A. L. Ungar

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.

Neuromodulation of acute-phase responses to interleukin-6 in guinea pigs

It is now generally recognized that interleukin-6 (IL6) is one of the cytokines that mediate the various nonspecific host defense responses to infectious pathogens. Among its now well-demonstrated effects on systemic administration are fever and acute-phase proteinemia. These effects are also activated by the cytokine, IL1, and it has been shown that they are modulated in the preoptic-anterior hypothalamus (POA). This study was undertaken to determine whether this brain region similarly drives the febrile and proteinemic responses to IL6. We compared, therefore, these responses of conscious guinea pigs to human recombinant (hr)IL6 administered intravenously (IV) and into the POA. hrIL6 given IV was not pyrogenic at 1 microgram/kg, caused low-grade, dose-independent fevers (0.4 +/- 0.1 degree C) at 5-20 micrograms/kg, and dose-related fevers at 50 and 100 micrograms/kg (0.6 +/- 0.0 and 0.9 +/- 0.1 degree C, respectively). However, all doses of hrIL6 induced elevations in the plasma levels of ceruloplasmin (as an indicator of acute-phase proteins), albeit not in a dose-dependent manner. Indomethacin (10 mg/kg, injected intramuscularly 20 min before hrIL6) abolished the febrile response, but did not prevent the rise in plasma ceruloplasmin levels. Fever and ceruloplasminemia were also evoked by 50 and 100 ng of hrIL6 injected into the POA (1 microliter bilaterally), but not by 25 ng. These results indicate that the inductions of fever and plasma ceruloplasmin by IL6 are, like those of IL1, modulated in the POA, albeit the effective doses are much higher than those of IL1.

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.

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

Genesis of biphasic thermal response to intrapreoptically microinjected clonidine

Intrapreoptic (IPO) microinjections of various agents cause unavoidable brain tissue injury, often resulting in prostaglandin (PG)-mediated core temperature (Tc) rises. However, IPO microinjection of the alpha 2-adrenoreceptor agonist clonidine (Clo) generally evokes a Tc fall, seemingly avoiding the influence of injury due to the microinjection procedure per se. To clarify this, we microinjected bilaterally into the preoptic/anterior hypothalamus of conscious guinea pigs various doses of Clo dissolved in pyrogen-free saline (PFS, 1 microliter/side). Clo caused biphasic hypo-/hyperthermic responses. The initial hypothermia was dose dependent: no decrease in Tc for 0.1 microgram of Clo, -0.4 +/- 0.1 degree C for 0.5 microgram, -0.9 +/- 0.1 degree C for 1.5 microgram, and -1.2 +/- 0.1 degree C for 5.0 micrograms. During the hyperthermic phase, Tc increased to a dose-independent level (1.0-1.5 degrees C), remaining there up to 5 h postinjection. PFS microinjected IPO also induced hyperthermia, but without any initial Tc decrease. This Tc rise was delayed by 100 min when the cyclooxygenase inhibitor indomethacin (Indo, 50 micrograms/microliters) was injected. Nontreated animals (time controls) maintained Tc at baseline levels during the whole experiment. The alpha 2-antagonist rauwolscine (2 micrograms/side), microinjected IPO 10 min before Clo (0.5 microgram/side), abolished the hypothermic without affecting the hyperthermic response phase; Indo (10 mg/kg), injected intramuscularly 20 min after the IPO microinjection of Clo (0.5 microgram), significantly attenuated the hyperthermic phase. These results confirm that an artifactitious, PG-mediated Tc rise consequent to nonspecific brain tissue injury contaminates the thermal response to agents (hyper- or hypothermizing) microinjected IPO.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of the hypothermic action of preoptic norepinephrine in guinea pigs

Conscious guinea pigs were either microinjected intrapreoptically (iPO) with various doses of norepinephrine (NE) bilaterally or microdialyzed with pyrogen-free saline (PFS) or 10 micrograms/microliters NE unilaterally immediately and unilaterally or bilaterally 2 days after probe insertion. Core temperature (Tco), skin temperature (Tsk), and rate of oxygen consumption (VO2) were monitored continuously. The microinjection of low doses of NE induced Tco rises, whereas that of the highest dose (10 micrograms/microliters) caused an initial Tco fall followed by a rise. The microdialysis of PFS or NE immediately after probe insertion caused Tco rises; the former was abolished and the latter was converted into a fall by indomethacin (Indo, a prostaglandin synthase inhibitor) pretreatment. Two days later, PFS evoked no thermal response whereas NE induced a Tco fall; neither response was affected by Indo pretreatment. The falls in Tco produced by NE microdialyzed uni- or bilaterally were similar. The microdialysis of NE induced a 15% reduction in metabolic rate but no change in Tsk. These results indicate that the Tco rise induced by NE microinjected iPO is a methodological artifact mediated by PGE2, whereas the Tco fall observed in its microdialysis appears to represent the authentic physiological action of this transmitter effected by a reduction in metabolic rate.