Thermoregulatory responses to lipopolysaccharide in the mouse: dependence on the dose and ambient temperature

Most published studies of thermoregulatory responses of mice to LPS involved a stressful injection of LPS, were run at a poorly controlled and often subneutral ambient temperature (T(a)), and paid little attention to the dependence of the response on the LPS dose. These pitfalls have been overcome in the present study. Male C57BL/6 mice implanted with jugular vein catheters were kept in an environmental chamber at a tightly controlled T(a). The relationship between the T(a)s used and the thermoneutral zone of the mice was verified by measuring tail skin temperature, either by infrared thermography or thermocouple thermometry. Escherichia coli LPS in a wide dose range (10(0)-10(4) microg/kg) was administered through an extension of the jugular catheter from outside the chamber. The responses observed were dose dependent. At a neutral T(a), low (just suprathreshold) doses of LPS (10(0)-10(1) microg/kg) caused a monophasic fever. To a slightly higher dose (10(1.5) microg/kg), the mice responded with a biphasic fever. To even higher doses (10(1.75)-10(4) microg/kg), they responded with a polyphasic fever, of which three distinct phases were identified. The dose dependence and dynamics of LPS fever in the mouse appeared to be remarkably similar to those seen in the rat. However, the thermoregulatory response of mice to LPS in a subthermoneutral environment is remarkably different from that of rats. Although very high doses of LPS (10(4) microg/kg) did cause a late (latency, approximately 3 h) hypothermic response in mice, the typical early (latency, 10-30 min) hypothermic response seen in rats did not occur. The present investigation identifies experimental conditions to study LPS-induced mono-, bi-, and polyphasic fevers and late hypothermia in mice and provides detailed characteristics of these responses.     

Thermoregulatory responses of rats to conventional preparations of lipopolysaccharide are caused by lipopolysaccharide per se-- not by lipoprotein contaminants

LPS preparations cause a variety of body temperature (T(b)) responses: monophasic fever, different phases of polyphasic fever, and hypothermia. Conventional (c) LPS preparations contain highly active lipoprotein contaminants (endotoxin proteins). Whereas LPS signals predominantly via the Toll-like receptor (TLR) 4, endotoxin proteins signal via TLR2. Several TLR2-dependent responses of immunocytes to cLPS in vitro are triggered by endotoxin proteins and not by LPS itself. We tested whether any T(b) response to cLPS from Escherichia coli 055:B5 is triggered by non-TLR4-signaling contaminants. A decontaminated (d) LPS preparation (free of endotoxin proteins) was produced by subjecting cLPS to phenol-water reextraction. The presence of non-TLR4-signaling contaminants in cLPS (and their absence in dLPS) was confirmed by showing that cLPS (but not dLPS) induced IL-1beta expression in the spleen and increased serum levels of TNF-alpha and IL-1beta of C3H/HeJ mice; these mice bear a nonfunctional TLR4. Yet, both cLPS and dLPS caused cytokine responses in C3H/HeOuJ mice; these mice bear a fully functional TLR4. We then studied the T(b) responses to cLPS and dLPS in Wistar rats preimplanted with jugular catheters. At a neutral ambient temperature (30 degrees C), a low (0.1 microg/kg iv) dose of cLPS caused a monophasic fever, whereas a moderate (10 microg/kg iv) dose produced a polyphasic fever. In the cold (20 degrees C), a high (500 microg/kg iv) dose of cLPS caused hypothermia. All T(b) responses to dLPS were identical to those of cLPS. We conclude that all known T(b) responses to LPS preparations are triggered by LPS per se and not by non-TLR4-signaling contaminants of such preparations.     

Threshold dissociation of thermoregulatory effector responses in febrile rabbits

When the core temperature stabilizes at a hyperthermic level after iv injection of lipopolysaccharide (LPS), the threshold core temperature for cutaneous vasoconstriction (Thcv) is significantly increased in hot and neutral environments, while the threshold core temperature for shivering (Thsh) is not significantly altered in hot or cold environments but is significantly reduced at thermoneutrality. This type of dissociated threshold alterations of thermoregulatory effector responses seems to be typical for the febrile response of rabbits to LPS. Because the same threshold dissociation can be demonstrated after icv injection of LPS, the systemic and the central effects of LPS in the generation of fever seem to be mediated by identical mechanisms. Prostaglandins of the E series (PGE), one of the mediators considered as important in fever generation, cause parallel increases in Thcv and Thsh when injected icv. This indicates that the mode of action of PGE on the central targets producing hyperthermia differs from that of the ensemble of mediators involved in the generation of LPS fever in rabbits. In rabbits pretreated with aspirin, the threshold dissociation after iv LPS injection still occurs. This indicates that factors other than PGE play an important role in the generation of the threshold dissociation of thermoregulatory effector responses, which is typical for LPS fever. These data indicate also that the states of activity of the thermoregulatory effectors involved in the febrile responses can be altered individually and that the activities of these effectors during LPS fever are quite different from their activities in the control state.     

Interleukin-1 receptor antagonist as a modulator of gender differences in the febrile response to lipopolysaccharide in rats

Febrile responses to bacterial pathogens are attenuated near term of pregnancy in several mammalian species. It is unknown, however, whether this reflects a fundamental physiological adaptation of female rats or whether it is specific to pregnancy. The aims of this study therefore were 1) to determine whether febrile responses to the bacterial endotoxin lipopolysaccharide (LPS) are attenuated in female vs. male rats and, if so, to identify possible mechanisms involved in modulating this and 2) to assess whether plasma concentrations of the anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), an important regulator of fever, are dependent on the physiological state of the female and could therefore be involved in modulating febrile responses. We found febrile responses were attenuated in cycling female vs. male rats and also in near-term pregnant dams vs. cycling females after intraperitoneal injection of LPS (0.05 mg/kg). Plasma levels of IL-1ra were significantly greater in female rats after injection of LPS, particularly during pregnancy, than in males. This was accompanied by attenuated levels of hypothalamic IL-1beta and cyclooxygenase-2 mRNA, two key mediators of the febrile response, in female rats. Furthermore, increasing plasma levels of IL-1ra in male rats by intraperitoneal administration of the recombinant antagonist attenuated hypothalamic mRNA levels of these mediators after LPS. These data suggest that there is a fundamental difference in febrile response to LPS between the genders that is likely regulated by IL-1ra. This may be an important mechanism that protects the developing fetus from potentially deleterious consequences of maternal fever during pregnancy.     

A specific antagonist of platelet-activating factor suppresses oedema formation in an Arthus reaction but not oedema induced by leukocyte chemoattractants in rabbit skin

The properties of a novel platelet-activating factor (PAF) antagonist, L-652731, on oedema responses in rabbit skin induced by exogenous inflammatory mediators and by mediators generated endogenously in a reversed passive Arthus reaction have been investigated. Oedema responses in the skin were measured by using the local accumulation of i.v. injected 125I-albumin. The antagonist, mixed with mediators before intradermal injection, caused a dose-dependent suppression of oedema responses to PAF. In contrast, responses induced by other directly acting mediators (bradykinin and histamine) and responses induced by PMN leukocyte-dependent mediators (C5a des Arg, N-formyl-methionyl-leucyl-phenylalanine, and leukotriene B4) were not suppressed. Thus, a secondary release of PAF does not appear to be involved in mediating the actions of these agents. In a reversed passive Arthus reaction, intradermal injection of L-652731 together with antibody resulted in a significant inhibition of the oedema formation measured for 2 hr after i.v. antigen challenge. In contrast, oedema responses induced by intradermal injection of preformed immune complexes were not affected by the antagonist. These results suggest that the endogenous production of PAF, in close proximity to microvascular endothelial cells, appears to be an important step in the development of an Arthus reaction. The cellular source of PAF is unknown, but one possibility is the PMN leukocyte, which releases PAF during phagocytosis of immune complexes.     

Kupffer cell-generated PGE2 triggers the febrile response of guinea pigs to intravenously injected LPS

Because the onset of fever induced by intravenously (i.v.) injected bacterial endotoxic lipopolysaccharides (LPS) precedes the appearance in the bloodstream of pyrogenic cytokines, the presumptive peripheral triggers of the febrile response, we have postulated previously that, in their stead, PGE2 could be the peripheral fever trigger because it appears in blood coincidentally with the initial body core temperature (Tc) rise. To test this hypothesis, we injected Salmonella enteritidis LPS (2 microg/kg body wt i.v.) into conscious guinea pigs and measured their plasma levels of LPS, PGE2, TNF-alpha, IL-1beta, and IL-6 before and 15, 30, 60, 90, and 120 min after LPS administration; Tc was monitored continuously. The animals were untreated or Kupffer cell (KC) depleted; the essential involvement of KCs in LPS fever was shown previously. LPS very promptly (<10 min) induced a rise of Tc that was temporally correlated with the elevation of plasma PGE2. KC depletion prevented the Tc and plasma PGE2 rises and slowed the clearance of LPS from the blood. TNF-alpha was not detectable in plasma until 30 min and in IL-1beta and IL-6 until 60 min after LPS injection. KC depletion did not alter the times of appearance or magnitudes of rises of these cytokines, except TNF-alpha, the maximal level of which was increased approximately twofold in the KC-depleted animals. In a follow-up experiment, PGE2 antiserum administered i.v. 10 min before LPS significantly attenuated the febrile response to LPS. Together, these results support the view that, in guinea pigs, PGE2 rather than pyrogenic cytokines is generated by KCs in immediate response to i.v. LPS and triggers the febrile response.     

Role of the anteroventral third ventricle region in fever in sheep

Ablation of the anteroventral third ventricle (AV3V) region, which includes the organum vasculosum laminae terminalis (OVLT), blocks the febrile response of guinea pigs to systemically injected endotoxin; by contrast, discrete lesions of the OVLT transiently enhance fever in rabbits and rats. To assess whether separate subdivisions of the AV3V may mediate these different effects, the thermal responses to Escherichia coli lipopolysaccharide (LPS, 0.25 micrograms/kg, i.v.) were measured in eight sheep before and 12-13 days after placement of lesions at various levels within the AV3V. The responses of four of these sheep to crude homologous endogenous pyrogen (EP, 1-2 mL, i.c.v.) were also evaluated. Additionally, five other sheep were tested with LPS 2-8 months postlesion. All the experiments were performed at thermoneutrality. Sheep were used because most of the frontal wall of their 3V forms an elongated OVLT consisting of an avascular body and a vascular base. The animals were classified postmortem according to the extent of tissue ablated. Lesion overlap analyses showed that (i) medial lesions which extended from the floor of the 3V to the anterior commissure and laterally into adjacent preoptic periventricular tissue were associated with significantly depressed fever after LPS (n = 2); (ii) comparable lesions, but which excluded the ventral portion of the AV3V, i.e., the base of the OVLT, did not alter the magnitude of the febrile response to LPS (n = 4); (iii) lesions of the lateral walls of the 3V and (or) of the adjacent medial preoptic and anterior hypothalamic areas but excluding the frontal 3V wall also did not affect fever height after LPS (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between platelet activating factor and eicosanoids during endotoxic shock in anaesthetized pigs

The effects of platelet activating factor (PAF) on eicosanoid release during endotoxic shock was investigated in anaesthetized pigs receiving 5 mug kg(-1) Escherichia coli endotoxin (LPS) into the superior mesenteric artery over a 60 min period, by measuring plasma levels of a variety of mediators. Fifteen of the 31 animals infused with LPS and not treated with BN 52021, a PAF receptor antagonist, died within 30 min after the commencement of LPS infusion (non-survivors), while the other 16 survived the experimental period of 3 h, though in a state of shock (survivors). No alterations were observed in plasma concentrations of eicosanoids in the non-survivors. A significant, though transient, increase in eicosanoid concentrations occurred only in the survivors. Treatment with BN 52021 (4 mg kg-1, i.v.) injected 5 min prior to LPS infusion, failed to exert any effect on the survival rate. However, pretreatment with BN 52021 prevented circulatory collapse in the survivors and reduced the concentration of cyclooxygenase enzyme products, without affecting LTB(4) release. Exogenous administration of PAF (0.01 mug kg(-1)) caused hypotension and increased TXB(2) levels although 6-keto PGF(1alpha) and LTB(4) concentrations were unchanged. The data suggest that prostanoid formation may be secondary to PAF release in circulatory collapse evoked by LPS infusion in survivors, and give further support to the suggestion that PAF prostanoid interaction is important during endotoxic shock. However, their role in early death seems to be negligible, indicating the importance of other mediators.     

Hypoxia primes endotoxin-induced tissue factor expression in human monocytes and endothelial cells by a PAF-dependent mechanism

Tissue factor (TF) is a glycoprotein which acts as a trigger of the coagulation cascade. TF expression may be induced at the surface of monocytes and endothelial cells by several stimuli including bacterial endotoxin (LPS) and cytokines (IL1β, TNFα) and there is a large body of evidence for the involvement of hypoxia as a primaring factor in the process leading to thrombosis. To define the molecular basis underlying this phenomenon, we evaluated the relative role of platelet activating factor (PAF). PAF primed human monocytes and human umbilical vein endothelial cells (HUVEC) for TF expression following exposure to E. coli LPS but was unable to enhance the induction of TF expression by IL1β. The priming effect of PAF with regard to LPS occurred in a time-and dose-dependent manner and was inhibited by the PAF receptor antagonist SR 27417. When HUVEC or monocytes were exposed to an hypoxic environment, a significant rise in LPS-induced TF expression was observed. Hypoxia had no effect on IL1-induced TF expression. The enhanced LPS-induced TF expression in both cell types was mediated by PAF as indicated by the inhibition obtained with SR 27417, added during hypoxia. Although the importance of hypoxia in the etiology of venous thrombosis has been acknowledged for a long time, evaluation of the relative importance of PAF in the process leading to thrombus formation is still lacking. Stasis-induced thrombosis performed in the rabbit jugular vein was enhanced in a dose-dependent manner by the prior i.v. administration of LPS (0.05 to 100 μg/kg, i.v.). SR 27417 administered simultaneously with LPS prevented thrombus formation with an ED50 value of 0.1 ± 0.04 mg/kg. These results therefore show that hypoxia promotes LPS-induced TF expression in HUVEC and human monocytes through a PAF-dependent mechanism in vitro and in vivo. © 1996 Wiley-Liss, Inc.     

Arginine vasopressin in fever: a still unsolved puzzle

(1) Administration of arginine vasopressin (AVP) in the ventral septal area (VSA) or intracerebroventricularly (i.c.v.) is thought to attenuate lipopolysaccharide (LPS) or prostaglandin (PG) E₂ fevers in rabbits and rats by acting on the V₁ receptor. (2) We found that the fever response of rabbits to intravenous LPS (200 ng/kg) or intra-VSA PGE₂ (500 ng) was not attenuated but enhanced by intra-VSA AVP (5 μg); a pharmacological analysis showed that this fever-enhancing effect was mediated by the V₂ receptor. (3) The febrile response of rats to intraperitoneal (50 μg/kg) or i.c.v. (100 ng) LPS was unaffected by i.c.v. AVP (2.5–100 ng). (4) The role of AVP in fever should be re-examined.     

Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.     

The spleen modulates the febrile response of guinea pigs to LPS

The febrile responses of splenectomized (Splex) or sham-operated (Sham) guinea pigs challenged intravenously or intraperitoneally with lipopolysaccharide (LPS) 7 and 30 days after surgery were evaluated. FITC-LPS uptake by Kupffer cells (KC) was additionally assessed 15, 30, and 60 min after injection. LPS at 0.05 microg/kg iv did not evoke fever in Sham animals but caused a 1.2 degrees C core temperature (T(c)) rise in the Splex animals. LPS at 2 microg/kg iv induced a 1.8 degrees C greater T(c) rise of the Splex animals than of their controls. LPS at 2 and 8 microg/kg ip 7 days postsurgery induced 1.4 and 1.8 degrees C higher fevers, respectively, in the Splex than Sham animals. LPS at 2 and 8 microg/kg ip 30 days postsurgery also increased the febrile responses of the asplenic animals by 1.6 and 1.8 degrees C, respectively. FITC-LPS at 7 days was detected in the controls within KC 15 min after its administration; the label density was reduced at 30 min and almost 0 at 60 min. In the Splex group, in contrast, the labeling was significantly denser and remained unchanged through all three time points; this effect was still present 30 days after surgery. Similar results were obtained at 60 min after FITC-LPS intraperitoneal injection. Gadolinium chloride pretreatment (-3 days) of the Splex group significantly reduced both their febrile responses to LPS (8 microg/kg ip) and their KC uptake of FITC-LPS 7 days postsurgery. Thus splenectomy increases the magnitude of the febrile response of guinea pigs and the uptake of systemically administered LPS.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

Hypothalamic neuronal histamine modulates febrile response but not anorexia induced by lipopolysaccharide

This study examined the contribution of hypothalamic neuronal histamine (HA) to the anorectic and febrile responses induced by lipopolysaccharide (LPS), an exogenous pyrogen, and the endogenous pyrogens interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Intraperitoneal (ip) injection of LPS, IL-1beta, or TNF-alpha suppressed 24-hr cumulative food intake and increased rectal temperature in rats.To analyze the histaminergic contribution, rats were pretreated with intracerebroventricular (icv) injection of 2.44 mmol/kg or ip injection of 244 mmol/kg of alpha-fluoromethylhistidine (FMH), a suicide inhibitor of histidine decarboxylase (HDC), to deplete neural HA. The depletion of neural HA augmented the febrile response to ip injection of LPS and IL-1beta and alleviated the anorectic response to ip injection of IL-1beta. However, the depletion of neural HA did not modify the LPS-induced anorectic response or TNF-alpha-induced febrile and anorectic responses. Consistent with these results, the rate of hypothalamic HA turnover, assessed by the accumulation of tele-methylhistamine (t-MH), was elevated with ip injections of LPS and IL-1beta, but unaffected by TNF-alpha at equivalent doses. This suggests that (i) LPS and IL-1beta activate hypothalamic neural HA turnover; (ii) hypothalamic neural HA suppresses the LPS- and IL-1beta-induced febrile responses and accelerates the IL-1beta-induced anorectic response; and (iii) TNF-alpha modulates the febrile and anorectic responses via a neural HA-independent pathway. Therefore, hypothalamic neural HA is involved in the IL-1beta-dominant pathway, rather than the TNF-alpha-dominant pathway, preceding the systemic inflammatory response induced by exogenous pyrogens, such as LPS. Further research on this is needed.     

Pharmacological modulation of the bronchopulmonary action of the vasoactive peptide, endothelin, administered by aerosol in the guinea-pig

Administration by aerosol for 1 min of solutions of endothelin (ENDO; 1, 5 or 10 micrograms/ml) to anaesthetized and ventilated guinea-pigs induced a dose-dependent bronchopulmonary response (BR) which was maximal within 4 to 5 min. In contrast, no significant change of the mean arterial blood pressure was observed. Pretreatment of guinea-pigs with propranolol (1 mg/kg, i.v.), mepyramine (1 mg/kg, i.v.), nifedipine (50 mg/kg, i.p.) or verapamil (0.3 mg/kg, i.v.) did not significantly affect the BR induced by an aerosol of a solution of 10 micrograms/ml ENDO. In contrast, BR was significantly reduced when the animals were pretreated with the cyclooxygenase inhibitor, indomethacin (10 mg/kg, i.v.) or the platelet-activating factor (PAF) receptor antagonist, BN 52021 (10 mg/kg, i.v.). These results indicate that aerosolized ENDO induces a BR via the generation of secondary mediators such as cyclooxygenase products and PAF in a process which is unaffected by the blockers of the voltage-dependent calcium channels.     

The amount of free corticosterone is increased during lipopolysaccharide-induced fever

The relation between lipopolysaccharide (LPS)-induced fever and bioavailability of corticosterone (B) was examined in male Wistar rats. Animals were injected with LPS (2.5 mg/kg i.p.) or saline and core temperature and heart rate were monitored continuously using a biotelemetry system. Blood samples were withdrawn from freely moving rats via jugular catheters for estimation of total and free plasma B. LPS induced a long-lasting increase (24-48 h) in core temperature and B secretion and a short-lasting increase (90 min) in heart rate. LPS-induced fever was accompanied by a significant increase in the free/total B ratio. In contrast, an acute injection of B, which resulted in circulating B levels similar to those found after LPS, did not affect the free/total B ratio. The important role of LPS-induced fever in the hormone secretion pattern and the equilibrium between free and total B was further demonstrated in an in vitro study showing that an increase in the temperature by 3 degrees C elevated the free B fraction and the free/total B ratio of plasma samples with concentrations of B in the physiological range (5-40 microg/dl). Taken together, these findings indicate that during LPS-induced fever there is an increase in the amount of biologically available B. Exposure of glucocorticoid-sensitive targets to elevated levels of free B could contribute to the restoration of homeostasis that is disturbed during inflammation.     

Putative antihyperpyretic factor induced by LPS in spleen of guinea pigs

We reported previously that the onset of LPS-induced fever, irrespective of its route of administration, is temporally correlated with the appearance of LPS in the liver and that splenectomy significantly increases both the febrile response to LPS and the uptake of LPS by Kupffer cells (KC). To further evaluate the role of the spleen in LPS fever production, we ligated the splenic vein and, 7 and 30 days later, monitored the core temperature changes over 6 h after intraperitoneal (ip) injection of LPS (2 microg/kg). Both the febrile response and the uptake of LPS by KC were significantly augmented. Like splenectomy, splenic vein ligation (SVL) increased the febrile response and LPS uptake by KC until the collateral circulation developed, suggesting that the spleen may normally contribute an inhibitory factor that limits KC uptake of LPS and thus affects the febrile response. Subsequently, to verify the presence of this factor, we prepared splenic extracts from guinea pigs pretreated with LPS (8 microg/kg ip) or pyrogen-free saline, homogenized and ultrafiltered them, and injected them intravenously into splenectomized (Splex) guinea pigs pretreated with LPS (8 microg/kg ip). The results confirmed our presumption that the splenic extract from LPS-treated guinea pigs inhibits the exaggerated febrile response and the LPS uptake by the liver of Splex guinea pigs, indicating the presence of a putative splenic inhibitory factor, confirming the participation of the spleen in LPS-induced fever, and suggesting the existence of a novel antihyperpyretic mechanism. Preliminary data indicate that this factor is a lipid.     

The GTPase Rap1 controls functional activation of macrophage integrin alphaMbeta2 by LPS and other inflammatory mediators

BACKGROUND: beta2 integrins mediate many aspects of the inflammatory and immune responses, including adhesion of leukocytes to the endothelium, complement-mediated phagocytosis in macrophages and neutrophils, and antigen-specific conjugate formation between cytotoxic T cells and their targets. A variety of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), and lipopolysaccharide (LPS) and other bacterial products induce the functional activation of beta2 integrins, but the signaling events that link membrane receptors to integrin activation are poorly understood. RESULTS: We report here that expression of the constitutively active small GTPases Rap1 or R-ras, but not Ras or RalA, is sufficient for functional activation of alphaMbeta2, the complement receptor 3 (CR3), in macrophages, allowing phagocytosis of C3bi-opsonized targets. Inhibition of Rap1, but not other Ras-like or Rho-like small GTPases, abolishes activation of alphaMbeta2 induced by phorbol esters, LPS, TNF-alpha or PAF. Finally, Rap1 activation specifically controls the binding properties of alphaMbeta2 towards its physiological ligand, namely the complement-opsonized phagocytic targets. CONCLUSIONS: In macrophages, the Rap1 GTPase regulates activation of the alphaMbeta2 integrin in response to a wide variety of inflammatory mediators.     

Cloricromene antagonizes antidipsogenic effects induced by endotoxin, but not by TNF alpha, in the rat.

Intravenous (640 micrograms/kg) or intracerebroventricular (0.5 and 1 microgram) injection of Escherichia coli endotoxin (LPS) causes inhibition of water intake induced by 24 hour period of water deprivation in the rat. Tumor necrosis factor alpha (TNF-alpha; 20 and 40 ng/rat) given into the lateral cerebral ventricle (i.c.v.) causes effects similar to those observed after LPS. Cloricromene, given either intravenously (1 and 2 mg/kg) or i.c.v. (250 and 500 ng), abolished the antidipsogenic effect induced by LPS (administered both i.v. and i.c.v.). Cloricromene (2 mg/kg, i.v. or 500 ng/rat, i.c.v.), on the contrary, did not modify the antidipsogenic effects induced by TNF-alpha. These data indicate that peripherally injected cloricromene (as well as that i.c.v. injected) antagonizes the effects of mediators of LPS on sites regulating thirst and suggest that cloricromene's action may be due to inhibition of brain TNF-alpha formation induced by LPS.     

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 microg/kg) or a low (10 microg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E(2) (PGE(2)) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 microg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE(2) formation (5 or 500 microg/kg diclofenac). Even the lowest dose of diclofenac (5 microg/kg) attenuated fever in response to 10 microg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE(2) at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 microg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 microg/kg LPS, whereas subcutaneous injections of 100 microg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 microg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.