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The Journal of Maternal-Fetal & Neonatal Medicine Volume 21, 2008 - Issue 11
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Original Article

A role for CXCL13 (BCA-1) in pregnancy and intra-amniotic infection/inflammation

Chia-Ling Nhan-Chang Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USACorrespondence[email protected]
, MD,
Roberto Romero Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA
, MD,
Juan Pedro Kusanovic Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA
,
Francesca Gotsch Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA
,
Samuel S. Edwin Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA
,
Offer Erez Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USA
,
Pooja Mittal Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USA
,
Chong Jai Kim Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Pathology, Wayne State University School of Medicine, Detroit, MI, USA
,
Mi Jeong Kim Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA
,
Jimmy Espinoza Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USA
,
Lara A. Friel Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USA
,
Edi Vaisbuch Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA
,
Nandor Gabor Than Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA
,
Shali Mazaki-Tovi Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USA
&
Sonia S. Hassan Perinatology Research Branch, Intramural Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NICHD/NIH/DHHS, Hutzel Women's Hospital, Bethesda, MD, Detroit, MI, USA; Department of Obstetrics and Gynecology, Wayne State University/Hutzel Women's Hospital, Detroit, MI, USA
 show all
Pages 763-775 | Received 21 Mar 2008, Accepted 30 May 2008, Published online: 07 Jul 2009

Abstract

Objectives. CXCL13 is a potent chemokine, produced by mature and recently recruited macrophages to sites of inflammation, which has antimicrobial and anti-angiogenic properties. The purpose of this study was to: (1) determine whether CXCL13 is present in maternal serum, umbilical cord blood, and amniotic fluid (AF); (2) to determine if AF concentration changes with intra-amniotic infection/inflammation (IAI); and (3) to localize the production of CXCL13 in chorioamniotic membranes and umbilical cord.

Study design. A cross-sectional study on maternal serum was performed including patients in the following groups: (1) non-pregnant women (n = 20), (2) normal pregnant women (n = 49), (3) patients at term not in labor (n = 30), and (4) patients in spontaneous labor at term (n = 29). Umbilical cord blood was collected from term neonates with (n = 30) and without labor (n = 28). Amniotic fluid was obtained from patients in the following groups: (1) midtrimester (n = 65); (2) term not in labor (n = 22); (3) term in labor (n = 47); (4) preterm labor (PTL) with intact membranes leading to term delivery (n = 70); and (5) PTL leading to preterm delivery with IAI (n = 79) and without IAI (n = 60). CXCL13 concentrations were determined by enzyme-linked immunosorbent assay. Chorioamniotic membranes and umbilical cords were examined with immunohistochemistry. Non-parametric statistics were used for analysis.

Results. (1) CXCL13 was present in 100% of serum and cord blood samples, and 99% of AF samples (339/343). (2) Serum CXCL13 concentration was significantly higher in pregnant women when compared to non-pregnant women (median 313.3 pg/mL (interquartile range (IQR) 197.2–646.9) vs. 40.5 pg/mL (IQR 29.5–93.5), respectively; p < 0.001). (3) Serum CXCL13 concentration decreased with advancing gestational age (Spearman's Rho = −0.424; p < 0.001). (4) There were no significant differences in the median serum CXCL13 concentration between women at term with and without labor (371.6 pg/mL (IQR 194.3–614.3) vs. 235.1 pg/mL (IQR 182.8–354.7), respectively; p = 0.6). (5) The concentration of CXCL13 in AF did not change with gestational age (p = 0.1). (6) Patients with PTL and delivery with IAI had a significantly higher median concentration of CXCL13 than those without IAI (median 513.2 pg/mL (IQR 199.7–2505.5) vs. 137.3 pg/mL (IQR 96.7–209.6), respectively; p < 0.001) and those who delivered at term (133.7 pg/mL (IQR 97.8–174.8); p < 0.001). (7) Spontaneous labor did not result in a change in the median AF concentration of CXCL13 (labor: 86.9 pg/mL (IQR 55.6–152.0) vs. no labor: 77.8 pg/mL (IQR 68.0–98.0); p = 0.8). (8) CXCL13 was immunolocalized to macrophages in fetal membranes and umbilical vein.

Conclusions. (1) We report for the first time the presence of CXCL13 in AF. (2) AF CXCL13 concentrations are dramatically increased in IAI. (3) Unlike other chemokines, AF and serum CXCL13 concentrations did not change with spontaneous parturition.

Introduction

The preterm parturition syndrome can be caused by multiple pathologic processes; however, the only process that has been proven to be causal is intrauterine infection/inflammation Citation[1-5]. The understanding of the specific roles and the identification of all the key components of the innate inflammatory defense system is still a work in progress, and preterm labor and its related adverse consequences are on the increase Citation[6].

Successful pregnancy requires tolerance to the fetal allograft as well as effective protection against infection. The mechanisms responsible for immune tolerance during pregnancy have been the subject of investigation for more than 30 years and remain controversial. On the other hand, a complex system exists to protect the conceptus and mother from infection. This system involves physical barriers to infection such as the mucous plug Citation[7-11], placenta Citation[12], fetal membranes Citation[13,14], and components of the innate and adaptive immune system such as neutrophils, macrophages, natural killer cells, and trophoblasts Citation[12],Citation[15-19]. Many of these cells require recruitment to the site of inflammation, which is accomplished by chemokines.

Chemokines are classified into four families by the structure of the conserved cysteine-motif in the amino-terminus of the proteins Citation[20]. Less than half of the recognized chemokines have been identified in amniotic fluid (AF), and several have been associated with preterm birth and intra-amniotic infection or inflammation, including CCL2 (MCP-1) Citation[17], CCL3 (MIP-1α), CCL5 (RANTES) Citation[21], CXCL1 (GRO-α) Citation[22], and CXCL8 (IL-8) Citation[23] among others Citation[24-29].

CXCL13, a chemokine previously named B-lymphocyte chemoattractant (BLC) in mice Citation[30] and B cell-attracting chemokine-1 (BCA-1) in humans Citation[31], was first identified in 1998 in liver and lymph nodes. CXCL13 is a potent chemokine secreted by monocytes, lymphocytes, and dendritic cells Citation[32] and is detected in serum Citation[33], normal lymphoid tissue, and in acute and chronically inflamed tissue Citation[34]. Its role in the inflammatory response is to attract B and T lymphocytes to areas of infection and inflammation Citation[35]. CXCL13 is involved in normal chemotaxis in response to routine physiological stress in the lymph nodes during development Citation[36]. In other tissues, parasitic and microbial exposure induces local production of CXCL13 leading to the formation of ectopic germinal centers Citation[37], which serve as tertiary lymphoid organs in inflamed and infected tissues.

In pregnancy, the identification of CXCL13 has been reported only in cases of placental malaria Citation[38]. Present in both acute and chronic placental malaria, CXCL13 expression in placenta was found to be up-regulated >130-fold in active malaria and >1000-fold in cases of massive malarial infection. In addition, CXCL13 has been observed to form lymphoid-like aggregates similar to ectopic germinal centers in other tissues.

The purpose of this study was to determine if: (1) CXCL13 is present in maternal serum and the amniotic cavity in normal pregnancy; (2) concentrations of CXCL13 in AF and maternal serum change with increasing gestational age and in the presence of spontaneous labor at term; (3) CXCL13 concentrations in AF are affected by the presence of intra-amniotic infection/inflammation (IAI); (4) CXCL13 is present in cord blood of term neonates; and (5) if the production of CXCL13 can be localized in chorioamniotic membranes and umbilical cord.

Materials and methods

Study design and population

A cross-sectional study was conducted by searching our clinical database and bank of biological samples including patients in the following groups: (1) non-pregnant women in the secretory phase of their cycle (n = 20), (2) normal pregnant women (n = 49), (3) patients at term not in labor (n = 30), and (4) patients in spontaneous labor at term (n = 29). Umbilical cord blood from term neonates was collected from mothers with (n = 30) and without labor (n = 28).

For the analysis of CXCL13 in AF, a cross-sectional study was conducted including patients in the following groups: (1) normal women in the midtrimester of pregnancy (16–18 weeks) who underwent amniocentesis for genetic indications and delivered normal neonates at term (n = 65); (2) normal pregnancies at term (≥37 weeks) with (n = 47) and without (n = 22) spontaneous labor; and (3) women with an episode of spontaneous preterm labor (PTL) and intact membranes, who were further subdivided into three groups: (a) PTL who delivered at term without IAI (n = 70), (b) PTL who delivered preterm without IAI (n = 60), and (c) PTL who delivered preterm with IAI (n = 79).

All patients provided written informed consent prior to the collection of samples. The collection and utilization of samples for research purposes was approved by the institutional review boards of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICH/NIH/DHHS, Bethesda, MD) and participating institutions. These samples have been previously utilized in published studies of other inflammatory markers and antimicrobial peptides in normal and abnormal pregnancies.

Definitions

Patients were considered to have a normal pregnancy if they did not have any medical, obstetrical, or surgical complications, and delivered a term neonate (≥37 weeks) of appropriate birth weight for gestational age Citation[39,40]. Spontaneous preterm labor was defined by the presence of at least two regular uterine contractions every 10 minutes associated with cervical changes before 37 completed weeks of gestation, requiring admission to the hospital. Intra-amniotic infection was defined as a positive AF culture for microorganisms. Intra-amniotic inflammation was defined as AF interleukin-6 (IL-6) concentration >2.6 ng/mL Citation[41].

Amniotic fluid collection

Amniotic fluid samples were obtained by transabdominal amniocentesis performed for genetic indications, evaluation of microbial status of the amniotic cavity, or assessment of fetal lung maturity in patients approaching term. The AF samples were transported to the laboratory in a sterile capped syringe. AF samples from patients with PTL were cultured for aerobic/anaerobic bacteria and genital mycoplasmas. White blood cell (WBC) count, glucose concentration, and Gram-stain were also performed after collection as previously described Citation[41-45]. The results of these tests were used for subsequent clinical management. In addition, IL-6 concentrations were determined from the AF of PTL patients; the results were used only for research purposes. AF not utilized for clinical assessment was centrifuged for 10 minutes at 4°C and the supernatant was aliquoted and stored at −70°C until analysis.

Human CXCL13 immunoassays

Concentrations of CXCL13 in AF and maternal and umbilical cord serum were determined using specific and sensitive enzyme-linked immunosorbent assays obtained from R&D Systems, Inc. (Minneapolis, MN, USA) as per the manufacturer's instructions. AF CXCL13 assays were validated in our laboratory prior to the conduction of this study. Validation included spike and recovery experiments, which produced parallel curves indicating that AF matrix constituents did not interfere with antigen–antibody binding in this assay system. The calculated inter-assay coefficients of variation for serum and AF in our laboratory were 7.2% and 3.9%, respectively. The calculated intra-assay coefficients of variation for serum and AF were 4.9% and 3.0%, respectively. The sensitivity was calculated to be 3.7 pg/mL for serum and 2.8 pg/mL for AF.

Immunofluorescence staining

Two frozen specimens with chorioamnionitis and funisitis and one control case with no evidence of inflammation were selected to determine the immunolocalization of CXCL13 in fetal membranes and umbilical cord. Immunofluorescence staining was performed on 5 μ-thick frozen sections after fixation with 4% paraformaldehyde. Following permeabilization with 0.25% TritonX-100 and blocking, sections were incubated with a rabbit polyclonal anti-CXCL13 antibody (ProteinTech Group, IL, USA). Alexa Fluor 594-labeled donkey anti-rabbit Ig (Invitrogen, CA, USA) was used as the secondary antibody. For double staining, sections were further incubated with a murine monoclonal anti-HLA-DR antibody (Dako, Glostrup, Denmark) and Alexa Fluor 488-labeled goat anti-mouse Ig (Invitrogen). The slides were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen), and images were taken using a Leica TCS SP5 spectral confocal system (Leica Microsystems GmbH, Wetzlar, Germany).

Statistical analysis

Amniotic fluid and serum CXCL13 concentrations were not normally distributed. Kruskal–Wallis with post-hoc analysis and Mann–Whitney U-tests were used for the evaluation of continuous variables. Spearman's Rho was utilized to assess correlations. Among patients with PTL and intact membranes, receiver operating characteristic (ROC) curve analysis was employed for the identification of patients who had IAI. A survival analysis with Kaplan–Meier was performed to examine the amniocentesis-to-delivery interval according to AF CXCL13 concentrations. A p-value of <0.05 was considered statistically significant. The statistical package utilized was SPSS v. 12.0 (SPSS Inc., Chicago, IL, USA).

Results

Detection of CXCL13 in serum

Serum CXCL13 was detected in all patients. The median serum CXCL13 concentration was significantly higher in pregnant women when compared to non-pregnant women (median 313.3 pg/mL (interquartile range (IQR) 197.2–646.9) vs. median 40.5 pg/mL (IQR 29.5–93.5), respectively; p < 0.001) (). In normal pregnancy, maternal serum CXCL13 concentration decreased with advancing gestational age from 8 to 40.7 weeks of gestation (Spearman's Rho = −0.424; p < 0.001). There were no significant differences in the median serum CXCL13 concentration between women at term with and without labor (median 371.6 pg/mL (IQR 194.3–614.3) vs. median 235.1 pg/mL (IQR 182.8–354.7), respectively; p = 0.6).

Figure 1. CXCL13 serum concentration in non-pregnant women and normal pregnancy. The median serum concentration of CXCL13 increases with pregnancy when compared to the non-pregnant state (non-pregnant: median 40.5 pg/mL (IQR 29.5–93.5) vs. normal pregnancy: median 313.3 pg/mL (IQR 197.2–646.9); p < 0.001).

Figure 1. CXCL13 serum concentration in non-pregnant women and normal pregnancy. The median serum concentration of CXCL13 increases with pregnancy when compared to the non-pregnant state (non-pregnant: median 40.5 pg/mL (IQR 29.5–93.5) vs. normal pregnancy: median 313.3 pg/mL (IQR 197.2–646.9); p < 0.001).

Detection of CXCL13 in amniotic fluid

displays the demographic and clinical characteristics of patients of the study groups. Amniotic fluid CXCL13 was detected in 98.8% (339/343) of patients. Four patients had CXCL13 concentrations below the limit of detection (three patients in the midtrimester group and one in the term in labor group). All four patients delivered at term without complication. The median AF concentration of CXCL13 was not different between women in the midtrimester of pregnancy and women at term not in labor (median 63.7 pg/mL (IQR 42.9–94.9) vs. median 77.8 pg/mL (IQR 68.0–98.0), respectively; p = 0.1) (). Similarly, no differences were found in the median AF CXCL13 concentration between women not in labor at term and those with spontaneous labor (median 77.8 pg/mL (IQR 68.0–98.0) vs. median 86.9 pg/mL (IQR 55.6–152.0), respectively; p = 0.8) ().

Figure 2. (a) Amniotic fluid concentration of CXCL13 in the midtrimester and in women at term without labor. There was no difference between the median amniotic fluid concentration of CXCL13 in women in the midtrimester and those at term (median 63.7 pg/mL (IQR 42.9–94.9) vs. median 77.8 pg/mL (IQR 68.0–98.0), respectively; p = 0.1). (b) Amniotic fluid concentration of CXCL13 in spontaneous labor at term. There was no difference in the median amniotic fluid CXCL13 concentration between women at term not in labor and those in labor (median 77.8 pg/mL (IQR 68.0–98.0) vs. median 86.9 pg/mL (IQR 55.6–152.0), respectively; p = 0.8).

Figure 2. (a) Amniotic fluid concentration of CXCL13 in the midtrimester and in women at term without labor. There was no difference between the median amniotic fluid concentration of CXCL13 in women in the midtrimester and those at term (median 63.7 pg/mL (IQR 42.9–94.9) vs. median 77.8 pg/mL (IQR 68.0–98.0), respectively; p = 0.1). (b) Amniotic fluid concentration of CXCL13 in spontaneous labor at term. There was no difference in the median amniotic fluid CXCL13 concentration between women at term not in labor and those in labor (median 77.8 pg/mL (IQR 68.0–98.0) vs. median 86.9 pg/mL (IQR 55.6–152.0), respectively; p = 0.8).

Table I.  Distribution of maternal characteristics in the study categories.

AF CXCL13 in preterm labor

displays the demographic and clinical characteristics of patients with spontaneous preterm labor with intact membranes. Among patients with spontaneous PTL with intact membranes, those with IAI had a significantly earlier gestational age at amniocentesis and delivery than those without IAI. In addition, patients with PTL with IAI had a significantly higher median AF concentration of CXCL13 than those without IAI who delivered preterm (median 513.2 pg/mL (IQR 199.7–2505.5) vs. median 137.3 pg/mL (IQR 96.7–209.6), respectively; p < 0.001). Similarly, patients with PTL and IAI had significantly higher median AF concentrations of CXCL13 than those with an episode of PTL who delivered at term (median 133.7 pg/mL (IQR 97.8–174.8), p < 0.001) (). The median AF CXCL13 concentrations in patients with PTL without evidence of IAI were not different between those who subsequently deliver preterm and at term (p = 0.8).

Figure 3. (a) Amniotic fluid concentration of CXCL13 in women with spontaneous preterm labor and intact membranes. The median amniotic fluid concentration of CXCL13 was significantly higher in women with preterm labor with intra-amniotic infection and inflammation (IAI) who delivered preterm than that of those without IAI who delivered at term (median 513.2 pg/mL (IQR 199.7–2505.5) vs. median 133.7 pg/mL (IQR 97.8–174.8), respectively; p < 0.001). Concurrently, in women who deliver preterm, the median amniotic fluid concentration of CXCL13 was significantly higher in women with evidence of IAI than in those without (median 513.2 pg/mL (IQR 199.7–2505.5) vs. median 137.3 pg/mL (IQR 96.7–209.6), respectively; p < 0.001). There was no difference in the median amniotic fluid concentration of CXCL13 between women with preterm labor without IAI who delivered preterm compared to those with preterm labor who delivered at term (p = 0.8). The vertical axis conforms to a logarithmic scale. (b) Amniotic fluid concentration of CXCL13 in patients with histological chorioamnionitis. The median amniotic fluid CXCL13 concentration was significantly higher in patients with histological chorioamnionitis who delivered within 72 hours after amniocentesis compared to those without histological chorioamnionitis (median 1201.1 pg/mL (IQR 246.5–4643.8) vs. median 216.0 pg/mL (IQR 113.4–430.3), respectively; p = 0.01).

Figure 3. (a) Amniotic fluid concentration of CXCL13 in women with spontaneous preterm labor and intact membranes. The median amniotic fluid concentration of CXCL13 was significantly higher in women with preterm labor with intra-amniotic infection and inflammation (IAI) who delivered preterm than that of those without IAI who delivered at term (median 513.2 pg/mL (IQR 199.7–2505.5) vs. median 133.7 pg/mL (IQR 97.8–174.8), respectively; p < 0.001). Concurrently, in women who deliver preterm, the median amniotic fluid concentration of CXCL13 was significantly higher in women with evidence of IAI than in those without (median 513.2 pg/mL (IQR 199.7–2505.5) vs. median 137.3 pg/mL (IQR 96.7–209.6), respectively; p < 0.001). There was no difference in the median amniotic fluid concentration of CXCL13 between women with preterm labor without IAI who delivered preterm compared to those with preterm labor who delivered at term (p = 0.8). The vertical axis conforms to a logarithmic scale. (b) Amniotic fluid concentration of CXCL13 in patients with histological chorioamnionitis. The median amniotic fluid CXCL13 concentration was significantly higher in patients with histological chorioamnionitis who delivered within 72 hours after amniocentesis compared to those without histological chorioamnionitis (median 1201.1 pg/mL (IQR 246.5–4643.8) vs. median 216.0 pg/mL (IQR 113.4–430.3), respectively; p = 0.01).

Table II.  Demographic and clinical characteristics of the preterm labor groups.

AF CXCL13 in preterm labor with evidence of histological chorioamnionitis

In patients with PTL who delivered within 72 hours of amniocentesis, placental pathology was available in 100% of them. Patients with histological chorioamnionitis and/or funisitis had higher median AF CXCL13 concentrations than those without (median 1201.1 pg/mL (IQR 246.5–4643.8) vs. median 216.0 pg/mL (IQR 113.4–430.3), respectively; p = 0.001) ().

Diagnostic cutoff and amniocentesis-to-delivery interval of an elevated AF CXCL13

The diagnostic efficacy of CXCL13 concentration in AF was assessed among patients with PTL and intact membranes. A cutoff of 260.2 pg/mL was derived from the ROC curve analysis for the identification of patients who had IAI (sensitivity 70.9%, specificity 89.2%, area under ROC curve 84%; p < 0.001) (). Survival analysis was employed to assess the relationship between IAI defined by CXCL13 concentration and the duration of the amniocentesis-to-delivery interval. Spontaneous labor and delivery was entered in the model as the event of interest and patients delivered for fetal or maternal indications were censored. The median amniocentesis-to-delivery interval was significantly shorter in patients whose AF CXCL13 concentrations were above 260.2 pg/mL (median amniocentesis-to-delivery interval: 4 days vs. 41 days; p < 0.001) ().

Figure 4. (a) Receiver operating characteristic (ROC) curve for identification of women with intra-amniotic infection or inflammation. An amniotic fluid CXCL13 concentration cutoff of 260.2 pg/mL was calculated (sensitivity 70.9%, specificity 89.2%, area under ROC curve 84%, p < 0.001). (b) Kaplan–Meier survival analysis of the amniocentesis-to-delivery interval (days) according to amniotic fluid CXCL13 concentration cutoff of 260.2 pg/mL in women with preterm labor and intact membranes. Women with amniotic fluid CXCL13 concentration of ≥260.2 pg/mL had a significantly shorter median amniocentesis-to-delivery interval than those with amniotic fluid concentrations of CXCL13 <260.2 pg/mL (amniocentesis-to-delivery interval 4 days, 95% confidence interval (CI) 2.1–5.9 vs. 41 days, 95% CI 33.2–48.8, respectively; p < 0.001). Patients delivered due to fetal or maternal indications were censored (circles).

Figure 4. (a) Receiver operating characteristic (ROC) curve for identification of women with intra-amniotic infection or inflammation. An amniotic fluid CXCL13 concentration cutoff of 260.2 pg/mL was calculated (sensitivity 70.9%, specificity 89.2%, area under ROC curve 84%, p < 0.001). (b) Kaplan–Meier survival analysis of the amniocentesis-to-delivery interval (days) according to amniotic fluid CXCL13 concentration cutoff of 260.2 pg/mL in women with preterm labor and intact membranes. Women with amniotic fluid CXCL13 concentration of ≥260.2 pg/mL had a significantly shorter median amniocentesis-to-delivery interval than those with amniotic fluid concentrations of CXCL13 <260.2 pg/mL (amniocentesis-to-delivery interval 4 days, 95% confidence interval (CI) 2.1–5.9 vs. 41 days, 95% CI 33.2–48.8, respectively; p < 0.001). Patients delivered due to fetal or maternal indications were censored (circles).

Immunohistochemistry of fetal membranes and umbilical cord

Immunohistochemical analysis of chorioamniotic membranes with histological inflammation demonstrated that CXCL13 was present only in HLA-DR positive macrophages in both chorion and decidua of fetal membranes (). In cases of funisitis, fetal macrophages in the umbilical vein were also immunoreactive to CXCL13 (–d). Control sections of umbilical vein without funisitis also demonstrated evidence of CXCL13 immunoreactivity (–h), but this was qualitatively diminished in comparison to cases with funisitis. In all cases, CXCL13 was localized mainly in the cytoplasm of the macrophages.

Figure 5. Immunohistochemistry of fetal membranes in a case of histologic chorioamnionitis. In fetal membranes, the amnion (AM) and the chorion (CH) of the fetal membranes are depicted in image (a) and the decidual layer is shown in image (b). Using HLA-DR labeling, resident macrophages are stained in green. Double-immunostaining with CXCL13 (in red) demonstrates CXCL13 within the macrophages. Nuclei of all cells are stained blue with DAPI. The arrow and arrowheads identify macrophages with CXCL13.

Figure 5. Immunohistochemistry of fetal membranes in a case of histologic chorioamnionitis. In fetal membranes, the amnion (AM) and the chorion (CH) of the fetal membranes are depicted in image (a) and the decidual layer is shown in image (b). Using HLA-DR labeling, resident macrophages are stained in green. Double-immunostaining with CXCL13 (in red) demonstrates CXCL13 within the macrophages. Nuclei of all cells are stained blue with DAPI. The arrow and arrowheads identify macrophages with CXCL13.

Figure 6. Immunohistochemistry of the umbilical cord vessel wall with (a) and without (b) funisitis. Nuclei of all cells are stained blue. Using HLA-DR labeling, macrophages are stained in green. Double-immunostaining with CXCL13 (in red) demonstrates CXCL13 within the cytoplasm of the macrophages. The localization of CXCL13 in the cytoplasm is apparent in the merged composite image.

Figure 6. Immunohistochemistry of the umbilical cord vessel wall with (a) and without (b) funisitis. Nuclei of all cells are stained blue. Using HLA-DR labeling, macrophages are stained in green. Double-immunostaining with CXCL13 (in red) demonstrates CXCL13 within the cytoplasm of the macrophages. The localization of CXCL13 in the cytoplasm is apparent in the merged composite image.

Detection of CXCL13 in umbilical cord blood

In term neonates, CXCL13 was detected in all umbilical cord samples. The median concentration of CXCL13 in neonates born to mothers with and without labor was not significantly different (median 79.0 pg/mL (IQR 62.5–103.8) vs. median 66.7 pg/mL (IQR 58.5–88.0), respectively; p = 0.2).

Discussion

Principal findings of this study

(1) Serum CXCL13 was increased with pregnancy and decreased as a function of gestational age, but did not change with spontaneous term labor. (2) CXCL13 was detectable in 98.8% of amniotic fluid samples. (3) CXCL13 concentration in AF was significantly increased in the presence of IAI. (4) No changes were observed in AF CXCL13 concentration with advancing gestation or in the presence of spontaneous labor at term. (5) CXCL13 was present in chorioamniotic, decidual, and umbilical cord macrophages, and the intracellular immunoreactivity of macrophages in the umbilical cord was increased in cases of histologic funisitis compared to those without evidence of histologic funisitis. (6) CXCL13 was present in the umbilical cord sera of neonates at term, and its concentration did not change with labor.

What is CXCL13?

Chemokines are a subgroup of chemo-attracting cytokines that include approximately 50 ligands involved in normal homeostasis in many organs and in the pathophysiology of infectious and inflammatory disease Citation[46]. Chemokines are classified into four structural families according to the structure of the cysteine residues in the amino-terminus (C, CC, CXC, CX3C). CXCL13 belongs to the CXC family of chemokines, all of whose genes are located on chromosome 4 Citation[47]. The only known receptor for CXCL13 is CXCR5, which exclusively binds CXCL13 Citation[48] and is expressed by mature B cells Citation[49], a subset of CD4+ and CD8+ T cells, in secondary lymphoid tissue follicles Citation[50] and immature dendritic cells Citation[51].

Originally thought to be involved only in B cell chemotaxis, CXCL13 was previously known as BLC and BCA-1. CXCL13 assists in B cell trafficking to high endothelial venules (HEVs) in mesenteric lymph nodes, Peyer's patches Citation[52], inducible bronchus-associated lymphoid tissues (iBALT) Citation[53], and various musoca-associated lymphoid tissues (MALT) Citation[54]. CXCL13 knockout mice have been used in various investigations involving lymph node development, and appear to have absent or reduced numbers of lymph nodes Citation[55], absence of B1 cells in the peritoneal and pleural cavities Citation[56], structurally abnormal nasal-associated lymphoid tissue that lack follicular dendritic cells Citation[57], and altered B cell adhesion in lymph nodes and Peyer's patches Citation[54]. Lymph nodes that are present in these animals, albeit mostly abnormal, are capable of mounting an immune response to microbial challenge although B cell migration into dendritic-rich zones of lymph nodes is critically impaired. In addition, CXCL13 does not appear to be essential for survival or reproduction since the CXCL13−/− mice used in these studies were able to mate and produce litters of pups without any reproductive complications, although the litter sizes or percentage of resorbed embryos were not reported. The presence of CXCL13 in both endothelium and epithelium-associated lymphoid tissues suggests an important role of CXCL13 on B cell function and the primary human defense response.

Recent studies have reported that CXCL13 also participates in T cell homing to germinal centers of lymphoid tissue Citation[58], induction of migration of immature dendritic cells Citation[51], and maintenance of epithelial cell angiostatic activity Citation[59]. In a study using human umbilical vein endothelial cells, CXCL13 interfered with angiogenesis through competitive binding to fibroblast growth factor (FGF)- 2 receptors on endothelium and through the formation of CXCL13–FGF-2 heterodimers Citation[60].

Role of CXCL13 in infection and inflammation

CXCL13 is implicated in chronic infectious diseases and is found in both the serum and tissues of infected individuals. CXCL13-expressing dendritic cells are found in high concentrations in granulomas in cat scratch disease (Bartonella henselae infections) Citation[61]. In neuroborreliosis, CXCL13 production is significantly correlated with spirochete load Citation[62], and measurement of serum CXCL13 has been proposed as a putative diagnostic or therapeutic marker for infection Citation[63]. In relapsing fever from Borrelia species, serum concentrations of CXCL13 have also been considered as a diagnostic marker for the disease Citation[64]. Serum concentrations of CXCL13 are elevated with human immunodeficiency virus (HIV) infection, and strongly correlate with inducible protein-10 (IP-10) and moderately correlate with viral loads Citation[33]. In addition, there is a down-regulation of CXCR5 on naïve B-cells in patients infected with HIV, which impairs the host's immune system to mount a proper response to the virus despite the elevated concentrations of CXCL13 Citation[65].

In several chronic infectious and inflammatory diseases, lymphoid neogenesis occurs in ectopic germinal centers in non-lymphoid tissue mediated by CXCL13 Citation[66]. These organized centers of local interaction of B cells, T cells, and dendritic cells serve to produce a local supply of immunoglobulin and chemokines. Ectopic germinal centers are located in bowel mucosa in inflammatory bowel disease Citation[34], synovial membranes in rheumatoid arthritis Citation[67,68], neural tissues in Lyme disease neuroborreliosis Citation[62], vascular endothelium of the CNS in lymphoma Citation[69], kidney in lupus nephritis Citation[70], salivary glands of patients with Sjogren's disease Citation[71,72], and lung in tuberculosis Citation[73].

While it is advantageous for lymphoid neogenesis to occur near the infection site in order to assist the host immune response in antigen presentation and antibody production, the resultant inflammation may exacerbate symptoms of the disease. For instance, in Lyme disease, the hallmark of chronic infection is marked tissue inflammation with resultant tissue damage. Patients frequently suffer from myositis Citation[74,75], and tissue biopsies of diseased skeletal muscles demonstrate increased expression of CXCL13 Citation[76].

There is paucity of literature on the role of CXCL13 in pregnancy. Muehlenbachs et al. Citation[38] investigated placentas from women with active Plasmodium falciparum infection and identified aggregates of B cells resembling ectopic germinal centers. The maternal macrophages in the intervillous space of malaria-positive placentas were positive for CXCL13, suggesting that up-regulation of CXCL13 was from the maternal inflammatory response. This local concentration of CXCL13 attracts naïve B cells and plasma cells and facilitates antibody production that results in extensive inflammatory intervillous infiltration that can occlude the maternal circulation. In these placentas, the pathologic finding of massive chronic intervillositis is hypothesized to cause adverse fetal outcomes Citation[77].

CXCL13 in normal pregnancy

We report for the first time, the presence of CXCL13 in the AF of normal pregnancy. In addition, serum CXCL13 concentration is increased in normal pregnancy when compared to the non-pregnant state, and decreases as a function of gestational age. CXCL13 is also detected in umbilical cord blood of term neonates. In all compartments, CXCL13 is detected in the majority of samples, and at term, the concentration does not change with spontaneous labor.

The precise biological action exerted by CXCL13 in the amniotic cavity is not clear because this is not a site of lymphoneogenesis. However, the finding of dramatic up-regulation of CXCL13 in IAI does not preclude its importance in the role of maternal or fetal immunity or in maternal tolerance of the fetal allograft. Regulatory T (Treg) cells have been implicated in autoimmune disease Citation[78], allergies Citation[79], transplant rejection Citation[80], and pregnancy Citation[81-83]. Regulatory T cells express CXCR5, the receptor for CXCL13. Blockade of CXCL13 with an antibody prevents migration of regulatory T cells into transplanted organs and may favor rejection Citation[84]. Under normal circumstances, there is up-regulation of CXCL13 in the organ shortly after transplantation. This suggests that anti-rejection T cells are homed to the allograft in the initial host response. In addition, the evaluation of renal transplant biopsies as a function of time reveal that CXCL13 expression is 27-fold higher in biopsies (with well formed aggregates of B cells) from acutely rejected transplants and below the limit of detection in non-rejected transplants Citation[85]. CXCL13 is also highly expressed in severe transplant vasculopathy Citation[86]. These findings suggest that there is a sensitive balance of CXCL13 expression and regulatory T cell chemotaxis in the maintenance of transplanted organs. The interaction between CXCL13 in the transplant and CXCR5 on the T cells plays an important role in allogeneic organ acceptance.

The rise in maternal serum CXCL13 concentration with pregnancy and the subsequent decrease throughout gestation parallels the chemokine changes that occur with successful transplanted allografts Citation[80],Citation[84,85]. The presence of CXCL13 in AF early in gestation and its stable concentration throughout pregnancy suggests that CXCL13 may also contribute to the homeostatic balance and that it may be important in the maintenance of a normal gestation.

CXCL13 in preterm labor

CXCL13 is expressed in biological fluids such as cerebrospinal fluid (CSF) Citation[64], plasma Citation[64], and serum Citation[33], and tissues in infection and inflammation Citation[38],Citation[61,62],Citation[73,74],Citation[76],Citation[87,88]. The findings of our study reported herein indicate that this can also occur in the amniotic cavity in cases of intra-amniotic infection. While microbial invasion of the amniotic cavity is an important cause of preterm labor, inflammation has also been implicated in the etiology of preterm labor and is associated with an equal rate of adverse fetal outcomes when compared with microbial invasion of the amniotic cavity Citation[41]. Numerous inflammatory peptides have been identified and have been proposed for use as diagnostic markers Citation[27],Citation[89] of increased risk of IAI and PTL. Of note, epithelial cell-derived neutrophil-activating peptide (ENA)-78 Citation[25], IL-6 Citation[90], and macrophage inhibitory factor Citation[91] are all pro-inflammatory proteins found to be elevated in IAI. Strong support of the involvement of CXCL13 in the inflammatory response is the stimulation of its production by TNF-α in the murine model Citation[92].

Lipopolysaccharide (LPS) can induce CXCL13 production by dendritic cells Citation[93]. This microbial product has been previously found in the amniotic cavity and decidua, which contain dendritic cells Citation[94]. It is possible that extra-amniotic infection stimulates the production of CXCL13 in the decidua. However, the precise mechanism responsible for the increased CXCL13 in AF in cases of intra-amniotic infection is unknown. We have demonstrated the presence of CXCL13 in the cytoplasm of macrophages in the fetal membranes and the umbilical cord. It is possible that microbial products or bacteria stimulate the production of CXCL13 by resident macrophages in the amniotic cavity, in the fetal membranes, or in the fetus. Indeed, there is evidence that LPS can stimulate monocytes and macrophages to produce CXCL13 Citation[34]. The same study indicated that macrophages secrete more CXCL13 than recently recruited monocytes or dendritic cells. It is possible that CXCL13 may be an important link between innate and adaptive immune responses because this chemokine would participate in the recruitment of B cells, which could produce antibodies.

What is unique about CXCL13 is that unlike other pro-inflammatory chemokines, such as IL-8 Citation[23] and CCL5/RANTES Citation[21], its concentration in AF does not change with normal term parturition and does not decrease with advancing gestation. Increased CXCL13 concentration in AF is specific to PTL with evidence of IAI. Indeed, the increase in AF CXCL13 is not only a marker for a disease process, but may also initiate a pro-inflammatory response to IAI that leads to the activation of preterm parturition pathways.

Origin of CXCL13 in the amniotic fluid

In placental malaria, maternal macrophages in the intervillous space were hypothesized to be the source of CXCL13 production Citation[38]. However, the identification of CXCL13 in the maternal compartment does not explain its presence in AF. Evaluation of cord blood demonstrates that the fetus mounts a response to infection by activation of monocytes and neutrophils Citation[95]. Specifically, elevations in fetal umbilical cord blood cytokines have been identified in IAI Citation[96], periventricular leukomalacia Citation[97,98], and a fetal systemic inflammatory response syndrome Citation[99]. Indeed, the leading hypothesis for the source of inflammatory cytokines in AF has been of fetal origin, from the fetal intravascular compartment Citation[100], or from fetal membranes Citation[101-103]. It is known that CXCL13 is present in normal umbilical endothelium Citation[104], and CXCL13 expression has been identified in fetal spleen and vascular smooth muscle as early as 14 weeks of gestation Citation[105]. Therefore, the source of the increase in CXCL13 concentrations in the AF with IAI may be a fetal response to IAI.

Conclusions

The chemokine CXCL13 is a physiologic constituent of AF. Unlike other known chemokines, CXCL13 concentration does not increase with advancing gestation or with spontaneous labor at term. Concentrations of this protein are significantly increased in the presence of IAI, indicating an acute response to microbial products in the amniotic cavity. Our findings suggest that CXCL13 may contribute to the innate immune response against intra-amniotic infection and/or inflammatory response. Further investigations are needed to elucidate the origin and biological actions of CXCL13 in normal and abnormal pregnancy.

Acknowledgements

This research was supported in part by the Intramural Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, DHHS.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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