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Endocrinology Vol. 139, No. 1 119-127
Copyright © 1998 by The Endocrine Society

ARTICLES

Intracerebroventricular Passive Immunization. I. The Effect of Intracerebroventricular Administration of an Antiserum to Tumor Necrosis Factor-{alpha} on the Plasma Adrenocorticotropin Response to Lipopolysaccharide in Rats1

Andrew V. Turnbull2 and Catherine L. Rivier3

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. Catherine L. Rivier, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study tested the hypothesis that the cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) is an important central nervous system mediator of the rat hypothalamo-pituitary-adrenal (HPA) axis response to the iv administration of lipopolysaccharide (LPS; 5 µg/kg). LPS produced a rapid (within 30 min) rise in plasma TNF-{alpha} levels, which preceded elevations in plasma ACTH (commencing at 45 min). Despite a lack of detectable TNF-{alpha} biological activity in the brain 30 min to 2 h after LPS administration, intracerebroventricular (icv) pretreatment (-20 h) with 5 µl anti-TNF-{alpha} antiserum significantly delayed the onset of the plasma ACTH response to LPS, suggesting that TNF-{alpha} acts within the brain. However, we also noted that the icv infusion of anti-TNF-{alpha} 20 h earlier produced experimentally significant concentrations of the same anti-TNF-{alpha} antibodies in systemic blood. This suggested the possibility that the effect of this antiserum was due to its leakage to the periphery. Indeed, 5 µl anti-TNF-{alpha} administered iv at -20 h produced an inhibition of the ACTH response to LPS that was temporally and quantitatively similar to that produced by icv anti-TNF-{alpha}. Intracerebroventricular administration of anti-TNF-{alpha} immediately before LPS produced only low systemic blood levels of corresponding anti-TNF-{alpha} antibodies and did not significantly alter the plasma ACTH response, whereas iv administration of anti-TNF-{alpha} immediately before LPS was clearly effective. Collectively, these results show that 1) biologically active levels of TNF-{alpha} in systemic plasma and the ensuing ACTH responses to LPS were always temporally and qualitatively related; and 2) even though icv administration of anti-TNF-{alpha} could inhibit the HPA axis response to LPS, this was apparent only when substantial amounts of anti-TNF-{alpha} antibodies had reached systemic blood. We, therefore, conclude that at the dose of LPS used in this study (5 µg/kg), TNF-{alpha} is an important mediator of the HPA axis response to LPS by an action within the periphery, but probably not within the brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TUMOR NECROSIS factor-{alpha} (TNF-{alpha}), along with other cytokines such as interleukin-1 (IL-1) and IL-6, are critical endogenous mediators of the physiological effects of endotoxin [lipopolysaccharide (LPS)]. The key role of TNF-{alpha} in response to LPS is demonstrated by its relative speed of elaboration (faster than IL-1 and IL-6) (1, 2, 3), its regulation of IL-1 and IL-6 production (4, 5), and the abrogation of response to LPS produced by inhibition of TNF-{alpha} synthesis (6) or action (7, 8). Although the major cells producing cytokines after peripheral administration of LPS are monocytes/macrophages within the periphery, these three cytokines are also key regulators of central nervous system (CNS)-mediated acute phase responses, such as fever, anorexia, neuroendocrine alterations, and behavioral changes (9, 10, 11). In particular, TNF-{alpha}, IL-1, and IL-6 play a critical role in endotoxin-stimulated activation of the hypothalamo-pituitary-adrenal (HPA) axis. Accordingly, in normal experimental subjects, the administration of TNF-{alpha}, IL-1, or IL-6 mimics the effects of LPS and increases the synthesis and/or secretion of hypothalamic CRF, which mediates consequent increases in pituitary ACTH and adrenal corticosteroid secretion (reviewed in Refs. 12 and 13). Furthermore, in LPS-treated laboratory animals, inhibition of TNF-{alpha}, IL-1, or IL-6 action within the periphery reduces the elevation in plasma ACTH concentrations (12, 13).

The mechanism through which elevated blood levels of these cytokines might influence CNS activities is unclear, as the large size of these polypeptides (17–26 kDa) precludes simple diffusion across the blood-brain barrier (BBB). Specific transport of cytokines across the BBB (14), cytokine activation of CNS regions that are relatively devoid of a BBB (such as the circumventricular organs) (15), induction of readily diffusible intermediates at the BBB interface (16, 17), and stimulation of peritoneal sensory afferents (18, 19) have all been proposed as potential mechanisms by which cytokines within the periphery may influence the CNS. However, in addition to elaboration within the periphery, TNF-{alpha}, IL-1, and IL-6 are synthesized within the brain, in astrocytes, microglia, endothelia, ependymal cells, and possibly neurons (9, 11, 20). Peripheral LPS administration produces rapid (within 1–2 h) increases in TNF-{alpha} messenger RNA (mRNA) in the rodent CNS (21, 22, 23, 24) and also elevates cerebral IL-1 and IL-6 mRNAs (23, 24, 25, 26, 27, 28, 29, 30). The possibility that increased production of cytokines within the CNS may influence HPA activity directly represents an attractive hypothesis given the importance of hypothalamic CRF secretion in HPA activation due to LPS or cytokine. Nevertheless, the physiological significance of elevated CNS cytokine synthesis has not been well studied. The present work sought to define the role of cerebral TNF-{alpha} in the mediation of LPS-induced HPA activation in the rat. First, we describe the time course of the elevation in plasma ACTH concentrations and plasma and brain biological activity of TNF-{alpha} in response to a moderate dose (5 µg/kg) of LPS given iv. Secondly, we determined the effect of inhibiting TNF-{alpha} action within the brain on the plasma ACTH response to iv LPS. To achieve inhibition of TNF-{alpha} within the brain, we adopted a protocol of prior intracerebroventricular (icv) passive immunoneutralization, an approach that has been used extensively in the investigation of neuropeptide function within the CNS (31, 32, 33, 34, 35, 36).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Rabbit anti-mouse TNF-{alpha} antiserum was donated by Dr. S. L. Kunkel (Department of Pathology, University of Michigan). This antiserum recognizes both recombinant and natural murine TNF-{alpha}, displays high cross-reactivity with rat TNF-{alpha}, does not cross-react with recombinant IL-1{alpha} or IL-1ß, and binds and neutralizes the biological effects of rat TNF-{alpha} both in vitro and in vivo (37, 38, 39, 40, 41, 42). In the present study, rats were passively immunized icv with 5 µl undiluted antiserum or iv with 500 µl antiserum (diluted 1:100 in sterile PBS-0.1% BSA). Similar volumes and dilutions of normal rabbit serum (NRS; Colorado Serum Co., Denver, CO) were used as control injections. Sheep anti-rabbit IgG antiserum was a gift from Dr. W. Vale (The Salk Institute, La Jolla, CA).

Recombinant murine TNF-{alpha} (rmTNF-{alpha}; code 88/532, First International Standard) was obtained from the National Institute for Biological Standards and Control (South Mimms, UK) and used as a standard in the analysis of TNF-{alpha} bioactivity. LPS (Escherichia coli serotype O26:B6; code L3755, lot. 20H4025) was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in sterile PBS (vehicle).

Animals
Male Sprague-Dawley rats (initial body weight, 170–240 g) were purchased from Harlan Sprague-Dawley Laboratories (Indianapolis, IN) and housed in animal facilities adjacent to experimental rooms (ambient temperature, 22 C). They were maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) and provided with rat chow (Harlan-Teklad, Madison, WI) and water ad libitum. All procedures described were approved by The Salk Institute animal use and care committee.

Surgical preparation
Rats were equipped with iv catheters 48 h before experimentation, as described previously (43). In experiments in which animals received infusions directly into the cerebroventricles, indwelling guide cannulas were implanted (44) 7–9 days before iv cannulation (at coordinates from Bregma with incisor bar set at -3.3 mm: anterior-posterior, -0.4 mm; lateral, -1.4 mm; dorsoventral, -3.5 mm). Intracerebroventricular treatments (5 µl) were infused via a connecting injection needle that extended 1 mm beyond the tip of the guide cannula (Plastics One, Roanoke, VA), and infusions were performed in freely moving rats over a period of 60 sec. After completion of experiments, animals were killed, and 5 µl India ink were injected via the icv assembly. Only data from animals that showed a distribution of ink throughout the ventricular system (i.e. third, fourth, and lateral ventricles and cerebral aqueduct) were included in subsequent analyses.

Sample collection
Blood samples were collected from undisturbed rats as described previously (43). For experiments in which repeated measurements were made, a maximum of 0.35 ml blood/sample was drawn and replaced with 0.2 ml sterile heparinized saline. Upon collection, each blood sample was divided into two chilled tubes, one containing EDTA (for measurement of ACTH) and the other containing preservative-free, sterile heparin (for measurement of TNF-{alpha}). Samples were centrifuged, and plasma was aliquoted and stored at -20 C (for ACTH) or -70 C (for TNF-{alpha}).

To collect specific brain regions for analysis of TNF-{alpha} biological activity, rats were deeply anesthetized (0.7 ml 35% chloral hydrate, ip) and transcardially perfused with 0.9% saline (60 ml/3 min) to remove contamination of samples with blood. Samples were collected 30 min, 1 h, and 2 h after iv administration of either vehicle (PBS) or LPS (5 µg/kg). The cerebral cortex, striatum, hypothalamus, and hippocampus were dissected from brains kept on wet ice and immediately frozen on dry ice. Supernatants of brain tissue homogenates were prepared as described previously (38) and assayed for TNF-{alpha} biological activity.

Assay of TNF-{alpha} biological activity
Plasma TNF-{alpha} biological activity was determined by comparing the cytotoxicity to L929 cells of samples to that of the WHO International Standard (code 88/532, First International Standard), as described previously (38). Briefly, L929 cells were cultured (7.5% CO2-92.5% O2; 37 C; 100% humidity) in RPMI 1640 medium (Cellgro, Herndon, VA) containing 5% FBS (Gemini BioProducts, Calabasas, CA), 2 mM L-glutamine (Sigma), 50 µM 2ß-mercaptoethanol (Sigma), 50 U penicillin, and 50 µg streptomycin/ml (Sigma) in 15-cm diameter tissue culture dishes (Becton Dickinson Co., Cockeysville, MD). Confluent cells were removed using approximately 15 ml trypsin-EDTA solution (IX, Sigma) and gentle agitation. Cells were washed and resuspended in medium containing 0.5 µg/ml mitomycin C (Sigma). One hundred microliters of cells were then plated in standard 96-well microtiter plates (Costar Corp., Cambridge, MA) at a concentration of 2 x 105 cells/ml and cultured overnight and incubated with serially diluted (1:2 to 1:3) standard or samples for approximately 24 h. Medium was removed from the cells by inverting the plates, and cell survival was determined colorimetrically using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) as previously described (43). All samples from the same experiment were analyzed for TNF-{alpha} bioactivity in the same assay. Lower detection limits varied between 4–135 IU/ml. Samples below or at the detection limit were assigned the activity of the detection limit for that sample.

Plasma ACTH measurements
Plasma ACTH concentrations were measured using a commercial immunoradiometric assay (Allegro, Nichols Institute, San Juan Capis-trano, CA), as described previously (45). In the present experiments, the lower limit of detection was 5 pg/ml, and within- and between-assay coefficients of variation were 9.3% and 7.6%, respectively, at 36 pg/ml and 7.6% and 15.2% at 310 pg/ml.

Recovery of TNF-{alpha} biological activity from plasma samples "spiked" ex vivo with rmTNF-{alpha}
To determine the recovery of TNF-{alpha} activity from plasma of rats infused with anti-TNF-{alpha} icv, blood samples were obtained from rats 30 min, 4 h, and 20 h after icv infusion of 5 µl of either NRS or anti-TNF-{alpha}. Twenty microliters of each plasma sample were incubated for 3 h at 37 C with 20 µl rmTNF-{alpha} (final concentration, 4000 IU/ml). These samples were then assayed (in duplicate) for TNF-{alpha} biological activity, as described above.

A separate series of experiments was performed to determine whether activity in plasma that masked the detection of TNF-{alpha} after icv anti-TNF-{alpha} was attributable to the rabbit anti-TNF-{alpha} antiserum that had been infused. Plasma samples were obtained from rats 20 h after icv infusion of 5 µl of either NRS or anti-TNF-{alpha}. Three 40-µl aliquots of each plasma sample were then incubated at room temperature for 3 h with 40 µl of 1) 0.5% NRS-0.9% sterile saline, 2) 0.5% NRS-5% polyethylene glycol (PEG)-0.9% sterile saline, or 3) 1:40 diluted sheep anti-rabbit IgG-0.5% NRS-5% PEG-0.9% sterile saline. Samples were washed with 50 µl saline and centrifuged (2000 x g) for 45 min. Seventy microliters of the resulting supernatants were incubated with 10 µl rmTNF-{alpha} (final concentration, 1000 IU/ml) for 3 h at 37 C and then assayed for TNF-{alpha} biological activity.

Data presentation and statistical analyses
The data are presented as the mean ± SEM, and the number of subjects in each experimental group is indicated in the figure legends. Statistical analyses of repeated ACTH and TNF-{alpha} measurements were performed on log-transformed data, using ANOVA with repeated measures followed by least squared means difference analysis as a post-hoc test. All other statistical analyses were performed using unpaired Student’s t test. A two-tailed probability of less than 5% (i.e. P < 0.05) was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of the changes in plasma ACTH concentration and plasma and brain TNF-{alpha} bioactivities produced by iv LPS
Immediately before iv injection (at 1130 h) of either vehicle (PBS) or LPS, plasma ACTH concentrations were low and similar in both groups of animals (vehicle, 14 ± 2 pg/ml; LPS, 15 ± 2 pg/ml). Plasma ACTH concentrations remained constant for 180 min after iv injection of vehicle, but rose slightly (to 36 ± 5 pg/ml) by 240 min (2.5 h before lights out). Injection of LPS (5 µg/kg, iv) produced a marked increase in plasma ACTH concentrations (Fig. 1Go), which commenced at 45 min (181 ± 34 pg/ml), peaked at 90 min (709 ± 94 pg/ml), and were returning toward preinjection values by 240 min (120 ± 37 pg/ml).



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  		Figure 1. The effect of an iv bolus of LPS (5 µg/kg) on plasma TNF-{alpha} biological activity (international units per ml; closed circles) and plasma ACTH concentration (picograms per ml; open squares) in the rat (n = 9 subjects). The plasma concentration of ACTH in vehicle-treated rats (n = 8; not shown) remained essentially constant, varying between a minimum of 14 ± 2 pg/ml at 0 min and a maximum of 36 ± 5 pg/ml at 240 min. Plasma TNF-{alpha} bioactivity in vehicle-treated animals remained below 150 IU/ml at all times.

 
Before injection of LPS or after treatment with vehicle, the majority (>70%) of plasma samples in this and subsequent experiments did not contain detectable TNF-{alpha} biological activity (detection limit varied between 4–135 IU/ml). In no experiment did injection of vehicle produce a measurable rise in plasma TNF-{alpha} bioactivity. Consequently, in all experiments only data from LPS-treated rats are represented graphically. Injection of LPS (5 µg/kg, iv) produced a marked rise in plasma TNF-{alpha} biological activity with a time course that preceded the rise in plasma ACTH concentrations (Fig. 1Go). The increase in plasma TNF-{alpha} biological activity was apparent 30 min after LPS (444 ± 85 IU/ml) and peaked at 60 min (12,603 ± 2,421 IU/ml); by 240 min, values had returned to around the detection limit of the assay (44 ± 11 IU/ml).

There was no detectable TNF-{alpha} biological activity (i.e. <100 IU/ml) in the supernatants prepared from homogenates of cerebral cortex, hippocampus, hypothalamus, striatum, or hippocampus of animals treated iv with only PBS (0.5, 1, or 2 h after injection). TNF-{alpha} activity was also undetectable in any brain region at any time after iv treatment with 5 µg/kg LPS. In contrast, icv injection of 1 µg LPS induced marked elevations in brain TNF-{alpha} biological activity within 1 h that lasted for at least 3 h (38).

Intracerebroventricular pretreatment 20 h earlier with anti-TNF-{alpha} antiserum delays the onset of the plasma ACTH response to LPS
Intracerebroventricular infusion of 5 µl anti-TNF-{alpha} antiserum 20 h earlier did not affect basal plasma ACTH concentrations, and similar basal values were observed in rats pretreated with NRS or anti-TNF-{alpha} (Fig. 2Go). In animals pretreated icv 20 h earlier with only NRS, iv LPS induced a similar elevation in the plasma ACTH concentration to that observed in the first experiment, with a significant increase apparent at 45 min, a peak at 90 min, and values returning toward baseline by 240 min. Infusion of 5 µl anti-TNF-{alpha} antiserum 20 h earlier delayed the onset of the plasma ACTH response to LPS (Fig. 2Go). Unlike NRS-pretreated rats, plasma ACTH concentrations in animals pretreated with icv anti-TNF-{alpha} remained unaltered at 45 min and were significantly reduced compared with those in NRS-treated rats at 60 min [anti-TNF-{alpha} (icv), 284 ± 41 pg/ml; NRS (icv), 508 ± 78 pg/ml; P < 0.05]. By 90 min after LPS injection, plasma ACTH concentrations were similar in rats treated with NRS (icv) and anti-TNF-{alpha} (icv), and no significant differences were observed between these two groups from 120–240 min after LPS injection (data not shown).



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  		Figure 2. The effect of icv anti-TNF-{alpha} antiserum pretreatment (5 µl; 20 h before) on the rise in plasma ACTH concentrations produced by LPS (5 µg/kg, iv; n = 6–9 subjects/experimental group). ANOVA with repeated measures indicated a significant interaction between anti-TNF-{alpha} and LPS treatments (P < 0.001). *, P < 0.05; **, P < 0.01 (vs. NRS/LPS-treated rats, by least squared means test).

 
In these experiments plasma TNF-{alpha} biological activity before injection of LPS was detectable in six of nine animals treated with NRS icv, but was below the detection limits in all animals pretreated with anti-TNF-{alpha} icv. LPS produced an increase in the plasma biological activity of TNF-{alpha} in icv NRS-pretreated rats similar to that observed in animals treated with LPS alone in the first experiment (see Figs. 1Go and 3Go). Pretreatment icv with 5 µl anti-TNF-{alpha} at -20 h inhibited the rise in measurable TNF-{alpha} biological activity in plasma produced by LPS (Fig. 3Go), preventing any significant rise in plasma TNF-{alpha} biological activity 30 min after LPS and significantly reducing the levels observed at 45 and 60 min. However, icv anti-TNF-{alpha} did not significantly affect the rise in TNF-{alpha} biological activity observed in plasma samples 90 min after LPS administration (Fig. 4Go) or at any time thereafter (data not shown).



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  		Figure 3. The effect of icv anti-TNF-{alpha} antiserum pretreatment (5 µl; 20 h before) on the rise in plasma TNF-{alpha} biological activity produced by LPS (5 µg/kg, iv; n = 6–9 subjects/experimental group). ANOVA with repeated measures indicated a significant interaction between icv anti-TNF-{alpha} and iv LPS treatments (P < 0.001). **, P < 0.01; ***, P < 0.001 (vs. NRS/LPS-treated rats, by least squared means test).

 


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  		Figure 4. A, Effect of anti-TNF-{alpha} icv (5 µl) on the recovery of TNF-{alpha} biological activity from plasma spiked ex vivo with rmTNF-{alpha}. Normal rats were injected icv with either NRS (open bars) or anti-TNF-{alpha} (solid bars), and blood samples were taken 30 min, 4 h, and 20 h later. Plasma samples were then incubated with recombinant mouse (rm) TNF-{alpha} for 3 h, and TNF-{alpha} biological activity was subsequently determined. n = 4 subjects/experimental group. The dotted line represents the calculated spiked TNF-{alpha} activity based on the amount of rmTNF-{alpha} added. B, Effect of preprecipitation with a sheep anti-rabbit IgG on the recovery of ex vivo spiked TNF-{alpha} biological activity from plasma samples of rats after icv infusion of anti-TNF-{alpha} (5 µl). Rats were injected icv with anti-TNF-{alpha}, and 20 h later blood samples were taken. Samples were then incubated with NRS/saline, NRS/PEG/saline, or sheep anti-rabbit IgG/NRS/PEG/saline and centrifuged at 2000 x g for 40 min. Supernatants were spiked with rmTNF-{alpha} and assayed for TNF-{alpha} biological activity. n = 4–5 subjects/experimental group. The dotted line represents the calculated spiked TNF-{alpha} activity based on the amount of rmTNF-{alpha} added. ns, Not significant; **, P < 0.01; ***, P < 0.001 (vs. icv NRS injected rats, by unpaired Student’s t test).

 
Anti-TNF-{alpha} administered icv reduces the recovery of TNF-{alpha} biological activity from plasma samples spiked ex vivo with rmTNF-{alpha}
That icv anti-TNF-{alpha} administration produced a reduction in the measurable biological activity of TNF-{alpha} in plasma after LPS treatment suggests that either neutralization of TNF-{alpha} within the CNS impairs peripheral TNF-{alpha} secretion, or significant amounts of anti-TNF-{alpha} antibodies are present in systemic blood after icv anti-TNF-{alpha} antiserum administration, thus inhibiting the detection of TNF-{alpha} biological activity in plasma. To ascertain whether anti-TNF-{alpha} antibodies were indeed present in blood after icv administration of anti-TNF-{alpha} antiserum, the recoveries of TNF-{alpha} biological activity from plasma samples of animals injected icv with 5 µl of either NRS or anti-TNF-{alpha} were compared. In the absence of spiked recombinant TNF-{alpha}, the TNF-{alpha} biological activity of all of these plasma samples was low (<150 IU/ml). In plasma samples from rats infused icv with NRS and subsequently spiked ex vivo with recombinant mouse TNF-{alpha} (final concentration, ~4000 IU/ml), virtually all (83–94%) TNF-{alpha} biological activity was recovered (Fig. 4AGo). However in animals treated icv with anti-TNF-{alpha}, a time-dependent reduction in the recovery of spiked TNF-{alpha} was apparent (Fig. 4AGo) that was statistically significant at 4 h (73% reduction compared with NRS-treated controls) and 20 h (76% reduction).

To determine whether the inhibitory activity of plasma from rats treated icv with anti-TNF-{alpha} was indeed attributable to the rabbit anti-TNF-{alpha} infused, a separate series of experiments was conducted. Animals were treated icv with either NRS or anti-TNF-{alpha}, and blood was collected 20 h later. Plasma samples were preprecipitated with an anti-rabbit IgG antisera, saline, or 5% PEG (i.e. unprecipitated) before spiking with rmTNF-{alpha}. As in the previous experiment, in unprecipitated samples from rats that received an icv infusion of anti-TNF-{alpha} 20 h earlier, a reduced (76–77%) recovery of spiked TNF-{alpha} biological activity was apparent (Fig. 4BGo). However, preprecipitation of plasma samples from icv anti-TNF-{alpha}-treated rats with the anti-rabbit IgG antiserum completely prevented the reduction in the recovery of subsequently spiked TNF-{alpha} (Fig. 4BGo).

Intravenous pretreatment 20 h earlier with anti-TNF-{alpha} antiserum produces a delay in the onset of LPS-induced elevations in plasma ACTH concentrations similar to that observed after anti-TNF-{alpha} administered icv
The data presented above indicate that anti-TNF-{alpha} administered icv 20 h earlier delays the onset of the plasma ACTH response to LPS, and that significant amounts of anti-TNF-{alpha} are present in systemic blood in this paradigm. To determine whether the observed effects of icv anti-TNF-{alpha} were due to inhibition of TNF-{alpha} action specifically within the brain, or whether it might be accounted for by leakage of anti-TNF-{alpha} from the brain and into the systemic circulation, we determined the effect of 5 µl (in a total volume of 500 µl) anti-TNF-{alpha} antiserum given iv 20 h before LPS.

Anti-TNF-{alpha} administered iv 20 h earlier inhibited the onset of the ACTH response to LPS; this effect was remarkably similar in both time course and magnitude to that observed when anti-TNF-{alpha} was administered via the icv route (compare Figs. 2Go and 5AGo). Intravenous anti-TNF-{alpha} significantly inhibited the rise in plasma ACTH concentrations at 45 min (iv anti-TNF-{alpha}, 19 ± 7 pg/ml; iv NRS, 164 ± 40 pg/ml; P < 0.001) and 60 min (iv anti-TNF-{alpha}, 172 ± 33 pg/ml; iv NRS, 484 ± 96 pg/ml; P < 0.05), but not at 90–240 min (Fig. 5AGo and data not shown).



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  		Figure 5. The effect of iv anti-TNF-{alpha} antiserum pretreatment (5 µl; 20 h before) on the rise in plasma ACTH concentration (A) and TNF-{alpha} biological activity (B) produced by LPS (5 µg/kg, iv; n = 4–7 subjects/experimental group). ANOVA with repeated measures indicated a significant interaction between anti-TNF-{alpha} and LPS treatments (P < 0.001 for both ACTH and TNF-{alpha} measurements). *, P < 0.05; ***, P < 0.001 (vs. NRS/LPS-treated rats, by least squared means test).

 
Before injection of LPS plasma, TNF-{alpha} biological activity was detectable in three of five rats injected with NRS iv, but was below detection limits in all animals pretreated iv with anti-TNF-{alpha}. Pretreatment iv with 5 µl anti-TNF-{alpha} at -20 h produced an inhibition of the LPS-induced rise in measurable biological activity of TNF-{alpha} in plasma very similar to that produced by anti-TNF-{alpha} administered icv (compare Figs. 3Go and 5BGo). Like the icv treatment, iv administration of anti-TNF-{alpha} 20 h earlier prevented any significant rise in plasma TNF-{alpha} biological activity 30 min after LPS and significantly reduced levels observed at 45 min (by 94%, compared with 88% by the icv route) and 60 min (by 70%, compared with 59% by the icv route). Neither route of anti-TNF-{alpha} administration significantly affected the rise in TNF-{alpha} biological activity observed in plasma samples 90 min after LPS treatment (Figs. 3Go and 5BGo) or at any time thereafter (data not shown).

Intracerebroventricular treatment with anti-TNF-{alpha} antiserum immediately before LPS does not affect the subsequent plasma ACTH response
Very low levels of corresponding antibodies are found in blood shortly (30 min) after icv infusion of antisera (Fig. 4Go). To determine whether icv treatment with anti-TNF-{alpha} antiserum influences the plasma ACTH response to LPS at a time when corresponding blood antibody levels are only low, animals were treated with anti-TNF-{alpha} immediately before LPS.

Either icv or iv anti-TNF-{alpha} immediately before LPS administration significantly reduced the increase in plasma TNF-{alpha} biological activity due to LPS, although the iv route appeared far more effective (Fig. 6Go). Intravenous anti-TNF-{alpha} administered immediately before LPS prevented any rise in plasma TNF-{alpha} activity until 60 min after LPS, at which time there was still an 85% inhibition of the rise in TNF-{alpha} biological activity. In contrast, icv anti-TNF-{alpha} did not significantly affect plasma TNF-{alpha} biological activity at 30 min and produced relatively smaller reductions in plasma TNF-{alpha} activity at 45 (68% reduction) and 60 min (55% reduction). As in previous experiments, neither icv nor iv anti-TNF-{alpha} affected TNF-{alpha} biological activity in plasma 90 min after LPS (Fig. 6Go) or at any time thereafter (data not shown).



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  		Figure 6. The effect of either icv (A) or iv (B) anti-TNF-{alpha} antiserum (5 µl), administered immediately before, on the rise in plasma TNF-{alpha} biological activity produced by LPS (5 µg/kg, iv; n = 6–10 subjects/experimental group). ANOVA with repeated measures indicated a significant interaction between anti-TNF-{alpha} and LPS treatments when anti-TNF-{alpha} was administered icv (A; P < 0.01) and iv (B; P < 0.001). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. respective NRS/LPS-treated rats, by least squared means test).

 
Pretreatment immediately before LPS with anti-TNF-{alpha} icv had no significant effect on the plasma ACTH response to LPS (see Fig. 7AGo), whereas the iv route of anti-TNF-{alpha} administration at this time remained effective and, as in earlier experiments, significantly inhibited the plasma ACTH response at 45 and 60 min (Fig. 7BGo).



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  		Figure 7. The effect of either icv (A) or iv (B) anti-TNF-{alpha} antiserum (5 µl), administered immediately before, on the rise in plasma ACTH concentrations produced by LPS (5 µg/kg, iv; n = 4–10 subjects/experimental group). ANOVA with repeated measures indicated a significant interaction between anti-TNF-{alpha} and LPS treatments when anti-TNF-{alpha} was administered iv (B; P < 0.001), but not when it was given icv (A; P = 0.60). *, P < 0.05: ***, P < 0.001 (vs. respective NRS/LPS-treated rats, by least squared means test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The original objective of this study was to define the role of cerebral TNF-{alpha} in the mediation of the HPA axis response produced by peripheral (iv) administration of a moderate dose of LPS (5 µg/kg). Recent reports showing marked induction of TNF-{alpha}, IL-1, and IL-6 mRNAs within the CNS in response to peripheral administration of LPS have suggested a novel mechanism by which cytokines may alter CNS activities, such as neuroendocrine secretions (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). However, the high doses of LPS used and the protracted time course of cytokine expression within the CNS have not permitted definitive conclusions as to whether cerebral elaboration of TNF-{alpha}, IL-1, or IL-6 plays an obligatory role in neuroendocrine secretory responses to LPS (12). Nevertheless, some studies have reported that intracerebral or icv antibodies/receptor antagonists to IL-1 inhibit some CNS responses produced by systemic LPS (46, 47). Consistent with the idea that TNF-{alpha} acts within the brain to produce HPA activation in response to peripheral LPS, the present study shows that icv pretreatment (at -20 h) with anti-TNF-{alpha} delays the onset of the plasma ACTH response to LPS, producing significant reductions in plasma ACTH concentrations 45–60 min after LPS administration. However, the data we present here indicate that the inhibitory effect of icv anti-TNF-{alpha} pretreatment is due to the escape/transport of anti-TNF-{alpha} into the general circulation and subsequent immunoneutralization of TNF-{alpha} within the periphery.

No increase in TNF-{alpha} biological activity was detectable in the brains of LPS-treated rats over a time course relevant to the HPA axis response (30 min to 2 h). These data alone, however, do not necessarily indicate that brain TNF-{alpha} is unimportant in the generation of the HPA axis response to LPS, as the levels of TNF-{alpha} produced may have been below the detection limits of the TNF-{alpha} bioassay. Furthermore, we previously reported (38) that localized inflammation caused by turpentine administration does not change TNF-{alpha} biological activity or mRNA in brain, yet inhibition of TNF-{alpha} by icv, but not iv, administration of either 5 µl anti-TNF-{alpha} antiserum or 1–50 µg soluble TNF-{alpha} construct reduces the ensuing HPA axis response.

In marked contrast to brain TNF-{alpha} biological activity, however, plasma levels of TNF-{alpha} showed a close temporal relationship to plasma ACTH concentrations, with the onset and peak of the plasma TNF-{alpha} response preceding those of ACTH by around 15 min. These data contrast with those reported by Givalois et al. (1), who showed that after intra- arterial administration (vs. iv in the present work) of similar doses of LPS, plasma ACTH concentrations increase before elevations in plasma TNF-{alpha}, IL-1, or IL-6 concentrations. The discrepancy between the present work and that of Givalois et al. (1) is probably due to the much more rapid increase in plasma ACTH concentrations produced by LPS administered directly into the carotid artery (15–30 min vs. 45 min in present study) and presumably reflects mechanisms of HPA activation that are specifically induced by this route of administration.

In addition to finding a close temporal correlation between plasma TNF-{alpha} biological activity and ACTH after iv LPS, the present work also showed that small amounts of anti-TNF-{alpha} antiserum administered iv produced a delay in the onset of the ACTH response to LPS. The early (45–60 min) inhibition of LPS-induced ACTH secretion produced by anti-TNF-{alpha} paralleled the neutralization of peripheral TNF-{alpha}, as assessed by the reduction in biologically active TNF-{alpha} in plasma at 30–60 min. It should be emphasized that the small dose (5 µl) of anti-TNF-{alpha} given iv was not intended to test the hypothesis that peripheral TNF-{alpha} mediates the increase in plasma ACTH concentrations after LPS, but served as a control experiment to test whether the effects we observed with this amount of antiserum administered icv were indeed due to actions of the antiserum within the CNS. This small amount of anti-TNF-{alpha} administered iv clearly became saturated, because although the increase in measurable TNF-{alpha} biological activity in plasma was prevented for the first 30–45 min after LPS, TNF-{alpha} bioactivity began to rise thereafter and was not significantly affected by 90 min after LPS. That the reduction in the neutralizing capacity of the iv administered anti-TNF-{alpha} antiserum over time was associated with a loss of inhibitory effect on plasma ACTH concentration further illustrates the key role of TNF-{alpha} within the periphery in eliciting the HPA response to LPS. Whether complete immunoneutralization of TNF-{alpha} action within the periphery would have totally prevented the plasma ACTH response to LPS was not investigated, but others (48) have reported a dramatic reduction in plasma ACTH concentrations due to iv LPS in rats injected iv with large doses (3.3 ml/kg) of a different anti-TNF-{alpha} antiserum.

We present here evidence that passive immunoneutralization via the icv route results in the appearance of significant quantities of corresponding antibodies in systemic blood within several hours. We chose to infuse anti-TNF-{alpha} antiserum the day before LPS administration, because this pretreatment regimen results in extensive penetration of antibodies throughout brain tissue (49, 50, 51), reduces the possibility of stress-induced hormone secretion due to the icv injection of immune serum, and has proved to be a successful means of inhibiting TNF-{alpha} action within the brain (38). However, the delay in onset of the ACTH response produced by anti-TNF-{alpha} administered icv at -20 h was paralleled by a reduction in biologically active TNF-{alpha} within systemic blood. This reduction in measurable TNF-{alpha} biological activity in plasma after LPS in icv anti-TNF-{alpha} antibody-pretreated rats appeared to be explained by the presence of experimentally significant amounts of corresponding anti-TNF-{alpha} antibodies within the systemic blood. The presence of rabbit anti-TNF-{alpha} in peripheral blood was demonstrated by the reduced recovery of biologically active TNF-{alpha} from the plasma of rats injected 4 and 20 h earlier with anti-TNF-{alpha} icv, and the removal of this inhibitory activity by preprecipitation of samples with sheep anti-rabbit IgG antibodies. We have subsequently compared the profiles of corresponding antibodies in systemic plasma after either icv infusion or iv injection of several different antisera. These data are presented in the following article (52), and a surprising finding pertinent to the present discussion is that although the levels of corresponding antibodies in blood 30 min after administration of antiserum are far lower after icv infusion (2–6% of iv injection), the levels 8–24 h after administration are virtually identical whether antiserum is infused icv or injected iv.

The effect of icv anti-TNF-{alpha} administered 20 h earlier on the plasma ACTH response to LPS was mimicked in both time course and magnitude by iv administration of the same dose (5 µl) of anti-TNF-{alpha} antiserum. The reduction in detectable TNF-{alpha} biological activity in plasma was also similar in both iv and icv anti-TNF-{alpha}-treated animals. Although these two routes of antiserum administration produced similar levels of corresponding antibodies in peripheral blood over this time frame (52), the limited entry of antibodies from blood to brain (53) means that only after the icv route were antibody levels markedly elevated within the brain parenchyma (49, 50, 51, 54). Consequently, that anti-TNF-{alpha} administered icv at -20 h produced inhibition of the plasma ACTH response to LPS only to an extent accounted for by the accompanying peripheral blood anti-TNF-{alpha} levels strongly argues that the effects of icv anti-TNF-{alpha} were due solely to actions within the periphery.

Consistent with this suggestion, when anti-TNF-{alpha} was administered icv immediately before LPS (thus producing only very low levels of antibodies in peripheral blood over the first hour after LPS), there was a far smaller impact on plasma TNF-{alpha} biological activity. Furthermore, the effects of icv anti-TNF-{alpha} on the plasma ACTH response to LPS were completely lost, suggesting that substantial neutralization of TNF-{alpha} within peripheral blood is necessary to observe the inhibitory effects of anti-TNF-{alpha} administered icv. It remains possible that anti-TNF-{alpha} administered icv immediately before LPS may not have penetrated brain tissue to reach potential sites of TNF-{alpha} action within the brain, thus having no effect on the HPA axis response. However, a number of studies have demonstrated either relatively widespread distribution of antiserum throughout the CNS or inhibitory effects of antiserum that could only be accounted for by actions specifically within the brain when the antisera are administered icv several (3.5–24) hours previously (38, 49, 50, 51, 54). Therefore, the absence of any demonstrable effect of icv anti-TNF-{alpha} (administered at either 0 or -20 h) that could be attributed to an action within the brain and the close correlation between the inhibition of the plasma ACTH response to LPS produced by icv anti-TNF-{alpha} and the levels of corresponding anti-TNF-{alpha} antibodies in blood strongly suggest that TNF-{alpha} does not act within the brain to elicit the increase in HPA axis activity. Rather, our data indicate that TNF-{alpha} is an important peripheral mediator of the HPA response to LPS. Previous studies have shown that blood-borne TNF-{alpha} stimulates pituitary ACTH secretion through PG (55)- and CRF (56)-dependent mechanisms. However, whether stimulation of PG and/or CRF release results from an action of TNF-{alpha} at the blood-brain interface, from the induction of other cytokines (such as IL-1 or IL-6), or via activation of sensory neural afferents (57) remains to be determined. It should be kept in mind that the present experiments relate to a single dose of LPS (5 µg/kg) administered iv, a dose that is extremely modest compared with those employed in most studies demonstrating induction of cytokine mRNA expression within the CNS (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Furthermore, the precise mechanism by which LPS induces activation of the HPA axis is dependent on the route of its administration (58, 59). Although our findings indicate that TNF-{alpha} action within the brain is not an obligatory step in the activation of the HPA axis in response to LPS, we cannot exclude the possibility that at other doses or other routes of LPS administration, cerebral TNF-{alpha} mechanisms may be recruited.

In conclusion, the present work indicates that TNF-{alpha} is an important mediator of the HPA axis response to LPS by an action within the periphery, but probably not within the brain. Furthermore, our data suggest that pretreatment with antiserum icv may lead to systemic blood levels of corresponding antibodies that are sufficient to neutralize peptides within peripheral blood/tissues. This latter hypothesis has now been rigorously tested using a panel of different antineuropeptide antisera, and the results are presented and discussed in the following article (52).


   Acknowledgments
 
We are grateful to Drs. Steve Kunkel (University of Michigan) and Wylie Vale (The Salk Institute, La Jolla, CA) for their generous gifts of reagents.


   Footnotes
 
1 This work was supported by NIH Grant DK-26741 (to C.L.R.) and the Foundation for Research. Back

2 Present address: North Western Injury Research Centre, Stopford Building, University of Manchester, Oxford Road, Manchester, United Kingdom M13 9PT. Back

3 Investigator with The Clayton Foundation. Back

Received June 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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