Mitogenic effect of prolactin on chicken lymphocytes in vitro

Immunology Letters, 24 (1990) 171-178

Elsevier IMLET 01381

Mitogenic effect of prolactin on chicken lymphocytes in vitro Krystyna Skwarlo-Sofita Department of Vertebrate Animal Physiology, Institute of Zoology, University of Warsaw, Warsaw, Poland

(Received 20 July 1989; revision received20 February 1990; accepted 22 February 1990)

1. Summary Lymphocytes obtained from thymus and spleens of 1-6 week old White Leghorn cockerels, untreated or immunized twice with sheep red blood cells (SRBC), were cultured with common T-cell mitogens in serial dilutions and/or different concentrations of bovine prolactin (PRL). [3H]Thymidine incorporation in newly synthesized DNA was used as a measure of lymphocyte mitogenic stimulation. Lymphocytes were stimulated by mitogens, as well as by P R L alone, in a dose-dependent way. Cell cultures prepared from immunized and nonimmunized donors differed in their response to mitogens or PRL. The present results demonstrate direct PRL action on avian lymphoid cells and resemble those found in the mammalian immune system. 2. Introduction A regulatory effect of prolactin (PRL) on the immune system has been shown in mammals [1, 2] and birds [3] and it appears to be highly complex. In mice, P R L plays a major role in the thymusregulated immune differentiation in early ontogeny [4] as well as in the developmental expression of Tand B-lymphocyte populations in the thymus and spleen [5]. In both mammals and birds P R L could stimulate [2, 3] or suppress [1, 6, 7] certain immune parameters. Key words: Chicken immunity; Prolactin; Mitogens; [3H]Thy-

midine incorporation; Thymocyte;Splenocyte Correspondence to: K. Skwarlo-Sotita,Dept. of VertebrateAni-

mal Physiology, Institute of Zoology, University of Warsaw, Zwirki i Wigury 93, PL-02 089 Warsaw, Poland.

Subsequently, P R L receptors on human peripheral blood lymphocytes and monocytes as well as on purified splenic T and B lymphocytes have been identified [8-10]. The lymphocyte P R L receptors seem to be involved in the regulation o f lymphocyte function through their action on ornithine decarboxylase (DOC), a key cell growth regulatory enzyme, which is strictly dependent on hormone concentration [8]. PRL can also down-regulate [11] or up-regulate [12] the number of its own receptors. Our own results have suggested that the effect of P R L on immunity o f chickens could be exerted either directly or indirectly, through its influence on concentration and on diurnal changes in plasma corticosterone [7, 13, 14]. Corticosterone might in turn modify the effect of PRL on immune parameters in chickens (Skwarlo-Sotita et al., in preparation). As mitogenic action of PRL on different tissues, including Nb 2 lymphoma cells and human lymphocytes, is already well known [8, 15-19], we sought to determine whether PRL could stimulate the in vitro blast transformation of chicken thymocytes and splenocytes. 3. Materials and Methods 3.1. C h i c k e n s

White Leghorn line G-99 cockerels served as donors of tissues for preparing lymphocyte cultures. After hatching, chickens were kept under controlled light (12 h light/12 h darkness) and temperature (initially 32 oC + 2 oC; after one week 24 ° C ___2 ° C). The birds had free access to the standard food and boiled water, supplemented with Polfamix Z vitamin mixture. At the age of 3 weeks, half of the birds were im-

0165-2478 / 90 / $ 3.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)

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munized i.p. with sheep red blood cells (SRBC) and reimmunized one week later, as described previously [201.

3.2. Cell cultures Lymphocyte cultures were prepared from thymus and spleens of 1-6-week-old chickens, nonimmunized or immunized with SRBC, according to our own modification of the previous methods employed in the study of chicken lymphocytes [21-26]. Chickens were killed by decapitation (always at the same time of day, i.e., between 4 and 6 h after light onset) and their thymuses and spleens were collected aseptically in culture medium (serum-flee Eagle's MEM medium; Biomed, Poland, supplemented with 10/xg/ml gentamycine, 2 mM L-glutamine and 5 × 10-5 M 2-mercaptoethanol). Lymphoid organs were cut into small pieces and homogenized gently in a glass homogenizer. Cell suspensions were transferred into test tubes and sedimented for 5 min to remove tissue debris. Supernatants were collected and centrifuged at 4 0 0 × g for 10 min. Sedimented cells were washed 4 times with culture medium. Aliquots of cell suspensions were diluted with NattHerrick diluent [27] and counted in a hemocytometer. Cell viability was determined by the trypan blue exclusion test and cell suspensions were adjusted to a final concentration 10× 10 6 cells/ml. Aliquots (100/zl) o f the cell suspension were added to 96-well flat-bottomed tissue culture plates (Falcon) and equal volumes of the mitogens or PRL, diluted in the same medium, were added. Control cultures contained additionally 100/zl of the culture medium alone. The cultures in duplicate or triplicate were incubated in a humidified atmosphere of 5% CO 2 in air at 41 °C for 48 h; subsequently, cultures were pulsed with 1/~Ci of tritiated thymidine ([63H]dTdR; UVVVR, Prague, Czechoslovakia; 40 MBq/ml) per well for another 18 h. The cells were collected on glass fiber filters with a multiple cell harvester (Scatron, Norway). Incorporated radioactivity was measured by conventional liquid scintillation spectrometry and expressed as counts per minute (cpm) per culture (1 × 106 cells).

172

3.3. Mitogens The following mitogens were used in serial dilutions: phytohemagglutinin P (PHA-P, Difco) in dilutions from 1:20 to 1:160; Concanavalin A (Con A, Serva) in concentrations from 50 to 6.25/zg per well; mitogen LF-7 (produced recently by Biomed, Krakow, Poland and used for stimulation of sheep [28] and human peripheral blood lymphocytes [29]) in serial dilutions from 1:2 to 1:32. Bovine prolactin obtained and purified as described previously [30] was dissolved in 0.1 M N a H C O 3 (30 IU/mg) and filtered through a millipore filter. PRL solution was diluted with culture medium to obtain final concentration ranging from 10-8 to 10-11 mol/100 tzl [8] which was added to the cultures in triplicate.

3.4. Statistical analysis Incorporation of radioactivity is given as the arithmetic mean + SEM o f triplicate cultures. Stimulation index was calculated as the ratio of the amount of the label incorporated in mitogen- or PRL-stimulated cells divided by the amount of label incorporated by unstimulated cells. Student's t-test was used to evaluate the difference between group means. 4. Results

4. I. Effect of PRL on chicken thymocytes, stimulated by T-cell mitogens In preliminary experiments with thymus cells from 1-week-old White Leghorn cockerels, the effect of T-cell mitogens, Con A and PHA, was examined without and with addition o f PRL in concentrations of 6.25 × 10-s mol per well (Table 1). It was found that both Con A and P H A in serial dilutions stimulated [3H]thymidine incorporation by chicken thymocytes in a dose-dependent way. PRL added to the cultures with the same mitogen concentrations markedly modified incorporation of radioactivity. In cultures stimulated by Con A, PRL addition caused an increase in cell proliferation in all concentrations of Con A used (not significant in cultures stimulated by lowest Con A concentration). Thymocyte stimulation by P H A was increased by PRL ad-

TABLE 1 Effect of PRL on chicken thymocyte stimulation by Con A and PHA. Mitogen conc.

3H-thymidine incorporation (cpm/culture) Without PRL

None

pa

5243± 556

With PRL

pa

8146± 867

AOTo (with - without PRL)

p~

+55

**

Con A 6.25 #g 12.5 #g 25.0 ~g

10524± 613 8632± 907 6539± 1735

*** ** NS

16376 ± 3158 33458±7032 20831 ± 5137

** ** **

+ 56 +288 + 219

NS ** *

PHAP 1:160 1:80 1:40

14796 ± 4190 16594±6679 11073 ± 882

* ** ***

8264± 879 7134± 913 31183 ± 9287

NS NS **

- 44 - 57 + 182

NS NS *

Thymocytes were prepared from 1-weak-old chickens. PRL was added at a concentration of 6.25 x 10-s tool per culture. Results are expressed as mean + SEM of triplicate cultures, a Statistical significance of the differences between the cpm with mitogen and those without mitogen; b statistical significance of the differences between the cpm without PRL and those with PRL. NS, not significant. * P<0.05; ** P<0.01; *** P<0.001.

d i t i o n o n l y in t h e highest P H A c o n c e n t r a t i o n ; in cultures s t i m u l a t e d by two lower P H A c o n c e n t r a t i o n s , the a d d i t i o n o f P R L resulted in a n o n - s i g n i f i c a n t decrease in [3H]thymidine i n c o r p o r a t i o n . P R L , alone, a d d e d to t h e culture increased by 55 % t h y m i d y n e inc o r p o r a t i o n by t h y m o c y t e s ( P < 0.01). 4.2. Response of chicken thymocyte and splenocyte

cultures to mitogens T h y m o c y t e s a n d splenocytes were s t i m u l a t e d b y m i t o g e n s e x a m i n e d in a d o s e - d e p e n d e n t m a n n e r (Figs. 1 a n d 2). I n l y m p h o c y t e cultures, o b t a i n e d f r o m t h y m u s a n d spleens o f chickens, i m m u n i z e d with S R B C , the r e s p o n s e to m i t o g e n s was also d o s e d e p e n d e n t (Figs. 1B a n d 2B). Nevertheless, s t i m u l a t i o n was g e n e r a l l y weaker t h a n in l y m p h o c y t e cultures f r o m n o n - i m m u n i z e d chickens. M a x i m a l s t i m u l a t i o n indices r a n g e d f r o m 13 to 24 in t h y m o cyte a n d f r o m 12 to 24 in splenocyte cultures, prep a r e d f r o m n o n - i m m u n i z e d chickens. In cultures m a d e o f cells o b t a i n e d f r o m i m m u n i z e d d o n o r s , c o m p a r a b l e values were 12-20 a n d f r o m 6-13, respectively; f u r t h e r m o r e , t h e s h a p e o f d o s e r e s p o n s e curves was s o m e w h a t different.

4.3. Effect of PRL on [3H]thymidine incorpora-

tion by chicken thymocytes and splenocytes T h e m i t o g e n i c a c t i o n o f P R L o n chicken t h y m o cytes a n d splenocytes is shown in Fig. 3. P R L in conc e n t r a t i o n 10 -9 m o l / w e l l causes m a x i m a l s t i m u l a t i o n o f b o t h t h y m o c y t e s a n d splenocytes f r o m n o n - i m m u n i z e d chickens. In cell cultures o b t a i n e d f r o m chickens, i m m u n i z e d with SRBC, s t i m u l a t i o n by P R L is weaker. Moreover, a statistically signific a n t decrease in [3H ] t h y m i d i n e i n c o r p o r a t i o n is o b served in cultures o f t h y m o c y t e s f r o m i m m u n i z e d chickens, i n c u b a t e d with P R L at a c o n c e n t r a t i o n o f 10 -n tool/well. O p t i m a l h o r m o n e c o n c e n t r a t i o n was f o u n d to be 10 -1° m o l / w e l l . 5. D i s c u s s i o n

Several p a p e r s d e s c r i b e different culture c o n d i t i o n s as being o p t i m a l for s t u d y i n g chicken l y m p h o c y t e response to mitogens, as a n i n d i c a t o r o f l y m p h o c y t e f u n c t i o n [21-23, 25, 26]. T h e r e s p o n s e o f chicken l y m p h o c y t e s to s t i m u l a t i o n with T - m i t o g e n s c o u l d be i n f l u e n c e d by m a n y factors. A m o n g these there are age a n d genetic line o f chickens, as well as the 173

16

B

¢'5 O X

E e, xx

12'

' 6.25 1:32

1;8o

1;4o

1~o-o- P~A

12.5 1:16

25.0 1:8

50.0 -x- ~qConA 1:/. -&- LF 7

6.25 1'32

~20

1;so

1;4o

1:io

12.5 1:16

25.0 1:8

50.0 1:/,

mitogen concentration

Fig. 1. Dose response of chicken thymocytesto mitogenic stimulation by phytohemagglutinin (PHA P, 9 ), Concanavalin A (Con A x ) and LF 7 ( ~ ). Cells were obtained from non-immunized chickens (A) or immunized with SRBC (B). Each point represents mean [3H]thymidine incorporation measured as cpm __ SEM. Numbers accompaning points indicate stimulation indices. Statistical significance (expressed as P) of the differences between the cultures with and without mitogen: *, P<0.05; **, P<0.01; ***, P<0.001.

medium used, serum addition, cell and mitogen concentration, etc. [23, 25, 26]. The present results have confirmed that our modification of culture conditions was adequate for in vitro chicken thymocyte and splenocyte transformation. Incorporation of radioactivity was relatively weak (maximal values ranged from 15 to 33 x 103 cpm per well), and was comparable with the results obtained by several other investigators o f chicken lymphocyte cultures [21, 25, 26]. It was somewhat weaker than that observed by Hovi et al. [22] and Nathanson [23]; this might be due to differences in culture conditions as well as to differences in age and genetic line of chickens used as lymphocyte donors. The possibility cannot be excluded that our source o f [3H]thymidine contributed to differences in results; it was obtained from the 174

Institute for Research, Production and Application o f Radioisotopes, UVVVR, Prague, Czechoslovakia, while others employed the product o f New England Nuclear, Boston, M A or Radiochemical Centre, Amersham, U.K. Nevertheless, stimulation of thymocyte and splenocyte cultures by the T-lymphocyte mitogens Con A and P H A P was dose-dependent and similar in extent to that found by other authors [21, 25, 26]. Dose-response curves were also obtained in cultures of lymphocytes stimulated by the mitogen LF-7, produced recently by Biomed, Krakow, Poland. This mitogen was previously used in genetical research for stimulation of sheep [28] and h u m a n peripheral lymphocyte cultures as effectively as P H A [29]. P R L has a marked effect on stimulation o f chick-

A

B

14'

13~=,xxx

0

x12 ED .

x I '

11~ xxx

l

10

10" XX

,

X

y

1 XXX

6

6.25 1;32

1".'80

12.5 146

1;40 25.0 1:8

1:20-o- PHA 50.0 -x-,~ConA 6.25 1:/, "&" LF 7 1=32 mitogen concentration

1/80 12.5 1:16

1:'/,0

25.0 1:8

1:20

50.0 1:/,

Fig. 2. Dose response of chicken splenocytesto mitogenic stimulation by PHA, Con A and LF 7. Symbolsas in Fig. 1.

en thymocytes with Con A and PHA. The effect of P R L on Con A-stimulated cells is additive and this phenomenon may be explained by the observation, that Con A-stimulated murine splenocytes release a substance with PRL-like activity, which is a factor essential for lymphocyte proliferation [31] and could probably cooperate with PRL added to the culture. It is difficult, however, to explain, why addition of P R L inhibits stimulation with low concentrations of PHA, and increases the mitogenic effect of its high concentration. Similar inhibition of the murine mixed lymphocyte reaction by addition of heterologous P R L observed by Hiestand et al. [32] was explained as due to a competitive antagonism of the added PRL preparation with the endogenous murine P R L bound to lymphocyte surface receptors. It is possible that mechanisms of these two phenomena could be similar. P R L alone added to the cultures affects DNA synthesis, measured as [3H]thymidine incorporation by chicken thymocytes and splenocytes. To our knowledge, this is the first report on the direct in vitro ef-

feet of PRL on avian lymphocytes, although mitogenie action of PRL on bursal lymphoid cells in vivo was already demonstrated [33]. In our experiments, we did not attempt to study the bursal ceils, because o f their known poor response to mitogens (LPS, PPD, DxS) normally active on mouse B cells [21, 26]. Effect of PRL on lymphocyte cultures was dosedependent. Similar PRL effect on human peripheral blood cells was reported by Russell et al. [8]. In the highest concentration examined (10-8 M) PRL inhibited DOC activity of human ceils [8]; in thymocyte cultures o f immunized chickens it is inhibitory only at a concentration o f 10TM M. The dose-dependent PRL effect on chicken thymocytes and splenocytes is similar but not identical. It could be explained by differences in cell populations and in immunological maturity of lymphocytes from the two lymphoid organs [34]. The present results indicate that the response of chicken lymphoid cells to stimulation with Tmitogens or PRL can be modified by the donors' immunization with SRBC, a T-dependent antigen. 175

A

lff

B

10"

B'

X

E XX

(..3

XXX

At"

•

XXX

molar prolactin concentration (I0 n )

Fig. 3. Incorporation of [3H]thymidineinto chicken thymocytesand splenocytescultured with varying PRL concentrations, expressed as: A, cpm (mean + SEM); B, stimulation indices; o, thymocytesfrom non-immunizedchickens;., thymocytesfrom chickensimmunized with SRBC; a, splenocytes from non-immunized chickens; A, splenocytes from chickens immunized with SRBC; other explanations as in Fig. 1.

Lymphocytes from immunized birds show weaker responses than those obtained from non-immunized donors. The optimal stimulatory dose of P R L was also different: 10-]° tool/well for cells from immunized donors vs. 10-9 mol/well for cells from non-immunized ones. We have previously found that exogenous P R L affects some immune parameters in vivo to a different degree in nontreated birds and in those immunized with SRBC [7, 13]. I m m u n e response itself changes the level of endogenous P R L [35]: in mice bacterial infection as well as immunization with SRBC caused a decrease in serum PRL. On the other hand, response of splenocytes to mitogens was diminished in mice in which endogenous P R L secretion was suppressed by bromocryptine treatment [36]. It means that the level of endogenous P R L is crucial for the lymphocytes to respond to mitogenic stimulation in vitro. This is probably the reason for weaker responses to mitogens and to P R L of splenocyte and thymocyte cultures obtained from chickens immunized with SRBC as compared with those obtained from non176

immunized donors. It could be concluded that regulatory effect of P R L on the chicken immune system, observed previously in viva [3, 7, 13, 33], might be exerted, at least in part, by direct h o r m o n e influence on Tlymphocyte function, which is in turn dependent on immune system activation by T-dependent antigen

(SRBC). Acknowledgements

Special thanks are due to dr T. H. Dzbefiski, head of Department of Medical Parasitology, National Institute of Hygiene, Warsaw, Poland, for helpful discussions and providing of technical facilities during cell culture preparation. The author is very grateful to her collegues Maria Waloch and Beata Lech for their excellent technical help. This work was supported by scientific program R-III-14 coordinated by the Jagiellonian University, Krakow, Poland.

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