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Increased inducibility of inflammatory mediators from peripheral blood mononuclear cells of women with salpingitis
Increased inducibility of inflammatory mediators from peripheral blood mononuclear cells of women with salpingitis Steven S. Witkin, PhD, Miklos Toth, MD, Jan Jeremias, AB, and William J. Ledger, MD New York, New York To investigate whether immune system activation may contribute to the tissue damage observed in salpingitis, we isolated peripheral blood mononuclear cells and quantitated production of the monocyte activation products tumor necrosis factor-a, interleukin-1, and interleukin-6. Unstimulated cells from 7 of 20 women with salpingitis spontaneously released tumor necrosis factor at a concentration >2 SO above the mean value produced by cells from 29 healthy donors. Interferon gamma (200 U/ml) further induced production of tumor necrosis factor from mononuclear cells of 11 women with salpingitis. In contrast, production of tumor necrosis factor by each of 23 other patients who lacked laparoscopic or clinical evidence of salpingitis was similar to that of the controls. In a subset of women whose cells were tested for production of other monokines, three of nine women with salpingitis spontaneously released interleukin-1 but none of the others did so. Four of nine patients with salpingitis also produced interleukin-6, but none of the others did so. None of the monokines were detected in serum from any subject. The results suggest that monocytes from women with salpingitis are primed in vivo and produce inflammatory mediators under conditions where monocytes from other women are poorly responsive. This increased monokine inducibility may contribute to the tubal damage that is the hallmark of salpingitiS. (AM J OBSTET GVNECOL 1991 ;165:719-23.)
Key words: Salpingitis, tumor necrosis factor, monocytes, interleukin-l, interleukin-6, Chlamydia trachomatis
Acute salpingitis is a m~or cause of occlusion of the fallopian tubes and the subsequent occurrence of infertility and ectopic pregnancy. The microorganisms responsible for this disease, primarily Neisseria gonorrhoeae and Chlamydia trachomatis, and possibly a variety of other aerobic and anaerobic bacteria, have been , identified. ,2 However, the mechanism of tubal damage, especially in nongonococcal pelvic inflammatory disease, remains unclear. In organ cultures C. trachomatis does not visibly damage the fallopian tubes. 3 However, extensive tubal damage often may be observed in vivo in chlamydial pelvic inflammatory disease! The immune response to infection might be responsible for the tubal damage in chlamydial pelvic inflammatory disease. A single inoculation of C. trachomatis into the fallopian tubes of monkeys induced a transient limited infection. Repetitive inoculations, however, resulted in permanent tubal damage.' A 57 kd chlamydial protein, which cross reacts with heat shock proteins present in other bacterial species, has been shown to induce a potent delayed hypersensitivity response in animals previously exposed to Chlamydia Sp.6 Antibodies
From the Immunology Division, Department of Obstetrics and Gynecology, Cornell University Medical College, Received for publication November 5, 1990; revised March 18, 1991; accepted March 22,1991, Reprint requests: Dr, Steven S. Witkin, Department of Obstetrics and Gynecology, Cornell University Medical College, 525 East 68th St., New York, NY 10021, 611 129783
to this protein were detected in women with tubal infertility 2 and correlated with the occurrence of ectopic pregnancy in women with pelvic inflammatory disease. 7 Interferon gamma, a potent activator of cell-mediated immunity, also has been identified in the circulation of women with pelvic inflammatory disease. S Products of activated monocytes and tissue macrophages, including tumor necrosis factor, interleukin-l, and interleukin-6, are potent mediators of inflammation and fibrosis. 9 Tumor necrosis factor and interleukin-l promote infiltration by granulocytes, monocytes, and neutrophils to inflammatory sites. Interleukin-l induces fibroblast growth and the synthesis of prostaglandin E2 and collagenase. Tumor necrosis factor stimulates the production of toxic oxygen radicals, augments phagocytosis, interferes with epithelial cell growth, and induces interleukin-6 synthesis. Interleukin-6 induces acute phase protein synthesis and differentiation of monocytes and T and B lymphocytes. Circulating unactivated (resting) monocytes from healthy individuals release little or no tumor necrosis factor, interleukin-l, or interleukin-6. However, if the monocytes are first exposed to interferon gamma, a product of activated T lymphocytes, or to microorganisms, they undergo a maturational change and become primed to produce monokines on subsequent exposure to low concentrations of Ii po poly sacca ride or bacteria that do not normally evoke a response in resting monocytes. lO We now report that peripheral blood mononuclear
720
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Fig. 1. Spontaneous in vitro production of tumor necrosis factor. Peripheral blood mononuclear cells from women with salpingitis or other disorders or from healthy controls were incubated for 20 hours at 37° C. Culture supernatants were collected and assayed for tumor necrosis factor by enzymelinked immunosorbent assay. Brahen horizontal line. Value 2 SDs above mean for healthy controls.
cells from women with salpingitis produce tumor necrosis factor-ex, interleukin-l, and interleukin-6 under conditions where cells from women without salpingitis remain normally unresponsive. The results suggest that monocytes from patients with salpingitis may be primed in vivo to readily produce inflammatory mediators that may contribute to subsequent fallopian tube damage. Material and methods Subjects. The study population consisted of 20 patients with a current diagnosis of salpingitis and 23 other patients diagnosed with other gynecologic problems, including pelvic pain (n = 4), recurrent miscarriage (n = 4), unexplained infertility (n = 4), urinary tract infection (n = 3), recurrent vaginitis (n = 2), menorrhagia (n = 2), ovarian cyst (n = 2), and postpartum endometritis (n = 2). Controls were 29 reproductive-age women in apparently good health with no history of pelvic inflammatory disease or infection with
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Fig. 2. Interferon gamma-induced production of tumor necrosis factor. Periperhal blood mononuclear cells from women with salpingitis or other disorders or from healthy controls were incubated in the presence of 200 U / ml of interferon gamma. Afer 20 hours, culture supernatants were collected and assayed for tumor necrosis factor. Broken horizontal line, Value 2 SDs above mean for healthy controls.
C. trachoma tis or N. gonon-hoeae. Salpingitis was diagnosed by laparoscopic surgery in 18 of the women who had visual evidence of tubal damage caused by an inflammatory process. Microbial cultures were not obtained during surgery. A differential diagnosis of salpingitis was made in two women who did not undergo laparoscopy, on the basis of the presence of adnexal tenderness , cervical motion tenderness, and identification of C. trachomatis in endocervical cultures. The four women with pelvic pain had no evidence of salpingitis during laparoscopy. The other patients did not undergo surgery. Peripheral blood mononuclear cell incubation. Mononuclear cells were isolated from heparinized peripheral blood by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) gradient centrifugation and added to multiple wells of a microtiter plate (2 X 10" cells per well) containing 10% heat-inactivated autologous serum in RPMI 1640 medium with 25 mmol HEPES buffer, Lglutamine, penicillin (100 UI ml), streptomycin (l00 pg/ml), and kanamycin (100 U Iml). Interferon gamma (Genzyme, Boston) was added to triplicate wells at a concentration of 200 unitslml. After incubation for 20
Monokine induction in salpingitis
Volume 165 Number 3
721
Table I. Production of interleukin-l and interleukin-6 by unstimulated peripheral blood mononuclear cells Salpingitis Subject
1 2 3 4 5 6 7 8 9 10 11 12
I nterleukin-l (pglml)
I
149 174
Other disorders
Interleukin-6 (nglml)
I nterleukin-l (pglml)
1.8 3.0
115 76 78 101 74 89 122 82 85
TO
T3 136 169 140 134 104 379
2.0 2.7 2.0 3.0
f7 6.3
I
Controls
I nterleukin-6 (nglml)
I nterleukin-l (pglml)
1.1 1.2 1.0 1.8 1.6 1.0 2.4 1.1 1.2
73 110 167 109 73 109 89 74 80 85 89 94
I
I nterleukin-6 (nglml)
1.9 1.6 2.7
l.8
1.6 1.6 1.0 1.5 1.3 1.3 1.1 ND
Peripheral blood mononuclear cells were incubated for 20 hours. Culture supernatants were collected and assayed for interleukin-l or interleukin-6. Underlined values are >2 SDs above mean values for control group. ND, Not determined.
hours at 37° C in a 5% carbon dioxide incubator, culture supernatants were collected, particulate components were removed by microcentrifugation, and the supernatants were stored at -70 C until used. Monokine assays. Culture supernatants were assayed for tumor necrosis factor-a (Cistron, Pine Brook, N.J.), interleukin-l (Cistron), and interleukin-6 (Genzyme) by enzyme-linked immunosorbent assays with commercially available kits. All samples were tested in triplicate and the mean value was determined. Replicates differed by <15%. Values were converted to picograms per milliliter by reference to a standard curve obtained by the simultaneous analysis of known concentrations of purified compounds in culture medium. The lower limit of sensitivity was 20 pg I ml for tumor necrosis factor and interleukin-l and 0.2 ng/ml for interleukin-6. Statistics. Differences in the numbers of women in each of the subject groups with elevated tumor necrosis factor production under the experimental conditions were evaluated by the X2 test, with the Yates correction factor. 0
Results
Spontaneous tumor necrosis factor production by peripheral blood mononuclear cells. The levels of tumor necrosis factor produced by unstimulated mononuclear cells from women with salpingitis, women with other disorders, and healthy controls are shown in Fig. 1. Only low levels of tumor necrosis factor were detected in the culture fluids of unstimulated cells from healthy women. A similar range of concentrations were detected in culture supernatants of mononuclear cells from women with disorders other than salpingitis. In marked contrast, mononuclear cells from seven of the
20 women with salpingitis spontaneously released tumor necrosis factor at levels >2 SDs above the control mean value. The difference in the number of women with elevated tumor necrosis factor production in the salpingitis and control groups was significant (P < 0.01). Tumor necrosis factor production by peripheral blood mononuclear cells incubated with interferon gamma. In the presence of interferon gamma, mononuclear cells from only one of 23 patients with a diagnosis of a disorder other than salpingitis and one of 29 healthy controls produced tumor necrosis factor at a level >2 SDs above the control mean value. In marked contrast, interferon gamma-stimulated cells from 11 of 20 women with salpingitis produced tumor necrosis factor at elevated levels (Fig. 2). The number of women in the salpingitis group releasing elevated levels of tumor necrosis factor under these conditions was significantly greater than the number of women in the other groups (p < 0.01). Interleukin-l and interleukin-6 production by peripheral blood mononuclear cells from women with salpingitis. Mononuclear cells from nine patients diagnosed as having salpingitis, nine patients with other diseases, and 12 healthy controls also were tested for their ability to release two other monocyte products, interleukin-l and interleukin-6. The upper limit of normal was designated as a value 2 SDs above the mean level of each mono kine produced by peripheral blood mononuclear cells from the healthy controls; the number of patients whose unstimulated cells produced elevated levels of these two monokines was evaluated (Table I). None of the nine patients without salpingitis produced an elevated level of interleukin-l or interleukin-6. In contrast, peripheral blood mononuclear
722
Witkin et al.
September 1991 Am J Obstet Gynecol
Table II. Production of interleukin-l and interleukin-6 by interferon-stimulated peripheral blood mononuclear cells Other disorders
Salpingitis Subject
1 2 3 4 5 6 7 8 9 10 11 12
I nterleukin-l (pglml)
139 163 106 1217 198 159 206 151 491
I
Interleukin-6 (nglml)
Interleukin-l (pglml)
2.1 2.2 1.0 6.5 2.3 2.0 2.2 1.9 3.7
126 109 87 134 88 102 147 118 91
I
Controls
Interleukin-6 (nglml)
I nterleukin-l (pglml)
1.1
115 12 165 148 143 121 108 91 80 94 105 114
2.1 1.1 1.8 1.7 1.0 1.9 1.2 1.1
I
Interleukin-6 (nglml)
1.9 1.7 2.2 1.5 1.6 1.9 1.1 1.5 1.3 1.5 1.9 ND
Peripheral blood mononuclear cells were incubated for 20 hours in the presence of 200 U I ml of interferon gamma. Culture supernatants were collected and assayed for interleukin-l or interleukin-6. Underlined values are >2 SDs above mean values for control group. ND, Not determined.
cells from three of the patients with salpingitis spontaneously produced elevated amounts of interleukin-l, and four produced elevated amounts of interleukin-6. Similar results were obtained with interferon gammastimulated cells (Table II). Four of the patients with salpingitis showed elevated interleukin-l levels and three had elevated interleukin-6 production but none of the other women did so. In contrast to the in vitro culture results, none of the sera from patients with salpingitis contained detectable tumor necrosis factor, interleukin-l, or interleukin-6. Comment
Peripheral blood mononuclear cells from women with salpingitis readily released products of activated monocytes in vitro under conditions where the corresponding cells from women with other gynecologic disorders or from healthy women remained relatively quiescent. Under our experimental conditions, adherence of the monocytes to the culture dishes could have been a sufficient stimulus to activate the primed monocytes to release monokines. II In contrast, none of the patient's sera contained detectable levels of tumor necrosis factor, interleukin-l, or interleukin-6. This is consistent with reports demonstrating that tumor necrosis factor has a short half-life and is detectable in the circulation only for a short period of time at the onset of an infection. I2 Thus mono kine production in vitro by unstimulated or interferon gamma-stimulated monocytes may be a more reliable indicator of infection than the presence of these compounds in serum. Primed monocytes present in the circulation of women with salpingitis would be highly susceptible to activation in vivo at infected tubal sites, even at loci
contammg low concentrations of microorganisms. Primed monocytes can be activated by levels of bacteria that are insufficient to stimulate unprimed (resting) monocytes. IO In addition, in cases of chlamydial salpingitis, nonproductive infections also may lead to the release of chlamydial antigens capable of activating monocytes. 6 Tumor necrosis factor has, in fact, been shown to induce the disordered synthesis of chlamydial antigens. I3 The resulting accumulation of monokines at specific sites would induce scarring and tissue damage. Furthermore, since tumor necrosis factor synergizes with bacterial endotoxin in the induction of tissue necrosis,I4 tumor necrosis factor production is most likely to initiate inflammatory tissue damage if a concomitant infection is also present. C. trachomatis infections, even those causing severe tubal damage, are often accompanied by mild or no symptoms I5 and may therefore remain undetected. In addition, the standard antibiotic treatments may not always completely clear the organism from the female genital tract.I6 Therefore low-level infections, even by micrroorganisms not normally considered to be pathogenic, may become capable of inducing monokine secretion from monocytes that were primed by "silent" chlamydial tubal infections. This might especially be a problem during pregnancy. Many studies have now shown that products of activated monocytes can induce preterm pregnant complications. I7 The reported increased incidence of first-trimester spontaneous abortion,IS preterm labor, or premature rupture of membranes I9. 2o in women with serologic or microbiologic evidence of C. trachomatis also may occur, at least partially, by this mechanism. Salpingitis is certainly not the only infectious condition in which primed monocytes are present in the
Volume 165 Number 3
circulation. Monocytes from patients with pulmonary tuberculosis· l or meningococcal meningitis 22 also were shown to be more responsive than monocytes from controls in releasing tumor necrosis factor. Acute localized infections at other body sites would be expected to produce similar findings. However, we have not observed primed monocytes in women with vaginal or urinary tract infections. This suggests that infections confined to mucosal surfaces may not prime circulating monocytes. The presence of a chlamydial infection is often difficult to detect clinically or by culture techniques. 2 .• 3 The methods described in this report may therefore be of value in the evaluation of women with suspected salpingitis. REFERENCES 1. Holmes KK, Eschenbach DA, Knapp ]S. Salpingitis: overview of etiology and epidemiology. AM] OBSTET GYNECOL 1980; 138:893-900. 2. Brunham RC, Maclean IW, Binns B, Peeling RW. Chlamydia trachomatis: its role in tubal infertility.] Infect Dis 1985; 152: 1275-82. 3. Hutchinson GR, Taylor-Robinson D, Dourmashkin RR. Growth and effect of chlamydiae in human and bovine oviduct organ cultures. Br] Venereol 1979;55: 194-202. 4. Gjonnaess H, Dalaker K, Anestad G, Mardh PA, Kvite G, Bergan T. Pelvic inflammatory disease: etiologic studies with emphasis on chlamydial infection. Obstet Gynecol 1984;59:550-55. 5. Patton DL, Wolner-Hanssen P, Cosgrove S], Holmes KK. The effects of Chlamydia trachomatis on the female reproductive tract of the Macaca nemestrina after a single tubal challenge and following repeated cervical inoculations. Obstet Gynecol 1990;76:643-50. 6. Morrison RP, Lyng K, Caldwell HD. Chlamydia disease pathogenesis. Ocular hypersensitivity elicited by a genusspecific 57kD protein.] Exp Med 1989;169:663-75. 7. Wagner EA, Schachter J, Bavoil P, Stephens RS. Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens. J Infect Dis 1990;162:922-7. 8. Grifo ]A, Jeremias], Ledger W], Witkin SS. Interferon'I in the diagnosis and pathogenesis of pelvic inflammatory disease. AM] OBSTET GYNECOL 1989;160;26-31. 9. Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL6 and related molecules (ILl and TNF). FASEB J 1990;4:2860-7.
Monokine induction in salpingitis
723
10. Adams DO, Hamilton TA. Phagocytic cells: cytotoxic activities of macrophages. In: Gallin]I, Goldstein 1M, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press, 1988:471. 11. Bodel PT, Nichols BA, Bainton DF. Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence. J Exp Med 1977; 145:264-74. 12. Michie HR, Manoque KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl] Med 1988;318:1481-6. 13. Shemer-Avni Y, Wallace LD, Sarov 1. Inhabition of Chlamydia trachomatis growth by recombinant tumor necrosis factor. Infect Immun 1988;56:2503-6. 14. Rothstein ]L, Schreiber H. Synergy between tumor necrosis factor and bacterial products causes hermorrhagic necrosis and lethal shock in normal mice. Proc Nat! Acad Sci USA 1988;85:607-11. 15. Henry-Suchet], Catalan F, Luffredo V, et al. Chlamydia trachomatis associated with chronic inflammation in abdominal specimens from women selected for tuboplasty. Ferti! Steril 1981 ;36:599-605. 16. Batteiger BE, Fraiz], Newhall W], Katz BP, Jones RB. Association of recurrent chlamydial infection with gonorrhea.] Infect Dis 1989;159:661-9. 17. Witkin SS, McGregor ]A. Infection-induced activation of cell-mediated immunity: possible mechanisms for preterm birth. Clin Obstet Gynecol 1991 ;34: 112-21. 18. Quinn PA, Petric M, Barkin M, et al. Prevalence of antibody to Chlamydia trachomatis in spontaneous abortion and infertility. AM] OBSTET GYNECOL 1987; 156:291-6. 19. Martin OH, Koutsky L, Eschenbach DA, et al. Prematurity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomatis infections. ]AMA 1982; 247:1585-8. 20. Gravett MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach D, Holmes KK. Independent associations of bacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. ]AMA 1986;256: 1899-903. 21. Takashima T, Ueta C, Tsuyuguchi I, Kishimoto S. Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis. Infect Immun 1990;58:3286-92. 22. Waage A, Halstensen A, Shalaby R, Brandtzarg P, Kierulf P, Espevik T. Local production of tumor necrosis factor, interleukin 1 and interleukin 6 in meningococcal meningitis. Relation to the inflammatory response.] Exp Med 1989; 170; 1859-67. 23. Schachter], Moncada J, Dawson CR, et al. Nonculture methods for diagnosing chlamydial infection in patients with trachoma: A clue to the pathogenesis of the disease? ] Infect Dis 1988;158:1347-52.
Key words: Salpingitis, tumor necrosis factor, monocytes, interleukin-l, interleukin-6, Chlamydia trachomatis
Acute salpingitis is a m~or cause of occlusion of the fallopian tubes and the subsequent occurrence of infertility and ectopic pregnancy. The microorganisms responsible for this disease, primarily Neisseria gonorrhoeae and Chlamydia trachomatis, and possibly a variety of other aerobic and anaerobic bacteria, have been , identified. ,2 However, the mechanism of tubal damage, especially in nongonococcal pelvic inflammatory disease, remains unclear. In organ cultures C. trachomatis does not visibly damage the fallopian tubes. 3 However, extensive tubal damage often may be observed in vivo in chlamydial pelvic inflammatory disease! The immune response to infection might be responsible for the tubal damage in chlamydial pelvic inflammatory disease. A single inoculation of C. trachomatis into the fallopian tubes of monkeys induced a transient limited infection. Repetitive inoculations, however, resulted in permanent tubal damage.' A 57 kd chlamydial protein, which cross reacts with heat shock proteins present in other bacterial species, has been shown to induce a potent delayed hypersensitivity response in animals previously exposed to Chlamydia Sp.6 Antibodies
From the Immunology Division, Department of Obstetrics and Gynecology, Cornell University Medical College, Received for publication November 5, 1990; revised March 18, 1991; accepted March 22,1991, Reprint requests: Dr, Steven S. Witkin, Department of Obstetrics and Gynecology, Cornell University Medical College, 525 East 68th St., New York, NY 10021, 611 129783
to this protein were detected in women with tubal infertility 2 and correlated with the occurrence of ectopic pregnancy in women with pelvic inflammatory disease. 7 Interferon gamma, a potent activator of cell-mediated immunity, also has been identified in the circulation of women with pelvic inflammatory disease. S Products of activated monocytes and tissue macrophages, including tumor necrosis factor, interleukin-l, and interleukin-6, are potent mediators of inflammation and fibrosis. 9 Tumor necrosis factor and interleukin-l promote infiltration by granulocytes, monocytes, and neutrophils to inflammatory sites. Interleukin-l induces fibroblast growth and the synthesis of prostaglandin E2 and collagenase. Tumor necrosis factor stimulates the production of toxic oxygen radicals, augments phagocytosis, interferes with epithelial cell growth, and induces interleukin-6 synthesis. Interleukin-6 induces acute phase protein synthesis and differentiation of monocytes and T and B lymphocytes. Circulating unactivated (resting) monocytes from healthy individuals release little or no tumor necrosis factor, interleukin-l, or interleukin-6. However, if the monocytes are first exposed to interferon gamma, a product of activated T lymphocytes, or to microorganisms, they undergo a maturational change and become primed to produce monokines on subsequent exposure to low concentrations of Ii po poly sacca ride or bacteria that do not normally evoke a response in resting monocytes. lO We now report that peripheral blood mononuclear
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Fig. 1. Spontaneous in vitro production of tumor necrosis factor. Peripheral blood mononuclear cells from women with salpingitis or other disorders or from healthy controls were incubated for 20 hours at 37° C. Culture supernatants were collected and assayed for tumor necrosis factor by enzymelinked immunosorbent assay. Brahen horizontal line. Value 2 SDs above mean for healthy controls.
cells from women with salpingitis produce tumor necrosis factor-ex, interleukin-l, and interleukin-6 under conditions where cells from women without salpingitis remain normally unresponsive. The results suggest that monocytes from patients with salpingitis may be primed in vivo to readily produce inflammatory mediators that may contribute to subsequent fallopian tube damage. Material and methods Subjects. The study population consisted of 20 patients with a current diagnosis of salpingitis and 23 other patients diagnosed with other gynecologic problems, including pelvic pain (n = 4), recurrent miscarriage (n = 4), unexplained infertility (n = 4), urinary tract infection (n = 3), recurrent vaginitis (n = 2), menorrhagia (n = 2), ovarian cyst (n = 2), and postpartum endometritis (n = 2). Controls were 29 reproductive-age women in apparently good health with no history of pelvic inflammatory disease or infection with
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Fig. 2. Interferon gamma-induced production of tumor necrosis factor. Periperhal blood mononuclear cells from women with salpingitis or other disorders or from healthy controls were incubated in the presence of 200 U / ml of interferon gamma. Afer 20 hours, culture supernatants were collected and assayed for tumor necrosis factor. Broken horizontal line, Value 2 SDs above mean for healthy controls.
C. trachoma tis or N. gonon-hoeae. Salpingitis was diagnosed by laparoscopic surgery in 18 of the women who had visual evidence of tubal damage caused by an inflammatory process. Microbial cultures were not obtained during surgery. A differential diagnosis of salpingitis was made in two women who did not undergo laparoscopy, on the basis of the presence of adnexal tenderness , cervical motion tenderness, and identification of C. trachomatis in endocervical cultures. The four women with pelvic pain had no evidence of salpingitis during laparoscopy. The other patients did not undergo surgery. Peripheral blood mononuclear cell incubation. Mononuclear cells were isolated from heparinized peripheral blood by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) gradient centrifugation and added to multiple wells of a microtiter plate (2 X 10" cells per well) containing 10% heat-inactivated autologous serum in RPMI 1640 medium with 25 mmol HEPES buffer, Lglutamine, penicillin (100 UI ml), streptomycin (l00 pg/ml), and kanamycin (100 U Iml). Interferon gamma (Genzyme, Boston) was added to triplicate wells at a concentration of 200 unitslml. After incubation for 20
Monokine induction in salpingitis
Volume 165 Number 3
721
Table I. Production of interleukin-l and interleukin-6 by unstimulated peripheral blood mononuclear cells Salpingitis Subject
1 2 3 4 5 6 7 8 9 10 11 12
I nterleukin-l (pglml)
I
149 174
Other disorders
Interleukin-6 (nglml)
I nterleukin-l (pglml)
1.8 3.0
115 76 78 101 74 89 122 82 85
TO
T3 136 169 140 134 104 379
2.0 2.7 2.0 3.0
f7 6.3
I
Controls
I nterleukin-6 (nglml)
I nterleukin-l (pglml)
1.1 1.2 1.0 1.8 1.6 1.0 2.4 1.1 1.2
73 110 167 109 73 109 89 74 80 85 89 94
I
I nterleukin-6 (nglml)
1.9 1.6 2.7
l.8
1.6 1.6 1.0 1.5 1.3 1.3 1.1 ND
Peripheral blood mononuclear cells were incubated for 20 hours. Culture supernatants were collected and assayed for interleukin-l or interleukin-6. Underlined values are >2 SDs above mean values for control group. ND, Not determined.
hours at 37° C in a 5% carbon dioxide incubator, culture supernatants were collected, particulate components were removed by microcentrifugation, and the supernatants were stored at -70 C until used. Monokine assays. Culture supernatants were assayed for tumor necrosis factor-a (Cistron, Pine Brook, N.J.), interleukin-l (Cistron), and interleukin-6 (Genzyme) by enzyme-linked immunosorbent assays with commercially available kits. All samples were tested in triplicate and the mean value was determined. Replicates differed by <15%. Values were converted to picograms per milliliter by reference to a standard curve obtained by the simultaneous analysis of known concentrations of purified compounds in culture medium. The lower limit of sensitivity was 20 pg I ml for tumor necrosis factor and interleukin-l and 0.2 ng/ml for interleukin-6. Statistics. Differences in the numbers of women in each of the subject groups with elevated tumor necrosis factor production under the experimental conditions were evaluated by the X2 test, with the Yates correction factor. 0
Results
Spontaneous tumor necrosis factor production by peripheral blood mononuclear cells. The levels of tumor necrosis factor produced by unstimulated mononuclear cells from women with salpingitis, women with other disorders, and healthy controls are shown in Fig. 1. Only low levels of tumor necrosis factor were detected in the culture fluids of unstimulated cells from healthy women. A similar range of concentrations were detected in culture supernatants of mononuclear cells from women with disorders other than salpingitis. In marked contrast, mononuclear cells from seven of the
20 women with salpingitis spontaneously released tumor necrosis factor at levels >2 SDs above the control mean value. The difference in the number of women with elevated tumor necrosis factor production in the salpingitis and control groups was significant (P < 0.01). Tumor necrosis factor production by peripheral blood mononuclear cells incubated with interferon gamma. In the presence of interferon gamma, mononuclear cells from only one of 23 patients with a diagnosis of a disorder other than salpingitis and one of 29 healthy controls produced tumor necrosis factor at a level >2 SDs above the control mean value. In marked contrast, interferon gamma-stimulated cells from 11 of 20 women with salpingitis produced tumor necrosis factor at elevated levels (Fig. 2). The number of women in the salpingitis group releasing elevated levels of tumor necrosis factor under these conditions was significantly greater than the number of women in the other groups (p < 0.01). Interleukin-l and interleukin-6 production by peripheral blood mononuclear cells from women with salpingitis. Mononuclear cells from nine patients diagnosed as having salpingitis, nine patients with other diseases, and 12 healthy controls also were tested for their ability to release two other monocyte products, interleukin-l and interleukin-6. The upper limit of normal was designated as a value 2 SDs above the mean level of each mono kine produced by peripheral blood mononuclear cells from the healthy controls; the number of patients whose unstimulated cells produced elevated levels of these two monokines was evaluated (Table I). None of the nine patients without salpingitis produced an elevated level of interleukin-l or interleukin-6. In contrast, peripheral blood mononuclear
722
Witkin et al.
September 1991 Am J Obstet Gynecol
Table II. Production of interleukin-l and interleukin-6 by interferon-stimulated peripheral blood mononuclear cells Other disorders
Salpingitis Subject
1 2 3 4 5 6 7 8 9 10 11 12
I nterleukin-l (pglml)
139 163 106 1217 198 159 206 151 491
I
Interleukin-6 (nglml)
Interleukin-l (pglml)
2.1 2.2 1.0 6.5 2.3 2.0 2.2 1.9 3.7
126 109 87 134 88 102 147 118 91
I
Controls
Interleukin-6 (nglml)
I nterleukin-l (pglml)
1.1
115 12 165 148 143 121 108 91 80 94 105 114
2.1 1.1 1.8 1.7 1.0 1.9 1.2 1.1
I
Interleukin-6 (nglml)
1.9 1.7 2.2 1.5 1.6 1.9 1.1 1.5 1.3 1.5 1.9 ND
Peripheral blood mononuclear cells were incubated for 20 hours in the presence of 200 U I ml of interferon gamma. Culture supernatants were collected and assayed for interleukin-l or interleukin-6. Underlined values are >2 SDs above mean values for control group. ND, Not determined.
cells from three of the patients with salpingitis spontaneously produced elevated amounts of interleukin-l, and four produced elevated amounts of interleukin-6. Similar results were obtained with interferon gammastimulated cells (Table II). Four of the patients with salpingitis showed elevated interleukin-l levels and three had elevated interleukin-6 production but none of the other women did so. In contrast to the in vitro culture results, none of the sera from patients with salpingitis contained detectable tumor necrosis factor, interleukin-l, or interleukin-6. Comment
Peripheral blood mononuclear cells from women with salpingitis readily released products of activated monocytes in vitro under conditions where the corresponding cells from women with other gynecologic disorders or from healthy women remained relatively quiescent. Under our experimental conditions, adherence of the monocytes to the culture dishes could have been a sufficient stimulus to activate the primed monocytes to release monokines. II In contrast, none of the patient's sera contained detectable levels of tumor necrosis factor, interleukin-l, or interleukin-6. This is consistent with reports demonstrating that tumor necrosis factor has a short half-life and is detectable in the circulation only for a short period of time at the onset of an infection. I2 Thus mono kine production in vitro by unstimulated or interferon gamma-stimulated monocytes may be a more reliable indicator of infection than the presence of these compounds in serum. Primed monocytes present in the circulation of women with salpingitis would be highly susceptible to activation in vivo at infected tubal sites, even at loci
contammg low concentrations of microorganisms. Primed monocytes can be activated by levels of bacteria that are insufficient to stimulate unprimed (resting) monocytes. IO In addition, in cases of chlamydial salpingitis, nonproductive infections also may lead to the release of chlamydial antigens capable of activating monocytes. 6 Tumor necrosis factor has, in fact, been shown to induce the disordered synthesis of chlamydial antigens. I3 The resulting accumulation of monokines at specific sites would induce scarring and tissue damage. Furthermore, since tumor necrosis factor synergizes with bacterial endotoxin in the induction of tissue necrosis,I4 tumor necrosis factor production is most likely to initiate inflammatory tissue damage if a concomitant infection is also present. C. trachomatis infections, even those causing severe tubal damage, are often accompanied by mild or no symptoms I5 and may therefore remain undetected. In addition, the standard antibiotic treatments may not always completely clear the organism from the female genital tract.I6 Therefore low-level infections, even by micrroorganisms not normally considered to be pathogenic, may become capable of inducing monokine secretion from monocytes that were primed by "silent" chlamydial tubal infections. This might especially be a problem during pregnancy. Many studies have now shown that products of activated monocytes can induce preterm pregnant complications. I7 The reported increased incidence of first-trimester spontaneous abortion,IS preterm labor, or premature rupture of membranes I9. 2o in women with serologic or microbiologic evidence of C. trachomatis also may occur, at least partially, by this mechanism. Salpingitis is certainly not the only infectious condition in which primed monocytes are present in the
Volume 165 Number 3
circulation. Monocytes from patients with pulmonary tuberculosis· l or meningococcal meningitis 22 also were shown to be more responsive than monocytes from controls in releasing tumor necrosis factor. Acute localized infections at other body sites would be expected to produce similar findings. However, we have not observed primed monocytes in women with vaginal or urinary tract infections. This suggests that infections confined to mucosal surfaces may not prime circulating monocytes. The presence of a chlamydial infection is often difficult to detect clinically or by culture techniques. 2 .• 3 The methods described in this report may therefore be of value in the evaluation of women with suspected salpingitis. REFERENCES 1. Holmes KK, Eschenbach DA, Knapp ]S. Salpingitis: overview of etiology and epidemiology. AM] OBSTET GYNECOL 1980; 138:893-900. 2. Brunham RC, Maclean IW, Binns B, Peeling RW. Chlamydia trachomatis: its role in tubal infertility.] Infect Dis 1985; 152: 1275-82. 3. Hutchinson GR, Taylor-Robinson D, Dourmashkin RR. Growth and effect of chlamydiae in human and bovine oviduct organ cultures. Br] Venereol 1979;55: 194-202. 4. Gjonnaess H, Dalaker K, Anestad G, Mardh PA, Kvite G, Bergan T. Pelvic inflammatory disease: etiologic studies with emphasis on chlamydial infection. Obstet Gynecol 1984;59:550-55. 5. Patton DL, Wolner-Hanssen P, Cosgrove S], Holmes KK. The effects of Chlamydia trachomatis on the female reproductive tract of the Macaca nemestrina after a single tubal challenge and following repeated cervical inoculations. Obstet Gynecol 1990;76:643-50. 6. Morrison RP, Lyng K, Caldwell HD. Chlamydia disease pathogenesis. Ocular hypersensitivity elicited by a genusspecific 57kD protein.] Exp Med 1989;169:663-75. 7. Wagner EA, Schachter J, Bavoil P, Stephens RS. Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens. J Infect Dis 1990;162:922-7. 8. Grifo ]A, Jeremias], Ledger W], Witkin SS. Interferon'I in the diagnosis and pathogenesis of pelvic inflammatory disease. AM] OBSTET GYNECOL 1989;160;26-31. 9. Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL6 and related molecules (ILl and TNF). FASEB J 1990;4:2860-7.
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10. Adams DO, Hamilton TA. Phagocytic cells: cytotoxic activities of macrophages. In: Gallin]I, Goldstein 1M, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press, 1988:471. 11. Bodel PT, Nichols BA, Bainton DF. Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence. J Exp Med 1977; 145:264-74. 12. Michie HR, Manoque KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl] Med 1988;318:1481-6. 13. Shemer-Avni Y, Wallace LD, Sarov 1. Inhabition of Chlamydia trachomatis growth by recombinant tumor necrosis factor. Infect Immun 1988;56:2503-6. 14. Rothstein ]L, Schreiber H. Synergy between tumor necrosis factor and bacterial products causes hermorrhagic necrosis and lethal shock in normal mice. Proc Nat! Acad Sci USA 1988;85:607-11. 15. Henry-Suchet], Catalan F, Luffredo V, et al. Chlamydia trachomatis associated with chronic inflammation in abdominal specimens from women selected for tuboplasty. Ferti! Steril 1981 ;36:599-605. 16. Batteiger BE, Fraiz], Newhall W], Katz BP, Jones RB. Association of recurrent chlamydial infection with gonorrhea.] Infect Dis 1989;159:661-9. 17. Witkin SS, McGregor ]A. Infection-induced activation of cell-mediated immunity: possible mechanisms for preterm birth. Clin Obstet Gynecol 1991 ;34: 112-21. 18. Quinn PA, Petric M, Barkin M, et al. Prevalence of antibody to Chlamydia trachomatis in spontaneous abortion and infertility. AM] OBSTET GYNECOL 1987; 156:291-6. 19. Martin OH, Koutsky L, Eschenbach DA, et al. Prematurity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomatis infections. ]AMA 1982; 247:1585-8. 20. Gravett MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach D, Holmes KK. Independent associations of bacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. ]AMA 1986;256: 1899-903. 21. Takashima T, Ueta C, Tsuyuguchi I, Kishimoto S. Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis. Infect Immun 1990;58:3286-92. 22. Waage A, Halstensen A, Shalaby R, Brandtzarg P, Kierulf P, Espevik T. Local production of tumor necrosis factor, interleukin 1 and interleukin 6 in meningococcal meningitis. Relation to the inflammatory response.] Exp Med 1989; 170; 1859-67. 23. Schachter], Moncada J, Dawson CR, et al. Nonculture methods for diagnosing chlamydial infection in patients with trachoma: A clue to the pathogenesis of the disease? ] Infect Dis 1988;158:1347-52.