Clinical Consequences of Immune Responses to Chlamydia in Men

halamydia trachomatis (C.t.) has been involved in variety of pathological conditions ofthe female and male genital tracts. Chronic female persistent infections due to this micro-organism have been recently investigated, but the role of C.t. has not been completely clarified especially in acute and chronic pathologies of the male genital tract. The immunocompetency of the female and male genital tract is well documentedl,Z; the production of secretory IgA (SIgA) can take place in the fallopian tubes, uterine cervix and vagina, prostate and the epididymis, resulting in local production of SIgA by submucosal plasma cells present in a quantity greater than plasma cells producing IgM and IgG. During infections, increased numbers of these three classes of plasma cells are present. Specific SIgA against various infectious agents, such as Escherichia coli and Chlamydia trachomatis,4’5’6 are produced in pathological conditions of the genital tract. SIgA mediates the protection of mucosal membrane by interfering with microbial adherence to mucosal surfaces and by inhibiting the penetration of potentially harmful micro-organisms into mucosal tissues. IgA occurs in serum and secretions as

genital tract is well documentedl,Z; the production of secretory IgA (SIgA) can take place in the fallopian tubes, uterine cervix and vagina, prostate and the epididymis, resulting in local production of SIgA by submucosal plasma cells present in a quantity greater than plasma cells producing IgM and IgG.
During infections, increased numbers of these three classes of plasma cells are present. Specific SIgA against various infectious agents, such as Escherichia coli and Chlamydia trachomatis, 4'5'6 are produced in pathological conditions of the genital tract.
SIgA mediates the protection of mucosal membrane by interfering with microbial adherence to mucosal surfaces and by inhibiting the penetration of potentially harmful micro-organisms into mucosal tissues. IgA occurs in serum and secretions as two subclasses that differ in amino acid sequences and glycosylation of the ot heavy chain. IgA1 predominates in serum whereas secretions may contain up to 60% IgA2. Subclass composition and response may be important parameters in understanding the host immune response, but the physiological significance of the two subclasses has not been explained.
It has been postulated that IgA1 and IgA2 antibodies may play different protective roles: IgA1 is susceptible to bacterial IgA1 proteases; the degrading effect of the IgA1 protease from N. gonorrhoeae on anti sperm antibodies suggested a predominant local IgA1 response at least to some antigens but the actual in vivo situation has never been proved. The role of IgA1 proteases has been demonstrated in relation to dental flora: they may promote the adherence of oral streptococci to tooth surface. In 1992, a very strong SIgA protease was demonstrated to be associated with the spermatic fraction of human semen. IgA2 is protease resistant and plays a true role in mucosal defense. The subtle functional differences between IgA1 and IgA2 became recently apparent but, their biological effective role is not clarified.
The appearance of elevated SIgA in seminal plasma, especially during infections, seems to be related to a pathological rather than a physiological situation of the male genital tract. The overproduc-to be present, in vivo, 1 in mice infected by MoPn Chlamydia trachomatis biovar, enhancing a protective role against the infection. TNFOt may play a determinant role in other cytokine pathways, leading to enhanced host defence and it is involved in the Tcell independent pathway of macrophage activation. Its production has been recently demonstrated as proportional to the intensity of the infection with high levels coincident with marked neutrophil influx. 11 Cytokines are important mediators of inflammation and their production may modulate immunity during Chlamydia trachomatis infections as proved by recent in vitro studies. The role of their induction, especially for IL-1 and IL-6, is actually unclear during several infections. A recent paper 1 concluded that ILl and IL6 production in vivo in mice could play a role in the pathogenesis and host defence of Chlamydia trachomatis infection. Additionally the immune response to Chlamydiae may be immunopathological, as was demonstrated in 1969 in trials of trachoma vaccination in children in Gambia 13 in which the severity of the disease in terms of clinical score was paradoxically greater in vaccinated than in unvaccinated controls. In addition, mice sensitized by immunization with a crude extract or with chlamydial heat shock protein suffered more severe inflammation when challenged intravaginally with live mouse pneumonitis agent organisms.
The cytokine content of patients' biological fluids may be a good indicator of the possible evolution and shift of the immune response, TH1 or TH2, mediated by IFN/ and IL-12 or IL-4. The TH1 Th2 shift itself may be involved in immunopathological conditions of the genital tract during Chlamydia infections, as demonstrated in human immunodeficienc virus pathogenesis studies in vitro. 14'5 Several recent studies suggest that TH cells are crucial in the resolution of established Chlamydial infections. 4,5 Additionally C. trachomatis was demonstrated to induce IL-8 secretion in in vitro infected HT29 cells, independent of ILia stimulation. 6 IFN/affects the IL-8 induction pathway that does not involve ILlot. This suggests that T-cell-derived cytokines may also control the host response to Chlamydia by altering the ability of nonimmune cells to respond to the infection, by secreting proinflammatory cytokines and thus increasing the number of inflammatory cells at the site of infection. Cellular immunity is, on the other hand, important in resolving and eradicating established chlamydial infections and pre-existing anti-MOMP antibodies are unable to protect against inital mucosal colonisation (25). In addition IL-10 is the major inhibitor of TH1 responses and of INF,/.

OBJECTIVE OF THE STUDY
Starting with the previous evidence and the observation of inflammatory symptoms in acute and chronic prostatitis patients, we investigated sera and mucosal secretions for the presence of IL-6 and other cytokines (IL-4, IL-10) in relation to anti-C.t.
IgA and IgG and to IgA subclasses in seminal fluids of selected men affected by "abacterial" prostatitis.
In addition we studied the concomitant presence of chlamydial DNA by PCR techniques; the global aim of our study was to possibly interrelate all these immunological parameters with the pathological situation and progression to chronicity in our patients.

MATERIALS AND METHODS
Our study population of 28 patients was selected starting from 290 outpatients admitted to our S.T.D. Centre, Infectious Diseases Unit, S. M. Annunziata Hospital, Florence Italy; the patients were affected by prostatitis or prostatovesciculitis.
The prevalence of anti C.t. specific SIgA in this population was 46.5% (135 positives). The patients' study inclusion criteria were the presence of acute or chronic prostatitis or prostatovesciculitis, clinically and echographically documented, the presence of a strongly positive screening test for species specific anti C.t. IgA in seminal fluids and serum.
Exclusion criteria were the presence of mycoplasmata, bacteria, protozoa, fungi in first morning urine and seminal fluids; negativity for sperm IgA, and antibiotic therapy carried out in the previous six months. 18/28 (64.2%) patients declared symptoms in the past 3 years and 4/28 patients (14.28%) for at least 5 years. All had symptoms since at least six months. Other signs and symptoms were present: prostatic microcalcifications, urethral discharge, stanguria, hematuria, perineal tenesmus, groin, pelvic or ejaculation pains, lymphoadenopathy. Previous S.T.D. infections were mainly H.P.V. infections (10/28). Patients ranged from 26-73 years old with a mean age of 39.1; 85% of patients were between 26-50 years old.
The patients' biological materials that were analysed were sera, semen and urethral swabs. Immedi-MAZZOL1 ET AL. ately after sampling, sera were centrifuged and frozen at -40C, if not immediately tested. To obtain idoneous urethral samples patients were required to return to the laboratory on at least 3 different subsequent days for 3 different samples: slides for Direct Immunofluorescence obtained by endourethral swabs, swabs in the idoneous transport medium for the ELISA test and samples from urethra for Amplicor PCR. All samples were immediately evaluated by the 3 methods. Our diagnostic protocol included a search for C.t. in patients' urethra by DIF ( Cell Culture Vero cells (African green monkey kidney) were grown in 150 cm tissue culture flasks (Falcon) with 199 medium (Biochrom, KG) supplemented with 5% heat inactivated calf fetal serum (Seromed), 0.22% NaHCO3 and 50 Ixg/ml of gentamicin at 37C.
The cells used in the culture were negative for mycoplasmas. When the cell monolayer was confluent, the growth medium was removed and the monolayer was trypsinized. Cells were suspended in growth medium and counted in a Burker glass chamber. For the assay 2 x 103 cells per well were seeded into a 24 well tissue culture plate (Falcon) containing cover slips. The culture was incubated at 37C for 24 hours in a COz incubator. The infection of the cells was performed by aspirating the culture medium and replacing it with Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum 0.17% NaHCO3, 0.4% glucose, 50 g/ml gentamicin, g/ml cyclohexamide (Sigma) containing Chlamydia trachornatis serotype LGV2-434 Bu strain at 1-5 inclusion forming units per cell, to insure infection of 80% of the cells. The infected cultures were incubated at 36.5C for 48 hours in a 5% COz thermostat. At this time 50-80% of the cells showed typical cytoplasmic inclusions detectable by phase-contrast inverted microscopy and stains. The medium was then aspirated and the cells washed 2 times with phosphate-buffered saline (PBS). The cover slips were fixed for 10 minutes with methyl alcohol cooled to -10C, dried and kept at -20C before use as antigens in indirect immunofluorescence test to detect anti-chlamydial secretory IgA.  4C). We collected the upper two-thirds of the supernatants (1 ml) and stored at -40C until assayed. Serum samples were aliquoted and one aliquot was decomplemented (20 min. 56C.) before testing to destroy the possible interfering action of the soluble IL-6 receptor, which binds IL-6 in solution. Bioassays of sera were performed in duplicate to verify if increments of the concentration could be present in the decomplemented fraction. Quantification of the IL-6 content was performed by using five standards of 10, 25, 50, 200 and 500 pg/ml IL-6. The standards were calibrated against the "Unclassified Interleukin-6 (recDNA human type) 88/514". One vial containing ng lyophilised IL-6 was used as a recovery control positive test and was reconstituted with a negative semen. Normal values in our population were previously calculated for semen starting from a selected normal population negative for signs and symptoms of prostatitis and for all bacterial, mycoplasmal and other microbial infections. IL-4, IL-10 were detected by quantitative ELISA tests (Biokine, Tcell Diagn. Inc. and Predicta Genzyme, USA).

Statistical Methods
The Wilcoxon test for paired data was used to determine the comparison between the detected values of all our immunological parameters by Labstat ver- important to verify if the local immunisation in these patients was locally produced or a translocated extension of humoral immunity, transudated from sera. IgA1, IgA2 subclasses were detected in seminal fluids and sera of all 28 patients except 2: IgAl was demonstrated in semen of 8/28 patients (28.5%). 93.7% of the patients declaring symptoms in the previous 3 years were positive for IgA2, making IgA2 immune response strictly related to the persistence of the infections. IgA2 was mainly present in our patients (92.8%) confirming, as in our previous observations, their presence as the mostexpressed and significant immunoglobulin subclass in semen. These data seem to be in agreement with the production of IgA1 proteases in this biologic fluid. Otherwise, the presence of IgA1, in the majority of the semen samples containing detectable levels of IgG, seems to correlate well with the possible transmembrane translocation or transudation of serum IgG, probably due to permeability damages of the blood-genital tract barrier, caused by inflammation and its powerful mediators. IL-4 was also assayed but this lymphokine was not detectable. IL-10 was present in high levels in 75% (25/28) of our patients and very high values were present in IgA2 positive patients. IL-6 was present at a fourfold higher concentration (>20 pg/ml) than normal serum levels (5 pg/ ml) in 75% (21/28) of our selected patients seminal fluids. Comparison of IL-6 detected in sera and semen showed a statistically significant difference (p 0.001) between mean concentrations, in favour of a local overproduction of this cytokine. If we plot IL-6 (semen) vs. IgG and IgA detected (Savyon Otherwise, 89.2% of these patients had evidence of secretory component. Thus the IgA immune response was a secretory related response. Mucosal surfaces in direct contact with the external environment, including the genital tract, have a common immune system with selective traffic of B and T lymphocytes from GALT and BALT. 16 The specific anti C.t. response in our patients is independent of the response in serum and mostly associated with secretory IgA. It was not possible to interrelate chlamydial DNA positivity with other markers of infection, because of the negative PCR results obtained in seminal fluids; we can only affirm that 28.5% (8/ 28) of our patients were PCR positive for C.t.DNA, confirming the presence of the micro-organism as a possible "primum movens" of the pathology and its presence. Another reason for negative PCR resuits could be the presence of very high values of SIgA: our finding seems to confirm animal model findings 17 in which an inverse correlation between specific SIgA titre in genital secretion and the presence of Chlamydia trachomatis isolated by culture in the cervix was found.

CONCLUSIONS
We conclude that our patients with anti Chlamydia trachomatis IgA positive "abacterial" prostatitis have "Chlamydial" prostatitis; they presented with specific local immunisation against this microorganism, a local overproduction of IgA confirming the proper immunocompetency of the genital tract, independent from the systemic humoral one, probably not protective (prevalence of DNA detection at a high rate) and continuously boostered by antigen elimination. Induction of IgA2 seems to be strictly related to the persistence of symptoms and, thus, of infection. Moreover, IgA2 is related to the type of antigenic stimulation 18 at the mucosal site and to repeated antigenic stimulation, more than to a biological shift in local IgA production, caused by destruction of IgA1 by bacterial proteases. This hypothesis seems to find confirmatory reports in a recent paper of Kawamura and co-workers, 19 demonstrating that the ancestral ot chain genes are closely related to the ot 2 and not ot ones of humans and hominoid primates. Future studies will be extremely useful in expanding our understanding of the biological role of these antibodies.
The presence of IgA2 in the case of chronic prostatitis in our population is a confirmatory marker of the persistence and pathology of the chlamydial infection. Secretory IgA themselves, in semen of our chronic prostatitis patients (symptoms since 3-5 years to at least 6 months), is an index of "active" infection and allows us to detect and to define "chronic active infections" of the prostate.
Our patients presenting with chronic prostatitis were very rich in symptoms, mainly those with corresponding high levels of secretory IgA in seminal fluids, confirming that anti C.t. IgA total content and SIgA concentration is a reliable index of infectious activity. These reports are, in addition, emphasized by the contemporary association of elevated values of IL-6, whose importance in inflammatory phase processes is well known, z The local production of IL-6 is well documented in our patients affected by prostatitis. Thus, we can confirm the production of this multifunctional cytokine in human chlamydial infection in vivo. IL-6 is strictly correlated to the anti Chlamydia trachomatis secretory and total IgA content in seminal fluids and doesn't seem to affect this immunoglobulin production, if not as a stimulatory factor. Like in the mouse el IL-6 production may reflect the CD4 + T cells helper function in genital tract mucosac by providing help to mucosal B cells for the production of Chlamydia-specific IgA. IL-4 was not detectable and this fact may be due to its rapid turnover in body fluids or to the fact we can detect only terminal TH2 cytokines. The recovery of IL-10 in patients with chronic symptoms and IgA2 was extremely interesting. IL-10 is the major inhibitor of IFNy which induces inhibition of Chlamydial growth in vitro zz'z3,z4 and resolution of Chlamydial infections, z5 Thus the presence of IL-10 in our chronic patients confirms that, in these patients too, cell mediated immunity is depleted and the TH2 immune shift seems correlated with the persistence of the infection and with the overproduction of IgA antibodies and with the adverse pathological events in the target organ, the prostate gland.
The biological role of IL-6, other cytokines and secretory IgA overproduction in modulating the passage from acute to chronic prostatic infection and micro-organism clearance, is emphasized by our findings, and the possibility that the pathogenesis of Chlamydial prostatitis could be immune complex related has to be thoroughly investigated in the future.