Complement component c5a is integral to the febrile response of mice to lipopolysaccharide.

OBJECTIVES: The complement system is critical to the febrile response of mice to intraperitoneally administered lipopolysaccharide (LPS). We previously identified C3 and C5 as two components potentially involved in this response. This study was designed to examine whether the complement system is also pivotal in the response of mice to intravenously or intracerebroventricularly injected LPS, to distinguish between C3 and C5 and their cognate derivatives as the essential mediator(s), and to determine whether the failure of complement-deficient mice to develop a fever could be due to their possible inability to secrete pyrogenic cytokines. METHODS: Wild-type (WT; C57BL/6J) mice, hypocomplemented or not by intravenously injected cobra venom factor (10 U/mouse), and C3-, CR3- and C5-sufficient and -deficient mice were intravenously challenged with LPS (0.25 mug/mouse); WT and C3-/- mice pretreated with a C5a receptor antagonist (C5aRa) were similarly challenged. In addition, the serum levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6 were compared in LPS-treated C5+/+ and C5-/- mice. RESULTS: LPS induced a 1 degrees C rise in core temperature in all the mice, except C5-/- mice and those pretreated with C5aRa. C5+/+ and C5-/- mice challenged intracerebroventricularly with LPS exhibited identical febrile responses. LPS induced similar increases in the serum levels of IL-1beta, TNFalpha and IL-6 in C5+/+ and C5-/- mice. CONCLUSIONS: C5a is crucial for the development of febrile responses to LPS in mice; its site of action is peripheral, not central. The possibility that an inability to produce cytokines could account for the failure of C5-/- mice to develop a fever is not supported.

Modulation of mouse endotoxic fever by complement.

It was recently reported that the complement system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneally (i.p.) injected, lipopolysaccharides (LPS). The present study was designed to identify which component(s) of the complement cascade may be specifically critical. To this end, we used mice with C3, C5, and CR2 gene deletions. To assess preliminarily the suitability of mice for such a study, we replicated our earlier studies with guinea pigs. Thus, to verify initially whether complement is similarly involved in the febrile response of wild-type (C57BL/6J) mice to i.p. LPS (Escherichia coli, 1 microg/mouse), we depleted complement with cobra venom factor (CVF; 7 U/mouse, intravenously [i.v.]). These animals did not develop fever, whereas the core temperature (T(c)) of CVF vehicle-treated controls rose approximately 1 degrees C by 80 min postinjection and then gradually abated over the following 2.5 h, confirming the involvement of complement in fever production after i.p. LPS injection and the suitability of this species for these studies. C3- and C5-sufficient (C3(+/+) and C5(+/+)) mice also developed 1 degrees C fevers within 80 min after i.p. LPS (1 or 2 microg/mouse) injection. These fevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment. C3- and C5-deficient (C3(-/-) and C5(-/-)) mice were also unable to develop T(c) rises after i.p. LPS. Both CR2(+/+) and CR2(-/-) mice responded normally to i.p. LPS (1 microg/mouse). These data indicate that C5, but not C3d acting through CR2, may play a critical role in the febrile response of mice to i.p. LPS.

Cyclooxygenase-2 mediates the febrile response of mice to interleukin-1beta.

Various lines of evidence have implicated cyclooxygenase (COX)-2 as a modulator of the fever induced by the exogenous pyrogen lipopolysaccharide (LPS). Thus, treatment with specific inhibitors of COX-2 suppresses the febrile response without affecting basal body (core) temperature (T(c)). Furthermore, COX-2 gene-ablated mice are unable to develop a febrile response to intraperitoneal (i.p.) LPS, whereas their COX-1-deficient counterparts produce fevers not different from their wild-type (WT) controls. To extend the apparently critical role of COX-2 for LPS-induced fevers to fevers produced by endogenous pyrogens, we studied the thermal responses of COX-1- and COX-2 congenitally deficient mice to i.p. and intracerebroventricular (i.c.v.) injections of recombinant murine (rm) interleukin (IL)-1beta. We also assessed the effects of one selective COX-1 inhibitor, SC-560, and two selective COX-2 inhibitors, nimesulide (NIM) and dimethylfuranone (DFU), on the febrile responses of WT and COX-1(-/-) mice to LPS and rmIL-1beta, i.p. Finally, we verified the integrity of the animals' responses to PGE2, i.c.v. I.p. and i.c.v. rmIL-1beta induced similar fevers in WT and COX-1 knockout mice, but provoked no rise in the T(c)s of COX-2 null mutants. The fever produced in WT mice by i.p. LPS was not affected by SC-560, but it was attenuated and abolished by NIM and DFU, respectively, while that caused by i.p. rmIL-1beta was converted into a T(c) fall by DFU. There were no differences in the responses to i.c.v. PGE2 among the WT and COX knockout mice. These results, therefore, further support the notion that the production of PGE2 in response to pyrogens is critically dependent on COX-2 expression.

Differential inhibition by nimesulide of the early and late phases of intravenous- and intracerebroventricular-LPS-induced fever in guinea pigs.

OBJECTIVES: The findings that inducible cyclooxygenase (COX)-2, but not constitutive COX-1, is upregulated in the brain of conscious rats approximately 1.5 h after intraperitoneal pyrogen administration, that the systemic administration of COX-2 inhibitors abolishes fever, and that COX-2-deficient mice do not develop fever in response to intraperitoneal lipopolysaccharide (LPS) have strongly implicated COX-2 in the mediation of the febrile response. However, the biosynthesis of COX-2 is significantly slower than the onset of the fever produced by intravenously injected LPS. It consequently seems improbable that inducible COX-2 could play a role in the initiation of this febrile response, but a role for COX-1 has not yet been categorically ruled out; or, alternatively, a constitutive isoform of COX-2 could have such a role. We have studied, therefore, the effects of the non-selective COX inhibitor indomethacin, the COX-1-selective inhibitor SC-560, and the COX-2-selective inhibitor nimesulide on the characteristically biphasic fever induced by intravenous LPS in conscious guinea pigs; it has an onset latency of approximately 10 min. METHODS: We injected the inhibitors 30 min before LPS, in various combinations of doses and routes; their respective vehicles were the control solutions. Core temperatures (T(c)) were monitored continuously, and plasma and brain PGE(2) levels were measured before and at 2-hour intervals after LPS administration. RESULTS: Intraperitoneal indomethacin at 10 mg kg(-1) attenuated both phases of intravenous LPS (2 microg kg(-1)) fever, but the first more so than the second; at 50 mg kg(-1), it inhibited the febrile response completely. Intraperitoneal SC-560 (5 mg kg(-1)) did not affect the febrile response to intravenous LPS (2 microg kg(-1)). Intraperitoneal nimesulide (0.3, 1.0, and 3.0 mg kg(-1)) dose dependently attenuated intravenous LPS (0.1 and 2 microg kg(-1)) fever; the second phase of the biphasic T(c) rise was affected significantly more than the first. Intraperitoneal nimesulide also prevented the associated rises in plasma and brain PGE(2) levels. Intracerebroventricular LPS (150 ng kg(-1)) evoked a monophasic fever with a long onset latency ( approximately 30 min); it was accompanied by a rise in brain PGE(2) only, implying that the febrigenic PGE(2) was generated directly in the brain. This response, however, was completely abolished by intraperitoneal nimesulide (3 mg kg(-1)), indicating that nimesulide crosses the blood-brain barrier. Intracerebroventricular nimesulide at 0.3 mg kg(-1) prevented the rise in plasma PGE(2) after intravenous LPS (2 microg kg(-1)) and again attenuated the second febrile peak significantly more than the first. CONCLUSIONS: COX-1 is not involved in intravenous LPS fever production, and COX-2 appears to play a greater role in the late than in the early phase of intravenous LPS fever in guinea pigs. The involvement of a constitutive COX-2 is inferred in the early phase.

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.

The afferent signalling of fever.

When infectious micro-organisms invade the body, fever often ensues. It is the most familiar and most manifest sign of infection. Yet, despite its ubiquity, little is definitively known regarding the detailed mechanism of its induction. The generally prevalent view is that entry into the body of such infectious micro-organisms first activates innate immune responses, which include the release of a complex variety of soluble mediators. Among these, the cytokines tumour necrosis factor (TNF) alpha, interleukin (IL)-1beta and IL-6 are thought to convey the pyrogenic message to the brain region where fever is regulated, namely the preoptic area (POA) of the anterior hypothalamus. The mechanism by which these peripheral signals may be transduced into central nervous signals is currently a matter of lively controversy. The issue is not trivial because, to the extent that these relatively large, hydrophilic peptides may be released into the circulatory system and transported to the brain by the bloodstream, they have to pass through the blood-brain barrier (BBB), which is impermeable to them. At least two routes are possible, and there is evidence for both: (1) active transport across the BBB by cytokine-specific carriers, and (2) message transfer where the BBB is 'leaky', i.e. in the 'sensory' circumventricular organs, particularly the organum vasculosum laminae terminalis (OVLT), on the midline of the POA, by the presumptive activation by, an as yet, indeterminate means of neurons projecting into the OVLT from the brain. But alternative pathways are also possible and support for some has been obtained: (1) the circulating cytokine-induced generation of BBB-permeable prostaglandin E2, the most proximal, putative mediator of fever, by endothelial cells of the cerebral microvasculature or perivascular microglia and meningeal macrophages, and (2) direct transmission to the POA of the pyrogenic messages via peripheral (largely vagal) afferent nerves activated by the cytokines. However, all four of these mechanisms have shortcomings (Blatteis & Sehic, 1997).

Thermoregulation in complex situations: combined heat exposure, infectious fever and water deprivation.

Heat exposure, infectious fever and water deprivation are stressors that, individually, produce disturbances in more than one regulated system, calling for diverse compensatory responses. A potential conflict is created when these stimuli are combined and impose concurrent stressful loads on the body because the homeostatic defenses mobilized against one are also partly needed against the other stressors. To learn how the competing demands of combined stressors for shared regulatory systems are met, rabbits were exposed to 32 degrees C and 37 degrees C (heat), administered lipopolysaccharide (Salmonella enteritidis LPS, 2 lg/kg, i.v.) in temperatures of 22 degrees C or 27 degrees C, or water-deprived for 1 or 2 days in 22 degrees C or 27 degrees C, in separate experiments. The corresponding controls were exposed to 22 degrees C or 27 degrees C, administered pyrogen-free saline i.v. in 22 degrees C or 27 degrees C, or normally hydrated in 22 degrees C or 27 degrees C. In subsequent experiments, two or all three of these treatments were applied concurrently. Core and ear skin temperatures and respiratory rates were monitored continuously. The results indicated that the concomitant needs of moderate heat exposure, fever and 1 day of water deprivation were generally met by the regulatory systems involved, but different patterns of thermoeffector activities were evoked and the eventual body temperature changes produced were different under each condition. However, when the test conditions were severe, their combined needs were not met adequately and the eventual compensatory response depended not only on the particular stimulus intensity, but also on the immediate importance for survival of the functions being defended. Thus, dehydration was the most dangerous factor to the physiological integrity of the animals. In sum, conflicting physiological stimuli appear to result in responses that are different from the responses to a single perturbation, the eventual output representing the resultant of the inputs rather than a singular output dictated by one dominant drive to the exclusion of the others.

Heat defense control in an experimental heat disorder.

Both whole-body heat exposure and intraperitoneal heating (IPH) result in a body temperature (T(b)) fall that occurs once heating is abated ("hyperthermia-induced hypothermia"). This phenomenon involves a decrease in the threshold T(b) (T(b-thresh)) for activation of metabolic heat production (cold defense). Whether the T(b-thresh) for ear skin vasodilation (heat defense) also changes during hyperthermia-induced hypothermia remains unknown. In experiment 1, we applied IPH to guinea pigs by perfusing water through a preimplanted intraperitoneal thermode and delivered the total heat load of either approximately 1.5 kJ ("short" IPH; perfusion duration: 14 min) or approximately 3.0 kJ ("long" IPH; 40 min). Short IPH caused skin vasodilation and a 1.1 degrees C rise in T(b); no hypothermia occurred when IPH ceased. Long IPH caused vasodilation and hyperthermia of a comparable magnitude (1.4 degrees C) that were followed by a T(b) fall to 1.9 degrees C below the preheating value. In experiment 2, the Tb-thresh for skin vasodilation was measured twice: at the beginning of long IPH and at the nadir of the post-IPH hypothermia. The two T(b-thresh) values were 39.0 (SEM 0.1)degrees C and 39.2 (SEM 0.2)degrees C respectively. In the controls, the T(b-thresh) was measured at the beginning and after short IPH; both control values were 39.0 (SEM 0.2)degrees C. We conclude that the hyperthermia-induced hypothermia, although previously shown to be coupled with a decrease in the T(b-thresh) for cold defense, occurs without any substantial change in the T(b-thresh) for heat defense. We speculate that postheating thermoregulatory disorders are associated with threshold dissociation, thus representing the poikilothermic (wide dead-band) type of T(b) control.

Pyrogenic signaling via vagal afferents: what stimulates their receptors?

Although there is good evidence that pyrogenic messages may be conveyed from the periphery to the brain via vagal afferents, the exact nature of the factors that activate their sensory terminals is unclear. Since IL-1beta and PGE2 have established roles in fever production and since their receptors have been identified on or near vagal nerves, they are potential candidate mediators. A difficulty, however, is that (1) IL-1beta is not expressed constitutively in mononuclear phagocytes, their presumed cell source upon stimulation by exogenous pyrogens, e.g. endotoxin, and (2) similarly, the isoform of the enzyme that selectively mediates the production and release of PGE2 by endotoxin-stimulated macrophages, COX-2, is also not constitutively expressed in these cells. Since the transcription and translation of these factors significantly lags the onset of fever induced by endotoxin administered intravenously, in particular, it is possible that a secondary, quickly-acting mediator evoked in almost immediate reaction to the presence of endotoxin excites, directly or indirectly, the sensory neurons. We have evidence that the complement component C5 contributes importantly to the initiation of the febrile response to endotoxin. This article briefly reviews the prevailing concepts of pyrogen sensing and signaling, examines their shortcomings particularly in terms of the temporal discrepancy between the very rapid onset of the febrile response to intravenously administered endotoxin and the significant delay in the elaboration of the putative mediators of fever, and presents newer data that may help to integrate the various preposed mechanisms.

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 (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.

The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2(-/-), but not in cyclooxygenase-1(-/-) mice.

Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1-/-) and COX-2 (COX-2-/-)-deficient C57BL/6J-derived mice. COX-1+/- and COX-2+/- mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 microgram/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (Tcs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1+/-, and COX-1-/- mice all responded to LPS with a 1 degrees C rise in Tc within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2+/- and COX-2-/- mice displayed no Tc rise after LPS. PFS did not affect the Tc of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.

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-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.

Cytokines and fever.

Fever is induced in response to the entrance of pathogenic microorganisms into the body and is thought to be mediated by cytokines. Because these pathogens most commonly invade the body through its natural barriers and because body temperature is regulated centrally, these mediators are presumed to be produced peripherally and transported by the bloodstream to the brain, to act. It is generally considered that their febrigenic messages are further modulated there by prostaglandin E2 (PGE2). However, the detailed mechanism by which these cytokines signal the brain and activate the febrile response is not yet clear. Indeed, the specific role of each cytokine has been difficult to establish due to complex interactions among them. Furthermore, recent evidence suggests that different pyrogens may induce different cytokines; for example, i.v. LPS (a model of systemic bacterial infection) induces large increases in IL-6, but only small rises in IL-1 and TNF alpha plasma levels. Moreover, their appearance lags the fever onset. We recently found that subdiaphragmatic vagotomy, decomplementation, and blockade of Kupffer cells suppress the febrile response of guinea pigs to i.v. LPS, and that i.v. LPS rapidly stimulates the release of norepinephrine (NE) and, hence, of PGE2 in their preoptic-anterior hypothalamus (POA, the brain region containing the thermoregulatory controller). Based on these and other data in the literature, we hypothesize that LPS fever may be initiated as follows: i.v., LPS-->complement-->Kupffer cells-->cytokines?-->vagal afferents -->n. tractus solitarius?-->A1/A2 cell groups?-->ventral noradrenergic bundle? -->POA-->NE-->PGE2-->fever.