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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 1 101-105
Copyright © 1997 by The Endocrine Society

Experimental Studies

Intercellular Adhesion Molecule-1 is Expressed on Human Granulosa Cells and Mediates Their Binding to Lymphoid Cells

Paola Viganò, Barbara Gaffuri, Guido Ragni, Anna Maria Di Blasio and Mario Vignali

II Department of Obstetrics and Gynecology (P.V., B.G., M.V.), I Department of Obstetrics and Gynecology (G.R.), University of Milano and Centro Auxologico Italiano (A.M.D.), 20135 Milano, Italy

Address all correspondence and requests for reprints to: Dr. Anna Maria Di Blasio, Molecular Biology Laboratory, Centro Auxologico Italiano, Viale Monte Nero 32, 20135 Milano, Italy.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Some immune cellular components have been recently demonstrated to play a critical role in ovarian physiology. Resident ovarian white blood cells are known to produce cytokines that modulate granulosa cell (GC) functions and differentiation. Moreover, it has been postulated that, during the formation of the corpus luteum and luteolysis, human luteal cells are able to interact with lymphocytes and macrophages through some adhesion molecules. This study was designed to examine, at messenger RNA and protein levels, whether intercellular adhesion molecule (ICAM)-1, known to be involved in leukocyte-cell binding, is expressed by human GCs. Furthermore, we also investigated whether this molecule could be involved in the complex events that allow the interaction between the ovary and the immune system. GCs, obtained from women undergoing in vitro fertilization procedures, were enzymatically dispersed with collagenase and cultured for different time periods. To assess the presence of ICAM-1 messenger RNA, total RNA obtained from freshly aspirated GCs and GCs luteinized in culture was reverse transcribed and then amplified using two oligonucleotide primers specific for the human ICAM-1 gene. A single major DNA band of the expected size (943 bp) was obtained. The identity of this material with the human ICAM-1 sequence was further confirmed by restriction enzyme analysis. Surface ICAM-1 protein was detected by flow cytometric analysis on luteinized GCs cultured for 7 and 15 days. Finally, to evaluate a possible functional activity of ICAM-1, a 51Cr-release-binding assay between peripheral blood lymphocytes and luteinized GCs was performed in the presence and absence of a monoclonal antibody against ICAM-1. As a result, lymphocyte adhesion to GC monolayers was significantly, but not completely, inhibited by the anti-ICAM-1 monoclonal antibody. These findings demonstrate that intercellular interactions between GCs and the immune system are, at least in part, mediated by the adhesion molecule ICAM-1. Based on this data, we might speculate that this molecule could participate in the remodeling processes of the ovarian endocrine compartment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RECENT literature related to ovarian function suggests the existence of a bidirectional communication network that links the immune and reproductive systems (1, 2). Resident ovarian representatives of the white blood series could constitute potential in situ modulators of ovarian function, acting through local secretion of regulatory soluble factors (2). Cytokines can modulate granulosa cell (GC) functions and differentiation and, in turn, human GCs can produce some cytokines whose secretion is under gonadotropin control (3, 4). Furthermore, it has been postulated that, during folliculogenesis, formation of the corpus luteum (CL) and luteolysis, lymphocyte, and macrophages could exert paracrine effects at the level of adjacent endocrine ovarian cells, with which they establish physical contacts through some adhesion-promoting receptors. In particular, two of these molecules, HLA-DR and Lymphocyte function-associated antigen (LFA)-3, both members of the Ig superfamily, were found to be highly expressed on human luteinizing granulosa and large luteal cells (5, 6, 7, 8). More importantly, their expression was shown to be differentially regulated during folliculogenesis and luteinization. Thus, it has been suggested that these antigens have a role in lymphoid-ovarian cell interaction and granulosa/luteal cell differentiation. However, whereas most attention has been directed, thus far, at the relevance of cytokines in ovarian physiology, the intercellular molecules involved in the close relationship between immune and ovarian cells are still largely unknown.

Among the cell recognition molecules known to play a crucial role in the interaction of immune cells with other tissues, one of the best examined is the intercellular adhesion molecule (ICAM)-1, a single chain 90–114 kDa sialoglyco-protein that is also a member of the Ig gene superfamily. ICAM-1 is present on various cell types, including bone marrow-derived cells. Constitutive expression varies with cells examined, but activation studies in humans demonstrated that inflammatory mediators (including IL-1, interferon {gamma}, and TNF{alpha}) cause strong induction of ICAM-1 in a wide variety of tissues and greatly increase binding of lymphocyte and monocyte through their cell surface LFA-1 (9, 10).

Accumulating evidence for a functional significance of adhesion receptors in the ovary led us to examine whether ICAM-1 expression occurs in the human ovary. In particular, we set up experiments to examine, at messenger RNA (mRNA) and protein levels, whether ICAM-1 is expressed by freshly aspirated human GCs and by GCs luteinized in culture. Moreover, we evaluated whether the molecule could be involved in the complex events that allow the interaction between ovarian and immune cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of human GCs

Human GCs were obtained from 12 women undergoing in vitro fertilization procedures and were cultured as previously described (11, 12). Briefly, the cells were washed and then dispersed at 37 C for 30 min in 0.1% collagenase (Boehringer-Mannheim, Milano, Italy). Thereafter, the dispersed cells were layered on 3.5 mL lymphocyte separation medium (Flow Laboratories, Milano, Italy) to separate GCs from red blood cells. To reduce contaminating adherent peripheral mononuclear cells, we first incubated the collected cells for 60 min at 37 C in humidified air containing 5% CO2 on a Petri culture dish (Falcon, Becton Dickinson, Milano, Italy). Nonadherent GCs were then collected, washed, and cultured in medium 199 EBSS supplemented with 10% FCS (BioWhittaker, PBI, Milano, Italy) and antibiotics. Medium was changed every other day. GCs in culture resembled the typical morphology of luteinizing cells as previously reported by McAllister et al. (13).

RNA extraction and DNA amplification

Total RNA was extracted from freshly aspirated human GCs and GCs luteinized in culture, according to the method of Chomczynski and Sacchi (14). The oligonucleotide primers designed to amplify a fragment of the human ICAM-1 gene are shown in Table 1Go. The primer set used to amplify an intron-spanning region of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene has been previously described (15). The latter gene provided a constitutively expressed internal control for complementary DNA (cDNA) quantity, integrity, and genomic DNA contamination. The presence of ICAM-1 and GAPDH mRNAs was demonstrated by amplifying respective target sequences using PCR according to the instructions provided with the GeneAmp Amplification Reagent Kit (Perkin-Elmer, Milano, Italy). One µg of total RNA was reverse transcribed to prepare cDNA. PCR was performed on the entire cDNA product using Thermus acquaticus (Taq) DNA polymerase with the manufacturer’s recommended buffers. Reaction conditions for reverse transcription were as follows: 1 mmol/L each deoxynucleotide triphosphate, 1 U RNasin, 100 pmol random hexamer, 200 U RT. The reaction was run at 42 C for 1 h. The reaction mixture was then heated at 99 C for 5 min and quick-chilled on ice.


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  		Table 1. Oligonucleotide primers for RT-PCR detection of human ICAM-1 and GADPH mRNA transcripts

 
To amplify ICAM-1 cDNA fragment, the amplification reaction mixture consisted of adding 10 pmol each of upstream and downstream primers and 1 U Taq DNA polymerase to the reverse transcription mixture. Samples were incubated for 35 PCR cycles. During each cycle, the samples were heated at 94 C (30 sec), cooled at 56 C (5 sec), and heated at 72 C (1 min). The predicted amplification product was 943 bp. PCR detection of GAPDH gene was performed as previously described (15). Each time PCR was carried out, the negative control was prepared using all reagents and substituting 1 µg of water for the RNA.

Flow cytometric analysis

On day 7 and day 15 of culture, 105 luteinized GCs were harvested from the culture dishes by a short incubation in 0.05% trypsin solution and stained with 10 µL anti-ICAM-1 (anti-CD54, Immunotech, Marseille, France) and anti-CD14 monoclonal antibodies (MoAbs) (Immunotech, Marseille, France) followed by fluoresceinated goat antimouse Ig. Negative controls were stained with the fluorescent reagent alone. Then all samples were analyzed on a flow cytometer (FACStar, Becton Dickinson & Co, Mountain View, CA) gated to exclude nonviable cells. The percentage of positive cells was calculated on a histogram displaying log10 of fluorescence (in arbitrary units) vs. number of cells. An electronic gate was positioned on the basis of 99% of autofluorescent-negative cells. Fluorescent cells trespassing the gate were considered as positive.

Binding assay

Leukocytes were obtained from buffy coats of blood donor, normal female volunteers and isolated on a Hypaque-Ficoll gradient as previously described (16). Lymphocytes were enriched and separated from monocytes by incubating mononuclear cells in RPMI-1640 (Bio Whittaker, PBI International, Mi, Italy) plus 10% FCS in tissue culture dishes for 1 h twice and saving the nonadherent cells. Peripheral blood lymphocytes (PBL) were then resuspended to a concentration of 1 x 106/mL in RPMI 1640 supplemented with 10% FCS.

Assays to detect binding of PBL to GCs were performed in triplicate in U-bottomed microtest wells. GC monolayers were pretreated with and without 5 µg/mL of an anti-CD54 MoAb and washed before the assay.

PBL (1 x 106) were labeled with 100 µCi sodium 51Cr for 2 h and washed three times before use. Lymphoid cells were layered on GC monolayers (30,000 GCs/well; 0.1 mL total vol) at a ratio of 10:1 and incubated at 37 C in a 5% CO2-95% air atmosphere for 1 h. Nonadherent cells were removed from the wells by gently washing with 100 mL aliquots of warmed medium for a total of four to five washes. Washes were identical for all wells within each experiment. Radioactivity was quantified after solubilization of the monolayer in 0.1 mL of 10% Triton X-100. Lymphoid cells also were incubated with 0.1 mL of medium alone for determination of the total amount of available 51Cr. The percentage of binding lymphocytes was calculated according to the following formula: Binding (%) = a/b x 100

where a = radioactivity from the wells with GC monolayers and b = total amount of available radioactivity.

Statistical analysis

Data are expressed as mean ± SEM. Differences among groups were compared by paired Student’s t test. P < 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT-PCR detection of ICAM-1 and restriction enzyme analysis

ICAM-1 mRNA in human GCs was detected by PCR analysis. Figure 1Go shows that freshly aspirated human GCs from preovulatory follicles express the gene coding for ICAM-1 because RT-PCR generated a DNA fragment corresponding to the predicted length, 943 bp, of the ICAM-1 amplification product. Moreover, the same results were obtained using cultured GCs that underwent luteinization. In each cell sample, all GAPDH amplification products were of 240-bp length whereas the 354-bp fragment, representing genomic DNA contamination, was never detected. The identity of the amplified product with the primers-defined ICAM-1 DNA sequence was further demonstrated by restriction enzyme analysis. The amplified fragment of the human ICAM-1 gene contains four HinfI sites. Consistent with this, digestion with HinfI cut the PCR product into five fragments of the expected size (Fig. 2Go).



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  		Figure 1. RT-PCR analysis of ICAM-1 and GAPDH mRNAs in human GCs. Total RNA extracted from freshly aspirated GCs (lane 2) and GCs cultured for 1, 4, 7, and 15 days (lanes 3–6) was reverse transcribed and amplified using specific oligonucleotide primers as described in Materials and Methods. Ten percent of the PCR mixture was resolved on a 4% agarose gel stained with ethidium bromide. Top panel, ICAM-1 amplification products; low panel, GAPDH amplification products obtained in the same samples. Size markers are indicated in lane 1. Lane 7, negative control with no RNA added.

 


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  		Figure 2. Restriction enzyme analysis of ICAM-1 PCR product obtained from GCs. Lane 1, size marker; lane 2, human ICAM-1 PCR products (943 bp); lane 3, DNA fragments after HinfI digestion. Expected size of the HinfI fragments = 89 bp, 143 bp, 155 bp, 242 bp, and 314 bp.

 
Flow cytometric analysis

The percent of luteinized GCs positive for ICAM-1 was detected by flow cytometry. Figure 3Go shows a histogram of a representative experiment. ICAM-1-positive cells ranged from 70–99% after either 7 or 15 days of culture. We were unable to perform the cytometric analysis on freshly aspirated GCs caused by the large contamination of dead GCs at the beginning of the culture, which may strongly affect the results. Less than 4% of the cells expressed CD-14 antigen, which represents the specific monocyte/macrophage marker (8).



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  		Figure 3. Histogram of a typical flow cytometric analysis of luteinized-GCs cultured for 7 days (left panels) and 15 days (right panels). ICAM-1 and CD14 expression profiles are shown. The percentage of positive cells was calculated as described in Materials and Methods. Upper panels represent negative controls stained only with the second fluorescent antibody.

 
Effect of an anti-ICAM-1 MoAb on PBL binding to GC monolayers

The LFA-1/ICAM-1 pathway is one the adhesion mechanisms through which LFA-1-bearing leukocytes can bind to ICAM-1-positive cells to display an effective immunological reaction (17). As shown in Fig. 4Go, incubation of GC monolayers with an anti-ICAM-1 MoAb resulted in a significant inhibition of PBL binding. In the presence and absence of the anti-ICAM-1 MoAb, the mean percentage binding ± SEM was 12.4 ± 2.5 and 8.9 ± 1.6, respectively, (P < 0.05). These values correspond to a binding inhibition of 28%. It is noteworthy, however, that the antibody could not completely abolish PBL adhesion.



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  		Figure 4. Binding assay between PBL and GC monolayers performed in presence and absence of an anti-ICAM-1 MoAb. GC monolayers were incubated with and without an anti-ICAM-1 MoAb for 1 h and then tested in a 51Cr-release-binding assay. Lymphoid to GC ratio was 10:1. Results represent the mean percentage binding ± SEM of 12 experiments; *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study examines for the first time the expression of the cell surface molecule ICAM-1 in human GCs. The results obtained by PCR analysis indicate that both freshly aspirated GCs and GCs luteinized in culture express the gene for ICAM-1. Moreover, as assessed by flow cytometry, immunoreactive ICAM-1 molecules are present on luteinized GCs cultured for 7 and 15 days. Interestingly, in each cell sample analyzed, approximately 80% of the single cells expressed the molecule. The low percentage of contaminating monocytes/macrophages in our cultures, detected by cytometric analysis of CD-14 antigen, allowed us to exclude the possibility that ICAM-1 expression might be attributed to these cell populations. Finally, a MoAb directed against ICAM-1 was able to partially inhibit lymphocyte binding to GC monolayers.

Cell-surface adhesion molecules are thought to play an important role in establishing the intercellular contacts that are essential for immunologic reactions and for the assembly and maintenance of the cellular architecture of complex tissues (10, 18). In humans, one of these adhesion pathways involves the ß-leukocytes integrin LFA-1 and its ligand ICAM-1 (17). This interaction seems critical for immunosurveillance during inflammation or neoplastic transformation. ICAM-1 induction by proinflammatory cytokines is an important means of regulating LFA-1/ICAM-1 interactions and, thereby, presumably inflammatory responses. In the absence of an inflammatory response, ICAM-1 is only expressed on few cell types (10).

For this reason, the observed constitutively high expression of ICAM-1 on endocrine ovarian cells and its functional involvement in the ovarian specific interaction with lymphocytes suggest important biological implications.

Indeed, this is the first report providing direct evidence that binding of lymphoid cells to steroid-producing cells of the ovary is mediated by adhesion molecules. Because the anti-ICAM-1 MoAb does not completely abolish the adherence, it seems likely that the adhesion structures of GCs include an heterogeneous group of molecules. However, ICAM-1 antigen may certainly play a role in the accumulation, distribution, and regulation of leukocytes in ovarian tissue.

Recent findings suggest that GCs have an extremely complex nature. It is now well established that several peptide hormones and growth factors are produced by these cells (2, 3, 11; also see Ref. 20). In both human and rat models, they have been demonstrated to express molecules, such as NCAM, typically present on neurons and peptide hormone-producing cells and cadherins, which mediate calcium-dependent cell adhesion (19, 20, 21, 22). Moreover, their biochemical and differentiated functions have been demonstrated to be regulated by immune cells and soluble immune products. Lymphocytes, macrophages, mast cells, and other lymphopoietic cells accumulate in perifollicular, paraluteal, and intraluteal sites at various times during the development and demise of Graafian follicles and CL. Lymphokines and monokines were suggested to inhibit both LH-stimulated and basal progesterone secretion and to contribute to the induction of ovulation by increasing PG synthesis by GCs (2, 3, 4). Taken together, these observations support the concept that follicular growth, antrum formation, and morphogenesis of CL are intercellular dynamic events whose regulation implies the participation of several mechanisms and molecules. In view of the data presented herein, we speculate that the surface protein ICAM-1 might be one of these molecules.

It should be considered that the cells in our system were exposed to high levels of gonadotropin releasing-hormone agonist, FSH, LH, hCG, and estradiol before their retrieval. These substances might directly or indirectly modulate ICAM-1 expression. Thus, further studies on the regulation of ICAM-1 will shed light on the relevance of this system to the process of follicular development and/or demise.

In conclusion, the present study shows that the adhesion molecule ICAM-1 is expressed at both RNA and protein levels by human GCs and is involved in their binding to lymphoid cells. To the best of our knowledge, these are the first data that could suggest an involvement of ICAM-1 in the processes of folliculogenesis and CL formation in the human ovary. Because this concept may become of potential clinical relevance, additional studies are required to precisely unravel the specific function of the molecule in ovarian physiology.

Received June 19, 1996.

Revised August 12, 1996.

Accepted August 19, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mori T. 1990 Immuno-endocrinology of cyclic ovarian function. Am J Reprod Immunol. 74:80–89.
  2. Adashi EY. 1990 The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr Rev. 11:454–464.[Abstract/Free Full Text]
  3. Ben-Rafael Z, Orvieto R. 1992 Cytokines-involvement in reproduction Fertil Steril. 1093–1099.
  4. Fukuoka M, Yasuda K, Emi N. 1992 Cytokine modulation of progesterone and estradiol secretion in cultures of luteinized human granulosa cells. J Clin Endocrinol Metab. 75:254–258.[Abstract]
  5. Khoury EL, Marshall LA. 1990 Luteinization of human granulosa cells in vivo is associated with expression of MHC class II antigens. Cell Tissue Res. 262:217–224.[CrossRef][Medline]
  6. Tidey GF, Nelson LM, Phillips TM, Stillman RJ. 1992 Gonadotropins enhance HLA-DR antigen expression in human granulosa cells. Am J Obstet Gynecol. 167:1768–1773.[Medline]
  7. Fujiwara H, Ueda M, Imai K, et al. 1993 Human leukocytes antigen-DR is a differentiation antigen for human granulosa cells. Biol Reprod. 49:705–715.[Abstract]
  8. Hattori N, Ueda M, Fujiwara H, Fukuoka M, Maeda M, Mori T. 1995 Human luteal cells express leukocyte functional antigen (LFA)-3. J Clin Endocrinol Metab. 80:78–84.[Abstract]
  9. Simmons D, Makgoba MW, Seed B. 1988 ICAM-1, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. Nature. 331:624–627.[CrossRef][Medline]
  10. Springer TA. 1990 Adhesion receptors of the immune system. Nature. 346:425–434.[CrossRef][Medline]
  11. Di Blasio AM, Viganò P, Cremonesi L, Carniti C, Ferrari M, Ferrari A. 1993 Expression of the genes encoding basic fibroblast growth factor and its receptor in human granulosa cells. Mol Cell Endocrinol. 96:R7–R11.
  12. Di Blasio AM, Viganò P, Ferrari A. 1994 Insulin-like growth factor-II stimulates human granulosa-luteal cell proliferation in vitro. Fertil Steril. 61:483–487.[Medline]
  13. McAllister JM, Mason JI, Byrd W, Trant JM, Waterman MR, Simpson ER. 1990 Proliferating granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage and 3 beta hydroxy-steroid dehydrogenase. J Clin Endocrinol Metab. 71:26–33.[Abstract/Free Full Text]
  14. Chomczynski P, Sacchi N. 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162:156–159.[Medline]
  15. Ryan IP, Schriock ED, Taylor RN. 1994 Isolation, characterization and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab. 78:642–648.[Abstract]
  16. Viganò P, Vercellini P, Di Blasio AM, Colombo A, Candiani GB, Vignali M. 1991 Deficient anti-endometrium lymphocyte-mediated cytotoxicity in patients with endometriosis. Fertil Steril. 56:894–899.[Medline]
  17. Dustin ML, Springer TA. 1988 Lymphocyte function-associated antigen-1 (LFA-1): interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol. 107:321–331.[Abstract/Free Full Text]
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  19. Blaschuk OW, Farookhi R. 1989 Estradiol stimulates cadherin expression in rat granulosa cells. Dev Biol. 136:564–567.[CrossRef][Medline]
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