Ve~rinaryImmuno~gyand Immunopathology, 8{1985) 351--362 351 Elsevier Science Publishers B.V., Amsterdam -- P r in t ed in The Netherlands

INABILITY TO DETECT A K CELL IN BOVINE PERIPHERAL BLOOD LEUKOCYTES

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. Inability to detect a K cell in bovine peripheral blood leukocytes. Vet. Immunol. Immunopathol., 8: 351-362.

Antibody-dependent cellular eytotoxicity to viral-infected cells, chicken red blood cells, and tumor cells was tested using different effector cell populations: polymorphonuclear cells, mononuclear cells, and mononuclear cells separated into adherent and nonadherent populations by Sephadex G-10. Polymorphonuclear cells were the most efficient mediators of antibody-dependent cellular cytotoxicity against most targets, although a combination of G-10 adherent and polymorphonuclear cells was more efficient in killing infectious bovine rhinotracheitis virus-infected cells than either single cell population. Removal of G-10 adherent cells from the mononuclear cell population removed all antibody-dependent cellular cytotoxieity from that population, indicating the lack of a typical K cell in bovine peripheral blood.

INTRODUCTION

Antlbody-dependent cellular cytotoxicity (ADCC) is a powerful cytolytic

mechanism in which antibody and Fc receptor-bearing effector cells cooperate.

Chicken red blood cells (CRBC), tumor cells, and virus-infected cells are

generally used as targets. Erythrocytes and virus-infected cells are lysed by

specific antibody and human lymphocytes, monocytes, and polymorphonuclear cells

(PMN), but tumor cells are lysed only by certain lymphocytes (K cells)

(Sanderson et al., 1975: Nelson et al., 1976; Berger and Amos, 1977; Melewicz et

al., 1977). K cells detected in human peripheral blood have been well

characterized (Trinchieri et al., 1975: Timonen et al , 1981. Bradley and

Bonavida, 1982), but their presence in other species has been controversial.

Mouse and rabbit cells from different sources have been reported to be very

inefficient or completely inactive against nucleated mammalian cells (Berger and

Amos, 1977; Tada et al., 1980: Karavodin et al., 1982). However, K cells have

been detected recently in the large granular lymphocyte population of mouse

spleen cells at a relatively low frequency (Kumagi et al., 1982).

Reports of ADCC in cattle have been limited to studies with infectious bovine

rhinotracheitis virus (IBRV)-infected cells and CRBC as targets.

0165-2427/85/$03.30 © 1985 Else~er Science Publishers B.V.

352

Polymorphonuclear c e l l s (PMN) ob ta ined from the l i p o p o l y s a c c h a r i d e - s t i m u l a t e d

mammary gland were the most efficient mediator of ADCC against these two target

cells (Rouse et al., 1976; Wardley et al., 1976b; Grewal et al., 1977).

Lymphocytes and macrophages mediated ADCC against CRBC, but macrophages (not

lymphocytes) could mediate ADCC against virus-infected cells (Rouse et al.,

1976). Because different target cells have different susceptibilities to

different effector cell populations and different effector populations are

present in tissues in different species, the present investigation was

undertaken to identify more thoroughly the cells in bovine peripheral blood that

mediate ADCC. A variety of target and effector cell populations were evaluated

in ADCC assays.

MATERIALS AND METHODS

Tarset cells

Bovine cells were cultured in monolayers from whole bovine embryos (BE),

bovine embryonic lung (BEL), bovine testicle (BT) or bovine embryonic kidney

(BEK). Cells were grown in Eagle's Minimal Essential Medium (MEM) with 10%

fetal bovine sertml (FBS), 200 U of penicillin/ml, i00 ug of streptomycin/ml, and

I00 ug of neomycin/ml. Human cell lines HSB-2, K562 and the mouse myeloma cell

line X63-Ag8.653 were maintained in suspension in RPMI 1640 containing 10%

heat-inactivated (56°C, 0.5 h) FBS and 5 ug of gentamicin/ml.

Freshly obtained CRBC were washed 3 times in Hanks' Balanced Salt Solution

before use.

Viruses

The following viruses were used: the Cooper strain of IBRV (3 x 107 TCID

50/ml), a herpesvirus; the SF-4 strain of paralnfluenza 3 virus (PI3V) (4 x

106 TCID 50/ml), a paramyxovirus; and a noncytopathogenic strain of bovine

viral diarrhea virus (NC-BVDV), a pestivirus, obtained from persistently

infected GBK cells.

Antiserum

Antisera produced in calves to IBRV, PI3V, BVDV-NADL, (which cross-reacted

with NC-BVDV), CRBC, the human tumor cell lines K562 and HSB-2, and murine

myeloma cell llne X63-AgS.653 were heat-lnactivated and stored at -70°C until

used.

Effector cells

Blood from 12- to 72-month-old crossbred cattle free of virus neutralizing

antibody to IBRV or PI3V was used as the source of effector cells. Whole blood

was diluted with an equal part of 0.014M citrate in phosphate buffered saline

353

(CPBS) and the buffy coat obtained by centrifugation. Buffy coat cells were

diluted 1:3 with CPBS and layered onto Histopaque (Sigma Chemical Co., St.

Louis, MO), density 1.077g/cm 3, and the tube was centrifuged at 600 x g for 12

min. Cells recovered from the interface washed 2 times in CPBS were 98-100%

mononuclear and had 97-99% viable cells.

Mononuclear cells were separated into adherent and nonadherent cells on

Sephadex G-10 (Pharmacia, Piscataway, NJ) columns as described by Jerrels et al.

(1980). A 10 ml plastic syringe barrel was plugged with a small piece of glass

wool and filled with 6-8 ml of Sephadex G-10. The column was washed with 50 ml

of RPMI 1640 containing 20% heat-lnactlvated FBS and incubated at 38°C for 0.5

h. Two ml of medium containing 5 x 107 mononuclear cells were placed on the

column and incubated at 38°C for 30 min. Nonadherent cells were eluted with 40

ml of prewarmed RPMI 1640 containing 20% heat-inactivated FBS. Adherent cells

were removed by adding 5 ml of 0.7% lidocaine hydrochloride (Lid-O-Cain 2%,

Butler Company, Columbus, OH), incubating the column for i0 min, and eluting the

cells with CPBS. Both cell preparations were greater than 95% viable. The

nonadherent population had less than i% phagocytic cells and the adherent

population had 15-20% phagocytic cells as determined by latex ingestion.

Phagocytic cells were identified by incubating 0.5 ml of 106 cells/ml with I0

ul of 10% latex particles (1.091 um diameter, Dow Diagnostics, Indianapolis, IN)

at 38°C 2-3 h.

PMN were obtained from the erythrocyte portion of peripheral blood after

centrifugation. (Chambers et al., 1982). Erythrocytes were lysed with a 0.83%

solution of ammonium chloride. The cells were washed once with CPBS,

resuspended in CPBS, and 8 ml of cell suspension was layered over 4 ml of 68%

Percoll solution (Pharmacia), density 1.0924g/cm 2. The tubes were centrifuged

at 400xg for 15 min and the enriched PMN pellet was washed in CPBS and then in

complete medium. Differential counts of the cell subpopulations were made on

cells centrifuged on a cytocentrifuge (Cytospln centrifuge, Southern

Instruments, Inc., Sewickley, PA) and stained with Diff Quick (Harleco,

Gibbstown, N.J.). Viability was determined by trypan blue exclusion. PMN

preparations contained 95-97% neutrophils and had 90-95% viable cells.

Cytotoxicity assay

Microcytotoxicity assays were performed in quadruplicate in round-bottom

microtiter plates at 38°C in a humidified atmosphere of 95% air and 5% CO 2.

The culture medium used throughout the assay was RPMI 1640 containing 5 ug of

gentamicin/ml, 2 mM L-glutamine, and 10% heat-lnactivated FBS. Adherent target

cells (BE, BEL, BT, BEK) were labeled with 51Cr in wells (104 cells/well)

as previously described (Campos et al., 1982): i00 ul of medium containing 1.5

to 2 uCi of sodium chromate (Na 2 51CrO4, New England Nuclear Corp.,

354

Boston, MA) were added to each well, and after 12 to 15 h of incubation the

51Cr-labeled medium was removed and 100 ul of RPMI 1640 containing 10%

heat-inactivated FBS was added to each well for 2-4 h more incubation. The

cells were washed 2 times with HBSS. The virus suspension (25 ul) or culture

medium was added to each well, and the microtiter plates were incubated for I to

1.5 h unless otherwise indicated. The solutions were removed by inverting the

plates; 25 ul of antiviral or normal serum was added; and the plates were

incubated for 0.5 h. Effector cells (i00 ul) were added and the total volume

was adjusted to 200ul/ well with culture medium.

Nonadherent tumor target cells K562, HSB-2 and X63-Ag 8.653 were labeled in

suspension (2 X 107/ml) with 1 ml of RPMI-1640 containing 10% FBS and I00 uCi

of Na251Cr04 for 1.5 h at 38°C. CRBC (2x107/ml) were labeled with 200

uCi of Na251Cr04 for 4 h at 38°C. All target cells labeled in

suspension were washed three times in HBSS and suspended at 2 x 105 cells/ml.

A 50 ul amount of cell suspension was incubated with 50 ul of specific antlsera

at 38°C for 0.5 h. Finally, i00 ul of effector cell suspension was added. In

each experiment, controls for each variable (with and without antibody, with and

without effector cells, virus-infected and uninfected cells) were run

simultaneously. The standard error for the replicates was always below 3%.

Microtiter plates were incubated with effector cells for different intervals,

usually 5-6 h (short-term) for target cells in suspension and 18 h (long-term)

for virus-infected cells unless otherwise indicated. At the termination of the

assay, total 51Cr release was obtained by adding I00 ul of 3% Triton X-100

(Sigma) to 8 wells/plate. Supernatant fluids were collected with the Titertek

harvesting system (Flow Laboratories, McLean, VA), and the amount of 51Cr

released was counted. Spontaneous release was always less than 1.5%/h.

ADCC was defined as the amount of lysis for target cells in the presence of

effector cells and immune sera minus the amount of lysis from target cells in

the presence of effector cells and non-immune serum, and was calculated based on

the amount of 51Cr released as follows:

Percent specific ( mean cpm experiment - mean cpm control ) XIO0 51Cr release = (mean cpm detergent lysls - mean cpm control)

RESULTS

ADCC a~ainst virus-infected ~ells

At an effector: target cell ratio of i00:I, a minimum of 12 h after IBRV-

inoculation was necessary before significant levels of cytotoxlcity (10%) with

mononuclear cells occurred while, maximal cytotoxlclty (42%) occurred at 23 h

when the background release of 51Cr was still below 30%.

355

Removal of adherent cells from the mononuclear cell population on Sephadex

G-10 columns completely removed the ADCC activity against IBRV-infected cells

while G-IO adherent cells retained this activity (Table I).

Mononuclear cellS, PMN, and mixtures of both were tested against

IBRV-infected cells in short-term cytotoxicity assays (Table 2). Under these

conditions ADCC was not found in the mononuclear cell population. However,

TABLE 1

Effect of mononuclear cell separation on Sephadex G-10 on ADCC to IBRV-infeeted

cells a

%Specific 51Cr release Target Cell Serum Mononuclear b G-10 nonadherent G-IO adherent

BE Normal 2.9 2.9 3.5 BE IBRV Normal -4.3 -5.3 -2.3 BE IBRV Ant i-IBRV 19.1 1.7 17.8

aSupernatants were collected 20 h after inoculation with IBRV, 18 h after the addition of effector cells.

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

TABLE 2

Synergism between PMN and mononuclear cells in mediating ADCC against IBRV-

infected BEL cells in 4-h assay a

% Specific 51Cr release

Calf Serum MC b PMN c PMN/MC d

82 Normal 0.2 1.5 7.2 Ant i-IBRV i. 7 61.0 62.5

308 Normal 0.9 -0.2 O. 5 Ant i-IBRV I. 8 60.5 74.7

84 Normal 0.8 2.1 I. I Anti-IBRV 0.2 56.6 57.3

13 Normal 0.6 5.1 7.2 Anti-IBRV 0.8 68.7 66.0

aSupernatants were collected 20 h after infection with IBRV, 4 h after the addition of effector cells.

bMononuclear cells after Histopaque separation at I00:I effector:target cell ratio.

CpMN after Percoll separation at i00:I effector: target cell ratio. d50% of mononuclear cells were added to 50% of PMN to have a final effector to target cell ratio of I00:i.

356

when mononuclear cells were mixed with an equal number of PMN, instead of a

reduction in PMN-mediated ADCC, the level of cytotoxicity remained the same. To

further investigate this ~enomenon, different effector:target cell ratios of

each cell type and their combination were compared in the same proportion. The

mononuclear cell:PMN combination was a more efficient mediator of ADCC than any

single cell type. At the same time, a slight increase in cytotoxicity in the

absence of specific antibody was noted (Fig. I).

80-

[] AN~ - mB~ ~ M

I0

0 ~ , , ,o,, ,~,, ~ " ~, , ~,, MC PMN MC

PMN

Fig. i. Comparison of ADCC against IBRY-infected BE cells mediated by mononuclear cells (MC), PMN, and their combination (MC/PMN) in a long-term cytotoxicity assay at effector: target cell ratios of I00:I and I0:i.

Mononuclear cells, G-10 nonadherent cells, G-10 adherent cells, PMN and

combinations of these cells were compared for their ability to kill IBRV-

infected cells. Cytotoxicity was always greater in the presence of antibody and

when G-IO adherent cells and PMN were combined (Table 3).

PMN were used as effector cells against Pl3V-infected, ~-BVDV-infected, and

IBRV-infected cells in the presence of immune serom (Table 4). The same PMN

population that effectively lysed IBRV-infected cells in the presence of

antibody was able to mediate only low levels of cytotoxicity against

NC-BVDV-infected and Pl3V-infected cells. Mononuclear cells did not show any

ADCC against Pl3V-infected cells in a long-term assay. However, when the high

level of natural cytotoxicity normally shown by mononuclear cells against

Pl3V-infected cells (Campos et al., 1982) was eliminated by usi~ a 5 h

incubation of effector and target cells in which target cells were infected 12 h

prior to addition of antiserum and effector cells, ADCC was found (Pl3V-infected

cells plus antibody, 15%; normal cells plus antibody 1%). Mononuclear cells

tested against bovine testicle cells persistently infected with NC-BVDV in a

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long-term ADCC assay were able to mediate only 15-17% ADCC in the presence of

antibody and no ADCC without antibody.

TABLE 3

ADCC of different effector cell fractions against IBRV-infected cellsa 51 b

%Speciflc Cr release

Target Specific C d e f n g g cell antiserum MC G-10NA G-10A PMN MC/PMN ~ G-IONA/PMN G-10A/PMN

BEK No 4.5 8.9 19.8 6.1 NT 7.1 0.5 BEK IBRV No 8.6 -7.0 19.7 -1.3 17.0 -2.8 1.5 BEK IBRV Yes NT -8.6 36.0 33.7 48.6 33.7 64.8

aMicrocytotoxicity assays were terminated 22 h after infection 18 h after addition of the effector cells.

bThe final effector: target cell ratio was 100:1. CMononuclear cells after Histopaque separation. dsephadex G-10 nonadherent cells. esephadex G-10 adherent cells. fpolymorphonuclear cells after Percoll separation. gcombination of effector cells were obtained by mixing 50% of each cell preparation before the assay.

TABLE 4

PMN-mediated ADCC against NC-BVDV-, IBRV-, AND Pl3V-infected cells

%Specific 5] Cr release

Specific Target cell antiserum 50:i b 25:1 I0:I

BEL NC-BVDV No 0.0 -5.0 -0.4 BEL NC-BVDV Yes 13.5 4.2 1.8 BEL IBRV No 15.8 -0.2 -0.9 BEL IBRV Yes 32.5 8.6 6.0 BEL PI3V No 0.3 1.5 -1.6 BEL PI3V Yes 5.5 -4.9 1.0

apMN cells were in contact with target cells for 18 h before the termination of the assay.

bEffector: target cell ratio.

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TABLE 5

ADCC a~ainst nonviral infected target cells a'~

%Specific5] Cr release

Target Specific cell antiserum c MC d PMN e

CRBC None 0.0 0.7 CRBC 1/50 15.6 87.6 K562 None -2.9 -2.1 K562 i/I0 3.3 13.1 HSB-2 None 0.7 8.9 HSB-2 Undil. 6.1 23.1 X63-Ag 8.653 None 10.4 13.2 X63-AG 8.653 i/I0 8.2 80.4

asupernatant fluids were collected after 6 h of incubation with the effector cells.

bThe effector cells were adjusted to a final effector:target cell ratio of 100:1

Coptlmal dilution of antibody to cause ADCC. dMononuclear cells after Histopaque separation. epolymorphonuclear cells after Percoll separation.

TABLE 6

ADCC of different effector cell fractions a~ainst CRBC

%Specific 5] Cr release

a b c MC G-10NA G-10A

d Calf No. Serum I00:i 50:1 I00:I 50:1 I00:I 50:1

22 Normal 2.3 2.6 0.6 -2.7 3.1 1.8 Ant i-CRBC 18.7 15.3 -1.8 -1.7 14.4 7.6

23 Normal 1.5 1.6 3.4 -2.3 0.4 ND Ant i-CRBC 36.1 24.5 3.9 -1.7 14.5 5.3

15 Normal -2.1 -0.9 0.I -0.4 -5.0 -0.I Ant i-CRBC 7.6 4.3 0.I 0.0 18.0 16.9

aMononuclear cells after Histopaque separation bSephadex G-IO nonadherent cells CSephadex G-10 adherent cells dEflector: target cell ratio.

ADCC against other target cells

To further investigate ADCC mechanisms in cattle, specific antibody against a

variety of target cells were produced and tested in short-term cytotoxlcity

assays. CRBC and the mouse myeloma cell line X63-Ag 8.653 were more susceptible

359

to lysis than either K562 or HSB-2, and in all cases PMN were more efficient

mediators of ADCC when compared with mononuclear cells (Table 5). Passage of

mononuclear cells through Sephadex G-10 columns removed the ADCC activity

against CRBC. The cell that mediated cytotoxicity adhered to the column and was

recovered by lidocaine treatment (Table 6).

DISCUSSION

Our results suggest that cattle lack a readily detectable nonadherent~

nonphagocytic K cell in peripheral blood, since removal of adherent mononuclear

cells by Sephadex G-10 removed cytotoxic effectors against all targets tested

(CRBC, tumor cells, and virus-infected cells). Rouse et al (1976) have

suggested that two kinds of bovine mononuclear cells, a plastic adherent and a

nonadherent cell, mediate ADCC against CRBC. However, the activity of the

nonadherent cell was much less than that of the adherent cell. We were unable

to detect a nonadherent mononuclear cell that was able to mediate ADCC against

CRBC. Because Sephadex G-10 columns are more effective in removing adherent

cells from human mononuclear cell preparations than other adherence techniques

(Jerrells et al., 1980; Kanski et al., 1981), we suggest that the minimal level

of ADCC detected by Rouse et al (1976) in the nonadherent population was due to

the lesser efficiency of plastic than G-10 in removing adherent cells. We did

not find any reduction in the number of immunoglobulin-bearing cells, but we did

find a consistent reduction of monocytes (Campos and Rossi, companion article).

The G-10 adherent cells recovered after treatment of the column with

lidocaine retained ADCC activity against CRBC, virus-infected cells, and tumor

cells. We also found PMN to be active against CRBC (Table 5) virus-infected

cells (Table 4) and tumor cells in ADCC.

Neutrophils have been shown to mediate ADCC in human beings (Fujimiya et al.,

1977), rabbits (Smith and Sheppard, 1982), guinea pigs (Hopkins and Dale, 1980),

and cattle (Wardley et al., 1976a; Wardley et al., 1976b; Grewal et al., 1977).

Grewal et al (1977) reported that PMN from the stimulated mammary gland of cows

were the most effective mediators of ADCC against IBRV-infected cells and CRBC°

Grewal et al., (1977) additionally reported that even though PMN from peripheral

blood were more effective than peripheral blood mononuclear cells, the former

were only 50% as effective as PMN obtained from the mammary gland. We found

that peripheral blood PMN were more efficient mediators of ADCC against all

targets tested than either mononuclear cells or G-10 adherent cells. The

combination of PMN and mononuclear cells was a better mediator of ADCC against

IBRV-infected targets than either cell fraction alone. Synergism was due to PMN

and G-10 adherent, rather than PMN and nonadherent, cells (Table 3). This

effect may be due to an increase in the viability of PMN by mononuclear cells or

to the production of l~phokines that activate PMN. Other factors that may

360

account for the synergism between mononuclear cells and PMN are the observations

that PMN are cytotoxic in the presence of complement (Grewal et al., 1980),

complement enhances ADCC to herpes virus-infected cells (Rouse et al., 1977),

and macrophages produce significant amounts of complement (Stecher, 1970). The

slight increase in cytotoxicity against IBRV-infected cells in the absence of

antibody (Fig. I) is in agreement with observations reported by Rouse et al.,

(1979, 1980) that PMN produce an interferon-like molecule in the presence of

herpes virus-infected cells that activates effector cells to mediate natural

cytotoxicity against IBRV-infected cells.

ACKNOWLEDGEMENTS

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.

The authors thank Roger Bridgman for technical assistance.

Publication No. 1619 School of Veterinary Medicine, and Alabama Agricultural

Experiment Station Journal Series No. 5-83390, Auburn University, AL.

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