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A comparison of the acute inflammatory response in adrenalectomised and sham‐operated rats
British Journal of Pharmacology
Abstract
Carrageenin pleurisy was induced in adrenalectomised (ADX) and sham‐operated (SHO) rats.
The magnitude and duration of inflammation, as estimated by fluid exudation and cell migration, was greatly increased (approximately doubled) in ADX rats compared with that in their SHO controls.
The content of eicosanoids (6‐keto‐prostaglandin F 1α (6‐keto‐PGF 1α ), thromboxane B 2 (TXB 2 ), and leukotriene B 4 (LTB 4 )) in inflammatory exudates from ADX rats was significantly (2–4 fold) greater than that of their SHO controls.
Resident macrophages obtained from ADX rats produced more eicosanoids per cell per unit time when stimulated in vitro with zymosan, than did cells from the SHO controls.
Administration of glucocorticoids blocked the inflammatory response and reduced the release of eicosanoids both in vitro and in vivo in both groups of rats.
These data are consistent with the notion that physiological amounts of glucocorticoids exert a tonic inhibitory action on phospholipase activity in normal animals and that the increased secretion of these hormones during the inflammatory response serves to check and control the development of inflammation.
... Adrenalectomy (ADX) or treatment with the GC receptor antagonist RU486 (Mifepristone) have been shown to exacerbate clinical disease in adjuvant arthritis [3] and experimental autoimmune encephalomyelitis [4]. Similar interventions in models of inflammation including zymosan-induced peritonitis, carrageenan-induced pleurisy and air-pouch inflammation have led to increases in infiltrating inflammatory cell number as well as measurable increases in a number of inflammatory mediators [5,6]. The studies of Sternberg et al. using hypothalamic-pituitaryadrenal axis (HPA)-deficient rats demonstrated that GC replacement in the physiologic range could prevent streptococcal cell wall (SCW)-induced arthritis [7]. ...
... There is evidence for regulation of inflammatory events, including natural killer (NK) cell cytotoxicity, nitric oxide production and T cell activation, expansion and programmed cell death in vivo by endogenous GC [35][36][37]. Endogenous GC blockade results in increased numbers of neutrophils at inflammatory sites in a number of animal models [5,6], implying an ability of these hormones to regulate factors involved in neutrophil transmigration, including leucocyte and endothelial activation. Increased local production of eicosanoids, prostaglandin E 2 and leukotriene B 4 has been demonstrated in inflammatory models following abrogation of endogenous GC [5]. ...
... Endogenous GC blockade results in increased numbers of neutrophils at inflammatory sites in a number of animal models [5,6], implying an ability of these hormones to regulate factors involved in neutrophil transmigration, including leucocyte and endothelial activation. Increased local production of eicosanoids, prostaglandin E 2 and leukotriene B 4 has been demonstrated in inflammatory models following abrogation of endogenous GC [5]. ADX has similarly been shown to increase IL-1 release in vivo and in vitro [38], thereby promoting neutrophil accumulation directly or indirectly through phospholipase A 2 activation. ...
The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 microgram/g) and subnephritogenic (50 microgram/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 +/- 32.8 versus 42.5 +/- 2.6 mg/24 h; P < 0.005). In ADX animals receiving subnephritogenic doses of anti-GBM injury was manifested by abnormal proteinuria (62.7 +/- 5.8 mg/24 h), accumulation of neutrophils which peaked at 6 h (7.2 +/- 1.37 neutrophils per glomerular cross-section (neut/gcs)) and macrophage accumulation in glomeruli at 24 h (6.8 +/- 1.2 macrophages/gcs). Sham-adrenalectomized animals given the same dose of anti-GBM globulin developed minimal or no glomerular injury: urinary protein excretion (8.7 +/- 1.5 mg/24 h, P < 0.001); neutrophils (0.2 +/- 0.04 neutrophils/gcs, P < 0.001); macrophages (1.2 +/- 0.5 macrophages/gcs, P < 0.001). The increased cellular recruitment to glomeruli in adrenalectomized animals was associated with glomerular endothelial P-selectin expression. P-selectin expression was not detected in sham-operated rats after anti-GBM injection. Complement deposition in glomeruli was minimal in both groups. Physiologic GC replacement of ADX rats receiving subnephritogenic-dose anti-GBM reversed the observed susceptibility to GN development, with urinary protein excretion (7.8 +/- 1.12, P < 0.005) and no detectable P-selectin expression or leucocyte accumulation in glomeruli. These results suggest that endogenous GC modulate heterologous anti-GBM nephritis in rats and that this may be attributable, in part, to regulation of P-selectin expression.
... inhibited production of PGEg, LTB4 and neutrophil accumulation at the inflammatory site induced by subcutaneous polyacrylamide gel in mice. Furthermore, Flower et al (1986) and Vincent et al (1986) found that fluid exudation, cell migration and eicosanoid production in a carrageenin-induced pleurisy model were greatly increased in adrenalectomised animals compared to sham-operated controls. Glucocorticosteroids have been shown to inhibit rat paw oedema induced by carrageenin which is thought to act principally by releasing eicosanoids, and local lipocortin inhibited the eicosanoid component of this oedema (Cirino et ah, 1989). ...
... Carrageenin rather than zymosan has often been used as the inflammatory stimulus where dexamethasone-induced inhibition of eicosanoid production has been demonstrated in vivo (Flower et al., 1986;Salmon et al., 1983). It is possible, therefore, that differences in stimuli used may explain, at least in part the conflicting results obtained in different studies, as might the variety of inflammatory models. ...
A characteristic feature of acute inflammation is increased microvascular permeability. Mediators that increase microvascular permeability can act in 2 ways; either directly on the endothelial cell eg. bradykinin, or by a mechanism dependent on circulating neutrophils eg. the chemoattractant FMLP. Oedema can be suppressed by both steroid and non-steroid anti-inflammatory drugs. These compounds may be able to act at several levels. This study was designed to investigate mechanisms of oedema formation and the possible sites of action of anti-inflammatory compounds. FMLP-induced oedema formation was not dependent on endogenous histamine release or pro-inflammatory products of the cyclo-oxygenase pathway. Intravenous infusion of zymosan-activated plasma produced transient neutropenia in rabbits which resulted in inhibition of oedema formation induced by FMLP, but not that induced by bradykinin. Ibuprofen, selectively inhibited FMLP-induced oedema formation when administered intravenously. This drug did not induce neutropenia and the effect was independent of cyclo-oxygenase inhibition. Ibuprofen may interfere with the interaction between circulating neutrophils and venular endothelial cells. The microtubule blocking agent colchicine also selectively inhibited FMLP- induced oedema formation even when it was administered at intervals after intradermal FMLP. This suggests that continuing interactions between functionally active neutrophils and endothelial cells are necessary for the protracted plasma protein leakage induced by this chemoattractant. There is evidence that anti-inflammatory steroids may owe some of their anti-inflammatory actions to effects on the target cells of inflammatory mediators eg. microvascular endothelial cells 2md neutrophils. In an attempt to investigate this possibility in vivo the ability of dexamethasone to modulate oedema formation and ¹¹¹In-neutrophil accumulation in rabbit skin was investigated. Dexamethasone was effective when administered in three ways. Firstly, local pretreatment of skin with dexamethasone inhibited oedema responses but not ¹¹¹In-neutrophil accumulation. Secondly, treatment of neutrophil recipient rabbits intravenously with dexamethasone inhibited both oedema responses and ¹¹¹In-neutrophil accumulation in response to exogenous chemoattractants. Thirdly, systemic treatment of neutrophil donor animals with dexamethasone resulted in suppression of ¹¹¹In-neutrophil accumulation in response to intradermal chemoattractants in recipients. Systemic treatment with dexamethasone suppressed neutrophil accumulation oedema formation and the generation of LTB₄ induced by intraperitoneal-injection of zymosan in the rabbit. The generation of TXB₂ and prostacyclin were not inhibited by dexamethasone. Depleting animals of circulating neutrophils inhibited the generation of LTB₄ suggesting that accumulating neutrophils are responsible for the generation of this chemoattractant. This study shows that anti-inflammatory compounds can inhibit oedema formation and neutrophil-accumulation by acting at several different sites. There is evidence for inhibition by effects on the microvascular endothelial cell and the neutrophil.
... Endogenous glucocorticoids play an important role in the inflammatory response, mediated by the synthesis of arachidonic acid derivatives (for review, see Garcia Leme 1989). Ablation of the adrenal glands has shown to increase the content of prostaglandin, tromboxane B2, and leukotriene B, in the inflammatory exudate (Flower et al. 1986). Resident macrophages obtained from adrenalectomized rats produced more eicosanoids per cell per unit of time when stimulated in vitro with zymosan than did the cells from controls (Flower et al. 1986). ...
... Ablation of the adrenal glands has shown to increase the content of prostaglandin, tromboxane B2, and leukotriene B, in the inflammatory exudate (Flower et al. 1986). Resident macrophages obtained from adrenalectomized rats produced more eicosanoids per cell per unit of time when stimulated in vitro with zymosan than did the cells from controls (Flower et al. 1986). These higher levels of eicosanoids may be involved in the threefold reduction in cell migration observed in Table 2. ...
Murine macrophages have been reported to utilize glutamine at high rates. However, the role of glutamine in macrophage function is still unknown. In the present study, the maximum glutaminase activity of macrophages was investigated under several endocrine dysfunctions that are known to cause alterations in macrophage function. The results obtained suggest that glutamine might play an important role in the onset of phagocytosis in inflammatory macrophages. Moreover, the studies show that insulin, glucocorticoids, and thyroid hormones may be responsible for the regulation of glutamine metabolism and, consequently, of macrophage function.
... The differing functions of Th1 and Th2 cytokines ultimately led to a proposed model for describing renal diseases as either Th1 or Th2 dominated [3,31]. Depletion of glucocorticoids (ADX) leads to lethal inflammatory reaction to endotoxin [32,33], which is prevented by dexamethasone [34,35]. Our results show significantly elevated IL-4 and significantly reduced IL-10 in untreated ADX rats compared to sham controls. ...
Physiological Glucocorticoids are important regulators of the immune system. Pharmacological GCs are in widespread use to treat inflammatory diseases. Adrenalectomy (ADX) has been shown to exacerbate renal injury through inflammation and oxidative stress that results in renal impairment due to depletion of GCs. In this study, the effect of myrcene to attenuate renal inflammation and oxidative stress was evaluated in the adrenalectomized rat model. Rats were adrenalectomized bilaterally or the adrenals were not removed after surgery (sham). Myrcene (50 mg/kg body weight, orally) was administered post ADX. Myrcene treatment resulted in significant downregulation of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) compared to untreated ADX rats. In addition, myrcene resulted in significant downregulation of immunomodulatory factors (IFNγ and NF-κB) and anti-inflammatory markers (IL-4 and IL-10) in treated ADX compared to untreated ADX. Myrcene significantly increased the antioxidant molecules (CAT, GSH, and SOD) and decreased MDA levels in treated ADX compared to untreated. Moreover, myrcene treatment reduced the expression of COX-2, iNOS, KIM-1, and kidney functional molecules (UREA, LDH, total protein, and creatinine) in ADX treated compared to ADX untreated. These results suggest that myrcene could be further developed as a therapeutic drug for treatment of kidney inflammation and injury.
... The background for paper II is the observation that in animal models of inflammation, treatment with metyrapone (an inhibitor of the cortisol synthesis), or a glucocorticoid antagonist or adrenalectomy, caused exacerbation of the inflammatory response, with increased concentrations of leucocytes, including enhanced adhesion and migration (Flower et al. 1986;Laue et al. 1988;Farsky et al. 1995). In RA patients, treatment with metyrapone resulted in decreased serum cortisol concentrations and significantly increased joint pain and tenderness (Saldanha et al. 1986). ...
... A number of cells in the human joint have the potential to generate nitric oxide (17), which has been shown to be a key mediator of tissue damage in experimental arthritis models (18). Current evidence indicates that endogenous glucocorticoids participate in the regulation of inflammatory processes (19)(20)(21)(22) and endocrine dysfunction involving the hypothalamic-pituitary-adrenal axis, and adrenal glucocorticoids has been postulated to contribute to the development and persistence of RA (23,24). This congruence of the described proinflammatory actions of MIF with the pathogenic mechanisms of RA, in the setting of powerful suppression of animal arthritis with anti-MIF strategies, led us to propose the hypothesis that MIF has a role in human RA. ...
Objective
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor α (TNFα) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue.MethodsMIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription–polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1β (IL-1β), TNFα, or interferon-γ (IFNγ), and dexamethasone (DEX). Mononuclear cell TNFα release after exposure to FLS-conditioned medium was measured by ELISA.ResultsMIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1β, TNFα, or IFNγ. In contrast, DEX 10−7M significantly reduced synoviocyte MIF, while DEX 10−10–10−12M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFα release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFα release.Conclusion
These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.
... Administration of RU 486, a GR antagonist, resulted in an increased inflammatory response compared to that in control animals (20). 'Carrageenin', a sulphated cell-wall polysaccharide found in certain red algae, induced non-specific inflammation in adrenalectomized rats that was substantially stronger than in normal animals (21). Both studies showed that endogenous GC are sufficient to mediate an anti-inflammatory effect. ...
Despite the importance of glucocorticoids (GCs) to modern medicine, the physiological role of endogenous GCs in immunomodulation is poorly understood. Evidence collected over the past decade convincingly shows that GCs at levels that can be reached physiologically affect the immune response in a more differentiated way than previously thought. This chapter discusses evidence suggesting that endogenous GCs not only suppress but also direct and enhance immune functions. Antigen-specific immunity, for example, can be either stimulated or inhibited by GCs depending on the dose and duration of GC exposure. At the molecular level, stimulating effects of GCs are reflected by their capacity to increase the expression of various cytokine receptors. Such stimulating actions are often overlooked but might well be equally important as the inhibitory functions during host defence and the maintenance of homeostasis.
... These phenomena are presumed to be expressions of an adaptive response that protect the host from inflammatory processes while creating an internal environment that reduces the likelihood of a proliferating infection (Berczi, 1998). For example, circulating corticosteroids in particular have long been attributed to protecting the vertebrate host from endotoxin shock (Dougherty, 1949;Flower et al., 1986). ...
Plasma cortisol responses of pallid sturgeon Scaphirhynchus albus and yellow perch Perca flavescens following injection with equal doses of lipopolysaccharide were compared. Concentrations of cortisol in plasma from pallid sturgeon did not change following injection (6·0–11·0 v. 6·4 ng l−1 pre-stress) while in yellow perch plasma they were shown to increase up to 6 h (117·0 v. 9·8 ng l−1 pre-stress) after the injection. These results are consistent with other reports for pallid sturgeon that illustrate a reduced cortisol response following other applied stressors relative to teleosts and suggest differences in the expression and regulation of their inflammatory responses.
... Second, there could be a blockade by dexamethasone of the hypothalamic-pituitaryadrenal axis suppressing, at the time of arthritis induction, the modulatory action of endogenous glucocorticoids upon inflammation. This modulatory role of endogenous glucocorticoids has already been demonstrated in a variety of experimental models [12,13] and seems to constitute a complete negative feedback loop. Inflammatory cells can release substances, like interleukin-1 [14], that increase ACTH secretion and serum corticosterone levels [15,16] which, in turn, can decrease inflammation. ...
Systemic dexamethasone phosphate administration to rabbits 2 h before induction of arthritis by antigen promoted a decrease
in cellular influx, vascular permeability and eicosanoid concentrations in the synovial fluids. When administered 24 h before
the articular challenge a significant increase in all parameters assessed was observed. A third group of animals receiving
an injection of dexamethansone before the intra-articular challenge. The results suggest that a blockade of the hypothalamic-pituitary-adrenal
axis could be responsible for the findings in animals treated 24 h before induction of arthritis, and that endogenous glucocorticoids
can be important for the down-regulation, of the articular inflammatory reaction.
... The correctly timed release of these hormones is essential to the organism's ability to respond to, and recover from, episodes of stress, inflammation, injury and infection. Adrenalectomy has long been known to exacerbate, prolong inflammatory responses, to delay recovery (Leme and Wilhelm, 1975;Flower et al., 1986;Moraes et al., 1987;Bertini et al., 1988;MacPhee et al., 1989;Masferrer et al., 1992Masferrer et al., , 1994 and to result in excessive cytokine production during the inflammatory response (Goujon et al., 1996). Removal of the adrenals from rodents challenged with even mild, sublethal inflammatory stimuli can have fatal consequences in adrenalectomized rodents. ...
The glucocorticoids are the most potent anti‐inflammatory drugs that we possess and are effective in a wide variety of diseases. Although their action is known to involve receptor mediated changes in gene transcription, the exact mechanisms whereby these bring about their pleiotropic action in inflammation are yet to be totally understood. Whilst many different genes are regulated by the glucocorticoids, we have identified one particular protein—annexin A1 (Anx‐A1)—whose synthesis and release is strongly regulated by the glucocorticoids in many cell types. The biology of this protein, as revealed by studies using transgenic animals, peptide mimetics and neutralizing antibodies, speaks to its role as a key modulator of both of the innate and adaptive immune systems. The mechanism whereby this protein exerts its effects is likely to be through the FPR receptor family—a hitherto rather enigmatic family of G protein coupled receptors, which are increasingly implicated in the regulation of many inflammatory processes. Here we review some of the key findings that have led up to the elucidation of this key pathway in inflammatory resolution.
British Journal of Pharmacology (2008) 155 , 152–169; doi: 10.1038/bjp.2008.252 ; published online 21 July 2008
... In order to investigate the possible participation of adrenal glucocorticoids in the oedema inhibition caused by peripheral benzodiazepine receptor ligands, both adrenal Ž . glands were removed as described by Flower et al. 1986 . After 7 days of surgery, animals received either PK11195 Ž . ...
This study investigates the anti-inflammatory effects of 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide and 7-chloro-5-(4-chlorophenyl)-1, 3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2-one in paw oedema induced by carrageenan in mice. Pretreatment (24 h) with both ligands inhibited oedema formation in at different doses (0.00001-10 mg/kg, i.p.) with range of inhibition of 25% to 70%, in animals with or without adrenal glands. These results demonstrate, for the first time, an in vivo anti-inflammatory property of peripheral benzodiazepine receptor ligands.
... In this context, vascular and cellular events of inflammatory reaction, cytokine secretion and antibody production are influenced by elevated levels of corticosteroids. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] For these reasons, it is plausible that elevated levels of corticosteroids after CTX injection could explain, at least in part, the absence of inflammatory effects during human or experimental envenomation and the impaired IgG 1 production described. To investigate this hypothesis in our experimental procedures, animals were pre-treated with metyrapone, a drug that blocks glucocorticosteroid production but does not interfere with mineralocorticoid activity. ...
Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions.
The aim of this paper was to investigate the mechanisms involved in these impaired responses.
Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment.
Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration.
Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.
... Peptides were assayed for PLA2 inhibitory activity according to Pepinsky et al. (1986) preparations to different agonists were recorded via isotonic transducers (type 7006, Basile, Comerio, Italy) coupled to a dual channel recorder (type 7070, Basile). All preparations were equilibrated for at least 1 h before starting any experimental procedure.Flower et al., 1986; Solito & Parente, 1989). At the end of the incubation, PGE2 concentrations in the supernatants were determined by specific radioimmunoassay (N.E.N., Dreieich, FRG). Cell viability was always >95% as measured by the trypan blue exclusion test. ...
A novel anti‐inflammatory peptide (residues 204–212) of human recombinant lipocortin 5 (hrLC5) found on the high similarity region with uteroglobin is described.
Peptide 204–212 dose‐dependently inhibited the contractions of rat isolated stomach strips elicited by porcine pancreatic phospholipase A 2 (PLA 2 ). Contractions caused by arachidonic acid (AA), prostaglandin E 2 (PGE 2 ) and 5‐hydroxytryptamine were not affected. No direct enzyme inhibition was observed in a radiochemical assay.
PGE 2 release by both human fibroblasts and rat macrophages was reduced by peptide 204–212 in a dose‐dependent manner.
The development of carrageenin‐induced oedema in rats was significantly inhibited by the local administration of peptide 204–212.
The pattern and potency of the biological effects of peptide 204–212 are similar to those of antiflammin 2, a lipocortin 1‐derived peptide.
It is suggested that peptide 204–212 may represent the active site responsible for the anti‐inflammatory properties of lipocortin 5.
... To discover how an absence of steroids, and by implication, lipocortin would alter the nature of the inflammatory response in vivo, the author and his colleagues compared the acute inflammatory response to carrageenin in the adrenalectomised and sham-operated rat (Flower et al., 1986). In a carefully matched experiment the irritant was introduced into the pleural cavity of the two groups of rats and the extent of the inflammation assessed at various time points. ...
The glucocorticoids inhibit our 'defence reactions' at many levels. One way in which they achieve this is by inhibiting the synthesis of chemicals involved in the promotion of the inflammatory response. The production of many mediators involved in the response to infection, injury, haemorrhage or metabolic disturbances are under glucocorticoid control such that elevated levels of hormone in the blood suppresses their formation. In many cases the action of these mediators is blocked as well. It might be thought that the glucocorticoids act simply by decreasing the synthesis rate of these protein regulators of inflammation such as the lymphokines, or of the enzymes which make prostaglandins. Whilst this undoubtedly does occur, another mechanism is also employed: that is, the glucocorticoid-induced synthesis of inhitibory proteins. Lipocortin (and possibly other related proteins) then is a sort of 'second messenger' of the glucocorticoids. It is only one of many such regulatory proteins but it is an important one, controlling as it does the mediators which promote development of the symptoms of the inflammatory response. It is undoubtedly the significant component of the inbuilt mechanism for terminating the inflammatory response which the physician exploits, for when he gives his patients relatively large doses of steroids to control an inflammatory response, he is in reality increasing the synthesis of these 'second mesenger' proteins such as lipocortin to a near maximum. All the early studies on lipocortin were performed in vitro, that is under conditions in which steroids were not normally present. Under these circumstances the generation and appearance of lipocortin seemed absolutely dependent upon the presence of glucocorticoids in the perfusing medium. These findings have led some to the erroneous notion that lipocortin was only present following treatment with exogenous steroids. Of course, all healthy mammals have circulating glucocorticoids and thus it is more rational to expect that lipocortin is a normal constituent of plasma and tissues (as indeed it appears to be), although the amount present in the cells can be increased by raising the concentration of endogenous or exogenous steroids. There has been a corresponding change in our appreciation of the function of lipocortin. Originally, it was regarded mainly as an 'anti-inflammatory protein' but today it seems more likely that this protein is present in most cells, and that its function is to control phospholipase A2 activity and to allow lipid hydrolysis only under strictly defined circumstances. This reversible inhibitory function of lipocortin could well be controlled by the phosphorylation and dephosphorylation cycle described above. Naturally, during inflammation, phospholipase is substantially activated and thus there is a requirement for a greater than normal supply of the inhibitory protein, hence the relationship between the rate of synthesis and the release of steroid hormones. The discovery, characterisation, islation, sequencing and cloning of lipocortin has opened up an entirely new and exciting chapter in cell biology and also holds out a strong promise for the future of anti-inflammatory therapy. In addition to their beneficial clinical effects, steroids produce a wide spectrum of side effects which preclude the use of these drugs for long periods of time except in the very seriously ill. These side effects are caused by changes in the transcription of specific genes in the same way as the anti-inflammatory effects. It has long been an article of a faith of scientists working in this are that if we could identify and isolate the 'second messengers' of steroid action that are responsible for the anti-inflammatory effects, it should be possible to produce drugs which possess many of the beneficial action of steroids without incurring the heavy penalty of side effects. The real value of this work is that it enables us to take our first stpes in that direction.
... Alterna tively, the poor growth of the tumor in the absence of glucocor ticoids may be attributable to immune suppression at physio logical or mildly increased glucocorticoid concentrations. In deed, in the rat, glucocorticoids at physiological levels have been shown to suppress the inflammatory response (24,25). Moreover, administration of RU 486 to either tumor-bearing or non-tumor-bearing animals resulted in significant splenic hypertrophy, with proliferation of myeloid elements, and thymic enlargement.3 ...
Glucocorticoid receptors are present in most normal and malignant mammalian cells. To examine the hypothesis that the growth of methylcholanthrene-induced malignant sarcoma is glucocorticoid dependent, we evaluated the behavior of malignant fibrosarcoma (MCA) in adrenalectomized rats treated with either normal saline or deoxycorticosterone acetate and in intact rats treated with placebo or with the glucocorticoid receptor antagonist RU 486. Survival, tumor weight, and loss of body weight (an index of cachexia) were measured. In MCA-bearing rats, neither survival nor loss of body weight was affected by bilateral adrenalectomy or by treatment with RU 486. Tumor weight and time-integrated tumor volume, however, were significantly less in bilaterally adrenalectomized rats without deoxycorticosterone acetate replacement than in animals treated with deoxycorticosterone acetate. Similarly, tumor weight and time-integrated tumor volume were less in intact animals treated with RU 486 than in intact animals treated with placebo. The glucocorticoid receptors in the tumor cells had similar binding capacity (Ro) and equilibrium dissociation constant (Kd) as in control rat fibroblasts. These results suggest that the growth of MCA sarcoma cells is partially dependent upon glucocorticoids. This effect of glucocorticoids, however, was not of sufficient magnitude to improve survival and prevent cachexia. We conclude that glucocorticoids appear to influence MCA sarcoma growth in the rat, and that glucocorticoid receptor blockade, perhaps in combination with other antitumor agents, merits future study in the treatment of malignant tumors.
... Moreover, IL-1 and TNFax are potent stimulators of the hypothalamo-pituitary-adrenal (HPA) axis leading to increased circulating corticosteroids ( ). The importance of this immunoregulatory feedback is indicated by enhanced cytokine release in adrenalectomized (ADX) animals (Perretti et al., 1989; Zuckerman et al., 1989) which show exaggerated inflammatory responses (Flower et al., 1986; Perretti et al., 1991). The Lewis strain of rat is particularly susceptible to such chronic inflammatory models as cell-wall arthritis and experimental allergic encephalomyelitis (EAE): this susceptibility has been attributed to a defect in the HPA axis in this strain which results in lower corticosterone (CCS) responses following challenge with various stimuli including IL-1 (Sternberg et al., 1989a; Mason, 1991). ...
Circulating corticosterone, interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNFα) activities in serum of Lewis and Wistar rats were measured following injection of lipopolysaccharide (LPS). IL‐1 was measured as ‘lymphocyte activation factor’ (LAF) activity following precipitation of inhibitory activity with polyethylene glycol. TNFα activity was measured as cytotoxic activity.
Compared to the Wistar, the Lewis rat had higher circulating LAF and TNF activities following LPS, and release of both cytokines was prolonged in this strain.
Corticosterone increases in response to LPS were less in the Lewis than in the Wistar rat following the initial peak at 1 h; basal corticosterone was lower in the Lewis rat.
Adrenalectomized Lewis rats had even greater amounts of circulating LAF and TNF activities following LPS than did intact animals; the effect of adrenalectomy was not however mimicked by acute treatment with the steroid receptor antagonist, RU486, suggesting that endogenous corticosteroids did not acutely control cytokine release.
Although in vivo administration of anti‐murine IL‐1α antiserum significantly lowered LAF activity of serum, circulating corticosterone in response to LPS was not affected. Similarly, treatment with anti‐murine TNFα monoclonal antibody (mAb) abrogated TNF activity without affecting corticosterone, suggesting that other mediators may be responsible for corticosterone release following LPS.
This ‘overproduction’ of inflammatory cytokines together with lower circulating corticosterone may contribute to the susceptibility of the Lewis rat to diseases such as adjuvant arthritis or experimental allergic encephalomyelitis.
Inflammation is a primordial response that organisms set up to counteract the invasion of pathogens and attack by any other xenobiotic; the symptoms characteristic of inflammation are well known, the classic rubor, dolor, calor, tumor and functio laesa being described since ancient times. This response of the host is complex and multifactorial, and crucial for survival: abnormal inflammation, as in the case of patients affected by leukocyte adhesion deficiency, a genetic disease whereby specific adhesion molecules are absent, is associated with poor life quality and precocious death. Fighting inflammation is a common problem that physicians have to face when dealing with a wide variety of diseases. Hence, understanding the molecular and cellular mechanism responsible for it is important for the design of a better therapy. Modern theories in this field of research have suggested that one way to achieve a better and more efficient anti-inflammatory therapy is to exploit the body's own arsenal of endogenous antinflammatory mediators. One such protein is Annexin-1 (AnxA1): an endogenous anti-inflammatory protein whose activity correlates to the pharmacological effects of glucocorticoids. In this review we will summarize the most recent studies on the biological effects of AnxA1 and in particular we will be focusing on the differential and yet complementary role of this protein in the innate and adaptive immune systems.
Understanding the way our body switches off host defence responses has yielded some of the most innovative recent discoveries in inflammation research. In reality the concept is not new, and was already implicit in early publications of the 1970s' which showed that during inflammation, glucocorticoids are increased in the circulation and that these protect the host from over-shooting and ensuing self-inflicted injury. Stemming from the first example of drugs developed on an endogenous anti-inflammatory pathway, that of the glucocorticoid, we have here touched upon other counterregulatory breakpoints, such as those centred on melanocortins; the annexin 1 system; the polyunsaturated fatty acid derivatives lipoxins and resolvins; galectin-1 and selected others, including novel chemical entities engineered to release anti-inflammatory gases and factors originally discovered in the developmental field. We propose that understanding the molecular mechanisms switched on by a given anti-inflammatory mediator and the events it modulates in target cells will be of great help in developing innovative ways to control inflammatory pathologies. This seems quite articulated and with a degree of complexity in the group of developmental axonal guidance factors. We propose that drugs discovered along this philosophy will have a better compliance and would be theoretically devoid of side effects since they will be acting by mimicking the way our own body assures the inflammatory response is restricted both spatially and temporally.
The effects of CBD, a potent analgesic cannabinoid on LTB4 and TXB2 production stimulated by A23187 was determined in mouse blood ex vivo and in human polymorphonuclear cells in vitro. At a dose of 10 mg/kg administered orally CBD inhibited LTB4 production in mouse blood and was equieffective to the dual lipoxygenase/cyclo-oxygenase inhibitor BW755C and the lipoxygenase inhibitor BWA4C used at a dose of 50 mg/kg. In the same blood samples CBD stimulated TXB2 production from between 20 to 30% over a 4 h period. A23187 stimulation of LTB4 synthesis in human polymorphonuclear cells was inhibited by CBD and Δ'-THC in a dose related manner (IC505.4 and 8.2 μm respectively). However, only CBD produced a 100% inhibition of LTB4 synthesis. The production of TXB2 in these cells was initially stimulated at low doses by CBD but at higher doses TXB2 synthesis was inhibited.
Endogenous glucocorticoids undoubtedly play a role in the control of immune responses: their contribution to inter-strain variation is unknown. The development of specific IgG and IgE was measured following inoculation with ovalbumin in Lewis, Fischer, Wistar and Brown Norway rats. The Lewis gives a smaller IgG and IgE response than the other strains and the responsein vivo to antigen injected into the paw correlates with the titre of specific antibody. Treatment with the steroid receptor antagonist RU486 (mifepristone) following inoculation reveals that in the Lewis, and to a lesser extent in the Brown Norway, the development of a specific IgG response is limited by endogenous corticosteroids. The IgG response in different strains is differently sensitive to treatment with the synthetic glucocorticoid dexamethasone, the Lewis being particularly resistant. The importance of control by endogenous corticosteroids should not be overlooked in contributing to strain differences in immune response.
It is now evident that several endogenous anti-inflammatory pathways are activated in parallel with the host inflammatory
response to maintain a homeostatic control. From this idea has arisen the concept of anti-inflammation, a term used to describe
the balance that exists between pro-inflammatory and anti-inflammatory mediators/pathways that operate in concert to initiate,
maintain and finally resolve the inflammatory reaction.
Glucocorticoids suppress the inflammatory response by altering leukocyte traffic and function, cytokine secretion and action, and phospholipid metabolism. We employed the glucocorticoid receptor antagonist RU 486, to examine whether glucocorticoids suppress the inflammatory response through a receptor-mediated mechanism and whether basal glucocorticoid secretion exerts antiinflammatory effects in the resting (non-stress) state. To test these hypotheses we evaluated the effects of increasing doses of dexamethasone, RU 486, or dexamethasome plus RU 486 on the exudate volume and concentrations of leukocytes, prostaglandin E2, (PGE2) and leukotriene B4 (LTB4) in intact rats that received subcutaneous carrageenin. Exudate volume, leukocyte concentration and LTB4 and PGE2 levels were all suppressed by dexamethasone in a dose-dependent fashion (P < 0.005). RU 486 was able to antagonize fully the suppressive effects of dexamethasone on the inflammatory response (P < 0.001) and to cause increases of exudate volume and leukocyte, PGE2 and LTB4 concentrations when given alone (P < 0.05). These increases ranged between 30 and 100% above the basal inflammatory response. We conclude that glucocorticoids most likely suppress the inflammatory response by a glucocorticoid receptor-mediated mechanism and under basal conditions exert tonic antiinflammatory effects.
Die negative Kontrolle von aktivierten Makrophagen ist ein wichtiger Mechanismus um pathologische Entzündungssyndrome zu vermeiden. Ein zentraler Regulator ist das Zytokin IL-10, welches die Produktion einer Vielzahl von Zytokinen und Chemokinen hemmt. In der vorliegenden Arbeit konnte die Phosphatase DUSP1 als ein Vermittler der inhibitorischen Wirkung von IL-10 identifiziert werden. Die DUSP1 abhängige Deaktivierung der MAPK p38 korrelierte mit der Inhibition proinflammatorischer Zytokine in Makrophagen. DUSP1 gendefiziente Mäuse reagierten auf die Injektion von LPS oder der Induktion einer polymikrobiellen Peritonitis mit erhöhter Produktion zahlreicher Zytokine und der Entwicklung eines letalen Schocksyndroms. Zusammenfassend konnte eine essentielle Rolle von DUSP1 in der Regulation von angeborenen Immunantworten gezeigt werden.
It is well known that histamine is found in high concentration in mast cell granules(1). The histamine content of these granules may be released to the extracellular space if an appropriate stimulus is provided(2). Besides histamine, other preformed active substances like enzymes, chemotatic factors and proteoglycans, as well as newly generated mediators like eicosanoids, platelet activating factor and adenosine are released during the secretion process of mast cells(3). The activation of mast cell degranulation has been associated with a number of pathologic disorders, most frequently, diseases derived from the atopic state(4). It is now evident that mast cells are the primary effector cells in the early reaction in both allergic and non-allergic asthma(5,6), although some authors doubt that the late reaction of asthma is a mast cell dependent event(6). Other studies point towards basophils as cellular elements involved in the secondary phase of inflammation in allergic diseases(7). Secretion would depend on a histamine releasing factor, and on the presence of IgE on the basophil's surface(8). There is also evidence suggesting involvement of mast cells in some non-allergic inflammatory processes like arthritis(9). The pharmacological management of these diseases basically consists in the use of methylxantines, beta 2-adrenergic agonists, glucocorticoids, sodium cromoglycate-like drugs, anticholinergic and antihistaminic H 1 antagonists(10). Their therapeutic effects include bronchodilatation, receptor and physiological antagonism, prevention of inflammatory responses induced by secondary cells, and finally, inhibition of mast cell activation(11). This review is concerned with compounds having inhibitory action on mast cell activation, and their possible importance on the pathophysiology of mast cell-related diseases.
Evidence suggests that prolactin (PRL) may have a role in immune function, but no data exist on the possible interference between PRL and inflammatory processes, in spite of the known correlation between inflammatory and immune reactions. In the present study the activity of prolactin on rat paw oedema was investigated. Repeated administrations of ovine PRL or a hyperprolactinaemia induced by pituitary gland graft provoked an evident increase of the inflammatory response induced by carrageenan. This effect was also present when adrenalectomised animals were used. Indomethacin completely suppressed the pro-inflammatory effect of ovine PRL; bromocriptine reduced the paw oedema, but when both bromocriptine and PRL were administered the two opposite effects seem to annual each other. Also phospholipase A2-induced paw oedema was potentiated by PRL pretreatment and inhibited by bromocriptine, whereas in dextran or serotonin-induced paw oedema both PRL and bromocriptine were ineffective. A possible involvement of prostaglandins and/or of phospholipase A2 in the pro-oedemigenic activity of PRL is suggested.
The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2- to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous injection of a sublethal dose of endotoxin (5 micrograms/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.
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Intraperitoneal and intra-articular (knee joint) injection of zymosan in the rat caused two phases of increased vascular permeability, a rapid increase (0.25–0.5 h) and a secondary increase (2–3 h) which was temporally associated with the onset of leukocyte infiltration. Intraperitoneal injection of zymosan led to a single peak of eicosanoid production (LTB4, C4, D4, E4 and 6-oxo-PGF1α
) which was maximal at 0.125–0.25 h. Intra-articular injection led to an initial peak of LTB4 production (maximal at 0.25 h) and a secondary peak of LTB4 and PGE2 production (maximal at 3 h). Oral administration of the 5-lipoxygenase (5-LO_ inhibitors phenidone, BW A4C (N-hydroxy-N-[3-(3-phenoxyphenyl)-2-propenyl] acetamide), A63162 (N-hydroxy-N-[1-(4-(phenylmethoxy) phenyl)ethyl] acetamide and ICI 207 968 (2-[3-pyridylmethyl]-indazolinone inhibited LTB4 production in A23187 stimulation bloodex vivo. The glucocorticosteroid dexamethasone had no effect in this model. The initial phase of increased vascular permeability in the peritoneal cavity and LTB4 production was dose dependently inhibited by the 5-LO inhibitors phenidone, BW A4C, A63162, and ICI 207 968 but not by dexamethasone or colchicine. The initial phase of increased permeability in the joint was unaffected by phenidone, BW A4C, dexamethasone or colchicine. However the latter two drugs inhibited the later phase of increased permeability and leukocyte infiltration in the joint and peritoneal cavity. These results demonstrate that zymosan induces eicosanoid productionin vivo but the relative importance of these mediators varies depending on the inflammatory site. Dexamethasone had a similar profile of anti-inflammatory activity to colchicine suggesting that inhibition of cell infiltration is the major mechanism of its therapeutic effect. No evidence for dexamethasone induced inhibition of eicosanoid production could be found suggesting that inhibition of the production or activity of other mediators is more important.
Prostaglandins (PGs) are believed to be involved in some of the manifestations of the acute phase response, which may be triggered by bacterial lipopolysaccharide (LPS). Glucocorticoids are part of this response and, among other things, regulate PG synthesis and are anti-inflammatory. We investigated the role of endogenous and exogenous glucocorticoids in the regulation of aortic prostacyclin synthesis and in its response to LPS. Rats were injected with LPS and their aorta incubated ex vivo. Aortic prostacyclin (PGI2) production declined 1 h after LPS injection and remained low for at least 96 h. On the other hand, LPS stimulated PGI2 production in adrenalectomized rats, although the latter had reduced capacity to synthesize PGI2, compared with sham-operated rats. Dexamethasone substitution restored synthesis. In intact rats only acute (2 h), but not repeated administration of dexamethasone, increased PGI2 production. In vitro IL-1 alpha stimulated aortic PGI2 synthesis. It is concluded that glucocorticoids may exert a biphasic influence on aortic PGI2 production, possibly through a dual action of lipocortins. Moreover, it is suggested that lipocortin requires activation before it can exert its full effect, and that agonists such as LPS may provide the stimulus for such activation.
Injection of bacterial lipopolysaccharide (LPS) into animals results in a transient increase in serum tumor necrosis factor (TNF). Maximal increases in TNF were detected by 1 h and 3-4 h serum TNF was no longer apparent. These animals were LPS tolerant and a repetitive LPS stimulus did not result in an additional peak in TNF. Regulation of TNF expression in LPS-tolerant animals was at the transcriptional level as TNF mRNA was not apparent in spleen or peritoneal macrophages following a second LPS stimulus. Adrenalectomized (adrex) mice, in contrast, did not become LPS tolerant and sera from these animals demonstrated an additional peak in TNF 1 h following a second LPS stimulus. Concomitant with the secondary rise in serum TNF in adrex mice was an increase in splenic TNF mRNA. The ability of adrex mice to become LPS tolerant was restored by exogenous glucocorticoids. LPS tolerance was also investigated in the galactosamine LPS model which like the adrex model is characterized by a thousandfold increase in the sensitivity of these animals to the lethal effects of LPS. Consistent with the absence of LPS tolerance in adrex mice, galactosamine-sensitized mice were also responsive to a second LPS stimulus and did not become LPS tolerant. While LPS-treated adrex mice had no significant increases in serum corticosterone, corticosterone levels in LPS-treated galactosamine-sensitized mice were comparable to LPS-stimulated normals suggesting that LPS tolerance involves both glucocorticoid-dependent and -independent components. Finally, prophylactic administration of a monoclonal antibody against murine TNF protected normal and galactosamine-sensitized mice from a lethal dose of LPS and yet had no protective effect in adrex animals.
IT IS well established that glucocorticoids exert catabolic effects on connective tissue and delay wound healing. They induce skin atrophy partly by decreasing the proliferative capacity of skin fibroblasts and the synthesis of the ground substance. Despite extensive investigations, the mechanisms of these in vivo steroid effects remain a matter of controversy, since the studies performed to elucidate the actions of adrenal steroids on the proliferation of various types of fibroblasts in vitro have led to contradictory results. Some authors have indeed described an inhibition of the proliferation of fibroblast cultures treated with glucocorticoids, whereas others have reported a stimulatory action of these drugs on cell division.
In the present paper, we have attempted to present a comprehensive review of the data and thus provide an explanation for the differences observed among in vitro experiments. In fact, many of the reported discrepancies may be due, in part, to the use of different experimental models, of different experimental schedules and culture conditions, and to the various methods employed to monitor cell proliferation. Moreover, the complexity of the actions of glucocorticoids in vivo may be explained by the demonstration that, in addition to their metabolic effects on fibroblasts, glucocorticoids also indirectly affect fibroblast proliferation by controlling the syntheses or actions of various factors produced by other cell types.
Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
1 Peritoneal macrophages (M phi) collected from adrenalectomized (ADX) rats released more interleukin-1 (IL-1) activity and prostaglandin E2 (PGE2) than macrophages from sham-operated (SHO) rats. 2 The increase in IL-1 activity in the supernatants was confirmed by the increase of the cell-associated 33 kD IL-1 alpha precursor in ADX macrophages stimulated by lipopolysaccharide (LPS). 3 After the injection of Complete Freund's Adjuvant (CFA) to induce adjuvant arthritis, 60% of the ADX rats died, while no deaths occurred in the SHO group. 4 The in vivo administration of dexamethasone inhibited both IL-1 and PGE2 release by macrophages as well as protecting ADX animals from CFA-induced death. Indomethacin and BW 755C partially protected the animals from this lethal effect. 5 These results suggest that adrenalectomy induces an increased release of IL-1 both in vitro and in vivo, and are consistent with a feedback mechanism between IL-1 and glucocorticoid hormones.
Familial Mediterranean fever is an autosomal recessively inherited disorder of unknown cause characterized by recurrent attacks of inflammation, involving mainly the peritoneum, pleura, synovia, and skin. Based on a phenotype analysis, we propose that its manifestations may be related to a genetic defect in one of the family of lipocortin proteins. Evidence is presented supporting an abnormality in the first step of prostaglandin/leukotriene synthesis.
We have investigated the in vivo regulatory network involving the neuroendocrine system, interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF). Adrenalectomy or hypophysectomy shifted the sensitivity curve to lipopolysaccharide (LPS)-induced lethal shock as well as TNF- and IL-1-induced deaths. Serum levels of IL-1 or TNF were altered in adrenalectomized or hypophysectomized mice following in vivo stimulation with LPS when compared to appropriate sham-operated control mice. Exogenous administration of either IL-1 or TNF could induce increases in serum corticosterone in sham-operated mice. Finally, treatment of adrenalectomized mice with corticosterone or dexamethasone could inhibit the induction of serum IL-1 and TNF and modified the pattern of these cytokine-induced deaths. Dexamethasone was more effective in these conditions than the natural glucocorticoid, corticosterone. Taken together, these data provide in vivo evidence for a feedback system involving the neuroendocrine axis (hypothalamus, pituitary and adrenal glands) leading to corticosterone production and subsequent regulation and/or modulation of IL-1 or TNF levels or activity.
Secretion of pituitary immunoreactive beta-endorphin is hypothesized to modulate the perception of pain. The present study examined this question by evaluating the effects of intravenous placebo or dexamethasone (0.1, 0.32, or 1.0 mg) on suppression of immunoreactive beta-endorphin secretion and development of postoperative pain after the surgical removal of impacted third molars in 48 patients. Compared with placebo, all doses of dexamethasone suppressed the postoperative increase in circulating levels of immunoreactive beta-endorphin. Patients administered 0.1 mg dexamethasone reported greater levels of pain, compared with those given placebo, from 60 through 120 minutes after surgery. Postoperative pain for the 0.32 and 1.0 mg doses did not differ from that for the placebo group. The increased pain after suppression of beta-endorphin release by the low dose of dexamethasone suggests that pituitary secretion of immunoreactive beta-endorphin alleviates postoperative pain under these conditions.
The mechanism of rat thrombocytopenia induced by i.v. injections of platelet-activating factor (PAF-acether) was investigated. Platelet counts performed after diluting the blood samples in 1% formalin in saline showed that PAF-acether (6 micrograms/kg i.v.) induced a significant thrombocytopenia in rats, which peaked within 1 h, followed by a drastic increase of platelet counts at 4 h and a return to basal levels at 24 h. At this time, it was not possible to induce thrombocytopenia with a second challenge with PAF-acether, indicating a clear state of desensitization which disappeared within five days after the first injection of PAF-acether. The pretreatment with the specific PAF-acether receptor antagonist, BN 52021 (2.5-15 mg/kg), 48740 RP (6-25 mg/kg) and WEB 2086 (0.25-1 mg/kg) blocked the thrombocytopenia dose dependently. The lipoxygenase inhibitor nordihydroguaiaretic acid, at 25-100 mg/kg, was also effective against the thrombocytopenia induced by PAF-acether, reinforcing the potential involvement of arachidonic acid derivatives in this process. Adrenal hormones may modulate this process, since adrenalectomized animals responded to PAF-acether with exacerbated thrombocytopenia. No reduction in the platelet counts was noted when the blood was diluted in formalin-free saline, indicating that unstable aggregates were formed in vivo, which tended to resolve in vitro. Our results suggest that the thrombocytopenia induced in rats by PAF results from a reversible process of intravascular platelet aggregation, probably following the secretion of platelet-activating substances released by a first-hit blood cell.
Glucocorticoids act on almost all organs and tissues. Their levels are rarely constant, showing both pronounced circadian variations and/or a rise during stressful situations. It is thought that glucocorticoids evoke an adaptation to stress via a variety of mechanisms including mobilization of fuel for metabolism, enhancement of cardiovascular function, and production of euphoria or behavioral activation. Glucocorticoids also suppress the immune system. It has been argued that this effect helps prevent autoimmune activation following “fight or flight” situations that result in trauma and inflammation.
- Since a direct effect of tixocortol pivalate (TP) has been described on cyclooxygenase pathway, local anti-inflammatory activities of some 21 thiol derivatives of steroids were investigated on the carrageenin-induced pleurisy model in comparison with dexamethasone (Dex) or other anti-inflammatory drugs. LTC4/D4 contents in pleural fluid were assayed by RIA as well as PGE2 levels to characterize the effects on arachidonate pathways.
- After oral administration, TP was inactive up to 1 g/kg on exudate volume and leukocyte migration as expected for this strict local anti-inflammatory steroid contrary to Dex (ID50=0.05–0.41 mg/kg).
- When administered locally, TP and tixocortol (T) exerted a dose dependent inhibitory activity on exudate volume (ID30=12.4 μg or 13.1 μg/pleural cavity) and leukocyte count (ID30=83 or 230 μg); in the same conditions, Dex was more active (ID30=0.7 and 2.6 μg).
- All these steroids decreased PGE2 and LTC4/D4 contents in exudate fluids, respectively TP (50 μg/pleural cavity) by 28 and 63%; T (100 μg) by 33 and 31%; Dex (5 μg) by 43 and 40%.
- Local co-administration of RU 486 (50 μg) with either TP, T or Dex reversed the anti-inflammatory effects of all steroids, indicating in these conditions a local activity through glucosteroid receptor occupancy.
The effect of purified crotapotin, a non‐toxic non‐enzymatic chaperon protein normally complexed to a phospholipase A 2 (PLA 2 ) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 μg/paw) and 5‐hydroxytryptamine (5‐HT) (3 μg/paw) in the rat hind‐paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea‐pig isolated lung were also investigated.
Subplantar co‐injection of crotapotin (1 and 10 μg/paw) with carrageenin or injection of crotapotin (10 μg/paw) into the contralateral paw significantly inhibited the carrageenin‐induced oedema. This inhibition was also observed when crotapotin (10–30 μg/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60°C) failed to inhibit carrageenin‐induced oedema. Subplantar injection of crotapotin (10 μg/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5‐HT‐induced oedema.
In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5‐HT stores.
Crotapotin (30 μg/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 m m ) and platelet activating factor (1 μ m ) in human platelet‐rich plasma. The platelet aggregation and thromboxane B 2 (TXB 2 ) release induced by thrombin (100 mu ml ⁻¹ ) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 μg/paw) did not affect the release of 6‐oxo‐prostaglandin F 1α and TXB 2 induced by ovalbumin in sensitized guinea‐pig isolated lungs.
Our results indicate that the anti‐inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo‐oxygenase activity. It is possible that crotapotin may interact with extracellular PLA 2 generated during the inflammatory process thereby reducing its hydrolytic activity.
Recent experimental evidence confirms the interrelationships between the central nervous, neuroendocrine and immune systems. Indeed, extensive duality exists in the use of neurotransmitters, hormones and receptors each system displays. In the present annotation, the effect of cytokines, soluble mediators of immune function, on the CNS and neuroendocrine systems is addressed and conversely, we discuss the modification of the immune compartment by the sympathetic nervous and neuroendocrine systems, with particular reference to the role of noradrenaline and corticosterone. Dysfunction between the systems is considered in the context of autoimmune conditions, with emphasis on experimental allergic encephalomyelitis and the contribution of corticosterone-driven T-cell apoptosis to recovery from the disease. Finally, we speculate on the relevance of neuroimmune interactions in the pathogenesis of multiple sclerosis.
To investigate the effects of nonsteroidal antiinflammatory drug (NSAID) therapy and water immersion on hypothalamic-pituitary-adrenal (HPA) axis function in rheumatoid arthritis (RA).
Plasma levels of adrenocorticotropic hormone (ACTH) and serum and urine levels of cortisol were compared in untreated RA patients, NSAID-treated RA patients, and healthy control subjects.
ACTH levels were significantly higher in untreated RA patients (mean +/- SEM integrated area 11,377 +/- 5,246 hours ng/liter) than in NSAID-treated RA patients (2,285 +/- 388 hours ng/liter) or healthy controls (1,845 +/- 35.5 hours ng/liter) (P < 0.001). Serum and urine cortisol levels were not significantly different between groups. Two-hour head-out water immersion had no effect.
Elevated ACTH levels without hypercortisolemia occur in untreated RA. NSAID therapy alters HPA axis response, but immersion has no effect.
The effects of long-term chronic stress (induced by repeated restraint, overcrowding or both), short-term chronic stress (induced by a triad of stressors over a short period of time early in life) and adrenalectomy were investigated on the prevalence, on the degree of insulitis and various physiological and immunological parameters in the NOD mouse, a spontaneous model of type I-insulin-dependent diabetes mellitus (IDDM). Long-term chronic stress, obtained by restraint once a week or overcrowding, significantly protected NOD females, while both applied concomitantly had only a tendency to protect against diabetes. In contrast, short-term chronic stress had no significant effect on diabetes expression, whereas adrenalectomy resulted in a trend toward accelerated diabetes onset. The various long-term chronic stress paradigms exerted different effects on the progression of insulitis: repeated restraint tended to protect against insulitis, overcrowding had no effect but, when associated with restraint, significantly counteracted the beneficial effect of restraint alone. Adrenalectomy and short-term chronic stress had no significant effect on the development of insulitis. Various parameters, such as body, thymus and spleen weights, thymus and spleen cellularities, mitogen-induced spleen cell proliferation and serum corticosterone levels were also studied under the various experimental conditions. Taken together, the observations suggest that stressors modulate the expression of spontaneous autoimmune diabetes by exerting pleiotropic effects on immune and/or inflammatory components at the pancreas level and on peripheral glucose metabolism.
YM‐26734 [4‐(3,5‐didodecanoyl‐2,4,6‐trihydroxyphenyl)‐7‐hydroxy‐2‐(4‐hydroxyphenyl)chroman] dose‐dependently inhibited the activities of extracellular phospholipase A 2 (PLA 2 ): rabbit platelet‐derived group II and porcine pancreas‐derived group I PLA 2 , with IC 50 values of 0.085 (0.056–0.129, n = 5) and 6.8 (5.0–9.6, n = 5) μ m , respectively.
In contrast, YM‐26734 did not reduce the activity of intracellular PLA 2 prepared from mouse macrophages, which preferentially hydrolyzed arachidonoyl phospholipids at concentrations up to 50 μ m . YM‐26734 also showed no effect against either sheep seminal vesicle cyclo‐oxygenase or rat leukocyte 5‐lipoxygenase.
Lineweaver‐Burk analysis showed that YM‐26567‐1 behaved as a competitive inhibitor of group II PLA 2 derived from rabbit platelets, with a K i value of 48 n m .
In mice, YM‐26734 inhibited 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA, 1 μg/ear)‐induced ear oedema in a dose‐dependent manner, with ED 50 values of 45 (30–67) μg/ear ( n = 5) and 11 (4–32) mg kg ⁻¹ , i.v. ( n = 5), but did not decrease arachidonic acid (4 mg/ear)‐induced ear oedema at 1 mg/ear and 30 mg kg ⁻¹ , i.v.
In rats, the accumulation of exudate fluids and leukocytes in the pleural cavity in response to carrageenin injection (2 mg) was significantly less in a group treated with YM‐26734 (20 mg kg ⁻¹ , i.v.) than in the control group (0.43 ± 0.02 vs 0.59 ± 0.03 g per cavity and 3.8 ± 0.2 vs 4.9 ± 0.3 × 10 ⁷ cells per cavity, respectively; n = 5).
These results suggest that YM‐26734 is a potent and competitive inhibitor of extracellular PLA 2 with selectivity for group II PLA 2 , and that the inhibition of group II enzymes activity may cause the suppression of inflammatory responses to TPA and carrageenin.
Endogenous glucocorticoids undoubtedly play a role in the control of immune responses: their contribution to inter-strain variation is unknown. The development of specific IgG and IgE was measured following inoculation with ovalbumin in Lewis, Fischer, Wistar and Brown Norway rats. The Lewis gives a smaller IgG and IgE response than the other strains and the response in vivo to antigen injected into the paw correlates with the titre of specific antibody. Treatment with the steroid receptor antagonist RU486 (mifepristone) following inoculation reveals that in the Lewis, and to a lesser extent in the Brown Norway, the development of a specific IgG response is limited by endogenous corticosteroids. The IgG response in different strains is differently sensitive to treatment with the synthetic glucocorticoid dexamethasone, the Lewis being particularly resistant. The importance of control by endogenous corticosteroids should not be overlooked in contributing to strain differences in immune response.
Lipomodulin, purified to near homogeneity from rabbit peritoneal neutrophils, was phosphorylated by cyclic AMP-dependent protein kinase from bovine heart with concomitant loss of its ability to inhibit phospholipase A2 from porcine pancreas. Phosphorylation of lipomodulin was confirmed by the incorporation of 32P from [gamma-32P]ATP. To demonstrate that lipomodulin undergoes phosphorylation in vivo, rabbit peritoneal neutrophils were incubated with 32P and lipomoculin was isolated by immunoprecipitation with serum from a patient with systemic lupus erythematosus which has anti-lipomodulin antibody. Analysis of 32P-labeled immunoprecipitates by sodium dodecyl sulfate electrophoresis revealed a single peak of radioactivity that comigrated with [35S]methionine-labeled lipomodulin. The administration of a chemoattractant, N-formyl-methionyl-leucyl-phenylalanine to intact rabbit neutrophils, resulted in a marked increase in arachidonate release from the cells and an increase in 32P incorporation into lipomodulin. A close correlation was found between the extent of phosphorylation of lipomodulin and the rate of arachidonate release. Phosphorylation of lipomodulin in neutrophils gradually returned to the control level with corresponding cessation of arachidonate release. In contrast to the in vitro system, phosphorylation of lipomodulin and release of arachidonic acid from peptide-stimulated neutrophils required Ca2+ entry into the cells. These results suggest that the phosphorylation-dephosphorylation of lipomodulin, phospholipase inhibitory protein, is an important mechanism for chemotactic receptor-mediated regulation of arachidonic acid release in rabbit neutrophils.
Basal levels of glucocorticoids maintained by negative feedback regulation are known to modulate a wide range of physiological processes, through a variety of effects such as those on carbohydrate metabolism and “permissive” actions on effects of other hormones. Glucocorticoid levels increase sharply in response to the stress of any kind of threat to homeostasis. The increased levels have traditionally been ascribed the function of enhancing the organism’s resistance to stress. How known physiological and pharmacological effects of high levels of glucocorticoids might accomplish this function, however, has been a mystery.
A generalization that is beginning to emerge is that many of these effects may be secondary to modulation by glucocorticoids of the actions of numerous intercellular mediators, including established hormones, prostanoids, neutral proteinases, and cytokines such as interferon. These mediators participate in physiological mechanisms — endocrine, renal, immune, neural, etc. — that mount a first line of defense against such challenges to homeostasis as hemorrhage, metabolic disturbances, infection, anxiety, and others.
Contrary to the traditional view that the role of glucocorticoids in stress is to enhance these defense mechanisms, it has become increasingly clear that glucocorticoids at moderate to high levels generally suppress them. This paradox first emerged when glucocorticoids were discovered to be antiinflammatory agents, and had remained a major obstacle to a unified picture of glucocorticoid function.
We have suggested that stress-induced increases in glucocorticoid levels protect not against the source of stress itself but rather against the body’s normal reactions to stress, preventing those reactions from overshooting and themselves threatening homeostasis. This hypothesis, the seeds of which are to be found in many earlier discussions of glucocorticoid effects, immediately accounts for the paradox noted above, and provides glucocorticoid physiology with a unified conceptual framework that can accommodate such apparently unrelated physiological and pharmacological effects as those on carbohydrate metabolism, inflammatory processes, shock and water balance. It also leads us to propose that some enzymes rapidly induced by glucocorticoids detoxify mediators released during stress-induced activation of primary defense mechanisms; those mediators could themselves cause damage if left unchecked.
When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [1-14C]arachidonic acid previously incorporated into phospholipids. The inhibitory potencies of glucocorticoids on phospholipase A2 activity correlated well with their anti-inflammatory activities and their abilities to bind to glucocorticoid receptors. Inhibitors of RNA and protein synthesis suppressed the inhibitory effect of glucocorticoids on phospholipase A2 activity. Digestion of the glucocorticoid-treated cells by Pronase overcame the inhibitory activity. Phospholipase A2 activity induced by Ca2+ ionophore A23187 was not affected by Pronase treatment. Gel filtration of proteins from neutrophil membranes labeled with [3H]lysine showed an induction of protein(s) (about 40,000 daltons) after glucocorticoid treatment. This protein inhibited a partially purified pancreatic phospholipase A2 and reduced the peptide-initiated chemotactic response of neutrophils.
During the last few years increasing experimental evidence has accumulated which indicates that PAF-acether (AGEPC, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is a potential mediator of inflammation and anaphylaxis (1,2). In chapter 6 of this book Page and co-workers give evidence that PAF-acether fulfils most of the criteria appropriate for an inflammatory mediator. Among these criteria, however, there are two points that await further support by experimental data. Firstly, the generation of PAF-acether during inflammation has been only scantily documented. Secondly, there is no evidence that the inhibition of the release or the actions of PAF-acether leads to an anti-inflammatory effect.
Two systems were used to test the effect of anti-inflammatory and anti-rheumatic drugs on chemotactic activity of cell-free exudates and also on the chemotactic responsiveness of exudate leucocytes. (1) Inflammatory cell-free exudates from treated rats were tested for their chemotactic activity on exudate leucocytes from untreated rats. (2) Polymorph and mononuclear cells from treated rats were tested for their responsiveness to the chemotactic activity of cell-free exudates from untreated rats. Levamisole, coumarin and D-penicillamine were ineffective in (1) and (2). Colchicine reduced chemotaxis of polymorphs in both systems (1) and (2), whereas no effect was observed on mononuclears. Naproxen was more effective in reducing the chemotaxis of polymorphs compared with mononuclears in systems (1) and (2). In contrast, indomethacin and dexamethasone reduced the chemotaxis of both polymorph and mononuclear cells in systems (1) and (21. Of the drugs tested dexamethasone exhibited the highest potency. These results emphasize the necessity for studying both cellular and humoral factors in the evaluation of the action of anti-inflammatory drugs on chemotaxis.
The accumulation of leucocytes at the site of inflammation may be brought about by chemotaxis or proliferation in the extravascular tissues. The present paper focuses on the chemotactic properties of different types of experimental inflammatory pleural exudates, using a modified Boyden chamber. The time-course of carrageenan-induced exudate chemotactic activity for polymorphs was maximal at 4 h, thereafter diminishing towards 24 and 48 h. Chemotactic activity for mononuclear cells remained unchanged throughout the 4--48 h time-course. Heating the exudates to 56 degrees C for 1 h partially reduced chemotactic activity. These results correlate well with the migration of polymorph and mononuclear cells into the pleural cavity during carrageenan-induced pleurisy. The potency of polymorph and mononuclear cell chemotactic activity of different exudates was of the following order: carrageenan greater than calcium pyrophosphate greater than reverse passive Arthus greater than dextran. The results are discussed in order to elucidate the differences between the underlying mechanisms responsible for leucocyte accumulation in different types of inflammatory reaction.
A simple double‐isotope assay for phospholipase A 2 activity of perfused organs is described.
Guinea‐pig lungs perfused through the pulmonary circulation exhibit a low background enzyme activity. This activity is blocked by dexamethasone, betamethasone and hydrocortisone, mepacrine, procaine or chlorpromazine. Aspirin and indomethacin are without effect.
Mechanical trauma, antigen challenge or injections of bradykinin, rabbit aorta contracting substance‐releasing factor (RCS‐RF) or histamine increase ‘basal’ phospholipase activity. The effect of these agents, except that of bradykinin, is blocked by dexamethasone or mepacrine.
The blocking effect of steroids is cumulative and dose‐dependent. They do not work in cell‐free systems. Inhibition by mepacrine is rapid and is effective in cell‐free lung homogenates.
It is suggested that agents which liberate prostaglandin endoperoxides and thromboxane A 2 from perfused lungs do so by activating phospholipase A 2 .
Dexamethasone and hydrocortisone induce the release of anti‐phospholipase proteins into the peritoneal cavities of rats.
Adrenocorticotropic hormone (ACTH) also releases these proteins in normal but not in adrenalectomized rats.
Peritoneal lavage proteins were separated by ion‐exchange and size exclusion chromatography. The anti‐phospholipase activity occurred in four separate fractions with the major component having an apparent mol. wt. of 40 k.
Column fractions containing these anti‐phospholipase proteins had anti‐inflammatory effects in the rat carrageenin pleurisy model whereas other fractions were inactive.
The proteins appear to be identical to macrocortin and lipomodulin, the ‘second messengers’ of glucocorticoid hormone action on the arachidonate system.
A radioimmunoassay for leukotriene B4 has been developed. The assay is sensitive; 5 pg LTB4 caused significant inhibition of binding of [3H]-LTB4 and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B2 and arachidonic acid do not exhibit detectable cross-reactions (less than 0.03%). However, some non-cyclic dihydroxy- and monohydroxy-eicosatetraenoic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic and 12-hydroxy-5,8,10,14-eicosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB4 from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB4 by reverse phase high pressure liquid chromatography following solvent extraction and silicic acid chromatography.
Glucocorticoid-induced anti-phospholipase proteins were partially purified by using ion-exchange and molecular sieve chromatography. These proteins, as well as dexamethasone itself, inhibited the hind-paw rat oedema induced by carrageenin. This inhibition was reversed by arachidonic acid, Anti-phospholipase proteins as well as hydrocortisone, also reduced the formation of prostaglandin E2 and leukotriene B4 by phagocytosing leucocytes. A specific monoclonal antibody was able to reverse the inhibition of eicosanoid formation. The mechanism of the anti-inflammatory effect of glucocorticoids and anti-phospholipase proteins is discussed in the light of these results.
Anti‐inflammatory steroids reduce prostaglandin E 2 (PGE 2 ) synthesis in rat renomedullary interstitial cells in culture by inhibiting the release of arachidonic acid from membranous phospholipid stores, exhibiting antiphospholipase‐like properties.
After treatment of the cells with dexamethasone 10 ⁻⁶ M , these cells release a protein in the supernatant.
This supernatant is able to inhibit PGE 2 secretion in untreated cells and to inhibit phospholipase A 2 activity in an in vitro system.
Using chromatofocusing separation, we showed that two distinct proteins exist with isoelectric points of 5.8 and 8.3.
Using gel permeation separation, we showed that two proteins exist with apparent molecular weights of 15,000 and 30,000 daltons.
We conclude that, in renal cells in culture, anti‐inflammatory steroids induce the synthesis and the release of two polypeptides which we have named ‘Renocortins’ (induced by corticoids in renal cells) causing the antiphospholipase‐like action of glucocorticoids.
Our results are in good agreement with others, but as renal cells are not directly involved in the inflammatory process, we suggest that this steroid‐induced phenomenon is not solely involved in the inflammatory reaction but is of more general physiological relevance.
Dexamethasone induces the synthesis of a phospholipase A2-inhibitory protein (PLIP) of molecular weight approximately equal to 55,000 from calf thymus and PLIPs of molecular weights 55,000, 40,000, 28,000, and 15,000 from A/J mouse thymus and from 12-day embryonic B10. A mouse palates. Sufficient quantities of calf thymus PLIP and of the 15,000 molecular weight mouse thymus and palate PLIPs were prepared and tested as inhibitors of programmed cell death in the medial-edge epithelium of single mouse embryonic palatal shelves in culture. All of the proteins tested prevent the loss of the medial-edge epithelium and, thus, produce the teratogenic effects of glucocorticoids in the palatal culture model. This teratogenic action of both PLIP and glucocorticoids is reversed by arachidonic acid, the precursor of prostaglandins and thromboxanes, suggesting that PLIP mediates the effects of glucocorticoids by inhibiting phospholipase A2.
Although cyclo‐oxygenase products have been detected at inflammatory sites the tissue of origin remains uncertain.
Inflammatory exudates were collected from rats 4, 6, 8, 12 or 24 h after subcutaneous implantation of carrageenin‐impregnated sponges.
Concentrations of the cyclo‐oxygenase products prostaglandin E 2 (PGE 2 ), 6‐oxo‐PGF 1α and thromboxane B 2 (TXB 2 ) in inflammatory exudates and serum (obtained from blood clotted at 37° C) were measured by specific radioimmunoassays.
TXB 2 concentrations in exudates increased to about 100 ng ml ⁻¹ at 8 h but decreased to less than 20 ng ml ⁻¹ after 24 h. PGE 2 concentrations increased from 4–12 h and remained between 80 and 120 ng ml ⁻¹ from 12–24 h. 6‐oxo‐PGF 1α had the same time course as that of PGE 2 but concentrations were approximately one third of PGE 2 values.
TXB 2 concentrations in serum from thrombocytopaenic rats were less than 5% of control values. Thrombocytopaenia did not affect TXB 2 , PGE 2 or 6‐oxo‐PGF 1α concentrations or total leukocyte numbers in inflammatory exudates.
Methotrexate‐induced neutropaenia did not affect serum TXB 2 concentrations but cyclo‐oxygenase products (including TXB 2 ) in 6 h inflammatory exudates were reduced by 60–95%.
Colchicine (1.0 mg kg ⁻¹ s.c.) prevented leukocyte accumulation in sponge exudates and this was accompanied by a reduction in TXB 2 , PGE 2 and 6‐oxo‐PGF 1α concentrations at 6 h.
These results indicate that platelets are the source of TXB 2 in clotting blood but do not contribute to cyclo‐oxygenase activity in experimental inflammation. The results also suggest that migrating leukocytes are the major source of thromboxane and to a lesser degree prostaglandins in acute 6 h inflammatory exudates.
Anti-inflammatory steroids induce the release in vivo of antiphospholipase proteins (APP) into the peritoneal cavities of rats. APP were partially purified by ion- exchange chromatography. The main anti-phospholipase activity was recovered in two zones of the elution gradient named APP I and APP II; their molecular weight (mol. wt) was determined with molecular sieve chromatography. Two phospholipase A2 (PLA2) activities were identified from rat peritoneal leucocytes, one with a pH optimum at 4.5 (a lysosomal enzyme) and one with pH optimum at 8.5 (a membrane-bound enzyme); the selective secretion of the former was observed when leucocytes were stimulated by phagocytosis. The effect of APP on both enzyme activities was studied on enzyme preparations from resting leucocytes. APP were also added to leucocytes incubated with or without phagocytozable material. After incubation, PLA2 activities were determined both inside the cells and in the culture medium. APP I revealed a mol. wt of 200 k with a small fragment of 15 k and inhibited membrane-bound PLA2; APP II revealed a mol. wt of 40 k and inhibited lysosomal PLA2.
Anti-inflammatory glucocorticoids inhibit prostaglandin (PG) biosynthesis by preventing arachidonic acid release from phospholipids rather than inhibiting the cyclooxygenase. As in other cells, this steroid action depends on receptor occupation and de novo protein/RNA biosynthesis. We have previously shown in guinea pig perfused lungs and rat peritoneal leukocytes that the effect of steroids in PG generation is mediated by an uncharacterized 'second messenger'. Now, we report that this factor (which we have named 'macrocortin') is an intracellular polypeptide whose release and synthesis are stimulated by steroids. Macrocortin derived from rat peritoneal leukocytes is very similar to that released from guinea pig lungs.
Macrocor-tin and the Mechanism of Action of the Glucocorticoids The adrenal medulla and adrenal cortex
- R J Flower
- J N Wood
- L Parente
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Nomenclature announcement, Anti-phospholipase proteins
- M Flower
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- L Russo-Marie
DI ROSA, M., FLOWER, R.J., HIRATA, F., PARENTE, L. & RUSSO-MARIE, F. (1984). Nomenclature announcement, Anti-phospholipase proteins. Prostaglandins, 28, 411-442.
A radio-immunoassay for leukotriene B4
- J A Simmons
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radio-immunoassay for leukotriene B4. Prostaglandins,
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Studies on cell motility in inflammation. I. The chemotactic activity of experimental inflammatory exudates
- PARENTE
A radio-immunoassay for leukotriene B4
- SALMON
Nomenclature announcement, Anti‐phospholipase proteins
- ROSA M.