Veterinary Immunology and Immunopathology, 25 (1990) 279-286 Elsewer Scmnce Pubhshers B V, Amsterdam - - Prmted m The Netherlands

279

Immunological Changes During Pregnancy in the Viviparous Lizard, Chalcides ocellatus A-H SAAD and S EL DEEB

Zoology Department, Faculty of Scwnce, Cairo Unwers~ty, Cairo 12613 (Egypt) (Accepted 7 December 1989)

ABSTRACT Saad, A-H and E1 Deeb, S , 1990 Immunological changes during pregnancy In the wvaparous hzard, Chalcrdes ocellatus Vet Immunol Immunopathol, 25 279-286 Splemc cells from pregnant and non-pregnant viviparous hzards (Chalc~des oceUatus) were stimulated m v:tro with the mltogens, concanavahn A (Con A), phytohemagglutmm (PHA) and Escher~ch~acohhpopolysaccharlde (LPS) Cellcultures from pregnant ammals were slgnlficantly less responsive to Con A and PHA than comparable cultures from non-pregnant ammals The response was depressed during the first permd of pregnancy and remained low m magnitude until partuntmn By contrast, the response of maternal splemc cells to LPS was reduced m pregnant hzards only during advanced pregnancy The drastm decrease m mltogemc responsiveness was assocmted with marked mvolutmn of the maternal spleen These findings strongly suggest that pregnancy impairs the lmmunoreact:wty of wv:parous hzards Possible mechamsms for th~s lmpa:rment and the relatmnshlp to circulating levels of sex hormones are discussed

INTRODUCTION

During pregnancy, decreased maternal cell-mediated lmmumty has been demonstrated in mammals, and this could be favourable for the m utero surv:val of the fetus (Bflhngham and Head, 1980) In VlVOstudms claimed a dimmutton of the delayed type hypersensitivity reaction m the skin and an mcreased tolerance to skin grafts, while the in vitro responses of maternal lymphocytes to mltogens, antigens and allogeneic lymphocytes were all depressed (Wilson, 1985) In ectothermw vertebrates, only a few studies m fish and salamanders have been devoted to feto-maternal immune relationships (Tamura and Honma, 1984, Badet, 1984), although interactions between the neuroendocrme and immune system are currently well known m reptiles, a pivotal group m the evolution of vertebrate immune systems (Zapata et al., 1983, Saad and E1 Rid:, 1988) The present study was undertaken to (1) examine structural and functional alterations occurring during pregnancy in the :mmune system of the

0165-2427/90/$03 50

© 1990 Elsevmr Science Pubhshers B V

280

A H SAAD AND S EL DEEB

wwparous hzard ChalcMes ocellatus, and (2) estabhsh a relatmnshlp between such lmmunologmal varmtmns and the circulating levels of feto-placental hormones MATERIALS AND METHODS

Animals C ocellatus is a vlwparous skmk with both chorm-allantom and amphaloplacentatmn There is only one annual breeding cycle, and pregnant females contain 3-8 embryos (Badlr, 1958) Pregnant females are available from the first week of May till mid-September Lizards were collected from local gardens and fields m the wcmlty of Cmro and maintained as described earher (El Deeb and Saad, 1987)

Determmatmn of gestation stage The duration of gestation m Chalc~des m the Cmro area is about two months (Badir, 1958). To determine the exact stage of gestation of each ammal used for hormonal and lmmunologmal studms, reference was made to the developmental stage of the embryos found m the animal Thus, at the t:me of samphng of spleen or blood, some embryos were taken and classified according to a table of development described prewously (El Deeb et al, 1985) The ammals were dlwded into three groups, comprising (1) those with embryos at stages 20-29, (2) those with embryos at stages 30-35, (3) those w~th embryos at stages 3641

Blood samphng Within 48 h of their armval at the laboratory, 3-5 hzards (pregnant and nonpregnant) were decapitated and blood was collected Blood samples from individual hzards were allowed to clot for 2 h at room temperature and then overmght at + 4 ° C, they were then centrifuged at 2500 rpm for 25 mm and 50100 #1 of each serum was stored at - 2 0 ° C until use A minimum of 3 sera/ group per stage of gestation were raeholmmunoassayed for determination of sex steroids

Sex sterotd determmatton Sex steroid hormones were measured following essentially the method descmbed by Arslan et al (1978), using a commercial radlolmmunoassay kit ( C o a t - A - C o u n t ) , Dlagnostm Products Corporat:on, CA, U S A ) :2~I-labelled steroids were used as the radioactive tracer Prehmmary tests confirmed the reproduclblhty and sensitivity of the method The mean intra- and raterassay coefficmnts of variation were m the ranges of 4-7% and 8-15%, respectively. All assays were done in duphcate

IMMUNOLOGICAL CHANGES DURING PREGNANCY IN T H E LIZARD CHALCIDES OCELLATUS

281

Cell preparatmn Spleens from 3-5 hzards at each embryonm stage were used for m v:tro mltogen response assays In&v:dual spleens were excised aseptmally and leucocyte suspensmns were prepared exactly as described prevmusly (Saad and E1 R:d:, 1988). After three washes m me-chilled, serum-free RPMI-1640 m e d m m (Sigma Chemical, St Louis, MO, U.S A.), leucocytes were counted using the trypan blue exclusmn test Lymphocyte blastogenes~s The ability to respond to mltogen, concanavahn A (Con A, Calbmchem., La Jolla, CA), phytohemagglutmm (PHA-p, Dffco Labs., Detroit, MI ) and Eschench~a coh hpopolysacchande (LPS, Dffco Labs.) were assessed by t n t m t e d thymMme ([3H] T d R ) uptake by the cells from md:wdual spleens as described m detail prewously {El Deeb and Saad, 1987) T n p h c a t e ahquots (0 1 ml, 0 5 × 106 cells) of each cell suspensmn were cocultured w:th 0.1 ml of mltogen suspensmn {Con A, 5/~g/ml, PHA-p, 10 ttg/ml; LPS, 10/~g/ml) m 96well round-bottom m:crot:ter plates (Nunk, Poskhd, Denmark). The cultures were maintained at 37°C m a 5% CO2 atmosphere for 96 h After mcubatmn with 0 5/tC1/well of [3H]TdR (5/tCl/ml, specific actlwty 2 Cl/mmol, Amersham Internatmnal, Bucks, Great Br:tam), they were harvested 8 h later w~th a multiple automated sample harvester (Mmlmash II, Dynatech, Switzerland ) and the incorporated radmactlv:ty was determined by scmtfllatmn spectrophotometry Control cultures were performed by culturing splemc cells with culture m e d m m only Normahzatmn o[ data To compare mltogen responses of cells from hzards :n different periods of pregnancy, data gathered from multiple experiments on ammals at the same stage {conducted ldentffically but separately) were normahzed so that they could be pooled for statlstmal analysis For each pemod, the mean background cpm values from mdiv:dual experiments were averaged to obtain a normahzatlon constant for each expemment. All raw cpm values for each experiment were multlphed by the normahzation constant determined for each group The normahzed data from a g:ven experiment were combined with the normahzed data from all expemments at that stage to obtain a grand average background cpm and a grand average stimulated cpm These normahzed values were compared using Student's t test RESULTS

Splenic cell numbers durmg pregnancy Leucocytes m the spleen were enumerated at different periods of pregnancy and at regular stages during embryomc development until stage 41, 1 e before

282

A

A H SAAD A N D S EL D E E B

6 I

O

,.=,

PERIODS

OF

PREGNANCY~

(15}

5

I

z

4

uu

o

o

3

(18)

z tu

(12)

.i ¢1. 2

NON PREGNANT

I

II

III

Fig 1 Changes m the number of wable splenic leucocytes/hzard at varmus permds of pregnancy Data are plotted as mean cell number per organ (mean _+ s e m ) Total number of ammals is gwen m parentheses, half-bar depmts standard error

birth (Fig. 1 ) A d u l t non-pregnant females were used as controls Control spleens ymlded about 5.2 + 1 2 × l0 s (mean + s e m ) cells per spleen As depicted m Fig. 1, the profile of change m splenic cell density showed no significant vamatlon during the first period of pregnancy, followed by a sharp decline to the lowest levels in the second period of pregnancy, and values remained at these low levels during the third period of pregnancy

M~togen~c responsweness durmg pregnancy A survey of about 56 adult female Chalc~des (17 non-pregnant and 39 pregnant) provided data on the influence of pregnancy on mltogen reactwlty As illustrated in Fig 2, control splenic cells showed a very high response to all mltogens tested Pregnant lizards as a whole (approximately equal numbers of animals from each period of pregnancy) demonstrated significantly lower m~togen responses than non-pregnant hzards The responses of splenic cells from pregnant lizards to Con A and P H A decreased significantly ( P < 0 01 ) with progression of pregnancy The decrease m the Con A response was large However, the response to L P S of splenic cells from pregnant lizards tended m most cases to be less than that m non-pregnant controls ( P < 0 01) In some instances, the response to L P S of pregnant lizards was similar to that of nonpregnant controls Generally, data grouped according to stage of gestation re-

IMMUNOLOGICALCHANGESDURING PREGNANCY IN THE LIZARDCHALCIDES OCELLATUS

283

PHA

lO

CON A LPS

Z - -

PERIODS

OF

PREGNANCY

I

:i D. ¢.)

I

.... NON PREGNANT

tilt

I

II

III

Fig 2 Prohferat]ve responses of h z a r d splemc cells (0 5 × 1 0 6 / w e l l ) during various periods of pregnancy to Con A (5 # g / m l ) , P H A ( 1 0 / z / m l ) a n d L P S (10/~g/ml) T h e cells were cocultured with the mltogens m m m r o t l t e r plates for 96 h, as descmbed m Matemals a n d Methods Each bar represents the m e a n response of 10-15 mdlv]dual spleens for each pemod of pregnancy Data are plotted as m e a n stimulated cpm (after normahzat]on, see Matemals a n d M e t h o d s ) plus s e m minus m e a n background cpm (after n o r m a h z a t l o n )

Pg/ml

ng/ml

300

120

100

20C

80~ 60

~

w z o w

o 10C

40

w

20

PREGNANT

~

PERIODS OF PREGNANCY---~

Fig 3 Changes m serum c o n c e n t r a t i o n of estrad]ol ( p g / m l ) a n d testosterone ( n g / m l ) during pregnancy m t h e hzard, Chalc~des oceUatus All values are means ( _+s e m ), numbers m parentheses represent n u m b e r of blood samples N u m b e r s below graph indicate pemods of pregnancy

284

A H SAAD AND S EL DEEB

vealed that maternal splenic cells were slgn:ficantly ( P < 0 01 ) hyporesponslve to all three types of mltogens (Fig 2 )

Sex steroid level dunng pregnancy As depicted m Fig 3, the serum estradlol (E2) concentration m adult nonpregnant hzards was relat:vely h:gh, being generally more than 90 + 0 72 p g / ml During pregnancy, E2 concentration Increased markedly, attaining a max1mum value of about 290 + 0 4 p g / m l during the first period Thereafter, beginning during the second period of pregnancy, it decreased sharply to about 65 _+1 1 p g / m l A low level was also recorded during the third permd of pregnancy (55 + 0 35 p g / m l ) On the other hand, serum testosterone (T) concentratmns m non-pregnant females reached about 70 + 0 35 n g / m l Earlier m pregnancy, T could be detected at low concentratmns, similar to those m nonpregnant controls Thereafter, during the second permd of pregnancy, it mcreased rapidly to a prominent peak (about 170 + 0 8 ng/ml, P < 0 01 ) and remained at high levels during the third period of pregnancy (Fig 3) DISCUSSION The results demonstrate that m the hzard C oceUatus some immune functions of the spleen were altered during pregnancy Concurrently, :t was shown that the number of splenic cells m Chalc~des during pregnancy decreased slgmficantly m comparison with intact non-pregnant controls These results are m contrast to the remarkable splemc enlargement that occurs during midpregnancy m some mammalian species (Tyndall et al, 1972, M a r o m and De Sousa, 1973) On the other hand, M c L e a n et al (1974) found no absolute increase m spleen weight during pregnancy m outbred rats, b u t did observe a significant increase m white cell density m the spleen However, m his experimental study, M a t t s o n et al (1979, 1984) found that the number of fetuses per mouse determines the degree of splenic enlargement, suggesting that the progressive growth of fetal tissues initiates and maintains a proliferative response m the maternal spleen These observations mdmate that hke thymlc involution, splenic enlargement during pregnancy may be common among m a m m a h a n specms The data reported above demonstrate that m our experimental conditions the mltogen respons:veness of lizard spleen cells was depressed during pregnancy Responses to Con A and P H A were decreased m cultures of splemc cells from pregnant ammals P r o h f e r a t m n m response to L P S was also decreased b u t to a somewhat more variable and less severe extent than the depression seen m the responses to Con A and P H A A wide var:ety of studms have demonstrated confhctmg results on mltogen-mduced transformation of splemc cells during pregnancy, especmlly m multlparous animals, showing depressed (Kryzych et a l , 1978), unchanged (Winter et a l , 1986) or even increased responses

IMMUNOLOGICAL CHANGES DURING PREGNANCY IN THE LIZARD CHALCIDES OCELLATUS

285

(Manak, 1982 ). The results obtained often depend on the strata combination used. However, it is still unclear whether the ~mpalrment of mltogemc response m the present work was caused by a decreased number of : m m u n o c o m p e t e n t cells a n d / o r by decreased functmn of the existing lymphocytes Although most authors report pregnancy-assocmted depressmn of immune lymphocytes m m a n y expemmental systems, the possible mechanisms responsible for such :mpairment of l m m u m t y are poorly defined (Noonan et a l , 1979, B a r n e t t et a l , 1983, Sano et al., 1984) Since one of the most typmal features of pregnancy is the progressive modlficatmn of hormonal balance, the observed alternatmns m the immune response during pregnancy may well depend on the day-to-day readjustment of hormonal e q m h b r m m Accorchng to present knowledge, the hormonal changes m different a m m a l specms involve primarily gonadotropms, estrogen and progesterone and, as a consequence of these hormonal mod~ficatmns, cortlcosterolds and thyroxine, with m m o r haemostatm readjustment of other hormones (cf in Blllingham and Head, 1980, Wilson, 1985) It may be t h a t circulating hormones regulate the varlatmn :n the immune response m pregnant hzards, and the explanation of some of our results could he m the role played by sex steroids Th~s prolonged contact with high, yet physmlogmal, endogenous levels of sex steroids (Fig 3) may account for the rapid : m p m r m e n t of mltogemc responses of pregnant Chalc~des Thus, sex steroids (testosterone and estradml) could be revolved :n the mechamsms resultlng m the depression of mltogemc actlv:ty m pregnant hzards, though ~t is still plaus:ble t h a t several other m e c h a m s m s are also revolved Further studms are needed to analyze the precise role and the possible mechamsms of such ~mmunologlcal depressmn m pregnant Chalc~des, since these non-specific :mmunologmal changes may be part of the overall p h e n o m e n o n leading to acceptance of the fetus m wwparous reptiles (Griffin, 1981 )

REFERENCES Arslan, M, Loba, J, Zaldl,A A, Jalah, S and Qazl, M H, 1978 Annual androgen rhythm m the spray-tailed hzard, Uromastlx hardwtckl Gen Comp Endocrmol, 36 16-23 Badlr, N, 1958 Seasonal vamatlonof the male urogemtalorgans of Sctncus scmcus L and Chalctdes ocellatus Forsk Z WlssenschZool, 160 290-352 Badet, M T, 1984 Specificand non-specific~mmunosuppresslvefactors m salamanderpregnancy serum J Reprod Immunol,6 299-311 Barnett, M A, Learmonth, R P, Phil, E and Wood,E C, 1983 T helper lymphocytedepression m early human pregnancy J Reprod Immunol, 5 55-57 Blllmgham,R E and Head,J R, 1980 Immunologyof reproduction present trends Ann Immunol (Inst Past ), 131 125-136 E1 Deeb, S and Saad, A-H, 1987 Ontogenyof Con A responsivenessand mixed leucocytereact:vlty m the hzard, Chalctdes ocellatus Dev Comp Immunol, 11 595-604 E1Deeb, S, Zada, S and El Rid1,R, 1985 Ontogenyof hemopoletmand lymphopoletmtissues m the hzard, Chalctdes ocellatus (Reptlha, Sauna, Sclncldae) J Morphol, 185 241-253

286

A H SAADAND S EL DEEB

Gr:ffm, J F T , 1981 Maternal-fetal tolerance nature's gift to v~vlpamty Ir Vet J , 35 194-204 Krzych, U , Strausser, H R , Bressler, J P and Goldstem, A L , 1978 Quant:tatlve differences m immune responses during the var:ous stages of the estrous cycle m female BALB/c mine J Immunol, 121 1603-1605 Manak, R C , 1982 Mltogemc responses of peripheral blood lymphocytes from pregnant and ov armctom:zed heifers and their modulation by serum J Reprod Immunol, 4 263-276 Marom, E S and De Sousa, M A B , 1973 The lympho:d organs durmg pregnancy in the mouse Chn Exp Immunol, 31 107-124 Mattsson, R , Nllson, B and Lmdahl-Kmsshng, K , 1979 An investigation of splemc enlargement m pregnant mine Dev Comp Immunol, 3 683-695 Mattsson, R and Mattsson, A, 1984 Immunoglobuhn-secretmg cells vamous maternal lymphoid tissues during syngene:c and allogenem murme pregnancy Dev Comp Immunol, 8 921-929 McLean, J M , Mosley, J G and Gibbs, A C C, 1974 Changes m the thymus, spleen and lymph nodes during pregnancy and lactatmn m the rat J Anat, 118 223-231 Noonan, K P Halhday, W J Morton, H and Clume, G J A, 1979 Early pregnancy factor is ~mmunosuppresslve Nature, 278 315-317 Saad, A -H, 1988 Cortmosterolds and :mmune systems of nonmammahan vertebrates a rewew Dev Comp Immunol, 12 481-494 Saad, A -H and El Rld~, R , 1988 Endogenous cortmostero~ds mediate seasonal cychc changes m lmmumty ofhzards Immunobmlogy, 177 390-403 Sano, M , Mmke, S , Yosh:km, Y and Nomoto, K , 1984 Existence of suppressor cells m the spleen ofallogenem and syngenem pnmlparous pregnant mice J Reprod Immunol, 6 239-251 Tamura, E and Honma, Y, 1984 Histological changes m the lympho:d system offish with respect to age, seasonal and endocrine changes Dev Comp Immunol, 3 239-243 Tyndall, R L , Otten, M R , Proffitt, J A , Bowles, N D and Tennant, R W , 1972 Some s:mllar~tms m the responses of mine to pregnancy and leukemogenesls Int J Cancer, 9 584-591 Wilson, M , 1985 Immunology of the fetus and newborn Lymphocyte phenotype and functmn Chn Immunol Allergy, 5 271-286 Winter, A J , Hall, C E , Jacobson, R H , Versterate, D R , Meredith, M P and Casteleman, W L 1986 Effect of pregnancy on the immune response of cattle to a Brucella vaccine J Reprod Immunot, 9 313-325 Zapata, A G , Garrldo, E , Leceta, J and Gomamz, R P , 1983 Relatmnsh:ps between neuroendocrme and :mmune systems m amphlbmns and reptiles Dev Comp Immunol, 7 771-775