Immunological studies of lactoferrin in human placentae

Journal of Reproductive Immunology, 23 (1993) 21-39

21

Elsevier Scientific Publishers Ireland Ltd.

JRI 00796

Immunological studies of lactoferrin in human placentae C h r i s t i a n J. T h a l e r a, C a r l o s A. L a b a r r e r e a, J o a n S. H u n t b, J o h n A. M c l n t y r e a a n d W. P a g e F a u l k a aCenterfor Reproduction and Transplantation Immunology, Methodist Hospital of lndiana, 1701 N. Senate Boulevard, Indianapolis, IN and bDepartment of Pathology and Oncology, The University of Kansas Medical Center, Kansas City, KS (U.S.A.) (Accepted for publication 13 July 1992)

Summary Lactoferrin (LF) and transferrin (Trf) are glycoproteins with strong affinities for ferric ions. Human syncytiotrophoblastic membranes analyzed by enzyme linked immunosorbent assay (ELISA) and immunoblotting were negative with monoclonal and polyclonal antibodies to LF. Immunohistological studies of 35 normal placentae showed that LF was absent from the trophoblast basement membranes, stroma and fetal stem vessel endothelium, but positive cells were occasionally noted in intervillous spaces and fetal stem vessels. In contrast, many LF-positive cells were identified within areas of immunopathology identified by the presence of T cells, HLA-DR-positive macrophages and platelets. Double-antibody experiments showed that the LF-positive cells in these areas reacted with CD 15 and CD 16 monoclonal antibodies (mAbs), indicating that the cells were polymorphonuclear neutrophils (PMN). PMN from peripheral blood analyzed by flow cytometry and immunocytology also showed reactivities with anti-LF, CD15 and CD16 and we consistently found that circulating PMN reacted better than placental PMN with antibodies to MHC class I antigens and gp 100, (CD67), which is a neutrophil activation marker. PMN adherent within placentae had no detectable MHC class I or CD67 antigens. These findings suggest PMN adherent to placental tissues downregulate or alter plasma membrane markers. LF appears to play a role in placental inflammation, for LF-positive cells were significantly enriched in areas of immunopathology. Correspondence to: W. Page Faulk, M.D., Methodist Center for Reproduction and Transplantation Immunology, 1701 N. Senate Boulevard, Indianapolis, Indiana 46202, U.S.A. 0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Key words: lactoferrin; transferrins; placenta; neutro_phils; immunohistology

Introduction

Lactoferrin (LF) and transferrin (Trf) are glycoproteins that show a high degree of primary and secondary structural homology and a strong affinity for ferric ions (Aisen and Listowsky, 1980; Mazurier and Spik, 1980; Metz-Boutigue et al., 1984; Legrand et al., 1988). In human placentae, Trf is present on the apical aspect of villous syncytiotrophoblast (King, 1976; Faulk and Johnson, 1978; Johnson and Faulk, 1978). This represents maternal Trf that is bound to Trf receptors (Galbraith et al., 1980). Trf binding to trophoblast has been implicated in materno-fetal iron transport, in trophoblast survival (Faulk and Galbraith, 1979) and in energy metabolism (Faulk et al., 1990; Berczi et al., 1991). In contrast, LF is present in mucosal epithelia and exocrine glands (Masson et al., 1966; Brock, 1980; Wichmann et al., 1989) and within secondary granules of PMN granulocytes (Esaguy et al., 1989) that enhance microbiocidal mechanisms (Ambruso and Johnston, 1981; Aruoma and Halliwell, 1987). We have investigated the distribution of LF in human term placentae. To do this we generated 3 monoclonal antibodies to LF and used them together with commercially available reagents. The results of this investigation form the basis of this report. Material and Methods

Lactoferrin Crystalline human LF (ICN Biochemicals, Cleveland, OH) was 99% pure according to the manufacturer's high pressure liquid chromatography analysis. When we analyzed these preparations by sodium-dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and staining of gels with comassie blue, we observed a single band at Mr of approximately 60 kDa corresponding to unreduced LF.

Antibodies Monoclonal antibodies to LF were generated in the Monoclonal Antibody Laboratory at the University of Kansas Medical Center, a facility supported by BRSG SO7 05373, Division of Research Resources, National Institute of Health. Balb/c mice were immunized by subcutaneous injection of 25/~g of LF in complete Freund's adjuvant (Gibco, Grand Island, NY). The mice were boosted 14 days later by subcutaneous injection of 15 /zg of the immunogen in incomplete Freund's adjuvant. A final boost of 25/~g of antigen in 0.01 M phosphate buffered 0.15 M saline (pH 7.2) (PBS) was given in-

23

traperitoneally 2 weeks later. Spleen cells were harvested and fused with the non-secretor Balb/c mouse myeloma cell line P3x63Ag8.653 by using standard methods (Zola, 1988). After 2 weeks in culture, the hybrid cells were screened for the presence of specific antibody by using an ELISA with LF coated plates. Antibody-producing hybrid cells were expanded, then were cloned by limiting dilution and retested by using the ELISA Technique. Specificity of rabbit anti-LF (Dakopatts, Santa Barbara, CA) was assessed by immunoelectrophoresis and immunoblotting (Thaler et al., 1990). Details of all antibodies used are summarized in Table 1. Enzyme linked immunosorbent assay (ELISA) Wells of Immulon II microtiter plates (Dynatech Lab Inc., Alexandria, VA) were coated as modified from Thaler et al. (1989a) by incubating with 50/zl of 0.1 M NaHCO3 buffer (pH 9.6), diluted LF (5/zg/ml), Trf (5/~g/ml), fibrinogen (20/~g/ml) and albumin (0.2 mg/ml) at 4°C overnight. All proteins were purchased from Sigma Chemical Company (St. Louis, MO). Wells were washed twice with PBS containing 0.05% Tween 20 (PBS-Tween) and blocked for 1 h at room temperature (RT) with PBS containing 20 mg/ml bovine serum albumin (BSA) (Sigma). After two washes, 50 /zl of primary antibodies, diluted as indicated in PBS-Tween were added for 30 min at room temperature (RT). Control incubations were done with hybridoma culture medium. Wells were washed three times and incubated for 30 min at RT with peroxidase conjugated rabbit antimouse Ig (1:2000). Wells were washed five times and developed by adding 100 /~1 of substrate buffer containing 0.42 mM tetramethylbenzidine (Miles Co., Nappersville, IL) and 0.0045% H202 in 0.1 M sodium acetate buffer (pH 6.0). The substrate reaction was stopped by adding 35/zl of 2 M H2SO 4 and optical densities (OD) of each well were measured at 450 nm by using the automated Biomek 1000 workstation (Beckman Instruments, Inc., Arlington Heights, IL). Mean OD values were calculated from triplicate determinations. The mean intraassay coefficient of variance for anti-lactoferrin antibodies (calculated from a total of 36 individual determinations) was 9.7%. To avoid the influence of interassay variability, only data within individual experiments were compared. The ELISA also was used to study LF in syncytiotrophoblastic membrane (STM) preparations. These were prepared according to Smith et al. (1974) and stored at -20°C. STM were thawed and resuspended in PBS by using a 5-s continuous sonication burst of the Sonicator W-375 (Heat Systems Ultrasonics, Inc., Plainview, NY) at number 2.5. STM suspensions were adjusted to protein concentrations of 50/~g/ml and coated to wells of microtiter plates by incubating 50/zl at 4°C overnight. LF used as positive control was diluted in PBS and coated at 5/~g/ml. Rabbit antisera and mAbs were diluted as indicated in PBS-Tween and peroxidase conjugated anti-species Ig antibodies were used at 1:2000. Plates were washed and developed as described.

Human transferrin Vimentin X-hapten carbohydrate (CDI5) 32-43 kDa, subunits of the CD8 glycoprotein 55 kDa, CD4 glycoprotein 19-28 kDa T cell receptor complex CD3 50-70 kDa glycoprotein IgGFc receptor Ili (CDI6) CDI9 glycoprotein

CD56 glycoprotein

Dako, anti-transferrin Dako, anti-vimentin Leu M1 Leu 2a

Leu 19

X-hepaten carbohydrate (CDI5) GIOF5 100 kDa glycoprotein (gpl00, CD67) TII 55 kDa glycoprotein E rosette receptor (CD2) EBM 11 Unidentified macrophage specific antigen Gpllb/IIIa Platelets, IIb/IIla complex Anti-/~2-microglobulin 12 kDa ~2-microglobulin light chain

MAB1223

Leu 12

Leu lla

Leu 3a Leu 4

Human Human Human Human

1A9b12 1A9dl IA9h6 Dako, anti-lactoferrin

lactoferrin lactoferrin lactoferrin lactoferrin

Specificity

Antibody

Definition of antibodies used in this study.

TABLE 1

Immunoblotting, ELISA Immunoblotting, ELISA lmmunoblotting, ELISA Immunoelectrophoresis Immunoblotting Immunoelect rophoresis Immunochemical Immunochemical Immunocytology FACS analysis Immunoprecipitation Functional-, inhibition-, and competition studies FACS analysis, Immunohistology FACS analysis, Immunoprecipitation FACS analysis

Dr. Joan S. Hunt Dr. Joan S. Hunt Dr. Joan S. Hunt Dakopatts Santa Barbara, CA Dakopatts Santa Barbara, CA Dakopatts, Santa Barbara, CA Becton-Dickinson, Mountain View, CA Becton-Dickinson, Mountain View, CA Becton-Dickinson, Mountain View, CA Becton-Dickinson, Mountain View, CA Becton-Dickinson, Mountain View, CA

Immunoprecipitation Immunohistology, Immunocytology Immunohistology, Immunocytology ELISA lmmunodiffusion, Immunoblotting

Chemicon, El Segundo, CA Dr. J.S. Thompson Dakopatts, Santa Barbara, CA Dakopatts, Santa Barbara, CA Chemicon, El Segundo, CA Hybritech, San Diego, CA

Mouse, IgM Mouse, lgG l Mouse, lgG I Mouse, lgG l Mouse, IgG 1

Becton-Dickinson, Mountain View, CA

Becton-Dickinson, Mountain View, CA

Specificity test

Supplier

Mouse, IgM

Mouse, lgG 1

Mouse, igGj

Mouse, lgG l

Mouse, IgG I Mouse, IgG I

Rabbit Mouse IgG l Mouse, IgM Mouse, IgG l

Mouse, IgG l Mouse, IgG 1 Mouse, IgG 1 Rabbit

Species Ig-class

Goat F(ab) 2 antimouse IgG, FITC conjugated Goat F(ab)2 antimouse IgG, RITC conjugated Goat F(ab) 2 antirabbit IgG FITC conjugated Goat F(ab)2 antirabbit IgG, RITC conjugated Swine anti-rabbit Ig peroxidase conjugated Rabbit anti-mouse Ig, peroxidase conjugated Goat anti-rabbit Ig, alkaline phosphatase conjugated

Fibrin

L-243

Q1/28

Dako, Anti microglobulin W6/32

TRA-2-10

Immunoprecipitation FACS analysis, Immunohistology Western Blot

Affinity purified

Affinity purified

Affinity purified

Immunochemical

Immunochemicai

Immunochemical

Seralab, Westbury, NY

Dr. T. Fuller Becton Dickinson Mountain View, CA American Diagnostics New York, NY Protos ImmunoResearch, San Francisco, CA Protos ImmunoResearch, San Francisco, CA Protos ImmunoResearch, San Francisco, CA Protos ImmunoResearch, San Francisco, CA Dakopatts, Santa Barbara, CA

Dakopatts, Santa Barbara, CA

Sigma, St. Louis, MO

Mouse, IgG2a

Mouse IgG Mouse, IgG2a

Goat

Goat

Goat

Goat

Swine

Rabbit

Goat

Mouse IgG (H & L chains)

Mouse IgG (H & L chains)

Rabbit IgG

Rabbit IgG

Rabbit Ig

Mouse Ig

Rabbit Ig

43 kDa class I heavy chain associated with ~2-microglobulin 43 kDa class I heavy chain HLA-DR (monomorphic) Human fibrin Affinity purified

Crossed Immunoelectrophoresis Immunoprecipitation

Dakopatts, Santa Barbara, CA

Rabbit

Mouse, IgG 1

Immunoblotting

Dr. Peter Andrews

Mouse, IgG l

Membrane cofactor protein, CD46 Human/~2-microglobulin

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SDS-PAGE and immunoblotting SDS-PAGE was done in 7.5% homogeneous separating gels and 4% stacking gels with 0.1% SDS according to Laemmli (1970). Samples were solubilized by boiling for 3 min in 2% SDS containing 125 mM Tris-HC1 (pH 6.8) and 20% (v/v) glycerol. For specificity controls of mAbs to LF, 3 ml of sample buffer containing 0.5 mg of LF were loaded on a 14 × 16 cm slab gel and electrophoresed. The MW-SDS-Blue Kit (Sigma) of prestained molecular weight markers was used to calibrate for relative molecular weights according to the technical bulletin provided by the supplier. Proteins were electrophoretically transferred to 0.2 /zm-pore-size nitrocellulose membranes (Bio-Rad, Rockville Center, NY) in 25 mM ethanolamine, 25 mM glycine and 20% methanol by using a transfer apparatus from Hoefer Scientific Instruments (San Francisco, CA) overnight at 0.15 A (Szewczyk and Kozloff, 1985). Membranes were blocked for 1 h with 20 mg/ml BSA in PBS, washed and incubated for 2 h with mAbs to LF by using an Immunetics Miniblotter 25 (Immunetics, Cambridge, MA). 200/zl of mAbs diluted 1:5 in PBS-Tween were used per lane. The membranes were washed for 1 h in PBS-Tween with several changes. Peroxidase conjugated rabbit antimouse Ig (1:500 in PBSTween) was incubated for 1 h. All incubations were done at RT. Membranes were washed extensively in PBS-Tween and developed in substrate buffer containing 60 mg of 4-chloro-1-naphthol (Sigma) in 20 ml methanol and 60 /zl of 30% H202 in 100 ml PBS. The presence of LF in solubilized STM preparations was studied by performing SDS-PAGE in 7.5% homogeneous minigels (7 x 9 cm) with 4% stacking gels. Solubilized STM proteins (20/~g) and 40 ng of LF were loaded per well. After electrotransfer, nitrocellulose membranes were cut, placed into 100 x 15 mm petri dishes (Becton Dickinson, Lincoln Park, NY) and blocked for 1 h with 20 mg/ml BSA in PBS. Membranes were incubated for 90 min with anti-LF mAbs, with mAb TRA 2-10 or mAb anti-vimentin (all diluted at 1:10 in PBS-Tween). Membranes were washed extensively in PBSTween and incubated for 1 h with alkaline phosphatase conjugated goat antimouse IgG and IgM (1:1000). Membranes were washed and color reactions visualized by using the Alkaline Phosphatase Conjugate Substrate Kit (BioRad) according to the supplier's recommendations.

Immunohistology and immunocytology Placentae from 35 normal term pregnancies were collected by vaginal delivery at Methodist Hospital, Indianapolis, placed in an ice bath and transported to the laboratory within 1 h after delivery. Ten tissue blocks (1 cm x 1 cm) of chorionic villi were dissected from the maternal side of the central cotyledon, including the basal plate. Three rolls of amnion/chorion membranes from each placenta were prepared according to Hsi et al. (1982).

27

All tissue blocks were washed in PBS (pH 7.2) and snap-frozen in liquid nitrogen. Tissues were kept at -20°C until 4.0-/~m sections were prepared with the use of a Tissue-Tek cryostat (Elkhart, Indiana). Tissue sections were removed from the cryostat blade by flash condensation onto glass microscope slides and air dried with an electric fan overnight at room temperature. No chemical fixation was used. For immunocytological analyses of peripheral blood leukocytes, blood from three healthy individuals was collected by venipuncture into Becton Dickinson vacutainer tubes containing heparin. Peripheral blood smears were prepared on glass microscope slides and air dried overnight at room temperature. Each tissue section was prewashed for 15 min in PBS and incubated for 15 min in a moisture box with 50 #1 of antibodies or fluorochrome conjugated proteins adequately diluted in PBS. After this incubation each section was rinsed in PBS and taken through three 10-min washes in PBS. After the third wash, sections were incubated for 15 min in a moisture box with 50/~1 of affinity purified F(ab')2 fragments of second antibodies conjugated with either fluorescein (FITC) or rhodamine (RITC). Double antibody experiments were done according to Labarrere et al. (1991) by expanding the above procedure between washing of the first antibody and addition of the antispecies fluorochrome conjugate. This was accomplished with the use of antibodies from different species and appropriately matched FITC and RITC secondary antibody conjugates. After the final wash each section was mounted with a coverslip containing PBS-buffered glycerol (60% v/v) adjusted to pH 8.0 with solid TRIS (Sigma). Each experiment was accompanied by PBS, isotype-matched irrelevant primary antibodies (Leu 12) and conjugate controls. Peripheral blood smears were studied with the same procedure. All antibodies were ultracentrifuged 100 000 × g at 4°C for 1 h before use. All sections and smears were studied by epi-illumination and interference optics according to Faulk and Hijmans (1972). A Leitz microscope fitted with an HBO-100 mercury-arc lamp was used. The Ploem epiilluminator in this microscope contained a 1-2 type FITC filter complex that consisted of a 450-490-nm excitation filter, a 510-nm dichroic mirror and a 515-nm barrier filter which allowed wavelengths greater than 515 nm to pass. The epi-illuminator also contained an N2.1 type RITC filter complex that consisted of a 515-560-nm excitation filter, a 580-nm dichroic mirror and a 580-nm barrier filter that allowed 580-nm wavelengths to pass. The microscope was connected to a Leitz camera which contained ASA 200 daylight 35 nun Ektachrome film.

Flow cytometric analysis of peripheral blood PMN PMN were prepared from acid citrate dextrose blood as previously described (Thaler et al., 1989b). Briefly, buffy coats were layered on Ficoll

28

Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged at 700 × g for 20 min. Pellets containing red blood cells and PMN were pooled to volumes of 4 ml and subjected to hypotonic lysis by diluting with distilled H20 to a total volume of 45 ml. After 20 s, isotonicity was restored by adding 5 mi of 0.1 M sodium phosphate buffer (pH 7.4) containing 1.5 M NaCI. Samples were centrifuged at 700 × g for 10 min and hypotonic lysis was repeated. P M N were washed twice in RPMI-1640 containing 20 mg/ml BSA (Sigma) and 0.2 mM colchicine (Sigma). Flow cytometric studies were done by using a modification of Thaler et al. (1991). Cells were adjusted at 1 × 10V/ml and aliquots of 100/A were incubated for 30 min with either 20 ~1 of undiluted mAbs, or 5 ~1 of undiluted rabbit anti-LF. Twenty microliters of balb/c control ascites fluid (Cedar Lane Laboratories, Ltd. Hornby, Canada) or 5/zl of normal rabbit serum were used as controls. Cells were washed twice and incubated 30 min with 5 1,1 of FITC conjugated goat F(ab')2 fragments to either mouse or rabbit IgG. Conjugate controls were done by substituting the first antibody with PBS. The second antibody incubation was omitted for directly conjugated primary antibodies. After two washes, cells were analyzed in a Beckon Dickinson FACStar plus flow cytometer with a 3-W argon laser set at 200 mW. 10 000 events were collected and logarithmically acquired FL-1 intensities were compared by changes in mean channel fluorescence on a linear scale. Results

Immunochemical studies with antibodies to lactoferrin mAbs 1A9b12, 1A9dl and 1A9h6 were incubated with human LF that was coated to ELISA plates at 5 t~g/ml, mAbs reacted dose dependently and were reactive to dilutions of 1:3125. No reactivities of anti-LF mAbs (used at 1:100) were found with Yrf, fibrinogen or albumin (Fig. 1). When tested by immunoblotting with LF, mAb produced bands of reactivities with Mr of approximately 60 kDa (Data not shown). This Mr corresponds to human LF analyzed under non-reducing conditions (Thaler et al., 1990). Medium control incubations did not produce bands. These studies indicated, that mAbs 1A9b12, 1A9dl and 1A9h6 specifically react with LF. The expression of LF on STM was investigated by using the ELISA and antibodies to LF. Rabbit anti-LF was strongly positive with human LF up to a dilution of 1:50 000 (Fig. 2B). When tested with STM preparations rabbit anti-LF was negative even when used at 1:500. Rabbit anti-Trf was strongly positive with STM (Fig. 2A). Similarly, mAbs to LF reacted strongly with human LF that was coated onto ELISA plates (Fig. 2B) but were negative with STM (Fig. 2A). mAb TRA-2-10 specific for membrane cofactor protein/CD46 that is expressed by STM (Vanderpuye et al., 1991) was

29 net OD 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 T

0

T

T__

T ~

Trf

LF 1A9d1

1

~

AIb ~

1A9b12

~

T

T Fbg

1A9h6

Fig. 1. Specificity control experiment with mAbs to lactoferrin done by ELISA with different proteins coated to the wells of microtiter plates, mAbs IA9d 1, 1A9b 12 and 1A9h6 (used at 1:100) are positive with lactoferrin (LF) and negative with transferrin (Trf), albumin (Alb) and fibrinogen (Fbg). Bars = S.D.

used as positive control and showed strongly positive reactions with STM and was negative with LF. These experiments indicated that LF is not expressed on STM membranes. To investigate ifLF, in spite of its absence on STM surfaces, could be present within these membranes, STM preparations were solubilized by boiling in 2% SDS. Solubilized proteins were separated by SDS-PAGE and analyzed by immunoblotting, mAbs 1A9dl, 1A9h6 and 1A9b12 were negative by immunoblotting with STM (Fig. 3). Positive reactivities were shown with purified LF, used as control, mAb TRA-2-10 used as positive control for STM preparations reacted with the reported (Vanderpuye et al., 1991) doublet with Mr of 55 kDa. These results indicate that LF is absent from STM.

Immunohistological studies of placental lactoferrin The trophoblastic mantle, trophoblastic basement membranes, villous stroma and fetal stem vessel endothelium were non-reactive with polyclonal and monoclonal antibodies to human LF. LF positive cells were occasionally (less than 3 per high power field, x 400) identified in normal chorionic villi and these cells were predominantly in contact with STM or fetal stem vessel endothelium (Fig. 4). In addition to their tissue adherence, LF positive cells were frequently found to adhere to each other. Double antibody experiments

30

A

OD 1450 nm]

0.8 0.6 0.4 0.2 8----.-.--..~-~

. ~

0

B

7-0 T

1 0.8 0.6 0.4 0.2

~-

1:100

1:1000

1:10000

(1:500)

(1:5000)

11:50000)

1A9b12

~

1A9dl

--~-

1Agh6

~

Anti Vlmentln

RC~LF

-~

NRS

~-

TRA2.10

~

RGTrf

Fig. 2. ELISA reactivities of STM with mAb and polyclonal antibodies to lactoferrin, mAbs IA9b12, 1A9d 1 and 1A9h6 used at 1:100, 1:1000 and 1:10 000 dilutions and rabbit antilactoferrin used at 1:500, 1:5000 and 1:50 000 were negative with STM (A) and positive with control wells that were coated with 5/~g/ml lactoferrin (B). Control mAb TRA-2-10 and rabbit antitransferrin were positive with STM (A). mAb anti-vimentin and normal rabbit serum (NRS) were used as negative controls.

showed that these cells were equally reactive with polyclonal and monoclonal antibodies to LF. LF positive cells were significantly enriched in areas of villous chronic inflammation (more than 20 per high power field, x 400) (Fig. 5). These lesions were identified by using double antibody studies with monoclonal and polyclonal antibodies to inflammatory cells and coagulation components as previously described (Labarrere et al., 1989; Labarrere et al., 1991). LF positive cells frequently were associated with T cells, platelets and HLA-DR positive macrophages in these areas. Large numbers of LF positive cells were observed in extensive areas of villous chronic inflammation having fibrinoid necrosis. This was confirmed by using a double antibody technique with antibodies to LF and fibrin. In these areas, anti-LF antibodies appeared to react also with placental tissues that were in close proximity to LF positive cells.

31

kD

A

B

CA

B

C

A

B

A B

CAB

116 --

U48.5-

36.526.6-

1Agdl

1Agh6

1A9 b12

TRA230

'Wlmentin

Fig. 3. Absence of lactoferrin from SDS-solubilized and PAGE separated STM proteins, mAbs lA9dl, IA9h6 and IA9b12 reacted with lactoferrin, loaded onto lanes A and were negative with solubilized STM, loaded onto lanes B. Control mAb TRA-2-10 reacted with a doublet at approximately 55 kDa in lane B that corresponds to CD46 in STM (Vanderpuye et al., 1991). Negative control mAb anti-vimentin did not produce specific bands. Lanes C represent prestained molecular weight markers.

In an effort to define LF positive cells in extraembryonic membranes, double antibody experiments were done with the use of rabbit anti-LF and mouse monoclonal antibodies to human leukocytes. The results of these experiments showed that LF positive cells did not react with mAbs to

Fig. 4. Cryostat section of snap-frozen unfixed normal chorionic villi reacted with polyclonal antibody to human lactoferrin. Isolated cells in the intervillous space and within or contiguous with fetal stem vessels are reactive (arrows). Note absence of reactivity of villous structures, x 250.

32

Fig. 5. Cryostat section of snap-frozen unfixed chorionic villi with chronic inflammation of unestablished etiology incubated with polyclonal antibody to human lactoferrin. Note many lactoferrin positive cells in areas of inflammation (arrows). × 250.

macrophages, T or B lymphocytes and NK cells. In contrast, all LF positive cells reacted with CD15 mAbs, MAB1223 and Leu M1 (Table 2). These mAbs reportedly are specific for the X-hapten present on neutrophilic granulocytes (Zola et al., 1981; Huang et al., 1983). A pattern of reactivity TABLE 2 Characteristics of lactoferrin positive cells determined by using double antibody experiments. Antibody

Cellular subset specificities

Reactivity with lactoferrin positive cells

Leu MI MAB 1223

Neutrophils, Monocytes Neutrophilic granulocytes Epithelial tissues Myeloid leukemia cells Neutrophilic granulocytes Natural killer cells Cultured monocytes Neutrophils, activated neutrophils Macrophages NK cells B cells T lymphocytes Helper/inducer T lymphocytes Cytotoxic/suppressor T lymphocytes Most nucleated cells Most nucleated cells Most nucleated cells

++

Leu 11a

GIOF5 EBM 11 Leu 19 Leu 12 Dako, T11 Leu 3a Leu 2a W6/32 Q1/28 /~2 microglobulin

+÷, strong positive reactivity +, decreased reactivity in intervillous cells and no reactivity in tissue adherent cells -, no detectable reactivity

++

++ +

+ + +

33

that closely resembled findings with anti-LF antibodies was observed with mAb Leu l l a (CD16). This mAb is specific for the IgG-Fc receptor III of neutrophils and NK cells (Phillips and Babcock, 1983). The reactivity of LF positive cells with mAbs to CD15 and CD16 suggests that these cells are PMN. Our immunohistological findings with LF positive cells in the placenta were compared to PMN from peripheral blood by using flow cytometry. PMN from peripheral blood also reacted with mAbs to CD15 and CD16 and with antibodies to LF (Fig. 6). During these studies we noticed that PMN from peripheral blood reacted consistently better than placental PMN with monoclonal and polyclonal antibodies to major histocompatibility complex (MHC) class I antigens. To compare intensities of antigen expression on PMN from peripheral blood and within placentae we simultaneously performed double antibody studies with peripheral blood smears and placental sections. Identical antibody dilutions and incubation times were used and photomicrographs were taken at identical magnifications and exposure times. Our studies clearly showed that PMN in placentae

AAB1223 I

neg. control Fig. 6. PMN from peripheral blood analyzed by flow cytometry. PMN are positive with rabbit antilactoferrin, mAbs Leu 1 la (CD16) and MAB 1223 (CD15). Note positive reactivities of mAb W6/32 to a monomorphic determinant of HLA class I antigens and mAb G10F5 that is specific for the neutrophil activation marker gp 100: (CD67). Balb/c ascites fluid and normal rabbit serum were used as negative controls.

Fig. 7. (a) Cryostat section of snap-frozen unfixed chorionic villi reacted with polyclonal antibody to human lactoferrin (left panel). Note that PMN are reactive (arrows). When the same section was incubated with mAb to/~2-microglobulin, PMN were negative (arrows, right panel), Note that fetal stern vessel endothelium and villous stroma are reactive. Similar findings were obtained by using mAb W6/32 and Q1/28. × 400. (b) Peripheral blood smear from a healthy individual incubated with polyclonal antibody to human lactoferrin. Note brightly reactive lactoferrin positive cells (left panel). When the same slide was incubated with mAb to 8~-micro~lobulin these cells also reacted (right panel). Note that •latelets are also reactive, x 400.

b

a

Fig. 8. (a) Peripheral blood smear from a healthy individual incubated with polyclonal antibody to human lactoferrin (left panel). Note many lactoferrin positive cells. When the same slide was incubated with mAb Gt0F5, these cells also were reactive (right panel), x 400. (b) Cryostat section of snap-frozen unfixed chorionic villi with chronic inflammation of unestablished etiology incubated with polyclonal antibody to human lactoferrin. Note many lactoferrin positive cells (arrows) in the inflamed area (left panel). When the same section was incubated with mAb GIOF5, most of these cells (arrows) were not reactive (right panel), x 400.

b

36

(i.e., both maternal and fetal PMN) had decreased reactivities with rabbit anti-/32-microglobulin and with mAbs W6/32 and Q1/28 that react with different epitopes of the MHC class I antigen complex (Parham et al., 1979; Quaranta et al., 1981). Indeed, PMN that were adherent to STM had no detectable expression of MHC class I antigens, while chorionic mesenchyme and fetal stem vessel endothelium were positive (Fig. 7a). In contrast, PMN from peripheral blood were positive for MHC class I antigens (Fig. 7b) and intravascular PMN in cryostat sections of normal myocardium and kidney tissues also were positive for Class I MHC antigens. To investigate if differences between PMN from peripheral blood and within placentae could be due to activation of placental PMN, we used mAb G10F5, specific for gp 100 (CD67) which is a PMN activation marker (van der Schoot et al., 1989). The samples we studied were from non-pregnant donors, mAb G10F5 was positive with PMN from peripheral blood that we examined by using flow cytometry (Fig. 6). Immunocytology studies with mAb G10F5 also showed that PMN in peripheral blood were positive with mAb G10F5 (Fig. 8a). In contrast, G10F5 reactivity with PMN that were located in the intervillous space was decreased and no reactivity was detectable with PMN that were adherent to placental tissues (Fig. 8b). These results indicate that the inability to detect MHC class I antigens on tissueadherent placental PMN is not due to activation, for they show no increased reactivity with antibody to the CD67 phenotypic marker. Discussion

In spite of its high degree of homology to Trf, the distribution of LF in human term placentae is quite different (Galbraith et al., 1980). In STM, where Trf is present in large quantities, (King, 1976; Faulk and Johnson, 1977; Faulk and Galbraith, 1979) we did not detect LF by immunohistology, ELISA or Western blot analyses. LF also was absent from trophoblastic basement membranes, villous stroma and fetal stem vessel endothelium. LF occasionally was identified in cells in intervillous spaces and within fetal stem vessels. These cells predominantly were adherent to the apical aspect of villous trophoblast and to fetal stem vessel endothelium. LF reactive cells have been described in placentae of 8-22 weeks gestation (Ogra et al., 1972). Many LF positive cells were found in areas of chronic inflammation that we identified with mAbs to inflammatory cells and coagulation components (Labarrere et al., 1989; Labarrere et al., 1991). Within these lesions, LF positive cells were associated predominantly with necrosis and fibrin deposition. Ogra et al. (1972) suggested that LF reactive cells in placentae represent neutrophils. The presence of PMN also was shown in mid-gestational mouse placentae, where these cells predominantly were adherent to vascular linings

37

of vessels near the trophoblast-decidua junction (Parr et al., 1990). We found LF cells bear CD15 and CD16 determinants that are expressed by neutrophils (Zola et al., 1981; Huang et al., 1983; Phillips and Babcock, 1983). Our immunocytological and flow cytometric studies showed that antibodies to LF and mAbs to CD 15 and CD16 determinants also reacted with PMN from peripheral blood. We do not know why PMN, when adherent to placental tissues, loose their reactivity with antibodies to MHC class I antigens. PMN adherent to placental tissues also failed to express the CD67 marker that is upregulated in activated PMN (van der Schoot et al., 1989). It is possible that these phenotypic changes are associated with PMN degranulation. Degranulating PMN release proteolytic enzymes from primary and secondary granules (Smolen, 1989), and such enzymes could alter surface antigens in such a way as to disallow their immunological recognition. Alternatively, MHC class I antigens could be down-regulated at a transcriptional level; it would be interesting to address this possibility by in situ hybridization experiments. Release of secondary granules by PMN also mobilizes LF to the surface (Wright and Gallin, 1979; Smolen, 1989) and this has been shown to change electrostatic membrane potentials in a way as to increase tissue adherence of PMN (Oseas et al., 1981; Boxer et al., 1982). That LF is released from PMN is supported by positive LF reactivities in tissues close to adherent PMN. After release from PMN, free LF would be expected to compete with Trf for ferric ions, because the affinity of LF for ferric ions is about 300 times stronger than that of Trf (Aisen and Leibman, 1972). A depletion of ferric ions in the microenvironment of degranulating PMN by LF could have dramatic effects on trophoblastic energy metabolism, because diferric Trf is needed as electron acceptor for the oxidation of NADH (Faulk et al., 1990; Berczi et al., 1991). Thus, LF in placentae could contribute to disruption of the trophoblastic mantle, which is so characteristic of placental inflammation (Altshuler and Russell, 1975; Labarrere et al., 1982; Labarrere and Faulk, 1990).

Acknowledgements This work was supported in part by the Research Department, Methodist Health Foundation and the National Kidney Foundation of Indiana. We thank Pat Gulley, David Wheaton and Harold Boldt for expert technical assistance and Sandy Hollowell for typing this manuscript. We are grateful to Dr. Tom Fuller, Harvard Medical School for mAb Q1/28, to Dr. Peter Andrews, The Wistar Institute, for mAb TRA-2-10 and to Dr. John Thompson, University of Kentucky for mAb G10F5.

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