Human amniotic fluid modulation of collagenase production in

cultured fibroblasts

A model of fetal membrane rupture

Felipe Vadillo-Ortega, MD, PhD,. Georgina Gonzalez-Avila, MD, MSc; Carlos Villaneuva-Diaz, MD,. Jose Luis Banales, BSC,b Moises Selman-Lama, MD, MSc,b and Alberto Alvarado Duran, MD'

Mexico City, Mexico

The participation of a mechanical factor as the only cause of rupture of fetal membranes during normal

labor or premature rupture has been criticized, and the involvement of an enzymatic mechanism has been proposed. In this study we analyzed the effect of human amniotic fluids at different gestational ages on the collagenase synthesis of cultured fibroblasts. Our results show that term amniotic fluids are capable of inducing the synthesis of collagenase and other proteases in fibroblasts, as revealed by selective

increases in collagenase activity and in immunoreactive collagenase. Nonterm amniotic fluids failed to do the same. This phenomenon is proposed as a model for studying the collagen degradation of fetal membranes during term gestation. (AM J OBSTET GVNECOL 1991 ;164:664-8.)

Key words: Collagenase, amniotic fluid, fibroblasts, premature rupture of membranes

Fetal membranes rupture is an event usually associ­ated with delivery. In normal circumstances these struc­tures remain intact until advanced cervical dilatation, 1

but in some women the rupture is not associated with labor and even occurs in the absence of contractions. Under these conditions there may be mother-newborn complications because prematurity and infections are strongly related to premature rupture of membranes (PROM)."'

A mechanical factor such as the unique determinant of normal or abnormal rupture has been criticized I.', and it has been suggested that an enzymatic mechanism may be involved. This resembles cervical ripening in which the active degradation of extracellular matrix components, mainly collagen, plays a well-documented role and is a prerequisite for cervical dilatation.'"

Two recent articles"' 10 reported the existence of local collagenolytic activity in fetal membranes. We showed in samples from PROM that there are extensive changes in collagen metabolism, including an increase in collagenolytic activity.](j The existence of collagen­olytic activity in fetal membranes allows us to postulate

From the Departamento de Bioquimica, Departamento de Fi.li%gia de /a Reproduccion, and Division de Cliniras Especiales, Instituto Nacional de Perinatologia." and Unidad de Investigaci6n. lnstituto Nacional de Enfermedades Re.lpiratorias, Secretaria de Salud.' Rncived for publication December 12. 1989; aae/)ted August 22. 1990. Reprint requests: Dr. Felipe Vadillo-Ortega. Departamento de Hio­quimica, Instituto Nacional de Perinatologia, Montes Umles 800. Col. Lomas Virreyes, Mexico, D.F. C.P. Mexico 1 JOOO. 6/1 124928 .

664

its possible partiCipation In chorioamnion ripening. The activity of this system must be switched on around the moment of labor, and its action must produce a diminution of the tensile strength of amnion mediated by a decrease in the collagen content.

This article presents evidence that indicates there are factor(s) that appear in term normal amniotic fluid that modulates collagenase synthesis in cultured fibroblasts.

Material and methods

Amniotic fluid sampling. Seventy-two amniotic fluid samples were collected from 12 to 40 weeks' gestation. Gestational age was determined by the patient's history. The nonterm samples were obtained from pregnant women undergoing diagnostic amniocentesis for an­tenatal diagnosis of congenital abnormalities. All sam­ples used were from women whose babies had no ge­netic alterations. Term gestation amniotic fluid samples (38 to 40 weeks) were obtained at the moment of de­livery. Grossly bloody samples were discarded.

Amniotic fluids were centrifuged at 10,000 g for 30 minutes in cold, sterilized by filtration through a 0.22 11m membrane (Millipore, Bedford, Mass.) and stored at - 70° C until used. Prior to use, protein determi­nation was performed with Bradford's method." U n­less otherwise indicated all reagents used were pur­chased from Sigma Chemical Co., St. Louis.

Fibroblast culture. Normal human fibroblasts (MRC-5 strain), obtained from the American Type Culture Collection, were seeded in 96 well plates and in 25 cm" tissue culture flasks (Flow, McLean, Va.). Cells were

Volume 164 Number 2

grown to confluency in Earle's modified Eagle medium (Biofluids, Rockville, Md.) with 10% fetal calf serum added, 100 U / ml penicillin, 100 J.Lg / ml streptomycin, and 250 ng/ml amphotericin B in 5% carbon dioxide and 95% air and a humid atmosphere for 5 to 7 days.

Before the experiments for collagenolytic activity or collagenase measurement were conducted, fetal calf se­rum was eliminated by replacing the culture media three times in 12 hours with fresh medium without fetal calf serum. During the last change, amniotic fluid was added at 15% to 20%, adjusting the volume by adding 300 ILg of protein per milliliter of medium. Incubation was continued 24 hours under the same conditions. Each amniotic fluid sample was evaluated in quadru­plicate. Four wells without addition of amniotic fluid were used as controls in each plate. As a positive control for stimulation of collagenase synthesis by fibroblasts, phorbol myristic acetate was used at a final concentra­tion of 10 ng/ml.'2 The incubation was stopped by add­ing Triton X-100 at a final concentration of 0.1 % and was maintained at 4° C for 2 hours. All supernatants were removed and each well or flask was individu­ally assayed for enzymatic activity or enzyme mea­surement.

Collagenolytic activity. Enzyme activity was mea­sured as described by Terato et al. 13. Type I collagen was extracted from human placenta with the pepsin­digestion method of Miller. 14 Radiolabeling of collagen was performed with a slight modification of Lefevere's method 15 with 5.0 mCi of acetic anhydride labeled with tritium (New England Nuclear, Boston, Mass.) instead of 25 mCi.

Labeled type I collagen (25 ILg) with a specific activity of 800,000 disintegration per minute per milligram were incubated in duplicate for 24 hours with 25 ILl of supernatant in each well. A buffer of Tris hydrochlo­ride 50 mmoliL, pH 7.4, was added to achieve a final concentration of 0.15 moli L sodium chloride 10 mmoliL calcium chloride, and 0.02% sodium azide. Collagen degradation was stopped by adding ethylene­diaminetetraacetic acid at a final concentration of 80 mmoliL; 50% dioxane was added cold and samples were centrifuged at 10,000 g for 10 minutes. Super­natants were counted in a liquid scintillation spectro­photometer (Beckman LS-100, 59% efficiency). Con­trol tubes were identical preparations with EDTA added at time zero. Collagenolytic activity was limited to EDTA-inhibitable tritium-labeled collagen degra­dation, and it was expressed as micrograms of degraded collagen per well per 24 hours.

The presence of latent collagenolytic activity was analyzed in 25 ILl of supernatant preincubated for 30 minutes at room temperature with 10 ILg/ml of L-1-Tosylamide-2-phenylethyl chloromethyl ketone­treated trypsin, then soybean trypsin inhibitor was added in tenfold excess (moles per liter). The optimum

Collagenase and fetal membranes 665

trypsin concentration for activation was documented previously with a range from 0.01 to 100 J.Lg/ml at eight different points, as suggested by Bauer.'6 Another 25 ILl aliquot was incubated in the presence of 1.0 mmol/L aminophenylmercuric acetate, a well-known collagen­ase activator. '7 These activated supernatants were in­cubated for collagenolytic activity as mentioned above.

Collagenolytic activity in each liquid used was assayed with the above methods.

A collagenase inhibitor has been isolated and well­characterized from amniotic fluid samples. lB. 19 To con­

trol the individual amniotic fluid inhibitory capacity on collagenolytic activity of media from stimulated fibro­blasts, 7.51Lg of amniotic fluid protein was incubated with 25 ILl of unstimulated fibroblast medium that was previously activated with trypsin-limited proteolysis, and assayed for collagenase activity as described. This amount of amniotic fluid protein was equivalent to that used in the conditioning experiments. Inhibition was calculated and used for correction of collageno­lytic activity in amniotic fluid-stimulated fibroblasts media.

Nonspecific protease activity. Heat-denatured tritium-labeled collagen was used as substrate for non­specific protease activity with the method of Sunada!O Supernatant from each well 25 ILl was incubated with 25 J.Lg of substrate (20,000 counts per minute) at 37° C for 24 hours. Activity was expressed as micrograms of substrate degraded per 24 hours per well. Each am­niotic fluid was also incubated alone, as a control, under these conditions.

_Collagenase measurement. Media of amniotic fluid­conditioned and Triton X-100-treated fibroblasts cul­tured in flasks were collected and concentrated 10 times by ultrafiltration. Immunoreactive collagenase protein was quantified with the indirect inhibition enzyme­linked immunosorbent assay described by Cooper et al!' The enzyme for development of the standard curve and the specific antiserum were provided by Dr. Eugene Bauer from Stanford University.

Statistics. Statistical differences were assessed with the Mann-Whitney U test.

Results

Fibroblasts under basal conditions produced only la­tent collagenase, which could be fully activated with 10ILg trypsin/ml. Activatable collagenolytic activity was 2.38 ± 1.32 ILg of degraded collagen per well per 24 hours (n = 14). This basal activity was substracted from the results of all experimental wells. The immuno­reactive collagenase in this media was 89.03 ± 9.79 ng/ml (n = 6). Fibroblast release of collagenase was 348.64 ± 36.32 ng/ml (n = 4) in response to stimu­lation by phorbyl myristic acetate.

Nonstimulated fibroblasts also released enzymes that degraded tritium-labeled gelatin. The values for this

666 Vadillo-Ortega et al.

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GESTATIONAL AGE (weeks)

Fig. 1. Amniotic fluids from 12 to 40 weeks' gestation were added to fibroblasts in culture and incubated for 24 hours. Later, collagenolytic activity was assayed in the media. A clear stimulation of collagenase activity was observed when terminal amniotic fluids were used. All terminal amniotic fluids (38 to 40 weeks' gestation) are represented at point 40 on X axis.

enzymatic activity averaged 1.05 ± 2.32 fLg of de­graded gelatin per well per 24 hours (n = 14), and these values were substracted from degradation values in their respective experimental wells.

Amniotic fluids did not show collagenolytic activity at any gestational age. However, amniotic fluid from women 2:37 weeks' pregnancy had a nonspecific pro­teolytic activity against gelatin labeled with tritium. These values were used to correct the activity in their corresponding conditioned media.

We found some inhibitory effect of amniotic fluid on collagenolytic activity; however. it was not >5% under the experimental conditions. These individual values of inhibition were used to correct the collagenolytic activity in media of amniotic fluid-stimulated fibro­

blasts. The addition of amniotic fluid to fibroblasts modified

their basal production of collagenase and nonspecific proteases. A clear stimulation of these enzyme activities was observed when amniotic fluids captured during delivery from term gestations (38 to 40 weeks) were used (Figs. 1 and 2). In contrast. the use of amniotic fluid from nonterm. nondelivering gestations pro­duced no effect because the values of active collagenase in media were similar to those from nonstimulated fi­broblasts. Despite the fact that collagenolytic activity increased around week 30 (Fig. 1), the capacity for collagen degradation was very similar in all the media stimulated with nonterm amniotic fluid. Collagenolytic

February 1991 Am.J Obstet Gynecol

activity in media from fibroblasts stimulated with term amniotic fluid was 6.56 ± 3.15 fLg of degraded collagen per well per 24 hours (n = 25). This activity was sig­nificantly higher (p < 0.001) when compared with the nonterm amniotic fluid induction (2.04 ± 1.11 fLg degraded collagen. n = 47). Collagenolytic activity was in the active form in the media from amniotic fluid­stimulated fibroblasts and no further activation was ob­tained with trypsin or aminophenylmercuric acetate.

Measurements of immunoreactive collagenase showed a pattern that was very similar to the results from collagenolytic activity assay (Fig. 3). The media from nonterm, nondelivering amniotic fluid-induced fibroblasts contained 51.91 ± 25.86 ng collagenase per milliliter (n = 12), a value lower than the basal syn­thesis of nonstimulated fibroblasts (p < 0.01) and was clearly different from term amniotic fluid-stimulation (156.52 ± 32.37 ng/ml) (p < 0.001).

The pattern of nonspecific protease activity induc­tion was similar to the collagenolytic activity. Term amniotic fluid-induced 11.35 ± 6.61 fLg degraded tritium-labeled gelatin per well per 24 hours. whereas nonterm amniotic fluid only induced 0.67 ± 1.24 fLg degraded tritium-labeled gelatin per well per 24 hours. These two groups were clearly different (p < 0.0001).

Comment Degradation of ex~racellular matrix components re­

quires a complex series of enzymatic activities. It is pos­tulated that at the least three groups of proteases are involved in extracellular matrix catabolism. A group of enzymes degrades collagen-associated molecules such as proteoglycans and fibronectin and forms monomeric collagen. A second group directly attacks collagen and the third group hydrolyzes the collagen-degradation product until free aminoacids are obtained. Members of the first group are stromelysin and telopeptidase. The second group is formed by collagenases. a group of metalloenzymes with affinity for collagen that under physiologic conditions are a unique class of proteases capable of degrading native collagen. They are secreted in a latent form. and later, in the extracellular space, they are subjected to strict control. Its activity is de­pendent on complex interactions between the enzyme and inhibitors and activators. The last group of en­zymes comprises several enzymes, such as gelatinase and PZ-peptide!2.23

The results of this study reveal that amniotic fluids from term pregnancies contain chemical signals that enhance the in vitro synthesis of collagenase and other proteases in cultured fibroblasts. The fact that such effect had a clear association with the end of gestation suggests that it may playa role in vivo in the degra­dation of the extracellular matrix of fetal membranes during normal labor. Because preformed collagenase

Volume 164 f\umber 2

is not stored inside fibroblasts," the effect of amniotic fluid was possible because of an induction of collage­nase synthesis, which is in agreement with the findin g of increased enzymatic activity and immunoreactive protein quantity. On the other hand , the possibility of latent enzyme activation was controlled with the use of the collagenase activators aminophenylmercuric ace­tate and trypsin.

Amniotic fluids from nonterm gestations appear to

inhibit the synthesis of collagenase as revealed by the immunoreactive measurement of this enzyme. How­ever, its activity in media did not show this behavior, probably because nonlimiting conditions for enzyme activity were used.

The media from amniotic fluid-conditioned fibro­blasts conta ined active collagenase only, as suggested by the fact that addition of the collagenase activators to the wells did not induce further collagenase activity. This could be explained by a collagenase activator al­ready present in the system, which may be ascribed to the action of some of the induced nonspecific proteases. Characterization of these enzymes is mandatory to clar­ify this point.

The use of a fibroblast cell line as a model of the biochemical events that may occur in fetal membranes during labor is acceptable because this cellular popu­lation is a normal constituent of these tissues. We hy­pothesized that activation of local fibroblasts by a signal present in term amniotic fluid induces the synthesis of collagenase and other proteases. The subsequent deg­radation of collagen, which is the main structural com­ponent of membranes, diminishes the mechanical sup­port of these tissues . At this moment the membranes are more fragile and a sudden increase in intrauterine pressure would lead to their rupture .

The identification of this mechanism opens the pos­sibility of stud yi ng its participation in PROM because, as we have shown, in PROM there a ppears to be an increase in collagen degradation.'o

It has been suggested that activation of local colla­genolytic activity in amnion may be promoted by ex­ternal factors such as inflammation and infection. In this context, it has been postulated that collagen deg­radation that occurs in PROM is caused by inflamma­tory cells, including polymorphonuclears and macro­phages, through the synthesis of collagenase and other proteases .'· ~5 However, as suggested by this study, it is also possible that under normal or pathologic condi­tions , collagenolysis may be activated by local normally resident cells such as fibroblasts. It has been shown that fibroblasts respond to macrophage and lymphocyte signals with the synthesis of collagenase, stromelysin , and other connective tissue-degrading metallopro­teases! g·26

Characterization of the molecular nature of the com-

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Collagenase and fetal membranes 667

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GESTATIONAL AGE (weeks)

Fig. 2. Proteolytic activity against gelatin tagged with tritium was assayed in media of fibroblasts stimulated by amniotic Auid . Pattern of these proteases was similar to collagenolytic activity and showed peak of activity when terminal amniotic Auid was used. Terminal amniotic Auids (38 to 40) are grouped at point 40 on X axis.

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Fig. 3. Immunoreactive collagenase in media from fibroblasts stimulated by amniotic Auid was quantified with indirect in­hibition enzyme-linked immunosorbent assay. Pattern of pro­tein was similar to collagenolytic activity (.). As controls of fibroblast functionality, nonstimulated fibrohlasts and phorbol myristic acetate-stimulated cells were included.

pounds with activity on fibroblasts may be crucial in the search for the factors involved in normal , and probably abnormal , rupture of the membranes. A possible can­didate for such a compound is prostaglandin E" be-

668 Vadillo-Ortega et al.

cause this molecule appears to be related to events in human labor under normal or abnormal circumstances, and it has been shown that this substance stimulates the production of collagenase.27

• 28

The initiation of human labor is associated with mo­bilization of arachidonic acid from fetal membranes; and increased concentrations of prostaglandin E2 in the amniotic fluid has been detected. 29 In addition, in­creased levels of this compound have been quantified in amniotic fluid of women with preterm labor,30 and in the presence of infection, the latter mediated through endotoxins that stimulate amnion cells to syn­thesize prostaglandin E2.31 In this way it is possible to hypothesize that an unidentified signal in normal cir­cumstances or bacterial products in pathologic condi­tions induce the synthesis of prostaglandins, especially prostaglandin E2 , and that this substance may then stim­ulate the synthesis of collagenase in local cell popula­tions, which in turn allows PROM.

We thank personnel of the Genetics Department of the Instituto Nacional de Perinatologia and Cesar Her­nandez for their expert technical assistance.

REFERENCES

1. Caldeyro-Barcia R, Schwarcz R, BelizanJM, et al. Adverse perinatal effects of early amniotomy during labor. In: Gluck L, ed. Modern perinatal medicine. Chicago: Year Book Medical Publishers, 1974:431.

2. Christensen K, Christensen P, Ingemarsson I, et al. A study of complications in preterm deliveries after pro­longed premature rupture of the membranes. Obstet Gy­necoI1976;48:670-7.

3. Naeye RL, Peters EC. Causes and consequences of pre­mature rupture of the fetal membranes. Lancet 1980; 1:192-4.

4. Artal R, Burgeson R, Hobel C, Hollister D. An in vitro model for the enzimatically mediated biochemical changes in chorioamniotic membranes. AM J OBSTET GYNECOL 1979; 133:656-9.

5. Skinner S J, Campos GA, Liggins GC. Collagen content of human amniotic membranes: effect of gestational length and premature rupture. Obstet Gynecol 1981;57:487-9.

6. Kitamura K, Ito A, Mori Y, Hirakawa S. Changes ill the human uterine cervical collagenase with special reference to cervical ripening. Biochem Med 1979;22:332-7.

7. Uldbjerg N, Ulmsten U, Ekman G. The ripening of the human cervix in terms of connective tissue biochemistry. Clin Obstet Gynecol 1983;26: 14-26.

8. Rajabi M, Dean D, Beydoun S, Woessner F. Elevated tissue levels of collagenase during dilatation of uterine cervix in human parturition. AM J OBSTET GYNECOL 1988;159: 971-6.

9. HalaburtJ, Uldbjerg N, Helmig R, Ohlsson K. The con­centration of collagen and the collagenolytic activity in the amnion and the chorion. Eur J Obstet Gynecol Reprod Bioi 1989;31:75-82.

10. Vadillo-Ortega F, Gonzalez-Avila G, Karchmer S, Meraz N, Ayala A, Selman M. Collagen metabolism in premature rupture of amniotic membranes. Obstet Gynecol 1990; 75:84-8.

11. Bradford M. A rapid and sensitive method for the quan­titation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-54.

February 1991 Am J Obstet Gynecol

12. Brinckerhoff C, Gross R, Nagase H, Sheldon L, Jackson D, Harris E. Increased levels of translatable collagenase messenger ribonucleic acid in rabbit synovial fibroblasts treated with phorbol myristate acetate or crystals of monosodium urate monohydrate. Biochemistry 1982;21: 2674-9.

13. Terato K, Nagai Y, Kawanishi K, Yamamoto S. A rapid assay method of collagenase activity using l4C-Iabeled sol­uble collagen as sustrate. Biochim Biophys Acta 1976; 445:753-62.

14. Miller E, Rhodes K. Preparation and characterization of the different types of collagen. Methods Enzymol 1982; 82:33-64.

15. Lefevere MF, Slegers GA, Claeys AE. Evaluation of a rapid, sensitive and specific assay for the determination of collagenolytic activity in biologic samples. Clin Chim Acta 1979;92:167-75.

16. Bauer EA, Stricklin Gp,Jeffrey JJ, Eisen AZ. Collagenase production by human skin fibroblasts. Biochem Biophys Res Commun 1975;64:232-40.

17. Stricklin G, Jeffrey J, Roswit W, Eisen A. Human skin fibroblasts procollagenase: mechanisms of activation by organomercurials and trypsin. Biochemistry 1983;22: 61-8.

18. Aggler J, Engvail E, Werb Z. An irreversible tissue inhib­itor of collagenase in human amniotic fluid: characteriza­tion and separation from fibronectin. Biochem Biophys Res Commun 1981;100:1195-201.

19. Stricklin G, Gast M, Welgus H. Amniotic fluid collagenase inhibitor: correlation with gestational age and fetal lung maturity. AMJ OBSTET GYNECOL 1986;154:134-8.

20. Sunada H, Nagai Y. A rapid microassay method for ge­latinolytic activity using tritium-labeled heat-denatured polymeric collagen as a sustrate and its application to the detection of enzymes involved in collagen metabolism. J Biochem 1980;87: 1765-71.

21. Cooper TW, Bauer EA, Eisen AZ. Enzyme-linked im­munoadsorbent assay for human skin collagenase. Coli Relat Res 1982;3:205-16.

22. Werb Z. Degradation of collagen: mechanisms. In: Weiss J,Jayson M, eds. Collagen in health and disease. London: Churchill Livingstone, 1982:121.

23. Reynolds JJ. The molecular and cellular interactions in­volved in connective tissue destruction. Br J Dermatol 1985; 112:715-23.

24. Harris ED, Wei gus HG, Krane SM. Regulation of the mammalian collagenases. Coli Relat Res 1984;4:493-512.

25. Sbarra AJ, Selvara RJ, Cetrulo CL, Feingold M, Newton E, Thomas G. Infection and phagocytosis as possible mechanism of rupture in premature rupture of the mem­branes. AMJ OBSTET GYNECOL 1985;153:38-43.

26. Vaes G, Huybrechts-Godin G, Peeters-Joris C, Laub R. Cell cooperation in collagen and proteoglycan degrada­tion. In: Dingle J, Gordon C, eds. Cellular interactions. North-Holland: Elsevier, Biomedical Press, 1981 :241.

27. Dayer JM, Krane SM, Russell RG, Robinson DR. Pro­duction of collagenase and prostaglandins by isolated ad­herent rheumatoid synovial cells. Proc Nat! Acad Sci USA 1976;73:945-9.

28. Wahl LM, Olsen CE, Sandberg AL, Mergenhagen SE. Prostaglandin regulation of macrophage collagenase pro­duction. Proc Nat! Acad Sci USA 1977;74:4955-8.

29. L6pez Bernal A, Hansell DJ, Alexander S, Turnbull AC. Prostaglandin E production by amniotic cells in relation to term and preterm labor. Br J Obstet Gynaecol 1987; 94':864-9.

30. Romero R, Emamian M, Wan M, Quintero R, HobbinsJ, Mitchell M. Prostaglandin concentrations in amniotic fluid of women with intra-amniotic infection and preterm la­bor. AMJ OBSTET GYNECOL 1987;157:1461-7.

31. Romero R, Hobbins J, Mitchell M. Endotoxin stimulates prostaglandin E2 production by human amnion. Obstet GynecoI1988;71:227-8.