Ve~rinaryImmunology and Immunopathology, 8 ( 1 9 8 5 ) 363--375 363 Elsevier Science Publishers B.V., A m s t e r d a m - - Pr in ted in The Netherlands

CELL-MEDIATED CYTOTOXICITY OF BOVINE MONONUCLEAR CELLS TO IBRV-INFECTED CELLS:

DEPENDENCE ON SEPHADEX G-IO ADHERENT CELLS

Manuel Campos and Charles R. Rossi

Animal Health Research, School of Veterinary Medicine and Alabama Agricultural

Experiment Station, Auburn University, AL 36849 (U.S.A.)

(Accepted 3 August 1984)

ABSTRACT

Campos, M. and Rossi, C.R., 1985. Cell-mediated cytotoxicity of bovine mononuclear cells to IBRV-infected ceils: dependance on Sephadex G-lO adherent cells. Vet. Irmnunol. Immunopathol., 8: 363-375.

Following intranasal inoculation of cattle with infectious bovine rhinotracheitis virus (IBRV) mononuelear cells that produced a genetically unrestricted cytotoxic response against IBRV-infected, but not against uninfected cells, were present in peripheral blood. Cytotoxicity was detected between 6 and 14 days after primary infection in a 20 h, but not in a 5 h, 51Cr-release assay. Cytotoxic activity was present in peripheral blood mononuclear cells from infected and subsequently hyperimmunized cattle for a considerably longer time. Neither natural cytotoxicity, antibody-dependent cell cytotoxicity, nor antibody produced during the assay was responsible for the cytotoxicity. However, cytotoxicity was dependent upon an adherent mononuclear cell that was partially removed by passage over nylon wool and completely removed by passage over Sephadex G-IO.

INTRODUCTION

Cell-mediated immunity to virus infections in mice and human beings is usually

due to the activity of genetically restricted cytotoxic T lymphocytes (CTL). In

those instances in which cytotoxicity has been reported to lack genetic

restriction, cytotoxicity has often been found to be due to the activity of

factors other than the activity of specifically sensitized CTL. However, little

information is available concerning genetic restriction in other species, and

there are several reports in which genetically unrestricted cytotoxicity has been

detected and has not been found to be due to antibody-dependent cellular

cytotoxicity or natural cytotoxicity (Fujimiya et al., 1979; Cremer et al.,

1982). In a study in cattle with infectious bovine rhinotracheitis virus (IBRV),

a bovine herpesvirus, Rouse and Babiuk (1977) reported that direct cellular

cytotoxicity was not genetically restricted to infected autologous target cells,

but extended to infected heterologous cells as well. Since the cells that

mediated this cytotoxicity were found in the nylon wool nonadherent cell

population, it was suggested that the cells might be T lymphocytes. Subsequent

studies have shown that cattle do mount genetically restricted antigen specific

0165-2427/85/$03 .30 © 1985 Else~er Science Publishers B.V.

364

lymphocyte responses to Theileria parva as shown by their preference to lyse

infected autologous as opposed to infected allogeneic cells (Emery et al., 1981;

Eugui and Emery, 1982).

In the present report we reinvestigated the genetic restriction of bovine

leukocytes in a direct cytotoxicity assay to IBRV-infected cells. Cytotoxicity

was found to be dependent on the presence of a cell that adhered to Sephadex

G-10. This cytotoxiclty was not genetically restricted and was present only in

animals that had been infected or infected and hyperimmunized with IBRV.

MATERIALS AND METHODS

V i r u s e s

The Cooper s t r a i n o f IBRV u s e d f o r p r i m a r y and s e c o n d a r y i n o c u l a t i o n o f

e x p e r i m e n t a l a n i m a l s and f o r i n f e c t i o n o f c e l l s in c y t o t o x l c i t y a s s a y s and t h e

SF-4 s t r a i n o f p a r a i n f l u e n z a t ype 3 v i r u s (PI3V) u s e d t o m e a s u r e n a t u r a l

c y t o t o x i c i t y (Campos e t a l . , 1982) a r e d e s c r i b e d in t h e compan ion a r t i c l e (Campos

and R o s s i , 1984) .

E x p e r i m e n t a l i n f e c t i o n o f s t e e r s

Twelve c r o s s b r e d s t e e r s 12-36 m o n t h s o l d f r e e o f n e u t r a l i z i n g a n t i b o d i e s to

1BRV were u s e d i n t h i s s t u d y . In 10 a n i m a l s (Nos . 17, 77 , 111, 16, 66 , 95 , 15,

72, 104, 84) t h e p r i m a r y immune r e s p o n s e was s t u d i e d . In t h r e e a n i m a l s (Nos . 13,

82 , 84) t h e s e c o n d a r y immune r e s p o n s e was s t u d i e d . Each a n i m a l was g i v e n 10 ml

(3 x 107 TCID50/ml) o f s t o c k IBRV i n t r a n a s a l l y and p r i m a r y immune r e s p o n s e s

were examined i n t h e d e s i g n a t e d a n i m a l s . S t e e r s i n wh ich t h e s e c o n d a r y immune

r e s p o n s e was s t u d y were a d d i t i o n a l l y g i v e n t he same d o s a g e i n t r a m u s c u l a r l y e v e r y

t h r e e m o n t h s i n o r d e r to m a i n t a i n m e a s u r a b l e l e v e l s o f c y t o t o x i c i t y .

T a r g e t c e l l s

C e l l c u l t u r e s p r e p a r e d from t he t e s t i c l e s o f t h e e x p e r i m e n t a l c a l v e s were u s e d

as t a r g e t s f o l l o w i n g p r i m a r y i n f e c t i o n . The c e l l s were f r o z e n and kep t in l i q u i d

n i t r o g e n u n t i l n e e d e d . P r i m a r y c e l l s grown f rom b o v i n e e m b r y o n i c k i d n e y (BEK),

whole b o v i n e embryos (BE), and b o v i n e embryon i c l u n g (BEL) were u s e d as t a r g e t s

f o r l e u k o c y t e s f rom h y p e r i m m u n l z e d s t e e r s . C e l l s were grown in E a g l e ' s Min imal

Essential Medium supplemented with 5% fetal bovine serum (FBS) and 5% calf serum

(CS), 200 U of penicillin/ml, i00 ug of streptomycin/ml, and I00 ug of

neomycin/ml.

Preparation of effector cells

Mononuclear cells, PMN and Sephadex G-10 adherent and non-adherent cells were

prepared as described in the companion article (Campns and Rossi, 1984). Cells

which did not adhere to nylon wool were obtained as previously described by

365

Julius et al., (1973). Briefly mononuclear cells were suspended in RPMI 1640

containing 20% heat-inactivated FBS, and 108 cells in 2 ml of culture medium

were applied to the column. The column was incubated at 38°C for 1.5 h and the

nonadherent cells were eluted with 40 ml of RPMI 1640 with 20% heat-inactivated

FBS. The percentage of immunoglubulin-bearing cells was determined using FITC

rabbit-antibovine sera reactive against bovine u, ~, and light chains.

Mononuclear cells contained 18-25% immunoglobulin-bearlng cells before and after

passage over Sephadex G-10 and 3-7% of immunoglobulin-bearing cells after passage

over nylon wool.

Cytotoxicity assay

Microcytotoxicity assays were performed in round-bottom microtiter plates

using IBRV-infected and noninfected cells as described in the companion article

(Campos and Rossi, 1984). After labeling the target cells with 51Cr,

effector cell preparations (I00 ul) were added and the total volume was adjusted

to 200 ul. Control wells consisting of vlrus-infected and noninfected target

cells without effector cells were prepared at the same time to determine the

amount of spontaneous 51Cr release. The total time of contact between IBRV

and target cells was 20-22 h while the total time of contact between effector and

target cells was 2 h less (18-20 h), unless otherwise indicated. Percentage

51Cr release was determined as described in the companion article (Campos and

Rossi, 1984).

RESULTS

Lysis of autolosous and allo~enelc testicle cells by IBRV-immune mononuclear cells

Two groups of 3 seronegative steers each were tested for their cytotoxlc

response against IBRV-infected and noninfected autologous and allogeneic testicle

cells. Cytotoxlcity before and 6 days after intranasal inoculation was

determined. Representative results of one group are shown (Table I). Although

IBRV-infected cells were more susceptible to lysis by immune mononuclear cells

than noninfected cells, no consistent differences were observed in the

susceptibility of autologous or allogeneic cells. In no instance were nonimmune

effector cells able to lyse IBRV-infected cells (Data not shown).

Characteristics of the primary response

Autologous and allogeneic testicle cells were infected for a total of 23 h.

Mononuclear cells from IBRV-infected cattle were then added and left in the assay

for the last 5 h (short assay) or 18 h (long assay) to determine whether a short

incubation of target and effector cells was adequate. Cytotoxicity was present

only after long-term incubation regardless of the target cell (Table 2). Since

autologous and allogeneic cells were lysed to the same extent, the remainder of

366

the results is expressed as the mean of the different targets used. The

cytotoxic response with time was studied in steer 84 using autologous and two

TABLE 1

Cytotoxicity of mononuclear cells against IBRV-infected autologous and allogeneic BT cells 6 days after intranasal inoculation with IBRVa, b

% Specific 51Cr release

Target Cell Steer 17 Steer 77 Steer iii

BT 17 3.1 1.0 3.8 BT 17-1BRV 26.9 13.6 16.4

BT 77 13.7 4.5 4.4 BT 77-1BRV 38.0 22.0 20.8

BT Iii 6.7 0.0 4.1 BT III-IBRV 31.7 11.6 17.6

asupernatant fluid was harvested after 22 h of inoculation with IBRV and 20 h after the addition of the effector cells.

bMononuclear cells were added at 100:1 effector:target cell ratio.

TABLE 2

Time required for cytotoxiclty of mononuclear cells against IBRV-infected cells a,

. % Specific 51Cr release d 5-h assay u 18-h assay

Target Steer Steer Steer Target Steer Steer Ste'er cell 17 77 iii cell 16 66 95

BT 17 1.6 2.1 1.4 BT 16 3.9 7.1 0.2 BT 17-1BRV 2.6 2.1 2.2 BT 16-1BRV 33.4 27.3 19.6

BT 77 3.4 0.6 0.2 BT 66 5.7 8.4 3.5 BT 77-1BRV -0.3 -1.3 -0.7 BT 66-1BRV 21.1 19.5 17.4

BT III 1.7 0.5 4.8 BT 95 4.9 2.2 4.3 BT IlI-IBRV 5.0 1.7 i0.0 BT 95-1BRV 11.5 19.5 28.8

aThis assay was done 8 days after intranasal inoculation with IBRV. bMononuclear cells were added at I00:I effector to target cell ratio. CTarget cells were inoculated with IBRV 18 h before addition of the effector cells. Mononuclear cells were left in the assay for 5 h.

dTarget cells were inoculated with IBRV 5 h before addition of effector cells.

367

8O

~ I B R V ~ IBRV + Ab G

65 W 4 ~

60 ~ : = I S R V

\ ' 30. ' ~5 , /

40 - , ~ 25.

35 , " U 2 0

2oJ " " I 0 -

5 ~ : / - - " , 3 6 9 i2 I5 18

I 2 S 4 5 6 l 8 9 '0 II 12 IS 14 H O U R S

DAYS

Fig. I. Cytotoxic response a f te r Fig. 2. Ef fect of contact time intranasal inoculat ion with IBRV. among e f fec to r ce l l s , IBRV, and target Mononuclear ce l ls from steer 84 were ce l ls in cy to tox i c i t y . BE ce l l s were tested for the i r a b i [ i t y to lyse infected with IBRV for a period of 22 h target cells at different days after before termination of the assay. inoculation. Supernatants were harvested at 3, 6, 9,

12, 15 and 18 h after the addition of mononuclear cells and assayed for released 51Cr. Results are the mean + standard error of the mean from 3 hyper immune a n i m a l s .

a l l o g e n e l c t a r g e t s . Peak c y t o t o x l c i t y o c c u r r e d b e t w e e n 7 and 14 days a f t e r =

primary intranasal inoculation. Cytotoxicity against noninfected or

Pl3V-infected cells (natural cytotoxiclty) did not vary significantly. Addition

of high-titer anti-IBRV serum did not increase the degree of lysis, but in some

instances, produced a low degree of blocking of cytotoxicity (Fig. i).

Characteristics of the in vivo secondary response

Three steers (Nos. 13, 82, 84) initially given IBRV intranasally that had

serum neutralizing antibodies to IBRV were injected intramuscularly with IBRV to

examine their secondary cytotoxic response. As in the primary response,

cytotoxicity to IBRV-infected cells was not present in a short-term inoubation

(<10% at 8 h) but was significant 13 h after infection of target cells (24%) and

reached a maximum at 18 h (40%). Cytotoxiclty against nonlnfected cells was less

than 5%. The requirement for a long-term incubation did not seem to be related

to a lack of viral antigen on the cell membrane since cells infected 22 h that

were in contact with effector cells for 6 h were not lysed, Whereas at 9 h there

was significant lysis which increased thereafter reaching a peak after't8 h.of

contact (Fig, 2). The requirement for a long contact between effector and

target cell suggests that something initiated early in the culture did not become

effective until later. To eliminate the possibility that cytophilic antibody

368

was involved in cytotoxicity against IBRV-infected cells, extensively washed

mononuclear cells were used as effectors and were compared with normally washed

mononuclear cells. Extensive washing of effector cells failed to reduce the

cytotoxicity (Data not shown).

Effect of nylon wool separation on cytotoxicity

In mononuclear cells from steers (Nos. 13, 82, 84) tested against three

IBRV-infected and noninfected bovine embryonic cells, we were unable to

demonstrate any increase in cytotoxlcity in the nylon wool nonadherent

population. On the contrary, most of the time a significant reduction in

cytotoxicity occurred (Data not shown). However, this reduction in cytotoxicity

did not correlate with the reduced number of B cells usually obtained by this

technique (>70%) and thus argues against B cell involvement.

0 ] STEER 8 4 ; t HYPERIMMUNE

I

4 0 EFFECTOR :

STEER 15

,3 t - _~ ~ I ~!i~g!i!iiii~i~i::iig::1:{::1::i::::::i::~ii::i::~i::::~i::!::!i::::gi::~}::{i:~g~i~i::i::ii 75: ~E!ii!iiii!i~i!2111!i!iiii~ ~ iiiiii [ ~i~ii~ Eli

30 STEER 72 ~ 5o=ll ~i!3!i~i;ii!~ii;;!;~!;!~!~!~i~i::i!;!~i~}i!i!~i~}}~;:iii:'{!::;i;i{;::i::i:;:.;:~i:.~:'!!i!:.!}:ii~i:i::iiii!::!i::::iii~i~i;i~}~]

7'5125: i 5 ° / 5 o = i

, o ,

2,o :

0 J ' - ' - - ~ " " ' = = " ~ ° ~ L ,o/~,~ 0 2 4 6 8 14 o ,o 20 ~o 4o so eo 70 e o

DAYS % SPECIFIC 51Cr RELEASE

Fig. 3. Effect of Sephadex G-IO sep- Fig. 4. Cytotoxicity of hyper- aration on cytotoxicity. Effectors immune steer against BK cells infected and target: mononuclear cells on IBRV- with IBRV. BK cells were infected with infected (B) and noninfected (i) IBRV and 2 h later mononuclear cells cells; nonadherent cells on IBRV- (MC), PMN and their combinations infected (~) and noninfected (~) (MC/PMN) were added at different

cells, effector: target cell ratio. The assay was terminated 18 h after the addition of the effector cells.

369

E f f e c t of Sephadex G-10 s e p a r a t i o n on c y t o t o x i c i t y

Mononuclear cells from hyperimmunized animals were passed through Sephadex

G-10 columns and nonadherent and adherent cells were compared with unfractionated

mononuclear cells. Whereas the nonadherent cells were not cytotoxic for

IBRV-infected cells, the adherent and combinations of adherent and nonadherent

cells were able to fully reconstitute cytotoxicity (Table 3).

Three steers (Nos. 15, 72, 104) were monitored at intervals during the course

of a primary infection and one steer (No. 84) was monitored following

hyperimmunization for a similar time period. Regardless of whether steers were

undergoing infection or were being hyperimmunized, removal of adherent cells by

Sephadex G-10 effectively removed the cytotoxicity (Fig. 3).

The role of antibody on cytotoxicity

To eliminate the possibility that antibody might be produced during long-term

in vitro incubation and might participate in an ADCC reaction with G-10 adherent

cells, cytotoxicity assays were done with PMN (the most efficient mediator of

ADCC in cattle against IBRV-infected cells) mixed at different ratios with

mononuclear cells from a hyperimmunized steer (No. 84) and, at the same time

specific antibody was added to cultures containing PMN. Addition of antibody to

45-

W 40- [ - ' ~ IBRV

W 35- ~ N O VIRUS W

30-

25-

20-

-- 15-

w

~ 5- I

MC Me PMNPMN + +

~glg Agglg

Fig. 5. Effect of aggregated immunoglobulin on cytotoxicity against IBRV-infected cells. ADCC of PMN was evaluated by the addition of specific antisera. No antibody was added to the mononuclear cells (MC). The final concentration of aggregated immunoglobulin (Agg.lg) was 3 mg/ml.

TABLE 3

Effect of Sephadex G-10 separation on cytotoxicity a % Specific 5] C

r release

Target

Calf

cell

Mononuclear b

NonadherentC

Adherent d

13

BT

0.4

0.0

-0.5

BT-IBR

11.5

4.8

12.2

82

BT

2.8

-3.2

ND

BT-IBR

20.8

6.7

20.3

84

BT

6.1

3.4

9.5

BT-IBR

33.1

7.3

32.5

aMicrocytotoxicity assay was terminated 18 h after the addition of effector c~

IBRV inoculation).

Effector cells were used at an effector:target cell ratic

bMononuclear cells obtained after Histopaque separation.

CNonadherent cells obtained by washing the Sephadex G-10 columns with RPMI 16L

20% heat-inactivated FBS.

dAdherent cells were eluted from Sephadex G-10 columns with 0.7% lidocaine ant

0.014M sodium citrate.

ecombination of adherent and nonadherent effector to target cell ratio so that

target cell ratio was 100:1.

371

the cultures containing PMN greatly increased the cytotoxiclty against

IBRV-infected cells, whereas in the absence of added antibody PMN failed to

enhance cytotoxicity of the mononuclear cells (Fig. 4). ~Pnen G-IO adherent cells

from three hyperimmunized calves (Nos. 13, 82, 84) were removed and the PMN mixed

with G-IO nonadherent cells, PMN that caused 62% specific 51Cr release in the

presence of specific antibody were not able to restore the cytotoxic response to

the G-IO effluent cells. Futhermore, the addition of aggregated immunoglobulin

to PMN reduced the high level of ADCC usually demonstrated by these cells against

IBRV-infected cells by 65%; however, the same concentration of aggregated

immunoglobulln was unable to inhibit the cytotoxicity of immune mononuclear cells

against the same target in the absence of specific antibody (Fig. 5).

DISCUSSION

The studies presented here have demonstrated that intranasal inoculation of

cattle with IBRV induces a genetically unrestricted cytotoxic response by

peripheral blood mononuclear cells against IBRV-infected cells that can be

detected 6 to 14 days after inoculation. These findings are in agreement with

previous studies by Rouse and Babiuk (1977) where cytotoxicity generated after

infection with either vaccinia virus or IBRV was directed against the respective

virus-infected autologous and allogeneic cell. In addition, we have demonstrated

that hyperimmunization with IBRV induced cytotoxic responses similar to those of

the primary response. However, unlike Rouse and Babluk (1977), we could not

ascribe cytotoxicity to T cells, but instead found that cytotoxicity was

dependent on a cell that adhered to Sephadex G-10.

The role of CTL in protection against viral infection has been the subject of

intensive studies, and it is now widely accepted that CTL are genetically

restricted to recognize viral antigens in association with major

histocompatibility antigens. Based on the report of Rouse and Babiuk (1977) on

the lack of genetic restriction of CTL to IBRV in cattle, there was some question

concerning the nature of CTL in cattle. However, other investigators

subsequently showed that genetically restricted lymphocytes were indeed produced

by cattle using Theileria parva transformed autologous and allogeneic cells as

targets for mononuclear cells obtained from T. parva-immunized cattle (Emery et

al., 1981; Eugui and Emery, 1982).

Besides the lack of genetic restriction in the cytotoxicity we have described,

additional evidence indicating that CTL were not involved is the requirement for

a long contact time between effector and target cell in our assay. This suggests

that either (i) activation of certain cells occurred during the incubation

period; (ii) the effector cell required a long time to kill celia; or (iii) only

a small population of cells was involved in lysis and that the cells required

372

considerable recycling before significant lysis could be detected. Also, whereas

CTL enriched populations are obtained after passage of mononuclear cells through

a nylon wool column (Lawman et al., 1980), we found that cells nonadherent to

nylon wool were not as efficient mediators of cytotoxicity as unfractionated

mononuclear cells. This result is in contrast to the report by Rouse and Babiuk

(1977) who detected genetically unrestricted killing associated with nylon wool

nonadherent cells. Furthermore, whereas vlrus-specific antiserum can often block

the interaction between CTL and virus-infected target cells (McFarland, 1974) we

found that addition of specific antibody against IBRV neither blocked nor

enhanced cytotoxicity (Fig. I).

That natural killer cells were not responsible for the cytotoxicity we

observed is suggested by the fact that though a typical natural killer cell has

not been detected in cattle, the natural cytotoxicity of bovine mononuclear cells

for parainfluenza virus type 3-1nfected cells is found in the Sephadex G-IO

nonadherent cell fraction (Campos et al., 1982). In contrast the cytotoxicity

to IBRV-infected cells was found in the adherent cell fraction. Furthermore, in

cases of activation of natural cytoxicity, the duration of enhancement is

generally short (Biron and Welsh, 1982) and activated natural killer cells are

not present in hyperimmunized animals (Biron and Welch, 1982). Nevertheless,

natural killer activity cannot be completely eliminated as a possible mechanism,

since failure to lyse one type of cell cannot be considered as an indicator of

all natural killer activity.

Being confident that typical CTL were not involved in the cytotoxicity we

observed, we were concerned about the role of ADCC in our assay. Although ADCC

is usually thought of as occurring by lysis of antlbody-sensitlzed target cells,

antibody-sensitlzed effector cells can also produce target cell lysis (Imir et

al., 1976). Human peripheral blood mononuclear cells that were cytotoxic for

influenza virus-lnfected allogeneic and xenogeneic cells in a 4-h assay were

dependent on the presence of antibodies on nonadherent effector cells. This

cytotoxicity was readily eliminated by incubating the effector cells at 37°C for

30 min prior to the assay (Greenberg et al., 1978). The cytotoxicity of human

peripheral blood mononuclear cells from seropositlve people for herpes simplex

virus-lnfected allogenelc cells was dependent on antibody that could be removed

by extensive washing of the effector cells (Moller-Larsen et al., 1977). In the

present experiment, extensive washing of mononuclear cells did not alter

cytotoxicity. Of further concern to us was the finding that peripheral blood

lymphocytes from vaccinia vlrus-immunized people were able to lyse allogeneic

target cells in 18 h, but not in 6 h, cytotoxicity assays due to the presence of

K cells and antibody produced during the incubation period (Perrin et al., 1977).

In contrast, Armerding and Rossiter (1980) found that cells from influenza

virus-infected mice present in a 16 h assay produced antibody, but ADCC was not

373

involved in lysis of allogeneic cells. However, addition of complement in the

system or the presence of complement in the fetal calf serum resulted in target

cell lysis. In order to avoid this complication we used heat-inactivated serum.

The above reports suggest that for antibody to mediate ADCC in assays designed

to measure direct cytotoxicity, efficient effector cells, such as K cells found

in the peripheral blood of human beings (Tada et al., 1980), but almost

undetectable in murine tissues (Tada et al., 1980), must be present. That ADCC

was not present in our assays is likely from the following evidence: (i) though,

among mononuclear cells, Sephadex G-IO adherent cells are the cells that mediate

ADCC to IBRV-infected cells (Campos and Rossi, 1984), they are not very efficient

mediators of ADCC; (ii) separation of bovine mononuclear cells on Sephadex G-IO

yielded a nonadherent, nonphagocytic, noncytotoxic cell population and an

adherent, phagocytic, cytotoxic cell population without altering the proportion

of B cells present in the nonadherent cell population; (iii) the number of free

virus particles in our assay was probably high since the target cells were not

washed after viral inoculation so that the majority of antibody produced during

incubation of the effector cells was probably bound by free virus and was not

available for ADCC; (iv) while passage of mononuclear cells through a nylon wool

column reduced the cytotoxicity, the reduction did not correlate with the marked

reduction in the number of B cells (80%); (v) PMN, the most efficient mediators

of ADCC against IBRV-infected cells (Grewal et al. 1977; Cempos and Rossi, 1984),

did not increase cytotoxicity when added to the cultures; (vi) finally, addition

of aggregated immunoglobulin was very effective in blocking PMN-mediated ADCC

against IBRV-infected cells, but was ineffective in blocking cytotoxicity of

mononuclear cells against the same targets (Fig. 5).

Macrophages activated by a variety of immunomodulators are capable of exerting

cytotoxic or cytostatic activity against tumor cells. In contrast to macrophages

activated by nonspecific immunomodulators, macrophages activated by alloantigens

and/or tumor antigens are, at least at some stage after immunization,

specifically cytotoxic for the immunizing cell (Gallily and Eliaha, 1976). Evans

and Alexander (1970, 1972) have suggested that macrophages become armed with

antigen-induced lymphocytic factors that allow specific recognition of target

cells. Similarly, Gallily and Eliahu (1976) found that the cytotoxic effect of

macrophages derived from alloimmunized mice (immune macrophages) were

immunologically specific and that sensitized T cells, but not B cells, were

capable of arming nonimmune macrophages and rendering them cytotoxic. Since the

cytotoxicity directed against IBRV-infected cells followed an immune pattern, and

at least one of the cells needed for cytotoxicity is adherent to Sephadex G-10

(probably a mononcyte/ macrophage), T lymphocyte specific arming and/or

activation of this cell must be considered as a possible cytotoxic mechanism.

Thus, we have shown that genetically unrestricted cytotoxicity develops in

374

cattle infected with IBRV, and the cell that is needed for cytotoxicity adheres

to Sephadex G-10. The cellular basis of this cytotoxicity remains undefined,

although the present evidence does not favor ADCC, natural cytotoxitity, or

cytotoxicity mediated by CTL.

ACKNOWLEDGEMENTS

This work was supported in part by the Alabama Agricultural Experiment Station

and U.S. Department of Agriculture Science and Education Administration grant

59-2011-0-2-047-0.

We thank Roger Bridgman for technical assistance.

Publication No. 1620 School of Veterinary Medicine, and Alabama Agricultural

Experiment Station Journal Series No. 5-83389, Auburn University, AL.

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