Modulation of Mouse Endotoxic Fever by Complement

It was recently reported that the complement system may be criticallyinvolved 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 thisend, we used mice with C3, C5, and CR2 gene deletions. To assesspreliminarily the suitability of mice for such a study, we replicatedour earlier studies with guinea pigs. Thus, to verify initiallywhether complement is similarly involved in the febrile responseof wild-type (C57BL/6J) mice to i.p. LPS (Escherichia coli,1 µg/mouse), we depleted complement with cobra venom factor(CVF; 7 U/mouse, intravenously [i.v.]). These animals did notdevelop fever, whereas the core temperature (T_c) of CVF vehicle-treatedcontrols rose $~$ 1°C by 80 min postinjection and then graduallyabated over the following 2.5 h, confirming the involvementof complement in fever production after i.p. LPS injection andthe suitability of this species for these studies. C3- and C5-sufficient(C3^+/+ and C5^+/+) mice also developed 1°C fevers within80 min after i.p. LPS (1 or 2 µg/mouse) injection. Thesefevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment.C3- and C5-deficient (C3^-/- and C5^-/-) mice were also unableto develop T_c rises after i.p. LPS. Both CR2^+/+ and CR2^-/- miceresponded normally to i.p. LPS (1 µg/mouse). These dataindicate that C5, but not C3d acting through CR2, may play acritical role in the febrile response of mice to i.p. LPS.

INTRODUCTION

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Introduction

Bacterial endotoxic lipopolysaccharide (LPS) triggers the complementcascade via both the classical and alternative pathways, resultingin the production of multiple bioactive fragments (reviewedin reference 49). It was recently found that the complementsystem may be important in the febrile response of guinea pigsto, particularly, intraperitoneally (i.p.) administered LPS,because serum complement depletion caused by cobra venom factor(CVF) dose-dependently blocked fever due to i.p. LPS and becausei.p. LPS per se promoted serum complement consumption (20, 38).However, it was not determined in those studies which specificcomponent(s) of the complement cascade may be especially criticalin this regard, because CVF activates the whole alternativepathway (8, 47) and the consequent serum complement level changeswere assessed as the total hemolytic activity of complement.

Among the complement components generated by LPS, C3 and C5are of particular interest in the context of fever because,among their various proinflammatory activities, they and theirderivatives independently induce the production by myeloid andcertain nonmyeloid cells of tumor necrosis factor alpha (TNF- ${alpha}$ ),interleukin 1ß (IL-1ß), and IL-6, all putativeendogenous fever mediators (2, 6, 7, 42). C3 and C5 also amplifythe secretions of TNF- ${alpha}$ and IL-1ß induced by LPS inmonocytes and macrophages (6, 7) and are necessary for the invivo acute-phase expression in mice of complement-reactive proteinand serum amyloid P component (41). It may logically be assumed,therefore, that to the extent that the production of these pyrogenicmediators may be impaired in C3- and C5-deficient animals, theirfebrile response to LPS may be correspondingly attenuated.

Since there are no specific antibodies against guinea pig complementfragments readily available commercially, it is difficult toidentify or to block specific guinea pig complement fragments.On the other hand, there are several complement fragment- orfragment receptor-deficient mice available for research. Therefore,in this study, we compared the febrile responses to i.p. LPSof C3- and C5-sufficient and -deficient mice to determine whethereither or both of these components might mediate LPS fever development.To this end, we first replicated with wild-type (WT) mice previousstudies with guinea pigs to verify whether depletion of complementby CVF also blocks LPS fever in mice, i.e., to confirm the involvementof complement in fever production in mice after i.p. LPS andthereby ensure the suitability of this species for the presentexperiments. We then compared the febrile responses of C3- andC5-sufficient and -deficient mice to i.p. LPS. CR2^+/+ and CR2^-/-mice were chosen as negative controls because CR2, as primarilya regulator of B-cell activation, was not expected to be involvedin the early phase of LPS-induced fever genesis; moreover, thesensitivity of B cells to LPS is 2 orders of magnitude lowerthan that of macrophages. The results implicated the C5 fragmentas the mediator of the fever caused by i.p. LPS in mice.

MATERIALS AND METHODS

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Materials and Methods

Animals. WT (C57BL/6J) mice and mice with congenital deletions of theC3, C5, and CR2 genes (C3^-/-, C5^-/-, and CR2^-/-, respectively)and their corresponding complement-sufficient congenic controls(C3^+/+, C5^+/+, and CR2^+/+) were used in these experiments. Allthe animals weighed 20 to 25 g on arrival. The WT and C5 (B10.D2H-2^d H2-T18^c Hc^o/oSnj [C5^-/-] and B10.D2 H-2^d H2-T18^c Hc¹/nSnj[C5^+/+]) mice were purchased from The Jackson Laboratory, BarHarbor, Maine. The C3 (129/C57BL/6) and CR2 mice were providedby two of us (V.M.H. and S.A.B.) (24, 27, 39). The targetedgene deletions of the knockout mice were verified by their suppliers.All the complement-deficient mice appeared to be clinicallywell under the conditions of our normal laboratory housing.

Following receipt, the animals were quarantined for 3 weeks,four to a cage, before any experimental use. Tap water and food(Agway Prolab mouse diet) were available ad libitum. The ambienttemperature (T_a) in the animal room was 22 ± 1°C;light and darkness were alternated, with light on from 0600to 1800 h. After quarantine, to moderate the psychological stressassociated with the experiments, the mice were trained to theexperimental procedures for at least 2 weeks (4 h daily) byhandling and placement in locally constructed, individual wire-meshconfiners designed to prevent their turning around and minimizetheir forward and backward movements, without causing restraintstress. To eliminate possible effects of circadian variations,all the experiments were begun at the same time of day (0830h). On the experimental day, the mice were connected to therelevant measuring devices, placed in their confiners, and allowedto stabilize for at least 3 h until their core temperatures(T_c) varied not more than ±0.1°C over five consecutive2-min periods. All the studies were performed in accordancewith National Institutes of Health and institutional guidelinesfor animal care and use.

Drugs. CVF (Naja naja kaouthia) was purchased from Calbiochem-Novabiochem(San Diego, Calif.). LPS was from Escherichia coli serotypeO111:B4 (lot 36F4019, prepared by trichloroacetic acid extraction;protein content, 0.5%; Sigma Chemicals, St. Louis, Mo.). Thevehicle for all the solutions was pyrogen-free saline (PFS;0.9% NaCl, USP; Abbott Laboratories, Chicago, Ill.).

Depletion of complement. To achieve virtually complete depletion of serum complement,based on previous experiments with guinea pigs (20, 38) andon the literature (10), CVF (7 or 10 U/animal) was injectedinto a tail vein of the mice. To minimize possible acute effectsof decomplementation, this treatment was administered in twoboluses. The first bolus of half the dose was given at 1430h, 21 h before the actual experiments; the second half-dosewas injected 2 h later. This schedule also served to minimizetemporal deviations from our usual experimental protocol, i.e.,beginning the experiments at 0830 h of the following day.

Temperature recording. The animals, fully conscious in their confiners, were placedunder a perforated hood (free airflow) to prevent undue disturbancesfrom noise and fluctuations in T_a (22 ± 1°C). TheT_cs of the mice were monitored constantly and recorded at 2-minintervals for the duration of the experiments on a MacintoshPlus 1-megabyte computer through an analog-to-digital converter,using precalibrated copper-constantan thermocouples inserted2 cm into the colon and taped to the tail. The data were displayedon a video monitor, printed digitally on an Imagewriter printer,and stored on a disk for subsequent statistical analysis. Controlmeasurements were begun when the animals' T_cs had become stabilized,as described above. The treatment pertinent to a given experiment,i.e., the i.p. injection of LPS (in PFS, 0.2 ml/mouse) or vehicle(PFS, 0.2 ml), was then administered, and the measurements continuedaccording to the same routine as before treatment for the following4 h.

Assay of total hemolytic complement activity. At the conclusion of the experimental periods, the mice weredeeply anesthetized with methoxyflurane (Metofane, lot 012485;Schering-Plough, Union, N.J.), and blood was collected fromtheir orbital sinuses, clotted on ice for 30 min, and centrifuged(Beckman Microfuge 12; 3,000 rpm, 4°C, 10 min). The resultingserum samples were stored at -70°C until assayed. For anassay, 5 µl of serum was added to wells placed in agarosegel containing standardized sheep erythrocytes sensitized withhemolysin (kit RC001.P; The Binding Site, San Diego, Calif.).The plates were incubated first for 18 h at 4°C and thenfor 1 h at 37°C. They were photographed with a charge-coupleddevice camera, and the images were stored and printed by usingNational Institutes of Health Image version 1.61 for analyzingelectrophoretic gels. Since the kit was not as sensitive tomouse serum complement as to guinea pig serum complement (20,38), it was not possible to assess serum complement quantitatively.Therefore, only qualitative, visual determinations of the presenceor absence of complement were made (Fig. 1).

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FIG. 1. Total hemolytic activities of mouse serum complement. Well 1, quality control. Wells 2 to 8, dilutions of the standard using the manufacturer's standard. Wells 9 to 11, serum from WT mice, showing positive reactions (complement is present). Similar positive reactions were obtained with serum from C3^+/+, C5^+/+ CR2^+/+, and CR2^-/- mice (not shown). Wells 12 to 14, serum from C5^-/- mice, showing no reaction (complement is absent). Similar negative reactions were obtained with serum from CVF-pretreated WT, C3^+/+, and C5^+/+ mice (not shown).

Experiments. (i) Experiment 1. To verify the involvement of complement in the febrile responseto i.p. LPS of WT mice, we administered CVF (7 U, in two half-doses)i.v. to these mice; the control solution was PFS. At 21 h afterthe first dose, the animals were injected i.p. with LPS (1 µg/animal)or vehicle (PFS).

(ii) Experiment 2. Prior to determining whether C3 and/or C5 may be the complementfragment(s) that mediates the febrile response to i.p. LPS,we verified that complement is indeed also critically involvedin the febrile response of C3^+/+ and C5^+/+ mice, since theseoriginated from different strains than the WT mice (see "Animals"above). To this end, these mice were pretreated with i.v. CVF(7 or 10 U/animal) or vehicle (PFS) as in experiment 1. At 21h after the first dose, they were injected i.p. with LPS (1or 2 µg/animal) or its PFS.

(iii) Experiment 3. To substantiate the role of C3 and/or C5 in the febrile responseof mice to i.p. LPS, we administered PFS and LPS (1 or 2 µg/animal,i.p.) to C3^+/+, C3^-/-, C5^+/+, and C5^-/- mice. CR2^+/+ and CR2^-/-mice were the negative controls for this experiment and weretreated similarly.

Statistical analyses. Results are presented herein as means ± standard errors.The values of T_c are reported as the changes from basal (initial)values (T_ci, the T_c at 2-min intervals averaged over the last10 min of the preceding 3-h stabilization period). Student'spaired t test was used to compare pre-LPS (basal) and post-LPS(maximal) data within a treatment. Differences between treatmentswere evaluated by a repeated-measure analysis of variance model,where factor 1 was the between-group factor (the experimentaltreatment) and factor 2 was the within-subject factor (the differentsampling periods). Each variable was considered to be independent.The 5% level of probability was accepted as statistically significant.

RESULTS

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Results

Experiment 1. The i.p. injection procedure and the associated handling perse rapidly induced a transient, ca. 0.7°C rise in T_c, despitethe prior training of these WT mice. It culminated within ca.10 to 15 min and then abated over the following 50 to 60 minin the PFS-treated group (Fig. 2A). LPS administration, on theother hand, caused a further rise of T_c that reached a peakof $~$ 1.1°C above T_ci by 30 min. T_c then declined slightly,rebounded, peaked a second time at ca. 80 min, and remainedat essentially the same level over the following 30 min (Fig.2B); it then gradually abated over the next 2 h. These responsesto PFS and LPS were consistent with those in our previous studiesusing the same strain of mice (19, 21) as well as with the dataof others for different strains in this T_a (17, 50). The firstT_c rise at 10 min in both treatment groups was handling-stressinduced; the superimposed T_c elevation and the second T_c riseof the LPS-treated group represented the pyrogenic responseto LPS. CVF (7 U/animal), which depleted the serum complementto below detection (Fig. 1, wells 12 to 14), had no effect onthe initial T_c rises of both groups but significantly attenuatedthe superimposed elevation and especially the second peak inthe LPS-treated mice.

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FIG. 2. Effects of i.p. injected PFS (0.2 ml) (A) and E. coli LPS (1 µg/animal in 0.2 ml of PFS) (B) on the T_c of WT C57BL/6J mice pretreated with PFS (CVF 0) or 7 U of CVF/animal. A 3-h stabilization period preceded collection of data. The T_cs are expressed as differences ( ${Delta}$ T_c) relative to their initial level (T_ci); the values are means ± standard errors. CVF was given i.v. 21 h before the measurements in two half-doses (see Materials and Methods). PFS and LPS were given at time 0. Numbers in parentheses are number of animals.

Experiment 2. The i.p. administration of PFS to C3- and C5-sufficient andCVF-pretreated C3- and C5-sufficient mice caused no demonstrablethermal effect other than the temporary, initial T_c rise associatedwith handling and the injection itself (Fig. 3A and C and 4A and C).CVF vehicle-pretreated C3^+/+ and C5^+/+ mice both developedtwo-peaked, ca. 1°C fevers at 30 and 80 min after LPS, respectively(Fig. 3 and 4B). These tended to abate more quickly ( $~$ 60 min)than the fevers of the WT mice (Fig. 2B). CVF (7 U/animal),which inconsistently reduced the serum complement of the C5^+/+mice (Table 1), had no significant effect on the fevers of theseanimals (Fig. 4B), whereas 10 U of CVF, which depleted the serumcomplement of both the C3^+/+ and C5^+/+ mice to below detection(Table 1), blocked their fevers in the same manner as in theWT mice (Fig. 3B and 4B) (P < 0.001 and F = 15.5968 [C3^+/+]and P < 0.001 and F = 16.5574 [C5^+/+] compared with micereceiving 0 U of CVF).

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FIG. 3. Effects of i.p. injected PFS (0.2 ml) and E. coli LPS (1 µg/animal in 0.2 ml of PFS) on the T_c of C3^+/+ mice pretreated i.v. with PFS (CVF 0) or 10 U of CVF/animal (A and B) and of C3^+/+ and C3^-/- mice (C and D). Conditions and notations are as described for Fig. 2.

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FIG. 4. Effects of i.p. injected PFS (0.2 ml) and E. coli LPS (2 µg/animal in 0.2 ml of PFS) on the T_c of C5^+/+ mice pretreated i.v. with PFS (CVF 0) or 7 or 10 U of CVF (A and B) and of C5^+/+ and C5^-/- mice (C and D). Conditions and notations are as described for Fig. 2.

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TABLE 1. Serum hemolytic complement activity of various strains of mice after LPS challenge

Experiment 3. In contrast to the C3^+/+ and C5^+/+ mice, the C3^-/- and C5^-/-mice did not develop febrile rises in response to i.p. LPS (Fig.3D and 4D) (P < 0.001 and F = 16.9416 compared with C3^+/+mice; P < 0.001 and F = 16.9768 compared with C5^+/+ mice).Indeed, the handling-stress hyperthermia appeared to be attenuatedin the C3^-/- mice (Fig. 3D), although not in the C5^-/- mice(Fig. 4D). CR2^-/- mice, which exhibited serum complement levelssimilar to those of their CR2^+/+controls (Table 1), developedT_c rises in response to handling and to i.p. PFS (Fig. 5A) andLPS (Fig. 5B) also not significantly different from those oftheir controls. After LPS, the first febrile peak occurred at30 min and the second occurred at $~$ 100 min. These fevers thenabated over the following 2 h (Fig. 5B).

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FIG. 5. Effects of i.p. injected PFS (0.2 ml) (A) and E. coli LPS (1 µg/animal in 0.2 ml of PFS) (B) on the T_c of CR2^+/+ and CR2^-/- mice. Conditions and notations are as described for Fig. 2.

DISCUSSION

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Discussion

The present results show that the complement system is criticallyinvolved in the febrile response to i.p. LPS of the three strainsof mice used in this study, since both decomplementation byCVF and complement fragment-targeted gene disruption inhibitedthe ability of these animals to develop fever after challengeby low doses of this exogenous pyrogen. These data further showthat C3 and/or C5 may be the fragment(s) that mediates i.p.-LPS-inducedfever, since, in contrast to their congenic, complement-sufficientcontrols, both C3- and C5-deficient mice were unable to mountfevers in response to this challenge. However, it may be speculatedthat C5 rather than C3 could be the key player in this instance,since C3 knockouts would not generate C5 whereas C5^-/- micewould not lack C3. On the other hand, both CR2^+/+ and CR2^-/-mice responded normally to i.p. LPS, indicating, as expected,that C3d acting through this receptor likely is not involvedin the febrile response to this pyrogen. Since macrophages cansynthesize most of the various components of the classical andalternative pathways of complement and little complement diffusesfrom the plasma into the peritoneal fluid under normal conditions,the complement initially activated by LPS in the peritoneumwas thus presumably derived principally from the peritonealmacrophages themselves (3).

It is well established that LPS almost immediately activatesthe complement cascade via both the classical and alternativepathways (reviewed in reference 49). The important involvementof complement in various LPS-induced host defense responsesis very well documented (40, 45, 48). Lately, complement hasalso been implicated in the LPS-induced expression of the criticalacute-phase proteins complement-reactive protein and serum amyloidP component (41), and other data have shown that under conditionsof complement deficiency, responses that are normally coactivatedwith the febrile response, e.g., phagocytosis, chemotaxis, andproduction and release of various inflammatory mediators, areimpaired (22, 40). Hence, a role for complement in LPS feverproduction would be congruent with these related host defenseresponses, as we demonstrated earlier in guinea pigs (20, 38).No other studies to date appear to have systematically examinedthe role of complement in fever, although it was noted in onestudy of canine endotoxic shock that congenitally C3-deficientBrittany spaniels exhibited reduced fevers during the first24 h after i.v. E. coli LPS (2 mg/kg) treatment compared withtheir congenic controls (34). Also, Mickenberg et al. (25, 26)found that the serum total hemolytic complement activity andC3 titers fell in rabbits within 5 min after the i.v. administrationof low-dose, soluble antigen-antibody complexes and that rabbitsdepleted of complement by pretreatment with CVF exhibited greatlydiminished febrile responses in comparison with untreated controls.On the other hand, congenitally complement-deficient, clinicallyinfected patients can present with fever (29), and, indeed,there is one interesting report of a C3-deficient child withan elevated T_c without a concurrent infection (32). Further,in one study of human subjects injected i.v. with relativelylow doses of E. coli LPS, no change in anaphylatoxin levelswas detected in the plasma and T_c rose normally 30 to 45 minafter the injection (46). In guinea pigs, by contrast, pyrogenicdoses of both i.v. and i.p. LPS are associated with early, transient,but significant decreases in serum CH₁₀₀ activity as well asfever (20). Thus, guinea pigs and mice seem to be similar intheir dependence on complement for the induction of their febrileresponse to LPS. The apparently discrepant independence of complementof human endotoxemic fever could be due to the fact that thepathogenesis of clinical infections is more complicated thanis shown by specific animal experimental models, generally involving,in addition to LPS, many inflammatory factors not incorporatingcomplement mediation. Moreover, the requirement for complementcan be bypassed with higher LPS loads (20).

Unfortunately, the present data do not indicate whether thea or the b cleavage products of C5 might be the critical mediatorsof these i.p. LPS-induced fevers or which cell type(s) in theperitoneum might be its specific target(s). A priori, C5a mightbe considered the more likely candidate mediator in the presentanimal model because it has been demonstrated to induce, synergisticallywith LPS, TNF- ${alpha}$ and IL-1ß production by mouse peritonealmacrophages (36). It also mediates the release of IL-6 by humanmonocytes (36). These cytokines are endogenous pyrogens, andputative IL-1ß binding sites have been identifiedon vagal sensory terminals in the abdomen that, according toone model, could convey the pyrogenic signals to the brain (reviewedin reference 4). The absence of C5a could therefore impair theproduction of these cytokines and, consequently, abolish thefebrile response. However, it seems unlikely that this effectcould solely and specifically account for the suppression offever observed here unless the amount of C5a required to activatethese macrophages under normocomplementemic conditions is verysmall and uniquely localized to the i.p. site of the injectedLPS bolus, since its concentration in uninflamed peritonealfluid is normally minimal and activation of C3 by CVF does notcause by itself a febrile response (20). On the contrary, itcauses a fall in T_c (38), presumably consequent to the inductionof coincident processes that combine to produce the systemicarterial hypotension typically observed after such a treatmentor following the systemic injection of complement (23, 44).C5a, moreover, is rather short-lived, and its degradation product,C5a desArg, is relatively inactive in this respect. The possibilitythat C5a could directly bind to its putative receptors on localsensory terminals (30) and relay the pyrogenic message to thebrain consequently also seems improbable in the present context,particularly since the induction of C5a receptors after LPSinjection lags behind the onset of the febrile response (11).

An alternative role of C5a in the mediation of i.p. LPS-inducedfever, however, may be more plausible. This role is relatedto the fact that C5a and C5a desArg have conventional, earlyfunctions of enhancing microvascular permeability and attractingleukocytes, thereby recruiting to the peritoneum factors thatare necessary to initiate host defense responses but are normallysparse in peritoneal fluid. Thus, as for LPS, these factorsinclude LPS-binding protein (LBP), which is synthesized mainlyin hepatocytes and secreted into the bloodstream constitutivelyin small amounts (1 µg/ml in mice) or in larger amounts(5 µg/ml) as a class I acute-phase protein induced hoursafter challenge (37). Indeed, the concentration of this proteinis minimal in uninflamed peritoneal fluid in vivo, althoughits abundant presence in inflamed peritoneal fluid in vivo (35)and its necessity for LPS activation of (murine) peritonealmacrophages have been demonstrated in vitro (15). LBP catalyzesthe monomerization of LPS micelles and transfers the monomersas LPS-LBP complexes to another protein, CD14.

CD14 is present in plasma in a soluble form (sCD14) and as amolecule that is glycosylphosphatidylinositol-anchored to themacrophage plasma membrane (mCD14); mCD14 exists on peritonealmacrophages, but sCD14 is scarce in normal peritoneal fluid.Since neither form possesses a transmembrane domain, signaltransduction is achieved by the LPS-LBP-CD14 complex cross-linking,in turn, to an ancillary cell membrane-associated receptor subunit(9), which in mouse peritoneal macrophages has been identifiedas the Toll-like receptor 4-MD-2 complex (1). If LBP is indeedabsent or minimally present in the peritoneums of normal miceand given that, in its absence, polymeric LPS at low concentrationsbinds poorly to mouse peritoneal macrophages and fails to stimulatethe production of TNF- ${alpha}$ , IL-1ß, and IL-6 (14, 31),the role of C5a might indeed be to increase vascular permeabilityand promote postcapillary venular leakage of plasma LBP intothe peritoneal locus of the injected LPS aggregates as wellas to attract neutrophils to this site. In fact, C3-deficientmice (hence also lacking C5) clear i.p. injected LPS, recruitneutrophils to the peritoneum more slowly, and produce lessTNF- ${alpha}$ than do C3-sufficient animals (33). The entry of sCD14into the peritoneum under these conditions would also be highlycomplementary to the febrigenic process because recent evidencehas suggested that the activation of polymorphonuclear leukocytesby LPS that results in the production of mediators involvesthe binding of LPS-sCD14 complexes to the cells' plasma membranes,followed by internalization and localization in the Golgi apparatus(43), in contrast to the delivery of LPS polymers via mCD14to phagosomes, which results in the lysosomal enzymatic degradationof LPS but not in the production of mediators (12, 16).

Moreover, since only minute amounts of complement diffuse fromthe plasma into the uninflamed peritoneum, the simultaneousgreater influx of complement under these conditions would amplifythe effects of the locally generated complement. Indeed, thistranslocation of complement could account for the initial, transientdecline in plasma complement levels previously observed in guineapigs after i.p. LPS (20). If this scenario is valid, fever inducedby i.v. LPS should not be complement dependent, due to the constitutivepresence of LBP and sCD14 in plasma in adequate concentrations.This seems indeed to be the case in guinea pigs, since hypocomplementemicguinea pigs developed normal febrile responses to i.v. LPS (38).The objection to this argument that LPS is detectable in plasma(18) within 15 min after its i.p. injection and, therefore,should produce a febrile response similar to that produced uponi.v. injection can be countered by the fact that LPS enteringthe circulation from the peritoneum does so slowly and incrementally,i.e., in amounts small enough to be readily cleared by the relevantcounteracting mechanisms available in plasma (sCD14, bactericidal/permeability-increasingprotein, lipoproteins, etc.). Indeed, 5 µg of LPS/kg,i.p., did not produce increased plasma IL-1ß concentrationsin rats (18), and a correspondingly low i.p. dose of fluoresceinisothiocyanate-labeled LPS was not detectable in guinea piglivers 15 min after injection (20). Higher i.p. doses of LPS,however, may appear in plasma more quickly (18, 28) and, therefore,be less dependent on complement (5, 20, 28). Finally, an involvementof C5b-9 is also possible, since membrane attack complex insublytic concentrations has been reported to activate monocytesto release IL-1ß (13).

Taken together, the present results are consistent with previousobservations obtained with guinea pigs that complement is importantlyinvolved in the febrile response to low-dose, i.p. injectedLPS; the cascade fragment C5 appears to be especially criticalfor this response. However, which of its transcripts, a and/orb, may mediate fever and by what mechanism(s) this may be accomplishedremain speculative. We suggest that C5a-enhanced microvascularpermeability and the consequent, rapid influx from plasma ofLBP, sCD14, and complement, all sparse in uninflamed peritonealfluid, may enable peritoneal macrophages to more efficientlytake up LPS and produce pyrogenic cytokines; their ability todo so is greatly limited in the absence of these plasma proteins.The associated recruitment of neutrophils augments their owncapacity in this host defense process. C5b-C9 may also separatelyand additionally stimulate the production of cytokines.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant NS-34857from the National Institutes of Neurological Disorders and Stroketo C. M. Blatteis.

We thank Easter Jenkins for her efficient typographic help andGregg Short and Daniel Morse for their excellent graphic artssupport.

FOOTNOTES

* Corresponding author. Mailing address: Department of Physiology, The University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Phone: (901) 448-5845. Fax: (901) 448-7126. E-mail: blatteis{at}physio1.utmem.edu.

Editor: W. A. Petri, Jr.

REFERENCES

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