CYTOKINES IN NORMAL AND ABNORMAL PARTURITION: ELEVATED AMNIOTIC FLUID INTERLEUKIN-6 LEVELS IN WOMEN WITH PREMATURE RUPTURE OF MEMBRANES ASSOCIATED WITH INTRAUTERINE INFECTION Uma Santhanam,l Cecilia Avila,’ Roberto Romero,’ Huguette Viguet,’ Nobuo Ida,3 Shingou Sakurai,4 Pravinkumar B. Sehgall,” The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pglml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AP from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p < 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM. Median AP IL-6 titers were 67 U/ml in women with microbial culture-negative AP who were not in active labor at the time of admission (group 4a, n = 17); 109 U/ml in those with culture-negative AP who were in active labor (group 4b, n = 25); 336 U/ml in those with culture-positive AF who were not in labor (group 4c, n = 21) and 4,330 U/ml in women with culture-positive AP who were in labor (group 4d, n = 33). The IL-6 ELISA provided data consistent with the B9 bioassay results. The median concentrations of AF IL-6 in women at midtrimester (n = 16) and in those at term who were not in labor (n = 15) were both 200 pg/ml; those in women with PROM were 800, 1,000,2,320, and 18,800 pg./ml in groups 4a (n = 12), 4b (n = 23), 4c (n = 14), and 4d (n = 31), respectively. Taken together, these data indicate that: (1) PROM per se may be associated with a modest elevation of AF IL-6 titers; (2) in the absence of infection, preterm parturition is not associated with a further elevation of AF IL-6; and most strikingly, (3) intraamniotic microbial infection is associated with marked elevations of AF IL-6 that are particularly high

‘The Rockefeller University, New York, NY 10021. *The Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06510. ‘Basic Research Laboratories, Toray Industries, Kamakura 248, Japan. 4Mishima Plants, Toray Industries, Mishima 411, Japan. *To whom correspondence should be addressed at The Rockefeller University, 1230 York Avenue, New York, NY 10021. Copyright o 1991 by W.B. Saunders Company 1043-4666/91/0302-0011$5.00/O KEY WORDS: amniotic fluid/B9 hybridoma IL-6 ELI&A/pregnancy and labor/premature

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growth factor assay/ rupture of membranes

1991: pp 155-163

Recent studies suggest that immunologically active cytokines participate in the pathophysiology of normal and abnormal pregnancy and parturition. Colonystimulating factor-l (CSF-1) has been implicated in the process of implantation,’ and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to stimulate placental growth.233 Interleukin-1 (IL-l) and tumor necrosis factor (TNF) have been implicated in the initiation of parturition in the setting of intrauterine infection.4-8 Indeed, IL-l and TNF have been detected in the amniotic fluid (AF) of women in 155

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when associated with active parturition (p < 0.001). A marked elevation in the AF IL-6 level was sometimes, but not always, accompanied by an elevation of the IL-6 level in the fetal circulation and vice versa, suggesting that separate pathologic events trigger IL-6 accumulation in these two intrauterine compartments. At best, only trace levels of IL-6 biological activity and immunoreactivity were observed in the maternal circulation, even in women with markedly elevated amniotic fluid and/or fetal plasma IL-6. The present data clearly implicate IL-6 in the physiology of normal human parturition and also point to a strong association between markedly elevated AF or fetal plasma IL-6 levels and abnormal labor that accompanies intrauterine infection. Copyright o 1991 by W.B. Saunders Company

preterm labor (PTL) with intraamniotic infection.4” These cytokines are produced by human decidua in response to endotoxin,6 and IL-l and TNF can stimulate the production of prostaglandins by human amnion and decidua.‘,’ These observations prompted us to investigate the participation of IL-6 in parturition, as this cytokine is a major mediator of the host response to infection and tissue injury. IL-6 is produced by endometrial stromal cells in response to IL-l and interferon-y (IFN-Y)~ and by decidual explants in response to endotoxin.” Moreover, we have previously reported that IL-6 is normally present in human amniotic fluid and that its concentrations increase during spontaneous labor at term and in women with preterm labor and intraamniotic infecti0n.l’ From our previous study, we were unable to dissect the relative contributions of parturition per se and intraamniotic infection to elevations in AF IL-6 concentrations, due to constraints of the study design. All patients in that study who had intraamniotic infection were in preterm labor. The current study addresses this issue by exploiting a naturally occurring pregnancy complication, preterm premature rupture of membranes (PROM), which is also the leading identifiable cause of prematurity.‘l We have evaluated AF IL-6 levels in patients with PROM in the presence or absence of intraamniotic infection. Since patients with PROM may present with or without active labor at the time of admission, these patients afford a unique opportunity to distinguish between the individual contributions of intraamniotic infection and labor to changes in AF IL-6 levels. The presented data suggest that while active labor and PROM each coiltribute a modest increase to AF IL-6 levels, intratiterine microbial infection is the dominant factor. In some instances, we were able to sample the AF and the maternal and fetal circulation in the same patient. We report marked elevations in IL-6 levels in fetal plasma associated with PTL and intrauterine infection. The.available data suggest that the amniotic cavity, the fetal circulation, and the maternal circulation behave as three separate compartments in their

IL-6 response to infection of intrauterine tissues. The marked elevations observed in AF IL-6 levels in the presence of microbial infection strongly suggest that, in principle, this cytokine is likely to represent a reporter cytokine of considerable prognostic value. We have used both a bioassay (the B9 hybridoma growth factor assay”) and a two-antibody sandwich ELISA (based on high affinity anti-IL-6 moAb’3z13a)to evaluate AF IL-6 levels. Overall, the two assays were comparable in their ability to distinguish between infected and non-infected patients. RESULTS We have previously reported the detection of low levels of IL-6 bioactivity in AF from women in the second trimester of pregnancy and at term, in the absence of labor, and a modest increase in IL-6 levels during spontaneous labor at term using the hepatocytestimulating factor assay in Hep3B cel1s.l’ In the present study, we employed the more convenient B9 assay for determination of titers of biologically active IL-6. The AF samples in these previous three groups were retested using the B9 assay. (Fig. 1). AF from women in the midtrimester (group 1) and third trimester at term (group 2) of normal pregnancy contained IL-6 bioactivity that could be detected in the B9 assay (median = 16 U/ml in group 1 and 15 U/ml in group 2). Also, AF IL-6 titers were higher in women in spontaneous labor at term (group 3) compared to women at term who were not in labor (group 2) (median = 74 U/ml, range: 5 to 1,383 U/ml for women in active labor versus median = 15 U/ml, range: < 3 to 105 U/ml for women not in labor; p < 0.05). The validity of determining AF IL-6 concentrations with an ELISA was tested by determining the concentration of IL-6 antigen in undiluted AF samples collected in midtrimester and at term in the presence or absence of labor (n = 16, 15, and 15, respectively; samples different from those depicted in Fig. 1). Women in active labor at term had significantly higher AF concentrations of IL-6 antigen than women at term

Amniotic fluid IL-6 in parturition

1200 1100 i

IL-6 U/ml

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600

400 t

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Midtrimester (n = 27)

Figure 1. Biologically and at term.

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Term No Labor (n = 33)

Term Labor (n = 40)

in AF of women

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the midtrimester (group 1) or at term (group 2) (group 4a, median = 67 U/ml; group 1, median = 16 U/ml; group 2, median = 15 U/ml; p < 0.05; for comparisons between group 4a and group 1 and for group 4a and group 2, see Fig. 3). A similar set of results was obtained when determining immunoreactive IL-6 (Fig. 4). AF IL-6 antigen concentrations were significantly higher in women with PROM and without infection and labor (group 4a) than in women in the midtrimester (group 1) at term who had intact membranes (group 2) (group 4a-median = 800 pg/ml, range: 200 to 1,240 pg/ml, Fig. 4; group l-median = 200 pg/ml, range: 60 to 1,600 pgiml; group 2-median = 200 pg/ml, range: 150 to 850 pg/ml; see Fig. 4). These data indicate that rupture of the membranes per se (in the absence of labor or intraamniotic infection) is associated with increased bioactive and immunoreactive IL-6 concentrations in the amniotic cavity. The group of patients with PROM provided us with an opportunity to dissect the relative contributions of parturition and intraamniotic infection on AF IL-6 titers. In these patients, in the absence of intraam-

in midtrimester

Scatter diagram illustrates three different groups of patients (AF samples used in this analysis are the same as in Fig. 5 in reference 10): (A) midtrimester (group 1: n = 27; median = 16 U/ml; range, 0.05; B compared to C: p < 0.05; C compared to A: p < 0.05 (Dunn’s test).

158 I Santhanam

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. 45000 F

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9000 7000 5000 IL-6 U/ml

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Figure

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No Labor No Infection (n = 17)

Biologically

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Labor No Infection (n = 25)

IL-6

No Labor Infection (n = 21)

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pg/ml, p < 0.05). These data indicate that in the setting of microbial invasion of the amniotic cavity, parturition is associated with a marked increase in AF IL-6 bioactivity and immunoreactivity. The possibility that IL-6 produced by infected intrauterine tissues may enter the maternal circulation, leading to systemic effects in the mother, or that IL-6 may be present in the fetal circulation in such patients, was evaluated by comparing biologically active IL-6 titers in AF and in maternal plasma of patients who presented with PTL and fever (Table 1). In some of these instances, we were also able to simultaneously evaluate IL-6 titers in fetal plasma and, depending upon availability of material, to verify the presence of IL-6 using the ELBA. Table 1 shows that many of the fetal plasma samples tested contained high levels of IL-6. Only low levels of IL-6, however, were observed in the maternal circulation even in the presence of high AF and fetal plasma IL-6 titers. An inspection of the

Labor Infection (n = 33)

with

2. 27000

PROM.

Scatter diagram illustrates four different groups of patients: (A) no infection and no delivery (group 4a; n = 17~;median = 67‘U)ml; range. 24 to 479 U/ml: mean = 101 U/ml: SD = 103 U/ml): (B) no infeition but active labor (group 4b: n = 25; median = lb;9‘U~ml; range, 10 to 967 U/ml; mean = 238 U/ml; SD = 260 U/ml); (C) infection but no delivery (group 4c: n = 21; median 336 U/ml; range, 8 to 56,978 U/ml; mean = 4,142 U/ml; SD = 12,367 U/ml); and (D) infection and labor (group 4d: n = 33; median = 4,330 U/ml; range, 27 to 67,140 U/ml; mean = 12,238 U/ml; SD = 17,435 U/ml). After logarithmic transformation, ANOVA indicated F = 18.7, p < 0.00001. Multiple comparisons were made with the Scheffe’s test. A p value of 0.05). Women with intraamniotic infection (Figs. 3 and 4, pooled data from groups 4c and 4d) had significantly higher AF IL-6 levels than women without infection (pooled data from groups 4a and 4b) (bioactive IL-6, intraamniotic infectionmedian = 1,557 U/ml; range: 8 to 67, 140 U/ml versus non-infected women-median = 90 U/ml; range: 10 to 967 U/ml; p < 0.00001, Mann-Whitney U test; immunoreactive IL-6, intraamniotic infection-median = 14,400 pg/ml; range: 200 to 28,800 pg/ml versus non-infected women-median = 800 pg/ml; range: 20026,800 pg/ml; p < 0.0001, Mann-Whitney U test). Among women with intraamniotic infections, those with spontaneous PTL (group 4d) had significantly higher AF IL-6 biological activity and IL-6 antigen (median = 4,330 U/ml and 18,800 pg/ml) than those without labor (group 4c, median = 336 U/ml and 2,320

500

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:

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.. .

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01 No

Labor No Infection (n = 12)

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Immunoreactive

.

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Labor No Infection (n = 23)

IL-6

No Labor Infection (n = 14)

in AF’ of women

Labor Infection (n =31)

with PROM.

Scatter diagram illustrates four different groups of patients (the AF samples analyzed were from within the groups described in Fig. 3): (A) no infection and no delivery (n = 12; median = 800 pg/ml; range, 5200 to 1,240 pgiml; mean = 703 pg./ml; SD = 401 pg/ml); (B) no infection but in labor (n = 23; median = 1,000 pg/ml; range, 200 to 45,000 pg/ml; mean = 2,923 pgiml; SD = 5,638 is/ml); CC) infection but no delivery (n = 14, median = 2,320; range, 5 200 to 16,000 pg/ml; mean = 5,020 pgiml; SD = 5,407 pgiml); and (D) infection and labor (n = 31: median = 18.800 Da/ml: range. 1.280 to 92,500 pgiml; mean ‘= 16,904 pgiml; SD 2 9,34

Cytokines in normal and abnormal parturition: elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection.

The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrat...
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