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
), 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
). 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 (, 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
). 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
). 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
). Exposure of fetal sheep to LPS mediated chorioamnionitis increases MCP-1 in cord blood, which may contribute to the fetal inflammatory response (18
). 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
). 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
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
). 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.