Flavio Coceani

Mechanisms of fever induced by recombinant human interferon.

Since the early trials using human interferon (hIFN) derived from blood leukocytes or cell lines, fever has been a prominent component of IFN therapy. Human protein impurities might account for the fever to cell-derived hIFN, but recombinant hIFN, free of extraneous human proteins, has produced fever in nearly all recipients during clinical trials. Our present studies were carried out to determine the mechanisms of fever due to recombinant hIFN currently being used in humans. Because recombinant hIFN is produced in Escherichia coli, in these experiments we considered contaminating endotoxin as the cause of fever. Polymyxin B, which blocks endotoxin, had no effect on the pyrogenicity of hIFN in rabbits. In addition, hIFN injected into an endotoxin-resistant strain of mice produced fever. The pyrogenicity of hIFN does not appear to involve production of leukocytic pyrogen (LP), since no circulating LP was detected in rabbits during IFN fever. Furthermore, human mononuclear cells incubated with hIFN in vitro at 10(4)-10(6) U/ml did not release LP. However, hIFN stimulated prostaglandin E2 (PGE2) release from rabbit hypothalamic tissue in vitro. Intracerebroventricular injection of hIFN into the awake cat also produced fever and a rise in PGE2 levels in the cerebrospinal fluid; both effects were reversed by treatment with indomethacin. We conclude that the fever of recombinant hIFN is not due to endotoxin but that hIFN is intrinsically pyrogenic by inducing PGE2 in the hypothalamus.

Occurrence of interleukin-1 in cerebrospinal fluid of the conscious cat

Interleukin-1 (IL-1) is synthesized and released in response to various pathogens, including bacterial endotoxin, and is assigned an intermediary function in the genesis of fever. Its site of action in the central nervous system (CNS), however, is uncertain because the polypeptide is seemingly unable to cross the blood-brain barrier. Since several cell types, including astroglial, microglial, and vascular cells, can generate IL-1 upon appropriate stimulation, we examined whether IL-1 is formed in the CNS and may therefore serve as a messenger for systemic noxae. Experiments were conducted in the conscious cat and IL-1 was assayed in cerebrospinal fluid (CSF) from the third ventricle using a highly sensitive murine helper T cell line, D10.G4.1. In general, IL-1 levels were barely detectable in the absence of fever and did not increase at any stage of the sustained fever following intravenous injection of endotoxin (bolus) or crude monocyte supernate containing IL-1 (bolus plus infusion). In contrast, intracerebroventricular injection of a pyrogenic dose of endotoxin led to the appearance of IL-1 in the CSF. IL-1 levels reached maximal elevation during the uprise phase of the fever and declined thereafter. By the same route, natural or recombinant human IL-1 had no effect on CSF-IL-1 levels, though both preparations were as effective as endotoxin in eliciting fever. These findings confirm earlier data with radiolabelled pyrogens and indicate that the blood-brain barrier is impermeable to IL-1. We conclude that blood-borne IL-1 is likely to act at a discrete site outside the blood-brain barrier, possibly the organum vasculosum laminae terminalis. Centrally formed IL-1 may instead act diffusely in promoting fever and fever-related events (e.g. sleep).

Interleukin-6 as an endogenous pyrogen: induction of prostaglandin E2 in brain but not in peripheral blood mononuclear cells.

Fever induced by endogenous as well as exogenous pyrogens is often prevented by cyclooxygenase inhibitors; endogenous pyrogens stimulate prostaglandin E2 (PGE2) in or near the thermoregulatory centers of the brain. The cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), are two pyrogens which stimulate brain PGE2 formation during fever and also increase PGE2 synthesis in human mononuclear cells in vitro. In the present study, we examined whether interleukin-6 (IL-6) stimulates PGE2 formation in a manner similar to IL-1 and TNF. Both glycosylated and non-glycosylated forms of recombinant human IL-6 were tested. Following intravenous injection into rabbits, the glycosylated IL-6 was more pyrogenic than the non-glycosylated form and there was no evidence of synergy in the production of fever when IL-6 and IL-1 were given simultaneously. IL-6 fever was blocked by prior administration of the cyclooxygenase inhibitor ibuprofen. IL-6 was also pyrogenic in the cat by either the systemic or the intraventricular route. However, in both species, IL-6 was less effective than IL-1 beta. When given intraventricularly to cats, IL-6 produced an increase in PGE2 levels of the cerebrospinal fluid in parallel with the rise in body temperature. In the latter respect, IL-6 imitated IL-1 beta; however, IL-6 from 0.15-15 micrograms/ml did not increase mononuclear cell PGE2 production in vitro whereas IL-1 beta induced 20-30-fold increases in PGE2 at 100 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone.

1 We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction controlling the formation of endothelin‐1 (ET‐1). In the present study, we have examined whether CO is formed naturally in the vessel. 2 The CO‐forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO‐2) and inducible (HO‐1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO‐1 was localized to endothelial and muscle cells, while HO‐2 was found only in muscle cells. Inside the muscle cells, HO‐1 and HO‐2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO‐1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3 CO was formed enzymatically from added substrate (hemin, 50 μm) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 μM). 4 ZnPP (10 μM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin‐treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5 ZnPP (10 μm) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 μM)‐contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6 We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.

The influence of lipoxygenase inhibitors on the in vitro production of human leukocytic pyrogen and lymphocyte activating factor (interleukin-1)☆

Leukocytic pyrogen (LP), the endogenous mediator of fever, is synthesized and released from mononuclear phagocytes following activation by several microbial and immunologically-derived substances. Purified fractions of LP also stimulate thymocyte proliferation and LP seems to be indistinguishable from lymphocyte activating factor (LAF) otherwise known as interleukin-1 (IL-1). In the present investigation, we have examined the effect on IL-1 production of drugs inhibiting both cyclooxygenase- and lipoxygenase-mediated transformations of arachidonic acid (ETYA, 5,8,11,14-eicosatetraynoic acid and compound BW755C, 3-amino-1-3-trifluoromethylphenyl-2-pyrazoline). Ibuprofen inhibited the production of PGE2 from stimulated human monocytes but had no effect on LP and LAF release. ETYA prevented LP production from human monocytes when added to the incubation fluid prior to activation by Staphylococcus albus. When added after cell activation, ETYA was ineffective. Similar results were obtained using BW755C. Prostaglandin E2 (PGE2) levels in cell supernates were markedly decreased in the presence of either drug when compared to supernates from untreated, stimulated cells. Low PGE2 levels were also demonstrated in supernates of cells in which either ETYA or BW755C were added 1 hour after stimulation. Pretreatment with BW755C also resulted in decreased LAF activity in the supernates of mononuclear cells stimulated with staphylococci, endotoxin, or muramyl dipeptide. Other experiments demonstrated that crude or purified human LP retains its activity following treatment with soybean lipoxidase. These findings indicate that a product of arachidonate lipoxygenase is important in the sequence of events underlying cell activation for the production of human LP/LAF/IL-1. The possibility that LP might be an eicosanoid-peptide conjugate structurally resembling the leukotrienes was ruled out.

Carbon monoxide-induced relaxation of the ductus arteriosus in the lamb: evidence against the prime role of guanylyl cyclase.

1 . We have previously found that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction which sustains contractile tone. Our proposal, however, has been questioned on the evidence of findings in other blood vessels implicating the guanylyl cyclase‐based relaxing mechanism as the target for CO. To investigate this issue further, we have carried out experiments in the isolated ductus from near‐term foetal lambs and have examined the effect of CO concomitantly on muscle tone and cyclic GMP content, both in the absence and presence of guanylyl cyclase inhibitors, or during exposure to monochromatic light at 450 nm. 2 . CO (65 μm) reversed completely, or nearly completely, the tone developed by the vessel in the presence of oxygen (30%) and indomethacin (2.8 μm). Cyclic GMP content tended to increase with the relaxation, but the change did not reach significance. Sodium nitroprusside (SNP), a NO donor, mimicked CO in relaxing the ductus. Contrary to CO, however, SNP caused a marked accumulation of cyclic GMP with levels being positively correlated with the relaxation. 3 . Methylene blue (10 μm) reduced marginally the CO relaxation, whilst LY‐83583 (10 μm) had an obvious, albeit variable, inhibitory effect. Basal cyclic GMP content was lower in tissues treated with either compound and rose upon exposure to CO. However, the levels attained were still within the range of values for tissues prior to any treatment. Furthermore, the elevation in cyclic GMP was not related to the magnitude of the CO relaxation. 4 . Illumination of the ductus with monochromatic light at 450 nm reversed the CO relaxation and any concomitant increase in cyclic GMP. In the absence of CO, light by itself had no effect. 5 . Ductal preparations with only muscle behaved as the intact preparations in reacting to CO, both in the absence and presence of guanylyl cyclase inhibitors, or during illumination. 6 . We conclude that the primary action of CO in the ductus arteriosus is not exerted on the guanylyl cyclase heme and that cyclic GMP may only have an accessory role in the relaxation to this agent. This finding reasserts the importance of a cytochrom P450‐based mono‐oxygenase reaction for generation of tone and as a target for CO in the ductus.

Involvement of endothelin‐1 in hypoxic pulmonary vasoconstriction in the lamb.

1. Using isolated pulmonary resistance vessels from mature fetal lamb and chronically instrumented lambs (8‐17 days old), we have examined whether hypoxic pulmonary vasoconstriction is sustained by activation of a constrictor mechanism or suppression of a dilator mechanism. 2. Hypoxia contracted both arteries and veins in vitro, and the contraction was greater with the former. After removing the endothelium, arteries responded faster to hypoxia, but the magnitude of the response remained unchanged. 3. Hypoxic arteries, unlike normally oxygenated arteries, did not contract with either indomethacin (2.8 microM) or N omega‐nitro‐L‐arginine methyl ester (L‐NAME, 100 microM). The same vessels relaxed with sodium nitroprusside (SNP, 0.001‐10 microM) but not with bradykinin (0.1‐100 nM). 4. Endothelin‐1 (ET‐1, 0.01‐10 nM) contracted isolated arteries and veins under normoxic and hypoxic conditions. In both vessels, the contraction was fast in onset and subsidence, and was inhibited by the ETA receptor antagonist BQ123 (1 microM). The ET‐1 precursor, big ET‐1 (100 nM), also contracted arteries and veins, but compared with ET‐1 its action was slower in development. Big ET‐1 contraction, unlike ET‐1 contraction, was curtailed by the inhibitor of the ET‐1‐converting enzyme, phosphoramidon (50 microM). 5. ET‐1 (0.1‐10 nM) had no effect on isolated arteries precontracted with a thromboxane A2 (TXA2) analogue (ONO‐11113) and treated with BQ123 (10 microM). Under the same conditions, ET‐1 relaxed the veins. Accordingly, in the absence of BQ123 treatment, the selective ETB receptor agonist IRL‐1620 (0.1‐100 nM) relaxed the contracted veins but not the arteries. 6. BQ123 (10 microM) inhibited the constriction of isolated arteries and veins to hypoxia. Likewise, in the conscious lamb a bolus of BQ123 (0.4 mg kg‐1, injected into the pulmonary artery) curtailed the rise in pulmonary vascular resistance (Rpa) brought about by alveolar hypoxia without changing significantly systemic vascular resistance (Rao). Under normoxia, Rpa was insignificantly affected by BQ123. 7. The results indicate that pulmonary resistance arteries are more susceptible to hypoxia than the veins, and that hypoxic vasoconstriction does not require an intact endothelium to occur. Hypoxic tone is ascribed primarily to intramural generation of ET‐1, while removal of the tonic action of a relaxant may only have an accessory role in the response.

Endothelin a receptor is necessary for O2 constriction but not closure of ductus arteriosus

In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O(2) mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ET(A) receptor -/-, +/-, and +/+ genotypes were used. Isolated DA from term ET(A) +/+ fetuses contracted to O(2) (5-95%) and a thromboxane A(2) analog (ONO-11113, 0.1 microM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1-100 nM). Indomethacin (2.8 microM) was also a constrictor. ET(A) -/- DA, unlike ET(A) +/+ DA, contracted marginally to O(2) and ET-1 but responded to ONO-11113. O(2) contraction was also reduced in ET(A) +/- DA. In vivo, DA constricted equally in tracheotomized ET(A) -/- and ET(A) +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ET(A) -/- fetuses in utero, although it occurred in ET(A) +/+ and +/- littermates. We conclude that ET-1 mediates the DA constrictor response to O(2). Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O2 mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ETA receptor -/-, +/-, and +/+ genotypes were used. Isolated DA from term ETA +/+ fetuses contracted to O2 (5-95%) and a thromboxane A2 analog (ONO-11113, 0.1 μM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1-100 nM). Indomethacin (2.8 μM) was also a constrictor. ETA-/- DA, unlike ETA +/+ DA, contracted marginally to O2and ET-1 but responded to ONO-11113. O2 contraction was also reduced in ETA +/- DA. In vivo, DA constricted equally in tracheotomized ETA -/- and ETA +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ETA -/- fetuses in utero, although it occurred in ETA+/+ and +/- littermates. We conclude that ET-1 mediates the DA constrictor response to O2. Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).

CD36 deficiency leads to choroidal involution via COX2 down-regulation in rodents.

Background In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the “wet” form of AMD. In contrast, very little is known about the mechanisms of the predominant, “dry” form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD. Methods and Findings We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency. Conclusions CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.

Mechanisms for Ductus Arteriosus Closure

Closure of the ductus arteriosus at birth is a complex phenomenon being conditioned by antenatal events and progressing in preprogrammed steps. Functional at first, narrowing of the vessel is determined by 2 overlapping processes--removal of the prostaglandin E(2)-based relaxation sustaining prenatal patency and activation of a constrictor mechanism by the natural rise in blood oxygen tension. Two schemes have been proposed for oxygen action--one involving a cytochrome P450 hemoprotein (sensor)/endothelin-1 (effector) complex and the other a set of voltage-gated K(+) channels. These proposals, however, are not mutually exclusive. Structural closure follows the constriction through a remodeling process initiated antenatally with the development of intimal cushions and completed postnatally by a host of humoral and mechanical stimuli. Research in this area has already provided clinical applications. Nevertheless, management of premature infants with persistent ductus remains troublesome and calls for an alternative approach to the prostaglandin E(2) inhibitors now in use. Studies in progress on the oxygen-sensing system may lead to a definitive solution for this problem.