Objective
17α-hydroxyprogesterone caproate (17P) may decrease risk of prematurity by suppressing maternal immunity. We hypothesized that in vivo 17P treatment attenuates immunoresponsiveness of peripheral blood mononuclear cells (PBMCs).
Study Design
Study subjects were gravidas receiving weekly prophylactic intramuscular 17P injections. Peripheral blood samples were obtained at 21-27 weeks’ gestation. Gestational age–matched, drug-naïve gravidas served as controls. To simulate infection, isolated PBMCs were stimulated with lipoteichoic acid (LTA) or lipopolysaccharide (LPS). Extracellular interleukin-6 (IL-6) concentrations were quantified by an enzyme-linked immunosorbent assay.
Results
Unstimulated IL-6 levels were comparable in PBMCs derived from drug-naïve and 17P-treated subjects. LPS and LTA induced a dose-dependent elevation of IL-6 in control PBMCs. In patients who received exogenous 17P, LPS, and LTA stimulated induction of IL-6 was significantly decreased compared with controls ( P = .005 and P = .02).
Conclusion
In vivo 17P attenuated immunoreactivity of PBMCs in our in vitro model of Gram-positive and Gram-negative bacterial infection.
Clinical trials have demonstrated that exogenous progestin therapy can lower the rate of recurrent preterm birth. Current American College of Obstetricians and Gynecologists guidelines recommend offering progestin therapy to women with a prior history of preterm birth or preterm premature rupture of membranes. Weekly intramuscular injections of the progestin analog 17α-hydroxyprogesterone caproate (17P) has become the most widely used mode of progestin treatment in current practice, but the underlying pharmacological mechanism of prevention of recurrent preterm delivery remains elusive.
Several mechanisms have been proposed to explain how progestins reduce the risk of preterm delivery, including modulation of the maternal inflammatory response. Because prematurity is positively correlated with maternal immune response to infection, the effect of progestins on maternal immunity is an active area of research. Progestin cross-regulation of host inflammatory response is well established in other gynecological disease states, including endometriosis and endometrial cancer. However, studies investigating progestin regulation of innate immunity have produced conflicting results.
Infection and immunity are mechanistically linked to premature parturition, and the clinical presentation of preterm labor may be in part caused by a maladaptation of the host’s immune response. In normal pregnancies, maternal inflammatory processes such as leukocyte activation are attenuated until just prior to parturition. Leukocyte migration to the myometrium and cervix promotes parturition at term by cytokine release resulting in up-regulation of prostaglandins and matrix metalloproteinases, leading to increased myometrial contractility, membrane rupture, and cervical ripening. Infection may contribute to a premature up-regulation of leukocyte migration and subsequent cytokine release, representing 1 mechanism in the complex cascade of signaling events triggering preterm labor.
Maternal infection is most commonly associated with mixed flora comprised of both Gram-positive and Gram-negative organisms. We and other investigators have demonstrated that in vitro progesterone attenuates interleukin (IL)-6 response in maternal and fetal peripheral blood mononuclear cells (PBMCs) following stimulation with lipopolysaccharide (LPS), a component of the cell wall in Gram-negative bacteria.
In this study, we hypothesized that in vivo 17P exposure attenuates in vitro cytokine responsiveness of maternal PBMCs to both Gram-positive and Gram-negative bacterial cell wall components.
Materials and Methods
This study was performed with the approval of the Madigan Army Medical Center Institutional Review Board. After informed consent, peripheral blood was obtained at 21-27 weeks’ gestation from women receiving 17P injections, 250 mg once a week, as clinically indicated by the obstetrical history. 17P therapy was initiated between 16 and 20 weeks’ gestation. Control samples were obtained from gestational-age matched patients. The 17P was obtained from a single compounding pharmacy.
Dexamethasone was from Phoenix Scientific (St Joseph, MO). Lipopolysaccharide (LPS) from Escherichia coli (serotype O55:B5) and lipoteichoic acid (LTA) from Bacillus subtilis were from Sigma (St Louis, MO). All reagents were prepared in working aliquots (1000 times) and stored frozen at between –20°C and –70°C until use. Dimethylsulfoxide for solubilizing progestins was obtained in single-use ampules (Sigma).
PBMCs were isolated by Histopaque gradient according to the manufacturer’s recommendations (Sigma) and plated as previously described. PBMCs were cultured for 48 hours. Cells were stimulated with LPS or LTA, incubated for 16 hours, and then supernatants were collected. They were stored at –70°C until an enzyme-linked immunosorbent assay (ELISA). The optimum dose of LPS or LTA was determined by generating a dose-response curve. The responsiveness of the assay was confirmed by the dexamethasone suppression of IL-6 secretion.
ELISAs were conducted according to the manufacturer’s recommendations using an IL-6 monoclonal capture antibody and biotinylated polyclonal secondary antibody (IL-6 Duoset DY206E; R&D Systems, Minneapolis, MN), with amplification in horseradish peroxidase–conjugated streptavidin and subsequent visualization using a colorimetric 3,3′,5,5′-tetramethylbenzidine substrate (Sigma Chemical) followed by 2 N sulfuric acid to terminate the reaction. Optical density measurements at 450 nm were measured using an HT Synergy plate reader coupled to Gen 5 software (Biotek Instruments, Inc, Winooski, VT). Purified recombinant IL-6 protein was used to generate a standard curve for assessing the approximate concentrations. The concentration of IL-6 was determined by normalization to purified IL-6 protein standards.
SPSS statistical software (version 14; SPSS Inc, Chicago, IL) was used to perform 1-way analysis of variance using a post hoc multiple comparisons test with Bonferroni correction to test the null hypothesis . A threshold of P < .05 was set a priori to determine the statistical significance.
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