Cytokine Patterns in Maternal Blood After Premature Rupture of Membranes Raj Raghupathy, PhD, FRCPath Ma’asoumah Makhseed, MD, MRCOG, Sherief El-Shazly, MSc, Fawaz Azizieh, MSc, Rashida Farhat, MBBS, and Laila Ashkanani, MBBCh OBJECTIVE: To compare two types of cytokines, type 1, which activate cell-mediated reactions and are important in cytotoxic and delayed-type hypersensitivity reactions, and type 2, which encourage vigorous antibody production and are commonly found in association with humoral immune responses, in blood of women with premature rupture of membranes (PROM). METHODS: Forty-four women with histories of at least three successful pregnancies and who currently delivered normally served as controls. The PROM group consisted of 30 women with spontaneous rupture of fetal membranes at term. Peripheral blood mononuclear cells were stimulated separately with a mitogen, placental cells, and a trophoblast antigen extract, and the supernatants examined for type 1 and type 2 cytokines. RESULTS: Mitogen-stimulated blood cells produced significantly higher levels of type 1 cytokines in PROM women than in normal controls. Higher levels of the type 1 cytokine interferon-␥ were produced by PROM samples stimulated with autologous placental cells and with trophoblast antigens. Ratios of type 1 to type 2 cytokines were higher in PROM compared with normal pregnancy, and in some cases as much as 25-fold higher. CONCLUSION: Women in the PROM group had a stronger type 1 reactivity whereas normal women were more predisposed to type 2 immunity; thus, PROM appears to be associated with a maternal type 1 bias. (Obstet Gynecol 2001;98:122– 6. © 2001 by the American College of Obstetricians and Gynecologists.)
The pathophysiology of premature rupture of membranes (PROM) is poorly understood, and its etiology is probably multifactorial, with maternal enzymes, maturational and mechanical forces, chorionic-amniotic phospholipid content, collagen disruption, amniotic cell cytokines induced by fetal signals, and bacterial phospholipases and collagenases having effects.1 A proportion of the cases of PROM From the Departments of Microbiology and Obstetrics and Gynecology, Faculty of Medicine, Kuwait University; and Maternity Hospital, Kuwait. Supported by the Kuwait Foundation for the Advancement of Sciences, Kuwait (Project 95-07-10).
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are of unexplained or unknown etiology, and even in PROM caused by infection, whereas it was previously thought that the pathogens were responsible for the complication, it has been suggested that many of the effects are actually mediated by endogenous host molecules such as cytokines.2 We propose that proinflammatory cytokines might play key roles in the events leading to PROM. Interleukin-1 bioactivity and concentrations were reported to be significantly higher in the presence of labor in women with preterm PROM and intraaminiotic infection. Similarly, in women with preterm PROM and intraaminiotic infection, interleukin-6 levels were higher in the presence of labor.2 Certain cytokines, such as granulocyte macrophage colony-stimulating factor, colony-stimulating factor-1, interleukin-3,3 and interleukin-10,4 appear to be propitious to the success of pregnancy, whereas cytokines such as interleukin-2, tumor necrosis factor-␣, and interferon-␥ are deleterious.5 Tumor necrosis factor-␣ stimulates the apoptosis of human primary villous trophoblast cells, and interferon-␥ augments tumor necrosis factor–mediated killing of trophoblast cells.6 Both tumor necrosis factor and interferon-␥ inhibit the outgrowth of human trophoblast cells in vitro.7 Interleukin-2, interferon-␥, and tumor necrosis factor-␣ terminate normal pregnancy after injection into pregnant mice.8 The placenta has been shown to secrete the anti-inflammatory cytokines interleukin-4, interleukin10,3,9,10 and transforming growth factor 2,11 and these cytokines could help prevent cytokine-mediated damage to the conceptus.3–5,9 –11 Interferon-␥, interleukin-2, and tumor necrosis factor-, ie, cytokines that have deleterious effects on the conceptus, are cytokines that are characteristic of T helper 1-type immunity. These type 1 cytokines induce several cytotoxic and inflammatory reactions and are thus responsible for the induction of cell-mediated inflammatory reactions. T helper 2-type cells secrete the type 2 cytokines interleukin-4, interleukin-5, interleukin-6, and interleukin-10 and are associated with help for humoral immunity.12,13 It has been proposed that normal pregnancy is associated with a maternal predisposition to type 2 immu-
VOL. 98, NO. 1, JULY 2001 © 2001 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
0029-7844/01/$20.00 PII S0029-7844(01)01408-9
Table 1. Demographic Data Characteristic
Normal pregnancy (mean ⫾ SE)
PROM (mean ⫾ SE)
Age (y) Parity Gravidity Gestational age (wk)
28.9 ⫾ 4.0 3.1 ⫾ 1.1 3.9 ⫾ 1.0 39.4 ⫾ 1.0
26.8 ⫾ 2.6 3.6 ⫾ 1.0 3.5 ⫾ 0.8 39.0 ⫾ 1.1
SE ⫽ standard error; PROM ⫽ premature rupture of membranes.
nity,3 whereas a preponderance of type 1 reactivity could lead to pregnancy failure.5,14 –16 Several studies of unexplained recurrent spontaneous abortion14 –19 and preeclampsia20 in humans have shown a close association between type 1 cytokines and pregnancy failure. However, an analysis of maternal type 1 and type 2 cytokine production vis-a`-vis PROM has not been reported. Based on the deleterious effects of type 1 cytokines on pregnancy, we hypothesized that women with PROM would be predisposed to type 1 reactivity compared with women with successful pregnancy. Here we report levels of cytokines produced by maternal peripheral blood mononuclear cells in women with PROM. MATERIALS AND METHODS Subjects were selected from women admitted to the Maternity Hospital, Kuwait, for normal delivery or upon spontaneous PROM at term. The control group in this study consisted of 44 women who had at least three normal pregnancies, without a history of PROM, abortion, stillbirth, or preterm delivery; these women went on to deliver normally. The PROM group consisted of 30 women. A demographic summary of the subjects is presented in Table 1. Blood samples and placentas were collected at the time of parturition. All subjects delivered spontaneously. The enrollment period of this study was 2 years. This study was approved by the Ethics Committees of Kuwait University and the Maternity Hospital, Kuwait. Peripheral blood mononuclear cells were separated from maternal peripheral blood by Ficoll-hypaque (Pharmacia Biotech, Sollentuna, Sweden) density gradient centrifugation, suspended in tissue culture medium (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal calf serum, aliquoted into 96-well tissue culture plates at a density of 105 cells per well and then challenged with the mitogen phytohemagglutinin (Sigma Chemicals, St. Louis, MO) at a concentration of 5 g/mL for 96 hours. Supernatants were harvested at 24 and 96 hours; some of the wells were pulsed with [3H]thymidine (Amersham International, Bucks, UK) for assessment of mitogeninduced proliferation. Thymidine-pulsed wells were har-
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vested 18 hours later, and the radioactivity was estimated. Stimulation indices were calculated as a ratio of thymidine uptake by mitogen-stimulated cells to that by nonstimulated cells. Placentas from normal controls and from women with PROM were obtained immediately after spontaneous vaginal delivery. Single-cell suspensions from placentas were prepared as described.18 One or two cotyledons were removed from the underlying fibrous tissues; the outermost (maternal face) tissue was removed, and tissue from the interior of the placenta was harvested aseptically, minced, and washed thoroughly in several changes of medium. Approximately 30 g of placental tissue was digested for 12 hours at 37C with 3 mL of 50 U/mL of dispase (Collaborative Research Inc., Bedford, MA). After digestion, the mixture was passed through gauze pads to remove undigested fragments and then subjected to Ficoll-hypaque density gradient sedimentation. The interface consisting of mononuclear cells was resuspended in medium for counting and viability testing. Maternal anti-placental cell reactivity was examined by coculturing maternal peripheral blood cells (responders) with irradiated placental cells (stimulators) as described previously.18 Placental cells were irradiated in a gamma chamber at 3000 rads, washed, and then resuspended at 106 cells/mL. One hundred microliters each of the suspension of peripheral blood cells and placental cell suspension were added to wells of 96-well tissue culture plates and the plates incubated for 96 hours at 37C, 5% carbon dioxide, and 90% humidity. Culture supernatants were collected 24 and 96 hours later. Some of the wells were pulsed with [3H]thymidine at 96 hours and the uptake of thymidine measured 18 hours later. Antigen extracts were prepared from the human gestational choriocarcinoma cell line JEG-3 (American Type Culture Collection, Rockville, MD), essentially as described by Hill et al.14 Cells were harvested and disrupted in a Dounce homogenizer (approximately 100 strokes). The suspension was then centrifuged at 3000 rpm for 10 minutes and the supernatant filtered through a 0.2-m filter, aliquoted, and stored at ⫺20C until use. This material is referred to as JEG antigen or JEG antigen extract. Maternal peripheral blood cells were cultured at a density of 105 cells per well with 30 g/mL of JEG antigen for 4 days after which supernatants were collected for cytokine estimation. Thymidine uptake was also performed on day 4 to ascertain the extent of proliferation. Sera and culture supernatants were frozen, stored, batched, and assayed together in duplicate. Interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-10, tumor necrosis factor-␣ and interferon-␥ were
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assayed by enzyme-linked immunosorbent assays (ELISA) (Immunotech SA, Marseilles, France); tumor necrosis factor- was estimated by ELISA kits obtained from R & D Systems (Minneapolis, MN). The manufacturer’s protocols were followed for the assays. The lower limit of sensitivity of each of the assays were as follows: 5 pg/mL of interleukin-2, 10 pg/mL of tumor necrosis factor-␣, 7 pg/mL of tumor necrosis factor-, 3 pg/mL of interferon-␥, 5 pg/mL of interleukin-4, 1 pg/mL of interleukin-5, 3 pg/mL of interleukin-6, and 5 pg/mL of interleukin-10. The standard Mann-Whitney test was used for nonparametric comparisons of median cytokine levels because the data were not distributed normally. RESULTS Interleukin-2, interleukin-4, interleukin-5, and tumor necrosis factor- were not detectable in the sera tested. However, the type 1 cytokine interferon-␥ was detected at significantly higher levels in women with PROM compared with normal controls (P ⬍ .001), whereas the type 2 cytokine interleukin-10 (P ⬍ .06) was found in higher levels in normal pregnancy than in PROM. Levels of interleukin-6 were higher in PROM than in normal pregnancy (P ⬍ .001). Background counts per minute, that is, thymidine uptake by unstimulated cells, was routinely under 600 cpm. The stimulation index of peripheral blood mononuclear cells from the normal pregnancy group stimulated with phytohemagglutinin was significantly higher than that in the PROM group. Stimulation indices of women in the normal pregnancy group ranged from 1 to 1252, with a median of 113, whereas the indices in PROM ranged from 1 to 212, with a median of 27; the difference in the medians of the two groups is statistically significant (P ⬍ .001). No significant differences were observed in the two groups when blood cells were stimulated with autologous placental cells and JEG antigen. When maternal mononuclear blood cells were stimulated with mitogen, two of the four type 1 cytokines tested, interferon-␥ and tumor necrosis factor-␣, were produced at significantly higher levels (P ⬍ .001) by the PROM group than by the normal group at both time points tested. Interleukin-2 levels were also significantly higher in PROM in 24-hour culture supernatants (P ⬍ .001). On the contrary, the type 2 cytokine interleukin-4 was secreted at significantly higher levels by the normal pregnancy group than by the PROM group. Levels of the other type 2 cytokines were not significantly different. As shown in Table 2, the ratios of mean values of the different type 1 to type 2 cytokines at the two time points were generally higher in PROM than in normal preg-
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Table 2. Cytokine Ratios in Mitogen-stimulated Peripheral Blood Mononuclear Cell Cultures Th1:Th2 ratio After 24-h culture IL-2:IL-4 IL-2:IL-5 IL-2:IL-6 IL-2:IL-10 IFN:IL-4 IFN:IL-5 IFN:IL-6 IFN:IL-10 TNF␣:IL-4 TNF␣:IL-5 TNF␣:IL-6 TNF␣:IL-10 After 96-h culture IL-2:IL-4 IL-2:IL-5 IL-2:IL-6 IL-2:IL-10 IFN:IL-4 IFN:IL-5 IFN:IL-6 IFN:IL-10 TNF␣:IL-4 TNF␣:IL-5 TNF␣:IL-6 TNF␣:IL-10 TNF:IL-4 TNF:IL-5 TNF:IL-6 TNF:IL-10
Normal pregnancy
PROM
6 6 0.07 0.7 11 11 0.1 1 10 10 0.1 1
28 32 0.2 3 31 35 0.2 3 33 38 0.2 3
7 2 0.07 0.7 116 35 1 12 21 6 0.2 2 27 8 0.3 3
8 0.9 0.03 0.7 1919 241 9 111 65 8.4 0.34 4 44 6 0.2 2.7
Th ⫽ T helper; PROM ⫽ premature rupture of membranes; IL ⫽ interleukin; IFN ⫽ interferon; TNF ⫽ tumor necrosis factor.
nancy, indicating a stronger type 1 bias in PROM. At 24 hours, the type 1 to type 2 ratio was higher in PROM in every combination, whereas at 96 hours the type 1:type 2 ratio was higher in PROM in 11 of 16 combinations. For example, the interferon:interleukin-4 ratio is about 17 times higher in PROM than in normal pregnancy, and the interferon:interleukin-10 ratio is about nine times higher. Maternal peripheral blood cells were cocultured with irradiated autologous placental cells and culture supernatants screened for cytokines at 24 and 96 hours. The levels of interleukin-6 and interleukin-10 were significantly elevated in normal pregnancy compared with PROM (P ⬍ .01 and P ⬍ .03, respectively). Conversely, levels of the type 1 cytokine interferon-␥ were significantly higher in PROM than in normal pregnancy (P ⬍ .001). Although there was a trend toward higher production of tumor necrosis factor-␣ in PROM, the differences were not statistically significant. A comparison of type 2:type 1 cytokine ratios (Table 3) shows that this ratio is
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Table 3. Cytokines in Placenta-stimulated and JEG-stimulated Maternal Peripheral Blood Mononuclear Cell Cultures MLPR
JEG
24-h IL-6:IFN␥ IL-6:TNF␣ IL-10:IFN␥ IL-10:TNF␣
96-h
96-h
Normal
PROM
Normal
PROM
Normal
PROM
444 17 31.5 1.2
79 12 5.9 0.8
505 31.3 29 1.8
76 16.4 6.8 1.4
3444 30.2 223 2
76 16.4 6.8 1.4
MLPR ⫽ mixed lymphocyte-placenta reaction; PROM ⫽ premature rupture of membranes; IL ⫽ interleukin; IFN ⫽ interferon; TNF ⫽ tumor necrosis factor.
higher in normal pregnancy in all combinations tested, indicating a predisposition to type 2 reactivity in normal pregnancy compared with PROM. In mononuclear cell cultures stimulated with the trophoblast antigen extract, interferon-␥ was produced at significantly higher levels by PROM samples than by normal pregnancy samples (P ⬍ .001). There was no significant difference in the levels of tumor necrosis factor-␣, interleukin-6, and interleukin-10 produced. Table 3 indicates that type 2:type 1 cytokine ratios were higher in normal pregnancy than in PROM in all combinations tested. For instance, the interleukin-6:interferon ratio in normal pregnancy was about 39 times higher in normal pregnancy, and the interleukin-10: interferon ratio was about 25-fold higher. This is suggestive of a stronger tendency toward type 2 reactivity in normal pregnancy and a type 1-dominance in PROM. DISCUSSION Although other studies have found elevated levels of certain cytokines in the amniotic fluid of women who had PROM,2 we are the first to describe the cytokine production patterns of maternal peripheral lymphocytes in women undergoing PROM. This is substantiated by our search of MEDLINE for the period 1966 to 2000, using combinations of search terms “premature rupture of membranes” and “lymphocytes,” “premature rupture of membranes” and “peripheral blood mononuclear cells,” and “premature rupture of membranes” and “cytokines.” We found that the stimulation of maternal peripheral blood mononuclear cells with mitogen elicited significantly higher production of type 1 cytokines and decreased production of the type 2 cytokine interleukin-4 compared with women with successful pregnancy who had normal deliveries. Lymphocyte proliferation as reflected by stimulation indices was significantly higher in normal pregnant women than in the PROM group. This is suggestive of greater proliferation of type 2 cells in normal pregnancy and might suggest a pregnancysupportive role for type 2 cytokines, which are known to downregulate type 1 reactivity.13
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Placenta-stimulated peripheral blood mononuclear cell cultures from the PROM group produced higher levels of interleukin-6 and interleukin-10 and lower levels of interferon-␥. Peripheral blood cells from women with PROM produced significantly higher levels of the interferon-␥. These data together point to a possible bias toward type 1 reactivity in women with PROM, and the analyses of ratios of T helper type 1 to T helper type 2 cytokines in the cultures strongly support that. The relative concentrations of type 1 versus type 2 cytokines are more important than the absolute levels of cytokines produced,21 and in almost all combinations of type 1:type 2 cytokine ratios, we found a skew toward type 1 bias in women with PROM. Serum levels of interleukin-10 were higher in normal pregnancy, whereas serum interferon-␥ levels were higher in PROM. Interestingly, interleukin-6 levels were higher in PROM than in normal pregnancy. Gomez et al2 reported elevated levels of interleukin-6 in the amniotic fluid of women with PROM, and that cytokine has been suggested to have important effects on physiologic mechanisms involved in the propagation of labor. T helper 1 and T helper 2 cells are probably just two among several subsets of T cells categorized on the bases of function and cytokine profiles.13 Although we found elevated levels of some of the type 2 cytokines in normal pregnancy compared with PROM, we did not observe uniformly elevated levels of all the type 2 cytokines; thus, it might be simplistic to refer to normal pregnancy and PROM as being exclusively type 2 oriented and type 1 oriented, respectively. However, a comparison of the ratios of type 1 and type 2 cytokines supports the contention that there is a skew toward type 1 reactivity in PROM. Furthermore, we found a possible association of type 1 cytokines with PROM, which does not prove a cause-and-effect relationship between the two. If type 1 cytokines are involved in the pathogenesis of PROM, the mechanisms underlying the events leading to the rupture of fetal membranes have yet to be determined. If proinflammatory cytokines make their appearance before infection, they might expose the membranes to
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bacteria, leading to bacterial invasion and PROM. Or if infection is the initial step, it could lead to a maternal immune response consisting of cytokine production among other events; these cytokines could then recruit other immune cells to the vicinity and mediate structural damage to fetal membranes. Even if the initial cascade of events is triggered by infection, subsequent steps in the cascade could involve cytokines that are antagonistic to the trophoblast or the fetus. Such steps, for instance, could involve increased production of prostaglandin E2 and increased uterine activity which could lead to PROM.22 More studies are required before we can draw conclusions about mechanistic and etiologic implications of maternal cytokines in PROM.
1. Mercer BM. Management of preterm premature rupture of the membranes. Clin Obstet Gynecol 1998;4:870 – 82. 2. Gomez R, Romero R, Edwin SS, David C. Pathogenesis of preterm labor and preterm premature rupture of membranes associated with intraaminiotic infection. Infect Dis Clin North Am 1997;11:135–75. 3. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: Is successful pregnancy a Th2 phenomenon? Immunol Today 1993;14:353– 6. 4. Chaouat G, Meliani AA, Martal J, Raghupathy R, Elliot J, Mosmann TR, et al. IL-10 prevents naturally occurring fetal loss in the CBAxDBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-r. J Immunol 1995;154:4261– 8. 5. Raghupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today 1997;18:478 – 82. 6. Yui J, Garcia-Lloret M, Wegmann TG, Guilbert LJ. Cytotoxicity of tumor necrosis factor-␣ (TNF-␣) and gamma interferon (IFN-␥) against primary human placental trophoblasts. Placenta 1994;15:819 –26. 7. Berkowitz R, Hill JA, Kurtz CB, Anderson DJ. Effects of products of activated leukocytes (lymphokines and monokines) on the growth of malignant trophoblast cells in vitro. Am J Obstet Gynecol 1988;151:199 –203. 8. Chaouat G, Menu E, Dy M, Minkowski M, Clark DA, Wegmann TG. Control of fetal survival in CBA x DBA/2 mice by lymphokine therapy. J Reprod Fertil 1990;89: 447–58. 9. Chaouat G, Cayol V, Mairovitz V, Dubanchet S. Localization of the Th2 cytokines IL-3, IL-4, IL-10 at the murine fetomaternal interface during pregnancy. In: Gupta SK, ed. Reproductive immunology. New Delhi: Narosa Publishing House, 1999:61–70. 10. Hanna N, Hanna I, Hleb M, Wagner E, Dougherty Balkundi D, Padbury J, et al. Gestational age-dependent expression of
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Address reprint requests to: Raj Raghupathy, PhD, Faculty of Medicine, Department of Microbiology, Kuwait University, PO Box 24923, Kuwait 13110; E-mail:
[email protected] Received October 19, 2000. Received in revised form February 8, 2001. Accepted March 14, 2001.
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