TNF and PGE2 in human monocyte-derived macrophages infected with Chlamydia trachomatis

In this study levels of prostaglandin E2 (PGE2), tumour necrosis factor (TNF) and interleukin-1 (IL-1) alpha in medium from monocyte derived macrophages (MdM) infected with Chlamydia trachomatis (L2/434/Bu or K biovars). TNF and PGE2 were found in both cases while IL-1 alpha was not detected. Both TNF and PGE2 levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE2 levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE2. Inhibition of PGE2 production by indomethacin resulted in increased production of TNF, while addition of PGE2 caused partial inhibition of TNF production from infected MdM.


Introduction
Different biovars of Chlamydia trachomatis, an obligate intracellular Gram-negative bacterium, have been associated with clinically distinct infections ranging from hyperendemic trachoma (serovars A, B, and C) to sexually transmitted infections and pneumonia (serovars D to K). Lymphogranuloma venereum is a sexually transmitted disease caused by C. trachomatis serovars El, L2 and L3, which are more invasive than biovars D to K. Lymphogranuloma venereum causes a systemic infection characterized primarily by gross lymphadenopathy, suppurative adenitis, and ulcerative genital tract and rectal diseases.
The mononuclear phagocytes, including both the tissue macrophages and their precursors, the circulating blood monocytes, act as effective microbicidal host defence cells against many pathogenic microorganisms. They have been implicated in regulating the functions of lymphoid and haematopoietic cells, and in most cases, these effects are mediated by soluble factors produced by circulating monocytes and tissue macrophages. 2 Endotoxin and lipopolysaccharide of the outer membrane of Gram-negative bacteria have been found to potently stimulate human monocytes to produce several substances with important biological activities, 3 including interleukin 1 (IL-1), 4,s tumour necrosis factor (TNF) 6'7 and prostaglandin E 2 (PGE2). 8 These factors induce a multitude of biological responses of importance in homeostasis, in host defence mechanisms, and, probably, in the pathogenesis of several diseases. 9 from the kinetic studies was subjected to RIA for PGE 2 analysis as described previously. 13 IL-1 alpha concentrations were determined by ELISA (Endogen Inc., Boston, MA, USA). The effect of dexamethasone and indomethacin on PGE2 production observed in MdM infected with the K biovar was much more pronounced than that observed in MdM infected with the L 2 biovar. This phenomenon was caused by the reduced ability of the L 2 biovar to stimulate PGE production in MdM compared to the K biovar. Fig. 2 3) and then declined. Daily replacement of the medium from the infected cells with fresh medium resulted in higher levels of TNF (data not shown).

Results
Infection at a higher M.O.I. of both L 2 and K strains resulted in higher TNF production (data not shown).
Addition of dexamethasone (10-6M) to the Chlamydia infected MdM resulted in inhibition of TNF production, while addition of indomethacin (10 -6 M) resulted in an increase of TNF production. Addition of PGE2 (10 -6 M) to the Chlamydia infected MdM resulted in partial inhibition of TNF production. The effect of dexamethasone and indomethacin on TNF production observed in MdM infected with the K biovar was much more pronounced than that observed in MdM infected with the t 2 biovar. Fig. 4

Discussion
Various biovars of C. trachomatis differ in their pathogenicity, but the mechanism is still obscure. One possible explanation might be the ability of the pathogen to turn on host defence mechanisms. The mechanisms of the host defence against diseases caused by chlamydial species are not clearly understood, but both humoral and cell mediated immunity are involved. TM Infection of macrophages by intracellular parasites might modulate production of TNF, PGE2 and IL-1 alpha, which, in turn, might have a profound effect on the outcome of the infection in vivo.
The present study shows clearly that L 2 and K biovars induced production of TNF from human MdM (Figs 1 and 3). This production increased when a higher multiplicity of infection of Chlamydia was used. Higher levels were observed in the case of K strain infected MdM. This might explain the inability of K strain to grow in MdM, as described by us (Schmitz, Manor, Sarov [Abstract, Germany], and Yong) as well as the difference in the severity of the outcome of the infection in vivo between the t 2 and K biovars--L, causing a more generalized infection than K. Recently, Williams et al. is showed that spleen cells from C. trachomatis, pneumonitis agent infected nu/+ and nu/nu mice produced TNF-. They suggested that TNFmight play a role in host defence in the murine model. It has been shown that TNFinhibits chlamydial growth in HEp-2 cells. 16 This suggests that in vivo, early in the chlamydial infection, TNF may play a protective role. However, it is also likely that TNF might cause some of the pathological effects seen in the course of chlamydial infection. The capability of Chlamydiae to induce TNF production from macrophages is not unique to chlamydiae, but has been demonstrated recently in a wide range of intracellular pathogens such as viruses, 7 bacteria, -2 eukaryotic parasites, 2 and fungi. 22 Various molecules have also been found to be able to induce TNF in macrophages, such as bacterial lipopolysaccharides and endotoxin. 23 Further studies are required to characterize the chlamydial component responsible for the induction of TNFin human macrophages. This study shows that L 2 and K also induced human MdM to produce PGE2 (Fig. 1) Dexamethasone inhibited TNF and PGE2 production (Figs 2 and 4). These results are in agreement with those described by Beutler et al. 3 and by Danon et al., 3 respectively who have shown that dexamethasone inhibits TNF and PGE2 production at the transcriptional level.
Only L 2 was found to replicate in MdM. 12 Treatment of the cells with dexamethasone enhanced the yield of infectious chlamydial particles in these cells. These results cannot be simply explained by the dexamethasone inhibition of TNF production from chlamydial infected MdM. This conclusion is based on the findings that addition of an excess of PGE2, which inhibits TNF production, or indomethacin, which enhances TNF production, did not affect the chlamydial yield in MdM. The mechanism by which dexamethasone enhances chlamydial yield in MdM needs further investigation. Corticosteroids have been found to enhance replication of viruses, 32 and certain intracellular parasites. 3-3s In contrast to PGE2 and TNF-, no IL-1 was detected in the media from MdM infected with either K or L 2 biovars. These results differ from those reported by Rothermel et al., 36 who showed that C. trachomatis induced production of IL-1 by human monocytes. This difference might be due to the difference in the M.O.I. used by Rothermel as compared to the M.O.I. in our system (1)(2) or to the differentiation state of the cells used by Rothermel (monocytes) compared to ours (MdM).
Roux Lombard et a/. 37 showed that blood monocytes cultured for several weeks, produced much less IL-1 than freshly isolated monocytes. Furthermore, they showed that monocytes cultured for a few weeks produced a specific IL-1 inhibitor.
The level of TNF detected in the medium of the t 2 or K strains infected MdM reached a maximum at 24 h post-infection and then declined (Fig. 2).
When the infected MdM were washed daily, the TNF level remained high throughout the entire experimental period (data not shown). A possible explanation is that the high level of PGE2 depressed TNF production, and that washing the cells eliminated the interference of PGE 2. This explanation is supported by Kunkel et al.8 who showed that PGE2 regulates macrophage derived TNF gene expression. These observations support the suggestion that TNF and PGE. may affect each other's production. 9'3 TNF produced by activated macrophages may be responsible for increased synthesis of PGE2, which, in turn, limits macrophage activation in an autoregulatory manner. 24'25 A delicate balance between TNF and PGE2 produced by macrophages might play a major role in the outcome and severity of chlamydial infection in vivo. Animal model studies are required to examine the possible therapeutic effect of prostaglandin inhibitors and antibodies to TNF on the outcome of chlamydial infections.