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ORIGINAL RESEARCH |
From the Department of Biochemical Sciences and Advanced Therapy, Section of Obstetrics and Gynaecology, the Department of Biology, and the Department of Clinical and Experimental Medicine, Section of Nuclear Medicine, University of Ferrara, Ferrara, Italy.
Address reprint requests to: Prof Fortunato Vesce, Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynaecology, University of Ferrara, 44100 Ferrara, Italy; e-mail: ves{at}dns.unife.it.
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ABSTRACT |
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METHODS: In an in vitro study, IL-6 and PGE2 release from amnion-like Wistar Institute Susan Hayflick cells was assayed under basal conditions, as well as after incubation with ampicillin. In an in vivo study, amniotic fluid IL-6 was assayed in a total of 212 patients submitted to genetic amniocentesis during the 17th week of their singleton physiological pregnancy. The study population was subdivided as follows: 92 patients sampled before ampicillin administration, 70 patients sampled 4 hours after administration of 1 g ampicillin, and 50 patients sampled 12 hours after administration of 1 g ampicillin.
RESULTS: At doses ranging from 10-7 to 10-4 M, ampicillin decreased IL-6 release from Wistar Institute Susan Hayflick cells. The drug effect was already statistically significant (-30%; P < .05) at the lowest concentration tested (10-7 M), reaching the maximum (-50%) at 10-6 M after 4 hours of incubation. Moreover, ampicillin concentrations ranging from 10-7 to 10-4 M decreased PGE2 release from Wistar Institute Susan Hayflick cells; maximal inhibition was reached at 10-6 M after 4 hours (-40%; P < .05). Finally, IL-6 levels measured in amniotic fluid of patients sampled 4 hours after ampicillin administration proved strongly and significantly reduced when compared with those sampled either before or 12 hours after treatment (P < .001).
CONCLUSION: The capacity of ampicillin to directly decrease amniotic IL-6 and PGE2 release should be considered in the management of bacterial and nonbacterial inflammatory complications of pregnancy mediated by the cytokine and prostanoid interaction.
LEVEL OF EVIDENCE: III
However, as the pregnancy progresses, IL-6 appears to play an opposite role. Its amniotic fluid concentration is increased in cases of spontaneous abortion secondary to second-trimester amniocentesis.5 In addition, cytokine plays an important role in the pathogenesis of preterm delivery by stimulating prostaglandin (PG) release.
The importance of a well balanced regulation of prostaglandin E2 (PGE2) and IL-6 levels for a successful pregnancy raises the question of whether it is possible to pharmacologically control their release. In this regard, it is worth noting that IL-6 is able to increase PG output from amnion cells6 and that PGs, in turn, can affect IL-6 release.7–9 Moreover, it has been reported that IL-10 is able to decrease both PGE210 and IL-611 release from fetal membranes.
Based on the close connection between the cytokine and the prostanoid, one can hypothesize that factors able to decrease PGE2 output could also impair IL-6. Because it has been demonstrated that some classes of antibiotics, of which ampicillin is the most effective, significantly inhibit PGE2 release from amnion tissue,12,13 the decision was made to test the effect of ampicillin on amniotic IL-6 release both in vitro and in vivo. The in vitro experiments were performed on amnion-like Wistar Institute Susan Hayflick cells, a suitable model for the study of amniotic prostanoid metabolism. In these experiments, along with IL-6, PGE2 release upon ampicillin treatment was also tested. The in vivo study consisted of assaying IL-6 amniotic levels before and after ampicillin administration at the 17th week of gestation.
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MATERIALS AND METHODS |
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Amnion-like Wistar Institute Susan Hayflick cells were obtained from the American Type Culture Collection (ATCC CCL-25; Manassas, VA) and maintained in the laboratory. Cells were grown at 37°C in an atmosphere of 5% CO2/95% air, in a mixture of Ham’s F12 and Dulbecco’s modified Eagle medium (1:1 vol/vol) supplemented with 10% fetal bovine serum (10%), 30 µg/mL gentamicin, and 0.25 µg/mL amphotericin B.
The cells were seeded in 24-well plates at 2 x 105 cells/well in Ham’s F12 and Dulbecco’s modified Eagle medium + 10% fetal bovine serum and grown to confluence (2–3 days).
For PGE2 and IL-6 determination, the medium was removed and replaced with fresh serum-free Ham’s F12 and Dulbecco’s modified Eagle medium containing ampicillin. Each point of both time-courses and dose-response relationships was performed in duplicate and obtained from 3 or 4 different cell culture plates.
After incubation of samples for the indicated time, the media were collected and stored at -80°C until PGE2 radioimmunoassay was performed.
Cell culture and PGE2 assay were performed at the Laboratory of Cellular Physiology of the Department of Biology of Ferrara University.
The study population consisted of a total of 212 consecutive white patients submitted to genetic amniocentesis during the 17th week of their singleton physiological pregnancy, as revealed by uncomplicated, either spontaneous or elective, cesarean delivery of an healthy fetus at term. Indication for cesarean birth was represented in all the cases by cesarean delivery in the previous pregnancy (12%). The sample was collected between December 2000 and December 2001 at the Section of Obstetrics and Gynaecology of Ferrara University. At this clinic, as part of a protocol aimed at decreasing the incidence of abortion secondary to the procedure, all patients to undergo amniotic fluid sampling are treated with an oral dose of 1 g ampicillin twice daily for 4 days. All the patients gave informed, verbal consent to the trial’s protocol. Approval for this study was obtained from the University of Ferrara institutional review board. The 212 cases were subdivided into the following 3 groups: 1) 92 amniotic fluids sampled before ampicillin administration (untreated group); 2) 70 amniotic fluids sampled 4 hours after administration of an oral dose of 1 g ampicillin (A group); and 3) 50 amniotic fluids sampled 12 hours after administration of 1 g oral ampicillin (B group).
Average maternal age was 36.2 years. The main indication for the diagnostic procedure was represented by advanced maternal age (66.5%); other indications were a positive triple test (21.4%), the presence of echographic markers of increased risk of fetal aneuploidy (10.3%), maternal anxiety (1%), and a family history of Down syndrome or other genetic disorders (0.8%).
Within 6 hours of withdrawal, all the samples were centrifuged at 800g for 10 minutes at 4°C to remove particulate materials and then stored at -80°C.
The assay procedure was performed at the Laboratory of Nuclear Medicine of Ferrara University.
Interleukin-6 was determined in duplicate on each collected medium by commercial enzyme-linked immunosorbent assay (ELISA; Bender Medsystem Diagnostic GmbH, Vienna, Austria) according to the manufacturer’s instructions.
The specific monoclonal antibody in this kit was able to detect IL-6 in cell culture supernatants, human serum, plasma, and body fluids in general. In addition, to ensure correct quantitative determination of the cytokine, all the samples were diluted 1:6 in assay buffer. A reference curve was obtained by the standard IL-6 concentrate (range 1.6–100 pg/mL) (1 ng/mL) adjusted to the International Reference Standard (NBSB 88/514).
Optical density (OD) values were obtained with an automated microplate reader (Model 550; Bio-Rad, Richmond, CA) at 450 nm (620 nm as optional reference wavelength). A linear regression was established between optical density (Y axis) and the corresponding standard concentration (X axis); IL-6 sample concentrations were calculated by interpolating from the reference curve and multiplying by the dilution factor. The results were expressed as picograms per milliliter for the amniotic samples and as picograms per 106 cells for the Wistar Institute Susan Hayflick cells experiments. Assay sensitivity was 1.4 pg/mL.
The amount of PGE2 was assayed in duplicate on each collected medium by a radioimmunoassay procedure, as previously reported.14 A specific antiserum for PGE1 and PGE2 (cross reactions 100% and 165%, respectively) was used. Label [3H] PGE2 (3 nCi) was added to each tube. The level of PGE2 in each serially diluted sample was determined by comparison with a standard displacement curve (15–250 pg/0.1 mL). Incubation was performed for 90 minutes at 4°C. Bound and free radioligands were separated by dextran-coated charcoal, followed by centrifugation of tubes for 10 minutes at 2,000g. Assay sensitivity was 15 pg/tube, and the intraassay or interassay coefficients of variation were less than 10%. Data were expressed as nanograms of PGE2 produced per 106 cells.
Cell culture and PGE2 assay were performed at the Laboratory of Cellular Physiology of the Department of Biology of Ferrara University.
IL-6 levels measured in amniotic fluid were normalized by using logarithmic transformation.
Kolmogorov-Smirnov goodness of fit test was computed on logarithmic transformation to assess whether the transformed variable was normally distributed (Kolmogorov-Smirnov d = 0.0665; P< .20).
Thus, to assess what effect both antibiotic administration and the amount of time elapsed between antibiotic treatment and amniocentesis have on IL-6 levels in amniotic fluid, one-way ANOVA was applied, followed by the Tukey honestly significant difference post hoc comparison test for unequal sample size. Descriptive statistics in Table 1
refer to the variables before logarithmic transformation. The significance levels for the rejection of the null hypothesis were set at P< .05. These statistical analyses were carried out by using Statistica Release 5.5 software (StatSoft Inc, Tulsa, OK).
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RESULTS |
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) showed a basal cytokine level that increased in time. The addition of 10-6 M ampicillin induced a significant and maximal inhibition after 4 hours (-50%; P< .05), although for longer incubation times this inhibitory effect was no longer observed. For this reason, subsequent dose-response relationships were tested at 4 hours (Figure 2). Concentrations of ampicillin, ranging from 10-7 to 10-4 M, decreased IL-6 levels; the drug effect was already statistically significant (-30%; P< .05) at the lowest concentration tested (10-7 M) and reached the maximum (-50%) at 10-6 M. The higher doses did not further inhibit IL-6 output.
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. The basal level of PGE2 increased with time. The addition of 10-6 M ampicillin induced a statistically significant inhibition after 30 minutes (-24%; P< .05) and maximal inhibition (-36%; P< .05) after 4 hours; with longer incubation times, the inhibitory effect disappeared. Therefore, we tested dose-response effects (Figure 4) at 30 minutes (A) and at 4 hours (B). At 30 minutes of incubation, concentrations of ampicillin, ranging from 10-7 to 10-4 M, decreased PGE2 release with significant effect (-26%; P< .05) only at 10-6 M. At 4 hours, the inhibition was significant and reached the maximum effect at 10-6 M (-40%; P< .05).
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shows the IL-6 levels measured in amniotic fluids along with the descriptive statistics. One-way ANOVA showed highly significant differences in IL-6 levels between the 3 groups (F = 18.04; P< .001). Post hoc comparisons revealed that IL-6 levels in amniotic fluids obtained from women treated with ampicillin 4 hours before amniocentesis were strongly and significantly reduced compared with the levels measured in women who were not treated or were treated 12 hours before the procedure (P< .001 for both comparisons). No significant difference was observed between IL-6 amniotic levels obtained from women not treated with ampicillin and those obtained from women treated 12 hours before amniocentesis (P= .866).
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DISCUSSION |
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On the basis of the above considerations, it is logical to think that, from the second trimester onward, a decrease in the amniotic level of IL-6 could play a protective role in preventing the cytokine-evoked prostaglandin release that leads to abortion, premature delivery, and perinatal morbidity. Several drugs are able to reduce IL-6 output,20–22 but a clinical study on their efficacy in the prevention of adverse events during pregnancy has never been performed.
Our data show that ampicillin has a clear inhibitory effect on IL-6 amniotic release, both in vitro and in vivo. As for the in vitro action, we found that, in amnion-like Wistar Institute Susan Hayflick cells, the maximal effect of the drug is reached at 10-6 M, 4 hours after treatment. Furthermore, we found that, in the same cells, ampicillin significantly inhibits PGE2 release, reaching the maximal effect at the same optimal dose and incubation time as for cytokine inhibition. This suggests a possible correlation between the PGE2 and IL-6 biosynthetic pathways. This correlation is further supported by the report that indomethacin is able to decrease the blood level of IL-6 by inhibiting PGE2 synthesis.20 On the contrary, along with IL-1ß and TNF
, the anti-inflammatory drug ibuprofen increases IL-6 levels in the mononuclear cells of preterm newborns.23 Such a dual effect of PGE2 on IL-6 release can be explained if one hypothesizes specific functions for each of the 4 prostanoid receptors. Indeed, it has been reported that, in mast cells, an increase in IL-6 is mediated by the interaction of PGE2 with its EP3 receptor. On the contrary, lack of the EP4 receptor in knockout mice lowers the level of circulating IL-6, as well as its macrophages and liver cell release.8,9
The present study also shows, for the first time, that ampicillin is able to decrease in vivo IL-6 amniotic fluid levels by a mechanism independent from its antibacterial property. Indeed, we found that an oral dose of 1 g of the drug is able to significantly reduce the amniotic cytokine level 4 hours after administration, a response fitting with the time course revealed by the in vitro study. The clinical implication of our finding deals with the management of IL-6–related inflammatory complications during pregnancy. Indeed, ampicillin, which is widely used in the treatment of bacterial-mediated complications, also appears to be indicated in nonbacterial inflammatory conditions. Based on our experience, we believe that its use is the main tool in our preventive arsenal for reducing spontaneous abortion secondary to second-trimester amniocentesis. In fact, in our 3,500 amniocenteses, all performed by the same operator, the total pregnancy loss was 0.3% (unpublished data), a percentage lower than that in the general population between the 17th and the 28th week of gestation. In our opinion, the only explanation for such a finding is that inflammatory conditions marked by increased IL-6 and PGE2 activity are effectively controlled by ampicillin through a mechanism independent of its antibacterial properties. Among other antibiotic candidates that lower the cytokine level, penicillin has been reported to inhibit IL-6 release from decidual cells,22 and erythromycin is known to reduce the Streptococcus pneumoniae–induced cytokine production in human whole blood.21 However, it is logical to believe that the efficacy of the latter is consequent to its antibacterial property, because it is unable to directly inhibit amniotic PGE2 release13.
In conclusion, given ampicillin’s ability to decrease amniotic IL-6 and PGE2 release, its use should be considered in the management of inflammatory complications, such as chorioamnionitis, premature labor, and premature rupture of the membranes mediated by the cytokine and prostanoid interaction.
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Footnotes |
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doi:10.1097/01.AOG.0000101282.38902.13
Received March 28, 2003. Received in revised form June 26, 2003. Accepted October 2, 2003.
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REFERENCES |
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