Penetration of interleukin-6 across the murine blood-brain barrier

ELSEVIER

Neuroscience Letters 179 (1994) 53-56

NEUROSClENCE IETT[RS

Penetration of interleukin-6 across the murine blood-brain barrier William A. Banks*, Abba J. Kastin, Enrique G. Gutierrez Veterans Affairs Medical Center and Tulane University School o f Medicine, New Orleans, LA 70146, USA Received 25 April 1994; Revised version received 12 June 1994; Accepted 10 July 1994

Abstract

Interleukin-6 (IL-6) can alter brain function after peripheral administration, suggesting that it, like IL-1 ~, IL-1B and TNF-a, might be able to cross the blood-brain barrier (BBB). We used multiple-time regression analysis to measure the unidirectional influx constant (Ki) into brain of radioactively labeled murine and human IL-6 given i.v. Ki values ranged from 3.05 to 4.54 (10 -4) ml/g/min and were inhibited by unlabeled IL-6 but not IL-la or TNF-a, showing that the transport system for IL-6 is distinct from those for IL-la and TNF-a. -0.2% of the dose injected i.v. entered each gram of brain. The capillary depletion method showed that most of the IL-6 taken up by brain entered the parenchyma. However, only ~ 16% of the radioactivity recovered eluted as intact I-IL-6 in brain and ~ 50% in CSF after chromatographic separation by HPLC/Sephadex. The efflux rate for IL-6 injected into the lateral ventricle of the brain suggests that it enters the blood with the reabsorption of CSF. These results suggest that blood-borne IL-6 can reach sites behind the BBB, but that susceptibility to enzymatic degradation may limit contact time within the CNS. Key words: Cytokine; HPLC; Capillary depletion; Saturable; Efflux; Cerebrospinal fluid

Interleukin-6 (IL-6) is a multifunctional cytokine with effects on both peripheral tissues and the CNS [17]. IL-6 can directly affect neurons [23] and may mediate fevers induced by lipopolysaccharide, endotoxin and IL-1 through a CNS pathway, with levels of IL-6 in the cerebrospinal fluid (CSF) correlating with fever responses induced by these substances [13,18]. The release of A C T H [20] by blood-borne IL-6 may be mediated in part at sites behind the blood-brain barrier (BBB). This suggests that IL-6, like I L - l a [5,8,10], IL-1B [11] and T N F - a [14], may be able to cross the BBB. Interactions between IL-6 and the BBB, comprised largely of the brain's vasculature, may be important in disease states. IL-6 levels are increased in the CSF of patients with multiple sclerosis [19], a disease in which disruption of the BBB is a major feature, and with CNS vasculitides [15]. We show here that IL-6 associates with and is transported across the BBB. Recombinant human IL-6 (hIL-6) and flagged murine

*Corresponding author. Address: Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, LA 70146, USA. Fax: (1) (504) 522 8559. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00590-7

IL-6 (fmlL-6) donated by Immunex (Seattle, WA) and murine IL-6 (mlL-6; R&D Systems, Minneapolis, MN) were radioactively labeled with 1251 by the enzymobead method (BioRad, Richmond, CA). The labeled cytokines (I-fmlL-6, I-mlL-6 and I-hlL-6) were separated from free iodide on a column of G-10 Sephadex and had specific activities of ~700 Ci/mmol. Unidirectional influx constants (Ki) in ml/g/min were determined by multiple-time regression analysis [12,21] as applied to cytokines [6]. Briefly, 2(106) cpm of I-IL-6 in 0.2 ml of lactated Ringer's solution (LRS) with 1% BSA was injected into the left jugular vein of 25 g pathogen-free male mice (Taconic, Germantown, NY) anesthetized with urethane. 2-45 min after injection, blood samples were obtained from the right carotid artery, the mouse immediately decapitated and the whole brain removed. Levels of radioactivity in serum and whole brain (without pituitary and pineal) were measured in a y counter. The K i and the apparent volume of distribution for brain, Vi, in ml/g, were determined from the equation: Arn/Cpt = K~[f0tCp(r)dr]/Cpt + Vi

(1)

where Am is cprn/g of brain, Cpt is cprn/ml of arterial

54

W.A. Banks et al./Neuroscience Letters 179 (1994) 53-56

30,0-

: ~,lli~,i,,, o

tagonist (Synergen, Boulder, CO) and MIP-10~ (R&D Systems) did not alter the K~of I-mlL-6 or I-fmlL-6. This shows that the transport system for IL-6 is distinct from those for the IL-l's and TNF-~. The percent of the i.v. injection present/g of brain (%Inj) was determined from the equation:

12SklL.6 ÷ unl.b4~y +

o

E 0

0

0

20.0

%Inj = Ki(expt)(Is)

(2)

c

lo.0

+'5

s'*

+'u

6'*

Exposure Time (rain)

Fig. I. Uptake by brain of i.v. l-fraiL-6 in the presence or absence of 50/lg/kg of unlabeled fmlL-6. Unlabeled fmlL-6 inhibited entry by ~48%.

serum and exposure time is measured by the term

[f otCp(r)dr]/Cpt. Fig. 1 shows the results for I-fmIL-6 with the brain/ blood ratios plotted against exposure time. The Ki, equal to the slope of this plot, was (3.83 + 0.44)10 -4 ml/g/min. This is similar to the Ki values for other cytokines with saturable transport across the BBB [11,14]. The similarity of the K~ for I-mIL-6 with that for I-hIL-6 is consistent with a low degree of species-specificity for IL-6 in contrast to differences in BBB transport rates of other, more species-specific, cytokines [11]. No differences existed between fmIL-6 and raiL-6. This is consistent with the previous finding that attachment to IL-6 of flag, a hydrophilic peptide of eight amino acids that aids in purification, does not alter the biological activities of IL-6 [16]. Saturable transport was tested by including 2.5, 10 or 50 pg/kg of an unlabeled IL-6 in the i.v. injection. ANOVA with the BMDP1R program (University of California Press, Berkeley, CA) showed statistically significant self-inhibition for fmIL-6:F6,32 = 5.48, P < 0.001 (Fig. 1) and for mIL-6: F6.3~ = 4.67, P < 0.005. Only the 50 pg/kg dose was tested for hIL-6 and this also showed self-inhibition: Fz.t6 = 3.84, P < 0.05 (Table 1). By contrast, 50 pg/kg of TNF-~ (Genentech, San Francisco, CA), IL-I~, IL-1B, IL-2 (Immunex), IL-1 receptor anTable 1 Effect of unlabeled I L-6's (50/~g/kg) on the K i value of the corresponding radioactively labeled IL-6's K i × 104 (ml/g.min) Cytokine

Label only (n)

+ Unlabeled (n)

Inhibition

fmIL-6 mlL-6 hlL-6

3.83 + 0.44 (11) 3.05 + 0.48 (11) 4.54 -+ 0.45 (13)

2.00 + 0.34 (8) 1.85 + 0.51 (7) 2.97 + 0.46 (7)

47.8% 39.3% 34.6%

For each cytokine, there was statistically significant self-inhibition.

where expt is exposure time and Is is the % of the injection/ml of arterial serum at that exposure time. For IfmIL-6, %Inj peaked at 0.225% 20 min after i.v. injection. Similar results were observed for I-mIL-6. The highest value for I-hIL-6 was 0.194% and occurred 30 min after injection. In five other mice, the entry of I-mIL-6 into the parenchyma of cerebral cortex depleted of capillaries was assessed as described for rats [22] and adapted to mice [14]. Briefly, mice received an i.v. injection of 2(106) cpm of I-mIL-6 and 99mTc-albumin (Medi + Physics albumin labeling kit; Paramus, NJ). 10 min later, blood was taken from the carotid artery, the mouse decapitated and the cerebral cortex removed, weighed and homogenized as previously descibed [22] at 4°C in < 1 min. Homogenates were centrifuged at 5400 x g for 15 min at 4°C in a swinging bucket rotor. The pellet, enriched in brain capillaries, and the supernatant, representing the parenchyma, were separated and the radioactivity measured. Values were corrected for vascular contamination as determined with 99mTc-albumin and expressed as volumes of distribution in pl/g. In capillaries, 3.4 + 0.5/A/g of I-mIL-6 was present and may reflect internalization, binding to endothelial receptors or the BBB transport system. For the parenchyma, 5.7 + 1.6/al/g of I-mIL-6 was present, indicating that the majority of IL-6 completely crossed the BBB. Identification of radioactivity entering the CNS was determined in mice given an i.v. injection of I-fmIL-6. Before decapitation, CSF was removed from the posterior fossa and brains washed free of blood by cardiac perfusion of 20 ml of LRS through the left ventricle of the heart with the abdominal aorta clamped and the jugular veins severed. Only completely clear CSF was analysed. Brain homogenates were centrifuged at 200,000 x g at 4°C for 20 min and the supernatant lyophilized until rehydrated with distilled water for analysis by HPLC. The material was eluted in a two-step process. Free iodide and iodinated proteins were separated on a G-10 column as described above. I-IL-6 fragments were separated from intact I-IL-6 by HPLC (C4 protein column, flow rate 1 ml/min with an eluant that increased linearly from 30 to 90% acetonitrile in water with 0.1% trifluoroacetic acid). A processing control for brains was obtained by the addition in vitro of I-IL-6 to a nonradioactive brain that was processed as above. For the processing control, 31.7% eluted as intact I-IL-6; this

W.A. Banks et al. / Neuroscience Letters 179 (1994) 53-56

4.S-

55

-3

i 4.0

Time (rain)

Fig. 2. Brain to blood efflux of I-fmlL-6 after i.c.v, injection. The half-time disappearance of 42.0 min is consistent with reabsorption of I-fmlL-6 with the CSF.

number was used to correct the brain samples for in vitro degradation. For CSF, no processing control was used. Most of the radioactivity in CSF eluted at the position of I-IL-6 even 30 min after i.v. injection (Table 2). This suggests that CSF may represent an enzymatically protected space for IL-6 or that IL-6 may be crossing the ependymal BBB (the blood-CSF barrier). By contrast, the amount of radioactivity from brain supernatant eluting as I-IL-6 was < 17% even as early as 2 min. This could reflect deiodination of the I-IL-6, but more likely represents degradation of IL-6 by endothelial or brain cells or by enzymes in the interstitial fluid. This level of enzymatic activity raises doubts about the relative contribution of blood-borne IL-6 to areas of the brain containing the endothelial barrier of the BBB. Brain to blood efflux after i.c.v, injection was measured as previously described [3]. Briefly, mice received an i.c.v, injection of 25,000 cpm of I-fraiL-6 in a volume of 1/11 of 1% BSA in LRS. Mice were decapitated 1, 2, 5, 10 or 20 min later (n = 6-7 mice/time point) and the radioactivity remaining in the brain measured. A '0' time point was determined in mice overdosed with anesthesia as previously described [3]. Fig. 2 shows the log(mean counts remaining in brain) vs. time. This relationship was statistically significant: r = -0.919, n -- 6, P < 0.01, Table 2 % of radioactivity obtained from CSF or brain interstitial fluid eluting as I-IL-6 Time (min)

CSF

Brain

2 10 30

51.3 57.6

16.6 13.1 10.9

Identification of I-IL-6 was by a two-step Sephadex/HPLC process. Brain, but not CSF, was corrected for in vitro processing degradation. A 2-min CSF sample was not obtained.

slope = (-7.16)10 and the I-fmlL-6 had a half-time disappearance from brain of 42.0 min. This is similar to the half-time disappearance from brain of albumin and suggests that IL-6 exits the CNS primarily by reabsorption of CSF [9], but is not transported by an efflux system as are some peptides [4] or sequestered by brain as are agglutinins [2] and TNF-~ [14]. In conclusion, these results show that IL-6 can penetrate the BBB by a saturable transport system to enter the CSF and the parenchyma of the brain as an intact molecule. The % of injected dose/g of brain of-0.2% is closer to that of~0.1% found for IL-10~ [5,8,11] than that of 0.02% for morphine [1,7]. However, unlike other cytokines found to cross the BBB, the amount of radioactivity recovered from brain interstitial fluid that could be identified as intact I-IL-6 did not exceed 17%. It is not known whether this degradation occurred in the circulation or within the brain after transport. Degradation could limit the contribution of blood-borne IL-6 to levels within the brain interstitial fluid. However, even small amounts of intact IL-6 may be sufficient to induce CNS effects. We thank Dr. Jeannine A. Majde for helpful ideas, Melita B. Fasold for aid with graphs and text, James L. Vannice (Synergen, Boulder, CO) for providing IL-1 receptor antagonist, Genentech for providing TNF-~ and Immunex for rmlL-6, hlL-6, IL-I~, IL-113 and IL-2. Supported by ONR (NODO14-92-J-1384) and the VA. [1] Advokat, C. and Gulati, A., Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats, Brain Res., 555 (1991) 251-258. [2] Banks, W.A. and Broadwell, R.D., Blood to brain and brain to blood passage of native horseradish peroxidase, wheatgerm agglutinin and albumin: pharmacokinetic and morphological assessments, J. Neurochem., 62 (1994) 2404-2419. [3] Banks, W.A. and Kastin, A.J., Quantifying carrier-mediated transport of peptides from the brain to the blood. In P.M. Conn (Ed.), Methods in Enzymology, Vol. 168, Academic Press, San Diego, CA,1989, pp. 652-660. [4] Banks, W.A. and Kastin, A.J., Editorial review: peptide transport systems for opiates across the blood-brain barrier, Am. J. Physiol., 259 (1990) El-El0. [5] Banks, W.A. and Kastin, A.J., Blood to brain transport of interleukin links the immune and central nervous systems, Life Sci., 48 (1991) PL117-PL121. [6] Banks, W.A. and Kastin, A.J., Measurement of transport of cytokines across the blood-brain barrier. In E.B. De Souza (Ed.), Methods in Neurosciences, Vol. 16, Academic Press, San Diego, CA, 1993, pp. 67-77. [7] Banks, W.A. and Kastin, A.J., Opposite direction of transport across the blood-brain barrier for Tyr-MIF-I and MIF-I: comparison with morphine, Peptides, 15 (1994) 23-29. [8] Banks, W.A., Kastin, A.J. and Durham, D.A., Bidirectional transport of interleukin-1 alpha across the blood-brain barrier, Brain Res. Bull., 23 (1989) 433437. [9] Banks, W.A., Kastin, A.J. and Fasold, M.B., Differential effect of aluminum on the blood-brain barrier transport of peptides, technetium and albumin, J. Pharmacol. Exp. Ther., 244 (1988) 579585.

56

W.A. Banks et al./Neuroscience Letters 179 (1994) 53-56

[10] Banks, W.A., Kastin, A.J. and Gutierrez, E.G., Interleukin-lct in blood has direct access to cortical brain cells, Neurosci. Lett., 163 (1993) 41-44. [11] Banks, W.A., Ortiz, L., Plotkin, S.R. and Kastin, A.J., Human interleukin (IL) 1~, murine IL-l~z and murine IL-lfl are transported from blood to brain in the mouse by a shared saturable mechanism, J. Pharmacol. Exp. Ther., 259 (1991) 988-996. [12] Blasberg, R.G., Fenstermacher, J.D. and Patlak, C.S., Transport of c~-aminoisobutyric acid across brain capillary and cellular membranes, J. Cereb. Blood Flow Metab., 3 (1983) 8-32. [13] Coceani, F., Lees, J., Mancilla, J., Belizario, J. and Dinarello, C.A., Interleukin~6 and tumor necrosis factor in cerebrospinal fluid: changes during pyrogen fever, Brain Res., 612 (1993) 165171. [14] Gutierrez, E.G., Banks, W.A. and Kastin, A.J., Murine tumor necrosis factor alpha is transported from blood to brain in the mouse, J. Neuroimmunol., 47 (1993) 169-176. [15] Hirohata, S., Tanimoto, K. and Ito, K., Elevation ofcerebrospinal fluid interleukin-6 activity in patients with vasculitides and central nervous system involvement, Clin. Immunol. Immunopathol., 66 (1993) 225-229. [16] Hopp, T.R, Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, D.R, Urdal, D.L. and Conlon, RJ., A short polypeptide

marker sequence useful for recombinant protein identification and purification, Bio-Technology, 6 (1988) 1204-1210. [17] Kishimoto, T., Akira, S. and Taga, T., lnterleukin-6 and its receptor: a paradigm for cytokines, Science, 258 (1992) 593-597. [18] LeMay, L.G., Vander, A.J. and Kluger, M.J., Role of interleukin 6 in fever in rats, Am. J. Physiol., 258 (1990) R798-R803. [19] Maimone, D., Gregory, S., Arnason, B.G.W. and Reder, A.T., Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis, J. Neuroimmunol., 32 (1991) 67-74. [20] Matta, S.G., Weatherbee, J. and Sharp, B.M., A central mechanism is involved in the secretion of ACTH in response to IL-6 in rats: comparison to and interaction with IL-lfl, Neuroendocrinology, 56 (1992) 516-525. [21] Patlak, C.S., Blasberg, R.G. and Fenstermacher, J.D., Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data, J. Cereb. Blood Flow Metab., 3 (1983) 14 7. [22] Triguero, D., Buciak, J. and Pardridge, W.M., Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins, J. Neurochem., 54 (1990) 1882-1888. [23] Xin, L. and Blatteis, C.M., Hypothalamic neuronal responses to interleukin-6 in tissue slices: effects of indomethacin and naloxone, Brain Res. Bull., 29 (1992) 27 35.