IL- ! activity.
Assays for IL- 1 inhibitor Mouse comitogen thymocyte assay. The mouse comitogen thymocyte assay was set up to measure bovine IL-l activity as described by Lederer and Czuprynski (1989). B6C3FI mice, 6-12 weeks old, were used as thymus donors.
254
J. JENSEN AND R.D. SCHULTZ
Mice were euthanized and the thymuses removed and minced in RPMI. Cell suspensions were washed twice by centrifugation in RPMI and resuspended in RPMI plus 10% FBS with 2.5 × 10-5M B-mercaptoethanol. Supernatants from monocyte cultures with LPS were used. Thymocytes (7 × 105, 50 gl) were added in duplicate to the wells of 96-well flat-bottom microtiter plates containing 0.5 gg ( 100/~1) concanavalin A (Con A; Sigma, St. Louis, MO) and 50 #1 supernatant diluted in RPMI. Control wells to measure thymocyte proliferation caused by Con A alone contained 50/ll RPMI instead of supernatant. Plates were incubated for 72 h at 37°C and then pulsed with 3H-thymidine (2/~Ci per well, 100 #1) for the last 6 h of incubation. Plates were frozen at - 2 0 ° C, thawed and cells harvested onto glass fiber filter strips using a semi-automatic cell harvester (Bellco Biotechnology, Vineland, NJ). 3H-thymidine incorporation was determined by scintillation counting in a Beckman LS 6800 liquid scintillation spectrophotometer. In experiments with LPS-stimulated cultures where exogenous human recombinant IL-1 (Genzyme, Boston, MA) was added, the IL-1 was diluted to the appropriate concentration in RPMI and 10/ll added to each well. Control wells without IL-1 contained 10/zl RPMI.
Test for IL-l-stimulated mouse thymocyte proliferation. Supematants from monocyte cultures without LPS were used. Thymocytes were prepared as described above and dispensed in duplicate into wells of 96-well fiat-bottom microtiter plates containing 0.5 gg ( 100 gl) Con A, 0.02 ng human recombinant IL-1 (10/~1) and 50/tl supernatant diluted in RPMI. Control wells to measure thymocyte proliferation caused by IL-1 contained 50 gl RPMI instead of supernatant. Plates were incubated at 37°C, pulsed with 3H-thymidine and harvested as described above.
Measurement of lL-1 mRNA by RNA-cDNA hybridization LPS-stimulated, BVDV-infected and uninfected monocytes were harvested by scraping at 18 h postinfection, resuspended in cold buffer (0.01 M TrisHCI, pH 8.0, 2 mM Mg C12, 0.01 M NaCI) and then lysed with 0.5% Nonidet P-40-0.5% deoxycholate on ice for 5 min (Maniatis et al., 1989). Following removal of the nuclear pellet by microcentrifugation, cytoplasmic RNA was phenol-extracted from the supernatant, ethanol-precipitated and quantified spectrophotometrically. RNA was blotted onto nitrocellulose and hybridized as described by Maniatis et al. ( 1989 ) using 32p labelled p 1301 (Lomedico et al., 1984) as the cDNA probe.
Lymphocyte blastogenesis test The lymphocyte blastogenesis test (LBT) was done as described previously (Jensen et al., 1988 ). Briefly, bovine PBML were harvested by centrifugation over Ficoll hypaque, resuspended at 5 × 106 cells/ml in RPMI with 20% FBS,
EFFECTOF BVDVON BOVINEMONOCYTES
255
and dispensed (50/~l) into 96-well flat-bottom microtiter plates containing phytohemagglutinin (PHA, 100/!!; GIBCO, Grand Island, NY) at various concentrations. Supernatants (50 ld) from BVDV-infected and uninfected monocytes were added, cells were incubated for 72 h and then labelled with 3H-thymidine for the last 18 h of incubation. Plates were frozen at - 2 0 ° C , thawed and the cells harvested on glass fiber filters. The incorporation of 3Hthymidine was determined by scintillation counting as described above.
Interferon assay Supernatants from monocytes were assayed for interferon activity by inhibition of replication of vesticu!ar stomatitis virus (VSV) in bovine turbinate cells (Bell et al., 1983 ). RESULTS
IL-1 inhibitor fiom B VD V-infected bovine monocytes The effect of supematants from BVDV-infected monocytes on the proliferation of mouse thymocytes stimulated with exogenous IL- 1 at the 50% maximal dose (0.02 ng) is shown in Table 1. Supernatants from monocytes infected with BVDV/NADL (CP) and BVDV/NY-I (NCP) significantly suppressed ( P < 0.05) IL- 1 stimulated proliferation of mouse thymocytes when compared with media control cultures without supernatant. Antiproliferative activity was not found in supernatants from monocytes exposed to yirradiated or heat-inactivated BVDV (data not shown). Antiproliferative acUVlCywas present co a unuuon ol 1:64 __-.L w l t n supematants ,,___ ~tom nD,v, ~L,, rv /,,~,, l s t ~,-,, .,~ infected monocytes and to a dilution of 1-32 with supet natants from BVDV/ .a_~ _
_-"
_11 -* 1 _ _ ~ _ _ "
_
TABLE 1 Effect of supernatants from BVDV-infected bovine monocytes on IL-l-stimulated mouse thymocyte proliferation Supernatant i
Media 14 _+4.2 BVDV-infected NADL NY-I Uninfected
Reciprocal dilution of supernatant (c.p.m. × 10- 3) 2 2
4
8
16
32
64
2.0_+0.9* 2 _+1.0" 19 _+4.3
2+_ I.I* 3_+1.2" 20_+3.8
4_+0.8* 6_+3.7* 15_+5.1
5 -L_,1.6" 6+_2.2* 16_+2.4
6_+ 1.3" 11_+3.4 16_+2.7
12_+2.8 14_+4.1 13_+4.1
150/iI media (RPMI) or 50/tl supernatant from BVDV-infected or uninfected bovine monocytes were added to mouse thymocytes stimulated with exogenous IL-I at the 50% maximal dose (0.02 ng). 2Results are expressed as mean counts per min_ s.e.m, from three experiments using mo~ocytes from three different cattle. Standard errors of duplicate cultures within each experiment w~ ~ _ 0.05 ) compared with control cultures when tested undiluted or diluted 1:2 or 1:4. Titration of supernatants from BVDV/NADLinfected monocytes showed that significant stimulatory (IL-1 ) activity over contro! cultures is restored at a 1:8 dilution (P 0.05 ) when tested undiluted or diluted 1:2 or 1:4, but stimulatory (IL-I) activity is restored upon further dilution. To test the hypothesis that the absence oflL- 1 activity in cell culture supernatants from LPS-stimulated BVDV-infected bovine monocytes is caused by ~l~,~ .,IP ..~ . . _ _ 1 . . 1 1 . 1 ~ TT I L l l ~ iJll~;al~;lll~,l~; U I gg ~ U l l . l U l l ~ I L ' I
.'__L211-2. llllllUlI,Ul~
_ ~ . _ _ _ a . - JP. . . . . . _'__~P__~._,.I _ _ _ 1 5Up~[[l~[[~[Il[~ l[UIll Ullllllgl,;U~fJ UllU
TABLE 2 Effect of supernatants from LPS-stimulated, BVDV-infected and uninfected monocytes on mouse thymocyte proliferation' Supernatant
Media4±l~5 BVDV-in~cted NADL NY-I Unin~cted
Reciprocal dilution of supernatant (c.p.m. × 10- 3) 2 1
2
4
8
2±1.3 2±1.4 21±6.8"
3±1.4 5±1.9 29±9.0*
7±2.3 5±2.0 23±5.2*
21±2.4" 17±7.8" 25±5.3*
50/d media (RPMI) or 50/zl supernatant from LPS-stimulated BVDV-infected or uninfected bovine monocytes were added to mouse thymocytes potentiated with Con A (0.5 #g). 2Results are e,~pressed as mean counts per rain + s.e.m, from five experiments using monocytes frr,n five different cattle. Standard errors of duplicate cultures within each experiment were < 10%. *Significantly different ( P < 0.05) from media controls without supernatant.
EFFEC'i Oi- tiv £,~ C , ~
VINEMONOCYTES
257
BVDV/NADL-infected monocytes were mixed and then assayed for stimulatory (IL-1 ) activity. The results are shown in Table 3. Supernatants from LPS-stimulated BVDV/NADL-infected monocytes inhibited IL-l activity present in supernatants from LPS-stimulated uninfected monocytes and the titration curve for IL- 1 activity for the mixture was not significantly different ( P > 0 . 0 5 ) than that for supernatants from BVDV/NADL-infected monocytes alone. TABLE 3 Effect of mixing supernatants from LPS-stimulated, BVDV-infected and uninfected bovine monocytes on mouse thymocyte proliferation Reciprocal dilution of supernatant (c.p.m. × 10- 3) 2
SupernatanP
Media 6 + 1.4 BVDV-NADL infected Uninfected Infected + uninfected
1
2
4
8
16
4-+2.4 23 _+4.8*
6+3.7 22_+ 8.4* 5-+ 1.7
8_+ 1.7 34_+ 4.9* 8 -+3.7
10_+2.8 21 + 7.8* 11 -+ 3.7
12_+3.9"
'50/~1 media (RPMI), 50/d supernatant from LPS-stimulated, BVDV-infected or uninfected bovine monocytes or 50 ~1 of a mixture ofsupernatants from LPS-stimulated, BVDV-infected and uninfected monocytes were added to mouse thymocytes potentiated with Con A (0.5/Lg). 2Results are expressed as mean counts per min_+ s.e.m, from three experiments using monocytes from three different cattle. Standard errors of duplicate cultures within each experiment were < 10%. *Significantly different (P< 0.05 ) from media controls without supernatant.
TABLE 4 Effect of exogenous IL-1 on IL-1 inhibitor activity in supematants from LPS-stimulated, BVDV-infected monocytes Supernatant'
Media BVDV-infected undiluted 1:2 1:4 1:8 Uninfected
Nanograms IL-I added (c.p.m. × 10-3) 2 20
2
0.2
0.02
33+ 12.1
38+ 7.6
37+8.1
19-+ 3.2
38+ 9.3 40+13.6 38+ 7.3 41+ 14.2 34+ 8.5
25+ 5.3* 30+ 8.4 37+ 8.5 39+ 9.8 38+10.2
12+3.4" 25+4.7* 34+8.9 36+7.4 35+4.8
7_+ 2.6* ll_+ 3.1" 27_+ 3.6 37 _+11.2 37.+_ !0.4
0.002 8_+1.7 9+1.9 10+3.2 12+4.1 35+8.7 35+5.2
0 7_+ 4.1 6+ 8+ 15+ 32+ 35+
2.4 2.9 4.6 12.4 6.3
m50/d media (RPMI) or 50/d supernatant from LPS-stimuiated, BVDV-infected or uninfected boo vine monocytes were added to mouse thymocytes potentiated with Con A (0.5/~g) and stimulated with varying concentrations of IL- 1. 2Results are expressed as mean counts per rain + s.e.m, of four experiments using monocytes from four different cattle. Standard errors of duplicate cultures within each experiment were < 12%. *Significantly different from media controls containing identical concentrations of IL- 1.
258
J. JENSEN AND R.D. SCHULTZ
The effect of the addition of exogenous human recombinant IL-1 on the proliferation of mouse thymocytes incubated with supernatants from LPSstimulated BVDV-infected or uninfected monocytes is shown in Table 4. Addition of IL-l in amounts less than or equal to the 50% maximal dose (0.02 ng) failed to overcome the suppressive effect of supernatants from BVDVinfected monocytes when the supernatants were undiluted or diluted 1:2 (P < 0.05 when compared with media controls) and suppression was still observed after addition of IL- l at a 100% stimulatory dose (0.2 ng). However, suppression was overcome by the addition of 20 ng IL-l. When BVDV-infected supernatants were diluted 1:4, suppression was overcome by the addition of 0.02 ng 1LoI. Addition of IL-l had no effect on the proliferation of mouse thymocytes incubated with supernatants from uninfected monocytes (Table 4), indicating that sufficient IL-l was present in these supernatants to stimulate maximal thymocyte proliferation.
IL- 1 mRNA in B VD V-infected monocytes The effect of BVDV infection on the amount of IL-1 mRNA in LPS-stimulated bovine monocytes was examined by RNA-cDNA hybridization (Fig. 1 ). No differences were seen between the intensities of the hybridization signals obtained using RNA extracted from BVDV -infected and uninfected monocytes, showing that IL-I mRNA is present in BVDV-infected and uninfected monocytes. IL-1 activity of monocytes from PI cattle The effect of in vitro BVDV infection on LPS-stimulated IL-1 activity of monocytes from three Pi cattle was examined (Table 5). The results were similar to those found using monocytes from cattle not persistently infected with BVDV (Table 5). Infection of monocytes from PI cattle with BVDV/ NADL in vitro resulted in the appearance of an inhibitor of IL-! activity in monocyte supernatants as evidenced by significant suppression of mouse thymocyte proliferation when compared with uninfected control supernatants. No IL-I inhibitor was detected in supernatants from monocytes from PI animals without in vitro infection with BVDV/NADL (Table 5), as evidenced by the stimulatory activity of these supernatants on mouse thymocyte proliferation. Effect of infection with BVDV isolates from PI cattle on IL-activity of bovine monocytes The ability of BVDV isolates from the three PI cattle to induce IL-1 inhibitory activity in monocytes from non-PI cattle was examined (Table 6). Infection of monocytes from non-PI cattle with each of the BVDV isolates from PI cattle resulted in the appearance of a soluble inhibitor of IL-l activity as determined by the mouse comitogen thymocyte assay. Supernatants from
EFFECT OF BVDV ON BOVINE MONOCYTES
259
3
4
Fig. 1. Detection of IL-1 mRNA in BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. RNA was extracted from LPS-stimulated uninfected monocytes (A-l, A-2) and LPS-~tlm,,l~tpd BVDV-infortad mnnneytes (A-3, A-4) and hlmted at 400 n~ RNA from uninfected monocytes without LPS stimulation is blotted in B 1 and B2. RNA from BVDV-infected monocytes without LPS stimulation is blotted in B3 and B4.
monocytes infected with each of the BVDV isolates suppressed mouse thymocyte proliferation when compared with supernatants from uninfected monocytes. No significant differences in anti-proliferative activity were found between supernatants from monocytes infected with the BVDV isolates from PI cattle and supernatants from monocytes infected with BVDV/NADL.
Effect of lL-1 inhibitor on bovine lymphocytes Supernatants from BVDV/NADL-infected and uninfected bovine monocytes were tested for their effect on the proliferative response of bovine PBMLs using the LBT. The results are shown in Table 7. The proliferative response of bovine PBMLs was evaluated at both optimal and suboptimal concentrations of PHA. In contrast to the inhibitory, effect on thymocyte proliferation, supernatants from BVDV/NADL-infected monocytes had no effect on the
260
J. JENSEN AND R.D. SCHULTZ
TABLE 5 IL-I activity in supernatants of LPS-stimulated monocytes from P! cattle infected with BVDV/NADL in vitro Supernatant'
Reciprocal dilution of supernatant ( c.p.m × I 0 - 3 )., !
Media 7 + 1.9 BVDV-infected Pl-! PI-2 PI-3 nonPl-! nonPI-2 Uninfected Pl-! PI-2 PI-3 nonPi-i non PI-2
2
4
8
16
6 + 1.6 5+ !.6 8+2.0 7+2.2 9+2.4
6+ 8+ 10+ 7+ il+
2.1 3.8 4.8 2.6 5.7
I 1 +4.6 8+2.6 19_+6.1" 11+5.1 8+3.8
21 + 15+ 27+ 24+ 32+
5.3* 4.0* 10.2" 8.7* 8.7*
20+9.9* 21+7.4" 22+6.4* 20+4.7* 29+7.1"
23+7.2* 27+4.3* 25+5.8* 17+3.3" 30+ 7.1"
26+ 32+ 31+ 18+ 27+
8.2* 8.1" 6.7* 3.4* ll*
28+5.1" 24+3.5* 25+3.1" 18+3.8" 24+4.2*
27+ 19+ 28+ 21+ 23+
3.0* 2.8* 6.3* 8.4* 3.4*
18+3.1" 19+3.1" 22+4.1" 22+3.0* 25+8.7*
50 Ill media ( RPMI ), 50 #! supernatant from LPS-stimulated, BVDV-NADL-infected monocytes from Pi or non-Pl cattle, or 50 gl supernatant from LPS-stimulated uninfected monocytes from Pi or non-PI cattle were added to mouse thymocytes potentiated with Con A (0.5 ,ug). -'Results are expressed as mean counts per min _+s.e.m, for three experiments. Standard errors of duplicate cultures were < 10%. *Significantly different ( P < 0.05 ) from media controls without supernatant.
TABLE 6 Effect of BVDV isolates from Pi cattle on LPS-stimulated IL-! activity of bovine monocytes SupernatanP
Media 9 + !.8 BVDV-PII BVDV-Pi 2 BVDV-PI3 BVDV-NADL Uninfected
Reciprocal dilution of supernatant (c.p.m. X l 0 - 3) 2 1
2
4
8
6+1.5 4 + !.4 3+4.9 7+3.9 35 + 5.3*
10+6.4 8 + 4. i 8+3.4 9+2.2 39 + 6.8*
12+3.0 26 + 4.7* 14+6.9 14+3.3" 36 + 7.4*
13+ 33 + 18+ 15+ 36 +
16 3.5* I 1.9" 3.1" 3.7* 8.3*
27+ 31 + 26+ 29+ 32 +
4.9* l 1.0" 7.2* 7.8* 6.6*
'50 gl media (RPM!), 50/tl supernatant from LPS-stimulated, BVDV-infected or unir.fected monocytes were added to mouse thymocytes potentiated with Con A (0.5 gg). -'Results are expressed as mean counts per min + s.e.m, for three experiments using monocytes from three different cattle. Standard errors of duplicate cultures were < 10%. *Significantly different ( P < 0.05 ) from media controls without supernatant.
EFFECTOF BVDVON BOVINEMONOCYTES
261
TABLE 7 Effect of IL- l inhibitor on mitogen-stimulated blastogenesis of bovine PBMLs Supernatant ~
Dilution of PHA (c.p.m. × l0 -3)2 50
Media BVDV infected undiluted 1:2 1:4 1:8 Uninfected
200
101 -+ 17
400
98.+ 18 42.+ 13
97.+14 104__+24 45.+ 7 112.+16 100+_25 38-+13 121 -+ 12 84_+27 52.+ 15 118.+ 17 91 .+ 14 41 .+ 19 121 .+ 18 89.+ 12 43.+ 12
800
1600
3200
no PHA
21 _+5
2.1 _+0.8
l.l -+0.3
0.5.+0.3
18__+6 24-+8 22.+9 20.+7 15.+5
2.3-+0.5 3.0_+1.3 2.5.+ l.l 2.7.+ l.l 2.8.+ 1.6
1.4__+0.4 0.3.+0.2 0.9+_0.5 0.7_+0.1 0.7.+0.3 0.4.+0.2 0.6.+0.5 0.4.+0.3 0.9.+0.5 0.3_+0.2
50/zl media ( R P M I ) or 50/A supernatant from BVDV-infected or uninfected bovine monocytes were added to bovine PBMLs stimulated with varying concentrations of PHA. -'Results are expressed as mean counts per min +_s.e.m, from three experiments using PBMLs from three different cattle, standard errors of duplicate cultures within each experiment were _
251
Elsevier Science Publishers B.V., Amsterdam
Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin- 1 activity of bovine monocytes* J. Jensen ~ and R.D. Schultz Department of Pathobiological Sciences, School of VeterinaryMedicine, Universityof WisconsinMadison, Madison, W153 706, USA (Accepted 12 November 1990)
ABSTRACT Jensen, J. and Schultz, R.D., 1991. Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin-1 activity of bovine monocytes. Vet. Immunol. Immunopathol., 29:251-265. The effect of bovine viral diarrhea virus (BVDV) infection in vitro on the interleukin-I (IL-I) activity of bovine monocytes was studied. Supernatants from BVDV-infected monocytes suppressed IL-l-stimulated proliferation of mouse thymocytes and masked lipopolysaccharide-stimulated IL-I activity of bovine monocytes in the mouse comitogen thymocyte assay. Suppression of mouse thymocyte proliferation was restored by the addition of IL-1. IL-1 inhibitory activity was induced both by the prototype variants BVDV/NADi. cytopathic and BVDV/NY-1 noncytopathic and by BVDV variants isolated from persistently infected cattle. Suppressed IL-I a ~ ; u v l t y w i a ~ a l a o found in supernatants from monocytes from persistently infected cattle following infection with BVDV in vitro. No differences in levels of IL-l mRNA synthesis were detected between BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. These results suggest that infection of bovine monocytes with BVDV results in the production and/or activation of a soluble inhibitor of IL-l activity. ABBREVIATIONS BVDV, bovine viral diarrhea virus; CMV, cytomegalovirus; Con A, concanavalin A; CP, cytopathic; FBS, fetal bovine serum; HIV, human immunodeficiency virus; IL-l, interleukin-1; LBT, lymphocyte blastogenesis test; LPS, lipopolysaccharide; NCP, non-cytopathic; PBML, peripheral blood mononuclear leukocyte; PHA, phytohemagglutinin; PI, persistently infected; RSV, respiratory syncytial virus; VSV, vesticular stomatitis virus. ~Present address: Department of Surgery, University of Wisconsin Medical School, 4679 Medical Science Center, 1300 University Avenue, Madison, WI 53706, U.S.A. *This work was supported in part by the large Animal Research Fund, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706 and by USDA-CSRA Grant 89-341164528.
0165-2427/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.
252
J. JENSEN AND R.D. SCHULTZ
INTRODUCTION
Infection of cattle by bovine viral diarrhea virus (BVDV) can cause generalized or specific immunosuppression. Postnatal infection with BVDV resuits in increased susceptibility of the infected host to secondary infection by bacteria or other viruses (Reggiardo and Kaebede, 1981; Potgeiter et al., 1984a,b). Infection with BVDV in utero can result in specific immunotolerance to the infecting strain of BVDV, depending on the age of the fetus at the time of infection and the biotype of the infecting BVDV (Brownlie et al., 1984; McClurkin et al., 1985; reviewed by Radostits and Littlejohns, 1988). In the infected host, BVDV replicates in lymphoid tissues, including B cells, T cells, monocytes and macrophages (Truitt and Schechmeister, 1973; Bolin et at., 1985). BVDV-induced suppression of several cellular immune functions, including T cell proliferation (Muscoplat et al., 1973; Pospisil et al., 1975), neutrophil phagocytic activity (Roth and Kaebefle, 1983; Roth et al., 1986) and monocyte chemotactic response (Akkina et al., 1979) has been observed in vitro and in vivo. Interleuidn-1 (IL-1 ) is an immunoregulatory molecule with a wide range of biological activities, including T cell maturation, B cell activation and stimulation of fibroblast and epidermal cell proliferation (Dinarello, 1985; Durum et al., 1985; reviewed by Oppenheim et al., 1986). BVDV infection of monocytes and macrophages and consequent perturbation of IL- 1 activity by BVDV could result in suppression of the host's immune response. Perturbation ofIL-1 activity following viral infection of monocytes or macrophages has been noted with several human viruses, including cytomegalovirus (CMV) (Kapasi and Rice, 1988 ), respiratory syncytial virus (RSV) and influenza virus [ Koberts et ai., i 986; McCarthy et ai., 1989) and human immunodeficiency virus (HIV) (Berman et al., 1987). Infection of macrophages with these viruses causes botih enhanced production of IL-I activity and induction of humoral inhibitors of IL-I activity. In some cases, virusinduced humoral inhibitors of IL-1 activity have been associated with antiproliferative effects of mitogen-stimulated T cell proliferation (McCarthy et al., 1989) and suppressed B cell maturation (Berman et al., 1986). Considering the immunosuppressive effect of BVDV infection on the host's immune response and the central immunomodulatory role of IL-1, the present study was designed to look at the effect of in vitro BVDV infection of bovine monocytes on IL- 1 activity. MATERIALS AND METHODS
Animals Adult cattle not infected with BVDV and three persistently infected (PI) cattle (Jensen et al., 1990) were used.
253
EFFECT OF BVDVON BOVINE MONOCYTES
Viruses and cell cultures BVDV/NADL was obtained from the stock virus collection at the James A. Baker Institute for Animal Health, Cornell University. BVDV/NY-I was obtained from Dr. S.R. Bolin, National Animal Disease Center, Ames, IA. Virus was propagated in mycop!asma-free primary bovine testicle cells in minimal essential media (MEM) with 10% fetal bovine serum (FBS) (low endotoxin lot 127F-0279, Sigma, St. Louis, MO) and 1% penicillin-streptomycin. The FBS used was free of noncytopathie (NCP) BVDV and did not have detectable antibody to BVDV. Cytopathic (CP) BVDV/NADL was titrated in bovine testicle cells by limiting dilution. NCP BVDV/NY-1 and the three NCP field isolates of BVDV from PI cattle were titrated in bovine testicle cells by limiting dilution, using the indirect fluorescent antibody technique as described previously (Jensen et al., 1990). For preparation of inactivated BVDV, virus was ~,-irradiated (3000 rads) or heat-inactivated at 60°C for 2 h (Atluru et al., 1979).
Preparation of monocyte-derived supernatants Bovine peripheral blood mononuclear leukocytes (PBML) were isolated by centrifugation over Ficoll-hypaque (Schultz, 1982) and resuspended in RPMI- 1640 media with 1% penicillin-streptomycin (RPMI) and 10% FBS (Jensen et al., 1988). PBML were plated in 24-well tissue culture plates (Coming Cell Wells, Coming, NY) at a cell density of 1 X 106 cells/cm 2 (2.6× 106 cells per well), incubated for 2 h at 37°C and nonadherent cells were removed by washing with RPMI. To prepare BVDV-infected monocyte cultures, monocytes were infected with 1 × 10s TCIDso BVDV (multiplicity of infection, two to five) in RPMI, the plates incubated for 1 h at 37°C and l~ g,.nL Aa~ ,rg . l~ ,c~ V,l ~ r hK,, .~l ~ ttJ Jt. .•, A n l c r,~mrLv,~tl hL , IvJ u~a©hlnt~ uV Vl l.e.t• h JLA ~ e , ~ m. ~ g A A ~ J • ~l~. vv V~AAAAA~
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more than 90% of adherent cells are infected with BVDV as determined by indirect fluorescent antibody staining (Jensen et al., 1990). Uninfected monocytes were incubated with RPMI for 1 h and then washed. RPMI with 10% FBS or RPMI with 10% FBS and i 0 gg/ml LPS (Escherichia coli lipopolysaccharide (LPS) 0.55:B5; Difco Laboratories, Detroit, MI) was then added to both infected and uninfected monocytes, the cultures incubated for 18 h at 37°C and the supernatants harvested by centrifugation (Lederer and Czuprynski, 1989). The 18 h incubation was chosen based on data from initial experiments showing optimal IL-l inhibitory activity in supernatants harvested at this time. Supernatants were stored frozen at - 2 0 ° C until assayed for IL- ! activity.
Assays for IL- 1 inhibitor Mouse comitogen thymocyte assay. The mouse comitogen thymocyte assay was set up to measure bovine IL-l activity as described by Lederer and Czuprynski (1989). B6C3FI mice, 6-12 weeks old, were used as thymus donors.
254
J. JENSEN AND R.D. SCHULTZ
Mice were euthanized and the thymuses removed and minced in RPMI. Cell suspensions were washed twice by centrifugation in RPMI and resuspended in RPMI plus 10% FBS with 2.5 × 10-5M B-mercaptoethanol. Supernatants from monocyte cultures with LPS were used. Thymocytes (7 × 105, 50 gl) were added in duplicate to the wells of 96-well flat-bottom microtiter plates containing 0.5 gg ( 100/~1) concanavalin A (Con A; Sigma, St. Louis, MO) and 50 #1 supernatant diluted in RPMI. Control wells to measure thymocyte proliferation caused by Con A alone contained 50/ll RPMI instead of supernatant. Plates were incubated for 72 h at 37°C and then pulsed with 3H-thymidine (2/~Ci per well, 100 #1) for the last 6 h of incubation. Plates were frozen at - 2 0 ° C, thawed and cells harvested onto glass fiber filter strips using a semi-automatic cell harvester (Bellco Biotechnology, Vineland, NJ). 3H-thymidine incorporation was determined by scintillation counting in a Beckman LS 6800 liquid scintillation spectrophotometer. In experiments with LPS-stimulated cultures where exogenous human recombinant IL-1 (Genzyme, Boston, MA) was added, the IL-1 was diluted to the appropriate concentration in RPMI and 10/ll added to each well. Control wells without IL-1 contained 10/zl RPMI.
Test for IL-l-stimulated mouse thymocyte proliferation. Supematants from monocyte cultures without LPS were used. Thymocytes were prepared as described above and dispensed in duplicate into wells of 96-well fiat-bottom microtiter plates containing 0.5 gg ( 100 gl) Con A, 0.02 ng human recombinant IL-1 (10/~1) and 50/tl supernatant diluted in RPMI. Control wells to measure thymocyte proliferation caused by IL-1 contained 50 gl RPMI instead of supernatant. Plates were incubated at 37°C, pulsed with 3H-thymidine and harvested as described above.
Measurement of lL-1 mRNA by RNA-cDNA hybridization LPS-stimulated, BVDV-infected and uninfected monocytes were harvested by scraping at 18 h postinfection, resuspended in cold buffer (0.01 M TrisHCI, pH 8.0, 2 mM Mg C12, 0.01 M NaCI) and then lysed with 0.5% Nonidet P-40-0.5% deoxycholate on ice for 5 min (Maniatis et al., 1989). Following removal of the nuclear pellet by microcentrifugation, cytoplasmic RNA was phenol-extracted from the supernatant, ethanol-precipitated and quantified spectrophotometrically. RNA was blotted onto nitrocellulose and hybridized as described by Maniatis et al. ( 1989 ) using 32p labelled p 1301 (Lomedico et al., 1984) as the cDNA probe.
Lymphocyte blastogenesis test The lymphocyte blastogenesis test (LBT) was done as described previously (Jensen et al., 1988 ). Briefly, bovine PBML were harvested by centrifugation over Ficoll hypaque, resuspended at 5 × 106 cells/ml in RPMI with 20% FBS,
EFFECTOF BVDVON BOVINEMONOCYTES
255
and dispensed (50/~l) into 96-well flat-bottom microtiter plates containing phytohemagglutinin (PHA, 100/!!; GIBCO, Grand Island, NY) at various concentrations. Supernatants (50 ld) from BVDV-infected and uninfected monocytes were added, cells were incubated for 72 h and then labelled with 3H-thymidine for the last 18 h of incubation. Plates were frozen at - 2 0 ° C , thawed and the cells harvested on glass fiber filters. The incorporation of 3Hthymidine was determined by scintillation counting as described above.
Interferon assay Supernatants from monocytes were assayed for interferon activity by inhibition of replication of vesticu!ar stomatitis virus (VSV) in bovine turbinate cells (Bell et al., 1983 ). RESULTS
IL-1 inhibitor fiom B VD V-infected bovine monocytes The effect of supematants from BVDV-infected monocytes on the proliferation of mouse thymocytes stimulated with exogenous IL- 1 at the 50% maximal dose (0.02 ng) is shown in Table 1. Supernatants from monocytes infected with BVDV/NADL (CP) and BVDV/NY-I (NCP) significantly suppressed ( P < 0.05) IL- 1 stimulated proliferation of mouse thymocytes when compared with media control cultures without supernatant. Antiproliferative activity was not found in supernatants from monocytes exposed to yirradiated or heat-inactivated BVDV (data not shown). Antiproliferative acUVlCywas present co a unuuon ol 1:64 __-.L w l t n supematants ,,___ ~tom nD,v, ~L,, rv /,,~,, l s t ~,-,, .,~ infected monocytes and to a dilution of 1-32 with supet natants from BVDV/ .a_~ _
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_11 -* 1 _ _ ~ _ _ "
_
TABLE 1 Effect of supernatants from BVDV-infected bovine monocytes on IL-l-stimulated mouse thymocyte proliferation Supernatant i
Media 14 _+4.2 BVDV-infected NADL NY-I Uninfected
Reciprocal dilution of supernatant (c.p.m. × 10- 3) 2 2
4
8
16
32
64
2.0_+0.9* 2 _+1.0" 19 _+4.3
2+_ I.I* 3_+1.2" 20_+3.8
4_+0.8* 6_+3.7* 15_+5.1
5 -L_,1.6" 6+_2.2* 16_+2.4
6_+ 1.3" 11_+3.4 16_+2.7
12_+2.8 14_+4.1 13_+4.1
150/iI media (RPMI) or 50/tl supernatant from BVDV-infected or uninfected bovine monocytes were added to mouse thymocytes stimulated with exogenous IL-I at the 50% maximal dose (0.02 ng). 2Results are expressed as mean counts per min_ s.e.m, from three experiments using mo~ocytes from three different cattle. Standard errors of duplicate cultures within each experiment w~ ~ _ 0.05 ) compared with control cultures when tested undiluted or diluted 1:2 or 1:4. Titration of supernatants from BVDV/NADLinfected monocytes showed that significant stimulatory (IL-1 ) activity over contro! cultures is restored at a 1:8 dilution (P 0.05 ) when tested undiluted or diluted 1:2 or 1:4, but stimulatory (IL-I) activity is restored upon further dilution. To test the hypothesis that the absence oflL- 1 activity in cell culture supernatants from LPS-stimulated BVDV-infected bovine monocytes is caused by ~l~,~ .,IP ..~ . . _ _ 1 . . 1 1 . 1 ~ TT I L l l ~ iJll~;al~;lll~,l~; U I gg ~ U l l . l U l l ~ I L ' I
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TABLE 2 Effect of supernatants from LPS-stimulated, BVDV-infected and uninfected monocytes on mouse thymocyte proliferation' Supernatant
Media4±l~5 BVDV-in~cted NADL NY-I Unin~cted
Reciprocal dilution of supernatant (c.p.m. × 10- 3) 2 1
2
4
8
2±1.3 2±1.4 21±6.8"
3±1.4 5±1.9 29±9.0*
7±2.3 5±2.0 23±5.2*
21±2.4" 17±7.8" 25±5.3*
50/d media (RPMI) or 50/zl supernatant from LPS-stimulated BVDV-infected or uninfected bovine monocytes were added to mouse thymocytes potentiated with Con A (0.5 #g). 2Results are e,~pressed as mean counts per rain + s.e.m, from five experiments using monocytes frr,n five different cattle. Standard errors of duplicate cultures within each experiment were < 10%. *Significantly different ( P < 0.05) from media controls without supernatant.
EFFEC'i Oi- tiv £,~ C , ~
VINEMONOCYTES
257
BVDV/NADL-infected monocytes were mixed and then assayed for stimulatory (IL-1 ) activity. The results are shown in Table 3. Supernatants from LPS-stimulated BVDV/NADL-infected monocytes inhibited IL-l activity present in supernatants from LPS-stimulated uninfected monocytes and the titration curve for IL- 1 activity for the mixture was not significantly different ( P > 0 . 0 5 ) than that for supernatants from BVDV/NADL-infected monocytes alone. TABLE 3 Effect of mixing supernatants from LPS-stimulated, BVDV-infected and uninfected bovine monocytes on mouse thymocyte proliferation Reciprocal dilution of supernatant (c.p.m. × 10- 3) 2
SupernatanP
Media 6 + 1.4 BVDV-NADL infected Uninfected Infected + uninfected
1
2
4
8
16
4-+2.4 23 _+4.8*
6+3.7 22_+ 8.4* 5-+ 1.7
8_+ 1.7 34_+ 4.9* 8 -+3.7
10_+2.8 21 + 7.8* 11 -+ 3.7
12_+3.9"
'50/~1 media (RPMI), 50/d supernatant from LPS-stimulated, BVDV-infected or uninfected bovine monocytes or 50 ~1 of a mixture ofsupernatants from LPS-stimulated, BVDV-infected and uninfected monocytes were added to mouse thymocytes potentiated with Con A (0.5/Lg). 2Results are expressed as mean counts per min_+ s.e.m, from three experiments using monocytes from three different cattle. Standard errors of duplicate cultures within each experiment were < 10%. *Significantly different (P< 0.05 ) from media controls without supernatant.
TABLE 4 Effect of exogenous IL-1 on IL-1 inhibitor activity in supematants from LPS-stimulated, BVDV-infected monocytes Supernatant'
Media BVDV-infected undiluted 1:2 1:4 1:8 Uninfected
Nanograms IL-I added (c.p.m. × 10-3) 2 20
2
0.2
0.02
33+ 12.1
38+ 7.6
37+8.1
19-+ 3.2
38+ 9.3 40+13.6 38+ 7.3 41+ 14.2 34+ 8.5
25+ 5.3* 30+ 8.4 37+ 8.5 39+ 9.8 38+10.2
12+3.4" 25+4.7* 34+8.9 36+7.4 35+4.8
7_+ 2.6* ll_+ 3.1" 27_+ 3.6 37 _+11.2 37.+_ !0.4
0.002 8_+1.7 9+1.9 10+3.2 12+4.1 35+8.7 35+5.2
0 7_+ 4.1 6+ 8+ 15+ 32+ 35+
2.4 2.9 4.6 12.4 6.3
m50/d media (RPMI) or 50/d supernatant from LPS-stimuiated, BVDV-infected or uninfected boo vine monocytes were added to mouse thymocytes potentiated with Con A (0.5/~g) and stimulated with varying concentrations of IL- 1. 2Results are expressed as mean counts per rain + s.e.m, of four experiments using monocytes from four different cattle. Standard errors of duplicate cultures within each experiment were < 12%. *Significantly different from media controls containing identical concentrations of IL- 1.
258
J. JENSEN AND R.D. SCHULTZ
The effect of the addition of exogenous human recombinant IL-1 on the proliferation of mouse thymocytes incubated with supernatants from LPSstimulated BVDV-infected or uninfected monocytes is shown in Table 4. Addition of IL-l in amounts less than or equal to the 50% maximal dose (0.02 ng) failed to overcome the suppressive effect of supernatants from BVDVinfected monocytes when the supernatants were undiluted or diluted 1:2 (P < 0.05 when compared with media controls) and suppression was still observed after addition of IL- l at a 100% stimulatory dose (0.2 ng). However, suppression was overcome by the addition of 20 ng IL-l. When BVDV-infected supernatants were diluted 1:4, suppression was overcome by the addition of 0.02 ng 1LoI. Addition of IL-l had no effect on the proliferation of mouse thymocytes incubated with supernatants from uninfected monocytes (Table 4), indicating that sufficient IL-l was present in these supernatants to stimulate maximal thymocyte proliferation.
IL- 1 mRNA in B VD V-infected monocytes The effect of BVDV infection on the amount of IL-1 mRNA in LPS-stimulated bovine monocytes was examined by RNA-cDNA hybridization (Fig. 1 ). No differences were seen between the intensities of the hybridization signals obtained using RNA extracted from BVDV -infected and uninfected monocytes, showing that IL-I mRNA is present in BVDV-infected and uninfected monocytes. IL-1 activity of monocytes from PI cattle The effect of in vitro BVDV infection on LPS-stimulated IL-1 activity of monocytes from three Pi cattle was examined (Table 5). The results were similar to those found using monocytes from cattle not persistently infected with BVDV (Table 5). Infection of monocytes from PI cattle with BVDV/ NADL in vitro resulted in the appearance of an inhibitor of IL-! activity in monocyte supernatants as evidenced by significant suppression of mouse thymocyte proliferation when compared with uninfected control supernatants. No IL-I inhibitor was detected in supernatants from monocytes from PI animals without in vitro infection with BVDV/NADL (Table 5), as evidenced by the stimulatory activity of these supernatants on mouse thymocyte proliferation. Effect of infection with BVDV isolates from PI cattle on IL-activity of bovine monocytes The ability of BVDV isolates from the three PI cattle to induce IL-1 inhibitory activity in monocytes from non-PI cattle was examined (Table 6). Infection of monocytes from non-PI cattle with each of the BVDV isolates from PI cattle resulted in the appearance of a soluble inhibitor of IL-l activity as determined by the mouse comitogen thymocyte assay. Supernatants from
EFFECT OF BVDV ON BOVINE MONOCYTES
259
3
4
Fig. 1. Detection of IL-1 mRNA in BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. RNA was extracted from LPS-stimulated uninfected monocytes (A-l, A-2) and LPS-~tlm,,l~tpd BVDV-infortad mnnneytes (A-3, A-4) and hlmted at 400 n~ RNA from uninfected monocytes without LPS stimulation is blotted in B 1 and B2. RNA from BVDV-infected monocytes without LPS stimulation is blotted in B3 and B4.
monocytes infected with each of the BVDV isolates suppressed mouse thymocyte proliferation when compared with supernatants from uninfected monocytes. No significant differences in anti-proliferative activity were found between supernatants from monocytes infected with the BVDV isolates from PI cattle and supernatants from monocytes infected with BVDV/NADL.
Effect of lL-1 inhibitor on bovine lymphocytes Supernatants from BVDV/NADL-infected and uninfected bovine monocytes were tested for their effect on the proliferative response of bovine PBMLs using the LBT. The results are shown in Table 7. The proliferative response of bovine PBMLs was evaluated at both optimal and suboptimal concentrations of PHA. In contrast to the inhibitory, effect on thymocyte proliferation, supernatants from BVDV/NADL-infected monocytes had no effect on the
260
J. JENSEN AND R.D. SCHULTZ
TABLE 5 IL-I activity in supernatants of LPS-stimulated monocytes from P! cattle infected with BVDV/NADL in vitro Supernatant'
Reciprocal dilution of supernatant ( c.p.m × I 0 - 3 )., !
Media 7 + 1.9 BVDV-infected Pl-! PI-2 PI-3 nonPl-! nonPI-2 Uninfected Pl-! PI-2 PI-3 nonPi-i non PI-2
2
4
8
16
6 + 1.6 5+ !.6 8+2.0 7+2.2 9+2.4
6+ 8+ 10+ 7+ il+
2.1 3.8 4.8 2.6 5.7
I 1 +4.6 8+2.6 19_+6.1" 11+5.1 8+3.8
21 + 15+ 27+ 24+ 32+
5.3* 4.0* 10.2" 8.7* 8.7*
20+9.9* 21+7.4" 22+6.4* 20+4.7* 29+7.1"
23+7.2* 27+4.3* 25+5.8* 17+3.3" 30+ 7.1"
26+ 32+ 31+ 18+ 27+
8.2* 8.1" 6.7* 3.4* ll*
28+5.1" 24+3.5* 25+3.1" 18+3.8" 24+4.2*
27+ 19+ 28+ 21+ 23+
3.0* 2.8* 6.3* 8.4* 3.4*
18+3.1" 19+3.1" 22+4.1" 22+3.0* 25+8.7*
50 Ill media ( RPMI ), 50 #! supernatant from LPS-stimulated, BVDV-NADL-infected monocytes from Pi or non-Pl cattle, or 50 gl supernatant from LPS-stimulated uninfected monocytes from Pi or non-PI cattle were added to mouse thymocytes potentiated with Con A (0.5 ,ug). -'Results are expressed as mean counts per min _+s.e.m, for three experiments. Standard errors of duplicate cultures were < 10%. *Significantly different ( P < 0.05 ) from media controls without supernatant.
TABLE 6 Effect of BVDV isolates from Pi cattle on LPS-stimulated IL-! activity of bovine monocytes SupernatanP
Media 9 + !.8 BVDV-PII BVDV-Pi 2 BVDV-PI3 BVDV-NADL Uninfected
Reciprocal dilution of supernatant (c.p.m. X l 0 - 3) 2 1
2
4
8
6+1.5 4 + !.4 3+4.9 7+3.9 35 + 5.3*
10+6.4 8 + 4. i 8+3.4 9+2.2 39 + 6.8*
12+3.0 26 + 4.7* 14+6.9 14+3.3" 36 + 7.4*
13+ 33 + 18+ 15+ 36 +
16 3.5* I 1.9" 3.1" 3.7* 8.3*
27+ 31 + 26+ 29+ 32 +
4.9* l 1.0" 7.2* 7.8* 6.6*
'50 gl media (RPM!), 50/tl supernatant from LPS-stimulated, BVDV-infected or unir.fected monocytes were added to mouse thymocytes potentiated with Con A (0.5 gg). -'Results are expressed as mean counts per min + s.e.m, for three experiments using monocytes from three different cattle. Standard errors of duplicate cultures were < 10%. *Significantly different ( P < 0.05 ) from media controls without supernatant.
EFFECTOF BVDVON BOVINEMONOCYTES
261
TABLE 7 Effect of IL- l inhibitor on mitogen-stimulated blastogenesis of bovine PBMLs Supernatant ~
Dilution of PHA (c.p.m. × l0 -3)2 50
Media BVDV infected undiluted 1:2 1:4 1:8 Uninfected
200
101 -+ 17
400
98.+ 18 42.+ 13
97.+14 104__+24 45.+ 7 112.+16 100+_25 38-+13 121 -+ 12 84_+27 52.+ 15 118.+ 17 91 .+ 14 41 .+ 19 121 .+ 18 89.+ 12 43.+ 12
800
1600
3200
no PHA
21 _+5
2.1 _+0.8
l.l -+0.3
0.5.+0.3
18__+6 24-+8 22.+9 20.+7 15.+5
2.3-+0.5 3.0_+1.3 2.5.+ l.l 2.7.+ l.l 2.8.+ 1.6
1.4__+0.4 0.3.+0.2 0.9+_0.5 0.7_+0.1 0.7.+0.3 0.4.+0.2 0.6.+0.5 0.4.+0.3 0.9.+0.5 0.3_+0.2
50/zl media ( R P M I ) or 50/A supernatant from BVDV-infected or uninfected bovine monocytes were added to bovine PBMLs stimulated with varying concentrations of PHA. -'Results are expressed as mean counts per min +_s.e.m, from three experiments using PBMLs from three different cattle, standard errors of duplicate cultures within each experiment were _
