Scand. J. Immunol., Vol. 5, 1976.
The Effect of Human Chorionic Gonadotropin and Placental Lactogen on Lymphocyte Transformation In Vitro J. H. MORSE Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA Morse, J. H. The Effect of Human Chorionic Gonadotropin and Placental Lactogen on Lymphocyte Transformation In Vitro. Scand. J. Immunol. 5, 779-787, 1976. Crude preparations of human chorionic gonadotropin (hCG) inhibited phytohemagglutinin (PHA) and allogeneic cell (MLC) lymphocyte stimulation of human peripheral blood lymphocytes (PBL) in vitro as measured by ["Hlthymidine incorporation, whereas purified hCG was not inhibitory except at very high concentrations. Sephadex G-lOO chromatography of crude hCG showed that the inhibitory efFect on PHA lymphocyte stimulation eluted in those fractions after hormonally active hCG. In addition, purified human placental lactogen (hPL) did not inhibit lymphocyte transformation except at very high concentrations. These data suggest that the immunosuppressive activities found in crude preparations are not due to the hormones but to contaminating material. ,7. H. Morse, M.D,, Columbia University, College of Physicians and SurgeonSt 630 W. 168th St., New York, NY 10032, USA
A decreased immune response during pregnancy has been postulated as important in preventing immunological rejection of the fetus. In the past the role of gestational hormones as immunosuppressive factors has mainly been confined to studies of estrogens, progesterone, and cortisone or adrenocorticotropin (17, 38). Recently, crude preparations of human chorionic gonadotropin (hCG) (2, 12, 14) and human placental lactogen (hPL) (12) have been shown to inhibit phytohemagglutinin {PHA) stimulation of human lymphocytes in vitro. These reports, implicating hCG and hPL as immunosuppressive factors, have been followed by conflicting reports regarding the capacity of hGG to inhibit stimulation of human lymphocytes by mitogens, antigens, and allogeneic cells. The inhibitory capacity of hPL has not been evaluated further. The present report describes the results of studies, in part reported previously (8, 24), suggesting
that crude hCG is more inhibitory than purified hCG on lymphocyte transformation and that the inhibitory activity found in crude preparations elutes on Sephadex G-lOO column chromatography in fractions distinct from purified hCG. Not reported previously was the failure of purified preparations of hPL to inhibit the response of peripheral blood lymphocytes to PHA and allogeneic cells,
MATERIALS AND METHODS Lymphocyte culture methods. Lymphocyte cultures were prepared by a modification of the method of Boyum (6). Forty to fifty ml of blood, obtained from normal individuals in phenol-free heparin, was allowed to sediment in a syringe at 37^C for 1—2 h. The plasma was expressed from the top of the syringe and gently mixed with an equal volume of sterile isotonic saline. Two volumes of the saline-
780
J. H. Morse
plasma solution were layered over one volume tjf Ficoll-Conray solution (10 ml 33.4% Conray plus 24 ml 9% Ficoll) and centrifuged for 30 min at 4"C (Ficoll was obtained from Pharmacia Fine Chemicals, Piscataway, N.J., and 60% Conray from Mallmckrodt Pharmaceuticals, St. Louis, Mo.). The interface containing lymphocytes was removed and washed three times with Eagle's minimal essential medium (MEM) containing 50 units/ml penicillin, 50 i^ig/ml streptomycin, 2mM L-glutamine. The cells were cultured in this medium with the addition of 10% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.), pooled human AB plasma, or autologous plasma. Unless otherwise specified, serum or plasma was heated to 56'C for 30 min before use. Triplicate cultures of 0.25 ml containing 1 or 2 X IO'* lymphocytes were placed in weils of microtiter plates (Linbro IS FB 96, Linbro Chemicals Co., New Haven, Conn.). After incubation at 37^C in 5% CO.y, 95% air for 5 days they were pulsed overnight with 1 [iCi/ well of I^H]tliymidine, 6.7 Ci/mmol (New England Nuclear Corp., Boston, Mass.). The cultures were harvested and washed extensively with saline on glass fiber filters (grade 934 AH, Reeve Angel, CHfton, N.J.) using a Mash 11 (Harvester Microbiological Associates) (15). The filters were dried and placed in 12 ml of Instabray (Yorktown Research, Hackensack, N.J.). '^H content was determined in a liquid scintillation spectrometer (Packard model 3320). The results were expressed as counts per minute (cpm) of [^H]thymidine per culture or as percentage of inhibition of thymidine incorporation of the mitogen control. Mitogens. Phytohemagglutinin (PHA-P, Difco Laboratories, Detroit, Micb.) was used at the optimal dose, usually either 0.001 or 0.005 per ml of culture. One-way mixed lymphocyte cultures (MLC) were prepared by incubating tbe stimulatmg lymphocytes at 2 X 10" cells/ml for 1 h at 37° with 25 ng/ml mitomycin C (Sigma Chemical Co., St. Louis, Mo.), followed by washing three times with large volumes of MEM. Preliminary cultures were performed with unrelated donors, and the most reactive ratios of stimulator to re-
sponder cell were seleceted for use with hCG —that is, 1.6 X 10" of mitoniycin-C-treated lymphocytes to 8 X 10* responder lymphocytes. Trypan blue dye viability studies showed about 82%—88% dye exclusion in most expernnents. hCG and hPL preparations. Both crude and purified hCG were generous gifts of Dr. Robert Canfield, Department of Medicine, Columbia University, College of Physicians and Surgeons. Crude preparations of hCG (purchased from Organon, West Orange, N.J.) were batches 91930, 92145, and 92666, which had estimated potencies of 3900 lU/mg, 2700 IU/mg, and 2680 lU/mg, respectively. The bioassay results, as well as the subsequent yields of purified hormone, indicated that approximately 75% of the bulk material in tbese crude preparations was not bCG. Purified bCGs 119 and 121 were prepared from crude hCG 91930 and 92145 by methods described previously (9, 10). Purified hCG appeared to contain less than 5% contamination with other material, and biological potency estimated by the rat ventral prostate weight bioassay (22) was 11,600 and 13,450 IU/mg. It is known that the biological potency of different preparations of purified hCG can vary significantly a.s a function of sialic acid content (39), and the range of bioassay results IS consistent with other chemical data, indicating tbat the preparations were greater than 95% hCG. hCG preparations were stenlizt-'d by passage through a 0.45-fim Millipore filter (Millipore Corp., Bedford, Mass.). Purified preparations of human placental lactogen were generously supplied by Dr. Frank Morgan, St. Vincent's School of Medical Restarch, Melbourne, Australia, and Dr. Louis Sherwood, Department of Medicine, Michael Reese Hospital, The University of Chicago, Pritzker School of Medicine. hPL 1 (Australia) was prepared by the method of Catt et al. (11) and considered to be 90% pure. A minor nonprotein contaminant was present. A bioassay of this preparation was not available. hPL 2 (Chicago) was biologically active in the radioreceptor assay of rabbit mammary gland membranes (36) and also in a radioreceptor
TmmuTWSuppressive Effects of hCG and hPL
assay using mammary tissue in organ culture. hPL 2 was prepared from placental extracts by methods described previously (35). Both hPL preparations had molecular weights of 22,000.
781
RESULTS Table I shows a representative experiment comparing the effect of crude and purified hCG on PHA-stimulated human peripheral
Table I. Effect of crude and purified human chorionic gonadotropin (hCG) on phytohemagglutinin (PHA) stimulatitin of peripheral blood lymphocytes* Type of hCG preparation Crude 91930
hCG
concentration (lU/ml) 3,900 1,950 975 390 195
I'urified CR-119
11,600 5,800 2,900 1,160 580
PHA control Cell control
["Hjthymidine incorporation (cpm)
Percentage inhibition
8,654 33,253 41,588 52,602 60,692
: 1,395 ,1:2,110 zi 5,941 ^ 1,239 ±13,979
85 44 30 12 0
46,358 36,937 56,680 53,275 50,665
±4,951 -!:2,744 f: 9,654 ±3,373 ±7,718
23 38 5 11 15
59,857 ; 2,142 _
102
.:_n
_
* Each culture contained 1 X 10" lymphocytes in 0.25 ml of culture medium with 15% heated AB plasma. Results are expressed as the arithmetic mean '•• SD of counts per minute of [^H]thymidinc incorporation per culture. Cmde hCG 91930 had a bioassay of 3900 IU/mg and purified hCG 119 had 11600 IU/mg.
I'able II. Effect of crude and purified human chorionic gonadotropin (hCG) on mixed lymphocyte reactivity of peripheral blood lymphocytes* Type of hCG Preparation Crude 92145
Purified CR-121
Responder - stimulator cells Responder cells alone
['H]thymidine incorporation (cpm)
Percentage inhibition
5,850 3,900 1,950
752 ±166 985 ih71
975 390 195
1,078 _ 236 1,205 -1.-155 1,333 ±191
55 40 47 35 27 19
743 1,333 1,592 1,682 1,389
55 19 4 0 16
hCG
concentration (IU/ml)
20,175 13,450 6,725 3,362 1,345
876 ±89
M42 ±83 fc357 1 675 ±101
1,651 i 353 217 -:6
* liach culture contained 1 X 10* lymphocytes in 0.25 ml of culture medium with 15% heated AB plasma. Results are expressed as the arithmetic mean ± SD of counts per minute of [^H]thymidine incorporation per culture. Crude hCG 92145 had a bioassay of 3900 IU/mg and purified hCG 121 had 13,450 IU/mg.
782
3- ff- Morse
blood lymphocytes (PBL). The concentrations of hCG in the cultures are expressed as IU/ml. In thi.s study 3900 IU/ml of the crude material produced 85%inhibition of the PHA control, whereas the purified hormone at 11,600 IU/ ml produced only 23% inhibition. Biologic variations did occur, but the crude hCG or parent preparation from which the purified hormone was made was always more inhibitory. Experience with four pairs of matched crude and purified hCG preparations has confirmed this observation. Since PHA is known to hind glycoproteins, crude hCG could have preferentially bound PHA, thus removing lymphocyte stimulation by glycoprotein-lectin Interaction (29). Allogeneic cell stimulation was used to circumvent this problem. Table II shows another representative experiment. Again the crude preparation produced 40% inhibition at 3900 IU/ml, whereas the purified hormone only gave 19% inhibition at 13,350 IU/ml. This experiment is included to show that occasionally very high concentrations of purified hCG, in this in-
CRUDE hCG
LOT
C
BO
92666
GLUCOSE
ISO
FRACTION NUMBER
Fig. 1. Sephadex G-lOO chromatography of crude 92666 in 0.05M NH^HCOs. The fractions shown on the abscissa were combined to form pools 1 through 11 and equalized to the same optical density. The degree of inhibition of phytohemagglutinin-stimulated lymphocytes by aiiquots of these pools and the bioassays for human chorionic gonadotropin (hCG) hormonal activity are illustrated in Table III.
Table III. Comparison of human chorionic gonadotropin (hCG) hormonal activity and lymphocyte inhibition in Sephadex G-lOO fractions of crude hCG Sephadex G-lOO pool no.
I 2 3 4 5 6 7 8 9 10 U
hCG concentration % Inhibition of (IU/mg)* PHA-stimulated lymphocytesf 11,889 11,889 12,189 1,983
25 9 3
N.D. 79
N.D. 99 97 77 0 15 0 0 0
* Bioassay of hCG by rat ventral prostate assay; courtesy of Dr. R. E. Canficld. All fraction pools were adjusted to O.D. 280. The confidence limits of these bioassays vary about 15%.
t PHA = phytohemagglutinin. stance 20,175 IU/ml, significantly inhibited (55%) the MLC reaction. Experiments were then initiated to ascertain if the immunosuppressive material found in crude hCG could be separated from the purified hormone. Crude hGG was placed on Sephadex G-lOO in 0.05M NH4HCO:i. The elution profile is shown in Fig. 1. The fractions were combined into 11 pools and the protein concentration equalized to an optical density of approximately 2. Table III compares the inhibitory effect on PHA stimulation with the hCG bioassay (IU/ml) of the fractions. Note that high hCG hormonal activity. above 11,000 IU/ml, was found in peaks 1-3. These are the peaks that are pooled for further purification of the hormone. However, lymphocyte inhibition was found in peaks 2, 4, 5, and 6. Peaks 5 and 6 have little hormonal activity, less than 25 IU/ml, and most of the immunosuppressive activity, greater than 95^,Interestingly, peak 2 had a lymphocyte inhibition comparable to that of peak 6, 79% compared with 77%. whereas the specific activity of the hormone was more than one thousand times greater in peak 2. Although only two different batches of
Immunosuppressive Effects of hCG and hPL 14000
12000
10000
STiM,+
8000
783
Fig. 2. The effect of human placental lactogen (hPL) 1 and hPL 2 on peripheral blood lymphocytes stimulated by mitomycin-Ctreated allogeneic lymphocytes. Each culture contained 2 X 10" stimulator (Stim.) and 2 X 10" responder (Resp.) lymphocytes in 5 % heated fetal calf scrum.
6000
4000
2000 STIM. RESR
° I
10
50
100
5 -
1000
l^q/ml)
crude hCG have been studied under slightly different buffer conditions, these data suggest that the substance(s) that inhibit stimulated lymphocytes eluted in a broad peak just after hCG. Preliminary evidence also showed lower
STIM.+ RESP,
I 10
50
100
1000
hPL (jJfl/mll
I'ig. 3. The effect of human placental lactogen (hPL) 1 on peripheral blood lymphocytes stimulated by mitomydn-C-treated allogeneic lymphocytes. Each culture contained 2 X 10'' stimulator (Stim.) and 2 X 10* responder (Resp.) lymphocytes in 5% heated fetal calf serum.
molecular weight inhibition in some batches of crude hCG (25). Because of its glycoprotein nature, purified hCG has an anomalous sedimentation behavior (8), eluting at around 70,000 daltons on Sephadex G-lOO rather than in the region of its actual molecular weight, which is 37,000 daltons. The main inhibitory activity elutes between 20,000 and 40,000 daltons. Because human placental lactogen, also called somatomammotropin (hCS), had been reported to inhibit PHA stimulation of lymphocytes, and because the molecular weight of its active form (22,000) coincided with that of the inhibitory activity in the crude hCG preparations, purified hPL preparations were evaluated for inhibition of lymphocyte stimui.ition by PHA and in the MLC. hPL also occurs as a dimer form termed 'Big' hPL with a molecular weight of 46,000 (33). Preparation 1 of hPL was judged to be 90% pure by weight but was known to have a nonprotein contaminant, whereas hPL preparation 2 was considered pure (hPL 2 was prepared in the same laboratory by the method that provided the amino acid sequence of hPL) and both contained only monomeric hPL. Fig. 2 shows that both purified preparations of
:84
J. H- Morse
g 10000
CELL CONTROL
nPL Ug/oil)
Fig. 4. The effect of human placental lactogen (hPL) 1 on peripheral blood lymphocytes stimulated by phytohemagglutinin (PHA). PHA-P was used at a concentration of 0.005 ml per ml of culture. Each contained 2 x 10* cells in 5"i, heated fetal calf serum.
hPL failed to inhibit MLC-induced lymphocyte transformation in concentrations ranging from 1 to 100 ng/tnl. hPL 1 did give 50% inhibition at 1000 (ig/ml and about 75% inhibition at 1000 (ig/ml in another MLC shown in Fig. 3. Fig. 4 shows the effect of increasing concentrations of hPL 1 on PHA-stimulated lymphocytes. Little change m inhibition occurred between 10 and 1000 fig/tnl. In another experiment, not illustrated here, both hPL 1 and 2 in concentrations of 1 to 1000 |ig/ml failed to inhibit PHA stimulation. Note in Fig. 4 that hPL, in concentrations of 1 to 1000 ng/ml, had no effect on resting unstimulated lymphocyte cultures. These data show that the purified hormone did not inhibit lymphocytes stimulated by either PHA or allogeneic cells. The inhibition tbat did occur was found in the more impure preparation.
DTSCUSSION The data indicate that both purified bCG and hPL have little inhibitory effect on PHAand MLC-induced lymphocyte transformation, except at very high concentrations—5000 IU/ml or greater for hCG and 1000 |ig/ml for
hPL. Crude preparations of hPL were not evaliited, but crude hCC showed greater inhibition than the purified hormone. The greater inhibition of PHA stimulation of lymphocytes by crude hCG compared with the purified hormone has previously been reported from this laboratory (8, 24) and also by others (7, 13). Similar results have been reported more completely elsewhere using lymphocytes stimulated by pokeweed, purified protein derivative, and allogeneic cells (8). However, as mentioned previously, conflict exists regarding the inhibitory role of hCG in the MLC reactions. Beling & Weksler (3) and Han (14) found significant inhibition by hCG at concentrations of 40-200 IU/ml and 10 IU/ml, respectively. These concentrations approach the physiologic concentrations, which can be as high as 10 IU/ml, in plasma taken during the first trimester (8). Caldwell et al. (7), using similar reagents, recently noted no inhibitory activity in the MLC reaction with purified hCG, as did Schiff et al. (31). Comparison of the hCG preparations used in these conflicting reports suggests that the crude preparations were more inhibitory than the purified hormone, regardless of the type of lymphocyte stimulation studied. The results of the present experiments on the immunosuppressive properties of hPL do not agree with those of Contractor & Davies (12), who, using commercially obtained hPL, occasionally noted inhibition of PHA lymphocyte stimulation with concentrations as low as 12.3 ng/ml and always when 50 to 100 \i%l ml was used. Again, the most likely explanation is that the purer reagents used here lack the inhibitory material found in crude placental extracts. The high concentration of purified hPL, 1000 f.tg/ml, required to inhibit stimulated lymphocytes is well above the concentration of 3—5 ng/ml found in pregnancy plasrna at term (34). Although the nature of the inhibitory material in the crude hCG preparations is unknown, several immunosuppressive substances found in urine and plasma from pregnant women should be considered. Other gestational hormones
Immwiosuppressive Effects of hCG and hPL
such as estrogen and progesterone are elevated during pregnancy. Both progesterone (23) and diethylstilbestrol (1) have been shown to inhibit the incorporation of [^HJthymidine by PHA-stimulated human PBL, whereas other investigators failed to find inhibition of the MLR and mitogen stimulation using physiologic concentrations of estrogen and progesterone (31). Even though estrogen, progesterone, and adrenocorticotropin have all been implicated as immunosuppressive substances with various degrees of certainty, their molecular weights are too low to be in the inhibitory peak on the Sephadex G-lOO column studied here unless they are aggregated or bound to heavier substances. It is also possible that hCG could act synergistically with another hormone or substance in the crude preparation to provide immunosuppression. Several plasma proteins that are elevated in or unique to pregnancy could inhibit stimulated lymphocyte cultures. These proteins include: immunoglobulins with antilymphocyte activity (30); pregnancy ;tf]-glycoprotein, described by Bohn (5); pregnancy-associated ag-macroglobulin (37), originally described by MacLaren et al. (21), which is identical to pregnancy-associated globulin (PAG) (16); PAPP-A. another recently described pregnancy-associated ti-globulin with a MW of 750,000, supposedly different from PAG (19); as well as a-fetoprotein. There is .ilso a third «.,-globulin, called pregnancy zone protein (32), which appears to be lmmunologically distinct from the other two a^,-globulins of pregnancy just described, namely PAG and PAPP-A (18). The presence of antilymphocyte antibodies in the maternal circulation has been documented in some patients, and certain commercial hGG preparations have recently been shown CO be contaminated with immunoglobulins, not necessarily antilymphocyte antibodies, in sufficient quantity to provide anticomplementary activity great enough to convert C3, the third component of complement (20). These immunoglobulins of complexes would be eluted from Sephadex G-lOO in a region of higher molecular weight than the inhibitory action
785
sliown in Fig. 1. As yet, pregnancy-specific ^[-glycoprotein and PAPP-A have not been tested in cell-mediated immune systems, and controversy exists regarding the ability of PAG or pregnancy-associated (ta-macroglobulin to inhibit the mitogenic responses of PPD- and PHA-stimulated lymphocytes (16). Mouse afetoprotein with a known MW of 49,000 (79,000 on sodium dodecyl sulfate gels) has definitely been shown to inhibit lymphocyte transformation (26) and antibody formation (27). There are also other immunosuppressive factors, not necessarily elevated during pregnancy, such as immunoregulatory alpha-globulin (IRA) and its peptide (28); SAA, an alpha-globulin found in amyloidosis (4); and a host of other less well characterized factors found associated with various clinical states. Although interesting possibilities, these are too numerous to review here. Similarly, hPL, which is isolated from placental extracts rather than pregnancy urine, may contain one or more of these immunosuppressive factors or perhaps an additional one. In summary, the possibility exists that the inhibitory activity found in hGG is related to these pregnancy-associated factors or other factors that show an influence in different tests for cell-mediated immunity. Whether these or similar substances have been concentrated sufficiently in commercial crude hCG preparations IS unknown and presently under investigation.
AGKNOWLEDGEMENTS The author is deeply grateful to Dr. Robert Ganfield for his collaboration in earlier related studies and for generously supplying crude and purified hCG, and to Drs. Frank Morgan and Louis Sherwood for their purified preparations of hPL. This work was supported by USPHS grant HL 10049 and the Systemic Lupus Erythematosus Foundation. The excellent technical assistance of Mr. Ken Miller is gratefully acknowledged.
786 .7- //. Morse REFERENCES 1. Ablin, R.U., Bruns, G.R., Guinan, P. & Bush, I.M. The effect of estrogen on the incorporation of 'H-thymidine by PHA stimulated human peripheral blood lymphocyles. J. Immunol. US, 705,1974. 2. Adcock, H.W., HI, Teasdale, F., August, C.S., Cox, S., Meschia, G., Battaglia, F.C. & Naughton, M.A. Human chorionic gonadotropin: its possible role in maternal lymphocyte suppression. Science 181, 845, 1973. 3. Beling, C G . & Weksler, M.E. Suppression of mixed lymphocyte reactivity by human chorionic gonadotropin. Clin. exp. Immunol. /5, 537, 1974. 4. Benson, M. D., Aldo-Benson, M.A., Shirahama, T., Borel, Y. & Cohen, A.S. Suppression of in vitro antibody response by a serum factor (SAA) in experimentally induced amyloidosis. J. exp. Med. 142, 236, 1975. 5. Bohn, H. Nachweis und cbaraklerisierung von schwangerschaftsproteinen in der menschlichcn placenta, sowie ihre quantative immunologische bestimmung im serum schwangerer frauen. Arch. Gyndk. 210, 440, 1972. 6. Beyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest. 21, Suppl. 97, 77, 1968. 7. Caldwell, J.L., Stites, D.P. & Fudenberg, H.H. Human chorionic gonadotropin: effects of crude and purified preparations on lymphocyte responses to phytohemagglutinin and allogeneic stimulation. J. Immunol. 115, 1249, 1975. 8. Canfield, R.E., Birken, S., Morse, J.H. & Morgan F.J. Human chorionic gonadotropin. pp. 299-315 in Parsons, J. (ed.) Proceedings of the Symposia on Drug AcHon-Peptide Hormones. Macmillan and Co., London, 1976. 9. Canfield, R.E. & Morgan, F.J. Human chorionic gonadotropin (hCG) I. Purification and biochemical characterization, p. 727 in Berson, S.A. & Yalow, R.S. (eds.) Methods of Invesiigaiive and Diagnostic Endocrinology. North-Holland Publishing Co., Amsterdam, 1973. 10. Canfield, R.E., Morgan, F.J., Kammerman, S., Bell, J.J. & Agosto, G.M. Studies of human chorionic gonadotropin. Recent. Progr. Hormone Res. 27, 121, 1971. 11. Catt, K.J., Moffat, B., Niall, H.D. & Preston, B.N. Purification and physiochemical properties of human placental lactogen. Biochem. J. 102, 27c, 1967. 12. Contractor, S.F. & Davies, H. Effect of human chorionic somatomammotrophin and human chorionic gonadotropin on phytohaemagglutinininduced lymphocyte transformation. Nature iLond.) 243, 284, 1973. 13. Gundert, D., Merz, W.E., Hilgenfeldt, U. &
Brossmer, R. Inability of highly purified preparations of human chorionic gonadotropin to inhibit the phytohemagglutinin-induced stimulation of lymphocytes. FEBS Letters 53, 309, 1975. 14. Han, T. Inhibitory effect of human chorionic gonadotropin on lymphocyte blastogenic response to mitogen, antigen and allogeneic cells. Clin. exp. Immunol. IS, 529, 1974. 15. Harrison, M.R., Thurman, G.R. & Thomas, G.M. A simple and versatile harvesting device for processing radio-active label incorporated into and/or released from cells in microculture. y. immunol. Methods 4, 11, 1974. 16. Home, C.H.W., McLay, A.L.C., Tavadia, H.B., Carmichael, I., Mallinson, A.C, Yeung Laiwah, A.A.C., Thomas, M.A. & MacSween. R.N.M. Studies on pregnancy associated globulin. Clin. exp. Immunol.13, 603, 1973. 17. Kuwata, T. Studies on the growth of Rous sarcoma and its variant strain in cortisone-treated hamsters. Cancer Res. 20, 170, 1960. 18. Lin, T.M. & Halbert, S.P. Immunological comparison of various human pregnancy-associated plasma proteins. Int. Arch. Allergy 48, 101, 1975. 19. Lin, T.M., Halbert S.P., Kiefer. D. & Spellacy, W.N. Three pregnancy-associated human plasma proteins; purification, monospecific antisera and immunological identification. !nt. Arch. Allertfv 47, 35, 1974. 20. Loke, Y.W. & Pepys, M.B. Effects of human chorionic gonadotropin preparations on complement IH vitro. Amer. J. Obstet. Gynec. 121, 37, 1975. 21. MacLaren, J.A., Reid, D.E., Konugres, A.A. & Allen, F.H., Jr. Pal, a new inherited alpha-2globulin of human serum. Vox Sang. {Basel) II, 553, 1966. 22. McArthur, J.W. The identification of pituitary interstitial cell stimulating hormone in human urine. Endocrinology 50, 304, 1952. 23. Mori, T., Kobayashi, H., Nishimura, T, Mori, T.S., Fujii, G. & Inou, T. Inhibitory effect of progesterone on the phytohaemagglutinin-induced transformation of human lymphocytes. Immunol. Commun. 4(6), 519, 1975. 24. Morse, J.H., Stearns, G., Arden. J., Agosto, G. & Canfield, R.E. The effects of crude and purified human gonadotropin on i« vitro stimulated human lymphocyte cultures. Cell Immunol. In press. 25. Muchmore, A.V. & Blaese, R.M. Immunoregulatory effects of human chorionic gonadotropin (hCG). Fed. Proc. 33, 750, 1974. 26. Murgita, R.A. & Tomasi, T.B. Suppression of the immune response by a-fetoprotein. II. The effect of mouse a-fetoprotein on mixed lymphocyte reactivity and mitogen-induced lymphocyte transformation. J. exp. Med. 141, 440, 1975. 27. Murgita, R.A. & Tomasi, T.B. Suppression of
Immunosuppressive Effects of hCG and hPL the immune response by a-fetoprotein. I. The efFect of mouse a-fctoprotein in the primary and secondary antibody response. J. exp. Med. 141, 269, 1975. 28. Occhino, j . C , Glasgow, A.H., Cooperband, S.R., Mannick, J.A. & Schmidj K. Isolation of an immunosuppressive peptide fraction from human plasma. J. ImmunoL HO, 685, 1973. 29. Powell, A.H. Maternal lymphocytes; Suppression b\' human chorionic gonadtropin. Science 184, 913, 1974. 30. Revillard, J.P., Robert, M., Betnel, H., Lajour, M,, Bonneau, M., Brochier, J. & Traeger, J. Inhibition of the mixed lymphocyte reaction by antibodies. Transplatil. Proc. 4, 173, 1972. 31. Schiff, R.I., Mercier, D. & Buckley, R.H. Inability of gestational hormones to account for the inhibitory effects of pregnancy plasma on lymphocyte responses in vitro. Cell. Immunol. 20, 69, 1975. 32. Schlontz, B. von, Stigbrand, T. & Tarnvik, A. Inhibition of PHA induced lymphocyte stimulation by pregnancy zone protein. FEBS Lellers 38, 23, 1973. 33. Schneider, A.B., Kowalski, K. & Sherwood, L.M. 'Big' human placental lactogen: disulfideReceived 4 May 1976 Received in revised form 15 June 1976
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linked peptide chains. Biochem. biophys. Res. Commun. 64, 717, 1975. 34. Sciarra, J.J., Sherwood, L.M., Varma, A.A. & Lundberg, W.B. Human placental lactogen (HPL) and placental weight. Amer. J. Obstet. Gynec. 101, 413, 1968. 35. Sherwood, L.M., Handwerger, S., McLaurin, W.D. & Lanner, M. Amino-acid sequence of human placental lactogen. Nature {New Bid.) 233, 59, 1971. 36. Shiu, R.P.C., Kelly, P.A. & Friesen, H.G. Radioreceptor assay for prolactin and other lactogenic hormones. Science ISO, 968, 1973. 37. Stimson, W.H. & Eubank-Scott, L. The isolation and partial characterization of a new a^-macroglobulin from human pregnancy serum. FEBS Letters 23, 298, 1972. 38. Thompson, J.S., Crawford, M.K., Reilly, R.W. & Severson, C D . The effect of estrogenic hormones on immune responses in normal and irradiated mice. J. Immunol. 98, 331, 1967. 39. Van Hell, H., Goverde, B.C., Schuurs, A.H.W. M., Dejager, E., Matthijscn, R. & Homan, J.D. H. Purification, characterization and immunochemical properties of human chorionic gonadotropin. Nature {Land.) 212, 261,1966.
The Effect of Human Chorionic Gonadotropin and Placental Lactogen on Lymphocyte Transformation In Vitro J. H. MORSE Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA Morse, J. H. The Effect of Human Chorionic Gonadotropin and Placental Lactogen on Lymphocyte Transformation In Vitro. Scand. J. Immunol. 5, 779-787, 1976. Crude preparations of human chorionic gonadotropin (hCG) inhibited phytohemagglutinin (PHA) and allogeneic cell (MLC) lymphocyte stimulation of human peripheral blood lymphocytes (PBL) in vitro as measured by ["Hlthymidine incorporation, whereas purified hCG was not inhibitory except at very high concentrations. Sephadex G-lOO chromatography of crude hCG showed that the inhibitory efFect on PHA lymphocyte stimulation eluted in those fractions after hormonally active hCG. In addition, purified human placental lactogen (hPL) did not inhibit lymphocyte transformation except at very high concentrations. These data suggest that the immunosuppressive activities found in crude preparations are not due to the hormones but to contaminating material. ,7. H. Morse, M.D,, Columbia University, College of Physicians and SurgeonSt 630 W. 168th St., New York, NY 10032, USA
A decreased immune response during pregnancy has been postulated as important in preventing immunological rejection of the fetus. In the past the role of gestational hormones as immunosuppressive factors has mainly been confined to studies of estrogens, progesterone, and cortisone or adrenocorticotropin (17, 38). Recently, crude preparations of human chorionic gonadotropin (hCG) (2, 12, 14) and human placental lactogen (hPL) (12) have been shown to inhibit phytohemagglutinin {PHA) stimulation of human lymphocytes in vitro. These reports, implicating hCG and hPL as immunosuppressive factors, have been followed by conflicting reports regarding the capacity of hGG to inhibit stimulation of human lymphocytes by mitogens, antigens, and allogeneic cells. The inhibitory capacity of hPL has not been evaluated further. The present report describes the results of studies, in part reported previously (8, 24), suggesting
that crude hCG is more inhibitory than purified hCG on lymphocyte transformation and that the inhibitory activity found in crude preparations elutes on Sephadex G-lOO column chromatography in fractions distinct from purified hCG. Not reported previously was the failure of purified preparations of hPL to inhibit the response of peripheral blood lymphocytes to PHA and allogeneic cells,
MATERIALS AND METHODS Lymphocyte culture methods. Lymphocyte cultures were prepared by a modification of the method of Boyum (6). Forty to fifty ml of blood, obtained from normal individuals in phenol-free heparin, was allowed to sediment in a syringe at 37^C for 1—2 h. The plasma was expressed from the top of the syringe and gently mixed with an equal volume of sterile isotonic saline. Two volumes of the saline-
780
J. H. Morse
plasma solution were layered over one volume tjf Ficoll-Conray solution (10 ml 33.4% Conray plus 24 ml 9% Ficoll) and centrifuged for 30 min at 4"C (Ficoll was obtained from Pharmacia Fine Chemicals, Piscataway, N.J., and 60% Conray from Mallmckrodt Pharmaceuticals, St. Louis, Mo.). The interface containing lymphocytes was removed and washed three times with Eagle's minimal essential medium (MEM) containing 50 units/ml penicillin, 50 i^ig/ml streptomycin, 2mM L-glutamine. The cells were cultured in this medium with the addition of 10% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.), pooled human AB plasma, or autologous plasma. Unless otherwise specified, serum or plasma was heated to 56'C for 30 min before use. Triplicate cultures of 0.25 ml containing 1 or 2 X IO'* lymphocytes were placed in weils of microtiter plates (Linbro IS FB 96, Linbro Chemicals Co., New Haven, Conn.). After incubation at 37^C in 5% CO.y, 95% air for 5 days they were pulsed overnight with 1 [iCi/ well of I^H]tliymidine, 6.7 Ci/mmol (New England Nuclear Corp., Boston, Mass.). The cultures were harvested and washed extensively with saline on glass fiber filters (grade 934 AH, Reeve Angel, CHfton, N.J.) using a Mash 11 (Harvester Microbiological Associates) (15). The filters were dried and placed in 12 ml of Instabray (Yorktown Research, Hackensack, N.J.). '^H content was determined in a liquid scintillation spectrometer (Packard model 3320). The results were expressed as counts per minute (cpm) of [^H]thymidine per culture or as percentage of inhibition of thymidine incorporation of the mitogen control. Mitogens. Phytohemagglutinin (PHA-P, Difco Laboratories, Detroit, Micb.) was used at the optimal dose, usually either 0.001 or 0.005 per ml of culture. One-way mixed lymphocyte cultures (MLC) were prepared by incubating tbe stimulatmg lymphocytes at 2 X 10" cells/ml for 1 h at 37° with 25 ng/ml mitomycin C (Sigma Chemical Co., St. Louis, Mo.), followed by washing three times with large volumes of MEM. Preliminary cultures were performed with unrelated donors, and the most reactive ratios of stimulator to re-
sponder cell were seleceted for use with hCG —that is, 1.6 X 10" of mitoniycin-C-treated lymphocytes to 8 X 10* responder lymphocytes. Trypan blue dye viability studies showed about 82%—88% dye exclusion in most expernnents. hCG and hPL preparations. Both crude and purified hCG were generous gifts of Dr. Robert Canfield, Department of Medicine, Columbia University, College of Physicians and Surgeons. Crude preparations of hCG (purchased from Organon, West Orange, N.J.) were batches 91930, 92145, and 92666, which had estimated potencies of 3900 lU/mg, 2700 IU/mg, and 2680 lU/mg, respectively. The bioassay results, as well as the subsequent yields of purified hormone, indicated that approximately 75% of the bulk material in tbese crude preparations was not bCG. Purified bCGs 119 and 121 were prepared from crude hCG 91930 and 92145 by methods described previously (9, 10). Purified hCG appeared to contain less than 5% contamination with other material, and biological potency estimated by the rat ventral prostate weight bioassay (22) was 11,600 and 13,450 IU/mg. It is known that the biological potency of different preparations of purified hCG can vary significantly a.s a function of sialic acid content (39), and the range of bioassay results IS consistent with other chemical data, indicating tbat the preparations were greater than 95% hCG. hCG preparations were stenlizt-'d by passage through a 0.45-fim Millipore filter (Millipore Corp., Bedford, Mass.). Purified preparations of human placental lactogen were generously supplied by Dr. Frank Morgan, St. Vincent's School of Medical Restarch, Melbourne, Australia, and Dr. Louis Sherwood, Department of Medicine, Michael Reese Hospital, The University of Chicago, Pritzker School of Medicine. hPL 1 (Australia) was prepared by the method of Catt et al. (11) and considered to be 90% pure. A minor nonprotein contaminant was present. A bioassay of this preparation was not available. hPL 2 (Chicago) was biologically active in the radioreceptor assay of rabbit mammary gland membranes (36) and also in a radioreceptor
TmmuTWSuppressive Effects of hCG and hPL
assay using mammary tissue in organ culture. hPL 2 was prepared from placental extracts by methods described previously (35). Both hPL preparations had molecular weights of 22,000.
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RESULTS Table I shows a representative experiment comparing the effect of crude and purified hCG on PHA-stimulated human peripheral
Table I. Effect of crude and purified human chorionic gonadotropin (hCG) on phytohemagglutinin (PHA) stimulatitin of peripheral blood lymphocytes* Type of hCG preparation Crude 91930
hCG
concentration (lU/ml) 3,900 1,950 975 390 195
I'urified CR-119
11,600 5,800 2,900 1,160 580
PHA control Cell control
["Hjthymidine incorporation (cpm)
Percentage inhibition
8,654 33,253 41,588 52,602 60,692
: 1,395 ,1:2,110 zi 5,941 ^ 1,239 ±13,979
85 44 30 12 0
46,358 36,937 56,680 53,275 50,665
±4,951 -!:2,744 f: 9,654 ±3,373 ±7,718
23 38 5 11 15
59,857 ; 2,142 _
102
.:_n
_
* Each culture contained 1 X 10" lymphocytes in 0.25 ml of culture medium with 15% heated AB plasma. Results are expressed as the arithmetic mean '•• SD of counts per minute of [^H]thymidinc incorporation per culture. Cmde hCG 91930 had a bioassay of 3900 IU/mg and purified hCG 119 had 11600 IU/mg.
I'able II. Effect of crude and purified human chorionic gonadotropin (hCG) on mixed lymphocyte reactivity of peripheral blood lymphocytes* Type of hCG Preparation Crude 92145
Purified CR-121
Responder - stimulator cells Responder cells alone
['H]thymidine incorporation (cpm)
Percentage inhibition
5,850 3,900 1,950
752 ±166 985 ih71
975 390 195
1,078 _ 236 1,205 -1.-155 1,333 ±191
55 40 47 35 27 19
743 1,333 1,592 1,682 1,389
55 19 4 0 16
hCG
concentration (IU/ml)
20,175 13,450 6,725 3,362 1,345
876 ±89
M42 ±83 fc357 1 675 ±101
1,651 i 353 217 -:6
* liach culture contained 1 X 10* lymphocytes in 0.25 ml of culture medium with 15% heated AB plasma. Results are expressed as the arithmetic mean ± SD of counts per minute of [^H]thymidine incorporation per culture. Crude hCG 92145 had a bioassay of 3900 IU/mg and purified hCG 121 had 13,450 IU/mg.
782
3- ff- Morse
blood lymphocytes (PBL). The concentrations of hCG in the cultures are expressed as IU/ml. In thi.s study 3900 IU/ml of the crude material produced 85%inhibition of the PHA control, whereas the purified hormone at 11,600 IU/ ml produced only 23% inhibition. Biologic variations did occur, but the crude hCG or parent preparation from which the purified hormone was made was always more inhibitory. Experience with four pairs of matched crude and purified hCG preparations has confirmed this observation. Since PHA is known to hind glycoproteins, crude hCG could have preferentially bound PHA, thus removing lymphocyte stimulation by glycoprotein-lectin Interaction (29). Allogeneic cell stimulation was used to circumvent this problem. Table II shows another representative experiment. Again the crude preparation produced 40% inhibition at 3900 IU/ml, whereas the purified hormone only gave 19% inhibition at 13,350 IU/ml. This experiment is included to show that occasionally very high concentrations of purified hCG, in this in-
CRUDE hCG
LOT
C
BO
92666
GLUCOSE
ISO
FRACTION NUMBER
Fig. 1. Sephadex G-lOO chromatography of crude 92666 in 0.05M NH^HCOs. The fractions shown on the abscissa were combined to form pools 1 through 11 and equalized to the same optical density. The degree of inhibition of phytohemagglutinin-stimulated lymphocytes by aiiquots of these pools and the bioassays for human chorionic gonadotropin (hCG) hormonal activity are illustrated in Table III.
Table III. Comparison of human chorionic gonadotropin (hCG) hormonal activity and lymphocyte inhibition in Sephadex G-lOO fractions of crude hCG Sephadex G-lOO pool no.
I 2 3 4 5 6 7 8 9 10 U
hCG concentration % Inhibition of (IU/mg)* PHA-stimulated lymphocytesf 11,889 11,889 12,189 1,983
25 9 3
N.D. 79
N.D. 99 97 77 0 15 0 0 0
* Bioassay of hCG by rat ventral prostate assay; courtesy of Dr. R. E. Canficld. All fraction pools were adjusted to O.D. 280. The confidence limits of these bioassays vary about 15%.
t PHA = phytohemagglutinin. stance 20,175 IU/ml, significantly inhibited (55%) the MLC reaction. Experiments were then initiated to ascertain if the immunosuppressive material found in crude hCG could be separated from the purified hormone. Crude hGG was placed on Sephadex G-lOO in 0.05M NH4HCO:i. The elution profile is shown in Fig. 1. The fractions were combined into 11 pools and the protein concentration equalized to an optical density of approximately 2. Table III compares the inhibitory effect on PHA stimulation with the hCG bioassay (IU/ml) of the fractions. Note that high hCG hormonal activity. above 11,000 IU/ml, was found in peaks 1-3. These are the peaks that are pooled for further purification of the hormone. However, lymphocyte inhibition was found in peaks 2, 4, 5, and 6. Peaks 5 and 6 have little hormonal activity, less than 25 IU/ml, and most of the immunosuppressive activity, greater than 95^,Interestingly, peak 2 had a lymphocyte inhibition comparable to that of peak 6, 79% compared with 77%. whereas the specific activity of the hormone was more than one thousand times greater in peak 2. Although only two different batches of
Immunosuppressive Effects of hCG and hPL 14000
12000
10000
STiM,+
8000
783
Fig. 2. The effect of human placental lactogen (hPL) 1 and hPL 2 on peripheral blood lymphocytes stimulated by mitomycin-Ctreated allogeneic lymphocytes. Each culture contained 2 X 10" stimulator (Stim.) and 2 X 10" responder (Resp.) lymphocytes in 5 % heated fetal calf scrum.
6000
4000
2000 STIM. RESR
° I
10
50
100
5 -
1000
l^q/ml)
crude hCG have been studied under slightly different buffer conditions, these data suggest that the substance(s) that inhibit stimulated lymphocytes eluted in a broad peak just after hCG. Preliminary evidence also showed lower
STIM.+ RESP,
I 10
50
100
1000
hPL (jJfl/mll
I'ig. 3. The effect of human placental lactogen (hPL) 1 on peripheral blood lymphocytes stimulated by mitomydn-C-treated allogeneic lymphocytes. Each culture contained 2 X 10'' stimulator (Stim.) and 2 X 10* responder (Resp.) lymphocytes in 5% heated fetal calf serum.
molecular weight inhibition in some batches of crude hCG (25). Because of its glycoprotein nature, purified hCG has an anomalous sedimentation behavior (8), eluting at around 70,000 daltons on Sephadex G-lOO rather than in the region of its actual molecular weight, which is 37,000 daltons. The main inhibitory activity elutes between 20,000 and 40,000 daltons. Because human placental lactogen, also called somatomammotropin (hCS), had been reported to inhibit PHA stimulation of lymphocytes, and because the molecular weight of its active form (22,000) coincided with that of the inhibitory activity in the crude hCG preparations, purified hPL preparations were evaluated for inhibition of lymphocyte stimui.ition by PHA and in the MLC. hPL also occurs as a dimer form termed 'Big' hPL with a molecular weight of 46,000 (33). Preparation 1 of hPL was judged to be 90% pure by weight but was known to have a nonprotein contaminant, whereas hPL preparation 2 was considered pure (hPL 2 was prepared in the same laboratory by the method that provided the amino acid sequence of hPL) and both contained only monomeric hPL. Fig. 2 shows that both purified preparations of
:84
J. H- Morse
g 10000
CELL CONTROL
nPL Ug/oil)
Fig. 4. The effect of human placental lactogen (hPL) 1 on peripheral blood lymphocytes stimulated by phytohemagglutinin (PHA). PHA-P was used at a concentration of 0.005 ml per ml of culture. Each contained 2 x 10* cells in 5"i, heated fetal calf serum.
hPL failed to inhibit MLC-induced lymphocyte transformation in concentrations ranging from 1 to 100 ng/tnl. hPL 1 did give 50% inhibition at 1000 (ig/ml and about 75% inhibition at 1000 (ig/ml in another MLC shown in Fig. 3. Fig. 4 shows the effect of increasing concentrations of hPL 1 on PHA-stimulated lymphocytes. Little change m inhibition occurred between 10 and 1000 fig/tnl. In another experiment, not illustrated here, both hPL 1 and 2 in concentrations of 1 to 1000 |ig/ml failed to inhibit PHA stimulation. Note in Fig. 4 that hPL, in concentrations of 1 to 1000 ng/ml, had no effect on resting unstimulated lymphocyte cultures. These data show that the purified hormone did not inhibit lymphocytes stimulated by either PHA or allogeneic cells. The inhibition tbat did occur was found in the more impure preparation.
DTSCUSSION The data indicate that both purified bCG and hPL have little inhibitory effect on PHAand MLC-induced lymphocyte transformation, except at very high concentrations—5000 IU/ml or greater for hCG and 1000 |ig/ml for
hPL. Crude preparations of hPL were not evaliited, but crude hCC showed greater inhibition than the purified hormone. The greater inhibition of PHA stimulation of lymphocytes by crude hCG compared with the purified hormone has previously been reported from this laboratory (8, 24) and also by others (7, 13). Similar results have been reported more completely elsewhere using lymphocytes stimulated by pokeweed, purified protein derivative, and allogeneic cells (8). However, as mentioned previously, conflict exists regarding the inhibitory role of hCG in the MLC reactions. Beling & Weksler (3) and Han (14) found significant inhibition by hCG at concentrations of 40-200 IU/ml and 10 IU/ml, respectively. These concentrations approach the physiologic concentrations, which can be as high as 10 IU/ml, in plasma taken during the first trimester (8). Caldwell et al. (7), using similar reagents, recently noted no inhibitory activity in the MLC reaction with purified hCG, as did Schiff et al. (31). Comparison of the hCG preparations used in these conflicting reports suggests that the crude preparations were more inhibitory than the purified hormone, regardless of the type of lymphocyte stimulation studied. The results of the present experiments on the immunosuppressive properties of hPL do not agree with those of Contractor & Davies (12), who, using commercially obtained hPL, occasionally noted inhibition of PHA lymphocyte stimulation with concentrations as low as 12.3 ng/ml and always when 50 to 100 \i%l ml was used. Again, the most likely explanation is that the purer reagents used here lack the inhibitory material found in crude placental extracts. The high concentration of purified hPL, 1000 f.tg/ml, required to inhibit stimulated lymphocytes is well above the concentration of 3—5 ng/ml found in pregnancy plasrna at term (34). Although the nature of the inhibitory material in the crude hCG preparations is unknown, several immunosuppressive substances found in urine and plasma from pregnant women should be considered. Other gestational hormones
Immwiosuppressive Effects of hCG and hPL
such as estrogen and progesterone are elevated during pregnancy. Both progesterone (23) and diethylstilbestrol (1) have been shown to inhibit the incorporation of [^HJthymidine by PHA-stimulated human PBL, whereas other investigators failed to find inhibition of the MLR and mitogen stimulation using physiologic concentrations of estrogen and progesterone (31). Even though estrogen, progesterone, and adrenocorticotropin have all been implicated as immunosuppressive substances with various degrees of certainty, their molecular weights are too low to be in the inhibitory peak on the Sephadex G-lOO column studied here unless they are aggregated or bound to heavier substances. It is also possible that hCG could act synergistically with another hormone or substance in the crude preparation to provide immunosuppression. Several plasma proteins that are elevated in or unique to pregnancy could inhibit stimulated lymphocyte cultures. These proteins include: immunoglobulins with antilymphocyte activity (30); pregnancy ;tf]-glycoprotein, described by Bohn (5); pregnancy-associated ag-macroglobulin (37), originally described by MacLaren et al. (21), which is identical to pregnancy-associated globulin (PAG) (16); PAPP-A. another recently described pregnancy-associated ti-globulin with a MW of 750,000, supposedly different from PAG (19); as well as a-fetoprotein. There is .ilso a third «.,-globulin, called pregnancy zone protein (32), which appears to be lmmunologically distinct from the other two a^,-globulins of pregnancy just described, namely PAG and PAPP-A (18). The presence of antilymphocyte antibodies in the maternal circulation has been documented in some patients, and certain commercial hGG preparations have recently been shown CO be contaminated with immunoglobulins, not necessarily antilymphocyte antibodies, in sufficient quantity to provide anticomplementary activity great enough to convert C3, the third component of complement (20). These immunoglobulins of complexes would be eluted from Sephadex G-lOO in a region of higher molecular weight than the inhibitory action
785
sliown in Fig. 1. As yet, pregnancy-specific ^[-glycoprotein and PAPP-A have not been tested in cell-mediated immune systems, and controversy exists regarding the ability of PAG or pregnancy-associated (ta-macroglobulin to inhibit the mitogenic responses of PPD- and PHA-stimulated lymphocytes (16). Mouse afetoprotein with a known MW of 49,000 (79,000 on sodium dodecyl sulfate gels) has definitely been shown to inhibit lymphocyte transformation (26) and antibody formation (27). There are also other immunosuppressive factors, not necessarily elevated during pregnancy, such as immunoregulatory alpha-globulin (IRA) and its peptide (28); SAA, an alpha-globulin found in amyloidosis (4); and a host of other less well characterized factors found associated with various clinical states. Although interesting possibilities, these are too numerous to review here. Similarly, hPL, which is isolated from placental extracts rather than pregnancy urine, may contain one or more of these immunosuppressive factors or perhaps an additional one. In summary, the possibility exists that the inhibitory activity found in hGG is related to these pregnancy-associated factors or other factors that show an influence in different tests for cell-mediated immunity. Whether these or similar substances have been concentrated sufficiently in commercial crude hCG preparations IS unknown and presently under investigation.
AGKNOWLEDGEMENTS The author is deeply grateful to Dr. Robert Ganfield for his collaboration in earlier related studies and for generously supplying crude and purified hCG, and to Drs. Frank Morgan and Louis Sherwood for their purified preparations of hPL. This work was supported by USPHS grant HL 10049 and the Systemic Lupus Erythematosus Foundation. The excellent technical assistance of Mr. Ken Miller is gratefully acknowledged.
786 .7- //. Morse REFERENCES 1. Ablin, R.U., Bruns, G.R., Guinan, P. & Bush, I.M. The effect of estrogen on the incorporation of 'H-thymidine by PHA stimulated human peripheral blood lymphocyles. J. Immunol. US, 705,1974. 2. Adcock, H.W., HI, Teasdale, F., August, C.S., Cox, S., Meschia, G., Battaglia, F.C. & Naughton, M.A. Human chorionic gonadotropin: its possible role in maternal lymphocyte suppression. Science 181, 845, 1973. 3. Beling, C G . & Weksler, M.E. Suppression of mixed lymphocyte reactivity by human chorionic gonadotropin. Clin. exp. Immunol. /5, 537, 1974. 4. Benson, M. D., Aldo-Benson, M.A., Shirahama, T., Borel, Y. & Cohen, A.S. Suppression of in vitro antibody response by a serum factor (SAA) in experimentally induced amyloidosis. J. exp. Med. 142, 236, 1975. 5. Bohn, H. Nachweis und cbaraklerisierung von schwangerschaftsproteinen in der menschlichcn placenta, sowie ihre quantative immunologische bestimmung im serum schwangerer frauen. Arch. Gyndk. 210, 440, 1972. 6. Beyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest. 21, Suppl. 97, 77, 1968. 7. Caldwell, J.L., Stites, D.P. & Fudenberg, H.H. Human chorionic gonadotropin: effects of crude and purified preparations on lymphocyte responses to phytohemagglutinin and allogeneic stimulation. J. Immunol. 115, 1249, 1975. 8. Canfield, R.E., Birken, S., Morse, J.H. & Morgan F.J. Human chorionic gonadotropin. pp. 299-315 in Parsons, J. (ed.) Proceedings of the Symposia on Drug AcHon-Peptide Hormones. Macmillan and Co., London, 1976. 9. Canfield, R.E. & Morgan, F.J. Human chorionic gonadotropin (hCG) I. Purification and biochemical characterization, p. 727 in Berson, S.A. & Yalow, R.S. (eds.) Methods of Invesiigaiive and Diagnostic Endocrinology. North-Holland Publishing Co., Amsterdam, 1973. 10. Canfield, R.E., Morgan, F.J., Kammerman, S., Bell, J.J. & Agosto, G.M. Studies of human chorionic gonadotropin. Recent. Progr. Hormone Res. 27, 121, 1971. 11. Catt, K.J., Moffat, B., Niall, H.D. & Preston, B.N. Purification and physiochemical properties of human placental lactogen. Biochem. J. 102, 27c, 1967. 12. Contractor, S.F. & Davies, H. Effect of human chorionic somatomammotrophin and human chorionic gonadotropin on phytohaemagglutinininduced lymphocyte transformation. Nature iLond.) 243, 284, 1973. 13. Gundert, D., Merz, W.E., Hilgenfeldt, U. &
Brossmer, R. Inability of highly purified preparations of human chorionic gonadotropin to inhibit the phytohemagglutinin-induced stimulation of lymphocytes. FEBS Letters 53, 309, 1975. 14. Han, T. Inhibitory effect of human chorionic gonadotropin on lymphocyte blastogenic response to mitogen, antigen and allogeneic cells. Clin. exp. Immunol. IS, 529, 1974. 15. Harrison, M.R., Thurman, G.R. & Thomas, G.M. A simple and versatile harvesting device for processing radio-active label incorporated into and/or released from cells in microculture. y. immunol. Methods 4, 11, 1974. 16. Home, C.H.W., McLay, A.L.C., Tavadia, H.B., Carmichael, I., Mallinson, A.C, Yeung Laiwah, A.A.C., Thomas, M.A. & MacSween. R.N.M. Studies on pregnancy associated globulin. Clin. exp. Immunol.13, 603, 1973. 17. Kuwata, T. Studies on the growth of Rous sarcoma and its variant strain in cortisone-treated hamsters. Cancer Res. 20, 170, 1960. 18. Lin, T.M. & Halbert, S.P. Immunological comparison of various human pregnancy-associated plasma proteins. Int. Arch. Allergy 48, 101, 1975. 19. Lin, T.M., Halbert S.P., Kiefer. D. & Spellacy, W.N. Three pregnancy-associated human plasma proteins; purification, monospecific antisera and immunological identification. !nt. Arch. Allertfv 47, 35, 1974. 20. Loke, Y.W. & Pepys, M.B. Effects of human chorionic gonadotropin preparations on complement IH vitro. Amer. J. Obstet. Gynec. 121, 37, 1975. 21. MacLaren, J.A., Reid, D.E., Konugres, A.A. & Allen, F.H., Jr. Pal, a new inherited alpha-2globulin of human serum. Vox Sang. {Basel) II, 553, 1966. 22. McArthur, J.W. The identification of pituitary interstitial cell stimulating hormone in human urine. Endocrinology 50, 304, 1952. 23. Mori, T., Kobayashi, H., Nishimura, T, Mori, T.S., Fujii, G. & Inou, T. Inhibitory effect of progesterone on the phytohaemagglutinin-induced transformation of human lymphocytes. Immunol. Commun. 4(6), 519, 1975. 24. Morse, J.H., Stearns, G., Arden. J., Agosto, G. & Canfield, R.E. The effects of crude and purified human gonadotropin on i« vitro stimulated human lymphocyte cultures. Cell Immunol. In press. 25. Muchmore, A.V. & Blaese, R.M. Immunoregulatory effects of human chorionic gonadotropin (hCG). Fed. Proc. 33, 750, 1974. 26. Murgita, R.A. & Tomasi, T.B. Suppression of the immune response by a-fetoprotein. II. The effect of mouse a-fetoprotein on mixed lymphocyte reactivity and mitogen-induced lymphocyte transformation. J. exp. Med. 141, 440, 1975. 27. Murgita, R.A. & Tomasi, T.B. Suppression of
Immunosuppressive Effects of hCG and hPL the immune response by a-fetoprotein. I. The efFect of mouse a-fctoprotein in the primary and secondary antibody response. J. exp. Med. 141, 269, 1975. 28. Occhino, j . C , Glasgow, A.H., Cooperband, S.R., Mannick, J.A. & Schmidj K. Isolation of an immunosuppressive peptide fraction from human plasma. J. ImmunoL HO, 685, 1973. 29. Powell, A.H. Maternal lymphocytes; Suppression b\' human chorionic gonadtropin. Science 184, 913, 1974. 30. Revillard, J.P., Robert, M., Betnel, H., Lajour, M,, Bonneau, M., Brochier, J. & Traeger, J. Inhibition of the mixed lymphocyte reaction by antibodies. Transplatil. Proc. 4, 173, 1972. 31. Schiff, R.I., Mercier, D. & Buckley, R.H. Inability of gestational hormones to account for the inhibitory effects of pregnancy plasma on lymphocyte responses in vitro. Cell. Immunol. 20, 69, 1975. 32. Schlontz, B. von, Stigbrand, T. & Tarnvik, A. Inhibition of PHA induced lymphocyte stimulation by pregnancy zone protein. FEBS Lellers 38, 23, 1973. 33. Schneider, A.B., Kowalski, K. & Sherwood, L.M. 'Big' human placental lactogen: disulfideReceived 4 May 1976 Received in revised form 15 June 1976
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linked peptide chains. Biochem. biophys. Res. Commun. 64, 717, 1975. 34. Sciarra, J.J., Sherwood, L.M., Varma, A.A. & Lundberg, W.B. Human placental lactogen (HPL) and placental weight. Amer. J. Obstet. Gynec. 101, 413, 1968. 35. Sherwood, L.M., Handwerger, S., McLaurin, W.D. & Lanner, M. Amino-acid sequence of human placental lactogen. Nature {New Bid.) 233, 59, 1971. 36. Shiu, R.P.C., Kelly, P.A. & Friesen, H.G. Radioreceptor assay for prolactin and other lactogenic hormones. Science ISO, 968, 1973. 37. Stimson, W.H. & Eubank-Scott, L. The isolation and partial characterization of a new a^-macroglobulin from human pregnancy serum. FEBS Letters 23, 298, 1972. 38. Thompson, J.S., Crawford, M.K., Reilly, R.W. & Severson, C D . The effect of estrogenic hormones on immune responses in normal and irradiated mice. J. Immunol. 98, 331, 1967. 39. Van Hell, H., Goverde, B.C., Schuurs, A.H.W. M., Dejager, E., Matthijscn, R. & Homan, J.D. H. Purification, characterization and immunochemical properties of human chorionic gonadotropin. Nature {Land.) 212, 261,1966.
