Elmir Sehic

Blockade of lipopolysaccharide-induced fever by subdiaphragmatic vagotomy in guinea pigs.

It is generally believed that fever is mediated by certain cytokines produced by immune cells activated by exogenous pyrogens, e.g., lipopolysaccharides (LPS), released into the circulation and transported to the brain There, the cytokines are thought to stimulate prostaglandin (PG) E2 production within the organum vasculosum laminae terminalis region. PGE2 then may act as a febrigenic mediator locally or in the surrounding preoptic area (POA). However, whereas the increases in preoptic PGE2 and body (core) temperature (Tc) following the intravenous (i.v.) administration of LPS correlate temporally, cytokine levels in blood lag both these increases. From recent data in the literature, we have conjectured that a possible, alternative communication pathway between the i.v. LPS-activated immune system and brain PGE2 may be provided by the vagi. To test this possibility, we measured the levels of PGE2 in the extracellular fluid of the POA (collected by microdialysis) of conscious, subdiaphragmatically vagotomized or sham-operated guinea pigs following LPS administration (2 micrograms/kg; i.v.); controls received pyrogen-free saline (PFS). The effluents from the microdialysis probes were collected over 30-min periods throughout the experiments and the samples analyzed by radioimmunoassay; Tc was monitored continuously using thermocouples inserted 5 cm into the colon. LPS induced a biphasic fall in Tc and failed to increase preoptic PGE2 levels in the vagotomized guinea pigs (n = 10), whereas in their sham-operated controls (n = 10) it induced increases in both preopitc PGE2 and Tc within 15 min after its injection; PFS (n = 13) had no effect on either variable. We postulate that peripheral immune cell-derived signals may be transmitted via the vagi to the medulla. From other data, we suggest further that they may be conveyed from here via the ventral noradrenergic bundle to the POA region, where the released norepinephrine induces the local synthesis of PGE2 and, hence, fever onset.

Pyrogen Sensing and Signaling: Old Views and New Concepts

Fever is thought to be caused by endogenous pyrogenic cytokines, which are elaborated and released into the circulation by systemic mononuclear phagocytes that are activated by exogenous inflammatory agents and transported to the preoptic-anterior hypothalamic area (POA) of the brain, where they act. Prostaglandin (PG) E2 is thought to be an essential, proximal mediator in the POA, and induced by these cytokines. It seems unlikely, however, that these factors could directly account for early production of PGE2 following the intravenous administration of bacterial endotoxic lipopolysaccharides (LPS), because PGE2 is generated before the cytokines that induce it are detectable in the blood and the before cyclooxygenase-2, the synthase that they stimulate, is expressed. Hence other, more quickly evoked mediators are presumed to be involved in initiating the febrile response; moreover, their message may be conveyed to the brain by a neural rather than a humoral pathway. This article reviews current conceptions of pyrogen signalling from the periphery to the brain and presents new, developing hypotheses about the mechanism by which LPS initiates fever.

Hypothalamic prostaglandin E2 during lipopolysaccharide-induced fever in guinea pigs

Prostaglandin E2 (PGE2) is postulated to be a central mediator of fever. It is generally believed that it is produced in the preoptic area of the anterior hypothalamus (POA) because, among other evidence, its level increases both in the third ventricle and in the POA in response to pyrogens. However, lately, the question has arisen whether PGE2 might, in fact, be formed outside of the brain substance and then penetrate it, in particular through the organum vasculosum laminae terminalis. If produced outside the brain substance, the peripheral blockade of its synthesis should prevent lipopolysaccharides (LPS)-induced fever, whereas the intracarotid infusion of PGE2 should produce an increase in core temperature (T(C)) as well as in preoptic PGE2. To verify this hypothesis, continuous measurements of T(C) and preoptic PGE2 levels were made in conscious guinea pigs administered the PGE2 synthase inhibitor, indomethacin (10 or 50 mg/kg, im) 30 min before S. enteritidis LPS (2 mu g/kg, iv) or before PGE2 microdialyzed into the POA (1 mu g/mu l at 2 mu g/min for 2.5 h) and during PGE2 infused into a carotid artery (1 mu g and 10 mu g/mu l at 2 mu g/min for 1 h). LPS induced a biphasic 1.4 degrees C fever that was consistently associated with an increase in the level of PGE2 in the POA. Indomethacin at 10 mg/kg attenuated the course of the LPS-induced fever and prevented the associated increase in preoptic PGE2 for 90 min after fever onset; thereafter, PGE2 was significantly reduced by comparison with controls. Indomethacin at 50 mg/kg completely abolished both the fever and the increased levels of PGE2 in the POA; the fever induced by PGE2 microdialyzed into the POA was not affected by indomethacin pretreatment The intracarotid infusion of PGE2 produced T(C) falls and no increase in preoptic PGE2 levels. The indomethacin-induced blockade of fever and inhibition of the associated increase in preoptic PGE2 levels further substantiates the presumptive link between PGE2 in the POA and fever caused by LPS. The failure of exogenous PGE2 infusion to induce increases in T(C) and preoptic PGE2 levels excludes the possibility that PGE2 formed outside of the brain penetrates the POA and induces fever. Thus, in guinea pigs, the PGE2 associated with LPS-induced fever may be synthesized in the POA.

Prostaglandins and the Release of the Adrenergic Transmitter

Prostaglandins (PG) are synthesized from arachidonic acid, which is deesterified from tissue lipids in response to various stimuli including adrenergic transmitter, consequent to activation of one or more lipase(s). The profile of arachidonic acid metabolites generated in response to sympathetic nerve stimulation or administration of norepinephrine (NE) may vary in different tissues. For example, in the kidney and spleen, PGE2, is the major and PGI2 and PGF2 alpha the minor products; whereas in the heart and blood vessels, PGI2 is the principal product of arachidonic acid generated in response to sympathetic nerve stimulation. PGE2 and PGI2 inhibit release of NE and/or the postjunctional actions of this neurotransmitter in several tissues. These observations and the findings that inhibitors of cyclooxygenase enhance NE release and the response of effector organs to nerve stimulation suggest that PGs act as physiological modulators of adrenergic transmission. The mechanism by which PGs modulate release of the adrenergic transmitter has not yet been established. NE appears to be released from sympathetic fibers during depolarization by influx of Na+, which is associated with entry of Ca++ through omega-conotoxin-sensitive Ca++ channels that are distinct from those in the vascular smooth muscle, which are sensitive to nifedipine. Ouabain in low external K+ activates the former, whereas external Na+ depletion activates the latter type of Ca++ channels in the nerve fiber and promotes release of NE. PGs (PGE2) may inhibit release of NE from nerve fibers by interfering with the availability of Ca++ through these Ca++ channels or promoting efflux of Ca++ from the nerve terminal.

Complement reduction impairs the febrile response of guinea pigs to endotoxin

Although it is generally believed that circulating exogenous pyrogens [e.g., lipopolysaccharides (LPS)] induce fever via the mediation of endogenous pyrogens (EP) such as cytokines, the first of these, tumor necrosis factor-α, is usually not detectable in blood until at least 30 min after intravenous administration of LPS, whereas the febrile rise begins within 15 min after its administration. Moreover, although abundant evidence indicates that circulating LPS is cleared primarily by liver macrophages [Kupffer cells (KC)], these do not secrete EP in immediate response. This would imply that other factors, presumably evoked earlier than EP, may mediate the onset of the febrile response to intravenous LPS. It is well known that blood-borne LPS very rapidly activates the intravascular complement (C) system, some components of which in turn stimulate the quick release into blood of various substances that have roles in the acute inflammatory reaction. KC contain receptors for C components and are in close contact with afferent vagal terminals in the liver; the involvement of hepatic vagal afferents in LPS-induced fever has recently been shown. In this study, we tested the hypothesis that the initiation of fever by intravenous LPS involves, sequentially, the C system and KC. To test this postulated mechanism, we measured directly the levels of prostaglandin E2(PGE2) in the interstitial fluid of the preoptic anterior hypothalamus (POA), the presumptive site of the fever-producing controller, of conscious guinea pigs over their entire febrile course, before and after C depletion by cobra venom factor (CVF) and before and after elimination of KC by gadolinium chloride (GdCl3). CVF and GdCl3 pretreatment each individually attenuated the first of the biphasic core temperature (Tc) rises after intravenous LPS, inverted the second into a Tcfall, and greatly reduced the usual fever-associated increase in POA PGE2. We conclude, therefore, that C activation may indeed be pivotal in the induction of fever by intravenous LPS and that substance(s) generated presumably by KC in almost immediate reaction to the presence of LPS and/or C may transmit pyrogenic signals via hepatic vagal afferents to the POA, where they rapidly induce the production of PGE2 and, hence, fever.Although it is generally believed that circulating exogenous pyrogens [e.g., lipopolysaccharides (LPS)] induce fever via the mediation of endogenous pyrogens (EP) such as cytokines, the first of these, tumor necrosis factor-alpha, is usually not detectable in blood until at least 30 min after intravenous administration of LPS, whereas the febrile rise begins within 15 min after its administration. Moreover, although abundant evidence indicates that circulating LPS is cleared primarily by liver macrophages [Kupffer cells (KC)], these do not secrete EP in immediate response. This would imply that other factors, presumably evoked earlier than EP, may mediate the onset of the febrile response to intravenous LPS. It is well known that blood-borne LPS very rapidly activates the intravascular complement (C) system, some components of which in turn stimulate the quick release into blood of various substances that have roles in the acute inflammatory reaction. KC contain receptors for C components and are in close contact with afferent vagal terminals in the liver; the involvement of hepatic vagal afferents in LPS-induced fever has recently been shown. In this study, we tested the hypothesis that the initiation of fever by intravenous LPS involves, sequentially, the C system and KC. To test this postulated mechanism, we measured directly the levels of prostaglandin E2 (PGE2) in the interstitial fluid of the preoptic anterior hypothalamus (POA), the presumptive site of the fever-producing controller, of conscious guinea pigs over their entire febrile course, before and after C depletion by cobra venom factor (CVF) and before and after elimination of KC by gadolinium chloride (GdCl3). CVF and GdCl3 pretreatment each individually attenuated the first of the biphasic core temperature (Tc) rises after intravenous LPS, inverted the second into a Tc fall, and greatly reduced the usual fever-associated increase in POA PGE2. We conclude, therefore, that C activation may indeed be pivotal in the induction of fever by intravenous LPS and that substance(s) generated presumably by KC in almost immediate reaction to the presence of LPS and/or C may transmit pyrogenic signals via hepatic vagal afferents to the POA, where they rapidly induce the production of PGE2 and, hence, fever.

Circulating Pyrogen Signaling of the Brain.: A New Working Hypothesis

It is generally believed that fever is caused by a cascade of host-generated endogenous pyrogens, members of the class of immunoregulatory hydrophilic polypeptides called cytokines, produced by various cell types (but, in the context of fever, primarily by mononuclear phagocytes) when activated by signals provided by infectious microorganisms and/or their products, e . g . , bacterial endotoxic lipopolysaccharides (LPS). Since such organisms most commonly invade the body by breaking one of its natural barriers, the cell sources of these cytokines are presumed to be located in the periphery. Since. furthermore, body temperature (Tb) is regulated centrally, it has been assumed that these cytokines are released into the bloodstream and transported to the preoptic area of the anterior hypothalamus (POA, the presumptive primary locus of the thermoregulatory controller), where they act. However, the presence of a blood-brain barrier (BBB) to peptides raises the question as to how systemically circulating cytokines could penetrate the brain and trigger fever production. Several mechanisms are plausible, but there is as yet no consensus.’ The currently prevalent concept is that the messages of cytokines are transduced where the BBB is leaky, specifically in the organum vasculosum laminae terminalis (OVLT, which lies on the midline of the medial POA, in the anteroventral wall of the third ventricle); i .e . , cytokines that have passed into the perivascular spaces of the OVLT are thought to stimulate cells within these spaces, triggering their release of prostaglandin E, (PGEJ which, being lipophilic, then diffuses from the OVLT into the adjacent neuropil, whence its signal is propagated further. However, the cell sources and targets of this fevermediating PGE2 are in dispute. Some new, recent data, moreover, are difficult to reconcile with the various, proposed OVLT paradigms.’ Recently, an alternative means of communication between peripheral cytokines and the brain was suggested, viz., via the activation of vagal afferents.’ We tested the possibility that LPS-induced fever may be mediated by this pathway by measuring the levels of PGE, in the preoptic fluid (collected by microdialysis) of conscious, subdiaphragmatically vagotomized or sham-operated guinea pigs following LPS administration (2 pg/kg, i ~ ) . ~ LPS induced a biphasic fall in Tb and failed to increase preoptic PGE, levels in the vagotomized animals, whereas it induced increases in both preoptic PGE, and Tb within 15 min after its injection in the

Relation between complement and the febrile response of guinea pigs to systemic endotoxin

We reported recently that the complement (C) system may play a role in the febrile response of guinea pigs to intravenous lipopolysaccharide (LPS) administration because C depletion abolished the LPS-induced rise in core temperature (Tc). The present study was designed to investigate further the relation between C reduction [induced by cobra venom factor (CVF); 20, 50, 100, and 200 U/animal iv] and the fever of adult, conscious guinea pigs produced by LPS injected intravenously (2 μg/kg) or intraperitoneally (8, 16, 32 μg/kg) 18 h after CVF; control animals received pyrogen-free saline. Serum C levels were measured as total hemolytic C activity before and 18 h after CVF injection and expressed as CH100 units. In other experiments, serum C levels were determined at various intervals after the intravenous and intraperitoneal injections at different doses of LPS alone. LPS produced fevers generally of similar heights but of different onset latencies and durations, depending on the dose and route of administration. CVF caused dose-related reductions in serum C, from ∼1,136 U to below detection. These reductions proportionately attenuated the fevers induced by intraperitoneal LPS, but not by intravenous LPS. Intravenous and intraperitoneal LPS per se caused reductions in serum C of 25 and 40%, respectively, indicating activation of the C cascade. These decreases were transient, however, occurring early during the febrile rise ∼30 min after LPS injection. These data thus support the notion that the C system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneal, LPS.We reported recently that the complement (C) system may play a role in the febrile response of guinea pigs to intravenous lipopolysaccharide (LPS) administration because C depletion abolished the LPS-induced rise in core temperature (T(c)). The present study was designed to investigate further the relation between C reduction [induced by cobra venom factor (CVF); 20, 50, 100, and 200 U/animal iv] and the fever of adult, conscious guinea pigs produced by LPS injected intravenously (2 microg/kg) or intraperitoneally (8, 16, 32 microg/kg) 18 h after CVF; control animals received pyrogen-free saline. Serum C levels were measured as total hemolytic C activity before and 18 h after CVF injection and expressed as CH(100) units. In other experiments, serum C levels were determined at various intervals after the intravenous and intraperitoneal injections at different doses of LPS alone. LPS produced fevers generally of similar heights but of different onset latencies and durations, depending on the dose and route of administration. CVF caused dose-related reductions in serum C, from approximately 1,136 U to below detection. These reductions proportionately attenuated the fevers induced by intraperitoneal LPS, but not by intravenous LPS. Intravenous and intraperitoneal LPS per se caused reductions in serum C of 25 and 40%, respectively, indicating activation of the C cascade. These decreases were transient, however, occurring early during the febrile rise approximately 30 min after LPS injection. These data thus support the notion that the C system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneal, LPS.

Blockade of Kupffer Cells Prevents the Febrile and Preoptic Prostaglandin E2 Responses to Intravenous Lipopolysaccharide in Guinea Pigsa

It is generally believed that fever is mediated by endogenous pyrogens (cytokines; e.g., interleukin-lp [IL-lp]) induced peripherally by exogenous pyrogen (e .g . , bacterial endotoxic lipopolysaccharide [LPSI)-activated immune cells, released into the bloodstream, and transported to the brain. Indeed, abundant evidence indicates that circulating LPS is cleared by various resident macrophages, particularly those in the liver (Kupffer cells), and that these cells produce cytokines. There is no direct evidence to date, however, that the contribution of Kupffer cells is critical to fever production. Because the presence of a blood-brain barrier (BBB) a priori precludes the free passage of cytokines into the brain, it has been hypothesized that they may reach the perivascular spaces of, in particular, the organum vasculosum laminae terminalis (OVLT), where the BBB is leaky, and bind to receptors distributed therein, thereby inducing new signals that transmit the original pyrogenic message further into the neuropile. However, it has also been speculated that, alternatively, circulating LPS may itself bind to macrophages in these spaces, thereby initiating the local production of cytokines which then could mediate the pyrogenic signal further into the brain. Indeed, macrophages and ramified microglia1*2 in the OVLT region have been shown to express dosedependently IL1/3 mRNA after the intravenous (iv) injection of LPS; however, the appearance of this message lags significantly behind the onset of fever. To ascertain whether systemic LPS activates macrophages preponderantly in the OVLT or in the liver to initiate the febrile response, we conducted two experiments. 1) On the assumption that LPS would bind to cells in either or both of these structures and that, therefore, its site of action might be differentiated, we injected fluorescein isothiocyanate (F1TC)-labeled E. coli LPS (Sigma Chemical, St. Louis, MO; 2.5 mg/kg) into the carotid artery of 6 rats, and 15 min later

Fever and the Organum Vasculosum Laminae Terminalis: Another Look

Lesions were made to compare in guinea pigs the previously demonstrated antipyretic effectiveness of ablation of the anteroventral third ventricle (AV3V) brain region, which contains the organum vasculosum laminae terminalis (OVLT), with that of the rostroventral lamina terminalis. Small lesions discretely localized to this site were reported to enhance the febrile responses of rats and rabbits. We found that both types of lesions blocked LPS-induced fever so long as the OVLT was completely ablated. Fever was not increased by the more discrete lesions in these guinea pigs. The present findings are consistent with the hypothesis that the pyrogenic signal is transduced from blood to brain via the OVLT.

Apparent Dissociation between Lipopolysaccharide-Induced Intrapreoptic Release of Prostaglandin E2 and Fever in Guinea Pigs

The i.v. administration of a lipopolysaccharide (LPS) regularly produced increases in both core temperatures (Tc) and levels of prostaglandin E2 (PGE2) in the intrapreoptic (iPO) extracellular fluid of guinea pigs with unilaterally implanted microdialysis probes, but these changes were not temporally related. The increase in PGE2 was greater in guinea pigs with bilaterally implanted microdialysis probes, although in half of the animals LPS caused a fall in Tc. The bilateral iPO microdialysis of cyclooxygenase inhibitors abrogated both the Tc and PGE2 changes induced by LPS. These results suggest that PGE2 may be produced in the preoptic area of conscious guinea pigs in response to LPS, but that its release and fever may be dissociated responses.