Ontogeny of natural killer cell activity and antibody dependent cell mediated cytotoxicity in pigs

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. i0, pp. 405-418, 0145-305X86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

1986.

ONTOGENY OF NAIURAL KILLER CELL ACTIVIIY AND ANIIBODY DEPENDENT CFI_L MEDIATED CYTOTOXICITY IN PIGS

W. C. Yangl and R. D. Schultzl Animal Health Research, Agricultural Experiment Station and Department of Microbiology School of Veterinary Medicine Auburn University, AL 36849

ABSTRACT Cells from pigs of various ages were collected from peripheral blood, mesenteric lymph node, spleen and thymus and t h e i r a b i l i t y to mediate antibody-dependent cell-mediated c y t o t o x i c i t y (ADCC) and natural k i l l e r (NK) cell a c t i v i t y was determined. ADCCagainst chicken red blood cell (CRBC) was present in ceils from peripheral blood, lymph node and spleen, but was absent in thymic c e l l s . There were no age-related differences in ADCC to CRBC and cells from fetal pigs had s i m i l a r a c t i v i t i e s to cells from adult pigs. Maximal c y t o t o x i c i t y against CRBC was found in the polymorphonuclear leukocyte (PMN) cell f r a c t i o n . In contrast to the good response against CRBC, PMN cells were not l y t i c in the ADCC assay when PI3 virus-infected cells were used as target c e l l s . Peripheral blood lymphocytes (PBL) had low but s i g n i f i c a n t l y r i c a c t i v i t y against PI3 virus-infected cells in the presence of high concentrations of specific antiserum. NK cell a c t i v i t y against K562 target cells was readily detected in PBL of pigs older than 2 weeks but was not observed with cells from spleen, lymph node or thymus from pigs of any age. PBL of pigs younger than 2 weeks of age had low but detectable NK a c t i v i t y : however, fetal pigs had no NK a c t i v i t y against K562 target c e l l s . In contrast, when PI3 virus infected Vero cells were used as target c e l l s , NK cells were detected in spleen and PBL, but not lymph nodes or thymus, of pigs greater than

]Current Address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53?06 4O5

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one day of age. Similar to the absence of a c t i v i t y to K562, none of the lymphoid cells from fetal pigs had NK a c t i v i t y against PI3 virusinfected Veto c e l l s . The present results suggest that the effector cells that mediate ADCC are d i s t i n c t from those that mediate NK a c t i v i t y in that ceils mediating ADCC develop e a r l i e r and are found in d i f f e r e n t organs than the NK c e l l s . Additionally, the cells that mediate NK a c t i v i t y against v i r a l infected cells may be d i f f e r e n t from those that mediate NK a c t i v i t y for K562 target c e l l s ; however, regardless of the target, NK cells are not present before b i r t h in the pig.

INTRODUCTION The role of natural k i l l e r (NK) cells and k i l l e r (K) cells mediating ADCC in 6atural immune defense mechanisms and immune surveillance has been investigated in mice, humans and certain other species ( I - 3 ) . NK ceils have spontaneous c y t o l y t l c effects against a variety of tumor ceils (2,3), v i r u s infected cells (1,3-6), and some normal cells (4,6). Certain characteristics of NK cells in man and mice have been well established. NK cells are a subpopulatlon of lymphocytes, null c e l l s , lacking the unique characteristic markers for i d e n t i f i c a t i o n of either T or B cells (1,6). NK cells are nonadherent and have receptors for the Fc portion of IgG (1,3,4). NK cell a c t i v i t y can be augmented by interferon and interleukln 2 (1,3,4). Some properties of activated NK cells are different from those of endogenous NK cells in that activated NK cells have an increased adherence to nylon wool, are enriched in Fc receptors, are enlarged in size, and have an expanded target range (4). ADCC has been suggested as a mechanism for natural resistance against tumors, certain v i r a l infections, and other microbial diseases (1,7). ADCCis mediated by a variety of c e l l s , such as neutrophils, macrophage-monocytes and lymphocytes. The specific interaction between target ceil-bound antibody and Fc receptor of the effector ceils is essential for the i n i t i a t i o n of ADCC. ADCC can be influenced by the substances that a l t e r metabolism of ceils (8). Lymphocyte effector cells for ADCC (K cells) and NK cells belong to overlapping populations of null cells (1,3,4). Owing to the presence of Fc receptors on NK cell membrane, NK cells are reported to mediate ADCC against targets r e l a t i v e l y resistant to endogenous NK a c t i v i t y (4,6). These NK cells are functionally defined as K lymphocytes when they are mediating ADCC, and can lyse a n t l v l r a l antlbody-coated vlrus-infected target c e l l s . Therefore, cells in the null cell population have the potential of mediating specific cytolysls of vlrus-lnfected cells (4,6). The ontogeny of NK cell a c t i v i t y against K562 tumor c e l l s , and K cell a c t i v i t y against TNP modified human B cell line SB has been reported in specific pathogen-free miniature pigs (9,10). The development of effector c e l l s , mainly neutrophils, mediating ADCC against chicken red blood cells (CRBC) in various organs has been investigated in cross bred conventional pigs ( l l ) . There are a few reports of NK or K cell mediated c y t o t o x l c i t y against virus infected cells in domestic animals (e.g. cattle and dogs) (5,12,13); however, l i t t l e or no information about NK and K cell a c t i v i t y to virus-lnfected cells is available for the pig.

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The present study was designed to evaluate the a b i l i t y of leukocytes of various organs from pigs of d i f f e r e n t ages ( f e t a l , neonatal and adult) to mediate natural k i l l e r a c t i v i t y against the tumor cell K562 and virus-lnfected target c e l l s . I t was also designed to examine the a b i l i t y of various cell types from pigs of d i f f e r e n t ages to function as k i l l e r cells in ADCC assay against heterologous red cells and vlrus-lnfected target c e i l s , with the expectation that these studies would better define the relationship between cells mediating NK and K cell a c t i v i t i e s .

MATERIALS AND METHODS

Animals Sixty cross bred (Landrace x Hampshire, Landrace x Duroc) pigs, one day of age to greater than 6 months old, and 6 fetal pigs (approx. llO days of gestation) obtained by hysterectomy were used in this study. Effector Cell Preparation Cells from peripheral blood, mesenterlc lymph node, spleen, and thymus were used as sources of effector c e l l s . Peripheral blood mononuclear cells (MN) were isolated by a modified density gradient method described by B#yum (14). Peripheral polymorphonuclear cells (PMN) were obtained from the red cell pellets after separation on Ficoll (Pharmacla Fine Chemicals, Piscataway, NJ) - Hypaque (Winthrop Laboratories Inc., New York, NY) (F-H) by hypotonic shock. Peripheral leukocytes (PMN & MN) were separated by sedimentation of red blood cells with 3% dextran (Dextran T-500, Pharmacla Fine Chemicals). Lymph node, spleen, and thymus were removed from animals immediately after death. The lymphoid cells were isolated by gently mincing the tissues in Ca2÷, Mg2+-free Hanks' balanced salt solution (HBSS, Gibco, Grand Island, NY) and centrifuging the cell suspension on a F-H gradient. All cells were washed three times in HBSS and resuspended in RPMI-1640 containing I0% fetal bovine serum (FBS, Gibco). Adherent cells were removed from MN cells by incubation of MN on plastic Petrl dishes for two hours at 37°C (15). The v i a b i l i t y of a l l cell suspensionswas determined by trypan-blue exclusion. V i a b i l i t y was always greater than 95%. Antlsera Antisera against CRBC and parainfluenza virus type 3 (PI3, strain SF-4) virus for the ADCC assay were prepared in pigs. Two adult pigs were inoculated with 30 ml of 20% CRBC suspensions Intraperltoneally every other day for five inoculations. Three weeks after the last inoculation, sera were collected and heat inactivated. Antibody t l t e r determined by the mlcrotlter hemagglutlnation technique was 1:256 (16). Antlsera to PI3 were prepared in two pigs by multiple Intranasa] inoculations of 5 ml of v i r a l suspension (lO 6.5 TCIDso/ml). A standard mlcrotlter neutralization test was used to determine the serum neutralization t l t e r which was l:l,O00 (17). Target Cells The human myeloblastic cell llne K562 was maintained in RPMI-1640 medium supplemented with I0% FBS and I% a n t i b i o t i c s ( p e n i c i l l i n lO0 U/ml, streptomycin lO0 ~g/ml, fungizone 0.25 ~g/ml) (Gibco). Vero cells were maintained as a monolayer in Eagle's minimal essential medium (EMEM) (Gibco)

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containing 2% FBS and I% a n t i b i o t i c s . Cultures were incubated in a humidified, 37°C, 5% CO2 in a i r chamber. The Vero cells were infected with PI3 virus prior to use in the assay. Cytotoxiclty Assay for NK and ADCC a c t i v i t y A modified 51Cr-release mlcrocytotoxlcity assay was performed as previously described (18) to detect NK and ADCC a c t i v i t y . The ADCC was performed as follows: l to 2 x I07 CRBC in 0.5 ml RPMI-1640 containing I0% FBS were labelled with lO0 to 200 ~Ci 51Cr Na2Cr04, ICN, Irvine California) and incubated at 39°C in a humidified 5% C02 in a i r incubator for 2 to 3 hours. Labelled CRBCwere washed 3 times in HBSS before use. 51Cr-CRBCwere resuspended in various concentrations of antiserum in RPMI-1640 containing 10% FBS; IXlO 4 CRBC in 50 ul of medium were placed into each well of 96-well round bottom mlcrotlter plates (Flow Laboratories Inc., McLean, VA) followed by the addition of effector cells at effector to target ratios lO0:l, 50:I, 20:I and l O : l . Dilutions of antibody used were 1/200, 1/500, I/l,O00, I/2,000, I/4,000, I/5,000, I/8,000, I/lO,O00, 1/50,000, I/lO0,O00 and 1/500,000. Each serum concentration and effector to target ratio were performed in t r i p l i c a t e . The plates were incubated at 39°C in a humidified incubator with 5% CO2 in a i r for 4 hours. After incubation, the supernatants were removed by a Titer-TekR supernate collecting system (Flow Lab). The radioactivity of each sample was determined in a gamma-counter (Beckman 5500, Beckman Instrument Inc., Fullerton, CA). The percentage of specific lysls was calculated as follows: % specific lysis =

test CPM - spontaneous CPM x I00 max CPM - spontaneous CPM

The max counts per minute (CPM) was obtained by adding 3% t r i t o n X-lO0 instead of medium (spontaneous CPM). Cytotoxicity to PI3 virus-lnfected cells - Vero cells were removed by trypslnization and resuspended In EMEMcontaining 5% FBS at a concentration of l to 2 X lO 5 cells/ml. Veto cells were infected with PI3 virus at a m u l t l p l l c l t y of infection of 5 : l and labelled with 51Cr (lO0 ,Ci/106 c e l l s ) . One hundred microliters of cell suspensions were placed into each well of round bottom microliter plates, and plates were incubated at 37°C in a humidified, 5% CO2 in a i r atmosphere for 16 to 18 hours. Plates were washed three times with HBSS containing I% FBS before assay. Antiserum at various concentrations in O.l ml of RPMI-1640 with I0% FBS were added into wells. The final dilutions of antibody used were 1:5, l : l O , 1:20, 1:50, l:IO0 and 1:200. Effector cells were tested in t r i p l i c a t e at the effector to target ratios above. Plates were incubated at 39°C for an additional 4 hours. Supernatants were harvested and specific c y t o t o x i c l t y was determined using the formula above. However, the specific ADCC against PI3 infected cells was determined by subtracting the specific percent lysis for cultures without antiserum from the specific percent lysis for cultures containing antiserum. In the NK cytotoxlcity assay for PI3 infected Vero cells the incubation time was increased to 18 hours. Antiserum was not added to the cultures. Natural k i l l i n g of K562 c e l l s K562 c e l l s at a c o n c e n t r a t i o n of 1 to 2XlO 7 in 0.5 ml RPMI 1640 c o n t a i n i n g 10% FBS were l a b e l l e d w i t h I00 to 200 c i S I C r and incubated at 39°C, in a h u m i d i f i e d 5% CO2 in a i r i n c u b a t o r f o r 3 to 4 hours. Labelled t a r g e t c e l l s were washed 3 times w i t h HBSS and adjusted to the d e s i r e d c o n c e n t r a t i o n w i t h medium. K562 suspensions In 0 . I ml

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were placed into each well of round bottom plate followed by adding O.l ml effector cell suspension at the concentrations of the effector to target ratios listed above. Each effector to target ratio was performed in triplicate. RESULTS Effect of Effector to Target Ratio and Concentration of Antibody on ADCC Activity Peripheral leukocyte populations were studied for ADCC a c t i v i t y against CRBC and PI3 virus-lnfected Vero cells in the presence of various concentrations of antiserum. The results of the effects of antiserum concentration and effector to target ratio on ADCC against CRBC are presented in Fig. l , 2, and 3. The degree of cytotoxlclty was dependent on the ratio of effector to target cells and the concentration of antiserum. At a constant dilution of antiserum, the most efficient cytotoxlc a c t i v i t y was seen in PMN preparation. ADCCa c t i v i t y with PMN was detected in the presence of an antiserum dilution as high as 1:500,000. In contrast, mononuclear (MN) cells had the lowest cytotoxlc a c t i v i t y at all levels of serum dilutions. The effector to target ratio effect was not seen in the PMN preparation; however, the difference of specific cytotoxlclty between effector to target ratio IO0:l or 50:I and 20:I was found at dilutions of serum greater than 1:5,000 (Fig. 2). L i t t l e or no cytotoxlc a c t i v i t y was seen ~n MN cells when a serum dilution greater than l:lO0,O00 was used. The results obtained for ADCC against virus infected Vero cells are presented in Table I . Peripheral lymphocytes (PBL) had specific cytotoxiclty when serum dilutions less than 1:50 were used. When mononuclear cells were used as effector cells, only low levels of ADCC to PI3 virus infected cells were seen. PMN cells were not Iytlc for PI3 infected ceils. Results in Table l suggest that monocytes (adherent cells) in the MN cell preparation inhibited PBL mediated ADCC against PI3 virus-lnfected cells. Tissue Distribution of Effector Cells for ADCC ADCC against CRBCwas determined in cells from peripheral blood, mesenterlc lymph node, spleen and thymus of adult pigs and fetal pigs. Maximal cytotoxlclty was seen with the peripheral blood PMN populations from fetal or adult pigs but splenic cells also were effective in lyslng CRBC. Cytotoxlc a c t i v i t y could only be detected for cells from lymph node when high concentrations of antiserum were present and cells from thymus had low or no ADCC a c t i v i t y . The relative a b i l i t y of effector cells to destroy CRBCwas peripheral blood PMN > peripheral blood MN > spleen cells > lymph node cells > thymlc cells. The cytotoxic a b i l i t y of effector cells from fetal pigs was the same as in adult pigs (Table 2). NK Actlvtty to Viral Infected and K562 Cells Peripheral blood lymphocytes from young pigs (5 to 6 wk) lysed PI3 virus infected Vero cells and human myeloblastlc cell llne K562. PMN cell populations did not have natural k i l l i n g a c t i v i t y (Table 3). The distribution of NK a c t i v i t y to K562 target cells ranged from 35.4 to ?5.0% at a lO0:l effector to target ratio (Fig. 4). The specific cytotoxlclty increased with the increase of effector ceil to target cell ratios. Simllar specific natural k i l l i n g a c t i v i t y was found (data not shown), when adherent cells (mainly monocytes) were depleted from mononuclear cell preparations, suggest that

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lymphocytes were the major cells mediating cytotoxiclty against PI3 virusinfected cells and K562. In preliminary studies (results not shown), NK a c t i v i t y was not detected in 4, 6, or lO hour assays, therefore, a 16 to 18 hour incubation time for the NK assay was established. Organ Distribution of NK Activity Lymphocytes derived from peripheral blood, mesenteric lymph node, spleen and thymus of fetal, neonatal and adult pigs were tested for their a b i l i t y to mediate natural cytotoxicity to K562 and virus |nfected Vero cells. NK a c t i v i t y against K562 was detected in PBL from pigs greater than a week old, but not in PBL from pigs less than one week of age. NK a c t i v i t y against K562 was not detected in the lymph node, spleen, or thymus (Table 4). The organ d i s t r i b u t i o n of c e l l s w i t h NK a c t i v i t y a g a i n s t PI3 v i r u s i n f e c t e d c e l l s is shown in Table 5. NK a c t i v i t y a g a i n s t v i r u s i n f e c t e d c e l l s was seen in PBL and s p l e n i c lymphocytes from pigs o l d e r than one day of age, but was not detected w i t h c e l l s from f e t a l p i g s . NK a c t i v i t y was not found in lymphocytes from lymph node or thymus. The amount of c y t o t o x i c i t y f o r PBL and s p l e n i c lymphocytes of d a y - o l d pigs was not as g r e a t as t h a t f o r pigs a week of age or o l d e r .

I¢1 <[ I.M ..i (n

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40

20

¢E uJ Q.

0

0.25

0.5

ANTIBODY

1

2

4

8

CONCENTRATiON(~2xl03)

FIG. 1 E f f e c t of a n t i s e r u m c o n c e n t r a t i o n on leukocyte (PMN ~ MN) mediated ADCC to CRBC at three d i f f e r e n t E:T r a t i o s , I00+I ( * * ) , 50:1 (o - o) and 20:1 (o - o).

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10(: u.I u) ,< ,.J w n-

8(

c3 F~

D.

60

40

z 0 n,,

20

a.

0

0.2 A N T I BODY

1.0

2.0

CONCENTRATION

10

20

(Xl05 )

FIG. 2 E f f e c t of antiserum concentration on PMN mediated ADCC to CRBC at three d i f f e r e n t E to T r a t i o s , lO0:l (* - * ) , 50:I (o - o) and 20:I (e - e).

uJ (n .d w el-

50

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20.

lZ

w (J

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1.0

2.0

CONCENTRATION

10

20

(y~105 )

FIG. 3 E f f e c t of concentration of antiserum on peripheral mononuclear c e l l mediated ADCC to CRBC at E to T r a t i o lO0:l (* - *) and 20:I (e - e).

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TABLE I. ADCCby Peripheral Leukocytes to PI3 Virus-infected Ceils in the Presence of Different Antiserum Concentrations (E:T ratio 100:I).

Effector Cell

Ab Concentrations 1:20 1:50

1:10

Lymphocyte (n=8)

10.8 ± 5.2a

Mononuclear ceils (MN) (n=8)

8.7 ± 3.8

l:lOO

6.2 ± 5.5

3.1 ± 2.1

4.3~± 4.7

1.6"*± 2.1

2.7 ± 3.5

1.3 +_ 2.2

MN+PMN (n = 8)

4.2 ± 4.5

0.6 ± 0.7

1.7 ± 2.3

0.2 +_ 0.2

PMN (n = 4)

0.1 ± 2.0

-1.5 ± 1.4

0.0 ± 2.0

-1.9 +_ 0.7

Specific % ADCC ± standard deviation Adherent cells depleted population S t a ti s t i c a l Significance at P < 0.05 as compared to lymphocyte preparation S t a t i s t i c a l Significance at P < 0.001 as compared to lymphocyte preparation

a

b "It

TABLE 2. Tissue distribution of effector cells from adult and fetal pigs for ADCCagainst CRBC (E/l ratio, 100:1).

I150 Effector cell Origin Blood

Aa

Serum Concentration 1/200

III00 Fb

A

F

PMN NTc NT NT 65.4+ 9.5 NT MN 59.8±6.9 NT 64.0±12.0 47.0±I0.0 63.8± 3.3

Mesenteric Lymph Node

15.1±18.0 NT 19.1±18.1

Spleen

59.9±11.7 NT 60.7±14.0 61.2± 6.9

Thymus

A

3.2±11.0 NT

5.5±10.3

a A: Adult Pigs (>4 mth) b F: Fetuses (approx. 110 day gestation) c NT: Not Tested

N1

NI

F

11500 A

0 F

A

F

NT 80.6+ 7.8 N1 -3.3+ 5.9 NT 48.3±11.9 NT -4.2± 5.0

-5.0 -5.0

17.3±13

NT -9.5± 1.7 NT -2.5±5.3

NT

59.2± 9.5

NT 14.2± 2.3 NT -4.3±5.2

-5.0

0.5± 9.2

NT -4.l±10.0 NT -2.8±4.8

-5.0

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80

,..i

60

7~ ee

: U

.':-40

U

r, ~, 20

N

t: i i

m

i

10:1 20:1

i

i

50:1 EFFECTOR :

|

100:1 TARGET

RATIO

FIG. 4 The natural c y t o t o x i c l t y of peripheral blood mononuclear cells to K562 at 4 d i f f e r e n t E:T ratios,

TABLE 3. The natural c y t o t o x l c l t y of peripheral blood leukocytes against PI3 vlrus-lnfected c e l l s .

% specific lysis at E:T ratios of Pig No.

1

Effector

lO:l

20 l

50:I

lO0:l

MN

20.0

48 8

56.5

68.8

PMN * MN

l .4

9 3

52.9

60.I

PMN

NT

28

NT

3.5

MN

20.5

30 4

43.3

78.0

PMN + MN

17.3

31 7

51 .I

78.4

NT

38

NT

4.5

PMN

NT = Not tested

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TABLE 4. The tissue distribution of effector cells from various aged pigs for natural cytotoxicity against tumor cell line K562. (Effector to target ratio I00:I)

AGE OF PIG

Cell Origin

Peripheral blood Spleen Lymph Node Thymus

Fetuses (110 dy) (n = 6) 1.5 ± 2.7 a

1 wk (n = 4)

2 wk (n = 2}

11.7 ± 3.8

38.8 ± 9.8

I-3 mth (n = 5)

>5 mth (n = 5)

29.8 ± 9.4

36.6 ± 12.3

-0.7 ± 4.4

NT

NT

-0.7 ± 3.5

NTb

NT

NT

-1.9 ± 0.5

-2.3 ± 3.5

NT

NT

-.26 ± 0.3

-2.6 ± 5.2

-1.6 ± 2.7

3.7 ±

1.3

a specific % 51Cr release ± SO b NT = Not tested

TABLE 5. Tissue distribution of effector cells from various aged pigs for natural cytotoxicity against PI3 infected Vero ceils. (Effector to target ratio, 100:1)

AGE OF PIG Ceil Origin

Fetuses (110 dy) (n = 6)

Peripheral blood

-2.7 ± 3.0 a

Spleen

-4.0 ± 2.0

Lymph Node Thymus

Day 01d (n = 6) 10.1 ± 7.1 5.9 ± 5.9

NTb -2.4 ± 2.6

NT 2.3 + 2.9

a S p e c i f i c p e r c e n t S1cr r e l e a s e ± SO b NT = Not t e s t e d

l wk (n = 5)

2 wk (n : 2)

28.6 + 10.3 43.6 ± I0.0

1-3 mth (n = 5)

>5 mth (n = 5)

44.6 ± 15.11 38.3 ± 9.9

NT

NT

33.4 + 10.6

17.4 ± 8.9

NT

NT

-0.4 + 0.1

3.8 ± 5.1

NT

NT

-1.9 ± 0.1

3.1 ± 1.3

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DISCUSSION I t is well established that white blood cells of different lineages can function as effector cells in ADCC (?,19). Different effectors, although all having Fc receptors, may not be able to lyse the same target cells (7). Therefore, to identify populations of effector cells, I t is important to experimentally determine the specific nature of the target cell. In the present study, two different target cell populations, CRBCand PI3 vlrus-lnfected Vero cells, were used to examine porcine effector cells mediating ADCC. Results demonstrated that when CRBCwere used as target cells, ADCCwas mediated by peripheral blood polymorphonuclear leukocytes, monocytes and lymphocytes, and only small amounts of antibody were required in the assay. In contrast, PMN were not able to k i l l antibody coated PI3 virus infected Vero cells. Similar findings with human effector cells were reported by Nelson et al. (20). Ho et al. have also reported that antlbody-sensltized canine distemper virus (CDV)-infected Vero cells are susceptible to the cytotoxlclty mediated by lymphocytes but not by PMN or monocytes (13). However, PMN were shown to be cytotoxlc to antibody treated Herpes virus infected cells (12,21). When adherent cells were depleted from the mononuclear cell population, the ADCC against PI3 virus infected cells mediated by lymphocytes was increased, suggesting that adherent cells (mainly monocytes) were inhibitory. This inhibitory effect of monocytes might be due to soluble mediators. I t has been suggested that monocytes release prostaglandin E (PGE) after membrane receptor activation (22). Elevation of cellular cAMP levels can inhibit or decrease ADCC a c t i v i t y of effector cells (7). PGE can increase cellular cAMP, therefore, PGE may inhibit ADCC by a mechanism involving an increase in cAMP. This hypothesis is currently being tested. ADCC against CRBC targets mediated by lymphocytes was present in the fetus, whereas, NK a c t i v i t y against K562 did not develop until the pig was one week old and NK cells to PI3 virus infected cells did not develop until one day or more after birth. The results of the present study suggest that the effector cells in ADCC are different from cells mediating NK, or, the effector cells for ADCC and NK a c t i v i t y are similar, but are in different stages of maturation. Also, i t is noteworthy that the effector lymphocytes for ADCC against CR8C targets from fetal pigs had the same amount of cytotoxicity as compared to the effector lymphocytes obtained from adult pigs. This finding is slmillar to the results reported by Huh et al. (lO). In the present study, NK a c t i v i t y against K562 target cells was present at adult levels in PBL of pigs older than one week of age, but at no age was a c t i v i t y found in spleen, lymph node or thymus. In contrast, i t has been reporte d that NK a c t i v i t y against tumor targets exists in PBL and spleen of rats (23), mice (3,24), and human beings (1,25,26). Previous findings in miniature pigs, reported by Kim et al. (1980), were consistent with the present results in that NK a c t i v i t y against K562 was only found in PBL and not spleen. However, when PI3 vlrus-infected cells were used in the present study as target cells, NK a c t i v i t y was present in PBL and spleen of pigs older than a day of age but not in fetal pigs. These results suggest that the effector cells with NK a c t i v i t y against K562 are different from those that mediate natural cytotoxlcity to virus infected cells, or the effector cells for both target cells are similar, but they mediate natural cytotoxlclty through different activation mechanisms. To suggest that the cells are different, we demonstrated that the effector cells for NK a c t i v i t y against PT3 virus infected ceils were more sensitive to suppression by cortlcosteroid than cells that mediated NK a c t i v i t y against K562 target cells (W.C. Yang and R.D. Schultz, unpublished

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results). Also, unlabelled (cold) K562 cells added to PI3 infected cultures in the present study did not decrease NK t o x i c i t y to virus infected cells (results not shown). These findings suggest that cells mediating K562 c y t o t o x i c l t y are d i s t i n c t from the effector cells with NK a c t i v i t y against PI3 virus-infected c e l l s . Heterogeneity among NK cells has been found in humans and mice (25-29). Studies are in progress with a panel of monoclonal antibodies to further define the cells involved in mediating NK c y t o t o x l c i t y of various target c e l l s .

ACKNOWLEDGEMENTS The authors thank Mr. Glenn Robertson and Mr. Stan Rogers for t h e i r assistance in collecting samples, Dr. C. Rossl for providing K562 cells and PI3 virus. This work was supported in part by a contract from FDA and by the USDA through grant from the Alabama Agricultural Experimental Station.

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