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(Journal of Leukocyte Biology. 2002;72:874-884.)
© 2002 by Society for Leukocyte Biology

Monocytes are progressively activated in the circulation of pregnant women

Patrizia Luppi^*, Catherine Haluszczak^*, Dawn Betters^*, Craig A. H. Richard, Massimo Trucco^* and Julie A. DeLoia

^* Division of Immunogenetics Department of Pediatrics, Rangos Research Center, Children’s Hospital of Pittsburgh, and
Department of Obstetrics, Gynecology, and Reproductive Sciences and Magee Women’s Research Institute, University of Pittsburgh, School of Medicine, Pennsylvania

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pregnancy is characterized by the presence of generalized leukocyte activation. We used flow cytometry to investigate changes in phenotype and intracellular cytokines of circulating granulocytes, monocytes, and T lymphocytes of pregnant women during gestation. We report that peripheral circulation of pregnancy is characterized by an increased percentage of granulocytes and a decrease in lymphocytes. The proportion of monocytes remains stable throughout gestation; however, a progressive up-regulation of surface markers CD11a, CD54, and CD64 was detected. Monocytes also showed higher production of interleukin (IL)-12 and IL-1ß compared with the nonpregnant state, and granulocytes had greater potential to synthesize IL-8. All these changes were particularly marked in late gestation. T lymphocytes did not have any characteristics of the activated state and showed a decreased IL-6 production. These findings demonstrate that activation of maternal monocytes and granulocytes increases during pregnancy and support the idea that pregnancy results in an elevation of the innate immune system and suppression of the adaptive immune system.

Key Words: leukocytes • intracellular cytokines • adhesion molecules • pregnancy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The maternal immune system is modulated in several ways during pregnancy such that a normal, robust response to pathogens is maintained and at the same time allows the fetus to survive and thrive. The concept that pregnancy is an immunosuppressive state permitting the fetal allograft to implant and grow held center stage for years [¹ ]. The physiological protection from fetal rejection is believed to be dependent on a T helper cell type 2 (TH2) immune response at the maternal-fetal junction [² ]. In fact, several groups have shown that in rodent models and humans, disruption of the cytokine balance at the interface can lead to activated leukocytes [³ ] and causes pregnancy failure [⁴ , ⁵ ]. Although there is little doubt that there can be down-regulation, deletion, or anergy of T cells at least in the pregnant rodent [^{6 7 8} ], new data shed doubt on the T(H)1/T(H)2 paradigm by demonstrating how interferon- (IFN-; a TH1 cytokine) but not interleukin (IL)-4 and IL-10 (TH2 cytokines) is crucial for a successful pregnancy [^{9 10 11 12 13} ]. Overall, these data suggest that fetal tolerance is a complex phenomenon, and numerous adaptations could operate in parallel to generate and maintain a state of immunosuppression [¹⁴ ].

However, this immunosuppressive condition does not exist throughout the entire body during pregnancy. In particular, circulating immune cells display many aspects of a proinflammatory state, as indicated by primed or activated leukocytes of the maternal innate immune system [^{15 16 17 18} ]. One of the most remarkable modifications of the immune system during pregnancy is the increase in the total number and proportion of granulocytes in the circulation [¹⁷ , ¹⁹ , ²⁰ ]. Together with monocytes, these cells are activated, as demonstrated by up-regulation of cell surface adhesion molecules, increased production of reactive oxygen species, and enhanced phagocytosis [^{16 17 18} , ²¹ ].

Sacks et al. [¹⁵ ] pooled these data and suggested that placental cellular products act systemically to prime and activate monocytes and other cells of the innate immune system. If this hypothesis is meritorious, then markers of activation of the innate system should rise concurrently with placental mass. Thus, the timing of the observed immune changes during gestation is an important test of this model.

In this report, we present longitudinal data of immune cell activation markers throughout gestation and in the nonpregnant state. We also present data showing a concordance of phenotypic characteristics with the functional capacity to produce cytokines. Modifications of phenotype and function are consistent with presence of a progressive increase in monocyte activation throughout pregnancy, reaching a peak at 29–36 weeks of gestation. Granulocytes also appeared to be activated during pregnancy, and T lymphocytes did not show any characteristics of the activated state. These findings support the belief that the innate immune system is activated, and the adaptive immune system is suppressed during pregnancy. Finally, we present data showing further activation of monocytes as a result of labor.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
A total of 17 pregnant women (mean age±SD; 31±4; range 21–37) entered the study during the first trimester of pregnancy. All the women were healthy and without any apparent coexisting disease. Of the 17 women participating in this study, one had a miscarriage at 11 weeks, and the remaining women had normal pregnancies without any complications. This study was approved by the Institutional Review Board of the Magee Women’s Hospital (Pittsburgh, PA).

Design
This was a prospective study in which changes in the proportion of the different subsets of circulating leukocytes, leukocyte adhesion molecule expression, and intracellular cytokine production were repeatedly investigated over gestation, from 3–36 weeks. The first blood sample was collected between 1 and 12 weeks of gestation in eight women; a second sample was collected between 13 and 28 weeks of gestation in 17 women; and a third sample was collected between 29 and 36 weeks of gestation in 13 women. For eight women, a blood sample was also collected before pregnancy or in the postpartum period (from 10 to 22 months after partum). None of the participants was in labor at the time of blood sampling.

Blood sampling
Peripheral venous blood (10 ml) was drawn in sodium heparin tubes, kept at room temperature, and processed for flow cytometry staining within 1 h from blood collection.

Whole blood assay for the analysis of adhesion molecules expression
Surface staining
Whole blood (50 µL) was incubated for 15 min in the dark at room temperature with 20 µl of the following anti-human monoclonal antibodies (mAb): fluorescein isothiocyanate (FITC)-conjugated mAb to -CD19, -CD4, -CD15, -CD56, -CD11a/lymphocyte function-associated antigen-1, -CD66b; phycoerythrin (PE)-conjugated mAb to -CD3, -CD8, -CD62L, -CD54, -CD49d; cy-chrome-conjugated mAb to -CD64, -CD11b/Mac-1; PE-cyanin 5.1-conjugated mAb to -CD45; peridin chlorophyll protein (PerCP)-conjugated mAb to -CD3; and allophycocyanin (APC)-conjugated mAb to -CD14. Each mAb was used at saturating concentrations optimized by initial titrations. The negative-control panels were comprised of mixtures of isotype-negative controls (Sigma Chemical Co., St. Louis, MO, and BD Pharmingen, San Diego, CA), diluted to an equivalent immunoglobulin concentration. Table 1 lists all antibodies used. Erythrocytes were lysed with 2 ml 1x PharM Lyse^TM (BD Pharmingen; cat. #35221E). Samples were then washed once with 2 ml washing buffer (phosphate-buffered saline with 0.5% bovine serum albumin and 0.1% sodium azide; Sigma Chemical Co.) and were stored in 1% paraformaldheyde at 4°C until measured by flow cytometry within 4 h.

Whole blood assay for the production of intracellular cytokines by circulating leukocytes
Intracellular staining
Blood (500 µl) was diluted 1:1 with RPMI 1640 (Gibco-BRL, Life Technologies Inc., Rockville, MD) containing 1 µg/ml phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.; cat. #P-8139), 1 µg/ml ionomycin (Sigma Chemical Co.; cat. #I-0634), and 1 µl brefeldin A (BFA; Golgiplug, Becton Dickinson; cat. #555029) in 5 ml Falcon tubes (Becton Dickinson; cat. #352063). A separate 500 µl blood was diluted 1:1 with RPMI 1640 (Gibco-BRL, Life Technologies) containing 1 µg/ml lipopolisaccharide (LPS) from Escherichia coli E26:B6 (Sigma Chemical Co.; cat. #L-2654) and 1 µl BFA in a separate 5 ml Falcon tube. For each subject, an unstimulated sample was set up in parallel with 1 µl BFA only to evaluate the spontaneous, intracellular cytokine production of the same cells at the time of collection. After 4 h incubation at 37°C in a 5% CO₂ humidified atmosphere, 100 µl blood was transferred to fresh tubes, each containing 20 µl of the following anti-human mAb: anti-CD3 mAb labeled with PerCP (Becton Dickinson); anti-CD14 mAb labeled with APC (Becton Dickinson) or with FITC (BD Pharmingen); or anti-CD15 mAb labeled with FITC (Dako Corp., Carpinteria, CA). The tubes were placed in the dark for 15 min. Red blood cells were lysed using 2 ml 1x FACS^TM lysing solution (Becton Dickinson; cat. #349202) for 10 min. The tubes were then centrifuged at 2000 rpm for 5 min, and the pellets were resuspended in 0.5 ml 1x FACS^TM permeabilizing solution (Becton Dickinson; cat. #340457) and incubated for 10 min in the dark. Cells were then washed with 2 ml washing buffer and centrifuged at 2000 rpm for 5 min. The pellets were then resuspended in the appropriate anticytokine mAb and incubated for 30 min in the dark. The anticytokine mAb were as follows: FITC-conjugated anti-human IL-2 (Becton Dickinson); PE-conjugated anti-human IFN- (Becton Dickinson), anti-human IL-1ß (Becton Dickinson), anti-human IL-6 (BD Pharmingen), and anti-human IL-8 (BD Pharmingen); and APC-conjugated anti-human tumor necrosis factor (TNF-; Becton Dickinson) and anti-human IL-12 (BD Pharmingen; Table 2 ). Each mAb (20 µl) was used at saturating concentration predetermined by titration experiments. For every subject tested, a set of negative-control panels comprised of isotype controls for the surface antibodies as well as for the intracellular anticytokine antibodies was used. The intracellular isotype controls were added after the permeabilization step. Cells were then washed twice with 2 ml washing buffer and centrifuged at 2000 rpm for 5 min. The pellets were resuspended in 200 µl washing buffer and immediately run on a Becton Dickinson FACSCalibur four-color instrument and analyzed by CELLQuest software (Becton Dickinson). For each sample, at least 30,000 events were acquired for each antibody combination, and data were saved for later analysis.

Blocking experiments
An additional control for the specificity of the antibodies for the cytoplasmic cytokines, specific binding was blocked in random samples of the unstimulated and PMA- and LPS-stimulated sets by using a molar excess of unlabeled (i.e., purified) anticytokine antibodies [²² ]. Purified mAb were added in each set of tubes after the permeabilization step and incubated for 1 h at room temperature. Identical amounts of the relevant, fluorescently conjugated, anticytokine antibody were added to the tubes, and the cells were washed twice with 2 ml washing buffer, centrifuged at 2000 rpm for 5 min, resuspended in 400 µl washing buffer, and immediately run on a Becton Dickinson FACSCalibur four-color instrument. Specificity of the labeled anticytokine mAb was demonstrated by the complete block of staining in the presence of the unlabeled anticytokine mAb.

Statistics
Changes in the percentage of the leukocyte subpopulations and in the geometric mean fluorescence values for the different leukocyte surface antigens between normal pregnant and nonpregnant women were compared using a two-tailed t-test with unequal variances. Changes in constitutive and stimulated intracellular cytokine production by CD3⁺, CD14⁺, and CD15⁺ cells between pregnant and nonpregnant women were compared using a two-tailed t-test with unequal variances. A P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Percentage distribution of circulating leukocytes throughout pregnancy
Circulating leukocytes in pregnant women showed a significant increase in the proportion of granulocytes and a decrease in lymphocytes (Fig. 1 ). The increase in granulocytes was detected at the earliest time-point analyzed (3 weeks), reaching significance at 13–28 weeks compared with nonpregnant women (P=0.002). After this stage, the proportion of granulocytes remained significantly higher compared with nonpregnant women (P=0.003). The decrease in lymphocytes was also observed as early as the first trimester (P=0.029) with the lowest proportion detected at 13–28 weeks (P=0.001). At 29–36 weeks of gestation, the proportion of lymphocytes was still significantly lower than in nonpregnant women (P=0.002) but was higher than what was observed at 13–28 weeks (Fig. 1) . Notably, the percentage of monocytes did not show any significant changes throughout pregnancy and was similar to nonpregnant controls.

View larger version (15K): [in this window] [in a new window]   Figure 1. Percentage distribution of circulating leukocytes throughout pregnancy. Shown is the proportion of lymphocytes, granulocytes, and monocytes in peripheral blood of women in the nonpregnant state (n=8), at 1–12 weeks (n=8), 13–28 weeks (n=17), and at 29–36 weeks of gestation (n=13), as determined by flow cytometry. Data are expressed as mean percentage ± SEM in the different groups. The percentage distribution of the different subsets of leukocytes was calculated by setting gates around the different circulating elements based on their SSC and FSC characteristics. *, Significant difference between nonpregnant and pregnant women.

Changes in adhesion molecules expression on circulating leukocytes throughout pregnancy
An outline of experimental design and antibody combinations used to determine expression of surface markers by flow cytometry in each experiment is shown in Figure 2A .

View larger version (42K): [in this window] [in a new window]   Figure 2. Outline of experimental design and antibody combinations for evaluation of the expression of surface antigens and intracellular cytokine productive capacity in circulating leukocyte subsets by four-color flow cytometry. In each experiment, an aliquot of peripheral blood from each individual was used for staining surface antigens (A) and for intracellular cytokines (B). Briefly, staining for surface antigens was performed by incubating whole blood (50 µl/tube) with the different combinations of mAb against leukocyte surface antigens and isotype controls, as depicted in A, described in Materials and Methods, and listed in Table 1 . In B, a schematic outline of the whole blood assay for intracellular cytokine evaluation is represented. Briefly, 500 µl whole blood from each individual was diluted with RPMI 1640 and incubated for 4 h at 37°C in the presence of BFA, an inhibitor of cytokines secretion, to evaluate spontaneous, intracellular cytokine production by the different subset of leukocytes. Two separate aliquots of 500 µl blood were diluted with RPMI 1640 and incubated for 4 h at 37°C with PMA/ionomycin or LPS in the presence of BFA. After incubation, unstimulated and stimulated blood (100 µl/tube) was stained with the different mAb for leukocyte surface markers, intracellular cytokines, and isotype controls, as shown in B, described in Materials and Methods, and listed in Tables 1 and 2 .

View larger version (10K): [in this window] [in a new window]   Figure 3. Increased expression of CD11a (A), CD64 (B), and CD54 antigens (C) on monocytes during pregnancy. The results show changes in mean fluorescence for CD11a (A), CD64 (B), and CD54 antigen (C) on monocytes in the nonpregnant state (n=8), at 1–12 weeks (n=8), 13–28 weeks (n=17), and 29–36 weeks of gestation (n=13). The analysis of the expression of the different surface markers on monocytes (CD14+) was performed by setting specific gates for the monocyte population based on dot-plot graphs of SSC versus CD14+ cells. Data are expressed as geometric mean fluorescence values ± SEM for each marker in the different groups of women. *, Significant difference in cell surface expression for the stated surface marker between nonpregnant and pregnant women.

T lymphocytes
The expression of adhesion molecules on T lymphocytes was essentially unchanged throughout pregnancy (data not shown).

Intracellular cytokine production in circulating leukocytes throughout pregnancy
To evaluate the cytokine productive capacity of circulating leukocytes, we used PMA and LPS activation, as they are the most robust stimuli for most cytokines [²³ , ²⁴ ]. PMA stimulates cytokine production by directly activating protein kinase C (PKC) [²⁵ ], and LPS, a glycolipid component of the outer membrane of the Gram-negative bacteria [²⁶ ], activates PKC after interaction with the CD14 antigen present on the surface of monocytes and granulocytes [²⁷ , ²⁸ ]. We used PMA to analyze the cytokine productive capacity of T lymphocytes, granulocytes, and monocytes, and LPS was used for monocytes and granulocytes. BFA, an inhibitor of Golgi-mediated protein secretion, was added to the samples to allow for the measurement of intracellular cytokine production [²⁹ , ³⁰ ]. The intracellular cytokine productive capacity of the different leukocyte subsets was then assessed by flow cytometry by setting gates encompassing the positive elements for each specificity on dot-plot graphs of SSC versus CD3⁺ (T lymphocytes), CD14⁺ (monocytes), and CD15⁺ (granulocytes) cells, as described in Materials and Methods. Expression of cytokines was not detected outside the gated leukocyte subset. An outline of experimental design and antibody combinations used in each experiment is shown in Figure 2B .

Monocytes
As monocytes generally serve as the principal reservoirs of inflammatory cytokines of the circulating leukocytes, we analyzed the unstimulated and stimulated intracellular production of IL-1ß, IL-6, IL-8, IL-12, and TNF- in women throughout pregnancy. Monocytes were identified by the CD14 membrane marker for this analysis. An example of flow cytometry detection of intracellular cytokine production is shown in Figure 4 . The results depicted in Figure 5A indicated that there was an increase in the percentage of cells spontaneously producing IL-12 throughout pregnancy with the highest frequency at 29–36 weeks. At this time period, the percentage of CD14⁺/IL-12⁺ cells (33.8%±11.14) was significantly higher when compared with the proportion of the same cells in nonpregnant women (5.4%±5.1; P=0.04; Fig. 5A ).

View larger version (32K): [in this window] [in a new window]   Figure 4. Cytokine production by monocytes of a pregnant woman at 34 weeks of gestation. Shown is a representative example of intracellular cytokine production by monocytes after stimulation with (B) or without (A) LPS for 4 h in the presence of the inhibitor of cytokine secretion BFA. After incubation, 100 µl blood was stained with anti-human mAb against CD14 labeled with FITC or with APC and was placed in the dark for 15 min. After lysis of red blood cells, samples were resuspended in saturating concentrations of the following anticytokine mAb and incubated for 30 min in the dark: PE-anti-human IL-1ß, IL-6, and IL-8 and APC-anti-human TNF- and IL-12. Cells were then washed and immediately run on a FACSCalibur instrument (Becton Dickinson). For each sample of the unstimulated and LPS-stimulated set, dot-plot graphs of SSC versus CD14+ cells were drawn, and a tight gate was created, encompassing the "bright" elements for this specificity. Within the CD14 gate, CD14+ elements expressing the different cytokines were analyzed by drawing separate dot-plots. Percentage of cytokine-positive cells was then defined by setting lower limits for cytokine positivity determined from each antibody combination using intracellular negative isotype controls.

View larger version (32K): [in this window] [in a new window]   Figure 5. Intracellular IL-1ß, IL-6, IL-8, IL-12, and TNF- production in unstimulated (A), PMA/ionomycin-stimulated (B), and LPS-stimulated (C) monocytes during pregnancy. Flow cytometry analysis of the constitutive, intracellular cytokine expression (A) and following PMA/ionomycin activation (B) or LPS activation (C) in monocytes of nonpregnant and pregnant women at 1–12 weeks (vertical-line bars), 13–28 weeks (open bars), and 29–36 weeks (horizontal-line bars) of gestation. Whole blood was cultured with no stimulus (A) or was activated with PMA/ionomycin (B) or LPS (C) for 4 h and stained with mAb against CD14 and against the different cytokines, as described in Materials and Methods. The inhibitor of cytokine secretion BFA was added in each sample to prevent cytokine secretion. Data are expressed as mean percentage ± SEM of CD14+ cells positive for the cytokines tested in the different groups of women. *, Significant difference of intracellular production for the stated cytokine between nonpregnant and pregnant women.

Upon LPS stimulation, almost all monocytes produced IL-1ß, IL-6, IL-8, and TNF- in all groups of women. We detected an increase in the production of IL-12 in late pregnancy with values at 13–28 weeks of 16.7%±7.4 cytokine-positive cells as compared with nonpregnant women (1%±0.6; P=0.025; Fig. 5C ). Moreover, there was a tendency toward a decrease in the potential to make IL-8 in late gestation, with a significant decline at 13–28 weeks (94.2%±1.8; P=0.011) and at 29–36 weeks (95.3%±1; P=0.019) as compared with nonpregnant women (98.5%±0.4; Fig. 5C ).

Granulocytes
Granulocytes were identified by the CD15 membrane marker. We focused on the analysis of IL-8 production, which is one of the most important chemokines produced by granulocytes [³¹ ] and on the proinflammatory cytokine IL-6. In the unstimulated state, granulocytes constitutively produced IL-6 and IL-8 (Fig. 6A ). There was a tendency toward an increase in the spontaneous and PMA-stimulated production of IL-8 in late gestation as compared with nonpregnant women, with significant values at 29–36 weeks (74.1%±5.1 vs. 46.8%±7.9; P=0.021). Conversely, the potential to produce IL-6 was decreased in granulocytes (Fig. 6B) .

View larger version (26K): [in this window] [in a new window]   Figure 6. Intracellular IL-6 and IL-8 production in unstimulated (A), PMA/ionomycin-stimulated (B), and LPS-stimulated (C) granulocytes during pregnancy. Flow cytometry analysis of the constitutive, intracellular cytokine expression (A) and following PMA/ionomycin activation (B) or LPS activation (C) in granulocytes of nonpregnant and pregnant women at 1–12 weeks (vertical-line bars), 13–28 weeks (open bars), and 29–36 weeks (horizontal-line bars) of pregnancy. Whole blood was cultured with no stimulus (A) or was activated with PMA/ionomycin (B) and LPS (C) for 4 h and stained with mAb against CD15 and against the different cytokines, as described in Materials and Methods. The inhibitor of cytokine secretion BFA was added in each sample. Data are expressed as mean percentage ± SEM of CD15+ cells positive for the cytokines tested in the different groups of women. *, Significant difference of intracellular production for the stated cytokine between nonpregnant and pregnant women.

T lymphocytes
Unstimulated peripheral blood T lymphocytes spontaneously produce negligible amount of cytokines. Activated T cells produce a variety of cytokines. In this study, we evaluated the PMA-stimulated production of IL-2, IFN-, TNF-, and IL-6. Analysis of T lymphocytes at different time-points throughout pregnancy did not show presence of significant changes in the percentage of cells positive for IL-2, IFN-, and TNF- compared with nonpregnant samples (Fig. 7 ). However, T cells had a reduced ability to produce IL-6 throughout pregnancy, with significant values at 1–12 weeks (30.8%±12; P=0.042), 13–28 weeks (25.3%±5.7; P=0.005), and at 29–36 weeks (27.3%±9.8; P=0.014) compared with nonpregnant women (67.3%±10.3; Fig. 7 ).

View larger version (26K): [in this window] [in a new window]   Figure 7. Intracellular IL-2, IFN-, TNF-, and IL-6 production in PMA/ionomycin-stimulated T lymphocytes during pregnancy. Flow cytometry analysis of the PMA/ionomycin-stimulated intracellular cytokine production in T lymphocytes of nonpregnant and pregnant women at 1–12 weeks (vertical-line bars), 13–28 weeks (open bars), and 29–36 weeks (horizontal-line bars) of pregnancy. Whole blood was stimulated with PMA/ionomycin for 4 h and stained with mAb against CD3 and against the different cytokines as described in Materials and Methods. The inhibitor of cytokine secretion BFA was added in each sample. Data are expressed as mean percentage ± SEM of CD3+ cells positive for the cytokines tested in the different groups of women. *, Significant difference of intracellular production for the stated cytokine between nonpregnant and pregnant women.

View larger version (34K): [in this window] [in a new window]   Figure 8. Demonstration of the specificity of the intracellular cytokine visualization method. Shown is a representative example of blocking of the specific binding of anticytokine antibodies in whole blood of a pregnant woman. Unstimulated, PMA/ionomycin-, and LPS-stimulated whole blood samples were analyzed for IL-6 intracellular production in CD14+ cells identified by a FITC-anti-human-CD14. The same analysis was performed on samples pre-blocked with unlabeled (i.e., purified) antibody (Block) as described in Materials and Methods. The specificity of the PE-anti-human-IL-6 mAb was demonstrated by the block of staining in the presence of the unlabeled, anticytokine mAb in the different conditions. Quadrant markers were set according to the negative intracellular isotype control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The analysis of the expression of adhesion molecules and of the unstimulated and stimulated, intracellular cytokine productive capacity of circulating leukocytes of pregnant women showed a concordance. The most marked modifications occurred in monocytes, which showed a progressive increase in activation throughout gestation, as evidenced by a significant up-regulation of CD11a, CD54, and CD64 surface antigens. Such a pattern of monocyte activation parallels the increase in placental mass. We did not find a significant increase in CD11b expression in monocytes throughout pregnancy. This finding is in apparent disagreement with our previous report [¹⁷ ], showing up-regulation of this surface marker during the third trimester of pregnancy. However, by dividing pregnant women who were or were not in labor at the time of blood draw, we found that the most significant up-regulation of this antigen appeared to be associated with the onset of labor, thus suggesting that further activation of monocytes may occur at this stage [³² ].

Cytokine expression in monocytes of pregnant women mirrored the observed, activated phenotype. The unstimulated and LPS-stimulated intracellular production of IL-12 showed a significant increase from 13 weeks of gestation onward. After PMA activation, a significant increase was detected for IL-1ß. IL-1ß and IL-12 are considered major proinflammatory cytokines secreted by activated macrophages. IL-12 is a major inducer of type 1 (cell-mediated) immune responses [³³ ], and IL-1ß up-regulates adhesion molecules, which are essential for the adhesion of leukocytes to the endothelial surface prior to emigration into the tissues [³⁴ , ³⁵ ]. IL-1ß has also been suggested to induce cervical ripening and massive infiltration of neutrophils in uterine tissues at the time of parturition [³⁶ , ³⁷ ]. These data demonstrate that in late pregnancy, monocytes have the ability to synthesize proinflammatory cytokines and are not immunosuppressed.

After 13 weeks of gestation, the one cytokine that appeared to be decreased in the PMA-stimulated samples was TNF-, suggesting tight regulation in the production of inflammatory cytokines. TNF- has potent, deleterious effects on pregnancy; administration to normal pregnant mice results in abortions [³⁸ ], programmed cell death of human trophoblasts [³⁹ ], and placental injury in the rat [⁴⁰ ]. Hormones, such as progesterone and estradiol, may modulate cytokine synthesis during pregnancy [⁴¹ ]. However, monocytes of pregnant women were fully responsive to LPS stimulation, as shown by the high number of stimulated, cytokine-positive cells. This suggests that in pregnancy, monocytes are able to respond to infections, e.g., caused by Gram-negative bacteria, and thus protect the mother’s and the baby’s health from pathogens. The only cytokine that showed a significant decrease after LPS stimulation was IL-8.

Our findings are in agreement with Sacks et al. [¹⁴ ], who reported that monocytes from normal pregnant women are primed to produce IL-12 but not TNF-. The biological significance of the increase in spontaneous and stimulated IL-12 during normal pregnancy needs further clarification. IL-12 production in monocytes is induced by many stimuli but particularly by bacteria and bacterial products (e.g., LPS) and intracellular parasites [³³ ]. The increased production of IL-12 during pregnancy may be considered as part of the generalized, inflammatory response associated with gestation, as it enhances monocyte activation and phagocytosis [³³ ], thus providing immediate protection to the mother in the event of an infection. An alternative view is that IL-12 may induce T cells for high IL-10 production [³³ ]. IL-10 is a type-2 cytokine whose levels have been shown to be elevated in normal pregnancy [⁴² ]. The fact that progesterone is able to enhance IL-10 production by T cells only in the presence of IL-12 [⁴³ ] further supports the idea that IL-12 during pregnancy may act as an immunomodulator.

Circulating granulocytes, another component of the innate immune system, are also activated during pregnancy and demonstrated greater spontaneous and PMA-stimulated production of IL-8 as compared with nonpregnant women. Granulocytes are not only producers but are also the primary targets for IL-8, responding to this mediator by chemotaxis, release of granule enzymes, respiratory burst activity, up-regulation of adhesion molecules expression, and increased adherence to endothelial cells [³¹ ]. Activated granulocytes, together with monocytes, have the potential to bind to the endothelium and migrate into tissues via interactions with endothelial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [⁴⁴ ]. ICAM-1 binds to CD11a and CD11b antigens [⁴⁵ , ⁴⁶ ], and VCAM-1 binds to CD49d [⁴⁷ ] on the surface of circulating leukocytes. Likely sites of leukocyte migration at term pregnancy and during labor are the myometrium and cervix [^{48 49 50} ], where these cells may locally release IL-8 as well other cytokines such as IFN- [⁵¹ ] and other mediators. IL-8 plays a role in tissue remodeling and cervical ripening that is important to facilitate passage of the fetus [⁵² , ⁵³ ]. Indeed, cervical ripening is considered a local inflammatory-like reaction [⁵⁴ ]. Conversely, we found that the percentage of IL-6⁺ granulocytes is decreased after PMA activation as compared with the unstimulated samples, although this decrease does not reach statistical significance. One explanation is that PMA stimulate the release of already synthesized IL-6, rather then stimulating new cytokine production. IL-6 is a proinflammatory cytokine that has been suggested to play a role in physiological mechanisms involved in uterine contractions and propagation of labor in normal pregnancy [⁵⁵ ].

In conclusion, our data demonstrate that during pregnancy, there is a progressive activation of the innate immune system, and monocytes are the most affected cell type. Through changes in surface antigen expression and selective cytokine production, monocytes could play a critical role in the modulation of the immune system [⁵⁶ ] during pregnancy. This modulation would assure immediate protection from pathogens and suppress specific immune responses (e.g., T cell functions) to tolerate the semi-allogenic conceptus. Monocytes with high CD64 antigen expression, such as has been described in patients with sepsis, are associated with high proinflammatory cytokine synthesis, release of the potent immunosuppressive prostaglandin E₂, and impaired antigen presentation, resulting in a profound disturbance of adequate interactions between monocytes and T cells [⁵⁷ , ⁵⁸ ]. Indeed, in this study, T lymphocytes did not show evidence of activation and demonstrated a significant decrease in their potential to synthesize IL-6, a potent proinflammatory cytokine.

Although the source of factor(s) responsible for the stimulation of monocytes is still uncertain, placental debris or other placental factors released into the maternal circulation stand as potential agents [¹⁵ ]. Given the fact that the state of activation of the innate immune system heightens with gestational age and shows a tendency to further increase during labor, it is tempting to speculate that inflammatory reactions may be involved in normal, spontaneous term delivery or in pre-term labor induction [³² ], as occurs with infections [¹⁸ ].

	ACKNOWLEDGEMENTS

 
This study was supported by NIH grant HD37862 to J. A. D., P. L., and M. T. The authors are indebted to Jill Brekosky for excellent technical help.

	FOOTNOTES

 
Correspondence: Patrizia Luppi, M.D., Children’s Hospital of Pittsburgh, Rangos Research Center, 3460 Fifth Avenue, Pittsburgh, PA 15213. E-mail: luppip+{at}pitt.edu

Received April 30, 2002; revised August 6, 2002; accepted August 12, 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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