ICAM-1 expression on immune cells in chronic villitis

Placenta 35 (2014) 1021e1026

Contents lists available at ScienceDirect

Placenta journal homepage: www.elsevier.com/locate/placenta

ICAM-1 expression on immune cells in chronic villitis ~ a c, V.A. Montalli d, O.P. Almeida c, E.S.A. Egal a, F.V. Mariano a, M.H. Blotta b, A.R. Pin a, * A.M. Altemani ~o Paulo, Brazil Department of Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Sa ~o Paulo, Brazil Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, Sa c ~o Paulo, Brazil Department of Pathology, Piracicaba Dental School, State University of Campinas, Sa d ~o Leopoldo Mandic Institute and Research Center, Campinas, Sa ~o Paulo, Brazil Department of Oral Pathology, Sa a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 8 October 2014

Introduction: ICAM-1 expression on the villous syncytiotrophoblast (ST) is believed to participate in migration of maternal cells into the inflamed villi regardless of villitis etiology. However, its expression on immune cells in chronic villitis (CV) has yet to be analyzed. ICAM-1 induces cellecell adhesion allowing intercellular communication, T cell-mediated defense mechanism, and inflammatory response. Material and methods: 21 cases of CV (all without an identifiable etiologic agent) and 3 control placentas were analyzed using ICAM-1, and for immune cells CD45, CD3 and CD68. These cells were subdivided according to their location in inflamed villi: a) within the inflamed villi and b) outside forming perivillous aggregates. Results: Large amounts of CD45, CD3 and CD68 were found within the inflamed villi and forming perivillous aggregates attached to areas of trophoblastic loss. Inflamed villi usually showed ICAM-1þ ST. The majority of immune cells surrounding areas of trophoblastic rupture presented marked expression of ICAM-1. In contrast, a small number of immune cells within the inflamed villi exhibited ICAM-1 expression. Only some (<5%) inflamed villi without trophoblastic rupture and with ICAM-1þ ST presented adherence of immune cells. Discussion: In inflamed villi of chronic villitis, the level of ICAM-1 expression on immune cells depends on their location: high in number of cells in the perivillous region and low within the villi. The strongest expression of ICAM-1 on immune cells attached to areas of trophoblastic rupture suggests that the loss of trophoblast can lead to an amplification of the inflammatory response. © 2014 Elsevier Ltd. All rights reserved.

Keywords: ICAM-1 Villitis Placenta T lymphocytes Macrophages Monocytes

1. Introduction Chronic villitis, an inflammatory lesion of placental villi, has been associated with the transmission of infection between mother and fetus (e.g. rubella, toxoplasmosis, etc) as well as with the maternal immune aggression of fetal tissues [1e3]. In inflamed villi, most of the immune cells are of maternal origin, suggesting that mononuclear cells have the capacity to migrate across the trophoblastic barrier into villous stroma [4e7]. Intercellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein, is expressed constitutively on the cell surface of a variety of cell types, such as immune cells, epithelial cells,

Abbreviations: ST, syncytiotrophoblast; CV, chronic villitis; ICAM-1, Intercellular adhesion molecule-1; LFA-1, leukocyte function-associated antigen-1. lia Vieira de Camargo, * Corresponding author. Faculty of Medical Sciences, Tessa ~o Paulo, Brazil. Tel.: þ55 19 3289 3897. 126-Zip Code: 13084-971, Campinas, Sa E-mail address: [email protected] (A.M. Altemani). http://dx.doi.org/10.1016/j.placenta.2014.10.004 0143-4004/© 2014 Elsevier Ltd. All rights reserved.

endothelial cells, fibroblasts, etc. Among immune cells, ICAM-1 is expressed on monocytes/macrophages, B lymphocytes, plasma cells, and on memory and activated T lymphocytes. In placental cells, ICAM-1 has been detected at low levels on the villous trophoblast [8]. However, ICAM-1 expression can be up-regulated by a number of factors, including proinflammatory cytokines and virus infection. In placentas, overexpression of ICAM-1 has been reported on syncytiotrophoblast (ST) in chronic villitis, massive chronic intervillositis and infections by Plasmodium falciparum, human cytomegalovirus and HIV-1 [9e14]. ICAM-1 plays important roles in the adhesion phenomena involved in the transendothelial migration of leukocytes and in the immune system. The ST of chorionic villi presents an endotheliallike function and in chronic villitis, ICAM-1 expression on the ST is believed to participate in migration of maternal cells into the placental villi [12,13]. In cultured ST, it has been described that monocytes adhered by ICAM-1 receptor LFA-1 (leukocyte functionassociated antigen-1) induce TNF-alpha-dependent apoptosis

1022

E.S.A. Egal et al. / Placenta 35 (2014) 1021e1026

accompanied by focal disruption of the trophoblast [15]. This focal damage of the placental barrier has been thought to be a route for maternal leukocyte infiltration into the villi and a possible mechanism of villitis onset [15]. In fact, in a previous study, our group showed that in the inflamed villi, regardless of the villitis etiology, there was trophoblast overexpression of ICAM-1 close to areas of rupture, which frequently presented leukocyte adherence [13]. In relation to the immune system, ICAM-1 induces cellecell adhesion allowing intercellular communication, T cell-mediated defense mechanism, and inflammatory response. In chronic villitis, to date, the studies on ICAM-1 have only focused on its expression on ST [12,13]. However, the inflammatory cascade initiated in the villitis onset and maintained during its evolution can potentially induce enhancement of ICAM-1 expression on other cell types that participate in the inflammatory process. In order to broaden our understanding of the mechanisms involved in the development of chronic villitis, we investigated ICAM-1 expression on immune cells in inflamed villi. 2. Material and methods 2.1. Cases This study was approved by the Institutional Ethics Committee. The surgical pathology archives of the Hospital of the University of Campinas (UNICAMP), S~ ao Paulo-Brazil were reviewed between 2008 and 2012 and contained 21 cases which had been diagnosed as chronic villitis without an identifiable etiologic agent. All cases were from term/near term singleton pregnancies and had hematoxylin and eosin (H&E) slides and/or paraffin blocks. Chronic inflammatory infiltrate was observed in the stroma of >one chorionic villi in all cases. Three term placentas without villitis were included as control. 2.2. Immunohistochemistry One paraffin block from each case was chosen for immunohistochemical studies. The following primary antibodies were used: anti-CD45 (IgG2a, clone 2B11 þ PD7/ 26, dilution 1/100), anti-CD68 (IgG2a, clone KP1, 1/1000), anti-CD3 (IgM, clone C3D1, 1/1000), anti CD34 (IgG1, clone QBEnd, 1/50) anti-ICAM-1 (IgG2a, SC-8439, 1/ 4000). All antibodies were from Dakopatts S/A, Denmark, except for anti-ICAM-1, which was from Santa Cruz Biotechnology Inc, USA. Briefly, the immunohistochemical staining was performed as follows: the 5-mm sections were deparaffinized, hydrated and endogenous peroxidase activity was quenched by immersion of the slides in 10% hydrogen peroxide. The antigen retrieval (AR) was achieved by boiling, in a steamer, in citrate buffer (pH: 6.0) or TriseEDTA buffer (pH: 8.9) according to the primary antibody used. After cooling, the sections were incubated at 4  C with the primary antibody overnight and then with the EnVision polymer for 1 h at 37  C. Subsequently, sections were stained for 5 min at 37  C with 3,30 -diaminobenzidine tetrahydrochloride (DAB) and counter-stained with hematoxylin. In selected cases (5 cases) double-labeling immunohistochemical staining was performed (EnVision doublestain, code K1395, DAKO, SA, Denmark). The antibodies CD45, CD68, CD3 and CD 34 were applied after antigen retrieval using citrate buffer (pH: 6.0) and incubated 30 min at 25  C; detection was achieved using the EnVision anti-mouse and anti-rabbit polymer HRP and DAB to visualize the binding of the first antibody. The sections were then incubated with an antibody against ICAM-1 at 25  C for 30 min. EnVision polymer linked to alkaline phosphatase and fast red as a substrate chromogen system were used to complete the second immunostaining. 2.3. Analysis of positive cells Serial tissue sections were used and each of the different antibodies (anti-CD45, anti-CD68, anti CD3, anti ICAM-1) was applied on a different section of the same block. Any CD3-positive cell was used as the criterion for villitis. Immune cells in villitis areas were subdivided according to their location: a) within the inflamed villi and b) outside forming perivillous aggregates. The relative numbers of stained leukocytes were considered in relation to all inflammatory cells seen in each location, i.e. a) all immune within the inflamed villi and b) all extravillous immune cells forming perivillous aggregates. Regarding ICAM-1 expression on ST, the number of positive villi was evaluated in areas with and without villitis in relation to all inflamed and normal villi, respectively. The immunoreactivity for ICAM-1 on leukocytes in both compartments (inside and outside the villi) was assessed using a three-tiered scale: a) 0 ¼ no reactive cells, b) 0.1e50% of cells, c) >50%.

3. Results In all cases, villitis was focal and involved only terminal and stem villi. A variable number of inflamed villi presented foci of

trophoblastic necrosis. The inflammatory infiltrate within the villi was composed of mononuclear cells and these also formed aggregates in the intervillous space around the areas of trophoblastic rupture (Fig. 1a). The immunohistochemical findings are in Table 1. All cases of villitis showed CD45 positive cells within the inflamed villi as well as in the perivillous region surrounding the areas of trophoblastic rupture (Fig. 1b). In the majority of cases, the inflamed villi exhibited numerous CD68 and CD3 positive cells (macrophages/ monocytes and T cells, respectively) within and outside the villi; the outside (extravillous) cells surrounded the foci of trophoblastic damage (Fig. 1c, d). The majority of cases (57.8%) presented a similar proportion of CD3þ and CD68þ cells inside and outside the villi (Table 2). In 26.3% of cases, CD3þ lymphocytes and CD68þ macrophages/monocytes were the predominant immune cells inside and outside the villi, respectively (Table 2). Regarding ICAM-1 expression on ST, the molecule was expressed on the ST of inflamed villi in variable extension in all cases of villitis (Fig. 2): in 76% (16 cases) in >50% of inflamed villi and 23.8% in <50%. In contrast, few non-inflamed villi showed ICAM-1 on ST (Fig. 2c). In inflamed villi, ICAM-1 expression on the ST was frequently associated with areas of trophoblastic rupture. In these areas, there usually was adherence of several perivillous immune cells (Fig. 2a, b; 3a). In inflamed villi without trophoblastic rupture and with ICAM-1 expression on the ST, we found in few villi (<5%) adherence of immune cells but the number of cells was usually small (Fig. 3b). Non-inflamed villi with ICAM-1 expression on the ST did not show such findings (Fig. 2c). In relation to ICAM-1 positive immune cells, the number varied according to their location in the inflamed villi. The majority of immune cells (>50%) surrounding the areas of trophoblastic rupture presented marked expression of ICAM-1 in all cases but one (Fig. 2a, b). In contrast, a small number of immune cells within the inflamed villi showed ICAM-1 expression (Fig. 2a, b). This expression was usually less marked than the one presented by immune cells in the perivillous region. Double-labeling immunohistochemical staining was performed to verify the quantity of CD45þ, CD68þ cells and CD3þ cells that was ICAM-1 positive in the two compartments (inside and outside the villi). In all cases the majority of CD45þ, CD68þ and CD3þ cells outside the villi (>50% of cells) was ICAM-1 positive whereas <50% of these cells expressed ICAM-1 inside the villi (Fig. 4a, b, c). As endothelial cells of villous fetal vessels were ICAM-1 positive as well, double-labeling immunohistochemical staining for CD34 (pan-endothelial marker) and ICAM-1 was performed. Inflamed villi frequently presented absence of CD34þ fetal vessels (Fig. 4d) and slight increase of ICAM-1 expression on stromal cells, which formed a thin network (see villous stroma in Fig. 4a,b,d). In control placentas, ICAM-1 expression was only detected on ST of rare terminal villi and on endothelial cells of fetal vessels (Fig. 2d). 4. Discussion This study further expands our previous investigations on trophoblastic ICAM-1 overexpression in human villitis showing that: a) in inflamed villi, immune cells expressed ICAM-1 and such expression was particularly marked on those adhered to foci of trophoblastic rupture, and b) inflamed villi with ICAM-1 overexpression on ST but without trophoblastic rupture rarely present adherence of immune cells. In relation to immune cells, ICAM-1 functions as a co-activation signal for activation of T cells [16]. ICAM-1 is an accessory molecule, which helps stabilize the interaction between the T cell receptor (TCR) and antigen in association with MHC class I (all

E.S.A. Egal et al. / Placenta 35 (2014) 1021e1026

1023

Fig. 1. Chronic villitis: A- Note immune cells within the stroma of the inflamed villus and forming an aggregate in the intervillous space around the site of trophoblastic rupture (hematoxylin and eosin (H&E) stained x40 objective). B- Numerous CD45þ leukocytes in the inflamed villi (x40). C- CD3þ T lymphocytes (x40). D- CD68þ macrophages/monocytes (x40). Note that CD3 lymphocytes and CD68 macrophages/monocytes are within the inflamed villi and in the perivillous space.

lymphocytes were the predominant cells in both types of villitis [19]. However, differently from our previous study, in the present series, immune cells were classified according to their location: a) within the inflamed villi and b) outside forming perivillous aggregates. This separation was carried out because a marked difference of proportion between maternal and fetal macrophages in these two regions has been reported. In the perivillous region, the overwhelming majority of CD68 macrophages is maternal cell, whereas within the inflamed villi, most of them (around 90%) are Hofbauer cells (fetal APC) [20,21]. In contrast, the majority of T cells within the inflamed villi is of maternal origin [20,21]. In the current series, we showed that the amount of ICAM-1 positive cells within the villi was smaller than that in the perivillous region. The level of ICAM-1 expression on the surface of cells depends on the concentrations of pro- and anti-inflammatory mediators and the availability of intracellular signal transduction pathways involved in the regulation of its expression [22]. A functional deficit of fetal cells could be a potential factor to the low ICAM-1 expression on the immune cells within the inflamed villi. Dendritic cells (which are a subtype of APCs) derived from fetal

nucleated cells) and class II (macrophages/antigen presenting cell -APCs) molecules [16]. In normal pregnancies, phagocytosis of apoptotic trophoblast debris by macrophages down-regulates the expression of both MHC-HLA class II molecules and ICAM-1 on these cells [17]. This ICAM-1 down-regulation is believed to reduce maternal immune responsiveness to paternally derived antigens so it would represent an anti-inflammatory/tolerazing response [17]. Indeed, it has been reported that expression of activation marker (such as ICAM-1) could mirror the functional capacity of immune cells to produce cytokines [18]. In this sense, in the current series, ICAM-1 overexpression on immune cells in inflamed villi can be considered an expected phenomenon since such cells are clearly involved in a pro-inflammatory response. We found ICAM-1 overexpression on cells with morphology of lymphocytes and monocytes/macrophages within the villi as well as in the perivillous region. In a previous study, our laboratory showed that immunohistochemical study of the inflammatory infiltrate was not helpful in distinguishing villitis of unknown etiology (VUE) from infectious villitis [19]. Macrophages (HAM 56/CD68 positive) and T

Table 1 Distribution of cases with villitis according to the percentage of reactive cells to the antibodies ICAM-1, CD45RO, CD68 and CD3. Quantity of positive immune cells

ICAM-1

CD45

CD68

CD3

IV

OV

IV

OV

IV

OV

IV

OV

No. cases (%)

No. cases (%)

No. cases (%)

No. cases (%)

No. cases (%)

No. cases (%)

No. cases (%)

No. cases (%)

0 <50% >50%

1 (4.7) 12 (57.3) 8 (38)

0 1 (4.7) 20 (95.2)

0 13 (61.9) 8 (38.1)

0 9 (42.8) 12 (57.1)

1 (5) 7 (35) 12 (60)

1 (5) 5 (25) 14 (70)

0 6 (28.5) 15 (71.4)

2 (9.5) 6 (28.5) 13 (61.9)

Total

21 (100)

21 (100)

21 (100)

21 (100)

20 (100)

20 (100)

21 (100)

21 (100)

IV: inside the villi; OV: outside the villi forming perivillous aggregates; CD45 labels lymphoid cells and macrophages; CD68 labels macrophages and monocytes; CD3 labels T lymphocytes.

1024

E.S.A. Egal et al. / Placenta 35 (2014) 1021e1026

Table 2 Proportion of CD68 and CD3 positive cells in inflamed villi according to their location: inside and outsidea the villi. Location of immune cells

Immune cells inside the villi Immune cells outside the villi a

Predominance of CD68þ cells

Predominance of CD3þ cells

Similar proportions of CD68þ and CD3þ cells

Number of cases

(%)

Number of cases

(%)

Number of cases

(%)

3 5

(15.7) (26.3)

5 3

(26.3) (15.7)

11 11

(57.8) (57.8)

Outside the villi forming a perivillous aggregate.

blood monocytes show lower HLA-DR expression than their adult counterparts following lipopolysaccharide activation [23]. In line with this phenomenon, it is likely that activated fetal macrophages (which are the majority of the macrophages within the villi [20,21].) might present lower ICAM-1 expression as well. On the other hand, it is also possible that maternal T regulatory cells, which are strongly increased during pregnancy [24] could enter the inflamed villi, react to foreign (fetal) antigens, inhibit the immune response and consequently lead to ICAM-1 down-regulation. As the methodology used in the present study does not allow clarifying the factors associated with the level of ICAM-1 expression on immune cells or the origin of cells (fetal or maternal), further investigations will be needed. In contrast to low ICAM-1 expression within the inflamed villi, we found that the majority of perivillous cells (>50% of cells) adhered to foci of trophoblastic rupture exhibited a strong ICAM-1 expression. Inflamed villi without trophoblastic rupture did not show adherence of immune cells, except for those with ICAM-1 overexpression on ST, which presented such phenomenon in some of them. However, in these villi, the number of immune cells was usually small in comparison to that observed in areas of trophoblast damage (see Fig. 3). In cultured ST, ICAM-1 expression was associated with monocyte adhesion that led to focal disruption of the trophoblast [15,25]. Our findings suggest that there is a marked

amplification of the inflammatory response associated with trophoblastic damage. It is likely that trophoblastic necrosis was initially caused by immune cells adhered on ST with ICAM-1 overexpression and the amplification of the inflammatory response would be a posterior event. In this sense, perivillous ICAM-1 positive cells could be maternal cells involved in allogenic recognition of fetal MHC antigens when there is loss of the trophoblastic barrier. Although in our study neither the mother nor the infant were investigated regarding clinical or serological evidence of infection, we believe that our series of villitis without an identifiable agent can be classified as villitis of unknown etiology (VUE). All of our cases presented histological characteristics consistent with those of VUE, i.e., a focal pattern of involvement affecting only the terminal and stem villi and inflammatory infiltrate of lymphohistiocytic type [2]. In the literature, two hypotheses have been considered for VUE etiology: an unidentified infection (probably by virus) or an allogeneic transplantation rejection reaction (maternal rejection of paternal antigens exposed within villi) [3,7,26]. The marked amplification of inflammatory response in the area of trophoblastic rupture detected in our series reinforces the hypothesis that VUE could represent a maternal immune aggression of fetal tissues. Finally, we showed that ICAM-1 was expressed usually on placental endothelial cells and rarely on ST of non-inflamed villi

Fig. 2. A and B e ICAM-1 is overexpressed on the ST and on the majority of the immune cells adhered to the area of trophoblastic rupture; note it is not expressed on immune cells within the villous stroma (40x objevtive). C e ICAM-1 is overexpressed on the ST of normal villi in a non-inflamed area (40x). D e Control placenta shows ICAM-1 expression on ST of a villus (arrow) (40x) and on fetal endothelial cells.

E.S.A. Egal et al. / Placenta 35 (2014) 1021e1026

1025

Fig. 3. A- ICAM-1 (red) is overexpressed on the ST and on several CD68þ cells (brown) in the perivillous region and adhered to an area with loss of ST (between *) (x100). B- ICAM-1 (red) is overexpressed on the ST and few ICAM-1/CD68þ cells (red/brown) are adhered on the ST (x100).

Fig. 4. A- CD3þ T lymphocytes (brown), B- CD68þ macrophages/monocytes (brown) and C- CD45þ leukocytes (brown) are in the intervillous space and within the villous stroma; ICAM-1 (red) is mainly overexpressed on the ones in the intervillous space (40x objective). D- Double-labeling immunohistochemical staining for ICAM-1 (red) and CD34 (brown): non-inflamed villi show juxtaposition of ICAM-1 (red) and CD34 (brown) on endothelial cells; in the inflamed villus, CD34þ vessels are not observed and there is ICAM-1 expression on the ST, several perivillous immune cells and some stromal cells (x40).

and control placentas. However, adherence of immune cells was not detected on the ST of these villi. Our findings confirm the one reported by Labarrere and colleagues (2005); the authors also found villi with such alteration in areas without villitis. The mechanisms associated with up-regulation of ICAM-1 on ST of noninflamed villi remain unclear and it is believed that this phenomenon may be linked to a physiologic process of trophoblast renewal [25]. Regarding endothelial cells, in our series, fetal vessels of control placentas as well as of non-inflamed villi expressed ICAM-1 and were positive for CD34. In contrast, the great majority of inflamed villi showed absence of CD34þ vessels and an increase of ICAM-1 in the villous stroma. As ICAM-1 can be expressed on fibroblasts and fibrosis is expected to occur with the progression of the lesion, this could be an explanation for the increased expression

of stromal ICAM-1. However, although CD34 is a robust panendothelial marker that can also detect newly formed vessels [27], it is a matter for further study whether CD34 negative vessels could be present in inflamed villi. In conclusion, in inflamed villi of chronic villitis, the level of ICAM-1 expression on immune cells depends on their location: high in number of cells in the perivillous region and low within the villi. The strongest expression on immune cells attached to areas of trophoblastic rupture suggests that the loss of trophoblast can lead to an amplification of the inflammatory response. Conflict of interest We do not have any actual or potential conflict of interest.

1026

E.S.A. Egal et al. / Placenta 35 (2014) 1021e1026

References [1] Altshuler G, Russell P. The human placental villitides: a review of chronic intrauterine infection. Curr Top Pathol 1975;60:63e112. [2] Redline RW. Villitis of unknown etiology noninfectious chronic villitis in the placenta. Hum Pathol 2007;30(10):1439e46. [3] Labarrere C, Althabe O, Telenta M. Chronic villitis of unknown aetiology in placentae of idiopathic small for gestational age infants. Placenta 1982;3:309e18. [4] Labarrere CA, Mcintyre JA, Faulk WP. Immunohistologic evidence that villitis in human normal term placentas is an immunologic lesion. Am J Obstet Gynecol 1990;162:515e22. [5] Labarrere CA, Faulk WP. Maternal cells in chorionic villi from placentae of normal and abnormal human pregnancies. Am J Reprod Immunol 1995;13: 54e9. [6] Kapur P, Rakheja D, Gomez AM, Sheffield J, Sanchez P, Rogers BB. Characterization of inflammation in syphilitic villitis and in villitis of unknown aetiology. Pediatr Dev Pathol 2004;4:453e8. [7] Redline RW, Patterson P. Villitis of unknown etiology is associated with major infiltration of fetal tissue by maternal inflammatory cells. Am J Pathol 1993;143:473e9. [8] Gaffuri B, Vigano P, Nozza A, Gornati G, Di Blasio AM, Vignali M. Expression of intercellular adhesion molecule-1 messenger ribonucleic acid and protein in human term placental cells and its modulation by pro-inflammatory cytokines (interleukin-1beta and tumor necrosis factor alpha). Biol Reprod 1998;58: 1003e8. [9] Sugiyama T, Cuevas LE, Bailey W, Makunde R, Kawamura K, Kobayashi M, et al. Expression of intercellular adhesion molecule 1 (ICAM-1) in Plasmodium falciparum-infected placenta. Placenta 2000;22:573e9. ~ oz LD, Mun ~ oz-Fern [10] Arias RA, Mun andez MA. Transmission of HIV-1 infection between trophoblast placental cells and T-cells take place via an LFA-1mediated cell to cell contact. Virology 2003;307(2):266e77. [11] Chan G, Stinski MF, Guilbert LJ. Human cytomegalovirus-induced upregulation of intercellular cell adhesion molecule-1 on villous syncytiotrophoblasts. Biol Reprod 2004;71(3):797e803. [12] Labarrere CA, Ortiz MA, Sosa MJ, Campana GL, Wernicke M, Baldridge LA, et al. Syncytiotrophoblast intercellular adhesion molecule-1 expression in placental villitis of unknown cause. Am J Obstet Gynecol 2005;193:483e8. [13] Juliano PB, Blotta MHSL, Altemani AMA. ICAM-1 is overexpressed by villous trophoblasts in placentitis. Placenta 2006;27:750e7. [14] Labarrere CA, Bammerlin E, Hardin JW, Dicarlo HL. Intercellular adhesion molecule-1 expression in massive chronic intervillositis: implications for the invasion of maternal cells into fetal tissues. Placenta 2014;35(5):311e7.

[15] Garcia-Lloret MI, Winkler-Lowen B, Guilbert LJ. Monocytes adhering by LFA-1 to placental syncytiotrophoblasts induce local apoptosis via release of TNF-a. A model for hematogenous initiation of placental inflammations. J Leukoc Biol 2000;68:903e8. [16] Fooksman DR, Vardhana S, Vasiliver-Shamis G, Liese J, Blair DA, Waite J, et al. Functional anatomy of T cell activation and synapse formation. Annu Rev Immunol 2010;28:79e105. [17] Abumaree MH, Stone PR, Chamley LW. The effects of apoptotic, deported human placental trophoblast on macrophages: possible consequences for pregnancy. J Reprod Immunol 2006;72(1e2):33e45. [18] Luppi P, Haluszczak C, Betters D, Richard CA, Trucco M, DeLoia JA. Monocytes are progressively activated in the circulation of pregnant women. J Leukoc Biol 2002;72:874e84. [19] Brito H, Juliano P, Altemani C, Altemani A. Is the immunohistochemical study of the inflammatory infiltrate helpful in distinguishing villitis of unknown etiology from non-specific infection villitis? Placenta 2005;26(10):839e41. [20] Myerson D, Parkin RK, Benirschke K, Tschetter CN, Hyde SR. The pathogenesis of villitis of unknown etiology: analysis with a new conjoint immunohistochemistrye in situ hybridization procedure to identify specific maternal and fetal cells. Pediatr Dev Pathol 2006;9:257e65. [21] Kim JS, Romero R, Kim MR, Kim YM, Friel L, Espinoza J, et al. Involvement of hofbauer cells and maternal T cells in villitis of unknown aetiology. Histopathology 2008;52:457e64. [22] Cornelius LA, Taylor JT, Degitz K, Li LJ, Lawley TJ, Caughman SW. A 5' portion of the ICAM-1 gene confers tissue-specific differential expression levels and cytokine responsiveness. J Invest Dermatol 1993;100:753e8. [23] Langrish CL, Buddle JC, Thrasher AJ, Goldblatt D. Neonatal dendritic cells are intrinsically biased against Th-1 immune responses. Clin Exp Immunol 2002;128:118e23. [24] Alijotas-Reig J, Llurba E, Gris JM. Potentiating maternal immune tolerance in pregnancy: a new challenging role for regulatory T cells. Placenta 2014;35: 241e8. http://www.ncbi.nlm.nih.gov/pubmed/24581729. [25] Xiao J, Garcia-Lloret M, Winkler-Lowen B, Miller R, Simpson K, Guilbert LJ. ICAM-1-mediated adhesion of peripheral blood monocytes to the maternal surface of placental syncytiotrophoblasts: implications for placental villitis. Am J Pathol 1997;150:1845e60. [26] Labarrere CA, Carson SD, Faulk SP. Tissue factor in chronic villitis of unestablished etiology. J Reprod Immunol 1991;19:225e35. [27] Fox SB, Harris AL. Histological quantitation of tumour angiogenesis. APMIS 2004;112:413e30.