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BOR - Papers in Press, published online ahead of print December 21, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044651
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BIOLOGY OF REPRODUCTION 74, 674–683 (2006)
DOI: 10.1095/biolreprod.105.044651
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Nerve Growth Factor Translates Stress Response and Subsequent Murine Abortion via Adhesion Molecule-Dependent Pathways1

Mareike Tometten , Sandra Blois , Arne Kuhlmei , Anna Stretz , Burghard F. Klapp , and Petra C. Arck 2 

Charité, Universitätsmedizin Berlin, Joint Institution of the Freie Universität and Humboldt Universität, Biomedical Research Center, Campus Virchow, 13353 Berlin, Germany

ABSTRACT

Spontaneous abortion is a frequent threat affecting 10%–25% of human pregnancies. Psychosocial stress has been suggested to be attributable for pregnancy losses by challenging the equilibrium of systems mandatory for pregnancy maintenance, including the nervous, endocrine, and immune system. Strong evidence indicates that stress-triggered abortion is mediated by adhesion molecules, i.e., intercellular adhesion molecule 1 (ICAM1) and leukocyte function associated molecule 1, now being referred to as integrin alpha L (ITGAL), which facilitate recruitment of inflammatory cells to the feto-maternal interface. The neurotrophin beta-nerve growth factor (NGFB), which has been shown to be upregulated in response to stress in multiple experimental settings including in the uterine lining (decidua) during pregnancy, increases ICAM1 expression on endothelial cells. Here, we investigated whether and how NGFB neutralization has a preventive effect on stress-triggered abortion in the murine CBA/J x DBA/2J model. We provide experimental evidence that stress exposure upregulates the frequency of abortion and the expression of uterine NGFB. Further, adhesion molecules ICAM1 and selectin platelet (SELP, formerly P-Selectin) and their ligands ITGAL and SELP ligand (SELPL, formerly P selectin glycoprotein ligand 1) respectively increase in murine deciduas in response to stress. Subsequently, decidual cytokines are biased toward a proinflammatory and abortogenic cytokine profile. Additionally, a decrease of pregnancy protective CD8{alpha}+ decidual cells is present. Strikingly, all such uterine stress responses are abrogated by NGFB neutralization. Hence, NGFB acts as a proximal mediator in the hierarchical network of immune rejection by mediating an abortogenic environment comprised of classical signs of neurogenic inflammation.

female reproductive tract, immunology, pregnancy, neuropeptides, stress

INTRODUCTION

Successful pregnancy is considered as a brilliant feat of nature, because it requires the delicate coordination and adjustment of complex biological processes to provide a nutritious environment for the fetus [1]. Concurrently, the maternal organism has to generate highly specialized adaptations to tolerate the fetus, which constitutes a "histoincompatible graft" based on the expression of paternal antigens [2].

Failure of pregnancy maintenance, usually occurring as spontaneous abortion during the first trimester in humans, is sadly a rather frequent outcome and affects 10–25% of pregnancies [1, 2]. Intriguingly, psychosocial stress aggravates the onset of such fetal losses, as pointed out by epidemiological studies [3, 4]. The murine model of DBA/2J-mated CBA/J females provides an established experimental approach to investigate the mechanisms involved in fetal rejection, particularly in pregnancies challenged by stress [5]. Based on insights derived from the CBA/J x DBA/2J model, the presence of a pregnancy-protective CD8{alpha}+ subpopulation in the uterus that is decreased in stressed mice could be identified [6]. Further, a shift from a pregnancy protective T helper 2-like (Th2l) cytokine predominance toward an abortive T helper 1-like (Th1l) cytokine profile and the loss of immunological tolerance mechanisms (expression of indoleamine-pyrrole 2,3 dioxygenase [INDO, formerly abbreviated as IDO], presence of CD4+CD25bright regulatory T cells, and synthesis of asymmetric IgG antibodies) could be observed after stress exposure [7]. Additionally, these stress-triggered pregnancy-threatening pathways are under the regulation of adhesion molecules, i.e., intercellular adhesion molecule 1 (ICAM1)/integrin alpha L (ITGAL, formerly leukocyte function associated antigen 1), and an ICAM1/ITGAL dependent recruitment of Th1l cells into the uterus leads to a subsequent Th1l polarization of decidual immune cells [7].

The response to stress perception is certainly not restricted to one single organ, i.e., the uterus. Stress generally attacks the homeostasis of the entire body and interferes with the equilibrium of nervous, endocrine, and immune systems [8–12]. Classical mediators of stress-induced immunological and endocrine alterations are neurohormones, cytokines, and catecholamines. Another potent molecule recently implicated in the pathophysiological response to stress and stress-related events is nerve growth factor (NGFB, formerly abbreviated as NGF), a member of the neurotrophin family. After exposure to stressful events, circulating and brain levels of NGFB significantly increase [13–15]. NGFB is also associated with the activation of the hypothalamic-pituitary-adrenal axis, hence representing a link between neuroendocrine and immune elements and translating environmental messages, such as stressful conditions, into pathophysiological responses [16, 17]. Recently, our group demonstrated the upregulation of NGFB and its functional receptor, neurotrophic tyrosine kinase 1 receptor type 1 (NTRK1, formerly known as tyrosine receptor kinase A), in uterine cells of stressed pregnant mice; this was associated with an increased number of abortions [18].

Interestingly, interactional processes between NGFB and endothelial cells and/or immune cells induce an upregulation of ICAM1 expression in response to NGFB and, subsequently, an increased recruitment of leukocytes into distinct tissues [19, 20]. Given the impact of NGFB on ICAM1 upregulation on the one hand, and the observation of an ICAM1-dependent homing of Th1l cells in failing pregnancies on the other hand, NGFB appears to be the candidate mediator to upregulate ICAM1 in stress-challenged pregnancies. Thus, within the hierarchical network regulating the immune integration at the feto-maternal interface, NGFB could be a proximate mediator interfering with the sensitive and complex adaptation system between the fetus and the uterine lining (decidua).

In the present study, we deliver direct evidence to support this hypothesis. In addition, we introduce a novel therapeutic approach to prevent stress-triggered miscarriages, aiming at neutralizing NGFB. We specifically investigated whether neutralization of NGFB before and after stress exposure 1) prevents abortion in stressed mice, 2) affects the local uterine NGFB expression, 3) affects the expression of adhesion molecules ICAM1/ITGAL and selectin platelet (SELP)/SELP ligand (SELPL, formerly P-Selectin glycoprotein ligand 1) and 4) has preventive effects on the decisive uterine cell subpopulation CD8{alpha}+ and the local Th1l/Th2l cytokine milieu.

MATERIALS AND METHODS

Animals

Mice (6–8 wk old) were purchased from Charles River and maintained in a barrier animal facility with a 12 h light/dark cycle. Animal care and experimental procedures were followed according to institutional guidelines and conformed to requirements of the state authority for animal research conduct (LaGetSi).

Experiment A: Dose Response of NGFB Neutralization in Stressed Pregnant Mice

Two independent experiments were performed. In experiment A, we identified the optimum dosage of intraperitoneally (i.p.) injected fractioned antiserum against NGFB (Sigma; used for monitoring beta-NGFB). We investigated different concentrations (1.6 µg/kg body weight [bdywt], 3.2 µg/kg bdywt and 4.8 µg/kg bdywt), which have been shown to be effective in other experimental settings in rodents [21–23]. After overnight cohabitation with DBA/2J males, CBA/J females with vaginal plugs (Gestational Day [gd] 0.5) were segregated and randomized to different treatment groups. The control group (n = 7) received i.p. injections of 200 µl sterile PBS on gd 5.5 and 6.5. A second group (stress group, n = 7) was exposed to stress on gd 5.5 and received i.p. injections of 200 µl sterile PBS on gd 5.5 and 6.5. Three more groups were injected with 1.6 µg/g bdywt (n = 5), 3.2 µg/g bdywt (n = 8) and 4.8 µg/g bdywt (n = 5) fractioned antiserum against NGFB in 200 µl PBS on gd 5.5 and 6.5 and were also exposed to stress on gd 5.5. To ensure the specificity of the NGFB-neutralizing antibody used, we injected additional stressed mice of the stress group with 3.2 µg/g bdywt rabbit serum (Sigma) in 200 µl PBS on gd 5.5 and 6.5. The NGFB antibody we used in these experiments is a fractioned antiserum that blocks biologically active NGFB. The manufacturer does not give any information about cross-reactivity of the investigated NGFB antiserum; however, because of the high sequence homology between members of the neurotrophin family, the rabbit sera that have been raised against NGFB may cross-react with, and possibly sequester, other neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3.

Implantations and resorption rate were determined on gd 13.5, revealing 3.2 µg/g bdywt to be the most efficient concentration for abolishing the stress effect on the resorption rate. Therefore, the subsequent experiment B was performed by using this concentration exclusively.

Application of Stress

The mice were exposed to sound stress for a duration of 24 h starting on gd 5.5. The sound stress was given by a rodent repellent device (Conrad Electronics), which randomly emitted sound 4 times per min lasting for 3 sec at a frequency of 300 Hertz and an intensity of 70 dB. The stress device was placed into the mouse cage so that the mice could not escape perception of the sound.

Resorption Rates

The mice were killed on gd 13.5, uteri were removed, and the total numbers of implantations and resorption sites (=abortions) were recorded. The resorption sites were identified by their small size and necrotic, hemorrhagic appearance compared to normal embryos and placentas. The percentage of resorption/abortion was calculated as the ratio of resorption sites and total implantation sites (resorptions plus normal implantation sites), as described previously [24].

Experiment B: Mechanisms of NGFB in Disturbed Pregnancy

In experiment B, we wished to further investigate the underlying mechanisms by which neutralization of NGFB protects pregnancy maintenance. Again, DBA/2J mated CBA/J females were segregated and randomized to different groups treated as described above (control, stress, and anti-NGFB + stress group). On gd 7.5, some of the mice of each group (control: n = 5; stress: n = 5; anti-NGFB + stress: n = 7) were narcotized, and blood cells were obtained by retro-orbital puncture collected in tubes containing heparin. Then, mice were immediately killed by neck dislocation, uteri were removed and divided into pieces. One section was frozen for immunohistochemistry and immunofluorescence (IF); from another section, uterus cells were isolated for flow cytometry. The rest of the mice (control: n = 4; stress: n = 4; anti-NGFB + stress: n = 4) were sacrificed on gd 13.5 to obtain the abortion rate calculated as described above. Because we did not observe a pregnancy-protective effect upon application of nonspecific IgG (rabbit serum) in stressed mice on gd 13.5, we refrained from analyzing uterine tissue for expression of ITGAL, SELPL, CD8 cells, and cytokine profiles by immunohistochemistry, immunofluorescence, and flow cytometry respectively on gd 7.5 in IgG-treated control and stressed mice, but performed these analyses in non-IgG-treated control and stressed mice.

Immunofluorescence Staining for NGFB and SELP

Cryostat sections (8 µm) were incubated overnight at 4°C in a humidity chamber with the primary antiserum to Nfgb and SELP, respectively (NGFB: 1:200, rabbit polyclonal antibody [Ab], Santa Cruz; SELP (purchased as anti P-Selectin: 1:100, monoclonal rat IgG1, Chemicon). This was followed by an incubation of 1 h at 37°C with tetramethylrhodamine-isothiocyanate conjugated F(ab)2 fragments of goat anti-rabbit (NGFB) and goat anti-rat IgG (SELP/P-Selectin), respectively, at a dilution of 1:200 (Jackson Immuno Research). After incubation, sections were rinsed in 0.1 mol/L TBS, pH 7.4, three times for 15 min each. Counterstaining of the tissue was performed by a 5 min incubation with 4',6-Diamidino-2-phenylindole (DAPI). After washing, all sections were mounted and stored at –20°C until analyzed. Negative controls with irrelevant goat IgG did not reveal specific immunoreactivity. Sections were examined by two independent persons blinded with regard to the treatment of the mice at x400 magnification under a Zeiss Axioscope fluorescence microscope. For each mouse, at least 10 microscopic fields (MF) of two tissue slices (thus 20 MF per mouse) were analyzed with special emphasis to uterine decidual tissue, feto-maternal interface, and placental tissue. The intensity of staining signals in distinct structural components of the pregnant uterus was scored: –, not present; (+), weak; +, moderate; and ++, intense positive staining. The amount of NGFB-expressing cells in the decidua and at the interface was scored as follows: (+), 0–3 cells per MF; +, ≤4 cells per MF; and ++, ≥5–10 cells per MF. The percentage of NGFB- and SELP-expressing vessels to the total number of vessels per MF was calculated by scoring 20 MF per mouse, then transferring scoring: +, ≤50 %; ++, 51%–70%; and +++, >70% positive vessels. Photo documentation was performed using digital image analysis system (Spot advanced software, version 3.5.2; Visitron Systems).

Immunohistochemical Staining for ICAM1

Cryostat sections (8 µm) were incubated with peroxidase avidin- and biotin-blocking solution (Vector), followed by another block using protein-blocking agent (Immunotech). The biotinylated hamster anti-mouse ICAM1 mAb (Pharmingen) was diluted 1:100 in TBS containing 1% fetal calf serum and applied for 1 h. As amplification and revealing system, we used peroxidase complex (Vector) 1:100 in TBS for 30 min. The signal was detected by incubating sections with 0.2 mg/ml diaminobenzidine (Sigma) and 0.05% hydrogen peroxide, followed by light counterstaining with 0.1% Meyer's hematoxylin. Again, 10 MF of two tissue slices for each mouse (thus 20 MF per mouse) were analyzed with special emphasis to uterine decidual tissue and placental tissue by two independent blinded persons. Slides were examined using a Zeiss Axioscope light microscope. The signal intensity was scored as follows: –, not present; (+), weak; +, moderate; and ++, intense positive staining. The percentage of ICAM1-expressing vessels to the total number of vessels per MF was calculated and scored as described above, again including 20 MF per mouse. Photo documentation was performed using a digital image analysis system (Zeiss KS400).

Preparation of Uterus Cell Suspensions

To obtain suspensions of uterine cells for characterization by flow cytometry, a method described previously [6] was used. Briefly, uteri were collected, washed with sterile PBS, carefully cut into small pieces, collected in tubes containing Hanks Balanced Salt Solution (HBSS) and digested for 20 min at 37°C under slight agitation with 200 U/ml hyaluronidase, 1 mg/ml collagenase, 1 mg/ml BSA/fraction V (all Sigma), and 0.2 mg/ml DNase I (Boehringer Mannheim GmbH). The isolated cells were then collected in a fresh tube through a 100-µm net (Becton Dickinson) and washed with RPMI 1640 containing 10% fetal bovine serum (FBS). The procedure was repeated twice, with HBSS medium containing no cocktail of enzymes. Cells were resuspended in a 1.080 g/cm3 HistoDenz solution (Sigma) and centrifuged at 800 x g for 20 min at room temperature. The low-density fraction at the interface was collected and washed several times.

Blood Cells

After treatment with ammonium chloride lysis buffer for 10 min to deplete erythrocytes, the cells were washed twice with PBS. Blood cells were used for determination of phenotype (surface expression) and adhesion molecule ligand expression by flow cytometry (staining protocol as described below).

Flow Cytometry Analysis

Flow cytometry was performed using our standard protocol [6, 25]. All monoclonal Abs were purchased from BD Bioscience and are listed in Table 1. For flow cytometry, the uterine and blood cells were incubated for 3 h with Brefeldin A (106 cells/ml medium with 1 µl/ml of Golgi Plug; BD Pharmingen) in RPMI with FBS in a humidified incubator at 37°C with 5% CO2. Brefeldin A is a commonly used agent that blocks cytokine secretion through inhibition of the Golgi apparatus. Flow cytometry was performed as follows: uterus cells were washed twice with buffer (PBS supplemented with 1% BSA (Sigma) and 0.1% sodium azide (Merck). Two percent of normal mouse serum was added to avoid nonspecific binding by Fc receptors. Cells were then incubated 30 min at 4°C with the respective monoclonal Ab against surface antigens. The cells were then washed and fixed using Fix solution (Becton Dickinson), incubating them for 30 min at 4°C in the dark. Subsequently, the cells were washed and permeabilized using FACS Permeabilizing Solution (Becton Dickinson), followed by incubation with the respective antibodies against intracellular antigens for 30 min at 4°C in the dark. Afterwards the cells were washed and then read. As controls, cells were stained with the corresponding isotype-matched monoclonal Ab. The acquisition was performed using a FACS Calibur (Becton Dickinson). Instrument compensation was set in each experiment using single-color stained samples. Data were analyzed by using Cell Quest software. FACS results were expressed as percentage of cells positive for the surface marker evaluated.


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  		TABLE 1. List of mAbs used for flow cytometry (BD Pharmingen).

Statistical Analysis

Statistical significance was determined using the nonparametric Mann-Whitney U-test as well as Bonferroni multiple comparison test. Significance was set at P < 0.05.

RESULTS

Stress-Triggered Increase of the Abortion Rate Could Be Abrogated by Neutralizing NGFB

Exposure to stress on gd 5.5 significantly boosts the abortion rate in DBA/2J mated CBA/J females analyzed on gd 13.5 when resorption sites are visible, supporting our previous findings in this experimental model on stress-triggered abortion (Fig. 1A). Interestingly, as investigated in experiment A, all of the different antibody concentrations used for neutralization of NGFB to find the best dose-response concentration significantly abrogated the stress-triggered increase of abortions. Stressed mice receiving NGFB antibody in the lowest concentration (1.6 µg/g bdywt) presented an abortion rate similar to the nonstressed control group, which was significantly reduced compared to the stress group receiving no anti-NGFB antibody. Application of anti-NGFB antibody at a concentration of 3.2 µg/g bdywt in stressed mice resulted in the lowest abortion rate observed in our setting, which was also significantly decreased compared to the stressed, non-anti-NGFB treated group. Strikingly, the highest anti-NGFB antibody concentration investigated in this experiment (4.8 µg/g bdywt) also had a preventive effect on the abortion rate; however, the abortion rate in these mice was higher compared to that of the mice receiving 3.2 µg/g bdywt anti-NGFB antibody. Injection of nonspecific antibody (rabbit immunoglobulins) had no preventive effect on stress-triggered abortions. Based on these dose-response analyses, the subsequent experiment B was performed by using neutralizing NGFB antibody at a concentration of 3.2 µg/g bdywt exclusively, and reproducible effects on the abortion rate could be observed, compared to experiment A (Fig. 1A).


Figure 1
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  		FIG. 1. A) Effect of stress exposure on gd 5.5 and the combination of stress exposure and neutralization of NGFB in different concentrations on the abortion rate, as determined on gd 13.5 in experiment A. Bars depict the mean percentage ± SEM. *P < 0.05, ** P < 0.01, and *** P < 0.005, as analyzed by nonparametric Mann-Whitney U-test and Bonferroni's multiple comparison test. B) Total number of implantations calculated by adding the number of normal implantation sites plus number of resorption sites. The data are depicted as the mean ± SEM; no significant differences were found between the groups, as analyzed by the nonparametric Mann-Whitney U-test and Bonferroni's multiple comparison test.

Noteworthy, neither stress, neutralization of NGFB, nor injection of rabbit immunoglobulins had a significant effect on the number of implantations, as investigated on gd 13.5 (Fig. 1B).

Stress-Triggered Increase of Uterine NGFB Expression is Abrogated by Neutralization of NGFB

Next, we analyzed the uterine expression of NGFB in the experimental groups by IF. This analysis was performed in tissue areas depicted in Figure 2. Representative examples of NGFB staining are shown in Figure 3, A–F. Taken together, control mice and NGFB-neutralized stressed mice presented a moderate NGFB expression, whereas an increased NGFB expression was observed in stressed mice (Table 2 and Fig. 3, A–F). In detail, stress exposure increased the expression of NGFB in the endothelium of decidual vessels when compared to the control group (Fig. 3, A–B). In stressed, NGFB-neutralized mice, the endothelial NGFB expression was similar to that of the control group, as shown in Figure 3C. Further, a differential NGFB expression pattern in stroma cells of the decidua and at the feto-maternal interface could be observed, because in control and NGFB-neutralized stressed mice sparse NGFB-expressing cells in the decidual compartment were present. Representative examples are shown in Figure 3, D and F. In addition, only isolated NGFB-positive cells could be observed at the feto-maternal interface in the respective groups. Conversely, stress exposure led to an increase of NGFB-expressing cells in both the decidual compartment (Fig. 3E), where no fetal cells invade (decidua parietalis), and at the feto-maternal interface. Trophoblast cells presented weak NGFB staining in all investigated groups.


Figure 2
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  		FIG. 2. Histology (left) and schematic drawing (right) of gd 7.5 mouse uterus. Histological analysis was performed in the area marked as trophoblast/decidua contact (=interface). Original magnification x50.


Figure 3
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  		FIG. 3. A–F) NGFB expression in the pregnant uterus on gd 7.5. NGFB staining resulted in bright red staining; cell nuclei were counterstained with DAPI for better orientation, resulting in bright blue staining. Moderate NGFB expression was seen in decidual vessel endothelium of control and NGFB-neutralized stressed mice (A and C); stress exposure increased endothelial NGFB expression (B). Further, stress increased the amount of NGFB expressing cells in the decidua (E) when compared to the control (D); NGFB neutralization abolished this effect (F). G–I) ICAM1 expression in the pregnant uterus on gd 7.5. ICAM1 staining resulted in brown staining. Compared to control mice, ICAM1-positive decidual vessels were increasingly expressed in stressed mice (G and H), whereas neutralization of NGFB decreased the endothelial expression of ICAM1 (I). Stroma cells in the decidual compartments of control mice presented a weak ICAM1 expression (G), stress increased ICAM1 expression in stroma cells (H, arrowhead), and NGFB neutralization abolished this effect (I). J–L) SELP (formerly known as P-Selectin) expression in the pregnant uterus on gd 7.5, staining resulted in bright red staining; cell nuclei were counterstained with DAPI, resulting in bright blue staining. Stressed mice presented a significant higher percentage of positive vessels and more intense staining compared to nonstressed mice (J and K); NGFB neutralization abolished this effect (L). Original magnification x400.


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  		TABLE 2. NGFB expression in pregnant uteri on gd 7.5.

Neutralization of NGFB Prevents Stress-Induced Upregulation of Uterine Adhesion Molecules and Their Ligands

To further determine the mechanisms by which NGFB affects the course of pregnancy, we analyzed the expression of adhesion molecules and their respective ligands ICAM1/ITGAL and SELP/SELPL. Specifically, we analyzed the expression patterns of ICAM and SELP by immunohistochemistry and immunfluorescence (Table 3 and Figs. 3 and 4), and the percentage of ITGAL- and SELPL-positive uterine cells by flow cytometry (Fig. 5).


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  		TABLE 3. ICAM1 and SELP distribution in the pregnant uterus.


Figure 4
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  		FIG. 4. Quantification of A) ICAM1-positive uterine (ut.) vessels and B) SELP-positive uterine vessels. The dark bars show the percentage of positive vessels, the light bars the percentage of negative vessels. Stress exposure increased the expression of ICAM1 and SELP, which was abrogated by neutralization of NGFB. *P < 0.05 as analyzed by nonparametric Mann-Whitney U-test and Bonferroni's multiple comparison test.


Figure 5
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  		FIG. 5. Neutralization of NGFB in stressed mice abrogates the stress-induced increase of A) the percentage of ITGAL-expressing uterine cells and B) the percentage of SELPL-expressing uterine cells. Bars depict the mean percentage ± SEM. *P < 0.05 as analyzed by nonparametric Mann-Whitney U-test and Bonferroni's multiple comparison test.

Compared to control mice, ICAM1-positive decidual vessels were increasingly expressed in stressed mice (Fig. 3, G and H, and Fig. 4A), whereas neutralization of NGFB decreased the endothelial expression of ICAM1 (Fig. 3I and Fig.. 4A). Increased expression of ICAM1 in stroma cells was observed in stressed mice (Fig. 3H), whereas control and NGFB-neutralized mice exhibited a weak ICAM1 expression pattern of stroma cells (Fig. 3, G and I). No ICAM1 expression in any group could be observed in uterine glands. Trophoblast cells derived from stressed mice revealed an intense ICAM1 expression, whereas trophoblast cells from control and NGFB-neutralized stressed mice had a moderate expression of ICAM1.

SELP was exclusively expressed in uterine vessels (see Table 3 and Figs. 3 and 4). Stressed mice presented a significantly higher percentage and intense staining in SELP-positive vessels compared to nonstressed mice, as shown in Figures 3, J and K, and 4. This effect was abrogated by NGFB neutralization (Figs. 3L and 4). No staining could be detected in stroma cells, glands, or trophoblast cells.

Concordant results could be obtained when analyzing the percentage of ITGAL- and SELPL-expressing uterine cells by flow cytometry (Fig. 5). Stress exposure significantly boosted the percentage of ITGAL+ (Fig. 5A) and SELPL+ (Fig. 5B) uterine cells compared to the control. Interestingly, neutralization of NGFB decreased the percentage of these ligand-expressing cells in stressed mice.

Differential Expression of CD8{alpha}+ Uterine Lymphocytes in Stressed Mice

Next, we determined the percentage of uterine CD8{alpha}+ cells, because a CD8{alpha}+ subpopulation has been suggested to be protective as regulatory cells in pregnancy maintenance [26]. As shown in Figure 6, stressed mice presented a significant decrease of the percentage of CD8{alpha}+ uterine cells when compared to control mice. Strikingly, in stressed, NGFB-neutralized mice, the percentage of uterine CD8{alpha}+ cells was restored to levels seen in control mice and was significantly different from levels seen in the stressed nonneutralized mice.


Figure 6
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  		FIG. 6. Effect of stress exposure and neutralization of NGFB on the percentage of CD8{alpha}+ uterine cells. Stress decreases the percentage, whereas neutralization of NGFB in stressed mice has a preventive effect with respect to CD8{alpha}+ uterine cells. Bars depict the mean percentage ± SEM. *P < 0.05 as analyzed by nonparametric Mann-Whitney U-test and Bonferroni's multiple comparison test.

Neutralization of NGFB Abrogates the Detrimental Effects of Stress on the Decidual Th1l/Th2l Balance

Because the Th1l/Th2l ratio has been proven to play an important role in the maintenance of pregnancy, and because a Th2l cytokine pattern is involved in successful pregnancy outcome [6, 27–29], we characterized the balance of Th1l/Th2l cytokines produced by subsets of uterine cells in our experimental groups. Upon examination of the intracellular expression of Th1l and Th2l cytokines, we calculated the mean value of the respective percentages for intracellular expression of tumor necrosis factor (TNF), interferon-{gamma} (IFNG) and interleukin (IL)12 as the "Th1l value" and the mean value of the respective percentages for intracellular expression of IL4 and IL10 as the "Th2l value." We then divided the mean Th1l value by the mean Th2l value to obtain the Th1l/Th2l ratio. We observed that the ratio was increased in favor of Th1l in stressed mice (2.2/1), compared to control mice (1/1) and stressed, anti-NGFB-treated mice (1/1). Intriguingly, upon NGFB neutralization in stressed mice, the pregnancy-protective Th1l/Th2l ratio could be restored. Figure 7 depicts the individual data per mouse for TNF as a representative Th1l cytokine and IL4 as a representative Th2l cytokine and reveals that stress exposure shifts the cytokine profile toward Th1l dominance, whereas NGFB neutralization abrogates this effect, because the mice treated with anti-NGFB show similar levels of TNF and IL4 to those seen in the control group.


Figure 7
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  		FIG. 7. Stress exposure provokes a shift of the cytokine profile toward a Th1l dominance, here shown for TNF as a representative for Th1l cytokines and IL4 as a representative for Th2l cytokines. Each symbol depicts the absolute number of cytokine-expressing uterine cells per mouse, as analyzed by flow cytometry and calculated from the total number of uterine cells. Presentation of the results as relative data (e.g., %TNF+/total lymphocytes) gives an almost identical graphic.

Subpopulations and Adhesion Molecule Ligand Expression in Peripheral Blood Cells

We further characterized subpopulations in peripheral blood cells by flow cytometry (see Table 4). No significant effect of stress or NGFB neutralization in stressed mice was found with respect to the percentage of CD8{alpha}+, CD4+ or CD3+ populations. Also, there were no differences found in the expression of ITGAL and SELPL on the peripheral blood cells.


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  		TABLE 4. Percentage of blood cell subpopulations and expression of adhesion molecules on peripheral blood cells on gd 7.5.

DISCUSSION

In the present study we provide convincing evidence that NGFB plays a major role as decisive mediator in stress-triggered abortions. We demonstrate for the first time that neutralization of NGFB abolishes the detrimental effects of stress on pregnancy maintenance in a murine model. Stress exposure upregulates uterine NGFB expression and expression of adhesion molecules ICAM1 and SELP and their ligands ITGAL and SELPL. It further decreases uterine CD8{alpha}+ cells and biases the cytokine profile toward inflammatory Th1l>Th2l upon implantation, all of which is abrogated by NGFB neutralization or a cross-reacting molecule. This profoundly extends the knowledge of mechanisms in stress-triggered abortion, showing that NGFB or a similar molecule seems to be a proximal mediator in the hierarchic network of immune rejection during pregnancy by mediating an abortogenic environment with the classical signs of neurogenic inflammation: 1) an inflammatory cell recruitment via upregulation of adhesion molecules, 2) the decidual cytokine imbalance with a predominance of Th1l cytokines, and 3) the decrease of the protective CD8{alpha}+ decidual cell subpopulation (see Fig. 8).


Figure 8
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  		FIG. 8. Hypothetical scenario. Stress provokes tachykinin 1 (TAC1, formerly known as Substance P) release, which mediates the local and endothelial increase of NGFB because of interactional processes (neurogenic inflammatory response, step I). Subsequently, increased expression of NGFB in the uterus skews the immune system toward an inflammatory Th1l response via upregulation of adhesion molecules (step II). Inflammatory cell influx aggravates NGFB levels even higher by migration of additional NGFB-expressing and -releasing cells. This inflammatory environment results in rejection of the fetus (step III).

Stress-Triggered Uterine Expression of NGFB Results in Neurogenic Inflammation

Neurogenic inflammation, a consequence of neuroimmune cross talk, represents local inflammatory reactions to threats, e.g., infection, toxins, trauma or stress, by involving nerves that contain inflammation-triggering neuropeptides. Potent mediators of this process are NGFB and the neuropeptide Tachykinin 1 (TAC1, formerly substance P), which dominantly contributes to the signs of neurogenic inflammation, e.g., extravasation, dilatation of vessels and recruitment of leukocytes [30–33]. Various interactional functions between NGFB and TAC1 are known including induction of NGFB expression and secretion by TAC1 [34] and activation of TAC1 synthesis and release by NGFB [35]. Based on these observations, the involvement of NGFB in neurogenic inflammation has been demonstrated in diverse pathological conditions [36, 37]. NGFB can be considered as a decisive mediator in neuronal-inflammatory cross talk, which can be inhibited by application of antagonists to tachykinin receptor 1 (formerly neurokinin-1 receptor) [38]. Vice versa, neutralization of NGFB inhibits TAC1 expression in peripheral neurons [39] and dorsal root ganglia (unpublished results).

Neurogenic inflammatory responses can also be caused by psychological or physical stress caused by release of TAC1 from sensory nerves and consecutive activation of mast cells and/or other immune cells [40, 41]. Mechanisms involved in stress-induced abortion in mice have been shown to be TAC1-dependent. Triggering the effect is the TAC1-mediated release of Th1l cytokine TNF-{alpha} by CD8{alpha}+ T lymphocytes [26, 29, 42]. Furthermore, stressful conditions are characterized by increased local and systemic NGFB levels [13, 43, 44] indicating NGFB as a translating factor in stress-induced neurogenic inflammatory states [39]. Recent results of our group depict increased NGFB and NTRK1 expression in uterine decidual tissue of both stressed pregnant mice and of mice that were TAC1 injected in order to mimic stress effects [18]. In the present study, we confirmed these observations for the peri-implantation period, affecting the expression of NGFB in the endothelium of vessels as well as the amount of NGFB-expressing cells in the decidua and at the feto-maternal interface. These observations are supported by other investigators, who demonstrated an enhanced NGFB secretion by endothelial cells of the brain after inflammatory stimulation, implicating the involvement of endothelial-derived NGFB in inflammatory immune processes [45]. Based on shape and distribution in the decidual tissue, our histomorphometric results let us suggest that the cells expressing NGFB in the decidua are immune cells, e.g., mast cells [46], macrophages [47], and T lymphocytes [48]. All of them exert local functional roles throughout both normal and disturbed gestation [49–51]. We speculate that NGFB-expressing immune cells migrate into the decidua and by release of NGFB further contribute to increased local NGFB levels, amplifying the neurogenic inflammatory response (Fig. 8, step I).

NGFB Affects Stress-Threatened Pregnancy via Adhesion Molecule-Dependent Pathways

Adhesion molecules and their ligands are crucial factors involved in leukocyte recruitment and generation of the inflammatory immune response that can be observed in failing pregnancies [7, 52]. The local microenvironment is characterized by influx of specific leukocyte subsets, e.g., natural killer cells, dendritic cells, T lymphocytes, and macrophages, generating an imbalance of the Th1l/Th2l cytokine profile with a preponderance of inflammatory Th1l cytokines. It is now well known that stress exposure in the abortion-prone CBA/J x DBA/2J mouse model leads to this characteristic inflammatory response [5, 6, 29]. We recently demonstrated that ICAM/ITGAL cross talk is a pivotal point in the generation of this pathological, Th1l-dominated tissue reaction [7]. Also, SELP has been shown to be involved in enhanced Th1l lymphocyte migration into decidual tissue, as observed in human tissue of spontaneous abortion [53].

In our study, ICAM1 expression was detected on endothelial cells, decidual stroma cells, and trophoblast cells, which conforms with published literature showing that ICAM1 is present on the cell surface of a wide variety of cell types, including fibroblasts and endothelial cells [54–56]. Moreover, ICAM1 is upregulated in response to a number of inflammatory mediators, e.g., proinflammatory cytokines, mediating the migration of leukocytes and further contributing to the inflammatory milieu. In addition, activated endothelial cells express SELP, which is regulated by inflammatory stimuli and is responsible for tethering of flowing leukocytes on the endothelium [57]. Because the typical stress response is characterized by inflammatory reactions, these data support the enhanced ICAM1/ITGAL and SELP/SELPL expression that we observed in stressed mice (Fig. 8, step II).

The present study shows that neutralization of NGFB abrogates the stress-induced upregulation of ICAM1 and SELP and their respective ligands, pointing to NGFB as inductor of adhesion molecule expression and, therefore, as proximate mediator in this networking organization. This is further confirmed by recent investigations that demonstrate the upregulation of endothelial ICAM1 and induction of endothelial cell proliferation induced by NGFB (or a cross-reacting molecule) and thus propose key functions for NGFB in the inflammatory response, i.e., inflammatory cell recruitment and angiogenesis [19, 20]. To our knowledge, this is the first study showing functional effects on the expression of SELP and its ligand SELPL by NGFB neutralization, expanding its interactional relationship with this adhesion molecule as well.

Moderate Levels of NGFB Level Seem to Be Mandatory for Pregnancy Maintenance

Based on the rather high abortion rate seen upon NGFB neutralization using a high dose of anti-NGFB, we propose that adequate or moderate levels of NGFB are mandatory for pregnancy maintenance. This hypothesis is complemented by independent experiments employing Balb/c-mated CBA/J females, which constitutes a low-abortion animal model, wherein application of anti-NGFB significantly increases the abortion rate (unpublished observations). A functional role of physiologic levels of NGFB during pregnancy is the acceleration of giant cell transformation in the developing trophoblast, which constitutes a crucial step in the placentation process [58]. Accordingly, neutralizing of functional NGFB besides the stress-triggered increase of NGFB with the administration of anti-NGFB at a high dose appears to have deleterious consequences for placentation, resulting in disturbance or termination of pregnancy. However, mice homozygous for NGFB gene disruption are not compromised with regard to fetal survival in utero [59]. Such unexpected reproductive outcomes in knockout mice are frequently observed, e.g., in INDO –/– mice [60] or IL4 –/– mice [61], which reproduce normally. Superficially regarded, these observations may appear puzzling. However, they clearly point toward compensatory mechanisms within the pregnancy-maintaining network of mediators, which should foster research providing therapeutical approaches supporting such compensatory mechanisms.

In summary, we propose NGFB as interposed mediator between stress perception and immune response in pregnancy. NGFB can be considered as a proximate mediator of a hierarchical network, capable of biasing immune responses toward inflammation. We hypothezise that the increase in NGFB after stress exposure is part of a neurogenic inflammatory response translating the threat of stress to the vulnerable site, the feto-maternal interface (Fig. 8, step I). Subsequently, increased expression of NGFB in the uterus skews the immune system toward an inflammatory Th1l response via upregulation of adhesion molecules (Fig. 8, step II). Inflammatory cell influx aggravates NGFB levels even higher by migration of additional NGFB-expressing and -releasing cells. This inflammatory environment results in rejection of the fetus (Fig 8, step III). The protective effect on the local cytokine balance with a well-balanced Th2l/Th1l profile and restored percentage of uterine CD8{alpha}+ cells in NGFB-neutralized stressed mice further supports this hypothesis. This opens clinical experimental approaches for therapeutic interventions by use of specific inhibitors and antagonists, which is the subject of investigations in a number of other pathological states involving NGFB, e.g., pain [62], asthma bronchiale [23], oncological disorders [63] or HIV infection [64].

ACKNOWLEDGMENTS

We are grateful for the excellent technical assistance of P. Busse, E. Hagen, C. Josties, P. Moschansky, and R. Pliet, and thank Bori Handjiski for his support in taking microscopic photographs.

FOOTNOTES

1 Supported by grants from the Charité to M.T., S.B., and P.A.; S.B. received a scholarship from the Schering Research Foundation. S.B., M.T., and P.A. are part of the EMBIC Network of Excellence, cofinanced by the European Commission through the FP6 framework program "Life Science, Genomics and Biotechnology for Health." Back

2 Correspondence: Petra Clara Arck, Biomedizinisches Forschungszentrum, Raum 2.0549, Augustenburger Platz 1, 13353 Berlin, Germany. FAX: 49 30 450 553962; petra.arck{at}charite.de Back

Received: 14 June 2005.

First decision: 7 July 2005.

Accepted: 20 December 2005.

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