Nonspecific immunity in pregnancy: monocyte surface Fcγ receptor expression and function

Journal of Reproductive Immunology40 (1998) 119–128

Nonspecific immunity in pregnancy: monocytesurface Fcg receptor expression and function

Diane Davis a,*, Robert Kaufmann b, Edward J. Moticka a

a Department of Medical Microbiology and Immunology, Southern Illinois Uni6ersity, School of Medicine,P.O. Box 19230, Springfield, IL 62794-1220, USA

b Department of Obstetrics and Gynecology, Southern Illinois Uni6ersity, School of Medicine,P.O. Box 19230, Springfield, IL 62794-1220, USA

Received 26 March 1998; received in revised form 17 July 1998; accepted 20 July 1998

Abstract

The state of pregnancy is an immunological enigma during which the body must preventrejection of the antigenically foreign fetus while at the same time maintain sufficient maternalhost defense mechanisms to combat infection. Although most studies on the immunology ofpregnancy focus on immune suppression, several studies have shown an increase in nonspe-cific host defense, which is postulated to be a compensatory mechanism for decreased specificimmunity during pregnancy. Studies in this laboratory have shown that monocyte surfaceFcgRI (CD64) and FcgRII (CD32) expression progressively increase throughout pregnancy,while surface MHC class II expression remains unchanged. Functional studies revealed thatthe number of phagocytic monocytes which could be isolated from pregnant women wasincreased. These cells exhibited an increased capacity to ingest IgG-opsonized humanerythrocytes. This study shows for the first time that monocyte surface FcgR expression andFcgR-mediated functions are increased during pregnancy. These results support the hypoth-esis that nonspecific immunity as represented by FcgR expression and function is increasedduring pregnancy. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Fcg receptors; Monocyte; Human pregnancy; Innate immunity

* Corresponding author. Fax: +1 217 5243227; e-mail: [email protected]

0165-0378/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.PII S0165-0378(98)00076-X

D. Da6is et al. / Journal of Reproducti6e Immunology 40 (1998) 119–128120

1. Introduction

A balance exists during pregnancy in which the body must preventrejection of the antigenically foreign fetus while at the same time maintainsufficient maternal host defense mechanisms to combat infection. Thus,while some immune functions are decreased during pregnancy, a compensa-tory mechanism may exist in which other mechanisms of immunity areincreased. A hypothesis regarding the immunology of pregnancy has beenformulated which states that to maintain sufficient maternal host defense,non-specific immunity is increased to compensate for the decrease in specificimmunity. This hypothesis is supported by several studies during pregnancy.Total T cell number (Maclean et al., 1991, 1992; Montcharmont andBonnard, 1992) and cytotoxic lymphocyte activity are both decreased(Nakamura et al., 1993); monocyte number and phagocytosis (Plum et al.,1978; Koumandakis et al., 1986; Shibuya et al., 1991), and serum levels ofIL2, IL1b, IL6, tumor necrosis factor and macrophage-colony stimulatingfactor (M-CSF) are increased (Hauser et al., 1987; Opsjn et al., 1993;Praloran et al., 1994).

The monocyte-macrophage plays a key role in linking the specific andnon-specific immune systems through MHC class II molecules and receptorsfor the Fc portion of antibody known as Fc gamma receptors (FcgR).Three classes of FcgR are present on monocytes, FcgRI (CD64), FcgRII(CD32) and FcgRIII (CD16) (Van de Winkel and Anderson, 1991). Thereare five primary functions mediated by FcgR; triggering of phagocytosis,production of reactive oxygen intermediates, participation in antibody-de-pendent cell-mediated cytotoxicity (ADCC), release of TNF-a, and releaseof IL6.

Pregnancy is associated with increased monocyte numbers in all threetrimesters of pregnancy (Plum et al., 1978), an enhanced ability of mono-cytes to migrate randomly through 3-mm filters (Hawes et al., 1980), and anincreased capacity of monocytes to ingest non-opsonized Staphylococcusepidermidis (Koumandakis et al., 1986). These data indicate that monocytesmay not only be increased in numbers, but they may also exhibit increasedfunction. Preliminary flow cytometric studies in this laboratory showed asimultaneous increase in monocyte CD64 and CD32 expression in the latestages of pregnancy. These results are consistent with those reported byShibuya et al. (1991), who observed an increase in CD32 in peripheral bloodneutrophils of pregnant women in the third trimester. These authors sug-gested that the phenomenon of increased nonspecific immunity as repre-sented by phagocytosis in pregnant women may be due, in part, to increasedFcgR-mediated function.

D. Da6is et al. / Journal of Reproducti6e Immunology 40 (1998) 119–128 121

This study was conducted to determine if the increase in CD64 and CD32expression by monocytes was a generalized phenomenon throughout preg-nancy. Simultaneously, the level of MHC class II expression was determinedin these same cells as an indicator of cell activation.

2. Materials and methods

2.1. Experimental subjects

Peripheral blood specimens from healthy volunteers on no medicationwere collected by venipuncture. Informed consent was obtained from allsubjects. For studies involving pregnant women, each group of subjectsconsisted of eight to ten healthy nulliparous or multiparous women aged21–39 years in one of the three trimesters of pregnancy; multiparoussubjects participated in the study only if previously normal pregnancies wererecorded. These studies were cross sectional in nature.

Control subjects were age-matched, premenopausal women. A previousstudy had demonstrated no difference in the mean channel fluorescence(MCF) of anti-CD64 staining of samples taken at various times during themenstrual cycle (Davis, unpublished).

2.2. Flow cytometric analysis of FcgR expression

Whole blood (100 m l) anticoagulated with sodium heparin was collectedfrom subjects and added to each tube of a panel of monoclonal antibodiesas illustrated in Table 1. The antibodies were directly conjugated withfluorescein (FITC) and/or phycoerythrin (PE) for the flow cytometric assayof FcgR expression. The HLA-DR monoclonal antibody was included toassess the activation state of the monocytes (Adams, 1991). Following a 1-hincubation at 4°C, the cells were washed once to remove unbound antibody,

Table 1Panel of monoclonal antibodies utilized

Monoclonal antibody Specificity

Isotype controlMsIgG1FITC/MsIgG2aPECD3FITC/CD14PE T cells/monocytes

FcgRICD64FITCCD32FITC FcgRIIHLA-DRFITC MHC class II


Fig. 1. Monocyte CD64 expression during pregnancy. Peripheral blood was drawn fromindividuals during the three trimesters of pregnancy and stained with FITC-conjugatedanti-CD64 monoclonal antibody (anti-32.2). The analysis gate was set on the monocytes andthese cells were analyzed on a Becton-Dickinson FACSvantage flow cytometer. The valuesrepresent the mean channel fluorescence (MCF)9SEM. *Represents a P value of less than0.05.

then subjected to hypotonic lysis using Immunolyse (Coulter Corporation,Hialeah, FL) to remove the erythrocytes. Specimens were analyzed on aBecton-Dickinson FACSvantage or Coulter Epics C flow cytometer. Mo-nocytes were gated initially by forward angle and right angle light scatter.Purity of monocytes was assessed by the CD3/CD14 monoclonal anti-body combination and all specimens were at least 90% pure (CD14+ );once the position of the monocytes in the scattergram was established,this antibody combination was eliminated from the panel. The meanfluorescent peak channels for CD64, CD32 and HLA-DR were recorded.As an additional means of quantitating FcgR expression, an antigenquantitation kit, Quantum Simply Cellular, was utilized (Sigma, St.Louis, MO).

2.3. Measurement of FcgR-mediated phagocytosis by human monocytes

The FcgR-mediated phagocytic capacity of monocytes from pregnantwomen was measured by adherent monocyte ingestion of anti-D-sensi-tized human Rh-positive erythrocytes. The assay is a variation of onedeveloped by Gallagher and Branch (1983). Monocytes were isolatedfrom peripheral blood by density gradient separation followed by adher-ence to glass coverslips for 1 h at 37°C and 5% CO2. The nonadherent


cells were removed by rinsing in phosphate-buffered saline (PBS). Cover-slips were placed in 35 mm Petri dishes and 1 ml of 0.25% Rh-positive R1rerythrocytes sensitized with a 1:10 dilution of anti-D serum (EA) waslayered over the monocytes. The cultures were incubated for 3 h at 37°Cand 5% CO2, rinsed to remove unassociated EA, fixed and stained withWright–Giemsa stain, then mounted onto standard microscope slides.Slides were coded and monocytes were examined for ingested erythrocytesusing a light microscope.

The following indices were determined by an investigator blinded to theexperimental groups: (1) the percentage of phagocytic monocytes based on200 monocytes scored; (2) the phagocytic index (PRBC), defined as the totalnumber of erythrocytes ingested per 200 monocytes scored.

2.4. Statistical analysis

Descriptive measures were used in the initial analysis of all data includingarithmetic mean and standard deviation. The mean values of the test groupswere compared to the nonpregnant control group using an independentt-test. In some cases, the data were converted to percent change as com-pared to the control group then subjected to a t-test. A P value of less than0.05 was considered significant.

Fig. 2. Monocyte CD32 expression during pregnancy. Peripheral blood was drawn fromindividuals during the three trimesters of pregnancy and stained with FITC-conjugatedanti-CD32 monoclonal antibody (anti-IV.3). The analysis gate was set on the monocytes andthese cells were analyzed on a Becton-Dickinson FACSvantage flow cytometer. The valuesrepresent the mean channel fluorescence (MCF)9SEM. *Represents a P value of less than0.05.


Table 2Expression of monocyte CD64 and CD32 during pregnancya

% Change in CD32CD32 ABCGroup % Change inn CD64 ABCCD64

—104 471Controlb —9 20 12037.8d 123 950d 18.6dTrimester 1c 10 27 743d

24.9d130 505dTrimester 2c 87.4d11 37 709d

105.3d 139 407d 33.4dTrimester 3c 9 41 307d

a Peripheral blood was collected from the various groups of women and stained withanti-CD64 and anti-CD32 monoclonal antibodies. The monocytes were analyzed on aBecton-Dickinson FACSvantage flow cytometer. ABC refers to the number of antibodiesbound per monocyte as determined by a standard curve of beads with different antibodybinding capacities (Quantum Simply Cellular) and approximates the number of Fc receptorsper cell.b Control group consists of nonpregnant non-contraceptive women.c CD64 and CD32 expression of monocytes from women in the three trimesters of preg-nancy.d PB0.05 as compared to control group.

3. Results

3.1. Monocyte surface FcgR expression is increased during pregnancy

Surface expression of FcgRI (CD64) and FcgRII (CD32) was measuredin pregnant women using monoclonal antibodies against CD64 (anti-32.2)and CD32 (anti-IV.3) and flow cytometric analysis. These values werecompared to monocyte FcgR expression in age-matched nonpregnant con-trol subjects. Fig. 1 illustrates the expression of monocyte CD64 as repre-sented by mean channel fluorescence (MCF). The elevation in CD64expression was significant in all three trimesters. A progressive increase inthe MCF of CD64 throughout the course of pregnancy was noted. Fig. 2shows the expression of CD32 in the three trimesters of pregnancy asrepresented by mean channel fluorescence. The expression of CD32 was alsoincreased in each of the three trimesters of pregnancy. The expression ofHLA-DR was also measured to assess the activation state of the monocytes,as monocytes may exhibit an increase in FcgR expression upon activation.There was no significant change in HLA-DR expression in any of thegroups tested (data not shown).

The mean channel fluorescence values are only a relative measure ofreceptor density. Therefore, an antigen quantitation kit, Quantum SimplyCellular (Sigma) was utilized to determine the amount of antibody bound tothe cell surface (ABC). In Table 2, the expression of monocyte CD64 and


CD32 as represented by the ABC is shown. These numbers approximate thenumber of Fc receptors per cell. A significant increase in the CD64 ABCwas found in all three trimesters as compared to nonpregnant controlsubjects with respective increases of 37.9, 87.4, and 105.3%. Monocytesfrom subjects in the three trimesters of pregnancy also exhibited a statisti-cally significant increase in the CD32 ABC with respective increases of 18.6,24.9, and 33.4%.

3.2. Monocyte FcgR-mediated phagocytosis is increased during pregnancy

Monocyte surface FcgR expression is increased during pregnancy (Figs. 1and 2). In other studies, an increase in FcgR expression has been shown toresult in elevated FcgR-mediated functions (TeVelde et al., 1992). Toconfirm if a similar correlation existed, monocytes from pregnant womenwere isolated and assayed for their ability to ingest anti-D-sensitized humanRh-positive erythrocytes. Initial analysis of the relationship between CD64and CD32 expression and phagocytosis revealed R2 values of greater than0.76 as illustrated in Fig. 3. These values indicate that the phagocytic assayprovides a good means of measuring FcgR-mediated function. The percent-age of monocytes from pregnant women and nonpregnant controls that had

Fig. 3. CD64 expression versus FcgR-mediated phagocytosis. Peripheral blood was drawnfrom 20 pregnant individuals and CD64 expression was correlated with adherent monocyteingestion of anti-D-sensitiized Rh-positive erythrocytes. The solid line represents the resultsof regression analysis and the dotted lines represent a 95% confidence interval. Regressionanalysis revealed an R2 value of 0.76.


Fig. 4. Percentage of phagocytic monocytes from the three trimesters of pregnancy. Thepercentage of monocytes which had ingested erythrocytes per 200 monocytes scored weredetermined by adherent monocyte ingestion of anti-D-sensitized Rh-positive erythrocytes.*Represents a P value of less than 0.05 as compared to a nonpregnant control group.

ingested erythrocytes was determined and is illustrated in Fig. 4. Anincreased percentage of phagocytic monocytes was observed in all threetrimesters of pregnancy as compared to the nonpregnant control group

Fig. 5. FcgR-mediated phagocytosis of anti-D-sensitized erythrocytes by human monocytesfrom women in the three trimesters of pregnancy. The number of erythrocytes ingested per200 monocytes scored from pregnant and postmenopausal women was compared to anonpregnant control group. *Represents a P value of less than 0.05.


(PB0.05). The total number of erythrocytes ingested/200 monocytes wasalso determined (phagocytic index). When these values were compared tothe nonpregnant control group, a significant increase in the phagocyticindex was observed in all three trimesters of pregnancy as shown in Fig. 5.

4. Discussion

Human leukocytes possess at least three different FcgR which bindvarious subclasses of human IgG (Unkeless et al., 1988). These receptors,which have unique cellular distribution patterns, allow immunocompetentand inflammatory cells to interact with antibody or antigen–antibodycomplexes. This interaction leads to cellular effector functions (ADCC;phagocytosis) and regulation of immune reactivity (cell differentiation;lymphokine secretion; cell surface receptor expression). These characteristicssuggest that FcgR may play an important role in the interaction betweeninnate and acquired immune responses. For example, an increase in thenumber of FcgR may enhance the phagogytic capability of the cells.

Our findings that monocyte surface FcgR expression progressively in-creases throughout pregnancy confirm and extend a recent study by Shibuyaet al. (1991), who reported that peripheral blood neutrophils show increasedexpression of CD32 during pregnancy. If these increases in receptorscorrelate with a simultaneous increase in phagocytosis, these results wouldargue that non-specific immune capabilities are enhanced during pregnancy.This enhancement may, in turn, reflect the presence of a mechanism bywhich the pregnant host deals with potential infections during a time whenspecific immune reactivity is blunted.

Our data further support the hypothesis that increased FcgR expressionresults in increased nonspecific immune functions. The phagocytic assayutilized in this study demonstrated that the percentage of phagocyticmonocytes was increased during pregnancy and that their phagocytic capac-ity was also increased. These data are in agreement with a previous reportin which the phagocytosis of S. epidermidis by monocytes from pregnantwomen was reported to increase gradually throughout pregnancy (Kouman-dakis et al., 1986). Although phagocytosis of non-opsonized S. epidermidisis mediated by the interaction between monocyte mannose–fucose receptorsand carbohydrate residues on the surface of the bacteria, rather thanthrough engagement of FcgR (Auger and Ross, 1992), both types ofphagocytosis are immunologically nonspecific. These two studies, then,support the hypothesis that nonspecific immunity as represented by phago-cytosis is increased during pregnancy.


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Nonspecific immunity in pregnancy: monocyte surface Fcγ receptor expression and function