Clin. exp. Immunol (1991) 86, 37-42

Lymphocyte proliferative responses to chlamydial antigens in human chlamydial eye infections D. C. W. MABEY, M. J. HOLLAND, N. D. VISWALINGAM*, B. T. GOH*, S. ESTREICH*, A. MACFARLANE, H. M. DOCKRELL & J. D. TREHARNE* Department of Clinical Sciences, London School of Hygiene and Tropical Medicine and *Institute of Ophthalmology and Moorfields Eye Hospital, London, UK

(Acceptedfor publication 14 May 1991)

SUMMARY In order to study the relationship between cell-mediated immune responses to Chlamydia trachomatis and the pathogenesis of human chlamydial eye disease, we have measured the peripheral blood lymphocyte proliferative responses to whole chlamydial elementary bodies in 40 subjects with oculogenital chlamydial infection of varying severity, 13 subjects with genital chlamydial infections and 12 healthy seronegative controls. The mean stimulation index was significantly higher in those with oculogenital infections than in controls. There was a strong correlation between the response to C. trachomatis serotypes B and L 1. We studied the relationship between proliferative responses and four clinical parameters: follicular conjunctivitis, papillary hypertrophy, corneal pannus and epithelial punctate keratitis, but were unable to show a significant association with any of these. Nor was there any association between proliferative response and serum antibody titre to C. trachomatis (pooled serotypes D-K), duration of disease or quantitative isolation of chlamydia from the conjunctiva. The depletion of CD8+ cells had no consistent effect on proliferative responses to serotype LI in 13 subjects. Keywords chlamydial infection pathogenesis cell mediated immunity

Wang, Grayston & Alexander (1967) were the first to note that in the Taiwan monkey (Macaca cyclops), certain features of the disease, notably corneal vascularization or pannus, were only seen in animals that had been repeatedly reinfected or previously immunized, implying that these had an immunopathological basis. More recently, Watkins et al. (1986) have found that a Triton X 100 extract of C. trachomatis gives rise to severe conjunctival inflammation when instilled into the eyes of previously infected guinea pigs, and that the kinetics and histopathological features of this response are typical of a delayed hypersensitivity reaction. A 57 kD chlamydial heat shock protein has been implicated as the immunogen in this model (Morrison, Lyng & Caldwell, 1 989b) and a similar phenomenon has been observed in the cynomolgus monkey (Taylor et al., 1987). The relevance of these findings to human disease remains to be determined. It is essential that the role of cell-mediated immunity (CMI) in human ocular chlamydial infection be defined if an effective vaccine is to be developed which does not stimulate hypersensitivity reactions, yet in vitro assays of CMI to chlamydial antigens in the course of such infections have not previously been reported. We report here the results of lymphocyte proliferation assays against chamydial antigens in patients with oculogenital chlamydial infections, and the relationship between these responses and clinical and microbiological find-

INTRODUCTION Trachoma, a chronic chlamydial eye infection transmitted from eye to eye in communities with poor hygienic standards, remains the commonest infectious cause of blindness. It has been estimated that 500 million people are affected by the disease, of whom 7 million are blind (Dawson, Jones & Tarizzo, 1981). Outside trachoma endemic areas, ocular chlamydial infections occur sporadically in association within genital tract infections; Chlamydia trachomatis is among the most prevalent genital tract pathogens worldwide (Schachter et al., 1975). Recent work has greatly increased our understanding of the immune response to infections with C. trachomatis. Monoclonal antibodies have been identified which neutralize infectivity both in tissue culture and in the primate eye, and the epitopes on the major outer membrane protein to which they bind have been mapped, suggesting that an effective subunit vaccine might be developed (Zhang et al., 1987; Baehr et al., 1988; Stephens, Wagar & Schoolnik, 1988); optimism has been tempered, however, by the fact that earlier chlamydial vaccines in some cases exacerbated the disease in animal models, presumably as a result of delayed hypersensitivity. Correspondence: Dr D. C. W. Mabey, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine (University of London), Keppel Street, London WC1E 7HT, UK.

ings.

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D. C. W. Mabey et al. PATIENTS AND METHODS

Patients Venous blood was obtained from 40 patients attending Moorfields Eye Hospital, London with oculogenital chlamydial infections (mean age 23-9 years, range 19-48, 20 male, 20 female) and 13 sexual contacts of such patients without ocular involvement (mean age 20-6 years, range 17-27, 6 male, 7 female). The eyes of all patients were examined by the same observer (N.D.V.) using an ocular microscope. Clinical findings were scored according to the classification of Dawson, Jones & Darougar (1975) for conjunctival follicles and papillary hypertrophy. Corneal pannus was measured in millimetres from the corneal margin. Corneal epithelial and subepithelial punctate keratitis were scored as absent or grade 1 (1 -3 spots), grade 2 (45 spots) or grade 3 ( > 5 spots). Patients' genital tracts were also examined by a genitourinary physician. This study was approved by the Ethical Committee of Moorfields Eye Hospital.

Controls Twelve healthy members of the Department of Clinical Sciences at the London School of Hygiene and Tropical Medicine, who were seronegative for chlamydial antibodies, acted as negative controls. Their mean age was 28-4 years, range 24-36; eight were male and four female. Diagnosis of chlamydial infection Swabs were taken for chlamydial isolation from the eye and urethra of all male patients and from the eye, cervix, urethra and rectum of all female patients.

Isolation Chlamydial isolation was carried out in mitomycin-treated McCoy cells according to the method of Woodland, Kirton & Darougar (1987). After 48 h incubation, monolayers were stained with a fluorescein-labelled antichlamydial monoclonal antibody (Microtrak, Syva Ltd, Palo Alto, CA) and examined with a fluorescent microscope for the presence of chlamydial inclusions.

Serology Sera from all study subjects were tested for the presence of IgG and IgM antichlamydial antibodies using a modified immunofluorescence test (Treharne, Darougar & Jones, 1977). Preparation of antigens Antigens were prepared from two serotypes of C. trachomatis. Serotype LI (440L TW3 strain) was obtained from the Institute of Ophthalmology, London, and a serotype B isolate (Jali 20/ OT) was obtained from a patient with trachoma in the Gambian village of Jali. Chlamydia were grown in cycloheximide-treated buffalo green monkey kidney cells according to conventional techniques; 48 h after inoculation cells were trypsinized, washed and resuspended in ice-cold isotonic phosphate-buffered saline (PBS) diluted I in 10 in distilled water, then passed through a tissue homogenizer (Yamato LSC, model LH21). Cell debris was pelleted by centrifugation at 300 g, and supernatants containing chlamydial elementary bodies (EB) were layered onto 30% triosil (Isopaque 440). Elementary bodies were

pelleted by centrifugation at 100 000 g for 1 h, washed and resuspended in PBS. They were inactivated by irradiation (100000 rads). Elementary bodies harvested from several bulk growths were pooled and the protein concentration measured according to the method of Lowry. Aliquots were stored at -70°C prior to use in proliferation assays. Lymphocyte proliferation assays Mononuclear cells were obtained from venous blood by centrifugation on a Ficoll-Hypaque gradient (Sigma Chemical Co., Poole, UK) according to conventional techniques. Mononuclear cells were washed, counted and cryopreserved in growth medium containing 10% DMSO. They were stored in liquid nitrogen until used in proliferation assays. Assays were carried out in RPMI 1640 tissue culture medium (GIBCO UK, Paisley, UK) and 5% autologous plasma. Cells were incubated in round-bottomed 96-well plates (Nunclon, Roskilde, Denmark) at 2 x 1 05 cells per well. Concanavalin A (Con A) (Sigma) was used as a positive mitogen control at a concentration of 5 ug/ml. Purified protein derivative (PPD) (Statenserum Institute, Copenhagen, Denmark) was used as a positive antigen control at a concentration of 10 ,ug/ml. Cells alone served as a negative control. Elementary bodies were diluted to give a final concentration of 5 jug protein/ml, preliminary experiments having shown that this concentration gave optimum discrimination between patients and healthy seronegative controls. All assays were carried out in triplicate. Mitogen-stimulated wells were pulsed with 1 yCi methyl 3Hthymidine (specific activity 5 mCi/l) after 3 days incubation; antigen-stimulated wells were pulsed after 7 days. Wells were harvested after overnight incubation with 3H-thymidine using a PHD cell harvester (Cambridge Technology Inc, MA). After addition of scintillation fluid, counts per minute were measured with a Beckman LS 1801 beta counter. Results are expressed as stimulation indices, that is the ratio of counts per minute (ct/ min) in antigen-stimulated wells to control wells. Peripheral blood mononuclear cells were depleted of CD8+ cells in selected subjects, using magnetic beads coated with antiCD8 monoclonal antibody (Dynal, Liverpool, UK). Following depletion, proliferation assays were set up as above with 2 x 10 cells per well. Immunofluorescent staining followed by FACS analysis confirmed that at least 90% of CD8+ cells had been removed in each case.

RESULTS

Chlamydial isolation Chlamydia trachomatis was isolated from the eyes of all patients with ocular disease, and from the genital tracts of 12/13 sexual contacts. The isolation-negative subject was seropositive. All subjects had clinical evidence of lower genital tract infection (pus cells present in urethral and/or cervical discharge), but only one female patient with oculogenital disease had signs of upper genital tract involvement. Lymphocyte proliferation assays Mean ct/min in unstimulated wells and wells stimulated with chlamydial EBs (serotype LI), PPD and Con A are shown for the three groups of subjects in Table 1. Similar counts were seen in all three groups in unstimulated wells and in wells stimulated with PPD and Con A. Significantly higher ct/min were seen in

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CMI and chlamydial disease pathogenesis Table 1. Counts per minute in control, antigen and mitogen stimulated wells in patients and controls

Category

n

Background

Chlamydial EBs

PPD

Con A

Controls Genital only Oculogenital

12 13

2442 (340) 2227 (446)

4563 (438)

5717(1244)

18929 (3744) 12 193 (2671)

31 732 (4851) 26553 (3814)

40

2721 (328)

7903 (700)

18430 (1530)

27260 (2158)

Results are expressed as mean counts per minute with s.e. in parentheses. EB, Elementary body; PPD, purified protein derivative; Con A, concanavalin A.

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88

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Seronegative controls Genital infections Oculogenital infections Fig. 1. Lymphocyte proliferative response to Chlamydia trachomatis serotype L 1 in patients and controls.

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Fig. 2. Lymphocyte proliferative response to Chiamydia trachomatis serotype Ll versus response to serotype B. EB, Elementary body.

chlamydial antigen-stimulated wells of subjects with oculogenital infections than in controls (t=4045; df=49; Pdia trachomatis. J. Immunol. 138, 575.