Inhibition of Intracellular Multiplication of Human Strains ofChlamydia trachomatisby Nitric Oxide

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

232, 595–601 (1997)

RC976335

Inhibition of Intracellular Multiplication of Human Strains of Chlamydia trachomatis by Nitric Oxide Joseph U. Igietseme,* Ijindah M. Uriri,* Marie Chow,† Etsuko Abe,† and Roger G. Rank† *Department of Microbiology and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310; and †University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Received January 22, 1997

It was previously shown that murine T cell clones could inhibit the intracellular growth of the mouse strain of Chlamydia trachomatis by cytokine-mediated induction of the inducible nitric oxide synthase (iNOS) system in epithelial cells, an effect enhanced by direct epithelial-T cell interaction via specific adhesion molecules. These findings and other recent reports showing that human mucosal epithelial cells secrete nitric oxide (NO) via iNOS expression would suggest that mucosal epithelial-derived NO may be involved in mucosal defense against Chlamydia and other pathogens that infect epithelial cells. As an initial approach to investigating whether NO contributes to chlamydial control in humans, the present studies evaluated the susceptibility of human isolates of C. trachomatis to NO delivered by chemical donors or via induction of the epithelial iNOS system by a cytokinesecreting T cell clone. It was found that a chlamydialspecific, cytokine-secreting, murine T lymphocyte clone (clone 2.14-0) could inhibit the intraepithelial growth of human strains of Chlamydia trachomatis (serovar E and H, and Lymphogranuloma venerum type L2) via the iNOS pathway when the clone was co-cultured with chlamydial-infected epithelial cells. Furthermore, treatment of infected epithelial cells with 50 mM of the NO donor, S-nitroso-L-glutathione, resulted in significant inhibition (approximately 70%) of chlamydial multiplication, while the NO scavenger, myoglobin plus ascorbate, could reverse the effect, demonstrating that NO could directly inhibit human strains of Chlamydia. The results are consistent with the hypothesis that the IFN-g-inducible iNOS pathway can contribute to chlamydial control in humans. q 1997 Academic Press

Abbreviations: iNOS, inducible nitric oxide synthase; NO, nitric oxide; MLA, NG-monomethyl-L-arginine monoacetate; GSNO, S-nitroso-L-glutathione; PELC, polarized epithelial-lymphocyte co-culture; GPIC, Chlamydia psittaci agent of guinea pig inclusion conjunctivitis; LGV, Lymphogranuloma venerum.

Genital chlamydial infection is common and in women the pathologic consequences, including pelvic inflammatory disease, ectopic pregnancy and infertility, have considerable psychological, public health and economic implications. The need for a reliable prophylactic vaccine as a preventive strategy (1) would require a thorough understanding of the pathogenesis and immunobiology of chlamydial disease, including the relevant host immune parameters that control Chlamydia, the mechanisms of chlamydial inhibition, and the antigens that elicit protective immunity. Experimental animal models have contributed to the present level of understanding of the immunobiology of genital chlamydial disease (2, 3). Studies in the murine model of chlamydial genital disease have shown that: (a) B cell-deficient mice could resolve the infection as efficiently as immunocompetent littermates (3); (b) productive reinfection could occur in the presence of relatively high titers of neutralizing serum antibodies (4, 5); and (c) immunodeficient (athymic) mice suffer persistent infection that can be cured by adoptive transfer of specific T cell lines and clones (6-8). These studies indicate that T cell-mediated immune (CMI) responses are sufficient for conferring protective chlamydial immunity in mice. The mouse model has therefore provided a unique opportunity to dissect and analyze the role of different effectors of CMI in chlamydial control, and to study in detail the molecular and biochemical mechanisms of chlamydial inhibition. In this respect, both in vitro and in vivo studies have revealed that T cell-derived IFN-g is crucial for chlamydial control in humans and in experimental animals (9-14). The biochemical basis of the anti-microbial action of IFN-g includes the activation of phagocytes (e.g., macrophages) to rapidly take up and degrade chlamydiae or infected cells; also, IFN-g can induce indoleamine 2,3-dioxygenase in phagocytes or non-phagocytic infected cells, an enzyme that catalyzes the decyclization of L-tryptophan into N-formylkynurenine (15-17), thereby limiting the availability of the essential

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amino acid, and consequent inhibition of chlamydial growth. In the murine system and in certain human cells, IFN-g induces iNOS, an enzyme that catalyzes the production of various anti-microbial reactive nitrogen intermediates, most notably nitric oxide (NO), from L-arginine (13, 18). By working in the murine model of genital chlamydial disease, we have generated a panel of MoPn-specific T cell lines and clones of both CD4 and CD8 subsets, that have been functionally and biologically analyzed for anti-chlamydial activity in vivo and in vitro, and for cytokine and activation characteristics (7, 8, 19, 20). The in vivo functional capability of established T cells was measured by the ability to cure chlamydial genital infection in mice upon adoptive transfer, thereby facilitating the establishment of several protective and nonprotective T cell lines and clones. Additional studies showed that in vivo protective T cell lines and clones could interact with infected epithelial cells to inhibit the intracellular growth of chlamydiae, via the iNOS system (19-21). Since the epithelial cell is the target of chlamydial infection and disease, the murine studies and recent reports by others (22-24) showing that various human mucosal epithelial cells secrete NO via expression of the iNOS, suggest that NO may be involved in local mucosal defense against pathogenic microbes that infect epithelial cells. However, it is unknown whether NO can inhibit the intraepithelial growth of human strains of C. trachomatis directly or under the conditions of epithelial-T cell interaction and iNOS induction. In the present study, we investigate the susceptibility of human isolates of C. trachomatis to NO either delivered by NO donors or induced via a IFN-gsecreting, chlamydial-specific murine T cell clone interacting with infected epithelial cells. Although the antimicrobial function of NO and other nitrogen intermediates associated with the iNOS pathway in humans is still being debated, these and other findings suggest that NO can contribute to the control of Chlamydia in humans, an important insight that could be considered when designing an intervention strategy. MATERIALS AND METHODS Animals Female //nu mice on BALB/c background (H-2d), 5-6 weeks old, were obtained from Harlan Sprague-Dawley, Inc., Indianapolis, IN, fed with food and water ad libitum, and maintained in Laminar flow racks under pathogen-free conditions of 12-hour light and 12-hour darkness.

Chlamydia Stocks and Antigens Chlamydial stocks for infecting epithelial monolayers were prepared in Hela cells, as previously described (4). Antigens of C. trachomatis agent of mouse pneumonitis (MoPn) used for T cell stimulation in culture were prepared by purification of elementary bodies (EBs)

from stocks with Renograffin gradients (4) and exposing the EBs to ultraviolet light for 3 hours.

Reagents The L-arginine analog and inhibitor of nitric oxide synthase, NGmonomethyl-L-arginine monoacetate (MLA) and the NO donor, Snitroso-L-glutathione, were purchased from CYCLO3PSS Biochem Corp., Inc., Salt Lake City, UT. Sodium salt of L-ascorbic acid (ascorbate) and myoglobin was obtained from Sigma Chemical Co., St. Louis, Mo.

Assessment of T Cell Inhibition of Chlamydia in the PELC System Polarized epithelial cell culture. The epithelial cell line employed for these studies, TM3 (ATCC CRL 1714), was derived from Leydig cells of BALB/c mice. TM3 cells have primary epithelial cell culture characteristics, non-tumorigenic and express receptors for estrogen, epidermal growth factor (EGF), androgen, and progesterone (25). TM3 cell line was maintained in culture with RPMI 1640 complete medium, composed of RPMI 1640 (Hazelton Research Products, Denver, PA) supplemented with 10 mM HEPES (GIBCO Labs., Grand Island, N.Y.), 10% heat-inactivated fetal bovine serum (GIBCO), 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM glutamine, 211005 M 2-mercaptoethanol (Sigma), 50 g/ml gentamycin (GIBCO). It has been established that chlamydial agents employed in these studies {MoPn, C. psittaci agent of guinea pig inclusion conjunctivitis (GPIC) and C. trachomatis serovars E and H} grow efficiently in TM3. To establish monolayers of polarized epithelial cell cultures that mimic the in vivo architecture of the mucosal epithelium, 21106 TM3 cells were seeded into the inner chamber of Costar’s multi-well Transwell plates (24mm diameter collagen-coated polycarbonate filter; pore size 0.45; Cat. #2435; Costar, Cambridge, Mass.), according to the method of Wyrick and co-workers (26). The polarized monolayer was attained after 24-48h of culture at 377C and 5% CO2 . Unless otherwise stated, infection of polarized epithelial monolayer was carried out with 106 inclusion-forming units (IFU) of the appropriate chlamydial agent in a volume of 0.25 ml of the growth medium. The outer chamber contained 2 ml of the growth medium. Infected epithelial cells were centrifuged at 20601g for 30 min. to facilitate infection, and centrifugation did not affect the polarization of the cells as determined by inverted microscopic observation. T cell clone 2.14-0. The isolation procedure and properties of clone 2.14-0 employed in these studies have been described (7, 8). Briefly, clone 2.14-0 is an MoPn-specific, CD4/, Th1 cell that was generated by limiting dilution technique from splenic cells of MoPn-infected mice. The clone is antigen-specific, MHC-restricted and biovar-specific. Antigen-stimulated 2.14-0 secretes IL-2, high levels of IFN-g and TNF-a. In addition, the clone is protective because it could cure mice of genital MoPn infection when adoptively transferred into infected syngeneic mice. Furthermore, clone 2.14-0 could interact with infected epithelial cells and inhibit the intracellular growth of MoPn through activation of the iNOS system (19-21, 27). Epithelial-T cell cultures. Exposure of infected epithelial cells to clone 2.14-0 cells was carried out by transferring 51106 T cells into the Transwell inserts containing infected epithelial cells (i.e., coculturing). It has been established that co-culturing epithelial cells and chlamydial-specific T cells leads to increased inhibition of intracellular growth of Chlamydia due to adhesion molecule-mediated enhancement of nitric oxide induction (20, 21). The various treatments of clone 2.14-0, including stimulation with antigen and antigen presenting cells (APCs), resting in culture medium alone, or treatment of cultures with NO donors, MLA and other reagents, have been previously described (7, 19, 20). At the end of a 48 h incubation period, the supernatants were collected from the inner and outer wells of the double-chambered cultures, placed in microvials and

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preserved at 0707C, for further cytokine and NO analyses. The productive growth of chlamydiae in polarized epithelial cells in the presence or absence of T cells or reagents was determined by isolation of MoPn from the filter samples in McCoy cells and detection of chlamydial inclusions by microimmunofluorescence assay (19, 20). Each set of isolation experiments was repeated at least 3 times in order to obtain a quantifiable and consistent pattern of results. Cultures for chlamydial isolation were established in triplicates and the results were expressed as the mean ({ S.D) of infectious forming units per milliliter (IFU/ml) values. The percentage inhibition was computed as follows: Mean IFU/ml control cultures 0 Mean IFU/ml of experimental cultures (/T cells) . Mean IFU/ml of control cultures

Gamma Interferon Assay The amounts of IFN-g elaborated by antigen-stimulated T cells in PELC cultures were quantitated with specific enzyme-linked immunosorbent assay (ELISA) kit (Cytoscreen Immunoassay Kit; BioSource Intl, Camarillo, CA). Assays were carried out on supernatants from cultures according to the supplier’s instruction and the amount of the cytokine in each sample was obtained by extrapolation on a standard calibration curve generated simultaneously. Data were calculated as the mean values ({ S.D) of quadruplicate cultures for each experiment. The results were derived from at least 3 independent experiments.

Measurement of Nitric Oxide Production The amounts of nitric oxide induced were quantitated in the supernatants collected from PELC cultures (as nitrite) by the Greiss reagent method (28). Briefly, equal volumes of 2-fold diluted supernatants and the Greiss reagent (1% sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/2.5% H3PO4) (50 mL each) were mixed in 96-well plates and the absorbance associated with color change was measured within 15 minutes at 550-nm wavelength in a Microplate Autoreader spectrophotometer (Bio-tex Instruments, Inc., Winooski, VT). Results represent the mean of quadruplicate wells for each set of samples obtained from different experiments.

Statistical Analysis The degree of inhibition of Chlamydia by T cell clone under different conditions was compared by performing a one-tailed t test and the relationship between different experimental groupings was assessed by analysis of variance (ANOVA) Minimal statistical significance was judged at P õ 0.05.

RESULTS Ability of MoPn-Specific Clone to Inhibit Human Serovars of C. trachomatis in the PELC System These studies extend: (a) previous reports revealing that the cytokine-producing clone 2.14-0 could interact with infected murine epithelial cells to inhibit the intracellular multiplication of the murine biovar of Chlamydia trachomatis (MoPn), by a mechanism involving IFN-g-mediated NO induction from infected epithelial cells (19-21); and (b) other recent reports that human mucosal epithelial cells express the iNOS enzyme with elevated NO production, suggesting a possible role of epithelial-derived NO in the defense against muco-

FIG. 1. Ability of MoPn-specific clone to inhibit human serovars of C. trachomatis in the PELC system. Duplicate cultures of chlamydial-infected polarized epithelial cells were co-cultures with 51106 cells of clone 2.14-0. The growth of each chlamydial agent was determined by tissue culture isolation from the excised filters by microimmunofluorescence method as previously described (19). Results represent the mean ({ S.E.M) of at least 3 separate experiments, calculated as percentage inhibition as given in the Materials and Methods section.

sally-acquired pathogens that colonize epithelial cells (22-24). Since the epithelial cell is the primary target of chlamydial infection and disease, we explored the possible biologic relevance of epithelial-derived NO in chlamydial control in the human genital epithelium, by investigating whether NO induction by clone 2.140 or from NO donors could inhibit the intracellular growth of human serovars of C. trachomatis. In initial studies, polarized epithelial cells were infected with MoPn, GPIC, or C. trachomatis serovars E or H, and co-cultured with clone 2.14-0 for 48h to assess the effect on the productive intracellular growth of chlamydiae, as previously described (19). Figure 1 shows that clone 2.14-0 could inhibit the intracellular multiplication of all strains of Chlamydia to approximately the same degree, when compared to cultures without T cells (P õ 0.0001). Since it was previously shown that the intracellular inhibition of MoPn by clone 2.14-0 correlated with the presence of relatively elevated levels of nitric oxide induction (20, 21), the nitrite levels in culture supernatants were also measured by the Greiss reagent method (28). Culture supernatant nitrite levels in the presence of T cells were

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42 { 3.4 mM (MoPn), 41.5 { 1.9 mM (Type E), 45.8 { 1.7 mM (Type H), 54.8 { 2.8 mM (LGV) and 41.3 { 4.1 mM (GPIC), as compared to 2.0 { 0.4, 1.2 { 0.5, 1.4 { 0.5, 5.0 { 0.7 and 1.3 { 0.5 mM in the absence of T cells, respectively. As previously reported (20), elevated levels of IFN-g were measured in co-cultures containing infected epithelial cells and clone 2.1-0 (data not shown), and the ability of anti-IFN-g antibodies to partially suppress the anti-chlamydial action of the clone (20) suggested that IFN-g was important for antimicrobial action. The results suggested that the murine T cell clone could inhibit both human and animal strains of Chlamydia, possibly by identical mechanism involving the induction of nitric oxide. Role of the Epithelial Inducible Nitric Oxide Synthase System in Chlamydial Inhibition It was previously established that clone 2.14-0 inhibited the growth of MoPn by cytokine- and adhesion molecule-mediated activation of epithelial iNOS system, a pathway sensitive to the L-arginine analog and specific inhibitor of the iNOS pathway, MLA (13, 20). To determine whether clone 2.14-0 inhibited the growth of human isolates of C. trachomatis by activating the epithelial iNOS system, the effect of MLA on chlamydial inhibition was investigated. Figure 2 shows that the ability of clone 2.14-0 to inhibit murine (MoPn), guinea pig (GPIC) and human (serovars E, H and LGV) strains of Chlamydia was drastically reduced by 70-90% in the presence of 5 mM MLA (P õ 0.00001 for each agent). Also, the presence of MLA was associated with at least 5-fold reduction in nitric oxide induction (P õ 0.00001 for each agent), as measured by culture nitrite (Figure 2), suggesting that chlamydial inhibition is directly related to nitric oxide production. As previously reported for MoPn (20), MLA had no effect on the growth of human strains of Chlamydia in epithelial cells in the absence of T cells (data not shown). The results indicated that human strains of Chlamydia are susceptible to the antimicrobial action of epithelial-derived nitric oxide. Therefore, if iNOS expression is a general property of mucosal epithelial cells, as recently reported for human lung, colonic and ocular epithelial cells (2224), NO production by genital mucosal epithelial cells may be involved in controlling chlamydiae following colonization. However, the foregoing results do not eliminate the possibility that other cellular substances or products may be involved in chlamydial inhibition, and so it was important to demonstrate that NO could directly inhibit Chlamydia in these studies. Direct Anti-chlamydial Action of Nitric Oxide To investigate the direct anti-chlamydial action of NO, infected epithelial cells were exposed to NO-

FIG. 2. Role of the epithelial inducible nitric oxide synthase system in chlamydial inhibition. Duplicate cultures of chlamydial-infected polarized epithelial cells were co-cultures with 51106 cells of clone 2.14-0, in the presence or absence of 5.0 mM MLA, as previously described (20). After 48h of incubation at 377 C the supernatants were assayed for NO by the Greiss reagent method (28) and the growth of each chlamydial agent was determined by tissue culture isolation from the excised filters by microimmunofluorescence method as previously described (19). Results represent the mean ({ S.E.M) of at least 3 separate experiments, calculated as percentage inhibition as given in the Materials and Methods section. The amounts of NO in supernatants were obtained from a standard calibration curve using NaNO2 in the range of 250 - 0.0 M, as described in the Materials and Methods section.

generating systems or NO donors and the effect on chlamydial growth was assessed. The NO donor employed in these studies, S-nitroso L-glutathione (GSNO), is a relatively stable nitrosothiol, which has been successfully used in vitro and in vivo as a reliable donor of NO with pharmacologic and physiologic activities (29). GSNO slowly releases NO spontaneously in aqueous solution and the release is enhanced by ascorbate and thiols, such as glutathione (29). Infected epithelial cells were treated with a range of concentrations of GSNO (5.0 - 50.0 M), incubated at 377C for 48h and the productive growth of chlamydiae was assessed by tissue culture isolation and microimmunofluorescence method, as previously described (19). Figure 3 reveals that the presence of 50 M GSNO could partially but significantly inhibit the multiplication of MoPn, C. trachomatis serovar H and LGV in epithelial cells by approximately 70%, as compared to either the presence of 5.0 M GSNO or the absence of the donor (P õ 0.00001 for each agent). Although the molar concentrations of GSNO used in culture were comparable to those of NO frequently detected in co-cultures containing

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show that NO possesses anti-chlamydial activity, which may suggest that NO production by chlamydialinfected epithelial cells could inhibit the multiplication of chlamydiae and control the disease. DISCUSSION

FIG. 3. Direct anti-chlamydial action of nitric oxide. Duplicate cultures of chlamydial-infected polarized epithelial cells were either co-cultured with clone 2.14-0 or treated with GSNO at the indicated concentrations, within 30 minutes of the infection. After 48h of incubation at 377 C the supernatants were assayed for NO by the Greiss reagent method (28) and the growth of each chlamydial agent was determined by tissue culture isolation from the excised filters by microimmunofluorescence method as previously described (19). Results represent the mean ({ S.E.M) of at least 2 separate experiments, calculated as percentage inhibition as given in the Materials and Methods section. The amounts of NO in supernatants were obtained from a standard calibration curve using NaNO2 in the range of 250 - 0.0 M, as described in the Materials and Methods section.

An understanding of the various cellular, molecular and biochemical mechanisms that are functionally available to the host for controling intraepithelial growth of chlamydiae will enhance present efforts to design a reliable anti-chlamydial prophylaxis or possibly a vaccine. T cell-mediated immunity is important for chlamydial control in humans and in experimental animal models, and T cell-derived cytokines, notably IFN-g, play a major role in microbial control by a combination of inflammatory, regulatory and effector mechanisms. The biochemical mechanisms of anti-microbial action of IFN-g include tryptophan (15-17) and iron (31) deprivation, induction of reactive oxygen species and the activation of the iNOS system leading to the production of elevated amounts of nitric oxide (30, 32). Nitric oxide has been shown to exert profound cytotoxic effects on intracellular pathogens, such as Mycobacteria (33), Leishmania, Schistosoma, Plasmodium, Cryptococcus, Toxoplasma, Rickettsia (34-36), and Chlamydia (13, 20). The action of NO is on critical

TABLE 1

An NO Scavenging System Can Reverse the AntiChlamydial Action of S-Nitroso-L-glutathione Culturea

infected epithelial cells and clone 2.14-0, the level of chlamydial inhibition by the NO donor was less than that obtained with the clone, which was over 90% (Figure 3). This may be due to the rate of release of NO by GSNO in culture or presently unknown biologic features of epithelial-derived nitric oxide. The observed effect of GSNO supported the hypothesis that following an infection, T cell-induced NO from epithelial cells could control the intracellular growth of Chlamydia. Additional studies were carried out to extend the results presented in Figure 3, that is, to confirm that NO released by GSNO was responsible for chlamydial inhibition observed. The ability of an NO-scavenging system, composed of 5.0 mg/ml myoglobin plus 1 mM ascorbate (a reducing agent) (30) to reverse the antichlamydial action of GSNO was tested. Table 1 reveals that the myoglobin scavenging system could reverse the anti-chlamydial action of GSNO. Myoglobin plus ascorbate alone had no significant effect on the growth of MoPn, C. trachomatis serovar H or LGV. The results

MoPn-infected TM3 (MT) only MT / GNSOc MT / GSNO / myoglobin / ascorbated MT / Myoglobin / ascorbate Serovar H-infected TM3 (SHT) only SHT / GNSO SHT / GSNO / myoglobin / ascorbate SHT / Myoglobin / ascorbate LGV-infected TM3 (LT) only LT / GNSO LT / GSNO / myoglobin / ascorbate LT / Myoglobin / ascorbate

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Nitrite (M) ({ S.E.M)b

0.00 74.19

4.44 (0.22) 25.38 (1.46)

6.69 3.06

7.50 (0.85) 11.68 (10.48)

0.00 72.06

3.30 (0.38) 34.00 (1.24)

8.24 2.51 0.00 72.15

9.00 0.89 6.25 39.63

6.58 4.21

(0.63) (0.11) (0.41) (0.91)

6.38 (0.53) 3.63 (0.32)

a PELC cultures, assessment of chlamydial growth in epithelial monolayers, and measurement of nitrite levels in supernatants were carried out as described in the Materials and Methods section (19– 21). b Standard error of mean. Results represent the mean of 3 independent experiments. c GSNO was used at 50.0 mM in culture. d Myoglobin/ascorbate NO scavenging system was used at 5 mg/ ml myoglobin plus 1 mM ascorbate.

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enzymes involved in energy and DNA syntheses (3032). Although the anti-microbial action of NO in humans is still actively debated (37), it appears that since the initial report of the anti-microbial action of NO in human cells (33), increasing reports are showing that various human cell types do secrete NO either actively or in response to diverse stimuli (22-24, 38, 39), and the production could be directly associated with antimicrobial function of the cells (34-36, 40). More so, the finding that human mucosal epithelial cells actively express the iNOS enzyme, in response to cytokines and bacterial components, and secrete elevated amounts of NO (22, 23, 39), suggests that NO may be involved in local mucosal defense against mucosally-acquired microbial pathogens that colonize the epithelium. In the present study, we tested the hypothesis that the epithelial iNOS system could contribute to the control of chlamydial disease in humans, by investigating whether human strains of C. trachomatis are susceptible to the anti-microbial action of NO delivered by NOgenerating systems or induced via the epithelial iNOS system. The background for the hypothesis investigated include the finding that human mucosal epithelial cells produce NO via the expression of the iNOS system, and our recent reports that murine epithelial cells could be stimulated by IFN-g to secrete NO and inhibit the intracellular growth of Chlamydia. These studies do not suggest that NO is the only anti-chlamydial effector in epithelial cells but that it may contribute to the protective mechanisms induced by cytokines, including tryptophan and iron deprivation, phagocyte activation and humoral immune responses. The results indicated that both epithelial-derived or NO released by chemical donors could inhibit the intracellular growth of both animal and human strains of C. trachomatis. It is possible that an active epithelial iNOS function is a mucosal phenomenon, where NO secreted has a role in host defense against mucosallyacquired pathogens and inflammation and under pathophysiologic conditions. The biologic and evolution significance of the epithelial iNOS system, the activation of which could control the intracellular growth of human strains of C. trachomatis, cannot be over-emphasized since the columnar epithelial cells of the cervix are the principal target cells of chlamydial infection. A role for the epithelial iNOS defense system in controlling mucosally-acquired pathogen that colonize the epithelium is an obvious biologic advantage of maintaining the system. The demonstration that NO from a chemical donor (GSNO) could significantly inhibit the intraepithelial growth of chlamydiae, and the reversal of the effect by an NO scavenger composed of myoglobin and ascorbate, indicated that NO may in deed play a role in controling the multiplication of human isolates of Chlamydia following epithelial colonization and immune initiation. Furthermore, it is pertinent to acknowledge that chlamydial inhibition by both

epithelial-derived NO and NO donor correlated with the detection of elevated amounts of nitrite in culture. In this respect, it has been established that a certain threshold level of NO secretion is required for antimicrobial activity (35, 40), and this has implication for the potential clinical use of NO in the management of infectious disease. However, there is the potential for NO to be a major pathophysiologic mediator of the immunopathogenesis of chlamydial disease, because of the association of excessive NO production with the pathogenesis of septic and inflammatory responses, including autoimmune diseases, diabetes and myocardial necrosis (32, 41). Although presently unknown, an understanding of the molecular target of NO that culminates in chlamydial inhibition will be useful for designing intervention strategies to control chlamydial disease, if the antimicrobial function of NO is established in humans. Future studies will investigate the biochemical mechanism and molecular target(s) of nitric oxide-mediated inhibition of intraepithelial multiplication of Chlamydia, as may be related to enzyme inactivation and functional impairment. ACKNOWLEDGMENTS This study was supported by grants from the Arkansas Science and Technology Authority and by PHS Grants AI41231 and AI26328 from the National Institutes of Health. We thank Dr. Harlan Caldwell for his excellent suggestions.

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