Immunology, 1975, 29, 509.
Human Chorionic Gonadotropin ITS INHIBITORY EFFECT ON CELL-MEDIATED IMMUNITY IN VIVO AND IN VITRO TIN HAN Department of Medicine B, Roswell Park Memorial Institute, New Buffalo, New rork, U.S.A.
rork State Department of Health,
(Received 30th December 1974; acceptedfor publication 26th February 1975) Summary. An inhibitory effect of human chorionic gonadotropin (HCG) on delayed hypersensitivity skin test responses in guinea-pigs given 4000 i.u. of HCG intraperitoneally, 1-7 days prior to skin testing, is presented. The effect of HCG on skin test responses lasted for at least 3 weeks. Inhibition of lymphocyte response to PHA or PPD in the HCG-treated guinea-pigs was also observed. Guinea-pig peripheral blood lymphocyte response to mitogen or antigen is unequivocally inhibited by HCG in vitro. The effect of this hormone is not due to direct cytotoxicity, and its inhibitory effect seems to be dose related. These observations support the hypothesis that the HCG and possibly other placental hormones might play an important role in protecting the foetus against maternal rejection. INTRODUCTION Maternal tolerance towards an antigenically foreign foetus is a phenomenon puzzling to all immunologists. The exact mechanism of this phenomenon is not well understood, but it is most likely controlled by an immunologically complex system. There have been many contradictory reports with regard to the cell-mediated immune status of pregnant women. Some investigators describe impaired lymphocyte blastogenic response to phytohaemagglutinin (PHA) or to purified protein derivative during pregnancy (Purtilo, Hallfren and Yunis, 1972; Finn, Hill, Govan, Ralfs and Gurney, 1972; Smith, Caspary and Field, 1972). However, others found no impairment of PHA response in women at all stages of normal pregnancy (Watkins, 1972). An inhibitory effect of plasma from healthy primigravidae on lymphocyte blastogenesis has been observed (Kasakura, 1971). However, no inhibitory effect of sera from healthy multigravidae on lymphocyte response has been seen (Purtilo et al., 1972). The trophoblast of the 10-day embryo could locally produce human chorionic gonadotropin (HCG), which is a glycoprotein, at a concentration far greater than 10,000 i.u./ml. An inhibitory effect of HCG on human lymphocyte responses to PHA has recently been described (Adcock, Teasdale, August, Cox, Meschia, Battaglia and Naughton, 1973; Kaye and Jones, 1971). An almost complete inhibition of PHA response by 4000-10,000 Correspondence: Dr Tin Han, Department of Medicine B, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203, U.S.A.
509
Tin Han
510 i.u./ml
concentration
addition,
the
of
inhibitory
HCG
effect
has of
been
HCG
observed
on
(Adcock
antigen-induced
al.,
et
Han, 1974). In
1973;
lymphocyte blastogenesis
and
the mixed lymphocyte reaction has also been reported (Han, 1974). The
present study
describes
the
in
vitro
as
well
as
the
in
cell-mediated immune
vivo
response in animals inhibited by the HCG. MATERIALS AND METHODS Animals employed were healthy adult Hartley guinea-pigs, and the effect of HCG on in vivo and in vitro cell-mediated immunity was studied. Guinea-pigs were immunized by injecting 0.1 ml of Freund's complete adjuvant H37 Ra
(FCA) (Difco Laboratories, Detroit, Michigan)
or
1000
yug
of
keyhole limpet
haemo-
cyanin (KLH) (Cal Biochem, Los Angeles, California) into each hind footpad. Intradermal skin tests with purified protein derivative (PPD) (250 TU) or KLH (100 yug) in 0 ml were performed and read at 24 and 48 hours. The maximum reaction (erythema) was recorded. Two to 4 weeks after FCA immunization, the animals were given 4000 i.u. of HCG in 10 ml of saline or 10 ml of saline only, intraperitoneally (i.p.). One to 7 days after injection, the animals were skin-tested with PPD. Eleven guinea-pigs immunized with FCA 1
in
either
HCG-treated
saline-treated groups
were
skin-tested
with
PPD
before
and1
day after intraperitoneal injection. In five guinea-pigs immunized with KLH, sequential skin tests with KLH were performed before and after two weekly i.p. injections of 4000 i.u. of HCG. Short-term guinea-pig lymphocyte cultures were set up using a whole-blood technique developed
in
heparin (10
our
laboratory (Han
u/ml)
was
obtained
and
Pauly, 1972).
aseptically
from
Blood
collected in
guinea-pigs
by
preservative-free of retro-orbital
means
punctures using capillary pipettes. The blood was diluted with 40 volumes of RPMI 1640 culture medium (Associated Biomedic Systems, Buffalo, New York) containing 100 units of penicillin and 50pug of streptomycin per millilitre. Three millilitres of cell suspension were transferred to 16 x 125 mm Falcon disposable plastic culture tubes. Ten micrograms of PHA (Burroughs Wellcome Company, Tuckahoe, New York) and 7'5 of PPD (Connaught Medical Research Laboratories, Canada) were added to each tube. Various amounts of phosphate-free HCG (Organon, West Orange, New Jersey) 100, 500, 1000, 2000 and 4000 i.u./ml, were added to duplicate culture tubes; control culture tubes received no HCG. The HCG was employed only in cultures containing cells of untreated guinea-pigs immunized with FCA. The hormone was not used in cultures containing cells
jug
of HCG-treated guinea-pigs immunized with FCA. Culture tubes with loose-fitting caps were incubated at 370 in a humidified atmosphere of 5 per centCO2 in air for 5 days (PHA) or 7 days (PPD). One microcurie of [3H]thymidine (specific activity 2-0 Ci/mmole) was added to each tube 24 hours prior to harvesting the cells. Incorporation[3H]thymidine of into DNA was determined according to the method previously described (Han and Pauly, 1972). In five experiments, the effect of 1-hour incubation of lymphocytes with HCG, prior to PHA addition, on lymphocyte blastogenesis was investigated as follows: lymphocytes were incubated with HCG, 4000 i.u./ml, or without HCG, for 1 hour; the cells were then exposed to PHA for 30 minutes, washed twice by centrifugation at 80 g for 5 minutes, and reconstituted with culture medium containing 10 per cent autologous plasma. The viability of lymphocytes at the time of harvest was studied in a few experiments by
511 HCG Efect on Cell-mediated Immunity the trypan blue dye exclusion method previously described (Han and Ohnuma, 1974). Statistical analyses were performed by Student's t-test.
RESULTS The in vivo inhibitory effect of HCG on PPD skin test responses in guinea-pigs is shown in Table 1. In the saline-treated group of guinea-pigs, the PPD responses ranged from 9 to 21 mm, with a mean value of 14-6 mm. On the other hand, skin test responses in the HCGtreated group of guinea-pigs ranged from 4 to 17 mm, with a mean value of 11 1 mm. The TABLE 1 INHIBITORY EFFECT OF HCG ON PPD SKIN
Group
Saline HCG
TEST RESPONSES IN GUINEA-PIGS*
PPD skin test response (mm)
20
Total
0 1
6 21
18 33
20 3
1 0
45 58
* Guinea-pigs were given 4000 i.u. of HCG or saline intraperitoneally, 1-7 days before skin tests were performed. The results are expressed as numbers of guinea-pigs.
TABLE 2 INHIBITORY EFFECT OF HCG ON PPD SKIN TEST RESPONSES IN GUINEA PIGS*
Group Saline
HCG
Guineapig 1 2 3 4 5 6 7 8 9 10 11 Mean 1 2 3 4 5 6 7 8 9 10 11 Mean
PPD skin test response (mm)
Before
After
18 18 17 16 15 15 14 13 13 12 12 14-8 18 18 16 16 16 15 15 14 13 13 10 14-9
17 16 17 21 19 16 16 17 12 17 15 16-6 14 11 12 10 9 11 9 11 12 10 4 10-3
* Skin tests were performed before and 1 day after HCG or saline injection.
Tin Han 512 difference in skin test response between these two groups of animals was statistically significant (P
Human Chorionic Gonadotropin ITS INHIBITORY EFFECT ON CELL-MEDIATED IMMUNITY IN VIVO AND IN VITRO TIN HAN Department of Medicine B, Roswell Park Memorial Institute, New Buffalo, New rork, U.S.A.
rork State Department of Health,
(Received 30th December 1974; acceptedfor publication 26th February 1975) Summary. An inhibitory effect of human chorionic gonadotropin (HCG) on delayed hypersensitivity skin test responses in guinea-pigs given 4000 i.u. of HCG intraperitoneally, 1-7 days prior to skin testing, is presented. The effect of HCG on skin test responses lasted for at least 3 weeks. Inhibition of lymphocyte response to PHA or PPD in the HCG-treated guinea-pigs was also observed. Guinea-pig peripheral blood lymphocyte response to mitogen or antigen is unequivocally inhibited by HCG in vitro. The effect of this hormone is not due to direct cytotoxicity, and its inhibitory effect seems to be dose related. These observations support the hypothesis that the HCG and possibly other placental hormones might play an important role in protecting the foetus against maternal rejection. INTRODUCTION Maternal tolerance towards an antigenically foreign foetus is a phenomenon puzzling to all immunologists. The exact mechanism of this phenomenon is not well understood, but it is most likely controlled by an immunologically complex system. There have been many contradictory reports with regard to the cell-mediated immune status of pregnant women. Some investigators describe impaired lymphocyte blastogenic response to phytohaemagglutinin (PHA) or to purified protein derivative during pregnancy (Purtilo, Hallfren and Yunis, 1972; Finn, Hill, Govan, Ralfs and Gurney, 1972; Smith, Caspary and Field, 1972). However, others found no impairment of PHA response in women at all stages of normal pregnancy (Watkins, 1972). An inhibitory effect of plasma from healthy primigravidae on lymphocyte blastogenesis has been observed (Kasakura, 1971). However, no inhibitory effect of sera from healthy multigravidae on lymphocyte response has been seen (Purtilo et al., 1972). The trophoblast of the 10-day embryo could locally produce human chorionic gonadotropin (HCG), which is a glycoprotein, at a concentration far greater than 10,000 i.u./ml. An inhibitory effect of HCG on human lymphocyte responses to PHA has recently been described (Adcock, Teasdale, August, Cox, Meschia, Battaglia and Naughton, 1973; Kaye and Jones, 1971). An almost complete inhibition of PHA response by 4000-10,000 Correspondence: Dr Tin Han, Department of Medicine B, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203, U.S.A.
509
Tin Han
510 i.u./ml
concentration
addition,
the
of
inhibitory
HCG
effect
has of
been
HCG
observed
on
(Adcock
antigen-induced
al.,
et
Han, 1974). In
1973;
lymphocyte blastogenesis
and
the mixed lymphocyte reaction has also been reported (Han, 1974). The
present study
describes
the
in
vitro
as
well
as
the
in
cell-mediated immune
vivo
response in animals inhibited by the HCG. MATERIALS AND METHODS Animals employed were healthy adult Hartley guinea-pigs, and the effect of HCG on in vivo and in vitro cell-mediated immunity was studied. Guinea-pigs were immunized by injecting 0.1 ml of Freund's complete adjuvant H37 Ra
(FCA) (Difco Laboratories, Detroit, Michigan)
or
1000
yug
of
keyhole limpet
haemo-
cyanin (KLH) (Cal Biochem, Los Angeles, California) into each hind footpad. Intradermal skin tests with purified protein derivative (PPD) (250 TU) or KLH (100 yug) in 0 ml were performed and read at 24 and 48 hours. The maximum reaction (erythema) was recorded. Two to 4 weeks after FCA immunization, the animals were given 4000 i.u. of HCG in 10 ml of saline or 10 ml of saline only, intraperitoneally (i.p.). One to 7 days after injection, the animals were skin-tested with PPD. Eleven guinea-pigs immunized with FCA 1
in
either
HCG-treated
saline-treated groups
were
skin-tested
with
PPD
before
and1
day after intraperitoneal injection. In five guinea-pigs immunized with KLH, sequential skin tests with KLH were performed before and after two weekly i.p. injections of 4000 i.u. of HCG. Short-term guinea-pig lymphocyte cultures were set up using a whole-blood technique developed
in
heparin (10
our
laboratory (Han
u/ml)
was
obtained
and
Pauly, 1972).
aseptically
from
Blood
collected in
guinea-pigs
by
preservative-free of retro-orbital
means
punctures using capillary pipettes. The blood was diluted with 40 volumes of RPMI 1640 culture medium (Associated Biomedic Systems, Buffalo, New York) containing 100 units of penicillin and 50pug of streptomycin per millilitre. Three millilitres of cell suspension were transferred to 16 x 125 mm Falcon disposable plastic culture tubes. Ten micrograms of PHA (Burroughs Wellcome Company, Tuckahoe, New York) and 7'5 of PPD (Connaught Medical Research Laboratories, Canada) were added to each tube. Various amounts of phosphate-free HCG (Organon, West Orange, New Jersey) 100, 500, 1000, 2000 and 4000 i.u./ml, were added to duplicate culture tubes; control culture tubes received no HCG. The HCG was employed only in cultures containing cells of untreated guinea-pigs immunized with FCA. The hormone was not used in cultures containing cells
jug
of HCG-treated guinea-pigs immunized with FCA. Culture tubes with loose-fitting caps were incubated at 370 in a humidified atmosphere of 5 per centCO2 in air for 5 days (PHA) or 7 days (PPD). One microcurie of [3H]thymidine (specific activity 2-0 Ci/mmole) was added to each tube 24 hours prior to harvesting the cells. Incorporation[3H]thymidine of into DNA was determined according to the method previously described (Han and Pauly, 1972). In five experiments, the effect of 1-hour incubation of lymphocytes with HCG, prior to PHA addition, on lymphocyte blastogenesis was investigated as follows: lymphocytes were incubated with HCG, 4000 i.u./ml, or without HCG, for 1 hour; the cells were then exposed to PHA for 30 minutes, washed twice by centrifugation at 80 g for 5 minutes, and reconstituted with culture medium containing 10 per cent autologous plasma. The viability of lymphocytes at the time of harvest was studied in a few experiments by
511 HCG Efect on Cell-mediated Immunity the trypan blue dye exclusion method previously described (Han and Ohnuma, 1974). Statistical analyses were performed by Student's t-test.
RESULTS The in vivo inhibitory effect of HCG on PPD skin test responses in guinea-pigs is shown in Table 1. In the saline-treated group of guinea-pigs, the PPD responses ranged from 9 to 21 mm, with a mean value of 14-6 mm. On the other hand, skin test responses in the HCGtreated group of guinea-pigs ranged from 4 to 17 mm, with a mean value of 11 1 mm. The TABLE 1 INHIBITORY EFFECT OF HCG ON PPD SKIN
Group
Saline HCG
TEST RESPONSES IN GUINEA-PIGS*
PPD skin test response (mm)
20
Total
0 1
6 21
18 33
20 3
1 0
45 58
* Guinea-pigs were given 4000 i.u. of HCG or saline intraperitoneally, 1-7 days before skin tests were performed. The results are expressed as numbers of guinea-pigs.
TABLE 2 INHIBITORY EFFECT OF HCG ON PPD SKIN TEST RESPONSES IN GUINEA PIGS*
Group Saline
HCG
Guineapig 1 2 3 4 5 6 7 8 9 10 11 Mean 1 2 3 4 5 6 7 8 9 10 11 Mean
PPD skin test response (mm)
Before
After
18 18 17 16 15 15 14 13 13 12 12 14-8 18 18 16 16 16 15 15 14 13 13 10 14-9
17 16 17 21 19 16 16 17 12 17 15 16-6 14 11 12 10 9 11 9 11 12 10 4 10-3
* Skin tests were performed before and 1 day after HCG or saline injection.
Tin Han 512 difference in skin test response between these two groups of animals was statistically significant (P
