CELLULARIMMUNOLOGY 118,337-344 (1989)

Early Human Decidual Ceils Exhibit NK Activity against the K562 Cell Line but Not against First Trimester Trophoblast

ASHLEY KING, CAROLINE BIRKBY, AND Y. W. LOKE

Division of Cellular and Genetic Pathology, Department of Pathology, University of Cambridge, Cambridge, United Kindgom

Received July II, 1988; accepted September 24, 1988

The susceptibility of cultured first trimester human trophoblast cells to lysis by NK cells has been studied. Trophoblast cells are resistant to lysis by NK cells from both peripheral blood and decidua. However, decidual cells extracted by enzymatic d&aggregation do exhibit cytotoxicity against the NK-sensitive cell line K562. The relevance of these findings to successful implanta- tion of the blastocyst is discussed. 0 1989 Academic Ress, Inc.

INTRODUCTION

In human pregnancy the fetally derived extravillous trophoblast needs to invade the maternal decidua and vessels for successful implantation and development of a normal blood supply to the fetoplacental unit (1). This invasion is finely monitored so that neither rejection nor undue penetration of the uterus by trophoblast ensues. The factors responsible for this control are unknown, but there is much interest in the possibility that this role may be assumed by the maternal bone marrow-derived cells in decidua which come into intimate contact with the invading trophoblast. These cells have been shown by immunohistology to be predominantly large granular lymphocytes (LGL) accompanied by macrophages and a few T cells (2, 3). Since LGLs are a subset of peripheral blood lymphocytes (PBL) which includes both NK cells and non-MHC-restricted T cells, it would be of interest to determine whether decidual LGLs are also capable of cytolytic activity against NK-sensitive cells like the K562 cell line. The additional question of whether human trophoblast is susceptible to NK lysis has also not been answered. Results from previous functional studies have been conflicting (4-7) due mainly to the lack of trophoblast cells with a sufficient degree of homogeneity for use as targets in in vitro assays. We have recently developed a culture method which consistently yields relatively pure cytotrophoblast cells (8) and this has provided us with the necessary trophoblast targets with which to test for NK cytolysis by both peripheral blood and decidual effecters.

MATERIALS AND METHODS

Trophoblast

First trimester placental tissue was obtained from vaginal termination of pregnancy and cultured as described previously (8). Trophoblast from three to four pregnancies

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338 KING, BIRKBY, AND LOKE

was mixed to prepare the cultured target cells. After 2 days the cultures were washed and the trophoblast cells were removed with trypsin. After further washing, the resul- tant cell suspension was resuspended at 2 X lo5 cells/ml in appropriate medium; 2 X lo4 cells were added to each well of a 96well flat-bottomed plate (Falcon) in a minimum volume of 160 ~1 of medium. The cells were left overnight to adhere at 37°C in 5% COZ.

K562

The K562 cell line (Flow Laboratories) was maintained in RPM1 1640 with 10% fetal calf serum (FCS) supplemented with 2 mM glutamine by twice weekly sub- culture.

Decidual Cells

Decidua was obtained from first trimester vaginal termination of pregnancy. De- cidual fragments were identified macroscopically from two to four pregnancies and pooled together. Decidua and trophoblast from a single conceptus were never used as effecters and targets in one experiment. The fragments were minced between two scalpel blades in a small volume of Hanks’ and were then treated in three different ways:

(1) Decidual fragments were placed in 75 ml of 0.25% trypsin (Difco) containing 0.02% ethylenediaminetetraacetic acid (EDTA, Sigma) and agitated with a magnetic stirrer at 37°C. After 6 min the cell suspension was filtered through two layers of muslin and spun at 400g for 5 min. The pellet was resuspended in RPM1 at 106/ml and 6 ml was layered onto 3 ml of Lymphoprep (Flow) and spun at 2000 rpm for 15 min. The band of cells at the interface was washed in RPM1 and resuspended at 2 x lO’/ml in full medium. Viability was assessed using trypan blue and was always more than 90%.

(2) Decidual fragments were incubated in 25 ml of collagenase/dispase (>O. 1 U collagenase/ml, >0.8 U dispase/ml) (Boehringer Cat No. 269638) for 40 min at 37°C with continual agitation. The suspension was filtered through gauze and then layered onto Lymphoprep as in ( 1).

(3) Decidual fragments were pushed through a 53-pm bronze sieve (Gallenkamp). After washing, the cell suspension was layered onto Lymphoprep as in (1) to remove the dead cells.

PBL

Heparinised venous blood was diluted to twice its volume in Hanks’ and layered onto Lymphoprep. After spinning at 2000 rpm for 15 min the cells at the interface were removed, washed twice, and resuspended at 2 X lO’/ml in RPM1 and 20% FCS.

Cytospin Smears

Cytospin smears of decidual cells were made using a Sandoz cytocentrifuge. The cells were suspended in RPM1 and 20% FCS at a concentration of 2 X 105/ml. The slides were air-dried, fixed in acetone at 4°C for 5 min, and stored at -20°C for immu-

HUMAN DECIDUAL NK ACTIVITY AGAINST TROPHOBLAST 339

nocytochemistry. Slides were also fixed in methanol for 10 min and stained with Giemsa to assess morphology.

Immunocytochemistry

An avidin-biotin peroxidase method was used to assess the numbers of bone mar- row-derived cells extracted in the decidual cell preparations. The following mono- clonal antibodies (McAb) were used:

(i) anti-leukocyte common antigen, l/50 (Dako)

(ii) Leu 19, l/50 (Becton-Dickinson)

(iii) Leu 5b (CD2), l/50 (Becton-Dickinson).

After air-drying and fixing in cold acetone for 10 min and rehydrating in PBS, the appropriate McAb was applied to the section for 30-60 min. After washing twice in PBS, biotin-conjugated rabbit anti-mouse IgG (Dako) diluted 1 in 100 for 30 min was applied. After further washing, avidin-horseradish peroxidase (Sera-Lab) diluted l/400 was added for 30 min. The peroxidase was developed with 3’-diaminobenzi- dine (DAB) (Sigma) with 0.03% hydrogen peroxidase. The slides were counter- stained with Carazzi’s haemotoxylin.

NK Cell Assay

Natural killer cell activity was measured using a chromium release assay. K562 cells were washed twice and labeled with 100 PCi of sodium “chromate

(51Cr) (Amersham CJS4) in a total volume of 200 ~1 at 37°C for 1 hr. After being washed three times in RPM1 the cells were left for 1 hr in RPM1 + 10% FCS. They were then washed again and resuspended at 1 X lO’/ml.

Cultured trophoblast cells which had been left overnight in 96-well plates were washed twice and 5 &i of 51Cr in 35 ~1 of RPM1 was added to each well. After leaving for 1 hr at 37°C the cells were washed three times and then left in appropriate trophoblast medium for 1 hr at 37°C. Before use they were washed again and 100 ~1 of medium was added. It was estimated that approximately half the trophoblast cells were lost during this labeling procedure leaving 1 X lo4 cells/well.

Effector cells from decidua or PBL were added at effector:target ratios of between 2OO:l and 12.5:1 in a volume of 100 ~1. After 5 hr incubation at 37°C in 5% CO:!, 100 ~1 of supematant was removed from each well and counted using a gamma counter. The percentage specific cytotoxicity was calculated using the standard for- mula:

% lysis = test cpm - spontaneous cpm max cpm - spontaneous cpm ’

Spontaneous cpm were obtained from wells containing target cells and medium only. Maximum cpm were obtained by adding 100 ~1 of 2.5% Triton to wells containing target cells only. Means were calculated from averages of three to six replicate cultures in all experiments. The spontaneous release varied between 10 and 15% for K562 and 15 and 25% for trophoblast.

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K562

Trophoblast

I Tmphoblast -

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2oo:i 1OO:l 5o:i 25:l

Effector-Target Ratio

FIG. 1. Chromium release assay using PBL against K562 and trophoblast.

RESULTS

(1) NK Activity of PBL against Cultured Trophoblast

In 10 experiments no significant lysis of cultured trophoblast cells was seen using PBL (Fig. l), although lysis of K562 cells was demonstrated.

(2) Decidual NK Activity against K562 and Trophoblast

In three experiments decidual cells extracted with both trypsin and collagenase/ dispase showed NK activity against K562 (Fig. 2), yet no lysis of trophoblast was seen.

(3) Eflect of Enzyme Treatment on NK Activity of PBL

As decidual NK activity against K562 was less than that seen with PBL (Fig. 2), the latter were treated with trypsin or collagenase/dispase in a similar manner. There was an obvious reduction in NK activity following trypsinisation of PBL (Fig. 3), but appreciable activity was still detectable very similar to that found with decidual cells. The results of treating PBL with collagenase/dispase are not shown as clumping of

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HUMAN DECIDUAL NK ACTIVITY AGAINST TROPHOBLAST 341

1 . ..““.*- pw -o- Decidua T

Decidua C/D

. 20b:l lob:1 5d:l

Effector-Target Ratio

FIG. 2. Decidual cells in chromium release assay against K562 and trophoblast. Decidua T = decidual cells extracted using trypsin. Decidua C/D = decidual cells extracted using collagenase/dispase.

cells made accurate dilution impossible. However, the results appeared essentially similar to those seen following trypsinisation of PBL.

(4) Efect of Sieved Decidual Cells on NK Activity against Trophoblast

In two experiments decidual cells were extracted by sieving and using in an NK assay against trophoblast. No lysis was observed which was, therefore, similar to our finding using enzymatically d&aggregated decidual cells.

(5) Immunohistochemistry of Cytospin Smears

Smears of decidual cells extracted with trypsin and collagenase/dispase were stained with LCA to assess the number of bone marrow-derived cells; 20-25% were LCA+ and of these cells 70-80% stained with Leu 19, a marker for large granular lymphocytes. Using immunohistochemistry on frozen sections of decidua we have also found that the majority of bone marrow-derived cells are Leu 19+ (2).

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KING, BIRKBY, AND LOKE

s-0- Untreated PBL

-49 PBL (Ttypsin)

1Oi 2OO:l 1OO:l 50:l 25:i

Effector-Target Ratio

FIG. 3. Effect of enzyme treatment of PBL on NK cytotoxicity of K562.

Smears of sieved decidual cells showed a higher proportion of LCA and Leu 19+ cells. The latter accounted for 50% of cells present. In addition, the morphology of these cells with Giemsa staining was characteristic of large granular lymphocytes. However, granules were not identified in Giemsa-stained cytospin smears extracted with enzymes.

DISCUSSION

Our experiments have shown that leukocytes isolated from early human decidua are not cytolytic to cultured trophoblast. This lack of reactivity is seen with leukocytes which have been obtained either by enzymatic disaggregation or by seiving. In con- trast, these decidual leukocytes have demonstrable cytolytic activity against an NK- sensitive cell line (K562) which suggests that the absence of activity against tropho- blast is unlikely to be due to damage incurred by the effecters during isolation. We have also shown that trophoblast is resistant to lysis by NK cells from peripheral blood so it would appear that this inertness is an inherent trophoblast characteristic. This could be a reflection of trophoblast membrane rigidity as K562 cells are reported to lose their susceptibility to NK lysis if their surface membrane is altered (9), or that trophoblast does not express NK targets (which are at present unknown). Alterna-

HUMAN DECIDUAL NK ACTIVITY AGAINST TROPHOBLAST 343

tively, trophoblast cells may have means of inhibiting NK lysis even if recognition occurs. The trophoblast cells used in our experiments are obtained from a culture technique recently developed in our laboratory which yields these cells with a high degree of homogeneity (8). In addition, these cultured cells are seen to exhibit many characteristics of extravillous trophoblast which is the relevant population interacting with decidua in vivo. In particular, almost all the cultured trophoblast cells express HLA Class I antigens detectable by McAb W6/32 like the interstitial trophoblast cells which invade decidua (8). In addition, staining for P-HCG and HPL on cultured mononuclear cells was negative. P-HCG and HPL are localised to villous syncytiotro- phoblast on tissue sections and are not found on extravillous trophoblast ( 10). Fur- thermore, although anti-trophoblast monoclonal antibodies A5 and C4 stained all the cultured trophoblast cells, the intensity of staining was much less than on sections of villous trophoblast. We have previously noted in tissue sections that the cells of the cytotrophoblast cell columns lose their characteristic staining pattern with McAb’s A5 and C4 as they begin to express HLA antigens (10). It would appear, therefore, that our culture system induces human trophoblast to develop along the extravillous pathway. We thus feel that this experimental system does mimic very closely the situation which occurs in the uterus during early pregnancy.

Studies of a similar nature so far reported in the literature are not directiy compara- ble to ours. Of the two reports utilising human material (4, 5) both used PBLs and not decidual leukocytes as effecters. As we have observed (2) the large granular lym- phocytes in decidua to which NK activity is attributed have an unusual phenotype (CD 16- CD3- Leu 19+). These account for the majority of bone marrow-derived cells in decidua while such cells comprise only a very small subset of the LGLs in peripheral blood (11). Also the trophoblast target used in these studies was full-term syncytiotrophoblast, so the relevance of these studies to early extravillous trophoblast migration in the first trimester uterus is not immediately apparent. Of the murine studies (6,7), the effecters used are generally splenic lymphocytes. Again, these cells may differ from those in decidua because it has been observed that in vivo perfusion of mice with an NK-specific asialo-GM1 antiserum resulted in total elimination of splenic NK activity, whereas decidual NK activity persisted, albeit at a reduced level (12). The study which most closely parallels ours is that of Croy and her colleagues ( 12), who showed no NK activity of murine decidual lymphocytes against blastocysts, although they were unable to purify trophoblast cells for use as targets. However, high levels of decidual NK activity against NK-sensitive YAC cells were found early in gestation.

NK cells are effector cells exhibiting non-MHC-restricted cytotoxicity without prior sensitisation, their main targets being undifferentiated or immature cells of nor- mal or neoplastic lineage. Since trophoblast cells are relatively undifferentiated and express low or aberrant Class I MHC antigens, it might be expected that they would be lysed by decidual effecters. Yet our present study detected no cytolysis of trophoblast. However, it must be remembered that proper implantation of the human placenta necessitates a certain degree of invasion including destructive infiltration of arterial walls (1). While these cells need to be controlled so that undue myometrial penetra- tion does not occur, they must not be completely eliminated. Perhaps decidual leuko- cytes exert a cytostatic influence rather than cytolysis. They may also inhibit the re- sponse of lymphocytes to immunogenic stimuli, acting as local natural suppressor cells which are known to be LGLs both phenotypically and morphologically (13).

344 KING, BIRKBY, AND LOKE

This is in accord with the work of Clark and his colleagues, who have demonstrated suppressor cell function in both human and murine decidua (14). It has even been suggested that these decidual cells may actually enhance trophoblast growth by the production of appropriate cytokines (15). We are testing these concepts with tropho- blast-decidua coculture experiments.

ACKNOWLEDGMENTS

We are grateful for financial support from the American Friends of Cambridge University, Cancer Re- search Campaign, East Anglian Regional Health Authority, Medical Research Council, and the Wellcome Trust. We thank our obstetric colleagues and staff at Addenbrooke’s Hospital for collecting the placental material.

This investigation also received financial support from the Special Programme of Research, Develop ment and Research Training in Human Reproduction, World Health Organization.

REFERENCES

1. Robertson, W. B., In “Obstetrical & Gynaecological Pathology” (H. Fox, Ed.), Chap. 44, p. 1149. Churchill Livingstone, London, 1987.

2. King, A., Wellings, V., and Loke, Y. W., Human Zmmunol., in press. 3. Bulmer, J. N., Hollings, D., and Ritson, A., J. Pathol. 153,281, 1987. 4. Paul, S., and Jailkani, B., Amer. J. Reprod. Zmmunol. 2,204, 1984. 5. Pross, H., Mitchell, H., and Werkmeister, J., Amer. J. Reprod. Zmmunof. 8, 1, 1985. 6. Zuckerman, F. A., and Head, J. R., Transplant. Proc. 19,554, 1987. 7. Tanaka, K., and Chang, K. S. S., Znt. J. Cancer 29,3 15, 1982. 8. Loke, Y. W., andBurland, K., PIacenta9, 173, 1988. 9. Roozemond, R. C., and Bonavida, B., J. Zmmunol. 134,2209, 1985.

10. Lake, Y. W., and Butterworth, B. H., In “Immunoregulation & Fetal Survival” (T. J. Gill and T. G. Wegmann, Eds.), p. 197. Oxford Univ. Press, London/New York, 1987.

11. Lanier, L. L., Le, A. M., Civin, C. I., Loken, M. R., and Phillips, J. H., J. Zmmunol. 136,4480, 1986. 12. Croy, B. A., Gambel, P., Rossant, J., and Wegmann, T. G., Cell. Zmmunol. 93,3 15, 1985. 13. Maier, T., Holda, J. H., and Claman, H. N., Zmmunol. Today 7,3 12, 1986. 14. Clark, D. A., Slapsys, R., Chaput, A., Walker, C., Brierley, J., Daya, S., and Rosenthal, K. L., Amer.

J. Reprod. Zmmunol. 10,100, 1986. 15. Wegmann, T. G., Amer. J. Reprod. Zmmunol. 15,67, 1987.