- Email: [email protected]
In vitro cytokine and prostaglandin production by amnion cells in the presence of bacteria
In vitro cytokine and prostaglandin production by amnion cells in the presence of bacteria Klaus Reisenberger, MD, Christian Egarter, MD, Ingrid Schiebel, Andreas Obermair, MD, Herbert Kiss, MD, and Rainer Lehner, MD Vienna, Austria OBJECTIVE: Our goal was to evaluate the effect of group B streptococci on cytokine and prostaglandin production by amnion cells in vitro. STUDY DESIGN: Amnion cells from placentas obtained immediately after primary cesarean section were incubated for 48 hours with heat-inactivated group B streptococci at increasing concentrations. Samples for quantification of interleukin-1 [3, interleukin-6, interleukin-8, tumor necrosis factor-a, and prostaglandin E2 were collected at 6, 12, 24, and 48 hours. RESULTS: Basal cytokine production was not demonstrable for any of the cytokines investigated. Incubation of amnion cells with bacterial antigen led to a significant increase in interleukin-6 and interleukin-8 production, whereas secretion of interleukin-1 [3 and tumor necrosis factor-a was not enhanced. In contrast to cytokines, basal prostaglandin E2 production was measurable but failed to increase after addition of antigen. CONCLUSION: Amnion cells can be stimulated to secrete interleukim6 and interleukin-8 in response to streptococcal antigen. However, this rise in cytokines does not induce an increase in prostaglandin E2, This may be explained by the lack of interleukin-1 and tumor necrosis factor-c~ production, two cytokines that have been shown to activate prostaglandin E 2 secretion by amnion cells. (Am J Qbstet Gynecol 1997;176:981-4.)
Key words; A m n i o n cell culture, prostaglandins, cytokines, bacterial infection, p r e t e r m labor
Ascension of bacteria f r o m the lower genital tract into the amniotic cavity is t h o u g h t to be a key event in the etiology of p r e t e r m ]abor. In their study on bacterial colonization of the lower extent of tile a m n i o n BrandtNiebelschfitz et al. 1 f o u n d that the microorganisms recovered most frequently were coagulase-negative staphylococci and g r o u p B streptococci, with less c o m m o n isolates including Ureaplasma and Mycoplasma species. W h e n bacteria colonize the cervix and the lower extent of the amnion, they are able to readily p e r m e a t e fetal m e m b r a n e s and invade the amniotic cavity, as recently d e m o n s t r a t e d by Gyr et al. ~ in an in vitro study. For example, Escherichia coli organisms p e r m e a t e chorioamniotic m e m b r a n e s within 6 hours after inoculation. The role of the a m n i o n in the initiation of parturition and the effects of infection on the biochemical processes in the a m n i o n are still n o t completely understood. A m n i o n ceils are an i m p o r t a n t source o f prostaglandin E 2 (PGE2) production. W h e n bacteria attach to or invade
From the Department of Obstetrics and @necology, University of Vienna Medical School. Receivedfor publication September5, 1996; revisedJanuary 2, 1997; acceptedJanua U 21, 1997. Reprint requests:Klaus Reisenberger, MD, Departmentof Obstetricsand Gynecology, Vienna Medical School, Wiihringer C_.4irtel18-20, 1090 Vienna, Austria. Copyright © 1997 by Mosby-Year Book, Inc. 0002-9378/97 $5.00 + 0 6/1/80541
chorioamniotic membranes, they c o m e into contact with a m n i o n cells. In 1955 Zitcer et al. s were the first to r e p o r t a m e t h o d for culture of a m n i o n cells. Since then n u m e r ous investigators have used a m n i o n culture systems to study physiologic and pathophysiologic events. < 5 Because the literature does n o t provide any data on the direct effects of bacterial antigen on cytokine and prostaglandin p r o d u c t i o n by a m n i o n cells, this study was designed to d e t e r m i n e the effects in a prima1T a m n i o n cell culture with use of a strain of Streptococcus because of the potential p a t h o g e n i c role in obstetrics. Material and m e t h o d s
A m n i o t i c m e m b r a n e s were obtained f r o m two w o m e n u n d e r g o i n g primary" cesarean section n e a r term to minimize the influence of labor on cytokine and prostaglandin production. W o m e n whose amniotic m e m b r a n e s were used had had an uneventful obstetric history, and they had b e e n taking no drugs. Patients with signs of a m n i o n infection syndrome, such as maternal fever, elevated maternal serum C-reactive protein levels, discolo r e d amniotic fluid, fetal tachycardia, or signs of gestosis were not i n c l u d e d in this study. Immediately after placentas had b e e n obtained, amn i o n tissue was dissected from the c h o r i o n laeve and placed in H a n k ' s balanced salt solution s u p p l e m e n t e d with 1% penicillin-streptomycin (Gibco, Paisly, Scotland) 981
982
Reisenberger et al.
May 1997 A m J Obstet Gyneco!
60
SO
40.
o.
I
I--,,--~o2} ~loa I
I 10 i
/'/"/
0S
...........
t2
-,o-.1041 --X---1O6! +106] -~-1071
o ........
24
48
hours
Fig. 1. Secretion of IL-6 by amnion cells in response to addition of increasing concentrations (10 tO 107) of heat-inactivated group B streptococci,
for transport to the laboratory, where a single cell suspension was prepared. 6-s The isolated ceils were resuspended in Dulbecco's m i n i m a l essential m e d i u m with n u t r i e n t mix F12 (1:1; Gibco) s u p p l e m e n t e d with 10% fetal calf serum, antibiotics, and antimycotics, seeded in 24-well culture dishes at a density of 3 × 105 per well and grown to confluence at 37°C in an atmosphere of 5% carbon dioxide in air for 3 to 4 days. Cell viability was assessed in control cultures with use of trypan blue. The microorganism chosen for this study, Streptococcus agalactiae, was placed in physiologic saline solution and heat inactivated in a water bath at 70°C for 10 minutes. Inactivation was considered necessary because, particularly with high microbial counts, live germs may have resulted in overgrowth. Media were aspirated from the cell culture, and fresh m e d i u m containing the S. agalactiae suspension was added in increasing concentrations (0 to 10 v colonybuilding u n i t s / m l ) . Analysis was done in triplicate, with cells plated in three wells for each microbial count and incubation period. The supernatants of these three wells were mixed and analyzed together. This procedure was used for financial reasons because testing each culture individually would have been three times as expensive. Samples were collected at 6, 12, 24, and 48 hours of i n c u b a t i o n and centrifuged for 10 minutes at 600g. The supernatants were stored frozen at -70 ° C until assayed at the e n d of the test series. As a control, u n i n o c u l a t e d m e d i u m was treated identically t h r o u g h o u t the incubation period, and samples for quantification of interleukin and prostaglandin were taken at the same intervals. The production of interleukin-l[3 (IL-I[3), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-ct (TNF-cQ, and PGE 2 from a m n i o n cells was measured from the supernatants by enzyme i m m u n o s o r b e n t assay (R&D Systems, Minneapolis).
Results In the control cultures not exposed to S. agalactiae antigen no basal cytokine production was observed. The incubation of amnion ceils with bacterial antigen did not result in the production of TNF-c~ and IL-l[3 during the 48-hour observation period. However, IL-6 and IL-8 concentrations showed a significant increase after stimulation with bacterial antigen (Figs. 1 and 2). Stimulation was similar for both cytokines, with IL-8 production being higher by the factor of 10. Synthesis of both interleukins was found to depend on the time of incubation and on the concentration of antigen added. At lower microbial counts measurable levels of IL-6 and IL-8 were not detected until 24 hours of incubation, whereas higher counts produced a significant increase within only 12 hours. In contrast to amnion cell cytokine production, a basal production of PGE 2 was demonstrable but showed no significant increase after addition of antigen. Also, there was no correlation between PGE 2 production and the concentrations of antigen added. Moreover, the production of PGE 2 started to decrease as early as 24 hours after inoculation (Fig. 3).
Comment Bacterial products such as phospholipases, lipopolysaccharides, and endotoxins that are released after disruption of gram-negative bacteria can liberate arachidonic acid from uterine tissues.9 Biotransformation of arachidonic acid by the cyclooxygenase pathway in turn gives rise to an obstetrically relevant compound, PGE 2, which is known to possess potent uterotonic properties. Moreover, bacterial infection activates immune mechanisms, resulting in an increase in cytokines that are secreted primarily by macrophages in the amnion, chorion, and decidua. The effects of these cytokines on prostaglandin production have already been investigated in numerous
Reisenberger et al.
Volume 176, Number 5 Am J Obstet Gynecol
983
600
600
4OO
~E. 3oo
-
2oo -
lOO q ol
12
24
48
hour~
Fig. 2. Secretion of IL-8 by amnion cells in response to addition of increasing concentrations (10 to 107) of heat-inactivated group B streptococci.
I........ I 0 6
12
24
48
hours
Fig. 3. PGE 2 concentration after addition of increasing concentrations (10 to 106) of heat-inactivated group B streptococci to an amnion cell culture.
studies. For example, it has b e e n shown that a m n i o n cells can be stimulated to p r o d u c e PGE 2 in response to TNF-a and Ibl{~. 4 Cytokines m o d u l a t e and control i m m u n o l o g i c processes, and it is likely that they also contribute to different aspects o f the hurnoral and cellular defense m e c h a n i s m s o f the fetal m e m b r a n e s . Thus inflammatory processes in the fetal m e m b r a n e s have b e e n f o u n d to trigger the secretion of IL-1 and IL-6, leading to significant accumulation o f these cytokines in the amniofic fluid, a° It is further believed that IL-8 causes degradation o f type I collagen by stimulating the release of collagenases f r o m chemotactically attracted p o l y r n o r p h o n u c l e a r leukocytes, thus contributing to cervical ripening, la In this in vitro investigation we have shown that bacterial antigen acts on a m n i o n cells to significantly increase secretion o f IL-6 a n d I b 8 . This rise in cytokine p r o d u c t i o n did n o t induce an increase in POE 2 secretion. Mitchell et al. 1~ have shown that IL-6 stimulates PGE 2 p r o d u c t i o n f r o m cultured a m n i o n cells. However, relatively high IL-6 concentrations (100 n g / m l ) are
r e q u i r e d to increase PGE 2 p r o d u c t i o n from amnion, and the IL-6 concentrations achieved in o u r study were well below these levels. In this context Bry and H a l l m a n as have d e m o n s t r a t e d in an in vitro m o d e l that the basal p r o d u c t i o n of PGE 2 by a m n i o n cells is increased after exposure to IL-l[3 and TNF-c~. In our study the production of these two cytokines was n o t e n h a n c e d after addition o f streptococcal antigen. This is in a g r e e m e n t with the results of M e n o n et al., 14 who f o u n d that a m n i o n cells appear to lack the capacity to p r o d u c e IL-I[3, although IL-I[3 can bind to receptors on the m e m b r a n e of a m n i o n cells. Prostaglandin p r o d u c t i o n in o u r investigation showed a significant decrease after about 24 hours o f incubation, d r o p p i n g even below the basal p r o d u c t i o n . This may be attributable to the lack o f prostaglandin H synthetase in cultured a m n i o n cells d e m o n s t r a t e d by Bala et al. a5 A major finding in the c u r r e n t study is that bacterial antigen stimulates cytokine p r o d u c t i o n in a concentrat i o n - d e p e n d e n t m a n n e r . At m e d i a n microbial concentrations the microbial counts were even directly correlated
984
Reisenberger et al.
with IL-6 and IL-8 synthesis (Figs. 1 and 2). However, further increases in bacterial antigen concentrations caused no further significant rise in the synthesis o f IL-6 and IL-8. Moreover, we observed a temporal p h e n o m e non, with h i g h e r microbial concentrations resulting in earlier stimulation of cytokine p r o d u c t i o n than lower concentrations (Figs. 1 and 2). In this study we were unable to assess w h e t h e r different microorganisms differ in their pattern of cytokine activation. It is conceivable that o t h e r bacterial antigens or even o t h e r streptococcal strains may cause earlier or m o r e a b u n d a n t stimulation of interleukins, leading to the p r o d u c t i o n of detectable amounts o f PGE 2. In vivo, the response of chorion, decidua, tissue macrophages, and neutrophils make for a m o r e c o m p l e x system, and interaction between the different tissues may occur. In conclusion, a m n i o n cell cultures may be a m o d e l to further evaluate which bacteria lead to a significant stimulation of cytokine and prostaglandin p r o d u c t i o n and which bacterial concentrations and combinations are most likely to cause p r e t e r m labor. REFERENCES
1. Brandt-Niebelschfitz S, Saling E, Kfichler R. Weitere Erfahrungen mit der Eipol-Lavage im Zusammenhang mit der Vermeidung yon Frfihgeburten. Z Geburtshilfe Perinat 1992;229-37. 2. Gyr TN, Malek A, Mathez Loic F, Altermatt HJ, Bodmer T, Nicolaides K, et al. Permeation of human chorioamniotic membranes by Escherichia coli in vitro. Am J Obstet Gynecol 1994;170:223-7. 3. Zitcer EM, Fogh J, Dunnebacke TH. Human amnion cells for large scale production of polio virus. Science 1955;122: 30. 4. Mitchell MD, Edwin SS, Lundin-Schiller S, Silver RM, Smotkin D, Trautman MS. Mechanisms of interleukin-lbeta stimulation of human amnion prostaglandin biosynthesis:
May 1997 Am J Obstet Gynecol
5. 6. 7.
8. 9.
10.
11.
12.
13.
14.
15.
mediation via a novel inducible cyclooxygenase. Placenta 1993;14:615-25. Zakar T, Olson DM. Stimulation of human amnion prostaglandin E 2 production by activators of protein kinase C. J Clin Endocrinol Metab 1988;67:915-23. Casey ML, MacDonald PC, Mitchell MD. Characterization of prostaglandin formation by human amnion cells in monolayer culture. Prostaglandins 1984;27:421-7. OkitaJR, Sagawa N, Casey ML, Snyder JM. A comparison of human amnion tissue and amnion cells in primary culture by morphological and biochemical criteria. In Vitro 1983; 19:117-26. Alitalo K, Kurkinen M, Vaheri A. Extracellular matrix components synthesized by human amniotic epithelial cells in culture. Cell 1980;19:1053-62. Lamont RF, Anthony F, Myatt L, Booth L, Furr PM, TaylorRobinson D. Production of prostaglandin E 2 by human amnion in vitro in response to addition of media conditioned by microorganisms associated with chorioamnionitis and preterm labor. A m J Obstet Gynecol 1990;162:819-25. Romero R, Brody DT, Oyarzun E, Mazor M, Wu YK, Hobbins JC, et al. Infection and labor, III: interleukin-l: a signal for the onset of parturition. Am J Obstet Gynecol 1989;160:1117-23. Lopez Bernal A, Hansell DJ, Khong TY, Keeling JW, Turnbull AC. Prostaglandin E production by the fetal membranes in unexplained preterm labour and preterm labour associated with chorioamnionitis. Br J Obstet Gynaecol 1989;96:1133-9. Mitchell MD, Dudley DJ, Edwin SS, Ludin-Schiller S. Interleukin-6 stimulates prostaglandin production by human amnion and decidual cells. E u r J Pharmacol 1991;192:18991. Bry K, Hallman M. Synergistic stimulation of amnion cell prostaglandin E 2 synthesis by interleukin-1, tumor necrosis factor and products from activated human granulocytes. Prostaglandins Leukot Essent Fatty Acids 1991;44:241-5. Menon R, Swan KF, Lyden TW, Rote NS, Formnato sJ. Expression of inflammatory cytokines (interleukin-l[3 and interleukin-6) in amniochorionic membranes. Am J Obstet Gynecol 1995;172:493-500. Bala GA, Thakur NR, Bleasdale JE. Characterization of the major phosphoinositide-specific phospholipase C of human amnion. Biol Reprod 1990;43:704-11.
Key words; A m n i o n cell culture, prostaglandins, cytokines, bacterial infection, p r e t e r m labor
Ascension of bacteria f r o m the lower genital tract into the amniotic cavity is t h o u g h t to be a key event in the etiology of p r e t e r m ]abor. In their study on bacterial colonization of the lower extent of tile a m n i o n BrandtNiebelschfitz et al. 1 f o u n d that the microorganisms recovered most frequently were coagulase-negative staphylococci and g r o u p B streptococci, with less c o m m o n isolates including Ureaplasma and Mycoplasma species. W h e n bacteria colonize the cervix and the lower extent of the amnion, they are able to readily p e r m e a t e fetal m e m b r a n e s and invade the amniotic cavity, as recently d e m o n s t r a t e d by Gyr et al. ~ in an in vitro study. For example, Escherichia coli organisms p e r m e a t e chorioamniotic m e m b r a n e s within 6 hours after inoculation. The role of the a m n i o n in the initiation of parturition and the effects of infection on the biochemical processes in the a m n i o n are still n o t completely understood. A m n i o n ceils are an i m p o r t a n t source o f prostaglandin E 2 (PGE2) production. W h e n bacteria attach to or invade
From the Department of Obstetrics and @necology, University of Vienna Medical School. Receivedfor publication September5, 1996; revisedJanuary 2, 1997; acceptedJanua U 21, 1997. Reprint requests:Klaus Reisenberger, MD, Departmentof Obstetricsand Gynecology, Vienna Medical School, Wiihringer C_.4irtel18-20, 1090 Vienna, Austria. Copyright © 1997 by Mosby-Year Book, Inc. 0002-9378/97 $5.00 + 0 6/1/80541
chorioamniotic membranes, they c o m e into contact with a m n i o n cells. In 1955 Zitcer et al. s were the first to r e p o r t a m e t h o d for culture of a m n i o n cells. Since then n u m e r ous investigators have used a m n i o n culture systems to study physiologic and pathophysiologic events. < 5 Because the literature does n o t provide any data on the direct effects of bacterial antigen on cytokine and prostaglandin p r o d u c t i o n by a m n i o n cells, this study was designed to d e t e r m i n e the effects in a prima1T a m n i o n cell culture with use of a strain of Streptococcus because of the potential p a t h o g e n i c role in obstetrics. Material and m e t h o d s
A m n i o t i c m e m b r a n e s were obtained f r o m two w o m e n u n d e r g o i n g primary" cesarean section n e a r term to minimize the influence of labor on cytokine and prostaglandin production. W o m e n whose amniotic m e m b r a n e s were used had had an uneventful obstetric history, and they had b e e n taking no drugs. Patients with signs of a m n i o n infection syndrome, such as maternal fever, elevated maternal serum C-reactive protein levels, discolo r e d amniotic fluid, fetal tachycardia, or signs of gestosis were not i n c l u d e d in this study. Immediately after placentas had b e e n obtained, amn i o n tissue was dissected from the c h o r i o n laeve and placed in H a n k ' s balanced salt solution s u p p l e m e n t e d with 1% penicillin-streptomycin (Gibco, Paisly, Scotland) 981
982
Reisenberger et al.
May 1997 A m J Obstet Gyneco!
60
SO
40.
o.
I
I--,,--~o2} ~loa I
I 10 i
/'/"/
0S
...........
t2
-,o-.1041 --X---1O6! +106] -~-1071
o ........
24
48
hours
Fig. 1. Secretion of IL-6 by amnion cells in response to addition of increasing concentrations (10 tO 107) of heat-inactivated group B streptococci,
for transport to the laboratory, where a single cell suspension was prepared. 6-s The isolated ceils were resuspended in Dulbecco's m i n i m a l essential m e d i u m with n u t r i e n t mix F12 (1:1; Gibco) s u p p l e m e n t e d with 10% fetal calf serum, antibiotics, and antimycotics, seeded in 24-well culture dishes at a density of 3 × 105 per well and grown to confluence at 37°C in an atmosphere of 5% carbon dioxide in air for 3 to 4 days. Cell viability was assessed in control cultures with use of trypan blue. The microorganism chosen for this study, Streptococcus agalactiae, was placed in physiologic saline solution and heat inactivated in a water bath at 70°C for 10 minutes. Inactivation was considered necessary because, particularly with high microbial counts, live germs may have resulted in overgrowth. Media were aspirated from the cell culture, and fresh m e d i u m containing the S. agalactiae suspension was added in increasing concentrations (0 to 10 v colonybuilding u n i t s / m l ) . Analysis was done in triplicate, with cells plated in three wells for each microbial count and incubation period. The supernatants of these three wells were mixed and analyzed together. This procedure was used for financial reasons because testing each culture individually would have been three times as expensive. Samples were collected at 6, 12, 24, and 48 hours of i n c u b a t i o n and centrifuged for 10 minutes at 600g. The supernatants were stored frozen at -70 ° C until assayed at the e n d of the test series. As a control, u n i n o c u l a t e d m e d i u m was treated identically t h r o u g h o u t the incubation period, and samples for quantification of interleukin and prostaglandin were taken at the same intervals. The production of interleukin-l[3 (IL-I[3), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-ct (TNF-cQ, and PGE 2 from a m n i o n cells was measured from the supernatants by enzyme i m m u n o s o r b e n t assay (R&D Systems, Minneapolis).
Results In the control cultures not exposed to S. agalactiae antigen no basal cytokine production was observed. The incubation of amnion ceils with bacterial antigen did not result in the production of TNF-c~ and IL-l[3 during the 48-hour observation period. However, IL-6 and IL-8 concentrations showed a significant increase after stimulation with bacterial antigen (Figs. 1 and 2). Stimulation was similar for both cytokines, with IL-8 production being higher by the factor of 10. Synthesis of both interleukins was found to depend on the time of incubation and on the concentration of antigen added. At lower microbial counts measurable levels of IL-6 and IL-8 were not detected until 24 hours of incubation, whereas higher counts produced a significant increase within only 12 hours. In contrast to amnion cell cytokine production, a basal production of PGE 2 was demonstrable but showed no significant increase after addition of antigen. Also, there was no correlation between PGE 2 production and the concentrations of antigen added. Moreover, the production of PGE 2 started to decrease as early as 24 hours after inoculation (Fig. 3).
Comment Bacterial products such as phospholipases, lipopolysaccharides, and endotoxins that are released after disruption of gram-negative bacteria can liberate arachidonic acid from uterine tissues.9 Biotransformation of arachidonic acid by the cyclooxygenase pathway in turn gives rise to an obstetrically relevant compound, PGE 2, which is known to possess potent uterotonic properties. Moreover, bacterial infection activates immune mechanisms, resulting in an increase in cytokines that are secreted primarily by macrophages in the amnion, chorion, and decidua. The effects of these cytokines on prostaglandin production have already been investigated in numerous
Reisenberger et al.
Volume 176, Number 5 Am J Obstet Gynecol
983
600
600
4OO
~E. 3oo
-
2oo -
lOO q ol
12
24
48
hour~
Fig. 2. Secretion of IL-8 by amnion cells in response to addition of increasing concentrations (10 to 107) of heat-inactivated group B streptococci.
I........ I 0 6
12
24
48
hours
Fig. 3. PGE 2 concentration after addition of increasing concentrations (10 to 106) of heat-inactivated group B streptococci to an amnion cell culture.
studies. For example, it has b e e n shown that a m n i o n cells can be stimulated to p r o d u c e PGE 2 in response to TNF-a and Ibl{~. 4 Cytokines m o d u l a t e and control i m m u n o l o g i c processes, and it is likely that they also contribute to different aspects o f the hurnoral and cellular defense m e c h a n i s m s o f the fetal m e m b r a n e s . Thus inflammatory processes in the fetal m e m b r a n e s have b e e n f o u n d to trigger the secretion of IL-1 and IL-6, leading to significant accumulation o f these cytokines in the amniofic fluid, a° It is further believed that IL-8 causes degradation o f type I collagen by stimulating the release of collagenases f r o m chemotactically attracted p o l y r n o r p h o n u c l e a r leukocytes, thus contributing to cervical ripening, la In this in vitro investigation we have shown that bacterial antigen acts on a m n i o n cells to significantly increase secretion o f IL-6 a n d I b 8 . This rise in cytokine p r o d u c t i o n did n o t induce an increase in POE 2 secretion. Mitchell et al. 1~ have shown that IL-6 stimulates PGE 2 p r o d u c t i o n f r o m cultured a m n i o n cells. However, relatively high IL-6 concentrations (100 n g / m l ) are
r e q u i r e d to increase PGE 2 p r o d u c t i o n from amnion, and the IL-6 concentrations achieved in o u r study were well below these levels. In this context Bry and H a l l m a n as have d e m o n s t r a t e d in an in vitro m o d e l that the basal p r o d u c t i o n of PGE 2 by a m n i o n cells is increased after exposure to IL-l[3 and TNF-c~. In our study the production of these two cytokines was n o t e n h a n c e d after addition o f streptococcal antigen. This is in a g r e e m e n t with the results of M e n o n et al., 14 who f o u n d that a m n i o n cells appear to lack the capacity to p r o d u c e IL-I[3, although IL-I[3 can bind to receptors on the m e m b r a n e of a m n i o n cells. Prostaglandin p r o d u c t i o n in o u r investigation showed a significant decrease after about 24 hours o f incubation, d r o p p i n g even below the basal p r o d u c t i o n . This may be attributable to the lack o f prostaglandin H synthetase in cultured a m n i o n cells d e m o n s t r a t e d by Bala et al. a5 A major finding in the c u r r e n t study is that bacterial antigen stimulates cytokine p r o d u c t i o n in a concentrat i o n - d e p e n d e n t m a n n e r . At m e d i a n microbial concentrations the microbial counts were even directly correlated
984
Reisenberger et al.
with IL-6 and IL-8 synthesis (Figs. 1 and 2). However, further increases in bacterial antigen concentrations caused no further significant rise in the synthesis o f IL-6 and IL-8. Moreover, we observed a temporal p h e n o m e non, with h i g h e r microbial concentrations resulting in earlier stimulation of cytokine p r o d u c t i o n than lower concentrations (Figs. 1 and 2). In this study we were unable to assess w h e t h e r different microorganisms differ in their pattern of cytokine activation. It is conceivable that o t h e r bacterial antigens or even o t h e r streptococcal strains may cause earlier or m o r e a b u n d a n t stimulation of interleukins, leading to the p r o d u c t i o n of detectable amounts o f PGE 2. In vivo, the response of chorion, decidua, tissue macrophages, and neutrophils make for a m o r e c o m p l e x system, and interaction between the different tissues may occur. In conclusion, a m n i o n cell cultures may be a m o d e l to further evaluate which bacteria lead to a significant stimulation of cytokine and prostaglandin p r o d u c t i o n and which bacterial concentrations and combinations are most likely to cause p r e t e r m labor. REFERENCES
1. Brandt-Niebelschfitz S, Saling E, Kfichler R. Weitere Erfahrungen mit der Eipol-Lavage im Zusammenhang mit der Vermeidung yon Frfihgeburten. Z Geburtshilfe Perinat 1992;229-37. 2. Gyr TN, Malek A, Mathez Loic F, Altermatt HJ, Bodmer T, Nicolaides K, et al. Permeation of human chorioamniotic membranes by Escherichia coli in vitro. Am J Obstet Gynecol 1994;170:223-7. 3. Zitcer EM, Fogh J, Dunnebacke TH. Human amnion cells for large scale production of polio virus. Science 1955;122: 30. 4. Mitchell MD, Edwin SS, Lundin-Schiller S, Silver RM, Smotkin D, Trautman MS. Mechanisms of interleukin-lbeta stimulation of human amnion prostaglandin biosynthesis:
May 1997 Am J Obstet Gynecol
5. 6. 7.
8. 9.
10.
11.
12.
13.
14.
15.
mediation via a novel inducible cyclooxygenase. Placenta 1993;14:615-25. Zakar T, Olson DM. Stimulation of human amnion prostaglandin E 2 production by activators of protein kinase C. J Clin Endocrinol Metab 1988;67:915-23. Casey ML, MacDonald PC, Mitchell MD. Characterization of prostaglandin formation by human amnion cells in monolayer culture. Prostaglandins 1984;27:421-7. OkitaJR, Sagawa N, Casey ML, Snyder JM. A comparison of human amnion tissue and amnion cells in primary culture by morphological and biochemical criteria. In Vitro 1983; 19:117-26. Alitalo K, Kurkinen M, Vaheri A. Extracellular matrix components synthesized by human amniotic epithelial cells in culture. Cell 1980;19:1053-62. Lamont RF, Anthony F, Myatt L, Booth L, Furr PM, TaylorRobinson D. Production of prostaglandin E 2 by human amnion in vitro in response to addition of media conditioned by microorganisms associated with chorioamnionitis and preterm labor. A m J Obstet Gynecol 1990;162:819-25. Romero R, Brody DT, Oyarzun E, Mazor M, Wu YK, Hobbins JC, et al. Infection and labor, III: interleukin-l: a signal for the onset of parturition. Am J Obstet Gynecol 1989;160:1117-23. Lopez Bernal A, Hansell DJ, Khong TY, Keeling JW, Turnbull AC. Prostaglandin E production by the fetal membranes in unexplained preterm labour and preterm labour associated with chorioamnionitis. Br J Obstet Gynaecol 1989;96:1133-9. Mitchell MD, Dudley DJ, Edwin SS, Ludin-Schiller S. Interleukin-6 stimulates prostaglandin production by human amnion and decidual cells. E u r J Pharmacol 1991;192:18991. Bry K, Hallman M. Synergistic stimulation of amnion cell prostaglandin E 2 synthesis by interleukin-1, tumor necrosis factor and products from activated human granulocytes. Prostaglandins Leukot Essent Fatty Acids 1991;44:241-5. Menon R, Swan KF, Lyden TW, Rote NS, Formnato sJ. Expression of inflammatory cytokines (interleukin-l[3 and interleukin-6) in amniochorionic membranes. Am J Obstet Gynecol 1995;172:493-500. Bala GA, Thakur NR, Bleasdale JE. Characterization of the major phosphoinositide-specific phospholipase C of human amnion. Biol Reprod 1990;43:704-11.