B lymphocyte differentiation and suppressor activity by T lymphocytes derived from neonatal and sucking piglets

Research in Veterinary Science 1986, 40, 400-405

B lymphocyte differentiation and suppressor activity by T lymphocytes derived from neonatal and sucking piglets A. SUGANUMA, Faculty of Veterinary Medicine, A. ISHIZUKA, Y. SAKIYAMA, School ofMedicine, Y. MAEDE, S. NAMIOKA, Faculty of Veterinary Medicine, Department of Veterinary Internal Medicine, Department of Pediatrics, Hokkaido University, Sapporo, Japan

The capacity of porcine neonatal T and B lymphocytes was studied employing a protein A haemolytic plaque assay. Only a few peripheral blood lymphocytes (POL) from newborn piglets differentiated into immunoglobulin (Ig)-producing cells on stimulation by pokeweed mitogen (PWM). Newborn POL also suppressed the differentiation of adult POL into Igproducing cells. This suppressive effect existed in T-cell enriched populations and appeared to be equally effective in the generation of IgG and IgMproducing cells. When newborn B lymphocytes were cocultured with T lymphocytes from adults in the PWM system, their differentiation into IgG and IgMproducing cells was enhanced. No such enhancement was seen in cocultures of newborn T and B lymphocytes. The generation of Ig-producing cells in POL from suckling piglets increased with ageing, and reached about half the adult mean at six weeks old. On the other hand, the suppressor activity of T lymphocytes was observed throughout the suckling period, although it gradually decreased with ageing and was not consistently demonstrated by five weeks of age. THE reasons why neonatal pigs are so susceptible to local infections has not been fully elucidated. A slow response to antigenic stimulation, immature lymphocyte function and active cellular suppressive mechanisms are some of the suggested explanations (Bourne 1973, Yabiki et a11974, Kim 1975, Allen and Porter 1977, Namioka et al 1983). Inability to make antibody could be the result of immaturity or suppression occurring at any or all levels of the monocyte, T or B lymphocyte interactions. These three cellular components of the immune system are all present in neonatal blood (Binns and Symons 1974, Escajadillo and Binns 1975, Schalm et al 1975, McCauley and Hartmann 1984a, 1984b). Peripheral blood lymphocytes (POL) from neonatal piglets respond well by proliferation to polyclonal T cell activators such as concanavalin A and phytohaemagglutinin (Namioka et al 1983) and splenic lymphocytes from neonatal piglets produce immunoglobulin (lg) with lipopoly-

saccharide stimulation (Symons and Clarkson 1979). Cells with cytoplasmic IgM formed the majority of 19 containing cells in the lamina propria of the small bowel in sucking pigs up to four weeks old; in other words, such pigs were in the early stage of immune development (Allen and Porter 1977). However, the concentration of serum Ig from sucking piglets is only about one third the adult mean at four weeks old (Curtis and Bourne 1971). The present study was undertaken to determine the regulatory role of T lymphocytes and the kinetics of maturation of B lymphocytes derived from newborn piglets using an in vitro 19 production system with pokeweed mitogen (PWM) as a T cell dependent B cell activator. Materials and methods

Animals Five six-month-old adult crossbred pigs (Landrace cross Large White) and 13 piglets (Landrace-Large White cross Duroc) derived from two crossbred sows (Landrace cross Large White) were used. All the pigs in this study had been kept on a specific pathogen free farm and throughout the experimental period were maintained in a minimal disease condition.

Blood samples Blood samples (20 to 40 ml) were collected from the jugular veins. Five randomly selected piglets derived from different litters were bled before receiving colostrum. Other blood samples (6 ml) were collected from eight piglets in the same litter after they had received colostrum at one, two, three, four, five and six weeks old, respectively. Heparin was added at 5 iu ml- I as an anticoagulant. PBL

preparation

Blood was diluted with an equal volume of phosphate buffered saline (pos) and lymphocytes were separated over Ficoll-Conray 400 solution by centri-

400

T cell suppressor activity in piglets fugation at 400 g for 30 minutes at 20°C. The FicollConray 400 solution was prepared by mixing 10 parts of 33·4 per cent Conray 400 solution (Daiichi Pharmaceutical Co) and 24 parts of 9 per cent Ficoll aqueous solution (Pharmacia Fine Chemicals); its gravity was 1·084±0·001. The use of this method yielded a recovery rate in excess of 60 per cent lymphocytes of 90 per cent purity. Cells from the interface were washed three times in PBS and suspended in RPMI-I640 (Grand Island Biological Co) containing 10 per cent heat-inactivated fetal calf serum (FCS; Filtron). The viability according to the trypan blue dye exclusion test was greater than 98 per cent.

Isolation of B lymphocytes The panning method was used (Wysocki and Sato 1978). Rabbit and anti-porcine IgG (E-Y Laboratories) was diluted to 40 /-Ig ml : I with 0·05 M Tris, pH 9·5 and 10 ml poured on to 90x 15 mm polystyrene petri dishes. After 40 minutes at room temperature the buffer was decanted and the dishes were washed four times with PBS and once with a few milli1itres of PBS containing I per cent FCS (PBs/I per cent FCS). A total of 3 x 107 PBL were suspended in 3 ml of PBS containing 5 per cent FCS and poured on to each antibody-coated dish. The plates were incubated at 4°C for 70 minutes. After 40 minutes the plates were gently agitated. At the end of incubation the plates were again gently agitated and the non-adherent cells removed by decanting the supernatant. The plates were washed by gently pouring 10 ml of PBS/I per cent FCS down the side wall of the dish and then swirling, tilting and decanting them. These non-adherent cells were regarded as non-B cells. The plates were then washed six more times in PBs/I per cent FCS before bound cells were recovered by the addition of 30 ml of PBs/I per cent FCS and the entire plate surface vigorously flushed by using a pasteur pipette. Indirect immunofluorescent staining showed that 78·9±5·5 per cent of the recovered cells were surface IgG positive cells, but 3·4±2·8 per cent of E-rosette forming cells were found in the recovered cells.

Isolation of T lymphocytes T lymphocytes were separated by density sedimentation of E-rosettes (Buschmann and Pawlas 1980). Sheep red blood cells (SRBC) were treated with neuraminidase (Type V; Sigma) at a concentration of 1 unit ml- I at 37°C for 30 minutes. A total of I x 107 ml- I of non-B cells (see above) were rosetted with 1 per cent of neuraminidase-treated SRBC at a ratio of 1:2. The mixtures were centrifuged at 600 g for five minutes and incubated at 37°C for 15minutes. After allowing them to stand for a further 60 minutes on ice,

401

the pelleted cells were gently resuspended, layered on Ficoll-Conray 400 solution and centrifuged at 400 g for 30 minutes. Rosetting cells in the bottom of the gradient were treated with O' 01 M, Tris-O: 84 per cent ammonium chloride buffer to lyse the erythrocytes, washed twice with PBS and used as T cell-enriched population. In addition, non-rosetting cells at the gradient interface were collected, washed twice with PBS and used as a non- T, non- B cell population. The T cell-enriched population contained 60 to 70 per cent of E-rosette forming cells, as judged by the total rosette formation with neuraminidase-treated SRBC and was contaminated with less than 2 per cent cells with membrane-bound IgG and about 10 per cent monocytes, as determined by morphology and peroxidase staining. The non-T, non-B cell population contained no surface lg positive cells, less than 4 per cent E-rosette forming cells and 30 per cent monocytes.

Culture conditions Lymphocytes were cultivated in plastic tubes (number 2058, Falcon Plastics) in a final volume of 2·0 ml of RPMI-I640 supplemented with penicillin (100 iu ml- I ) , streptomycin (100 /-Ig ml : ') and 10 per cent heat-inactivated FCS. A preliminary experiment employing different doses of PWM (Grand Island Biological Co) indicated that three-day cultures with 5 /-II ml I of PWM gave optimal results with regard to the differentiation of peripheral lymphocytes from adult pigs into Ig-producing cells. The cultures mentioned above were performed at a density of 0·25 to 1·0 x 1()6 in 2·0 ml respectively, and then incubated at 37°C for three days in a humidified atmosphere of 5 per cent carbon dioxide and 95 per cent air.

Coupling ofprotein A to the erythrocytes Protein A (Pharmacia Fine Chemicals) was coupled SRBC using chromium chloride as described previously (Gronwicz et aI1976). SRBC stored in Alsever's solution were washed five times in 0·9 per cent sodium chloride before use. Thereafter one part of Protein A (I mg ml- I ) was mixed with 18 parts of chromium chloride (2·5 x 10- 4 M) and two parts of packed SRBC. All reagents were diluted in 0·9 per cent sodium chloride. The mixture was incubated at 30°C for 60 minutes, washed once in 0·9 per cent sodium chloride and twice in Hanks' balanced salt solution (HBSS) and kept at 4°C until use. to

Plaque assay One hundred microlitres of cells suspended in HBSS were added together with 20/-11 of Protein A coupled

A. Suganuma, A. Ishizuka, Y. Sakiyama, Y. Maede, S. Namioka

402

pWM(n) 0 15 _ _---L_ 20 . ----_ _........._ 5 _- - -10 - ' ' - -_ _........... _ Newborn pel

- (5)

Newborn pel

+

(5)

Adult pel Adult pel Adult pel + newborn pel Adult pel + newborn T

+ (3)

Adult pel + newborn non-T non-B +

(3)~~~~~~:~~~~~~~=~=~"'T"""o

10

15

IgM -pfc x 1(}'/culture

c=J----I

IgG-pfc x 1(}'/culture

~

FIG 1: Generation of PWM-induced pfc in cultures of PBl from adult pigs and from newborn piglets, and in cocultures of their subpopulations. PBl (1 x 1()6cells) from adult pigs and newborn piglets were cultured for three days in the presence of PWM. 1()6 cells of newborn PBl and their subpopulations were cocultured with 1()6cells of adult PBl in the PWM. Isolated Tand non- T, non-B lymphocytes from newborn piglets did not generate pfc in the presence of PWM. Data represent the mean (±SD)

(diluted 1:2'5),20 J.l1 of the antiserum diluted I :25 (rabbit anti-porcine IgG and IgM; Miles Scientific) and 20 J.l1 of saac-absorbed guinea pig complement (diluted I :4) into 300 J.l1 of HBSS containing 0·6 per cent agarose (Litex). This mixture was plated on to a 9 em plastic petri dish and incubated at 37°C for eight to 10 hours in a humidified incubator under 5 per cent carbon dioxide. Plaques were counted under indirect light. SRBC

Results

Differentiation of B lymphocytes and suppressor activity of T lymphocytes from newborn piglets

PBL (I x 1()6) from newborn piglets and from adult pigs were separately cultured for three days in the presence of PWM. After three days, the number of viable cells recovered from each culture tube was not significantly different (8·4±2·5 x 105 for newborn piglets and 8' I ±2· I x 105 for adult pigs). However, as shown in Fig I, newborn PBL generated only a few IgEvaluation ofsuppressor activity of T lymphocytes producing cells of IgG and IgM upon PWMT lymphocytes (1 x 1()6) from sucking piglets were stimulation as compared to adults. When cocultures added to cultures of PBL (1 x 1()6) from adult pigs and of adult PBL and newborn PBL were prepared, the stimulated with PWM. The potential suppressive number of pfc of IgG and IgM was lower than that in effects of these T lymphocytes on the differentiation the culture of adult PBL alone. This effect was most of adult PBL into Ig-producing cells were evaluated by marked when newborn T lymphocytes were used and using the plaque assay. The degree of suppression was was dose-dependent (Fig 2). The addition of allogenic determined according to the following formula adult T lymphocytes enhanced the differentiation of adult PBL into Ig-producing cells. (Miyawaki et aI1979): To evaluate the ability of newborn B lymphocytes Number of plaque to differentiate into Ig-producing cells, B lymphoforming cells (pfc) cytes from newborn piglets and from adult pigs were in cocultures cocultured with allogenic T lymphocytes from Per cent suppression = 1- - - - - - - - x 100 newborn animals and from adults for three days in the Number of pfc in presence of PWM. As shown in Table I, when adult T cultures of adult lymphocytes were added to newborn B lymphocytes, PBL alone the number of IgG- and IgM-pfc in newborn Iympho-

T cell suppressor activity in piglets IgG

b

~ 200

x o

~ "S

C.

o

e co o

o

0

150

Q;

.0

4

403

~ IgG

CJ

IgM

3 2

E

100

:J

Z

t< 50

o

0·25 0·5

1·0

0

0·25 0·5

1·0

Number of T Iympocytes added x 10· FIG 2: The suppressive effect of T lymphocytes from newborn piglets on the generation of Ig-producing cells by adult PBL. Different numbers of T lymphocytes from adult pigs (-0-1 and from newborn piglets ( - . -I were cocultured with 1()6 adult PBL for three days in the presence of PWM. Isolated T lymphocytes from newborn and adult pigs did not generate pte in the presence of PWM. Results are expressed as the percentage of control culture without added T cells. Data represent the mean (±SO) of four separate experiments

cytes increased, amounting to roughly half the number when allogenic adult T lymphocytes were added to adult B lymphocytes. Moreover, when newborn T lymphocytes were added to adult B lymphocytes, the number of IgG- and IgM-pfc was markedly suppressed (Table I).

Generation of Ig-producing cells in PBL and suppressor activity of T lymphocytes in the suckling period

FIG 3: Generation of PWM-induced Ig-producing cells in PBL from sucking piglets. 1()6cells of PBLfrom a litter of eight sucking piglets and from three unrelated adult pigs were cultured for three days in the presence of PWM. Data represent the mean (±SO) of the total number of Ig-producing cells per culture. With a t test, in which the results from adults are the controls: IgG = P
number of IgG- and IgM-producing cells at six weeks of age reached 60 per cent and 35 per cent respectively of those in cultures of adult PBL (Fig 3). The mean suppressions by T lymphocytes from sucking piglets at one and two weeks old were 62 per cent and 44 per cent respectively (Fig 4). This suppressor activity of T lymphocytes diminished gradually with advancing age and was not observed by five weeks old. However, the addition of adult T lymphocytes enhanced the differentiation of adult PBL more strongly than that of T lymphocytes at six weeks old.

PBL from sucking piglets of various ages and from unrelated adult pigs were cultured for three days in the presence of PWM. The number of Ig-producing cells increased gradually with advancing age, and the

60 40

§ 20

.~

TABLE 1: Differentiation of B lymphocytes from newborn and adult pigs cultured with newborn or adult T lymphocytes in the PWM-stimulation system*

Cell combinations

n

Newborn B cells Newborn B + newborn Tt Newborn B + adult Tt Adult B cells Adult B;t newborn T Adult B + adult T

5 3 5 5 5 7

Number of pfc per culture x 10- 2 IgG IgM

23± 36 70± 57 1047± 748 209± 146 210± 203 1930±105O

28± 33 75± 63 836:+:514. 323±223 84± 51 1573±559

• 5 x 1()5cells of B lymphocytes from newborn piglets and adult pigs cultured with 5 x 1()5cells of allogenic T lymphocytes from newborn and adult pigs for three days in the presence of PWM t Isolated T lymphocytes from newborn and adult pigs did not generate pfc in the presence of PWM Data represent the mean (±sol

e

0. 0. :J

0

(/)

t< -20 -40 -60

2

3

4 Weeks

5

6

Adult

FIG 4: The suppressive effect of T lymphocytes on the generation of Ig-producing cells by adult PBL in the PWM system. 1()6cells of T lymphocytes from sucking piglets were added to cultures containing 1()6cells of adult PBLin the presence of PWM. Results are expressed as the percentage of suppression compared with the control culture, without the addition of T lymphocytes. Negative result indicates an increase in number of pfc. Each column represents the mean (±SO) of suppression (P
404

A. Suganuma, A. Ishizuka, Y. Sakiyama, Y. Maede, S. Namioka

Discussion The suppressor activity of T lymphocytes, as evaluated by the coculture with PBL from unrelated adult pigs in the PWM-system, was consistently demonstrated not only in the newborn period but also throughout the sucking period. This suppressor activity of T lymphocytes decreased with age and had disappeared after five weeks. These findings are very similar to those reported for neonatal infants (Hayward and Lawton 1977, Oldstone et al 1977, Miyawaki et al 1979, Andersson et al 1980), and to those of other animals (Folch and Waksman 1974, Mosier and Johnson 1975, Droege 1976, Calkins and Stutman 1978). According to the results of Miyawaki et al (1979), the suppressor activity of human T lymphocytes in cocultures stimulated by PWM persists in children for up to two years after birth. Though the population of B lymphocytes in neonatal PBL almost reaches that of the adult mean (McCauley and Hartmann 1984b), the differentiation of B lymphocytes into plasma cells in vivo is relatively deficient in neonatal piglets at birth and up to about four weeks old (Namioka et aI1983). In this study it was clear that the suppressor activity of newborn T lymphocytes was still observed at four weeks old and this period closely correlated to the duration of poor antibody production. Judging from the results that the neonatal T lymphocytes induced suppressor activity by pWM-stimulation, the T lymphocytes might suppress the differentiation of B lymphocytes into plasma cells in vivo. In the present study, only a few PBL from colostrum-deprived newborn piglets differentiated into Ig-producing cells of IgG and IgM in the PWMstimulated cultures and, moreover, newborn PBL remarkably suppressed differentiation of adult PBL into Ig-producing cells. This suppressive effect existed in the T cell-enriched population and the suppressor activity of newborn T lymphocytes appeared to be equally effective in the generation of Ig-producing cells for IgG and IgM. On the other hand, newborn B lymphocytes differentiated into Ig-producing cells of IgG and IgM by the addition of adult T lymphocytes in the PWM system. . These results suggest that pWM-induced differentiation of B lymphocytes in newborn PBL was strongly suppressed by the T lymphocytes of the newborn piglets. No enhancement of differentiation of adult B lymphocytes into Ig-producing cells occurred following the addition of newborn T lymphocytes, which may be due to newborn T lymphocytes inducing excessive suppressor activity after PWM stimulation. Therefore it is unclear whether or not newborn T lymphocytes have a helper function. However, the existence of a helper function of newborn T lymphocytes has been shown by using a Nocardia water soluble mitogen-driven system in

which no suppressor activity of T lymphocytes is induced (Nagaoki et al 1981). As was mentioned previously, the existence of suppresssor T lymphocytes in experimental animals and humans in the neonatal period has been reported (Folch and Waksman 1974, Mosier and Johnson 1975, Droege 1976, Hayward and Lawton 1977, Calkins and Stutman 1978, Miyawaki et al 1979, Andersson et al 1980). Fetuses of these mammals receive maternal IgG through the placenta. In contrast, the porcine fetus is prevented from receiving maternal Ig by an epitheliochorial placenta (Kim et al 1966, Chapman et al 1974). Consequently, newborn piglets do not possess maternal Ig and thus their agarnmaglobulinaernia continues until they can suck colostrum, which contains a large amount of maternal Ig (Yabiki et al 1974). In this study, the existence of suppressor T lymphocytes in newborn piglets without maternal Ig was demonstrated, which suggests that pWM-induced suppressor activity of newborn T lymphocytes might not depend upon the existence of maternal Ig, It is known that suppressive immunoresponsiveness is seen in a fetus with which the graft versus host reaction between mother and fetus might be prohibited. On the other hand, since the serum concentration of e-fetoprotein is very high in neonatal and sucking piglets, suggesting that sucking piglets may remain at a fetal stage (Fujimoto et al 1984), the suppressor activity could continue for some time. Though the surface markers of T lymphocytes derived from the cord blood were almost identical to those of adult T lymphocytes, there are considerable differences between the two populations concerning the mechanism of activities (Yachie et al 1981, 1982, Miyawaki et al 1982). Recently, monoclonal antibodies for the subsets of pig T lymphocytes have been reported (Jonjic and Koszinowski 1984, Pescovitz et al 1984, Hammerberg and Schurig 1986). However, additional studies are necessary to elucidate the kinetics of the possible functions of these T cell subsets in p i g s . ' The generation of differentiating PBL from sucking piglets into Ig-producing cells of IgG and IgM increased gradually with advancing age. This generation reached roughly half the adult mean at six weeks old, although no suppressor activity of T lymphocytes was observed at this age. The differentiation of adult PBL into Ig-producing cells by the addition of adult T lymphocytes was marked, while the addition of T lymphocytes from six-week-old sucking piglets only slightly enhanced the differentiation of adult PBL. These findings could be due to the lack of a helper function in T lymphocytes at six weeks old in the pWM-stimulation system. Further studies are required to elucidate possible helper functions of T''Iymphocytes in the suckling piglet.

T cell suppressor activity in piglets Acknowledgements

The authors are grateful to Professor S. Matsumoto of the department of pediatrics, Faculty of Medicine, Hokkaido University for useful suggestions. This study was supported by a grant-in-aid for scientific research (grant number 59560279) from the Ministry of Education, Science and Culture, Japan. References ALLEN, W. D. & PORTER, P. (1977) Immunology 32,819-824 ANDERSSON, U., BIRD, G. & BRITTON, S. (1980) European Journal of Immunology 10, 888-894 BINNS, R. M. & SYMONS, D. B. A. (1974) Research in Veterinary Science 16, 260-262 BOURNE, F. J. (1973) Proceedings of the Nutrition Society 32, 205-215 BUSCHMANN, H. & PAWLAS, D. (1980) Veterinary Immunology and Immunopathology I, 215-224 CALKINS, C. E. & STUTMAN, O. (1978) Journal ofExperimental Medicine 147, 87-97 CHAPMAN, H. A., JOHNSON, J. S. & COOPER, M. D. (1974) Journal of Immunology 112, 555-563 CURTIS, J. & BOURNE, F. J. (1971) Biochimica et Biophysica Acta 236,319-332 DROEGE, W. (1976) European Journal of Immunology 6,279-287 ESCAJADILLO, C. & BINNS, R. M. (1975) International Archives of Allergy and Applied Immunology 48,261-275 FOLCH, H. & WAKSMAN, B. H. (1974) Journal of Immunology 113, 127-139 FUJIMOTO, T., HARA, A., MAEDE, Y. & NAMIOKA, S. (1984) Research in Veterinary Science 36,212-216 GRONWICZ, E., COUTINHO, A. & MELCHERS, F. (1976) European Journal of Immunology 6, 588-590 HAMMERBERG, C. & SCHURIG, G. G. (1986) Veterinary Immunology and Immunopathology II, 107-121 HAYWARD, A. R. & LAWTON, A. R. (1977) Journal of Immunology 119, 1213-1217 JONJlC, S. & KOSZINOWSKI, U. H. (1984) Journal of Im-

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Accepted September 20, 1985