Intra-amniotic infection upregulates neutrophil gelatinase-associated lipocalin (NGAL) expression at the maternal-fetal interface at term: implications for infection-related preterm birth

Abstract

Objective: Neutrophil gelatinase-associated lipocalin (NGAL) is a ubiquitous lipocalin that serves as a critical component of innate immunity and a transport shuttle for numerous substances (retinoids, arachidonic acid, prostaglandins, fatty acids, steroids, iron, and MMPs). Despite the well-documented association between intra-amniotic infection/inflammation (IAI) and preterm birth, NGAL expression in the uterus has not previously been examined. This study investigates NGAL expression at the maternal-fetal interface in vivo and in vitro.

Methods: Neutrophil gelatinase-associated lipocalin expression in term placenta with/without IAI was examined by immunohistochemistry. Trophoblast and decidual stromal cells were retrieved from elective cesarean, purified, and depleted of leukocytes. On days 1 (cytotrophoblast cells) and 4 (syncytiotrophoblast), cells were stimulated with/without interleukin 1β (IL-1β; 1 ng/mL), tumor necrosis factor α (TNF-α; 1 ng/mL), or lipopolysaccharide (LPS; 1 μg/mL). Neutrophil gelatinase-associated lipocalin messenger RNA (mRNA) and protein expression were measured by immunocytochemistry/Western blot and RT-qPCR, respectively.

Results: Under basal conditions, NGAL is expressed in trophoblast, but not decidua. Trophoblast NGAL is significantly upregulated in tissues with evidence of IAI vs controls. NGAL expression was increased after stimulation with all 3 pro-inflammatory mediators in day 1 (cytotrophoblast) but not day 4 cells (syncytiotrophoblast). IL-1β and TNF-α (not LPS) upregulated NGAL gene expression in cytotrophoblast (not syncytiotrophoblast) cells.

Conclusions: Intra-amniotic infection/inflammation is associated with increased expression of NGAL in trophoblast tissues in vivo. IL-1β, TNF-α, and LPS stimulated NGAL in cytotrophoblast cells (not syncytiotrophoblast and decidua) in vitro. These data suggest that, in keeping with its role as a mediator of innate immunity, NGAL may have a central role to play in IAI-induced preterm birth.

Figure 1.

Immunohistochemical localization of neutrophil gelatinase-associated lipocalin (NGAL) at the maternal-fetal interface. Serial sections of placental tissues collected from women with or without clinical and histologic evidence of intra-amniotic infection (IAI) were stained for cytokeratin-7 (to identify trophoblast; B and F), vimentin (to identify decidual stromal cells [DSCs]; C and G), and NGAL (D and H) as described in the Materials and Methods section. Representative images are shown. In the absence of IAI (A–D), NGAL staining was seen consistently in cytokeratin-positive trophoblast cells, but only in occasional vimentin-positive decidual stromal cells (DSCs). In the presence of IAI (E–H), cytokeratin-positive trophoblast cells (especially extravillous cytotrophoblast cells) showed strong staining for NGAL with no apparent increase in staining in syncytiotrophoblast and decidual cells (H). Negative controls using second antibody only showed the absence of nonspecific binding (A and E). In all tissues, NGAL expression is localized to the cytoplasm of the trophoblast cells and is not present in the nucleus. Magnification is ×40.

Figure 2.

Quantitative analysis of immunohistochemical studies of neutrophil gelatinase-associated lipocalin (NGAL) expression at the maternal-fetal interface with or without intra-amniotic infection. As described in the Materials and Methods section, serial sections of placental tissues with or without clinical and histologic evidence of intra-amniotic infection (IAI) were stained for NGAL and analyzed using AxioVision (Carl Zeiss Microimaging, Inc., Thornwood, New York). A computer-generated H-score (in relative light units) was assigned for a representative tissue section and corrected for background light intensity. Results are reported as mean + SEM from at least 3 separate tissue sections with measurements performed in triplicate. Statistical differences are shown. NS indicates not significant.

Figure 3.

Effects of pro-inflammatory mediators on neutrophil gelatinase-associated lipocalin (NGAL) expression by term trophoblast cells as determined by immunocytochemistry. Term trophoblast cells on day 1 (cytotrophoblast) and day 4 of culture (syncytiotrophoblast) were treated for 24 h with or without IL-1β (1 ng/mL), TNF-α (1 ng/mL), or LPS (1 μg/mL), and NGAL expression measured by immunocytochemistry as described in the Materials and Methods section. Representative images are shown. Trophoblast cells on days 1 (cytotrophoblast; A) and (syncytiotrophoblast; B) were fixed and labeled with cytokeratin 7 (a trophoblast-specific marker) and NGAL. DAPI staining was used to identify the cell nuclei. The images were analyzed using AxioVision (Carl Zeiss Microimaging, Inc., Thornwood, New York). A computer-generated H-score (in relative light units) was assigned for a representative section and corrected for background light intensity. Data are presented as fold change compared with no stimulation (control), and results are reported as mean + SEM from at least 3 separate sections with measurements performed in triplicate (C). Statistical differences are shown. Abbreviations: IL-1β indicates interleukin-1β; LPS, lipopolysaccharide; NS, not significant; TNF-α, tumor necrosis factor-α.

Figure 4.

Effects of pro-inflammatory mediators on NGAL expression by term trophoblast cells as measured by western blot analysis. Term trophoblast cells on day 1 (cytotrophoblast) and day 4 of culture (syncytiotrophoblast) were treated for 24 hours with or without IL-1β (1 ng/mL), TNFα (1 ng/mL), or LPS (2.5 U/mL), and NGAL expression determined by western blot analysis as described in the Materials and Methods section. A representative western blot is shown [A]. To quantify the results, the intensity of the NGAL bands was corrected for expression of the housekeeping protein, heat shock protein-90 (HSP-90). Data are presented as mean ± SEM from 4 separate experiments [B]. Statistical differences are shown. Abbreviations: IL-1b, interleukin-1b; LPS, lipopolysaccharide; NS, not significant; TNFα, tumor necrosis factor-α.

Figure 5.

Effects of preeclampsia on neutrophil gelatinase-associated lipocalin (NGAL) gene expression. Term trophoblast cells on day 1 (cytotrophoblast) and day 4 of culture (syncytiotrophoblast) were treated for 6 hours with or without IL-1β (1 ng/mL), TNF-α (1 ng/mL), or LPS (1 μg/mL). RNA was extracted and subjected to RT-qPCR using specific primers as described in the Materials and Methods section. NGAL mRNA levels were corrected for expression of the housekeeping gene, β-actin. Data are expressed as mean ± SEM from 3 separate experiments performed in duplicate. Statistical differences are shown. Abbreviations: IL-1β indicates interleukin-1β; LPS, lipopolysaccharide; NS, not significant; TNF-α, tumor necrosis factor-α.

Figure 6.

NGAL expression by term decidual stromal cells as determined by immunocytochemistry. Term decidual stromal cells (DSCs) were isolated, purified, and cultured as described in the Materials and Methods section. NGAL expression was measured by immunocytochemistry. Representative images are shown from cells without IL-1b or thrombin stimulation. Fixed DSCs were stained with DAPI to identify the cell nuclei [A], the stromal cell-specific marker vimentin [B], and NGAL [C]. A composite image is included [D]. The images were analyzed using AxioVision (Carl Zeiss Microimaging, Inc, Thornwood, NY).