Human trophoblast interferons: Production, purification, and biochemical characterization

Trophoblast Research 8:315-329, 1994

HUMAN TROPHOBLAST INTERFERONS: PRODUCTION, PURIFICATION, AND BIOCHEMICAL CHARACTERIZATION George Aboagye-Mathiesen 1, Ferenc D. T6th 1,2, Peter M. Petersen~, Niels Norskov-Lauritsen ~, Milan Zdravkovic ~, and Peter Ebbesen 1~ 1The Danish Cancer Society Department of Virus and Cancer Gustav Wieds Vej 10 DK-8000 Aarhus C, Denmark 2Institute of Microbiology Medical University H-4012 Debrecen, Hungary

INTRODUCTION The trophoblast layer of the h u m a n placenta performs m a n y important functions during pregnancy. It mediates vectorial transport of nutrients and oxygen between maternal and fetal circulations, secretes steroid and protein hormones (Loke and Whyte (1983), is believed to play a role in immunoglobulin transport from mother to fetus (Wood et al., 1982), prevents the rejection of the fetus by the mother (Lala et al., 1983), and also act as a barrier to virus traffic from mother to fetus. In the very early stages of pregnancy the cytotrophoblast cells are highly proliferative and invades into the endometrium and forms an essential element of the embryo implantation, a property that resembles the invasion of malignant tumors in m a n y respects. Most of the physiological and biochemical functions of the human placenta are carried out by the syncytiotrophoblast which is composed of large multinuclear cells which are formed by the fusion of mononuclear cytotrophoblast, a process which takes place continuously throughout the gestational periods of pregnancy. The regulation of the physiological and biochemical functions of the trophoblast layer is, however, not well understood. IFNs are a family of proteins (or glycoproteins) produced by vertebrate cells after exposure either to viral infections or various synthetic and biological inducers. In humans, four functionally related but antigenically distinct IFNs (~, ~, y, and c0) have been identified and are recognized by the Committee on the Nomenclature of Interferons. They bind to receptors on the cell surface and stimulate an antiviral state, inhibit cell growth, stimulate natural killer cell activity and cytotoxicity activities of lymphocytes and macrophages, and induce cell differentiation in normal cells as well as some neoplastic cells (Pestka et al. ,1987). IFNs have been detected immunohistochemically in human placental interface (Bulmer et al., 1990) and in trophoblast in placental sections (Howatson et al., 1988; :3To Whom Correspondence Should Be Addressed.

315

316

Aboagye-Mathiesen et al.

Paulesu et al., 1991) but these placental proteins has not been fully characterized. Chard et al. (1986) have reported significant levels of IFN-0c in human amniotic fluid, placental tissue, fetal membranes, and maternal decidua from all three trimesters of pregnancy, although IFN-cz was not detected in maternal blood during pregnancy. IFNs produced in the maternal-fetal interface may play an important role in protecting the fetus from virus infections and regulate the growth and differentiation of trophoblast cells, as demonstrated to stimulate or inhibit the proliferation of malignant human trophoblast in vitro (Berkowitz et a1.,1988; Athanassakis et al., 1987). In other species such as cattle and sheep, trophoblast cells have been shown to produce unique IFNs [bovine trophoblast protein-1 (bTP-1) and ovine trophoblast protein- ! (oTP-1)] in the early stage of pregnancy which apart from their antiviral and antiproliferative properties, similar to the already known IFNs, also initiate maternal recognition of pregnancy (Roberts et al., 1989; Imakawa et al., 1989; Flint et al., 1988). Despite reports indicating the presence of IFNs and their possible production by the feto-placental unit, there has been few attempts to produce these proteins from the individual placental cells in vitro. We recently reported (Aboagye-Mathiesen et al., 1990; T6th et al., 1990) in vitro production of IFNs from pure cultures of human term trophoblasts after challenge with poly(I:C) and Sendai virus. Here we report the production of IFNs in first and third trimester trophoblast and syncytiotrophoblast cultures infected with viruses. Furthermore, the purification and biochemical characterization are also reported. MATERIALS AND METHODS Viruses NDV (Strain B1) and Sendai virus (Parainfluenza 1), the most widely used virus inducers of IFN, were obtained from American Type Culture Collection (ATCC) and were propagated in allantoic cavities of 10-day-old embryonated eggs b y inoculation of a 10-2 or 10-3 dilution of infected allantoic fluids. After the eggs were incubated at 37~ for 3 days, the allantoic fluids were harvested and the infectivity was titrated in chicken erythrocytes. Antibodies Polyclonal goat anti-human IFN-~ and monoclonal mouse anti-human IFN-[3 used for antiviral neutralization assays and the preparation of HPLC sorbents were obtained from Janssen Biochimica, Belgium, and Boehringer Mannheim GmbH, respectively. The polyclonal anti-human IFN-cc, produced in goat using human IFN-(x (consisting of more than 20 different IFN-c~ subtypes) from Sendai virus-stimulated white blood cells, neutralize all IFN-cc subtypes and all recombinant human IFN-(zs but do not react with human IFN-[3 and -T. The monoclonal human IFN-13 does not react with human IFN-~ and -T. Polyclonal anti-human IFN-T (Serotec, Oxford, UK) used for the neutralization test do not neutralize human IFN-c~ and IFN-13.

Isolation of First and Third Trimester Trophoblasts Third trimester trophoblast cells were isolated from placentae from spontaneous deliveries by immunomagnetic microspheres as described by Douglas and King (1989) and was analyzed with FACStar Plus Flow Cytometer (Becton Dickinson

Trophoblast Interferons

317

Immunocytometry Systems, Mountain View, CA) using specific fluorescin-conjugated monoclonal antibodies showed the isolated third trimester cells to contain 99.9% cytokeratin positive cells. These cells did not react with antibodies to porcine vimentin and human CD14. First trimester trophoblast cells were isolated from placental tissues (5 to 12 weeks) of legal termination of pregnancy first by the method of Fisher (1989) (yielding 80% trophoblast) and secondly by selective detachment of trophoblast cells with EDTA on ice from the mixture after culturing for 24 hours. Briefly, after enzymatic and percoll gradient isolation as described by Fisher (1989), 1 x 106 cells/ml were seeded for 24 hours in RPMI 1640 containing 10% FCS. First trimester trophoblast cells were detached from the mixture by adding 0.02% EDTA to the cells after aspiration of the medium. The flask containing the cells were kept on ice for 10-12 minutes. Detached trophoblast cells were washed twice with RPMI 1640 medium and was seeded 1 x 106 cells/ml in RPMI 1640 medium containing 10% FCS. Flow Cytometry analysis of the purified first trimester cells using fluorescin-conjugated monoclonal antibodies revealed >94% cytokeratin positive and <5.7% vimentin positive cells. These ceils did not react with antibodies to human CD14. Induction of IFNs First and third trimester mononuclear trophoblast were seeded in RPMI 1640 medium (1 x 106 cells/mi) supplemented with 10% FCS at 37~ and 5% CO 2 in air. After 24 hours of culture the medium was removed and the cells were infected with NDV (30 HAU/106 cells) or Sendai virus (200 HAU/106 cells) for 1 hour in serum-free RPMI 1640 medium. The unadsorbed virus was removed by washing the cells three times with serum-free medium. The virus-stimulated cells were incubated in RPMI 1640 medium containing 5% FCS for 18 hours and the supernatants which contained the produced IFNs were harvested and acidified to pH 2.0 for 48 hours at 4~ to inactivate virus and then neutralized to p H 7.2. Syncytiotrophoblast cultures were established by culturing first and third trimester mononuclear trophoblast cells in RPMI 1640 medium containing 10% FCS or keratinocyte growth medium (KGM) (Clonetics Corporation, San Diego, CA) for 3 to 4 days until they flattened out and aggregated to form multinucleated cells. They were characterized by secretion of human chorionic gonadotropin (hCG), a biochemical marker of differentiation (Douglas and King, 1990) and then were infected to produce IFNs under similar conditions with NDV and Sendai virus. Control trophoblast ceils and syncytiotrophoblast cultures were established similarly except with the omission of virus stimulation. Purification of Tro-IFNs Tro-IFNs were purified by tandem high-performance affinity chromatography (HPAC) on Cibacron blue F3GA, anti-IFN-13 and anti-IFN-R columns connected in tandem (HEMA-BIO 1000 VS-F3GA, HEMA 1000 VS-anti-IFN-~, and HEMA 1000 VSanti-IFN-a, respectively). The HPLC sorbents were prepared as described previously (Aboagye-Mathiesen et a1.,1991) and were packed in PEEK columns (50 m m x 7.5 m m internal diameter). Concentrated crude IFNs were injected onto column A (HEMA-BIO 1000 VS F3GA), equilibrated with 20 mM sodium phosphate buffer p H 7.2 containing 200 mM NaCI, at a flow rate of 1 ml/minute. The column was washed with the same buffer until the absorbance at 280 nm returned to baseline. The IFNs adsorbed to column A were eluted with 20 mM sodium phosphate buffer, pH 7.2 containing 1.0 M NaCI, and

318

Aboagye-Mathiesen et al.

50% ethylene glycol at a flow rate of 0.5 ml/minute onto columns B (HEMA 1000 VSanti-IFN-[3) and C (HEMA 1000 VS-anti-IFN-a). Columns B and C were further washed with 20 mM sodium phosphate buffer pH 7.2 containing 0.5 M NaCl. IFNs bound to columns B and C were eluted separately by a step elution with 0.1 M glycineHCI, p H 2.4 or by pH gradient (from 7.2 to 2.4) at a flow rate of 0.5 ml/minute. The elution from the columns were monitored at 280 nm (0.16 absorbance units full scale) and fractions of 250 ~l were collected. All fractions were neutralized to pH 7.2 and were assayed for IFN antiviral activity. Fifty ~l of the fractions containing the highest IFN antiviral activity were analyzed on SDS-PAGE.

Biochemical Characterization IFN Antiviral and Neutralization Assays IFN antiviral activity was assayed by inhibition of vesicular stomatitis virus (VSV), Indiana strain, plaque formation in human amniotic cell line WISH (ATCC). Cells were seeded in 96-well microplates (40,000 cells/well) and were cultured for 24 hours at 37~ and 5% CO2 in air. IFN preparations were then titrated by adding 100 ml of two- or four-fold serial dilutions to each well. The IFN-treated cells were incubated for 24 hours and were infected with VSV (50 plaque-forming units per well). The titrations were scored microscopically 24 hours after virus inoculation. The highest dilution giving 50% reduction of the viral plaques was considered as the end point. The IFN titers were standardized by comparison with the National Institute of Health Standard for human IFN-c~ (G-023-902-530) and -~ (G-023-902-527). IFN titers, using human trisomic 21 fibroblast cell line GM 2767 (Cell Repository, Camden, NJ, USA) and bovine (MDBK) cells were performed similarly using vesicular stomatitis virus as the challenge; results were expressed as the highest dilution giving 50% protection. IFN antiviral neutralization assays were performed by incubating IFN samples, diluted to a concentration of 100 IU, with 10-fold excess human anti-IFN-(~, -~, or -~' antibodies. After 1 hour incubation at 37~ residual IFN antiviral activity was determined as described above. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Proteins were resolved by SDS-PAGE (12.5%) by the method of Laemmli (1970) and were rendered visible by silver staining using Bio-Rad silver staining kit.

Chemical Stability Purified tro-IFNs preparations dialyzed against 0.02 M sodium phosphate buffer pH 7.2 were tested for their stability towards sodium dodecyl sulphate (SDS), ~mercaptoethanol and urea. Samples were constituted to 1% SDS or 1% SDS, 1% bmercaptoethanol, and 5 M urea or 1% ~-mercaptoethanol and 5 M urea. Incubation times and temperature were 1 hour at 37~ or 1 minute at 100~ respectively. After treatment, samples were diluted in assay medium (Eagle's minimal essential medium containing 5% FCS) before assaying for residual antiviral activity.

Trophoblast Interferons

319

Effect of Tro-IFNs on PGE2 Secretion Trophoblast cells, seeded at a density of 1 x 106/ml, were incubated with 1000 IU/ml of purified tro-IFN-0~ or -13 in keratinocyte growth medium supplemented with 5% FCS at 37~ 5% CO2. Control cells were incubated under the same conditions without addition of IFN. Culture medium from IFN-treated and untreated cultures were harvested at 12 hour intervals. The concentrations of PGE2 in the culture medium was determined using PGE2 enzyme immunoassay kit (Cayman Chemical Company, MI, USA) according to the manufacture's instructions. Glycoprotein Analysis To determine the presence of glycosylation in the purified tro-IFN components we tested for their binding to concanavalin A affinity column (HEMA 1000 VS-Con A). After injection of the IFN samples, the column was washed with column buffer (20 mM sodium phosphate buffer pH 7.2, containing 1 M NaCl and MnC12, CaC12, and MgCl2. Bound IFNs were then eluted with 0.1 M ot-methyl-D-mannopyranoside and 50% ethylene glycol in the column buffer.

Protein Determination Protein concentrations were determined by the dye-binding assay of Bradford (1976) or by absorbance at 280 run using known concentrations of bovine serum albumin as a standard.

Statistical Analysis The IFN antiviral activity of each IFN preparation was evaluated with six replication assays. The results are expressed as the mean + s.d and the coefficient of variation for N = number of IFN preparations and n = number of replication of assays. The results of the PGE2 were expressed as the mean + s.d. RESULTS Table 1 shows the differential IFN yields and compositions from first- and third trimester trophoblast and syncytiotrophoblast cultures stimulated with Sendai virus and NDV. The data demonstrate that the first trimester trophoblast produced higher levels (about 5 to 6-fold) of IFNs than the third trimester trophoblast. The syncytiotrophoblast cultures established by culturing third trimester mononuclear cytotrophoblast for 3 to 4 days, however, produced upto 2-fold (in the case of NDV) more IFN than the mononuclear trophoblast at term. The syncytiotrophoblast cultures from the first trimester showed only a small increase in IFN production as compared to the mononuclear first trimester trophoblast when infected with the two viruses. Furthermore, the magnitude of the IFN production was depended on the inducer. NDV induced higher levels of IFN than Sendai virus in both first- and third trimester trophoblast and syncytiotrophoblast cultures. No IFN activity was detected in all the control trophoblast cultures which were not stimulated with virus but cultured under the same conditions. Table 1 further shows the compositions of the different IFN preparations determined by antiviral neutralization assay using specific human IFN antisera. The

320

Aboagye-Mathiesen et al.

data demonstrate that Sendai virus and NDV produced mixtures of IFN-0t and -13. Sendai virus induced 75% IFN-[3 and 25% IFN-0t whereas NDV induced 65% IFN-I3 and 35% IFN-0t. Although the compositions of the produced IFNs varied with the two inducers the levels of IFN-ct and -[3 were the same for each inducer in the first and third trimester trophoblast and syncytiotrophoblast cultures. Tandem High-Performance Affinity Purification of IFNs Figure I shows the purification of the crude tro-IFN preparations by HPAC on columns A (cibacron blue), B (anti-IFN-13), and C (anti-IFN-ct) connected in tandem. All the IFNs bound to column A when applied at low ionic strength (200 mM NaCl). Greater than 95% of the serum proteins were unretained on column A and were washed out. Complete elution of IFNs were obtained from column A with 20 mM sodium phosphate buffer, pH 7.2 containing 1.0 M NaCl, and 50% ethylene glycol. The eluted IFNs specifically adsorbed on columns B and C depending on their antigenicity. IFN antiviral activity assay of the fractions eluted from columns B and C showed two peaks of IFN activity (Figure 1A and B). The summary of the purification is shown in Table 2. The application of pH gradient from 7.2 to 2.4 resulted in the separation of the tro-IFN-0c components into two peaks. Figure 2 shows a gradient elution (from p H 7.2 to 2.4) profile for the separation of tro-IFN-cc components from columns C. Characterization of tro-IFNs The types of IFN eluted from columns B and C were identified by IFN antiviral neutralization test using specific human IFN antibodies (Table 3). The antiviral activity of the IFNs eluted from columns B and C (Figure 1A and B) were completely neutralized by anti-IFN-~ and anti-IFN-ct, respectively. This showed that the troIFNs eluted from columns B and C were exclusively of the 13and 0c types, respectively. Furthermore, the two peak fractions eluted from column C by the pH gradient were neutralized by polyclonal anti-IFN-a. Biochemical characterization of the tandem HPAC-purified IFNs by SDSPAGE is shown in Figure 3. The molecular masses of the tro-IFN-a subtypes were 16 and 22 kDa whiles that of tro-IFN-13 was 24 kDa. The antiviral activity of the purified tro-IFN preparations were stable at pH 2 for 2 weeks. The tro-IFN-13 and a fraction of tro-IFN-cz (peak I in Figure 2) bound to Con A affinity column (Figure not shown) whiles a fraction of tro-IFN-0t (peak II) did not bind. These suggested that a fraction of tro-IFN-ct and tro-IFN-13 are glycoproteins. The properties of the tro-IFNs are shown in Table 4. The antiviral activities of the tro-IFN components were tested on different human and bovine cell species. It was observed that the tro-IFN components exhibited a broad spectrum of antiviral activities on the human cell species (WISH and GM 2767) tested. However, the troIFN-0t subtypes were more active with respect to human WISH cells in protecting bovine MDBK cells. In contrast, tro-IFN-[3 was inactive in protecting bovine MDBK cells but active on all the human cell species tested.

Trophoblast Interferons

321

Table 1

IFN Yields and Compositions of Sendai- and NDV-Stimulated First and Third Trimester Trophoblast and Syncytiotrophoblast Cultures

First trimester trophoblast Third trimester trophoblast First trimester trophoblast Third trimester trophoblast

Inducer

IFN yield (IU/106 cells)

Sendai virus

42440 + 4220 (9.9%) 7400 _+880 (11.9%)

25

75

25

75

81200 + 6340 (7.8%) 16000+ 1480 (9.3%)

35

65

35

65

Sendai virus

NDV NDV

Composition* IFN-(z (%) IFN-~ (%)

Syncytiotrophoblast (first trimester)

Sendai virus

64240 + 4820 (7.5%)

25

75

Syncytiotrophoblast (third trimester)

Sendai virus

12800_+1240 (9.7%)

25

75

Syncytiotrophoblast NDV 86680 + 5560 35 65 (first trimester) (6.4%) Syncytiotrophoblast NDV 32860 + 2400 35 65 (third trimester) (7.3%) Data are the mean + s. d. and the coefficient of variation of IFN antiviral activities in parentheses; number of IFN preparations (N) is 9 and replication of assay (n) is 6 (P < 0.05). *Determined by IFN antiviral neutralization test Table 2 Purification of Sendai- and NDV-induced Tro-IFNs by Tandem HPAC Total Activity (IU)

Specific Activity (IU/m~)

Purification (-fold)

Recovery (%)

Crude to IFN (Sendai) Fractions 40 - 51 (Column B, Figure 1A) Fractions 69-78 (Column C, Figure 1)

4.25 x 106 2.99 x 106

9.05 x 103 1.15 x 108

1 12707.1

9.80 x 105

7.01 x 107

7745.8

23

Crude-tro-IFN (NDV) Fractions 40-52 (Column B, Figure 1B) Fractions 70-79 (Column C, Figure 1B)

5.50 x 106 3.18 x 106

9.98 x 103 2.70 x 108

1 27054.1

100 57.8

1.60 x 106

9.10 x 107

9118.2

100 70.3

29

322

A b o a g y e - M a t h i e s e n et al.

A. 20

50~EG

pH24(,

l

6-

3

O2S

2 z

Fmct~

2( ;=C~J.SG%EG

pH2& 6

05

< 025

25 30 35 ~0 ~S ~ 55 60 ~

~ ~

Fret )on number

Figure 1. Tandem HPAC of crude (A) Sendai-induced tro-IFN and (B) NDV-induced tro-IFN preparations. Individual IFN preparations were applied to column A and was displaced onto columns B and C with 50% ethylene glycol. IFN activity ( I ) was eluted from columns B and C separately with glycine-HCl, pH 2.4 ( ~ ). Table 3 Antiviral Neutralization* of Tandem HPAC-Purified Sendai- and NDV-Induced tro-IFNs IFN fractions 20 - 27 (Figure 1A) 40 - 51 (Figure 1A) 69 - 78 (Figure 1A)

Residual antiviral activity (%) after incubation with anti-IFN-oc anti-IFN-~ anti-IFN- 7 100 100 100 100 0 100 0 100 100

20 - 30 (Figure 1B) 100 100 100 40 - 52 (Figure 1B) 100 0 100 70 - 79 ( F i b r e 1B) 0 100 100 *Neutralization tests were performed with a 10-fold excess of respective antibodies

Trophoblast Interferons

323

Table 4 Properties of Purified tro-IFNs

Protection of cells lines from different species against VSV infection Species

Cell line

Human Human Bovine

W IS H GM2767 MDBK

A n t i v i r a l activity* tro-IFN-(x (Peak I) tro-IFN-c~ (Peak II) 256 256 784 768 1024 512

tro-IFN-~ 256 1024 <2

Chemical stability Treatment None 1% SDS 1% SDS + 1% b-ME + 5 M urea 1% b-ME + 5 M urea

Incubation 37~ 1 h

None 100~ 1% SDS 1% SDS + 1% b-ME + 5 M urea 1% b-ME + 5 M urea

1 min

tro-IFN-ix 2560 (100%) 2816 (110%) 640 (25%)

tro-IFN-~ 4000 (100%) 640 (16%) 2560 (64%)

24 (0.93%)

64 (1.6%)

22 (0.86%) 2400 (93.7%) 256 (10%)

32 (0.8%) 640 (16%) 2560 (64%)

16 (0.63%)

768 (19.2%)

*Antiviral activity is expressed as the highest dilution giving 50% protection against VSV. ~-ME = ~-mercaptoethanol

Table 5 Inhibition of PGE 2 Secretion by tro-IFNs p~ PGE2/106 cells/ml Incubation time (h) 12 24 36 48 60 72

No treatment 3.6 + 0.44 4.7 + 0.12 10.8 + 0.56 12.4 + 0.88 14.0 + 0.64 14.9 + 0.77

tro-IFN-(x 3.2 + 0.42 3.6 + 0.14 4.6 + 0.48 5.3 -+ 0.92 6.3 -+ 0.52 6.7 _+0.41

tro-IFN-~ 3.1 + 0.36 3.5 + 0.16 4.4 + 0.52 5.1 + 0.90 5.9 + 0.46 6.2 + 0.56

Data represent the mean + s. d. (N = 3). Cells were treated with 1000 I U / m l of tro-IFN0~ o r -~.

324

Aboagye-Mathiesen et al.

Peak~

8.0

0.2 pH

172 ~'~'\~,~, Peok 4.0

|'~

6.0C o x

I ,w,

! 1.2o

o <

0.,~

0.1

z

2.0

8

12

20

16

Froction number

to

Figure 2. Separation of tro-IFN-(~ components on column C (HEMA 1000 VS-anti-IFNcz) by p H gradient elution (..... ). Two peaks of IFN activity (11) was eluted at p H 3.6 and 2.4.

kOa g4~-

t

2

3

4

kOo -94 ~!i -67

~ 3~

-43

'!b -30 .91,-O/

20.1I

.,~Ol

14,4- i l l

~

41b -14.4

Figure 3. Silver-stained SDS-polyacrylamide gel of tandem HPAC-purifed tro-IFNs. Lanes 1 and 4 are standard protein markers; lanes 2 and 3 are tro-IFN-~ (24 kDa) and tro-IFN-o~ subtypes (16 and 22 kDa), respectively.

Trophoblast Interferons

325

The antivirai activities of the tro-IFNs were stabilized to different degrees by SDS under reducing and non-reducing conditions at 37~ and 100~ As shown in Table 4, the antiviral activity of tro-IFN-(x was stable in 1% SDS at 37~ and 100~ but less stable under reducing (1% SDS, 1% ~-mercaptoethanol, and 5 M urea) conditions whereas tro-IFN-~ was more stable under reducing conditions at 37~ and 100~

Inhibition of PGEz Secretion by Tro-IFNs In the absence of tro-IFNs, PGE 2 secretion by trophoblast cultures increased from 3.6 + 0.44 pg/106 cells/ml at 12 hour incubation to 14.9 +_0.77 pg/106 cells/ml at 72 hours (Table 5). As also shown in Table 5, 1000 I U / m l of tro-IFN-a and -13 reduced the secretion of PGE2 in trophoblast cultures by 48.9% and 58.8%, respectively as compared to the secretion by untreated cultures after 72 hours of incubation. DISCUSSION The data presented demonstrate that h u m a n trophoblast cells produce different levels of IFNs at different stages of pregnancy. The highest IFN production is in the first trimester as compared to term cytotrophoblast cells. However, the syncytiotrophoblast at term produced higher level of IFNs than the mononuclear trophoblast when challenged with the two viruses. The high levels of IFN production in the first trimester trophoblast as compared to term trophoblast and syncytiotrophoblast cells, and the diverse biological effects of IFNs together suggest a role of tro-IFNs in a complex series of events in the early pregnancy. Most of the physiological and biochemical functions of the placenta are carried out by the syncytiotrophoblast which is formed continuously during the whole period of pregnancy by fusion of cytotrophoblast cells. The syncytiotrophoblast is the first fetally-derived cell layer which an invading agent such as virus has to transverse and its ability to produce higher levels of IFNs may represent a barrier to viral infection. In cytopathic inhibition assay with human amniotic WISH cells challenged with VSV, the purified tro-IFN-(~ and -~ had a specific activity between 0.7 - 2.7 x 108 I U / m g of protein. The importance of the antiviral effect is further supported by the fact that, in some cases, maternal infection may spread to the placenta but fail to progress to the fetus (Yamauchi et al., 1974; Klein et al.,1976; Remington and Desmonts, 1976). The syncytia formation (differentiation) which was accompanied by enhanced IFN responsiveness support the notion of IFNs as important for placenta biology. The higher IFN response from first versus third trimester trophoblast cells furthermore suggest a central role of tro-IFNs in the antiviral defense at a time when the fetal immune system is not yet developed. It is also noteworthy that the period of high IFN responsiveness is also the period of invasive behavior of the trophoblasts. We are presently testing the hypothesis that tro-IFNs in an autocrine way, is involved in regulating trophoblast invasiveness. Evidence has accumulated suggesting an immunomodulatory function for IFNs (Minato et al., 1980; Metz, 1975). At least one manner in which tro-IFN might be immunoregulatory is by an effect on prostaglandin metabolism. During pregnancy the survival of the semiallogenic fetus in a potentially harmful maternal immunologic environment has been suggested (Wood et al., 1987) to be due to the local

326

Aboagye-Mathiesen et al.

immunosuppression generated in the tissues around the fetus. Human trophoblast has been demonstrated to produce arachidonic acid metabolites (Rose et al., 1987) with direct immunosuppressive effects (Parhar et al., 1989). In particular, PGE2 has been implicated in the immunosuppression at the feto-placental interface (Lala et al., 1989). The tro-IFNs has a proven capacity to modulate PGE2 production in human trophoblast cultures. For example, tro-IFN-ct and -~ caused inhibition of PGE2 production by cultured trophoblast cells. This suggests that the tro-IFNs are potent local regulatory factors and that their actions on prostaglandin secretion may be one of their fundamental properties. More direct evidence derives from the observation that infection in pregnancy leads to an increased generation of prostaglandins, by intrauterine tissues and can cause preterm labor, apparently exerting the same functions as with normal for term labor (Mitchell et al., 1990). This and the high levels of IFN production by trophoblast stimulated with viruses suggest the notion that the tro-IFNs may regulate prostaglandin production during infection in the feto-placental interface during pregnancy. SUMMARY Stimulation of h u m a n first and third trimester trophoblast and syncytiotrophoblast cultures with viruses (Sendai and Newcastle Disease virus) led to the production of a mixture of IFN-ct subtypes and -~. The magnitude and compositions of the IFNs produced were dependent on the gestational age of the trophoblast, the type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produce 5- to 6-fold more IFNs than the third trimester trophoblast on per cell basis whereas syncytiotrophoblast produced 2-fold more IFNs than the mononuclear trophoblast at term when challenged with viruses. The viruses induced different levels and compositions of IFN-0t and -[3 in both first and third trimester trophoblast and syncytiotrophoblast cultures. Tandem high-performance affinity chromatography (HPAC) of the virusinduced trophoblast interferon (tro-IFN) preparations resulted in the isolation of IFNr subtypes with molecular masses of 16 and 22 kDa and J3 with molecular mass 24 kDa on SDS-PAGE. The antiviral activity of the tro-IFNs were stable at pH 2.0 and a fraction of the tro-IFN-tx and -~ were demonstrated to be glycoproteins. The tro-IFNs showed different antiviral activities when assayed on human and heterologous bovine cell species. Tro-IFN-tx subtypes protected both human and bovine (MDBK) cells from virus infection whereas tro-IFN-j3 showed a high degree of species specificity being only active in protecting the human cell types tested. The tro-IFN-tx and -~ inhibited the secretion of prostaglandin E2 (PGE2) in trophoblast cultures in vitro. REFERENCES Aboagye-Mathiesen, G., T6th, F.D., Juhl, C., Norskov-Lauritsen, N., Petersen, P.M., and Ebbesen, P. (1990) Purification and initial characterization of human placental trophoblast interferon induced by polyriboinosinic-polyribocytidylic acid. J. Gen. Virol. 71, 3061-3066. Aboagye-Mathiesen, G., T6th, F.D., Juhl, C., Norskov-Lauritsen, N., Petersen, P.M., Zachar, V., and Ebbesen, P. (1991) Characterization of human placental trophoblast interferons. J. Gen. Virol. 72, 1871-1876.

Trophoblast Interferons

327

Athanassakis, I., Bleackley, R.C., Paetkav, V., Guilbert L., Barr, P.J. and Wegmann, T. G. (1987) The immunostimulating effect of a T cell and T cell lymphokines on murine fetally derived placental cells. J. Immunol. 138, 37-44. Berkowitz, R.S., Hill, J.A., Kurtz, C.C., and Anderson, D. (1988) Effects of activated leukocytes (lymphokines and monokines) on the growth of malignant trophoblast cells in vitro. Am. J. Obstet. Gynecol. 158, 199-203. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Bulmer, J.N., Morrison, L., Johnson, P.M., and Meager, A. (1990) Immunohistochemical localization of interferons in human placental tissues in normal, ectopic and molar pregnancy. Am. J. Reprod. Immunol. 22, 109-116. Chard, T., Craig, P.H., Menabawey, M., and Lee, C. (1986) Alpha interferons in human pregnancy. Br. J. Obstet. Gynaecol. 93, 1145-1149. Douglas, G.C. and King, B.F. (1989) Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J. Immunol. Methods 119, 259-268. Douglas, G.C. and King, B.F. (1990) Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei. J. Cell Sci. 96, 131-141. Duc-Goiran, P., Robert-Galliot, B., Lopez, J., and Chany, C. (1985) Unusually apparently constitutive interferons and antagonists in human placental blood. Proc. Natl. Acad. Sci. (USA)82, 5010-5014. Fisher, S.J., Cui, T.-Y., Zhang, L., Hartman, L., Grahl, K., Guo-Yang, Z., Tarpey, J., and Damsky, C.H. (1989) Adhesive and degradative properties of human placental cytotrophoblast cells in vitro. J. Cell Biol. 109, 891-902. Flint, A.P.F., Lamming, G.E., and Stewart, H.J. (1988) A role of interferons in the maternal recognition of pregnancy. Mol. Cell. Endocr. 58, 109-111. Howatson, A.G., Farquharson, M., Maeger, A., McNicol, A.M., and Foulis, A.K. (1988) Localization of s-interferon in the human feto-placental unit. J. Endocr. 119, 531-534. Imakawa, K., Hansen, T.R., Malathy, P.-V, Anthony, R.V., Polites, H.G., Marotti, K. R., and Roberts, R.M. (1989) Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-I; a comparison with ovine trophoblastic protein-1 and bovine interferon-czH. Mol. Endocr. 3,127- 139. Klein, J.O., Remington, J.S., and Marcy, S.M. (1976) An introduction to infection of the and newborn infants. In: Infectious Disease of the Fetus and Newborn, (eds.) J.S. Remington and J.O. Klein, W.B. Saunders:Philadelphia, pp. 1-32.

328

Aboagye-Mathiesen et al.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685. Lala, P.K., Chatterjee-Hasrouni, S., Kearns, M., Montgomery, B., and Colavincenzo, V. (1983) Immunobiology of the feto-maternal interface. Immunol. Rev. 75, 87-116. Lala, P.K. (1989) Similarities between immunoregulation in pregnancy and in malignancy: The role of prostaglandin E2. Am. J. Reprod. Immunol. 20, 147-152. Lebon, P., Girard, S., Thepot, F., and Chany, C. (1982) The presence of interferon in human amniotic fluid. J. Gen. Virol. 59, 393-396. Loke, Y.W. and Whyte, A. (eds.) (1983) Biology of Trophoblast. Elsevier, New York. Metz, D.H. (1975) The mechanism of action of interferons. Cell 6, 429-439. Minato, N., Reid, L., Cantor, H., Lengyel, P., and Bloom, B.R. (1980) Mode of regulation of natural killer cell activity by interferon. J. Exp. Med. 152, 124-137. Mitchell, M.D., Edwin S., and Remero, R.J. (1990) Prostaglandin biosynthesis by human decidual cells: Effects of inflammatory mediators. Prost. Leuko. Essent. Fatty Acids 41, 35-38. Parhar, R.S., Yagel, S., and Lala, P.K. (1989) PGE2-mediated immunosuppression by first trimester human decidual cells blocks activation of maternal leukocytes in the decidua with potential anti-trophoblast activity. Cell. Immunol. 120, 6174. Paulesu, L., Romagnoli, R., Bellizzi, E., Cintorino, M., Ricci, M.G., and La Rosa, R. (1991) Immunohistochemical localization of interferon-7 and interferon-7 receptors in human placental tissues. Placenta 12, 427 (Abstract). Pestka, S. Langer, J.A., Zoon, K.C., and Samuel, C.E. (1987) Interferons and their actions. Annu. Rev. Biochem. 56, 727-777. Remington, J.S. and Desmonts, G. (1976) Toxoplasmosis. In: Infectious Disease of the Fetus and Newborn, (eds.) J.S. Remington and J.O. Klein, W.B. Saunders:Philadelphia, pp. 191-332. Roberts, R.M., Imakawa, K., Niwano, Y., Kazemi, M., Malathy, P.-V., Hansen, T. H., Glass, A.A., and Kronenberg, L.H. (1989) Interferon production by the preimplantation sheep embryo. J. Interferon Res. 9, 175-187. Rose, M.P., Myatt, L., and Elder, M.G. (1987) Arachidonic acid metabolites in the human placenta. Tropho. Res. 2, 71-83. T6th, F.D., Norskov-Lauritsen N., Juhl C.B, Aboagye-Mathiesen, G., and Ebbesen, P. (1990) Interferon production by cultured human trophoblasts and choriocarcinoma cell lines induced by Sendai virus. J. Gen.Virol. 71, 3067-3069.

Trophoblast Interferons

329

Wood, G.W., Bjerrum, K., and Johnson, B. (1982) Detection of IgG bound within human trophoblast. J. Immunol. 129, 1478-1484. Wood, G.W., Ossama, W., and Hunt, J.S. (1987) Regulation of maternal anti-paternal immune response in vitro by uterine macrophages. Tropho. Res. 2,161-171. Yamauchi, T., Wilson, C., and St. Geme Jr., J.W. (1974) Transmission of live, attenuated mumps virus to the human placenta. N. Engl. J. Med. 290, 710-712.