M. J. P. Adolfs

Reduced exudation and increased tissue proliferation during chronic inflammation in rats deprived of endogenous prostaglandin precursors

Two models of chronic inflammation were studied in rats deprived of endogenous precursors of prostaglandins by feeding the animals on essential fatty acid deficient (EFAD) food. During kaolin-induced pouch-granuloma, exudate production was markedly reduced in EFAD rats, when compared with normal animals. The exudates from normal rats contained large amounts of PGE, but in the exudates from EFAD rats the amount of PGE was very markedly reduced. Similarly, with carrageenan-impregnated polyether sponges, the exudative component of inflammation was reduced in EFAD rats. However, the proliferative component was significantly increased, particularly in relation to the stunted growth of EFAD rats. Sponge exudates from EFAD rats contained fewer leucocytes than those from normal animals but the fall in leucocyte count was much smaller than the very marked reduction in PGE activity. EFAD rats also exhibited a significant increase in adrenal weights. The results are discussed in the light of the ambivalent (pro- or anti-inflammatory) role of endogenous PGS. It appears that, in the proliferative phase of inflammation, the anti-inflammatory role of PGs is more dominant.

Prostaglandin E2 elevation of cyclic-AMP in granuloma macrophages at various stages of inflammation: Relevance to anti-inflammatory and immunomodulatory functions

In the carrageenin-induced granuloma of rats the inflammatory tissue growth and macrophage invasion on the one hand and the cyclic-AMP content of the macrophages on the other, display opposite directional changes. Macrophages, isolated from this tissue at different stages of inflammation, were used to examine the effect of prostaglandin E2 on intracellular levels of c-AMP. It appears that during infiltration of the macrophages into the inflammatory tissue, the sensitivity of adenylate cyclase to activation by PGE2 increases. Arguments are presented that these observations made in vitro, are in direct relevance to the previous described anti-inflammatory effect of PGE on granuloma tissue in vivo.

Cannulated sponge implants in rats for the study of time-dependent pharmacological influences on inflammatory granulomata

Abstract An experimental granuloma method is described which consists essentially of implanting carrageenin-impregnated polyether sponges, provided with a polythene cannula, into dorsal subdermal tissue of rats. The cannula is exteriorized at the back of the neck and allows the administration of drugs into the sponge implant at any chosen time during the different phases of granuloma development. The value of the method is illustrated by brief reference to the effects of PGEs and the phenolic antioxidant, MK-447, on exudative and proliferative components of 8-day inflammatory granulomata. With each of the substances, the effects were either facilitary or inhibitory depending on the time at which the administration into the inflamed site took place.

Distinction between prostaglandin E2 and prostacyclin as inhibitors of granulomatous inflammation

Prostaglandins (PGs) of the E series, are detectable at inflamed sites and exert pro-inflammatory actions, such as vasodilatation and potentiation of plasma exudation induced by other mediators (see Flower 1977). However, with animal models of chronic inflammation, high doses of PGEs exert anti-inflammatory actions in vivo (see Bonta & Parnham 1978). Recently, we observed that local administration of PGE,, at 1000-fold lower doses than those used by other authors, during the early phase of spongeinduced granulomatous inflammation, enhances subsequent granuloma formation, whereas administration during the later phase inhibits granuloma formation (Bonta & Parnham 1979). In fact, in rats which were deficient in the fatty acid precursors of endogenous PGs, exogenous PGEl inhibited granuloma formation when given at the low daily dose of 50ng. We now report similar proand anti-granuloma effects with low doses of PGE, (the most commonly detected PG at inflamed sites) and additionally show that the later inhibitory effects of PGE, on granuloma formation are capable of counteracting the early stimulatory effects. We have also compared the inhibitory action of PGE, with other PGs including prostacyclin (PGIJ, a novel PG (Moncada et al 1976) which has been found to exert similar actions to PGE,, including vasodilatation and increased vascular permeability (Peck & Williams 1978; Higgs et al 1978b; Murota et al 1978). Granulomatous inflammation was induced in rats, using subcutaneously implanted, carrageenan-soaked, polyether sponges (2 per rat) with indwelling cannulae (Bonta et a1 1979), and the PGs were injected either with the 1 ml 2% Na carrageenan on implantation (day 1) and/or through the cannulae (in 0.51111) at different times, as described earlier (Bonta & Parnham (Moncada et a1 1976). All rats were killed on day 8, sponges removed and granuloma formation expressed as the dry weight of tissue surrounding the sponges (Bonta et a1 1979). In some experiments, both adrenals from each rat were removed and weighed together, their weights being expressed in terms of 1OOg body weight. Table 1 shows that, while injection of PGE, (2 pg) into sponges on implantation (day 1) had no effect on granuloma formation, measured after 8 days, the same daily dose administered on days 1-3 enhanced granuloma formation after 8 days. This action is similar to the granuloma stimulation produced by PGE, (1 pg), which was thought to be secondary to an initial vasodilating action, following injection on day 1 (Bonta & Parnham 1979). Furthermore, the inhibition of 8 day granuloma formation caused by administration of PGEa on days 4-7 (Table 1) was identical to the effect of PGE, administered over the same period (Bonta & Parnham 1979). It should be noted that the increase in control (saline-treated) granuloma weights with progressively later treatment periods, is coinci dental, since, in a large number of control experiments, granuloma weight was unrelated to timing of saline treatment, despite considerable variation in granuloma weights (Bonta et al 1979). Furthermore, it was previously found that this variation in granuloma weights did not alter the effects of added PGEl (Bonta & Parnham 1979). Thus, PGE, injected on day 1 enhanced granuloma weight when control values ranged from 0.32 to 0.78 g and when injected on days 4-7, PGE, inhibited granuloma weight in experiments in which control values ranged from 0.35 to 1.03 g. In the present study PGE,, administered on days 4-7, inhibited granuloma weight in two experiments in which

Interaction between PAF-acether and drugs that stimulate cyclic AMP in guinea-pig alveolar macrophages

The PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-induced arachidonate release from alveolar macrophages was significantly reduced by prostaglandin E2 (PGE2) and by the beta-adrenoceptor agonist salbutamol. In addition, PAF-acether markedly reduced the increase in intracellular cyclic AMP (cAMP) concentrations induced by PGE2 and salbutamol. Our data indicate an inverse relationship between intracellular cAMP levels and free arachidonate availability in alveolar macrophages treated with PAF-acether. A rise in intracellular cAMP therefore represents an important alternative route for controlling the effects of PAF-acether and the resulting inflammatory alterations in the respiratory system.

Atriopeptins and nitroprusside provoke opposite changes in cGMP and cAMP levels in human macrophages

Atrial natriuretic factor (ANF, 10(-7) M) and, even more potently, sodium nitroprusside (SNP, 10(-5)-10(-3) M) stimulated cGMP formation in human peritoneal macrophages. This suggests that the two forms of guanylate cyclase, the particulate form stimulated by ANF and the soluble form activated by SNP, coexist in this cell type. A fall in cAMP levels in parallel with the rise of cGMP levels provoked by ANF and SNP was noticed that was amplified by an increase in the concentration of the phosphodiesterase inhibitor, IBMX. Our finding that ANF, contrary to its action in other tissues, was unable to exert direct inhibitory effects on the adenylate cyclase activity in isolated macrophage membranes, together with the observation that SNP was able to mimic the effect of ANF on cAMP levels indicates that the cAMP-lowering effect of ANF is most likely mediated through the cGMP signal.

Continuous monitoring of prostacyclin production by the isolated, intact, rat aorta using a bioassay technique

Isolated rat aortae were perfused with Krebs buffer in vitro and the synthesis of prostacyclin-like material (PGI2-L) was continuously monitored by measuring the contraction of a superperfused rat stomach strip exposed to the aortic perfusate. PGI2-L release was high after initiation of the aorta perfusion but then gradually declined and stabilized at a basal rate of production that was maintained for at least 180 min. Levels of 6 keto prostaglandin F1 alpha(6ketoPGF1 alpha), the stable breakdown product of PGI2, in the aortic perfusate reflected the changes in biological activity. The concentration of PGE2 in the aortic perfusate remained constant throughout the experiment while the level of thromboxane (Tx)B2 (the stable product of TxA2) decreased with time to below the level of detection of the radioimmunoassay (RIA) used. All biological activity was abolished by heating the aortic perfusate for 30 min at 37 degrees C, while perfusing with 30 microM indomethacin inhibited aorta PGI-L formation. Analysis (by linear regression) of the relationship between PGI2-L formation and the body weight or age of the animals used revealed that PGI2-L synthesis was better related to body weight (r2 = 0.90) than age (r2 = 0.75).

Low concentrations of prostaglandin E2 inhibit the prostacyclin-induced elevation of cyclic adenosine 3',5'-monophosphate in elicited populations of rat peritoneal macrophages.

1 Elevation of cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) in elicited populations of rat peritoneal macrophages was used as a parameter to examine the influence of prostaglandin E2 (PGE2) on the effects of prostacyclin (PGI2) and (±)‐5E‐13,14‐didehydro‐carbo‐prostacyclin (DDH‐carbo‐PGI2) in vitro. 2 PGE2, within the range of 1.4 × 10−9 to 1.2 × 10−8 m, caused a concentration‐dependent inhibition of the rise in cyclic AMP induced by 2.8 × 10−6 m PGI2 or DDH‐carbo‐PGI2. 3 With higher concentrations of PGE2 the inhibition was either non‐existent or masked by the effect of PGE2 per se on cyclic AMP levels. 4 The present findings suggest that the earlier observed low responsiveness of granuloma macrophages to PGI2, in terms of rise in cyclic AMP, is possibly due to permanent exposure of these cells to environmental endogenous PGE2.

Antigen challenge modifies the cyclic AMP response of inflammatory mediators and β-adrenergic drugs in alveolar macrophages

Adenylate cyclase activity was determined in alveolar macrophages (AMs) obtained from bronchoalveolar lavage (BAL) fluids of naive and antigen-challenged guinea pigs. After the anaphylactic reaction in ovalbumin-sensitized guinea pigs, the basal levels of cyclic AMP in AMs were significantly increased compared to the levels in naive AMS (1.87 +/- 0.22 versus 5.26 +/- 0.45 pmol cyclic AMP/5.10(6) cells). Prostaglandin E2 (PGE2), prostacyclin (DC-PGI2), histamine, isoprenaline and salbutamol stimulated adenylate cyclase activity more effectively in AMs obtained from sensitized guinea pigs after the booster injection compared to AMs obtained from non-treated animals. Moreover, DC-PGI2 and histamine, which were hardly able to induce a rise in cyclic AMP levels in naive AMs, become effective activators in AMs obtained after antigen challenge (100 and 60% increase in the response, respectively). Using selective receptor ligands, we have shown that beta 2-adrenoceptors and H2-subtype histamine receptors are functionally coupled to macrophage adenylate cyclase activity. The present data indicate that sensitization does not affect the configuration of the receptor on the outer membrane (no change in affinity constants), but affects other parts of the transmembrane signal system leading to the intracellular production of cyclic AMP (e.g. regulatory binding proteins or increases in the number of receptors).

A polyether sponge constituent which is antioxidant, anti-inflammatory and increases prostaglandin concentrations at the inflamed site.

Granuloma formation was studied in rats following s.c. implantation of polyether sponges soaked in carrageenin. Sponges, which had been boiled in ethanol, produced larger granulomata and more exudate than unboiled sponges, but prostaglandin (PG) concentrations were lower in exudates from boiled sponges.Sub-cutaneous injection of the ethanolic sponge extract inhibited the delayed phase of carrageenin-induced rat hind paw oedema. A counter irritant action was ruled out by the demonstration that local administration of the extract also inhibited hind paw oedema.Ultra-violet spectrophotometry of the anti-inflammatory extract indicated that the main constituent was probably a phenolic material, commonly used as an antioxidant in the manufacture of polymers. The antioxidant nature of the extract was confirmed by its inhibition of the autoxidation of adrenaline.The results are discussed in the light of recent reports on the anti-inflammatory properties of phenolic antioxidants and their effects on the PG biosynthetic pathway.