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Endocrinology Vol. 138, No. 11 4830-4836
Copyright © 1997 by The Endocrine Society

ARTICLES

Clearance of 125I-Labeled Interleukin-6 from Brain into Blood Following Intracerebroventricular Injection in Rats1

Guanjie Chen, W. Lester Castro, Hsiao-Hui Chow and Seymour Reichlin

Department of Medicine (G.C., W.L.C., S.R.), University of Arizona College of Medicine, Tucson, Arizona 85724; Department of Pharmacy Practice and Science (H.-H.C.), University of Arizona College of Pharmacy, Tucson, Arizona 85721; and Enid and Mel Zuckerman Fellow in Psychoneuroimmunology (W.L.C.), Arthritis Division, University of Arizona College of Medicine, Tucson, Arizona 85724

Address all correspondence and requests for reprints to: Seymour Reichlin, University of Arizona College of Medicine, Department of Medicine, Arizona Health Sciences Center, Room 7338, 1501 North Campbell Avenue, Box 245021, Tucson, Arizona 85724-5021. E-mail: reichlin{at}u.arizona.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To test the hypothesis that interleukin-6 (IL-6) induced within the brain can be released into peripheral blood, 125I-labeled IL-6 was injected into the lateral cerebral ventricle of rats, and its concentration in peripheral blood followed serially. Acid-precipitable tracer appeared within 5 min of injection and entered the blood following first-order kinetics (fractional rate, 0.0116 ± 0.0022/min). Comparison of areas under the curve of intracerebroventricular (icv) vs. iv injection showed that 37.1–46.5% of tracer injected into the lateral cerebral ventricle appeared in the blood over a 4-h period. icv IL-6 exits at least in part via venous drainage (superior sagittal sinus/aortic concentration gradient was 1.47 ± 0.23 and 3.05 ± 0.87 in two separate groups). Prior icv injection of human IL-1ß (100 ng) did not alter rate of degradation or of exit of radioiodine-labeled IL-6 from the brain. These studies indicate that a relatively high proportion of IL-6 that arises in the brain enters the peripheral circulation. Direct secretion of IL-6 from brain to blood may be a mechanism by which the brain modifies peripheral metabolic, endocrine, and immune activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRACEREBROVENTRICULAR (icv) injection of interleukin (IL)-1 leads to elevation of peripheral blood levels of IL-6 in the rat (1, 2, 3, 4) and in the monkey (5). Similar changes have been reported to occur in rats following icv injection of Escherichia coli endotoxin, which is a lipopolysaccharide (LPS) (6, 7). The means by which this change is brought about has not been determined with certainty. That the brain itself could be a source of circulating IL-6 after central administration of IL-1 was suggested by the finding in several laboratories that IL-6 and several other inflammatory cytokines appear in the brain and cerebrospinal fluid (CSF) after icv injection of IL-1 and LPS (3, 7, 8, 9, 10, 11, 12, 13, 14), and by the demonstration in this laboratory that the concentration of IL-6 in the superior sagittal sinus (SSS) blood (the principal venous drainage route of CSF and brain) was higher than that in aortic blood entering the brain (3, 4). Further evidence that IL-1 and endotoxin can activate expression of inflammatory cytokines in the brain after icv or iv injection is the appearance of messenger RNAs (mRNAs) coding for several inflammatory cytokines (7, 11, 15) in CNS structures, including the meninges, choroid plexus, cerebral vascular endothelium, glia, and neurons. That inflammatory cytokines can leave the brain after icv injection has been shown using radioiodinated tracers of IL-6 in the mouse (16) and tumor necrosis factor-{alpha} (TNF-{alpha}) in the rat (17). Immunoreactive human IL-1 receptor antagonist has also been shown to appear in the peripheral blood after icv injection in rats (18).

To further test the hypothesis that IL-6 arising in the brain is secreted at least in part into peripheral blood by way of the SSS, measurements were made of SSS/aortic blood gradients after icv injection of 125I-labeled IL-6. The use of an exogenous IL-6 tracer also made it possible to measure the proportion of IL-6 that enters the peripheral circulation from the cerebral ventricles and the rate of transfer from brain to peripheral blood, and to determine whether brain to blood clearance of IL-6 is affected by prior exposure to IL-1. Because peripheral concentrations of IL-6 after central administration are influenced by the rate of clearance of the cytokine after it enters the peripheral circulation, measurements were also made of 125I-labeled IL-6 clearance after iv injection. As independent measures of rate of clearance of IL-6 from the brain after icv injection, residual brain radioactivity was assayed at the termination of the experiment, and the extent of diffusion of tracer from CSF to brain parenchyma determined by comparing the concentration of labeled tracer in CSF with that in whole brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
As in an earlier study (3, 4), male rats, Sprague-Dawley strain, Taconic Farms (Germantown, NY), weighing 280–386 g were maintained at an ambient temperature of 20–24 C, exposed to a 12 h light/12 h dark lighting schedule, fed Harlan Teklad (Madison, WI) 4% Diet 700, and given water ad lib. Intracerebroventricular injections were carried out under sodium pentobarbital anesthesia (initial dose, 35–55 mg/kg ip). To minimize the extent of leakage of injected material from the ventricle by way of the needle track, the radiolabeled test material 125I-labeled IL-6 (labeled by the Bolton-Hunter method, specific activity 88.1 µCi/µg purchased from New England Nuclear, Boston, MA) was injected through a guide cannula placed 5–7 days before the experimental procedure. Animals were placed in a Kopf stereotaxic instrument (David Kopf Instrument, Tujunga, CA), the scalp incised, and the cranium stripped with a cotton gauze pledget. A guide cannula was placed under stereotaxic control, 1.5 mm lateral to the midline and 0.5 mm posterior to the bregma and fastened to the skull by means of a stainless steel calvarial screw and dental acrylic cement. The tip of the guide cannula was inserted 2.98 mm below the surface of the cranium, a point shown in preliminary studies to be approximately 0.5 mm above the ventricular roof. At the time of injection, animals were reanesthetized, and icv injections made over a 2-min period through a 26-gauge inner cannula whose tip was placed 3.48 mm below the cranial surface. This point had been shown in previous studies to be within the ventricle as demonstrated by the appearance of Evans Blue dye in the cerebral ventricle or by demonstration of free flow of saline at a pressure of 10 cm or less of saline. In one group of animals (series 1, n = 10), the inner cannula was removed 2 min after the injection. In another group (series 2, n = 10) the polyethylene collecting tubing was sealed with a heated hemostat just above its connection to the cannula, and the cannula was not removed until the termination of the experiment, 4 h after injection. As noted in Results, the two procedures gave similar results. To verify the precise amount of radioactive material that had been delivered to the brain, the radioactivity of the inner cannula and tubing when filled with the standard 10-µl injection volume was counted in a well-type scintillation counter, and at the time of death, the residual radioactivity in the inner cannula, guide cannula, and connecting plastic tubing was determined and subtracted from initial content of radioactivity.

To determine the rate of appearance in blood of IL-6 after icv injection, venous blood samples were removed through a polyethylene cannula (od 0.965 mm, id 0.58 mm; PE Intramedic Tubing, Clay Adams Co., Mountain View, CA) placed via the external jugular vein into the right atrium. The catheter was kept filled with heparin solution (200 U/ml) between samplings to prevent clotting. Blood (0.2 ml) for determination of counts in 125I-labeled IL-6 was removed at 5, 10, and 20 min and then every 20 min for a total of 4 h, and blood volume replaced with equivalent amounts of 5% crystalline BSA in normal saline. Animals were maintained in the anesthetized state throughout, receiving repeated injections of sodium pentobarbital as needed, and were kept on a heated pad.

In a separate series of experiments, the peripheral clearance of 125I-labeled IL-6 was determined after injection of at least 106 counts of tracer (contained in 0.5 ml of 5% bovine albumin in normal saline) into the right atrium via a cannula placed in the contralateral external jugular vein (n = 9).

In a third series of experiments, the effect of IL-1ß on brain-to-blood clearance of 125I-labeled IL-6 was determined. The inner cannula was loaded in sequence so that the initial 10 µl of fluid extruded contained 100 ng of human IL-1ß (Collaborative Biomedical Products, Bedford, MA), and the second 10 µl contained the tracer IL-6 solution. The amount of IL-I used had been shown in earlier studies to induce high CSF levels of IL-6, which reached their peak between 2–4 h (3). Control animals were given saline instead of IL-1. The study was performed by injecting the first 10 µl of fluid (containing IL-1ß), followed by the injection, after an interval of 2 h, with 10 µl of labeled IL-6 tracer. The cannula was left in place throughout the experiment. Serial measurements of venous blood were made over a 2-h period.

At the end of the experiment, samples of CSF (70–250 µl) were taken by puncture of the cisterna magna, 1 ml blood was removed from the SSS after exposing it with a dental drill, the animals were exsanguinated via the abdominal aorta, and the whole brain (except for the olfactory lobes) removed. To separate labeled 125I-labeled IL-6 from 125I (arising from degraded tracer), 0.2 ml whole blood was added to 0.2 ml of cold 10% trichloracetic acid (TCA), and the precipitated radiolabeled tracer centrifuged (12,000 rpm x 15 min in a Beckman microfuge; Beckman Instruments, Inc., Fullerton, CA). The pellet was counted in a well-type {gamma}-scintillation counter. Whole brain was extruded through a 16-gauge hypodermic needle into equal parts of ice-cold 10% trichloracetic acid to remove degraded tracer, the suspension centrifuged, and supernatant removed before determination of radioactivity. It is possible that the acid precipitable fraction of blood and brain includes partially degraded IL-6 that does not have biological activity. Preliminary studies showed that at least 95% of the radioactivity in CSF was acid precipitable. In subsequent calculations therefore, total counts in CSF were taken as the measure of undegraded tracer.

Statistical analysis
Counts of radioactivity were compared in groups by Fishers t tests as indicated in Results. Estimates of apparent IL-6 pool size and fractional turnover of the tracer from blood after iv injection were made by fitting a two compartment model to the blood clearance data using Winnonlin Non Linear Estimation Program Version 01.0 Core Version 26 March, 1996 (Scientific Consulting, Apex, NC). Estimates of the amount of tracer that entered the blood after icv injection were made by measuring the area under the curve (AUC) of radioactivity by the trapezoidal method (19) and comparing it with AUC after iv injection. Estimates of the rate of transfer of tracer from the brain to the blood in individual rats were made from empirically derived absorption models using Simusolv Executive Version 3.0 software (Dow Chemical Corp., Midland, MI). As detailed in Results, blood radioactivity appearance curves were adequately explained by a simple first-order absorption model in 10 of the 20 animals studied, and this subgroup was used to calculate brain to blood clearance rates. In the remainder, scatter was too large to accurately assess clearance parameters in individual animals. Estimates of rate of loss of tracer from the brain were determined directly by measurements of residual activity in brain from rats killed 2 h and 4 h after icv injection. Estimates of passage of tracer from CSF to brain parenchyma were made by comparing the concentration of tracer in the CSF (cpm/ml) to the concentration of tracer in the whole brain (cpm/g). Based on preliminary studies, average brain weight was taken to be 1.8 g.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clearance of 125I-labeled IL-6 from blood after iv injection
Following iv injection, labeled IL-6 disappeared rapidly from the blood over the first 20 min and then more slowly thereafter, apparently following a biexponential decline over time (Fig. 1Go). Estimated apparent volume of distribution was 39.1 ± 2.3% of body weight, t1/2 of the terminal linear portion of the disappearance curve was 107.3 ± 3.2 min, and fractional clearance of the body pool was 0.0065 ± 0.0002/min. AUC of blood radioactivity over a 4-h period (expressed as percent initial dose per milliliters x time in minutes) was 88.70 ± 6.76, a figure used to calculate the fraction of labeled IL-6 reaching the blood after icv injection (see below).



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  		Figure 1. Time course of disappearance from venous blood of 125I-labeled IL-6 following iv injection of labeled tracer compared with time course of appearance of tracer following icv injection. Values shown are mean ± SE; n = 9 for iv, n = 10 for icv (series 2). Disappearance from blood followed a biexponential pattern with a rapid early phase and a slower rate of disappearance.

 
Clearance of 125I-labeled IL-6 from CSF into blood
Precipitable radioactivity in blood was detected as early as 5 min after icv injection (Figs. 1Go and 2Go) and increased progressively to reach a peak between 40–140 min (mean ± SE, 70 ± 7.8 min). In some individual animals, blood levels remained relatively constant throughout the 1- to 4-h observation period, whereas in others blood precipitable radioactivity disappeared apparently following an exponential decline. The AUC of blood concentration (AUC expressed as percent initial dose per milliliter x time in minutes ± SE) was 32.86 ± 4.49 (series 1) and 41.21 ± 5.00 (series 2) compared with 88.70 ± 6.76 after iv injection. Thus, in the two series, 37.1% and 46.5% of the icv administered dose respectively appeared in peripheral blood over a 4-h period. The total amount of labeled IL-6 reaching the peripheral blood would be greater than this estimate because at 4 h all of the rats had appreciable amounts of labeled tracer in the blood and residual tracer in the brain.



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  		Figure 2. Time course of appearance in peripheral venous blood of 125I-labeled IL-6 following icv injection in a single rat. This pattern is compatible with a two compartment model that follows first-order kinetics of clearance from brain to blood.

 
Fractional clearance of brain-to-blood labeled tracer determined by pharmacokinetic analysis of blood radioactivity curves
As determined by a curve-fitting program, the pattern of appearance of radioactivity into the blood after icv injection fits best with a two compartment model in which tracer was absorbed from brain following first-order kinetics. In the 10 rats whose individual data points followed this pattern with minimum scattering, the mean rate of IL-6 appearance in the blood following icv injection was 0.0116 ± 0.0022/min. In the remaining rats analyzed individually, there was too much scatter to differentiate between a clearance model with one or with two fractional rates of clearance from brain to blood.

Fractional clearance of 125I-labeled IL-6 from brain after ICV injection determined from residual radioactivity in brain
The difference between administered and residual brain radioactivity at the completion of the experiment is the amount of tracer-labeled IL-6 that had been cleared from the brain. This value can be used to calculate its fractional rate of disappearance assuming a constant rate of disappearance. At 4 h, residual brain radioactivity (percent of dose ± SE) in series 1 rats was 24.14 ± 4.57, and in series 2 rats was 25.47 ± 7.94. These values were used to calculate t1/2 and fractional clearance rates. Mean t1/2 ± SE in minutes was 73.9 ± 4.35 in series 1 and 72.06 ± 3.58 in series 2. Fractional clearance rates in series 1 and 2, calculated by the formula K = 0.693/t1/2, were 0.0094 and 0.0096, respectively. Corresponding values obtained at 2 h from a group of rats doubly injected with saline followed by tracer (see below) were: t1/2 60 ± 13 min, fractional clearance, 0.01268 ± 0.00151. Values for clearance of tracer from brain based on measurements of residual radioactivity corresponded well to estimates of brain to blood clearance made by analysis of blood radioactivity assuming first-order kinetics of disappearance which was 0.0116 ± 0.0022 (see above).

Rate of degradation of 125I-labeled IL-6
The fraction of blood radioactivity that is not precipitated by cold trichloracetic acid was 1.05% ± 0.08 after iv injection in the first 10 min, a proportion that was much less than the value of 10.4 ± 3.8% in rats given 125I-labeled IL-6 icv (P = 0.05) (series 2). These findings suggest a first pass degradation (Fig. 3Go) of the tracer, but the difference could be due at least in part to selective release from the brain into the blood of free Na125I contaminating the labeled preparation. By 40 min, the degraded fraction after either route was indistinguishable and had reached a steady state of increasing degradation, to reach a mean of 20.58 ± 2.6% at 4 h.



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  		Figure 3. Time course of appearance of degraded (acid-soluble) radioactivity in plasma following either iv or icv administration of 125I-labeled IL-6. Significant amounts of degraded tracer were detected in blood at 5 and 10 min after icv injection but not iv injection suggesting a first pass degradation, but thereafter proportion of degraded tracer was same after either route of administration (iv group, n = 8, icv group, second series, n = 10).

 
Concentration of radioactive IL-6 in SSS and aorta after icv injection of tracer (Table 1Go).
In animals injected iv with tracer, SSS concentrations were virtually identical with those in the aorta. In contrast, SSS levels were significantly higher than aortic levels in icv-injected animals. SSS/aortic ratios in series 1 were 1.47 ± 0.23 and in series 2 were 3.05 ± 0.87. When compared with paired concentration ratios for vena caval blood to aortic blood, P values were 0.0005 and 0.027, respectively.


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  		Table 1. Ratio of 125I-labeled IL-6 concentrations, SSS blood to aortic blood (SSS/A), and vena caval blood to aortic blood (V/A) 4 h after icv injection in rats

 
Distribution of radioactivity within brain after icv injection (Table 2Go).
By 4 h after icv injection of labeled IL-6 tracer, the cerebral substance showed much higher radioactivity per unit mass than that of CSF. This distribution was estimated from concentration of counts per minute per gram of brain (assumed to be 1.8 g) compared with counts per minute per milliliter of CSF. For this calculation the acid-precipitable fraction was assumed to be the undegraded radioactivity associated with IL-6 in brain. In contrast, because preliminary studies showed that 95% or more of radioactivity in CSF was precipitated with TCA, total counts in CSF were used to calculate CSF to brain distribution of radioactive tracer. In both series 1 and 2, the ratio of concentration of brain/CSF was 4.0 ± 1.1. The ratio between brain and CSF radioactivity concentration was so high that it can be concluded that diffusion of tracer into the brain substance was far advanced by 4 h. This inference was supported by visual examination of brain and ventricular space after icv injection of Evans Blue dye tracer carried out in preliminary studies. In contrast, at 2 h (using data from doubly injected rats, which differed in that the tracer dose was administered 2 h after an initial saline injection; see below) the ratio was 0.71 ± 0. 2, indicating that much less tracer had passed into the cerebral substance at this earlier time interval. The change in distribution was not due to a selective loss of radioactivity from the CSF, because total brain residual radioactivity at 4 h was not significantly less in the 4-h as compared with 2-h animals.


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  		Table 2. Distribution of acid-precipitable radioiodine-labeled IL-6 between CSF and brain parenchyma following icv injection

 
Effect of icv injection of IL-1ß on brain-to-blood clearance of 125I-labeled IL-6
The injection of IL-1ß 2 h before the injection of tracer had no detectable effect on any of the measures of clearance of 125I-labeled IL-6 from the brain when compared with rats given saline control injections. Values for saline-injected vs. IL-1-injected rats (n = 8/group) were: AUC (14.92 ± 2.07 vs. 14.19 ± 2.28); residual brain radioactivity (24.29 ± 3.57 vs. 26.83 ± 3.17); brain/CSF ratio (0.71 ± 0.2 vs. 0.63 ± 0.24.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was carried out to test the hypothesis that IL-6 induced within the brain can be released into peripheral blood, to determine the magnitude of transfer, and to confirm our earlier finding that one pathway of passage of IL-6 in the brain into the peripheral blood was by way of structures draining into the SSS. We found that icv-injected, tracer-labeled IL-6 appears in the peripheral blood within 5 min of injection. Over the ensuing 4 h, tracer-labeled IL-6 is cleared from the brain to the blood following a pattern of first-order kinetics (in 10 of 20 animals analyzed) at a fractional rate of 0.0116 ± 0.0022/min (1.16% of brain pool/min). This value for clearance into blood based on kinetic analysis corresponds reasonably well to values obtained from direct measurements of residual radioactivity at 2 h (0.01268/min) and at 4 h (0.0095/min), indicating that virtually all of the labeled tracer that leaves the brain can be accounted for by its appearance in the blood. Based on comparisons of the AUC after iv and icv injection, it can be concluded that between a third and a half of icv-injected tracer (37.1–46.5%) appeared in the blood over a 4-h period in acid-precipitable form. It is possible that some of the labeled material, though still bound to iodide, was partially degraded and therefore inactive. The total amount reaching the blood over time after icv injection would be somewhat larger than this figure, because at 4 h all of the icv injected animals still had appreciable concentrations of residual labeled tracer in the brain. The fraction of labeled IL-6 that does not reach the peripheral blood is probably degraded in the brain before, or in the course of, its exit as indicated by the relatively large fraction of nonprotein-bound tracer that appears in the blood within 5 min of icv injection and which reached a value of more than 20.58 ± 2.6% at 4 h. The relatively high fraction of nonprecipitated radiodide found in the blood within 20 min of injection into the brain could be due in part to the more rapid efflux from brain to blood of free Na125I contaminating the labeled IL-6 preparation. The labeled tracer contains less than 1% free iodide, and after iv injection the nonprecipitable fraction in venous blood was 1.047 ± 0.084%. That icv IL-6 enters the cerebral venous circulation is shown by the high concentration gradient between SSS and aorta, which is in contrast to the gradient between peripheral venous blood and aorta, which approaches unity. Finally, measurements of the concentration of radioactivity in CSF with that in brain parenchyma indicate that by 4 h almost all of the injected tracer left in the brain has been transferred from cerebral ventricle to the parenchymal compartment.

That labeled IL-6 introduced into the cerebral ventricle is lost from the brain has previously been shown by Banks et al. (16) in experiments in which brains of mice were examined at intervals up to 20 min after icv injection. The reported fractional rate of clearance over the first 20 min (0.0163/min) after injection was approximately 50% higher than was observed in the present experiments, a difference that may be due to species differences (mouse vs. rat), differences in anesthetic, markedly different brain sizes, use of different tracer preparations, or the fact that the time interval over which the study was conducted was much shorter than was used in our experiment (20 vs. 240 min). 125I-labeled TNF-{alpha} tracer also is reported to enter the blood rapidly after icv injection in the rat (17), a finding that we have confirmed (Ref. 4 and our unpublished observations), as is human IL-1 receptor antagonist (18). Several other cytokines introduced into the brain (of mice) also decline exponentially over time at rates roughly comparable with values obtained for IL-6 in the mouse. These include murine IL-1a (t1/2 38.1 min), human IL-1a (t1/2 33.8 min), and murine IL-1a (t1/2 33.8 min) (20), murine TNF-{alpha} (t1/2 59.5 min) (21), and IL-2 (t1/2 23.8 min) (22).

The mechanism by which the IL-6 is cleared from the cerebral ventricle is probably by bulk transport as suggested by comparison with measurements of clearance of the inert marker inulin from the CSF of the rat. A number of earlier studies have estimated rates of disappearance by repeated sampling of the cisternal CSF at intervals after icv injection of tracer. If one assumes a CSF volume of 250 µl as determined by Bass and Lundborg (23), one can calculate the fractional clearance per minute in previously published work as 0.00752 (23), 0.0088 (24), 0.011 (25), and 0.0160 (26). These values compare reasonably well with the values obtained in our experiment: fractional clearance from brain to blood was 0.0116/min, fractional loss of tracer from brain determined at 2 h was 0.01268, and at 4 h was 0.0095. The modest differences observed in estimated CSF clearance obtained in these studies can be attributed to factors such as type of anesthesia, the size of the brain, the age of the animal, and the assumption of a CSF volume of 250 µl. Rate of disappearance of IL-6 from the mouse brain observed by Banks et al. (16) were also interpreted by them to indicate that passage out was by bulk transport, because it corresponded to the rate with which albumin tracer exits the brain.

The finding that the concentration of radiolabeled IL-6 in the SSS is significantly greater than levels in the aorta (ratio of SSS to aortic blood 1.47 ± 0.23 and 3.05 ± 0.88 in the two series of rats at 4 h) indicates that the labeled compound is leaving the brain at least in part by way of the CSF drainage into the venous system. This observation confirms earlier reports from our laboratory that showed higher levels of bioactive IL-6 in the SSS blood after icv injection of IL-1ß (3, 4). Enrichment of IL-6 in sagittal sinus blood over aortic blood supports the hypothesis that transport from CSF into the venous blood is by way of pores in the arachnoid villi draining into the SSS (27).

Other potential routes of drainage of CSF from the brain are not excluded by the demonstration of an SSS/aortic IL-6 gradient. Indeed, there is convincing evidence to indicate that CSF (including its macromolecular constituents) can exit the brain by way of perineuronal sheaths of cranial nerves, which are extensions of the subarachnoidal space (28, 29, 30, 31, 32). This drainage system is best developed for the olfactory neurons that pass through the cribriform plate and terminate in the nasal mucosa. Substances contained in CSF entering the submucosal space can be resorbed through the rich vascular plexus of this region or pass into lymphatic channels that drain into the deep cervical lymphatic chain of nodes. Cserr and colleagues (28, 29, 30, 31) have proposed that this route of exit of macromolecules from the brain is an efficient means by which antigens arising in the brain can be presented to the immune system. They have shown that the intensity of the immune response to human albumin and ovalbumin was greater after icv than after systemic injection (34, 35). Our data suggest that the brain can also serve as a direct secretory source of immunoregulatory cytokines.

Passage of IL-6 tracer out of the brain was not modified by prior injection of IL-1ß. Il-1 does not disrupt the blood-brain barrier as measured in mice by uptake into the brain of macromolecules (36). Our data suggest that bulk transport out of the brain is not affected by IL-1 either.

Although this experiment clearly shows that an IL-6 tracer introduced into the cerebral ventricle diffuses throughout the brain and enters the SSS and peripheral blood, it does not mean that IL-6 found in the SSS after central inflammatory stimuli comes only from CSF. Histological analysis shows that soon after introduction of IL-1 or LPS into the brain, cytokine production by vascular endothelium and choroid plexus (8, 13) are activated. Isolated cerebral microvessels (37) and choroid plexus explants (4) secrete IL-6 after exposure to toxin and/or IL-1. Systemic injection of LPS is followed by the appearance of bioactive IL-6 in the anterior hypothalamus (of guinea pigs) within 1 h (38), and expression of IL-6 mRNA in the hypothalamus (of rats) also within 1 h (11). Brain parenchymal sources of cytokines such as activated astrocytes and microglia (9, 10, 12, 13, 15, 38, 39, 40) and neurons (14, 41) apparently respond relatively late to local inflammatory stimuli (8, 13). It is likely therefore that early after central inflammatory stimulation, venous drainage from the brain contains components secreted by circumventricular structures (including ependyma), endothelia, vascular smooth muscle, and various elements of the choroid plexus (which drain directly into the SSS). Later in the course of central inflammation, IL-6 (and other cytokines) arising from brain parenchymal elements could enter the brain interstitial space, which is in continuity with the CSF (42), and enter the blood via the SSS and by perineuronal cranial nerve channels.

The fact that IL-6 has been shown to enter the peripheral blood from the cerebral ventricle, by what appears to be bulk transport, and that other macromolecules are similarly transported, makes it likely that virtually any cytokine arising in the brain could enter the periphery, and depending on its concentration materially influence both peripheral metabolic and immunological activity. Such may be the mechanism by which closed head injury (43, 44, 45, 46, 47, 48, 49), ischemic stroke (50, 51), experimentally induced encephalomyelitis (52), meningitis (53), and brain death (54) can induce the circulating inflammatory cytokines and metabolic changes that are characteristic of the acute phase response (44, 47, 48, 50, 54, 55).

In our experiment, clearance of labeled IL-6 from the blood after iv injection appeared to follow an exponential rate of decay following a rapid phase of disappearance from the blood over the first 40 min after injection. In similar previous studies reported by Castell and collaborators (56), t[1/2] for the interval from 5–20 min (the longest time observed) was reported to be 55 min, which is considerably shorter than the value reported in our study of 107.3 ± 3.2 min for the interval of 60–240 min. It is probable that the difference in the two estimates is due to the inclusion in their analysis of the earlier rapid phase of blood clearance. These workers also measured organ uptake of IL-6, and found that a very large proportion of the labeled tracer was initially taken up by liver and later by the skin (as much as 35% of total injected counts were found in the skin at 5 h) compared with only 3.2% in plasma (57). This large extravascular distribution of tracer is in accordance with findings in the present study that show that under steady state conditions, the apparent volume of distribution of the tracer is 39.1 ± 2.3% of body weight indicative of a large extravascular distribution space.

These studies were motivated initially to determine the mechanism by which icv injection of IL-1 brings about an elevation of IL-6 in peripheral circulation. Although they show that IL-6 passes from brain to peripheral blood, this mechanism does not fully explain the observed time course of appearance and disappearance of IL-6 from peripheral blood after icv injection of IL-1. In the studies of De Simoni et al. (2), circulating bioactive IL-6 levels were not significantly elevated at 1 h, were maximally increased at 2 h, and had dropped markedly by 4 h, which is a time course that has been observed consistently in our laboratory (Refs. 3, 4, and our unpublished observations). In contrast, CSF levels of IL-6 are not detectable at 1 h, are barely detectable at 2 h, reach their peak at 4 h, and remain elevated for at least 8 h (2, 3, 4). It is likely therefore that other mechanisms are responsible for the acute burst of IL-6 found in peripheral blood after central IL-1 administration. Based on analogous work using acute stress of various types, including restraint and hemorrhage, which are known to increase peripheral IL-6 levels (58, 59), it is possible that the acute changes seen between 2–4 h are due to mobilization of IL-6 from peripheral sources such as the liver by the sympathetic nervous system (58, 59).

Because a relatively large amount of icv-administered cytokine tracer gradually enters the peripheral circulation, it is important to differentiate central from peripheral effects of centrally administered materials. Bodnar and colleagues (17) suggested that the metabolic and possibly even the behavioral effects of TNF-{alpha} administered by intracerebral route could be mediated by the entry of the cytokine into peripheral blood, and we have shown (G. Chen, R. S. McKuskey, and S. Reichlin, unpublished observations) that the peripheral acute immune effects of LPS administered by icv injection are due, at least in part, to circulating endotoxin that has passed into the blood from the brain.


   Acknowledgments
 
We gratefully acknowledge the helpful suggestions of Drs. W. A. Banks, R. F. Keep, and Michael Mayersohn. Miss Teresa Sherman provided valuable technical assistance.


   Footnotes
 
1 This work was supported by United States Public Health Service NIH Grant 16684. Back

Received June 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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