|Home | About | Journals | Submit | Contact Us | Français|
Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis, and IL-1 contributes to the pathogenesis of chorioamnionitis-induced lung inflammation and fetal inflammatory responses. We tested the hypothesis that IL-1 mediates the systemic and pulmonary induction of MCP-1 and MCP-2 in response to lipopolysaccharide (LPS) induced chorioamnionitis. MCP-1 mRNA, MCP-2 mRNA and MCP-1 protein expression were measured in two models: 1) intra-amniotic LPS and 2) intra-amniotic recombinant sheep IL-1α given at varying intervals prior to preterm delivery at 124d gestational age. Intra-amniotic LPS or IL-1α induced MCP-1 mRNA and protein and MCP-2 mRNA in fetal lung many fold at 1-2d. LPS induced intense MCP-1 expression in sub-epithelial mesenchymal cells and interstitial inflammatory cells in the lung. Inhibition of IL-1 signaling with recombinant human IL-1 receptor antagonist (rhIL-1ra) did not attenuate LPS induced increase in MCP-1 or MCP-2 expression. MCP-1 and MCP-2 were not induced in liver or chorioamnion, but MCP-1 increased in cord plasma. LPS or IL-1 can induce robust expression of MCP-1 or MCP-2 in the fetal lung. LPS induction of MCP-1 is not IL-1 dependent in fetal sheep. MCP-1 and MCP-2 may be significant contributors to fetal inflammation.
Chorioamnionitis is associated with the majority of preterm births prior to 28 weeks gestation in the US (1, 2). Although preterm infants less than 30 weeks gestation are only about 1.5% of the approximately 4 million births in the US, they account for 70% of the neonatal mortality. Although multiple cytokines are thought to contribute to preterm labor and delivery (3), MCP-1 is the most predictive cytokine for preterm birth for women with a short cervix (4). MCP-1 is also elevated in the amniotic fluid of women who deliver preterm after mid-trimester amniocentesis (5). This cytokine is increased in cervical secretions, amniotic fluid and placental tissues from preterm deliveries associated with chorioamnionitis (6–8). MCP-1 is also increased in the bronchoalveolar lavage fluid (BALF) and plasma of infants with oxidant injury, who subsequently develop bronchopulmonary dysplasia (BPD) (9, 10). BPD develops in about 48% of infants born at <28 wks gestation (11). Chorioamnionitis is associated with a systemic inflammatory response and adverse neurological and gastrointestinal outcomes in premature infants (12, 13).
The subfamily of CC chemokines includes MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, and eotaxin. Both MCP-1 and MCP-2 bind and signal through a 7-transmembrane spanning G protein-coupled receptor, CCR2 (14). MCP-1 is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages (15). Both MCP-1 and its receptor CCR2 are important mediators in inflammatory diseases such as multiple sclerosis and atherosclerosis (16, 17).
Intra-amniotic injection of LPS in sheep recruits neutrophils and monocytes to the fetal lung and results in lung injury responses similar to BPD (18). This LPS mediated recruitment is CD18 and IL-1 dependent (18, 19). Intra-amniotic IL-1 also causes recruitment of monocytes and neutrophils to the fetal lung (20, 21). IL-1 transcriptionally controls induction of MCP-1 and MCP-2 in in-vitro systems (22).
Given the importance of MCP in monocyte chemotaxis in adult inflammatory diseases and its prominent association with prematurity, we explored induction of MCP-1 and MCP-2 by LPS and IL-1 in preterm fetal sheep. To test the hypothesis that MCP-1 and MCP-2 induction by intra-amniotic LPS is IL-1 dependent, we used 2 models of chorioamnionitis: intra-amniotic injection of LPS or IL-1. IL-1 signaling also was inhibited with a recombinant human IL-1 receptor antagonist (rhIL-1ra) (19). We measured the expression of MCP-1 mRNA and protein, and MCP-2 mRNA in the lung, liver, chorioamnion and plasma and localized MCP-1 in the lung.
Fetal sheep were sampled in Western Australia with approval from the animal care and use committees of Cincinnati Children’s Hospital (Cincinnati, OH), and the University of Western Australia (Perth, WA, Australia). In separate protocols, date-mated Merino ewes with singleton fetuses were randomly assigned to the following groups (n=5–9/group): 1) 10 mg of LPS (E. coli 055:B5; Sigma, St. Louis, MO), given by intra-amniotic injections at 2 h, 5 h, 2 d or 7 d before delivery. 2) 100 μg of recombinant sheep IL-1 α (Protein Express, Cincinnati, OH), given by intra-amniotic injection 1, 2, 4 or 7 d before delivery, 3) 100 mg rhIL-1ra (Anakinra [Kineret]; Amgen, Inc., Thousand Oaks, CA) or saline given by intra-amniotic injection 3 h before intra-amniotic LPS (10 mg) or saline injection (19). Animals delivered 2 d after LPS exposure received only 1 dose of rhIL-1ra, and animals delivered 6 or 7 d after LPS exposure received two additional intra-amniotic 100-mg doses of rhIL-1ra or saline treatment at 2 and 4 d (19). All animals were surgically delivered at 124d gestational age (83% term gestation), at which the fetal sheep is in an early alveolar stage of lung development, roughly corresponding structurally to a human equivalent of 32–34 weeks (23). Control animals were injected with an equivalent 2 ml volume of saline. The lung maturational and inflammatory responses to LPS, IL-1α, and IL-1ra for these animals were previously reported (19). Intra-amniotic injections were given with ultrasound guidancewith verification of needle placement by electrolyte analysisof amniotic fluid (19). The biological efficacy of the dose and route of administration of rhIL-1ra in the sheep was verified by demonstrating that the same lot of rhIL-1ra completely inhibited intraamniotic and intravenous IL-1α induced lung and systemic inflammation (reported in the online section of ref (19)).
Plasma from heparinized cord blood was collected. BALF was recovered from the leftlung using 3 normal saline lavages to total lung capacity (19). Theright upper lobe of lung was inflation fixed with 10% bufferedformalin at 30 cm H2O pressure. Pieces of the liver, chorioamnion and theright lower lobe of the lung were snap-frozen for RNA analysis.
Total RNA was isolated using a modified Chomczynski method from lung, liver and chorioamnion (24). The sheep MCP-1 cDNA clone [pGEMT-MCP-1] was cut with Spe1 and an antisense riboprobe with a 268 nucleotide-protected fragment was synthesized using T7 RNA polymerase. The sheep MCP-2 cDNA clone [pGEMT-MCP-2] was cut with Bsa1 and an antisense riboprobe with a 190 nucleotide-protected fragment was synthesized using T7 RNA polymerase. Ten micrograms of RNA were used for RNase protection assays with the antisense MCP-1 or MCP-2 riboprobes. Solution hybridization was performed as reported (24). The probe for L32, a ribosomal protein mRNA, was included in each assay as an internal standard. Protected fragments were resolved on 6% polyacrylamide 8 M urea gels, visualized by autoradiography and quantified on a Phosphor Imager using Image Quant version 1.2 software (Molecular Dynamics, Sunnyvale, CA).
In situ hybridization was used to localize MCP-1 mRNA expression in paraffin embedded sections with a digoxigenin-labeled antisense riboprobe ([pGEMT-MCP-1] cut with Not1). The plasmid MCP-1 was digested with Not1 forthe antisense probe (T7 polymerase; Promega) and with NcoI forthe sense probe (SP6 polymerase; Promega). The riboprobe wasgenerated in an in vitro transcription reaction using digoxigenin-UTP(Roche). For hybridization, the sense and antisense probes werediluted in hybridization buffer to a final concentration of1 μg/ml (100 μl/section) and incubated at 57°C.After hybridization, sections were washed with a buffer containing50% formamide for 30 min at 65°C. Sections were then treatedwith RNase A/T1 to reduce nonspecific binding followed by washes.An anti-digoxigenin antibody conjugated with alkaline phosphatasewas applied (1/2000; Roche) overnight at 4°C followed byreacting with an alkaline phosphatase substrate containing nitroblue tetrazolium chloride (Roche). Controls for specificityof riboprobe binding included lung tissues obtained fromlambs exposed to intra-amniotic saline and homologous(sense) probes.
Recombinant sheep MCP-1 protein was synthesized using a cloned full-length sheep MCP-1 sub-cloned into an expression vector (Protein Express, Cincinnati). Anti MCP-1 antibodies were generated in house in guinea pigs and rabbits by injecting the recombinant sheep MCP-1 protein using standard techniques for generation of polyclonal antibodies (25).
Immunostaining was done on paraffin embedded sections using antigen retrieval(0.1 M citrate with micro-wave boiling) as previously reported (26). The following primary antibodies were used: a) Goat polyclonal antibody against vimentin (Santacruz biotechnology, CA, catalogue #sc7557, dilution 1:50), b) Guinea pig anti-MCP1 antiserum (1:250) and c) Anti-smooth muscle actin (Sigma, St. Louis MO, catalogue# 5228, dilution 1:5000). After the slides were washed withPBS to remove unbound antibody, they were incubated with thesecondary biotinylated antibody against rabbit IgG (Vector Laboratories, Burlingame,CA, dilution 1:200), or the appropriate fluorescently labeled secondary antibodies for co-localization experiments (dilution 1:200). For the experiments using biotinylated antibodies, avidin (Vector Laboratories) was added and stainingfor positive cells was developed with diaminobenzidine and cobaltwith a nuclear fast red counterstain. Specificity of staining was demonstrated by the ability of excess peptide to compete with the antibody signal, and by using lung sections from control animals.
MCP-1 protein was quantified in undiluted alveolar wash and cord plasma. We developed a sandwich ELISA assay for measuring MCP-1 in fluids using a) IgG fraction of rabbit anti-sheep MCP-1 serum as a coating antibody, and b) guineapig anti-sheep MCP-1 serum for the detection antibody (19). Standard curves were constructed using serial dilutions of recombinant sheep MCP-1. The lowest detection limit of this assay was 0.1ng/ml with a dynamic range of 0.1–80 ng/ml, and a correlation coefficient of 0.99 for all assays.
All values were expressed as means ± SEM. ANOVA was used for comparison of differences between groups with Dunn’s multiple comparison test used for post hoc analysis. The unpaired t-test with Welch correction was used for comparison between two groups. Significance was accepted at p < 0.05 (2-tailed).
MCP-1 mRNA expression increased 4 fold at 2 h, 7 fold at 5 h, by over 50-fold at 1 and 2 d and returned to control levels at 7d after intra-amniotic LPS (Fig. 1A). Intra-amniotic IL-1 induced MCP-1 mRNA 56 fold in the lung at 1d with inter-animal variability and returned to baseline levels at 4–7 d after exposure (Fig. 1B). Consistent with the mRNA expression MCP-1 protein increased in BALF after intra-amniotic LPS (Fig. 1C) and after intra-amniotic IL-1 (Figure 1D).
The mRNA for MCP-1 was not detected in chorioamnion or fetal liver following intra-amniotic injections of LPS or IL-1α (data not shown). In contrast, MCP-1 protein was not present in cord plasma from control lambs, but increased in cord plasma after intra-amniotic LPS (Fig. 1E) or IL-1 (Fig. 1F).
Intra-amniotic LPS induced intense MCP-1 mRNA expression in the mesenchyme around large airways within 5 h, with more diffuse expression by 1 and 2 d throughout the mesenchyme and in inflammatory cells (Fig. 2). Consistent with the mRNA expression, MCP-1 protein was detected in the mesenchymal cells surrounding the airways at 5 h (Fig. 3B) and 1 d (Fig. 3C), with protein localized primarily to inflammatory cells by 2 d (Fig. 3D). To further localize the mesenchymal cells expressing MCP-1 at 5h post LPS, we performed co-localization immunostaining experiments using the combination antibodies against: MCP1+alpha-smooth muscle actin or MCP1+vimentin. The cells expressing MCP-1 did not express alpha-smooth muscle actin (data not shown) indicating that the cells were not of smooth muscle or myofibroblast origin. However, a large number of the MCP-1 expressing cells also expressed vimentin (Fig. 3E–G). The results are consistent with the predominant expression of MCP-1 in the fibroblasts early after LPS exposure.
In separate groups of animals given intra-amniotic IL-1ra followed by LPS, the LPS exposure greatly increased the mRNA for MCP-1 in lung tissue, the amount of MCP-1 protein in BALF and the MCP-1 protein in cord plasma (Fig. 4). Prior exposure to intra-amniotic rhIL-1ra did not change the LPS induced increases in MCP-1 in the lung or the cord plasma.
In patterns similar to MCP-1, MCP-2 expression greatly increased after intra-amniotic LPS or IL-1 (Fig 5A–B). In a separate experiment, the IL-1ra inhibition of IL-1 signaling did not change the increase in MCP-2 mRNA at 2 d (Fig 5C).
We initially identified MCP-1 as a gene that was highly induced in fetal lung after intra-amniotic LPS in a subtraction hybridization screen (27). We have now explored the expression of MCP-1 and MCP-2 in preterm fetal sheep models of chorioamnionitis. MCP-1 and MCP-2 are robustly induced in the fetal lung after intra-amniotic LPS or IL-1. Soon after fetal exposure to LPS, expression of MCP-1 was detected in resident lung cells, primarily the mesenchymal fibroblasts. The LPS induced MCP-1 and MCP-2 expression was not dependent on IL-1 because blocking the IL-1 receptor did not change MCP-1 or MCP-2 expression. There was no significant MCP-1 or MCP-2 expression in the fetal liver or chorioamnion, although MCP-1 was increased in cord plasma. The origin of the plasma MCP-1 is not known, but could be from lung or circulating inflammatory cells. MCP-1 is implicated in the pathogenesis of preterm labor and bronchopulmonary dysplasia (BPD) (6–10), diseases commonly associated with infants born prematurely. However, in these diseases many other cytokines are also upregulated. Understanding the hierarchical regulation of the cytokine networks will enable study of the pathogenesis of inflammation in these diseases. The results of the present study demonstrate that either signaling via a TLR4 receptor or IL-1 receptor in the setting of chorioamnionitis can induce MCP expression in the lung.
MCP-1 and MCP-2 are mediators of monocyte recruitment in the lung and other organs. Experiments using CCR2 −/ − & MCP-1−/ − mice demonstrate that MCP-1 is required for monocyte recruitment. (15, 28, 29). A neutralizing antibody to MCP-1 inhibited monocyte and neutrophil recruitment to the injured lung (30). In fetal sheep, intra-amniotic LPS induced pulmonary recruitment of neutrophils and monocytes is CD18 dependent (18). IL-1 is a major down-stream contributor to LPS induced lung inflammation In fetal sheep, because inhibition of rhIL-1 signaling by IL-1ra decreased LPS mediated pro-inflammatory cytokine expression and the recruitment of monocytes and neutrophils to the fetal lungs by about 70% (19).
In the present experiments, there was a trend towards decreased LPS induced MCP-1 expression in the bronchoalveolar lavage fluid (BALF) of the lambs after blockade of IL-1 signaling (Fig 4B, p=0.08 for LPS vs LPS+rhIL-1ra). Although, the result could be due to a type-II error, the more likely explanation is that there were fewer cells in the BALF for the LPS+rhIL-1ra group compared to the LPS group as reported previously (19). Indeed, when BALF MCP-1 expression was normalized to the number of inflammatory cells, the corresponding values for MCP-1 levels were 46 ± 6 ng/106 cells (2d LPS group) vs 198 ± 119 ng/106 cells (2d LPS+rhIL-1ra)(p=0.3). These results are consistent with the interpretation that while IL-1 signaling partially mediates LPS induced pulmonary inflammatory cell recruitment, it does not mediate pulmonary MCP-1 expression. It is interesting that at 2d after LPS exposure, there was a correlation between BALF MCP-1 level and neutrophil counts (R2=0.76) and a more modest correlation with monocyte counts (R2=0.17). However, the precise role of MCP-1 in fetal monocyte or neutrophil recruitment is not known since an inhibitor of MCP signaling for testing in sheep is not yet available. Similarly, we could not characterize the expression and modulation of the MCP-2 protein expression or the MCP receptor CCR2 in these experiments because of lack of appropriate reagents. Taken together, our data suggest that MCP-1 and IL-1 may both contribute to inflammatory cell recruitment in response to a TLR4 agonist.
LPS signals via TLR4 whereas IL-1 signals via IL-1R, but both agonists share similar intracellular signaling pathways (31). While LPS and IL-1 induce many genes in common, they also induce distinct sets of genes in the fetal lung. For example, the interferon inducible genes IP-10 and MIG are induced by LPS but not IL-1 (27), while IL- 1β, IL-6, IL-8 and SAA3 are induced by both LPS and IL-1 (19). SAA3 and MCP-1 induction by LPS is not IL-1 dependent in the fetal sheep while recruitment of inflammatory cells is partially IL-1 dependent (19, 32). These experiments inform us of the cytokine regulatory networks induced by a TLR agonist in vivo.
Fetal inflammatory response syndrome is a poorly characterized entity that is associated with umbilical cord inflammation and increased plasma cytokine levels in the absence of bacteremia (33, 34). A systemic inflammatory response induced by chorioamnionitis is postulated to be the proximate cause of fetal organ injuries such as periventricular leukomalacia (35), necrotizing enterocolitis (12), or BPD (12, 35). Exposure of fetal sheep to LPS mediated chorioamnionitis increases MCP-1 in cord blood, which may contribute to the fetal inflammatory response (18, 19). A caveat of the LPS-chorioamnionitis model is that the pro-inflammatory agonist used in these experiments is the LPS derived from gram-negative organisms, while the most common bacteria isolated from placenta of preterm infants with chorioamnionitis are the Ureaplasma species (36). Despite these limitations, the present experiments are an initial attempt to understand the mechanisms of regulation of MCP-1, a commonly identified chemokine in the perinatal period of preterm infants (6–10).
Chorioamnionitis is associated with pulmonary expression of cytokines that have different sites of expression. IL-1 is expressed in inflammatory cells of the lung, while SAA3 and IP-10 are expressed mostly in the lung epithelium (32). We find that MCP-1 is expressed initially in the lung mesenchyme, predominantly fibroblasts, followed by expression in the inflammatory cells. These specific sites of expression may have implications for lung injury and repair.
Hyperoxia-induced pulmonary leukocyte accumulation in newborn mice is partly prevented with an anti-MCP-1 antibody (37), thus macrophage chemokine blockade may attenuate the inflammation that can impair lung development and result in BPD (38). Concentrations of MCP-1 in amniotic fluid are increased in women in preterm labor with or without chorioamnionitis, suggesting that MCP-1 may play a role in preterm labor regardless of the presence of chorioamnionitis (5, 7). Plasma MCP-1 levels are elevated for the first 21 d after birth in infants who developed BPD (9). Blood MCP-1 levels are elevated in adults with atherosclerosis, acute myocardial infarction and unstable angina (39). Thus, MCP-1 may be a non-specific inflammatory marker and is a potential diagnostic biomarker for BPD. The expression profile of MCP-1 and MCP-2 in fetal sheep in response to LPS or IL-1 suggests that these cytokines may contribute to multiple inflammation associated pathologies in the preterm and deserve further study.
The authors wish to thank Angela Keiser, Bill Hull, and Amy Whitescarver for their expert technical assistance.
Grant Support: This work was supported by grants HL-65397 and HD-57869 from the National Institutes of Health.
Disclosures: None of the authors have a commercial interest in any entity related to subject of the manuscript or have a conflict of interest relative to the work.