Prostaglandins

and

Medicine

1:

183-199,

1978.

PROSTAGLANDINS AS REGULATORS IN CELLULAR IMMUNITY M. A. Bray, D. Gordon, J. Morley. Department of Clinical Pharmacology, Cardiothoracic Institute, Fulham Road, London, SW3 6HP, UK. (reprint requests to MAB). ABSTRACT Guinea-pig peritoneal macrophages have been shown to generate substantial concentrations of E-type prostaglandins during culture in vitro. Partially purified lymphokine preparations stimulate PGE production by macrophages. Exogenous PGEl and PGE2 are potent inhibitors of lymphocyte activation and secretion of lymphokines causing lymphocyte mitogenesis and macrophage migration inhibition. Also indomethacin augments the secretion of these lymphokines in vitro. It is therefore suggested that production of PGE by 'des a physiological mechanism for the regulation of macrophages prove lymphocyte activation in reactions of delayed hypersensitivity. INTRODUCTION Lymphokines, which are produced by in vitro culture of sensitised lymphoid cells with specific antigen, exhibit a range of biological actions appropriate to mediators of delayed hypersensitivity reaction (1). Especially noteworthy is their capacity to induce DNA synthesis in non-sensitised lymphocytes, for this provides an amplification or positive feed back system in the delayed hypersensitivity reaction. Lymphokine formation thus accommodates the observation that only a small proportion of responding cells in animals sensitised by cell transfer delayed hypersensitivity reactions of animals are cells specifically sensitive to the eliciting antigen (2). The operation of such amplification devices in the immune response implies the existence of regulatory mechanisms tolimit the extent and duration of reactions of delayed hypersensitivity. Several control mechanisms with possible relevance to cellular immunity have been described either involving production of soluble mediators by lymphocytes and macrophages or depending upon cell/cell contact. Thus both specific and non-specific suppressor cell populations have been proposed to account for in vitro reduction of the immune response to the sensitising antigen (3) and to additional antigens following cell transfer experiments (4). The cell types involved in these responses are known to

include a T-cell sub-population as well as glass adherent, macrophage-like cells (5). In tissue culture experiments low molecular weight materials released from lymphoid tissue spontaneously, or in response to specific antigen stimulation, have been shown to reduce the in vitro mitogenic response of target cells as measured by increased 3H-thymidine incorporation in vitro (6, 7). Additionally non-dialysable lymphocyte chalones have been reported to inhibit the release of lymphokines (8) and to reduce 3H-thymidine uptake by mitogen stimulated human lymphocytes (9). The macrophage has also been reported to release a soluble inhibitor of lymphocyte activation (10) and both high and low molecular weight factors have been demonstrated to originate in macrophage cultures, with the former causing stimulation and the latter inhibition of lymphocyte DNA and proteinsynthesis (11). Our own observation that lymphokines are capable of stimulating macrophages to produce substantial amounts of E-type prostaglandins (12) suggests a regulatory mechanism, in which the controlling agent would be of known chemical structure for it is well established that PGEl is a potent inhibitor of lymphocyte activation by phytohaemagglutinin (13) and that E-type prostaglandins are capable of synergistically enhancing the inhibition of the PHA response produced by hydrocortisone (14). Accordingly we have investigated the capacity of prostaglandins to modify lymphocyte activation and lymphokine production in the guinea-pig. MATERIALS

AND METHODS

Animals: Male outbread Dunkin-Hartley -0

strain guinea-pigs were used throughout

9).

Sensitisation: (a) BGG/FCA: Guinea-pigs were sensitised to bovine gamma globulin (BGG) by intradermal (id) injection of an emulsion of equal volumes of BGG (2.0 mg/ml) and Freunds Complete Adjuvant (FCA) into multiple skin sites (either hips and shoulders or the four foot pads) to give a total Animals were used 3 weeks after sensitisfinal dose of 200 pg BGG/animal. ation. (b) BCG: Guinea-pigs were sensitised to tuberculin by intraperitoneal (IP) injection of 0.1 ml commercial Bacillus CalmetteGuerin (BCG). Animals were used 4 weeks after sensitisation. Lymphokine preparation: Lymphgkine was prepared from specifically sensitised lymph node lymphocytes (1 x 10 viable cells/ml) incubated in the presence of specific antigen with or without drugs in Eagles Minimal Essential Medium (EKM: supplemented with 28 mM sodium bicarbonate, 200 mM l-glutamine and antibiotics) for 24 h at 37 C in an atmosphere of 5% CO2 in air. Supernatants were collected and lymphokine activity separated from the inducing antigen by precipitation of antigen using ammonium sulphate (AS) at 40% saturation (15). The resulting lymphokine-enriched supernatants were dialysed for 24 h against running water to remove salts and against volatile buffer for a further 24 h prior to freeze drying and storage at -2O'C. (a) Lymphocyte DNA synthesis. Lymph node lymphocytes Lymphokine assay: obtainBd from teased guinea-pig peripheral lymph nodes were incubated at 1 x 10 viable cells/ml in the presence of lymphokine in ElJEM with added 184

5% heat decomplemented normal guinea-pig serum. After 48 h culture 1 (ICi 3H-thymidine (SCi/rnnol: Radiochemical Centre, Amersham, UK) was added to each culture followed by a further 24 h incubation. Cultures were harvested and the protein precipitated with 5% tricholoacetic acid. 3H-thymidine incorporation was measured via a liquid scintillation counter and the results were expressed as quench corrected disintegrations per minute. (b) Cell migration inhibition. Guinea-pig peritoneal exudate cells were collected 3 days after IP injection of 20 mls sterile72% Cells were washed, counted and resuspended to 4-5 x 10 starch solution. viable cells/ml in EMEM supplemented with 10% heat decomplemented foetal calf serum (FCS). Cells were packed into capillary tubes (100 pl haematocrit tubes) and alaowed to migrate into medium containing lymphokine. After 40 h culture at 37 C the extent of cell migration was measured by image projection. Results were expressed as area of migration in sq mm. (c) Increased vascular permeability. Guinea-pigs were used in groups of three, each being carefully shaved over the dorsal flank skin. Animals received an intravenous injection via the hind foot vein of l-2 pCi 1251-guinea-pig serum albumin (GPSA) in Evans blue dye, together with 1.5 mg/kg mepyramine maleate immediately prior to use. Three animals were used per assay with intradermal (i d ) injections of saline solutions of lymphokine being allocated to skin sites according to a balanced, incomplete Latin square which permitted estimation of variance between animals, skin sites and treatments. After 4 h animals were sacrificed and discs of skin, wholly including the lesions , were excised from the flank skin. One ml of venous blood was also removed from each animal to permit calculation of isotope accumulation in terms of equivalent blood volume. 1251-GPSA incorporation into the lesions was measured on an automatic gamma spectrometer and results expressed as ul of whole blood equivalent (16). Measurement of the response to antigen: Measurement of lymphocyte trans formation and cell migration inhibition was undertaken as in the lymphok ine assay, but using sensitised cells cultured in the presence of specific antigen, with or without drug addition. In vivo studies utilised sensit ised animals which had received intradermal injections of specific antigen 24 h prior to intravenous injection of labelled albumin and dye. Drugs were injected intradermally 2 h prior to sacrifice. *production Starchinduced peritoneal exudate cells were collected as described for cell migration experiments, and resuspended at 1 x 10' viable cells/ml in EMEM supplemented with 10% heat-decomplemented FCS. Aliquots (0.5 ml) of the cell suspension were dispensed into sterile, disposabae .12 x 75 mm plastic culture tubes (Falcon, USA) and incubated for 24 h at 37 C in an atmosphere of 5% CO2 in air. Cells from BCG-sensitised animals were cultured in the presence of purified protein derivative of tuberculin (PPD: O-100 pg/ml), and cells from non-sensitised animals were cultured in the presence and absence of seven lymphokine preparations with established migration inhibitory, mitogenic, chemotactic and vascular permeability - increasing activities (17, 18). Supernatants were collected and stored at -20 C prior to prostaglandin radioimmunoassay.

185

Prostaglandin radioimmunoassay: Prostaglandin E-like immunoreactivity on culture supernatants was measured using sheep anti-PGEZ-bovine serum albumin antiserum, which cross-reacted 100% with PGE2:55% with PGEl:ll% with 13, 14dihydro-PGEE:l.5% with PGFZr(:O.Z%with 15-keto-PGE2:0.8% with 13, 14-dihydro15-keto-PGE2:0.6% with PGA2:0.3% with thromboxane 82 and 0.2% with PGB2. Briefly the assay procedure consisted of equilibration of antiserum (0.1 ml 1 in 2500 dilution) with 3H-PGE2 (circa 10,000 dpm in 0.1 ml) and unknown or standard prostaglandin solutions (15-2000 pg PGE2 in 0.1 ml) in 0.1 M phosphate buffer pH 7.2 at 4 C overnight. Antibody-bound 3H-prostaglandin was obtained by ammonium sulphate precipitation, using bovine gamma globulin as carrier protein, and radioactivity determined using standard scintillation counting techniques. Unknownswere derived by comparison with the standard displacement curve, and expressed as PGEE equivalents. Antigensand drugs: BCG:

Bacillus Calmette-Guerin (Glaxo, UK).

BGG:

Bovine gamma globulin (.FractionII

FCA:

Freunds complete adjuvant prepared by addition of 2 mgs/ml ground tubercle bacilli (Min. Ag. Fish., UK) to light mineral oil (Gallenkamp, UK).

PPD:

Freeze-dried purified protein derivative of tuberculin (Min. Ag. Fish., UK).

from bovine plasma) (Armour, UK).

Lymphokines: Prepared as described and dissolved in EMEM immediately before use. Lymphokine preparations LK-1 to LK-7 were kindly provided by R. A. Wolstencroft of the Kennedy Institute, London. Prostaglandins: Synthetdc compounds (Cambrian, UK) stored as stock solutions (200 pg/ml) at -20 C in 95% ethanol and diluted in EMEM or saline imnediately prior to use. Indomethacin: Synthet c compound (Merck, Sharp & Dohme, UK) stored in powder was dissolved in 0.1 N NaOH and titrated to pH 7.0 with 0.1 N HCl prior to dilution with EMEM or saline. Statistical analysis: The experimental results depicted in Tables 1-3 have been subjected to a fu 1 analysis of variance. This has enabled the overall variance to be partitioned between treatments, groups and separate experiments. Where overall differences between treatments have been found to be statistically significant a one-tailed test for significance has been applied to the means, results being expressed as a probability (P) of less than 5% (PCO.O5), 1% (PLO.01) or 0.1% (PcO.001).

186

RESULTS Effect of PGs on antigen induced responses of cellular itnnunity: The effect of PGs upon antigen induced responses of cellular immunity has been determined by challenge of cell populations from tuberculin sensitive guinea-pigs with PPD in the presence of PGs and by the local injection of PGs intradermally in lesions of established tuberculin type hypersensitivity In the case of the in vitro test systems of macrophage migration inhibition and of lymphocyte D?IAsynthesis there was highly significant suppression of the antigen induced responses by PGEl and PGE2 (1.0 ug/ml) whilst PGAl (1.0 pg/ml) and PGF2&(1.0 pg/ml) were without effect in the cell migration inhibition test (Table 1). This inhibitory action of PG cannot be attributable to modification of target cell reactivity for both PGEl and PGE2 did not modify the inhibitory response to preformed lymphokine on these cells and were without effect upon the migration of peritoneal exudate cells in the absence of antigen (Fig. 1, Table 1). In the case of lymphocyte DNA synthesis these doses of PGEl and E2 similarly fail to modify the reaction of lymphocytes to preformed lymphokine (Table 1). Action of prostaglandin El (100 ng/ml) on (A) antigen (PPD) and ymphokine induced cell migration inhibition. Mean 5 SEM (n=5). By analysis of varience: slopes are not significantly different. p values compares the dose response data ? PGEl. Antigen: p

Prostaglandins as regulators in cellular immunity.

Prostaglandins and Medicine 1: 183-199, 1978. PROSTAGLANDINS AS REGULATORS IN CELLULAR IMMUNITY M. A. Bray, D. Gordon, J. Morley. Department of...
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