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Gelsolin down-regulates lipopolysaccharide-induced intraamniotic tumor necrosis factor-α production in the midtrimester of pregnancy

Published:December 29, 2008DOI:https://doi.org/10.1016/j.ajog.2008.09.011

      Objective

      The purpose of this study was to identify gelsolin in midtrimester amniotic fluid and evaluate its interaction with lipopolysaccharide (LPS).

      Study Design

      Supernatants from 40 midtrimester amniotic fluid samples were incubated with Escherichia coli LPS, and gelsolin binding was measured by enzyme-linked immunosorbent assay. Unfractionated aliquots of 25 of the fluids were cultured ex vivo for 24 hours in the presence of LPS and supernatants tested for tumor necrosis factor (TNF)-α and interleukin (IL)-10 production, and the influence of antigelsolin antibody was evaluated.

      Results

      Each amniotic fluid was positive for gelsolin that bound to LPS. LPS-induced TNF-α production was inversely proportional to the amniotic fluid concentrations of LPS-bound gelsolin (r = -0.5047; P = .006). Preincubation with monoclonal antibody to gelsolin led to an increase in LPS-induced TNF-α production (P = .01). There was no relationship between gelsolin and IL-10 production.

      Conclusion

      Gelsolin is present in midtrimester amniotic fluid, binds to LPS, and inhibits the induction of TNF-α.

      Key words

      Gelsolin is a protein found both in human plasma and in the cytoplasm.
      • Sun H.Q.
      • Yamamoto M.
      • Mejillano M.
      • Yin H.L.
      Gelsolin, a multifunctional actin regulatory protein.
      Intracellularly, gelsolin functions to cleave actin filaments, thereby aiding in chemotaxis and movement of intracellular structures. Plasma gelsolin is a component of an actin-scavenging system that prevents capillary plug formation by depolymerizing actin filaments that have been released from injured tissues and are present in the circulation.
      • Rothenbach P.A.
      • Dahl D.
      • Schwartz J.J.
      • et al.
      Recombinant plasma gelsolin infusion attenuates burn-induced pulmonary microvascular dysfunction.
      Clinically, reduced circulating gelsolin concentrations have been associated with increased mortality in patients with trauma and burn.
      • Mouzner K.C.
      • Moncure M.
      • Smith Y.R.
      • Dinubile M.J.
      Relationship of admission plasma gelsolin levels to clinical outcomes in patients after major trauma.
      Gelsolin also functions as a lipid carrier and mediator of inflammatory responses by virtue of its strong binding affinity for several cytosolic and plasma lipids, such as phosphatidylinositol and lysophosphatidic acid.
      • Goetzl E.L.
      • Lee H.
      • Azuma T.
      • Stossel T.P.
      • Turck C.W.
      • Karliner J.S.
      Gelsolin binding and cellular presentation of lysophosphatidic acid.
      • Karliner J.S.
      • Honbo N.
      • Summers K.
      • Gray M.O.
      • Goetzl E.J.
      The lysophospholipids sphingosone-1-phosphate and lysophosphatic acid enhance survival during hypoxia in neonatal rat cardiac myocytes.
      Of specific interest to studies of infection-related preterm birth is the recent finding that gelsolin binds to lipopolysaccharide (LPS) from various bacteria with high affinity.
      • Bucki R.
      • Georges P.C.
      • Espinassous Q.
      • et al.
      Inactivation of endotoxin by human plasma gelsolin.
      For Editors' Commentary, see Table of Contents
      The intraamniotic cavity at midtrimester is not always sterile; asymptomatic carriage of microorganisms can be present in up to 10% of pregnant women.
      • Gerber S.
      • Vial Y.
      • Hohlfeld P.
      • Witkin S.S.
      Detection of ureaplasma urealyticum in second-trimester amniotic fluid by polymerase chain reaction correlates with subsequent preterm labor and delivery.
      • Perni S.C.
      • Vardhana S.
      • Korneeva I.
      • et al.
      Mycoplasma hominis and Ureaplasma urealyticum in midtrimester amniotic fluid: association with amniotic fluid cytokine levels and pregnancy outcome.
      • Wenstrom K.D.
      • Andrews W.W.
      • Bowles N.E.
      • Towbin J.A.
      • Hauth J.C.
      • Goldenberg R.L.
      Intrauterine viral infection at the time of second trimester genetic amniocentesis.
      However, the majority of these women have uneventful pregnancies and deliver healthy neonates. Microbial products such as LPS from the cell wall of Gram-negative bacteria may also sometimes be present in amniotic fluid. We have hypothesized that mechanisms exist within the amniotic cavity to down-regulate induction of proinflammatory immunity that may place the ongoing pregnancy at risk. In previous studies we demonstrated that ex vivo–cultured whole midtrimester amniotic fluid samples can be induced to produce cytokines, including tumor necrosis factor (TNF)-α,
      • Jean-Pierre C.
      • Bongiovanni A.M.
      • Perni S.A.
      • et al.
      Extracellular 70-kD heat shock protein in mid-trimester amniotic fluid and its effect on cytokine production by ex vivo–cultured amniotic fluid cells.
      • Sezen D.
      • Bongiovanni A.M.
      • Jean-Pierre C.
      • Linhares I.M.
      • Skupski D.
      • Witkin S.S.
      Ex vivo cytokine production by whole mid-trimester amniotic fluid.
      a major trigger of preterm labor,
      • Gravett M.G.
      • Witkin S.S.
      • Haluska G.J.
      • Edwards J.L.
      • Cook M.J.
      • Novy M.J.
      An experimental model for intraamniotic infection and preterm labor in rhesus monkeys.
      • Romero R.
      • Gomez R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazur M.
      • Berry S.M.
      A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition.
      and that intraamniotic 70-kDa heat shock protein modulated this process.
      • Jean-Pierre C.
      • Bongiovanni A.M.
      • Perni S.A.
      • et al.
      Extracellular 70-kD heat shock protein in mid-trimester amniotic fluid and its effect on cytokine production by ex vivo–cultured amniotic fluid cells.
      The objectives of the present study were to evaluate midtrimester amniotic fluid for the presence of gelsolin that reacted with LPS and determine whether this interaction influenced LPS-induced cytokine production.

      Materials and Methods

      Forty women undergoing routine transabdominal amniocentesis during weeks 15-19 of gestation, between December 2006 and June 2007, were enrolled. Indications for amniocentesis were advanced maternal age, abnormal values on first- or second-trimester maternal serum screening, or maternal request. All patients were negative for vaginal and cervical infections as assessed by routine cultures during a previous clinical examination. Subsequent analysis revealed the absence of chromosome abnormalities in all subjects. The study was approved by our institutional review boards, and written consent was obtained from all participants.
      Excess unprocessed amniotic fluid was transported to the laboratory within 3 hours of collection. Aliquots were removed for immediate ex vivo culture; the remainder of the samples were centrifuged, and supernatant fractions stored at -80°C until analyzed. All amniotic fluid samples were visibly free of blood contamination.
      Binding of gelsolin in amniotic fluid supernatants to LPS was quantitated by enzyme-linked immunosorbent assay (ELISA). Microtiter plate wells were coated with LPS from Escherichia coli serotype 0111:B4 (Sigma, St Louis, MO) by adding 100 μL of a 30-μg/mL solution in sodium carbonate-bicarbonate buffer, pH 10, and incubating for 24 hours at 4°C. The LPS solution was removed, and wells were washed 3 times with 0.05% Tween-20 in phosphate-buffered saline (TPBS). For measurement of LPS-bound gelsolin, amniotic fluid supernatants were diluted 1:2 in TPBS and added in duplicate to the LPS-containing wells, incubated for 1 hour at room temperature, and then the wells were washed 3 times with TPBS. Antigelsolin mouse monoclonal antibody (Novus Biologicals, Littleton, CO) at a concentration of 3.2 μg/mL in TPBS was then added to each well and incubated for 1 hour at room temperature. The wells were again washed 3 times with TPBS, and 100 μL/well of a peroxidase-conjugated antimouse IgG (1:500 dilution in TPBS) (KPL Laboratories, Gaithersburg, MD) was added for a 1-hour incubation at room temperature. After washing of the wells 3 times with TPBS, 100 μL of the peroxidase substrate, 3,3′,5′,5-tetramethylbenzidine (Sigma), was added. The reaction was stopped after 30 minutes by addition of 100 μL of 0.18 mol/L of sulfuric acid, and the absorbance at 450 nm was determined using a microtiter plate reader.
      Blank wells lacking LPS and LPS-containing wells lacking amniotic fluid were always assayed in parallel to the test samples and their absorbance values subtracted from the experimental values. The optical density was proportional to amniotic fluid concentration from undiluted samples to a 1:10 dilution. Replicate samples assayed on the same day or on different days had an assay variability of < 10%.
      The total protein concentration in the amniotic fluid supernatants was measured by the bicinchoninic acid protein assay (Pierce, Rockford, IL). The LPS-bound gelsolin optical density value was adjusted to the total protein concentration in each amniotic fluid supernatant.
      For ex vivo culture, 0.1-mL aliquots of fresh unfractionated amniotic fluid samples were added to multiple wells of a sterile microtiter plate. To quadruplicate wells, 0.02 mL of either Roswell Park Memorial Institute (RPMI) culture medium alone or culture medium containing a final concentration of 50 ng/mL E coli serotype 0111:B4 LPS was added. The plates were incubated for 24 hours in 5% CO2 at 37°C and centrifuged; supernatants of like aliquots were pooled and frozen at -80°C until assayed.
      To further directly assess the influence of gelsolin on LPS-induced TNF-α and interleukin (IL)-10 production, 0.1 mL of fresh unfractionated amniotic fluid samples was incubated for 1 hour at 37°C in the presence of control mouse IgG 2a antibody or antigelsolin mouse IgG 2a monoclonal antibody at a concentration of 50 μg/mL prior to addition of either RPMI culture medium alone or culture medium containing 50 ng/mL E coli LPS. After a 24-hour incubation period at 37°C, supernatants were collected and frozen at -80°C until assayed for TNF-α and IL-10.
      Amniotic fluid supernatant fractions were tested in duplicate for TNF-α and IL-10 by ultrasensitive commercial ELISA tests (BioSource International Inc, Camarillo, CA). The lower limits of sensitivity were < 0.20 pg/mL for IL-10 and < 0.09 pg/mL for TNF-α.
      For determination of gelsolin by Western blot, amniotic fluid supernatants were mixed with sample buffer (0.5 mol/L Tris HCl pH 6.8; 10% glycerol, 2% sodium dodecyl sulfate; 0.01% bromphenol blue; and 4% beta-mercaptoethanol), boiled for 10 minutes, and 100 μg was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to a nitrocellulose membrane using a semidry blot device (Transblot; BioRad, Hercules, CA). After transfer, the membrane was blocked with 5% dry milk in Tris-buffered saline and incubated with the gelsolin-specific monoclonal antibody overnight at 4°C. The membrane was then blotted with horse radish peroxidase-conjugated secondary antimouse antibody, and images were developed using a chemiluminescence device (BioRad).
      Associations between gelsolin detection and LPS-induced ex vivo TNF-α and IL-10 production were assessed by the Spearman rank correlation test. The effect of antigelsolin antibodies on TNF-α and IL-10 production was analyzed by the Student's t-test using the Welch correction. A P value < .05 was considered significant.

      Results

      All subjects proceeded to have an uneventful term delivery of a healthy neonate. Amniotic fluid samples were all positive for gelsolin that reacted with LPS, as determined by optical density values that exceeded those obtained in control wells. The range of optical density values (minus background) were 0.096 to 0.315/mg amniotic fluid protein. There was no relation between gelsolin level and reason for amniocentesis or gestational age at the time of amniocentesis.
      Because we lacked purified gelsolin for use as a standard in the ELISA tests, Western blot analyses were performed to verify that the antigelsolin monoclonal antibody was detecting amniotic fluid-derived gelsolin of the correct molecular weight. The results of 7 randomly selected amniotic fluid samples (Figure 1) confirmed that all were positive for the 86-kDa gelsolin protein.
      Figure thumbnail gr1
      FIGURE 1Western blot of amniotic fluid for gelsolin
      Seven amniotic fluid samples were subjected to polyacrylamide gel electrophoresis, proteins transferred to nitrocellulose membranes, and location of gelsolin identified with specific monoclonal antibody.
      Sezen. Gelsolin down-regulates LPS-induced TNF-α production. Am J Obstet Gynecol 2009.
      Sufficient amniotic fluid volumes were available from 25 of the samples to permit ex vivo culture analysis. The relationship between LPS-induced TNF-α production and amniotic fluid gelsolin concentration is shown in Figure 2. There was a strong negative association between the level of intraamniotic gelsolin and LPS-induced TNF-α production by cells in amniotic fluid (r = -0.5047; P = .0062). In marked contrast, there was no relationship between the intraamniotic gelsolin level and IL-10 production by the amniotic fluid cultures (data not shown).
      Figure thumbnail gr2
      FIGURE 2Association between intraamniotic gelsolin levels and lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production by ex vivo–cultured amniotic fluid
      Gelsolin levels in amniotic fluid samples that bound to LPS were determined by enzyme-linked immunosorbent assay (ELISA). Aliquots of same amniotic fluids were cultured ex vivo in presence or absence of LPS, and TNF-α release into culture supernatant was assessed by ELISA. TNF-α production in absence of LPS was subtracted from experimental values.
      Sezen. Gelsolin down-regulates LPS-induced TNF-α production. Am J Obstet Gynecol 2009.
      To further assess whether gelsolin was inhibiting LPS-induced TNF-α and/or IL-10 production, the influence of antibody to gelsolin on these activities was assessed (Table). A 1-hour preincubation of whole amniotic fluids with monoclonal antibody to gelsolin prior to addition of LPS led to a significant increase in TNF-α production, as opposed to amniotic fluids preincubated with a control monoclonal antibody (P = .011). In contrast, the mean production of IL-10 was unaffected (P > .05).
      TABLEInfluence of monoclonal Ab to gelsolin on LPS-induced TNF-α and IL-10 production by ex vivo–cultured midtrimester amniotic fluid
      Amniotic fluidTNF-α production (pg/mL)IL-10 production (pg/mL)
      Control AbAntigelsolin AbControl AbAntigelsolin Ab
      11461503.05.3
      21001199.37.7
      39612223.910.2
      4732221.11.6
      530942.31.8
      613025013.915.6
      774797.77.2
      81371722.39.2
      91102118.05.2
      101002503.84.6
      Mean100.5166.9
      P = .011 vs control Ab.
      7.56.8
      P = .792 vs control Ab.
      SD32.763.67.04.2
      SE10.320.12.21.3
      Ab, antibody; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor.
      Whole unprocessed amniotic fluids were incubated in duplicate for 1 hour at 37°C in the presence of either 50 μg/mL mouse monoclonal Ab to gelsolin or a control monoclonal Ab. LPS was then added to the ex vivo cultures and incubation continued for 24 hours. The culture supernatants were then collected and assayed for TNF-α and IL-10 levels by enzyme-linked immunosorbent assay.
      Sezen. Gelsolin down-regulates LPS-induced TNF-α production. Am J Obstet Gynecol 2009.
      a P = .011 vs control Ab.
      b P = .792 vs control Ab.

      Comment

      Using a unique LPS-binding assay, we report for the first time the presence of gelsolin in midtrimester amniotic fluid from apparently healthy women and its ability to react with LPS from E coli. Furthermore, we present data implicating gelsolin in limiting the proinflammatory immune response to LPS by cells present in the amniotic cavity. The ability of gelsolin to bind to LPS and to inhibit TNF-α production by cells in the amniotic cavity strongly suggests that this protein participates in the down-regulation of intraamniotic proinflammatory cytokine production that would be potentially damaging to the fetus and to the ongoing pregnancy. Prolonged or excessive production of proinflammatory mediators has been implicated in the etiology of neonatal neural defects
      • Hagberg H.
      • Mallard C.
      • Jacobsson B.
      Role of cytokines in preterm labor and brain injury.
      and the induction of preterm birth.
      • Hillier S.L.
      • Witkin S.S.
      • Krohn M.A.
      • Watts D.H.
      • Kiviat N.B.
      • Eschenbach D.A.
      The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection.
      LPS-mediated induction of proinflammatory immunity proceeds via activation of Toll-like receptor (TLR) 4.
      • Janssens S.
      • Beyaert R.
      Role of Toll-like receptors in pathogen recognition.
      The binding of LPS to gelsolin apparently interferes with this LPS-TLR 4 interaction and, thereby, prevents activation of genes coding for inflammatory mediators. Gelsolin has been reported to compete with LPS binding protein for reactivity with LPS,
      • Bucki R.
      • Georges P.C.
      • Espinassous Q.
      • et al.
      Inactivation of endotoxin by human plasma gelsolin.
      and this may be the mechanism of gelsolin anti-TNF-α activity within the amniotic cavity. In contrast, gelsolin appeared to have no consistent effect on IL-10 production. The lack of a correlation between gelsolin-related influence on LPS-induced TNF-α and IL-10 production suggests that these 2 mediators may function independently in response to the presence of endotoxin in the amniotic cavity. Recent studies have suggested that regulation of IL-10 production is more complex than previously thought. This antiinflammatory mediator is not only produced by Th2 lymphocytes but also by Th1 cells, B lymphocytes, dendritic cells, and macrophages.
      • O'Garra A.
      • Vieira P.
      TH1 cells control themselves by producing interleukin-10.
      Therefore, it is not surprising that TNF-α and IL-10 may be differentially regulated under specific environmental conditions. Our sample number was small and, therefore, further studies are required to explore mechanisms influencing the relative intraamniotic production of TNF-α and IL-10.
      Squamous epithelial cells exfoliate from the amnion, skin, and the urogenital, respiratory, and digestive tracts of the fetus and are present in second-trimester amniotic fluid. The cell concentration varies between 103 and 106 cells/mL.
      • Hoehn H.
      • Salk D.
      Morphological and biochemical heterogeneity of amniotic fluid cells in culture.
      • Prusa A.R.
      • Hengstschlager M.
      Amniotic fluid cells and human stem cell research–a new connection.
      We recently reported on experiments using ex vivo–cultured midtrimester amniotic fluid. The addition of a TLR 2 agonist (the peptidoglycan component of Gram-positive bacterial cell wall) to whole amniotic fluid cultures stimulated the release of the inducible 70-kDa heat shock protein. The addition of exogenous 70-kDa heat shock protein was also shown to potentiate TNF-α production by the cultured amniotic fluid cells.
      • Jean-Pierre C.
      • Bongiovanni A.M.
      • Perni S.A.
      • et al.
      Extracellular 70-kD heat shock protein in mid-trimester amniotic fluid and its effect on cytokine production by ex vivo–cultured amniotic fluid cells.
      Thus, amniotic fluid cells possess TLRs, and TLR activation leads to release of an endogenous mediator that promotes an antimicrobial inflammatory response. In addition, we have also shown that the intraamniotic concentration of Clara cell protein 16, a major immunomodulatory protein, is elevated in women who subsequently deliver preterm after premature rupture of membranes.
      • Perni S.C.
      • Vardhana S.
      • Kalish R.
      • Chasen S.
      • Witkin S.S.
      Clara cell protein 16 concentration in mid-trimester amniotic fluid: association with fetal gender, fetal GA +38 CC16 gene polymorphism and pregnancy outcome.
      Together with the present report, these observations highlight the existence of multiple immune regulatory mechanisms apparently operative within the midtrimester amniotic cavity. Further investigations are necessary to elucidate the involved pathways.
      It remains to be determined whether midtrimester intraamniotic levels of gelsolin vary with race/ethnicity, fetal sex, gravidity and parity, or the subsequent development of neonatal morbidities or adverse pregnancy outcomes. Studies to address these issues are now in progress. The binding of gelsolin to LPS would also be expected to reduce its actin-severing function. Whether this is pertinent to regulatory mechanisms within the amniotic cavity is presently unknown.
      In summary, gelsolin may be a component of a fetal immunoregulatory system that recognizes the presence of bacterial LPS and modulates the amount and duration of its interaction with cellular receptors.

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