Effects of human chorionic gonadotropin preparations on complement in vitro Y.

W.

LOKE,

M.

B.

PEPYS,

M.A.,

M.D.*

M.A.,

M.B.,

M.R.C.P.**

Cambridge,

England

The effects gonadotropin plementary

on complement in vitro by several commercial (HCG) were investigated. These preparations and produced marked conversion of the third

preparations of human chorionic were demonstrably anticomcomponent of complement (Cl,).

However, detailed analyses showed that the anticomplementary activity was because of the presence immunoglobulins in the HCG preparations. In view of the known modifying effects of C’,-reactive agents on skin allograft survival and of antigen-antibody complexes on lymphocyte transformation, it may be necessary to reconsider previous work on the immunosuppressive effects of HCG preparations.

of

A L T E R E I) M A T E R N A L immunologic reactivity caused by endocrinologic changes during pregnancy may play a role in the survival of the fetal allograft. Recently, human chorionic gonadotropin (HCG) preparations have been reported to have various immunologic activities including prolongation of skin allograft survivaI,1 alteration of the maternal thymolymphatic system,’ a depressant effect on antibody production,” and a local immunosuppression of maternal lymphocytes. 4 We have investigated the effect of HCG on the complement system in vitro. Material

and

I.U. per milligram,* purified by sequential chromatography on diethylaminoethyl cellulose and Sephadex G-100. Complement. The preparations used were: ( 1) rabbit complement-fresh normal rabbit serum absorbed at 4O C, with human AB and 0 red cells and buffy coat; (2) guinea pig complement-fresh normal guinea pig serum absorbed at O” C. with sheep erythrocytes; (3) human complement-fresh normal human serum absorbed at O” C. with sheep erythrocytes. All sources of complement were used fresh or stored at -197O C. and thawed only once. Cytotoxicity tests. Human trophoblast cells were cultured as previously described.5 The cells were suspended to a concentration of 1 x lo6 cells per milliliter in Hanks solution and in Hanks solution containing HCG Preparation 1 in a concentration of 16,500 I.U. per milliliter. Cytotoxic tests were performed under oil in Microtest tissue culture plates? with 1 ~1 unit volumes delivered by a syringe.$ The serum used was a multispecific antihuman lymphocyte antigen (HL-A) serums which

methods

HCG preparations. (1) 3,300 milligramt;

The preparations used were: I.U. per milligram+; (2) 2,000 I.U. per (3) 5,255 I.U. per ampules; (4) 13,000

From the Department Cambridge.

of Pathology,

Received

for

publication

Accepted *Supported

May

8, 1974. by the Cancer

Feb.

**Supported by the Medical Present address: Department Postgraduate Medical School, Hospital, London, England. tPaines $Koch-Light $Second Medical Division,

B

Byrne,

Greenford,

Laboratories,

University

of

7, 1974. Research

Campaign.

Research Council. of Medicine, Royal Hammersmith

“Serona Roma, Rome, Italy. tFalcon Plastics, Div. of Bioquest, 1950 Oxnard, California 93030. *Hamilton, Whittier, California. $Burke, National Institutes of Health.

England. Colnbrook,

England.

International Standard, provided by the Research Council, Biological Standards London, England. 37

Williams

Dr.,

38

Loke

and

Pepys

Am

Table I. Inhibitory

effect of HCG

on cytotoxic

action

of Burke

serum __ ---

Dilution

it-1

Trophoblast cells (T27) suspended in Hanks solution plus Burke serum plus complement

Z/2

++

1

++

Z/4

1

(++)

Z/8

(++)

January 1, 147: J. Obstct. Gywxd

) l/16

(+)

of Burke

serum

/ Z/32

1 Z/64

w

w

jl/i2B1/216

w

1

w

,“““;

w

Trophoblast cells (T27) suspended in HCG plus Burke ++ serum plus complement W (W) w w w w W (+) w ++ = 100 per cent kill; (++) = 75 per cent kill; (+) = 30 per cent kill; w = 15 per cent kill; (W) = 5 per cent kill.

Table II. Reduction with

dilution

Dilution of HCG

of inhibitory

Conversion of the third component of complement (C’,). Fresh normal human serum was incu-

effect

of HCG of Burke

Dilution Undiluted

1

l/2

(

serum l/4

1

l/8

++ iUndiluted W ++ +t + l/2 (; St ++ + l/4 (++) ++ ++ ++ l/8 ++ ++ = 100 per cent kill; (++) = 75 per cent kill; + = 50 per cent kill; (+) = 30 per cent kill; w = 15 per cent kill. was found to give a strong cytotoxic reaction with human trophoblast cells.” One microliter volumes of cells and serum were incubated at 37’ C. for one hour after the addition of the same volume of rabbit complement. Each test was then stained with 0.4 per cent trypan blue and read with an inverted phase-contrast microscope. Immunofluorescent tests. Trophoblast cells were incubated with anti-HL-A serum for 30 minutes at room temperature. Some of the cells were suspended in Hanks solution while others were suspended in Hanks solution containing HCG Preparation 1 in a concentration of 16,500 I.U. per milliliter. After 2 washes, the cells were incubated with a fluorescent-labeled anti-human globulin* diluted y5 for 30 minutes at room temperature. The cells were washed twice, mounted on slides prepared with silicone, and examined under ultraviolet light. Complement lysis. Sheep erythrocytes sensitized with 10 minimum hemolytic doses of the immunoglobulin M (IgM) fraction of rabbit anti-sheep red cell antiserum were incubated for 30 minutes at 37’ C. with either guinea pig (l/50) or human (l/20) complement and different dilutions of the HCG preparations. After incubation, the tubes were centrifuged, and the degree of lysis was estimated visually. *Nordic,

Tilburg,

Holland.

bated with HCG, phosphate-buffered saline (PBS) or the C’,-cleaving protein of cobra venom factor (COF)~, 8 for 2 hours at 37O C. The extent of C’s conversion was then estimated by antigen-antibody with a monospecific sheep crossed electrophoresiss anti-human C’, serum.8 The effect of ethylenediaminetetra-acetic acid (EDTA) upon this C’, conversion was also tested. Gel diffusion. The antigenic purity of HCG preparations was tested by double diffusion and immunoelectrophoresis against sheep anti-whole human serum, sheep anti-human light chain serum and goat anti-HCG serum.* All tests were in 1.25 per cent agarose gel in pH 8.6 Verona1 buffer, 0.06M. Absorption of gamma globulin. Polyvalent rabbit anti-human globulin serum was insolubilized with ethyl chloroformate. lo HCG Preparation 1 was absorbed overnight at 4’ C. with this material. Results HCG in a high concentration inhibited the cytotoxic action of a multispecific anti-HL-A serum toward human trophoblast cells in vitro. (Tables I and II). Antibody was still demonstrable on the cells by immunofluorescence in the presence of HCG which indicated that the mode of action was not because of interference with fixation of antibody. However, HCG preparations were demonstrably anticomplementary and inhibited the lysis of sensitized sheep red cells by guinea pig or human complement (Tables III and IV). They also produced marked conversion of C’s on incubation with normal human serum (Fig. 1) . This effect was inhibited by EDTA, suggesting that it was the result of complement activation rather than, for example, merely *Nordic.

Volume Number

121 1

Effects of HCG preparations

on complement

39

Fig. 1. C’, conversion by HCG preparations. 2 to 6: HCG (Preparation 1)) PBS, or CoF was incubated for 2 hours at 37” C. with fresh normal human serum (NHS) ; the cIs profile was then analyzed by crossed immunoelectrophoresis. I, HCG (Preparation 1) producing marked C’s conversion; 2, PBS control with slight conversion; 3, CoF producing complete conversion; 4-6, same as 2 to 3 but with 0.01M EDTA present throughout, C’, conversion inhibited. 7 to IO: HCG, PBS, or CoF was incubated for 10 minutes at 37” C. with NHS. An excess of NHS containing O.OlM EDTA was then added, and, after a further 2 hours at 37” C., the C’, profile was analyzed. 7, HCG; 8 and 10, PBS showing no CYn conversion; 9, CoF producing marked C’, conversion.

Table III. Inhibition HCG preparation 1 2 3

(per cent)

by HCG

of lysis by guinea

preparations

Inhibition I.U.

8,000 HCG/ml. 100 100 100

I.U.

4,000 HCG/ml.

I.U.

2,000 HCG/ml.

50 50 50

proteolytic enzyme activity. Brief incubation of HCG with serum did not generate an EDTA-stable C’:, convertase, in contrast to observations with CoF.ll Analyses of the antigenic composition of the present HCG preparations showed the presence of serum proteins on immunoelectrophoresis (Fig. 2) which were confirmed as immunoglobulins by double diffusion against an anti-light chain serum. All three crude preparations of HCG were found to contain immunoglobulins. When the immunoglobulin was absorbed from the HCG preparations with an insoluble polyvalent rabbit anti-human immunoglobulin serum, there was a marked reduction in the inhibition of lysis (Table V). Furthermore, a high-

25 25 25

of lysis

I.U.

pig complement

(%) 1,000 HCG/ml. 0 0 0

-

I.U.

500 HCG/ml.

I.U.

0 0 0

ly purified HCG preparation mentary activity (Table V) .

0 HCG/ml. 0 0 0

had

no anticomple-

Comment The present studies indicate that the observed anticomplementary effects of HCG preparations are because of the presence of immunoglobulin, presumably in an aggregated or complexed and, therefore, complement-fixing form. It has recently been demonstrated that the C’,-cleaving protein (CoF) of cobra venom suppresses thymus-dependent antibody production in vivo and prolongs skin allograft survival? Other C’,-reactive agents, including heataggregated human IgG, have similar effect?“; therefore, it may be necessary to reconsider previ-

40

Loke

and

January 1, 1975 Am. J. Obstet. Gynecol.

Pepys

Fig. 2. Immunoelectrophoresis of HCG Preparation 1. HCG and NHS were electrophoresed in parallel and then developed with anti-HCG serum and anti-NHS serum. There was precipitation between anti-HCG and NHS and between HCG and anti-NHS. Lines in the gamma globulin region are indicated with arrows.

Table IV. HCG inhibition

of lysis by human complement Inhibition 16,000 Z.U. HCG/ml.

HCG

Preparation

1

/

H;;%&

100

i

Hz;Tml.

50

preparation

1 (Unabsorbed) 1 (Absorbed with gamma globulin) 4

17,000 Z.U. HCG/ml.

100

8300 Z.U. HCG/ml.

100

100 0

50 0

0 0

10%

H~x?l.

of lysis

1 H;LYJkl.0

0

activity (%)

I

2,125 Z.U. HCG/ml.

I

1,062.5 Z.U. HCG/ml.

0 Z.U. HCG/ml.

50

0

0

0

0 0

0 0

0 0

0 0

tion in view of the demonstrated impurity of commercial HCG and the known modifying effect of antigen-antibody complexes on lymphocyte transformation.15

7.

Pearse, W. H., and Kaiman, H.: AM. J. OBSTET. GYNECOL. 98: 572, 1967. Nelson, J. H., Hall, J. E., Manuel-Limson, G., Freidberg, H., and O’Brien, F. J.: AM. J. OBSTET. GYNECOL. 98: 895, 1967. Younger, J. B., St. Pierre, R. L., and Zmijewski, C. M.: AM. J. OBSTET. GYNECOL. 105: 9, 1969. Kaye, M. D., and Jones, W. R.: AM. J. OBSTET. GYNECOL.

1

0

4,250 Z.U. HCG/ml.

100

REFERENCES

2.

H~;%?l.

anti-

ous published works on the immunosuppressive effects of HCG preparations in view of these and the present findings. A recent report of inhibition of the in vitro lymphocyte response to phytohemagglutinin by HCG14 must also be accepted with cau-

1.

1

(%,I

of HCG on its inhibitory Inhibition

HCG

Iysis

25

Table V. The effects of absorption and purification

34,000 Z.U. HCG/ml.

of

1029,1971.

Loke, Y. W., and 1970. Loke, Y. W., Joysey, 232: 403, 1971.

Borland, V.

C.,

R.: and

Nature Borland,

228: R.:

561,

8. 9. 10. 11. 12. 13. 14.

Nature 15.

Ballow, M., and Cochrane, C. G.: J. Immunol. 103: 944, 1969. Pepys, M. B.: Ph.D. Thesis, University of Cambridge, 1973. Laurell, C.-B.: Anal. Biochem. 15: 45, 1965. Avrameas, S., and Ternynck, T.: J. Biol. Chem. 242: 1651, 1967. Miiller-Eberhard, H. J., and Fjellstriim, K.-E.: J. Immunol. 107: 1666, 1971. Pepys, M. B.: Nature New Biol. 237: 157, 1972. Pepys, M. B.: Submitted for publication. Contractor, S. F., and Davies, H.: Nature New Biol. 243: 284, 1973. Moller, G.: Clin. Exp. Immunol. 4: 65, 1969.

Effects of human chorionic gonadotropin preparations on complement in vitro.

Effects of human chorionic gonadotropin preparations on complement in vitro Y. W. LOKE, M. B. PEPYS, M.A., M.D.* M.A., M.B., M.R.C.P.** Cam...
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