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blood–spinal cord barrier
Advanced Drug Delivery Reviews 36 (1999) 291–298
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Penetration of neurotrophins and cytokines across the blood–brain / blood–spinal cord barrier Weihong Pan*, Abba J. Kastin VA Medical Center and Tulane University School of Medicine, 1601 Perdido Street, New Orleans, LA 70146, USA
Abstract Now that peptides are no longer considered too large to cross the blood–brain barrier, attention has turned to the possibility that larger substances like polypeptides might also enter the central nervous system (CNS). This review summarizes evidence showing that many cytokines and neurotrophins not only enter the brain but also enter the spinal cord, sometimes faster than into the brain. 1999 Elsevier Science B.V. All rights reserved. Keywords: Neurotrophins; Cytokines; Blood–brain barrier; Spinal cord
Contents 1. Introduction ............................................................................................................................................................................ 2. Neurotrophins ......................................................................................................................................................................... 2.1. Nerve growth factor (NGF) and bNGF.............................................................................................................................. 2.2. Neurotrophin (NT)-5 ........................................................................................................................................................ 2.3. NT-3 ............................................................................................................................................................................... 2.4. Brain-derived neurotrophic factor (BDNF)......................................................................................................................... 3. Cytokines ............................................................................................................................................................................... 3.1. Tumor necrosis factor-a (TNF)......................................................................................................................................... 3.1.1. TNF entry into the brain ......................................................................................................................................... 3.1.2. TNF entry into the spinal cord in EAE..................................................................................................................... 3.1.3. TNF and ebiratide entry into the transected spinal cord ............................................................................................. 3.1.4. Rationale of upregulation of the saturable transport system for TNF .......................................................................... 3.2. Interferon (IFN)a and IFNg.............................................................................................................................................. 3.3. Interleukin (IL)-1a and IL-1b ........................................................................................................................................... 3.3.1. IL-1a entry into the spinal cord............................................................................................................................... 3.3.2. Circadian variation of IL-1a entry ........................................................................................................................... 3.4. IL-2 ................................................................................................................................................................................ 3.5. IL-6 ................................................................................................................................................................................ 3.6. Macrophage inflammatory protein (MIP)-1a and MIP-1b ................................................................................................... 3.7. Granulocyte-macrophage colony-stimulating factor (GM-CSF) entry into the spinal cord ...................................................... 3.8. Fibroblast growth factors (FGF)........................................................................................................................................ 4. Comparison with neurotrophic peptides .................................................................................................................................... 4.1. Pituitary adenylate cyclase-activating polypeptide (PACAP) ............................................................................................... *Corresponding author. Tel.: 1 1-504-5895928; fax: 1 1-504-5228559; e-mail: [email protected] 0169-409X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 98 )00086-6
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4.2. Ebiratide ......................................................................................................................................................................... 5. Conclusions ............................................................................................................................................................................ Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................
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1. Introduction
2. Neurotrophins
The blood–brain / blood–spinal cord barrier (BBB) serves as a dynamic interface between the periphery and CNS for the neurotrophins and cytokines, substances generally fitting the definition of polypeptides [1]. BBB research with these substances is relatively new. Just as our initial work showing that peptides can cross the BBB was met with skepticism, even among a few prominent researchers in the field, so there are researchers in the newer areas of neurotrophins and cytokines who are skeptical about these larger substances entering the CNS from the periphery. This review will summarize the overwhelming evidence now existing for the penetration of neurotrophins and cytokines into the brain and spinal cord. It will be restricted to studies of the endogenous forms of the neurotrophins and cytokines, not discussing studies involving synthetic chemically modified carriers for which neither mechanisms of transport nor toxicity has been determined. The review will be organized according to the individual endogenous substance. Most of the studies of entry into the CNS involve determination of the unidirectional influx rate by multiple-time regression analysis after i.v. bolus injection of femtomole quantities of the labeled neurotrophin (or cytokine). The details of this method, with emphasis on cytokines, as well as for exit from the brain are provided elsewhere [2–4]. The stability and distribution in brain parenchyma are tested for each compound with standard methods. By use of cerebral cortex, the possibility of leakage through circumventricular organs (CVOs) to bypass the BBB is excluded. The permeability of the BBB shows regional variation, as measured by radiolabeled albumin and sucrose. In general, the penetration of most cytokines and neurotrophic proteins across the BBB is much higher than what would be expected for simple diffusion.
Most members of the mammalian neurotrophin family cross the BBB through saturable transport systems to arrive intact in the CNS [5]. The radioactively labeled neurotrophins are relatively stable in blood and CNS and have variable permeability across the BBB. The rates of transport differ among the neurotrophins tested and among the regions examined. The initial volume of distribution in brain, which usually reflects the association with cerebral endothelial cells, also varies among neurotrophins and among regions. The studies rely on the use of 125 I for detection. It has been demonstrated by more than one group that neurotrophins labeled with 125 I remain biologically active [6,7]. In a study involving transport across the BBB of a peptide, which is much smaller than a neurotrophin and hence might be more susceptible to change by addition of an iodine, we found no difference in rate of entry or saturability between the tritiated and iodinated forms [8].
2.1. Nerve growth factor ( NGF) and b NGF The entry of the 7s NGF pentomer into the brain is rapid [5]. High-performance liquid chromatography (HPLC) shows that most of the injected [ 125 I]NGF remains intact in brain and serum 20 min after i.v. injection. Capillary depletion studies shows that iv injected NGF reaches the parenchyma of the brain. The rate of influx of NGF into the brain and the entire spinal cord is about twice as fast as for bNGF [5]. That the rate of entry into the spinal cord of the considerably larger NGF is much faster than that of its smaller bNGF subunit represents an unexpected finding with therapeutic implications. There are no significant differences in lipophilicity, so that neither molecular weight nor lipid solubility, key factors in simple diffusion, could explain this finding. Entry into spinal cord for both NGF and bNGF is more
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than twice as fast as into the brain. Entry of NGF is not inhibited by excess NGF when given i.v., but is saturable when perfused in buffer free of blood elements, including possible binding proteins.
2.2. Neurotrophin ( NT)-5 For NT5 the rate of entry into the brain and spinal cord is much slower than for NGF and slightly slower than for bNGF [5]. As with NGF and bNGF, the influx rate into the spinal cord is more than twice as fast as into the brain. The entry rate into the cervical region of the spinal cord is higher than for the lumbar or thoracic regions, in that order.
2.3. NT-3 For NT3 the entry rate is slower than that for NGF, bNGF, or NT5 [5]. Again, the entry is more than twice as fast as into brain and faster in the cervical than in the lumbar or thoracic areas. Entry is saturable in the brain but not in the spinal cord at the dose injected i.v.
2.4. Brain-derived neurotrophic factor ( BDNF) In contrast to a report finding no transport of unconjugated BDNF into brain [9], Poduslo and Curran [10] showed that BDNF enters brain at a rate comparable to the rate they found earlier for insulin and somewhat faster than for NGF, NT-3, and CNTF. Insulin enters the CNS relatively fast, saturably, and in a physiologically meaningful way [11–13]. Saturable transport of BDNF into the brain and spinal cord was found recently [14]. Influx is much greater in the cervical than in the thoracic or lumbar regions of the spinal cord, raising the possibility of differential entry not only by different neurotrophins but also by region of the cord. SDS–polyacrylamide gel electrophoresis (PAGE) shows the entering protein is intact, as had been found by Poduslo and Curran 60 min after uptake [10]. Capillary depletion studies confirm that most of the BDNF reaches the brain parenchyma. Efflux of BDNF from brain to blood occurs at the rate of the usual reabsorption of cerebrospinal fluid (CSF) and is not saturable. We found evidence in blood not only for protein
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binding but also for self-aggregation [14]. This might explain another unusual finding with BDNF, the paradoxical increase in the entry of [ 125 I]BDNF into the brain with co-injection of a small amount of unlabeled BDNF. It is possible that the excess unlabeled BDNF displaces the iodinated material from binding proteins or dissociated aggregates, among other explanations.
3. Cytokines
3.1. Tumor necrosis factor-a ( TNF) 3.1.1. TNF entry into the brain TNF enters the brain and CSF by a saturable system not shared with other cytokines [15]. Entry in intact form into the CSF, as shown by HPLC, confirms the results of capillary depletion studies in which it has been found not to be sequestered by capillaries. There is an age-related difference in permeability of the BBB to TNF. In neonatal rats, the entry rate of TNF is more than three times as fast as in adult mice. This is probably not explained by species differences since mice would be expected to transport the murine TNF used in the studies faster than rats [15]. In normal animals, there are reports that TNF may increase, decrease or cause no change in the BBB permeability [16]. This implies that TNF could affect its own transport system. 3.1.2. TNF entry into the spinal cord in EAE In a system with known disruption of the BBB— acute experimental autoimmune encephalomyelitis (EAE)—we showed that the transport of TNF into the spinal cord of these mice is greater than can be accounted for by disruption of the BBB measured with albumin and sucrose [17]. Although TNF circulates as a trimer similar in size to albumin, it penetrates much faster than albumin at a rate comparable to that of sucrose, a molecule more than 50 times smaller in size. The complete self-inhibition of the increased entry suggests an enhanced saturable transport system for TNF in EAE. The general disruption of the BBB in the acutely ill EAE mice, as measured by the ratio of TNF in
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CNS over blood at 10 min after i.v. injection, is greater in the thoracic and lumbar regions of the spinal cord than in the cervical region or the brain [17]. TNF does not further damage the BBB since the entry of albumin is not changed additionally. The saturability of TNF entry in all three regions of the spinal cord suggests an upregulation of the transport system.
3.1.3. TNF and ebiratide entry into the transected spinal cord During the acute phase of spinal cord injury by transection in the lumbar region, permeability to TNF is increased in different regions proximal to the lesion [18]. The increase of TNF penetration is greater than can be explained by simple disruption of the BBB and extends beyond the area injured. Specifically, increased entry of TNF occurs in regions (cervical and thoracic) and at times (24 h in the proximal lumbar region) unaccompanied by disruption of the BBB as measured by albumin and sucrose. In addition, the increased transport of TNF from blood to spinal cord proximal to the lumbar lesion is saturable. As would be expected by the selectivity of the saturation process, the excess TNF did not affect the entry of albumin. There is regional distribution of the enhanced permeability (expressed as the ratio of TNF / albumin) 2 h after spinal cord injury, and the correlating inhibition by excess unlabeled TNF. 3.1.4. Rationale of upregulation of the saturable transport system for TNF TNF is one of the few cytokines for which attention has been directed to the dual effects that may occur in many disease states [16]. Increased production of TNF and / or increased demand for it from the periphery may be essential in the early response of the CNS to insults, although excess TNF has been related to pathogenesis. It also is possible that TNF available in the early stages of disease can serve as a negative feedback to abolish the later overproduction that causes neuronal death. We found dual effects of TNF after exogenous administration in EAE. Low doses seem to have a positive modulatory effect, whereas higher doses aggravate the severity of EAE. Therefore, upregulation of the
transport system on the BBB may have a role in confining CNS disorders.
3.2. Interferon ( IFN)a and IFNg Intact IFNs are found in the brain up to 1 h after i.v. injection. The spinal cord has greater permeability to the IFNs than does the brain [19]. For each region of the spinal cord, the permeability to IFNa is greater than that for IFNg. In contrast to the saturable penetration of TNF into all regions of the brain and spinal cord, passage of IFNg is saturable only in the brain and cervical region of the spinal cord but not in the thoracic or lumbosacral area, and that of IFNa is not saturable in any region at the dose tested. This emphasizes the necessity of examining all regions of the spinal cord when studying the penetration of the BBB by different compounds and testing saturability, even when the entry rate is reasonably high.
3.3. Interleukin ( IL)-1a and IL-1b IL-1a and IL-1b enter the brain by a shared, saturable transport system [20]. There is no saturable egress of these cytokines from the brain. Intact human IL-1a has a faster rate of entry into brain tissue of the mouse than of the rat. In the mouse, the entry rate is faster for murine IL-1a than for human IL-1a, and is faster for murine IL-1a than for murine IL-1b [20,21]. As with all such studies, the inclusion of albumin eliminates non-specific leakage during the period of study [20]. Specific study of larger doses (2 mg / mouse) of IL-1a, IL-1b, and also IL-2 for up to 2 h after i.v. injection shows no increased permeability of the BBB [22]. An antibody directed toward the portion of the interleukin molecule that binds to the murine T lymphocyte receptor severely blocks entry of IL-1a into the brain and inhibits its clearance from blood [20]. In contrast, the antibody directed towards a nonbinding area of IL-1a does not inhibit the transport. Two different antibodies, directed at the sites on the murine T lymphocyte receptor that either bind or do not bind IL-1 show partial inhibition of the entry of IL-1a into the brain. The soluble murine receptor to IL-1 only partially
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blocks the entry of IL-1a into brain [23] and is not transported across the BBB. This suggests that the BBB transporter for IL-1 can strip IL-1 from its soluble receptor. In contrast, the soluble p75 human receptor to TNF totally blocks the entry of both human and murine TNF into brain. The endogenous IL-1 receptor antagonist is itself saturably transported across the BBB to enter the brain and CSF from the blood in intact form [24]. It shares the transport system for IL-1a and IL-1b, each of the three cytokines inhibiting each other’s transport. For the capillary depletion studies, showing penetration into brain parenchyma, portions of cortex devoid of CVOs were used. Although CVOs can concentrate IL-1a from the blood to a greater extent than cortex, this route cannot account for more than 5% of the total brain uptake of the cytokine [25]. IL-1a injected i.v. is saturably transported into the posterior division of the septum of the brain, on the pathway between the hippocampal formation and the midbrain [26]. IL-1 receptor antagonist, IL-1b, and TNFa are not concentrated in the septum and none of these three cytokines inhibit transport of IL-1a there.
3.3.1. IL-1a entry into the spinal cord IL-1a is a proinflammatory cytokine that may be involved in the early phase of spinal cord injury. In a previous study we found that intact IL-1a enters all three regions of the spinal cord, without significant differences [27]. In addition, it was determined that the material entering the brain and diffusing caudally could only account for about 1% of the total radioactivity found in the spinal cord after i.v. administration. 3.3.2. Circadian variation of IL-1a entry Recently, we found a circadian variation in the entry of IL-1a in several tissues [28]. The variation in entry into the spinal cord is greater than that into brain, testis, or muscle. Entry of IL-1a into spinal cord is fastest at 08:00 h and slowest at 24:00 h, the difference between these times being 10-fold. By contrast, there is no circadian rhythm for the disappearance curves in blood or for the spinal entry of human TNF, which in contrast to murine TNF does not cross the BBB. Thus, IL-1a shows a circadian
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pattern of entry into the spinal cord that is dependent on its specific uptake rather than on non-specific factors that would also have affected hTNF.
3.4. IL-2 Unlike the other cytokines, IL-2 is not saturably transported into the brain under the conditions studied [29]. Nevertheless, it does enter the CNS in intact form 10 times faster than albumin. Its entry is not affected by antibodies to IL-2, compatible with a nonspecific method of entry. At the doses used, it does not damage the BBB, and damage to the BBB secondary to hypertension elicited by epinephrine is not enhanced or prolonged by IL-2 [22].
3.5. IL-6 Although IL-6 is saturable transported into the brain, most of this cytokine is degraded before reaching the parenchyma of the brain [30]. In contrast, most of the IL-6 entering the CSF remains intact even 30 min after peripheral injection.
3.6. Macrophage inflammatory protein ( MIP)-1a and MIP-1b MIPs behave differently from the other cytokines studied. They bind reversibly with the luminal surface of the endothelial cells comprising the BBB [31]. This binding is greatly reduced by perfusion of the vascular space of the brain. The association with the vascular space does not increase over time nor is it self-inhibitable.
3.7. Granulocyte-macrophage colony-stimulating factor ( GM-CSF) entry into the spinal cord GM-CSF is a peripherally produced compound that can exert CNS effects in addition to hematopoietic effects. These include neuroregenerative actions [32]. We found that GM-CSF enters the spinal cord from the blood significantly faster than does the albumin control [33]. There is no apparent gender difference and species variation (mice and rats). The intact nature of the GM-CSF arriving in the CNS was shown by acid precipitation and HPLC, an effect that capillary depletion with perfusion showed could
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not be accounted for by any GM-CSF residual in the vasculature.
3.8. Fibroblast growth factors ( FGF) We measured entry into brain and cervical, thoracic, and lumbar regions of the spinal cord for acidic (a)FGF and basic (b)FGF (McLay and Kastin, unpublished results). The pattern of BBB permeability is different from the other compounds discussed in this review. Although both aFGF and bFGF enter faster than the albumin control, this entry is not self-inhibited by a large dose of unlabeled cytokine which is usually sufficient to suppress a transport system, indicating that entry occurs by non-saturable diffusion. Moreover, unlike most of the other cytokines, there seems to be considerable amount of reversible binding to the capillary endothelial cells that comprise the BBB; this was shown by the inclusion of a perfusion step in the capillary depletion procedure to wash out the vasculature.
4. Comparison with neurotrophic peptides
4.1. Pituitary adenylate cyclase-activating polypeptide ( PACAP) PACAP has neurotrophic properties such as preventing gp120-induced death of hippocampal neurons and CA1 neurons after global ischemia [34,35]. We found that, unlike FGF, the influx of iodinated PACAP is saturable for the cervical and thoracic regions of the spinal cord [36]. The use of radioactively labeled albumin again showed that the spinal cord provides a significant barrier to serum proteins. This means that the tissue / serum ratios of albumin reflect the vascular space of the tissues, which vary with region. The cervical cord shows the highest degree of vascularity and the thoracic the lowest. These ratios for the vascular marker albumin are not affected by PACAP. In the same study [36], complete transection of the spinal cord results in long-lasting effects on the uptake of PACAP that cannot be explained by disruption of the vascular barrier [5]. For example, unlike BDNF no change in the entry of PACAP into the lumbar cord, the site of transection, is found until
a week after injury when increased influx occurs in the cervical, thoracic, and distal, but not proximal, lumbar regions. If the effects are only due to traumatic disruption of the vascular barrier, they would have been expected to occur much earlier. Thirty minutes after transection there is a marked decrease in the transport of PACAP into the cervical cord that lasts for at least 3 days, changes not seen with albumin. A slower entry of PACAP, but not albumin, is found in the thoracic cord by 24 h but not in the lumbar cord. One speculative explanation for the transient decrease in entry of PACAP might involve a circulating factor that would be released after spinal cord injury. If this is related to the inflammatory phase of the trauma, it might be expected to decrease with time while PACAP transport is allowed to increase. This circulatory factor, such as a cytokine similar to those tested in some of the other studies (e.g. IFNg or IL-1a), could act on the BBB generally, only in specific areas, or only on selective transport systems. The difficulty with such an explanation, however, involves our observations that spinal cord injury does not decrease entry of TNFa or affect the entry of ebiratide in either direction. The transport of PACAP into the CNS after transection of the spinal cord is different from that found with TNF and ebiratide, a melanocortin analog with potent neurotrophic properties [17,18,37].
4.2. Ebiratide Ebiratide is a six-amino acid analog of an ACTH / a-MSH fragment that has neurotrophic properties [37–39]. Ebiratide has a faster transport rate than cytokines and neurotrophins in normal mice, and this rapid entry is unaffected by transection of the spinal cord in the lumbar region.
5. Conclusions Like peptides [40,41], most neurotrophins and cytokines are transported across the BBB from blood to brain. This transport is usually saturable and not confined to endothelial cells of the cerebral vasculature. It reflects intact material entering the CNS. Many neurotrophins and cytokines enter the spinal
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cord, where they could exert therapeutic effects, at a faster rate than into brain. Such transport represents further support for the dynamic regulatory functions of the BBB.
[11]
[12]
Acknowledgements [13]
The authors thank William A. Banks, M.D. for long-term collaboration and kind help, and Melita B. Fasold for editorial assistance.
[14]
[15]
References [16] [1] A.J. Kastin, J.E. Zadina, R.D. Olson, W.A. Banks, The history of neuropeptide research: version 5.a, in: J.N. Crawley, S. McLean (Eds.), Neuropeptides: Basic and Clinical Advances, Ann. NY Acad. Sci., New York, 1996, pp. 1–18. [2] W.A. Banks, A.J. Kastin, Measurement of transport of cytokines across the blood–brain barrier, in: P.M. Conn, E.B. De Souza (Eds.), Neurobiology of Cytokines, Part A, Academic Press, San Diego, 1993, pp. 67–77. [3] W.A. Banks, A.J. Kastin, 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, 1989, pp. 652–660. [4] W.A. Banks, M.B. Fasold, A.J. Kastin, Measurement of efflux rates from brain to blood, in: G.B. Irvine, C.H. Williams (Eds.), Methods in Molecular Biology, Neuropeptide Protocols, vol. 73, Humana Press, Totowa, NJ, 1997, pp. 353–360. [5] W. Pan, W.A. Banks, A.J. Kastin, Permeability of the blood– brain / spinal cord barrier to neurotrophins, Brain Res. 788 (1998) 87–94. [6] P.S. DiStefano, B. Friedman, C. Radziejewski, C. Alexander, P. Boland, C.M. Schick, R.M. Linsay, S.J. Wjegand, The neurotrophins BDNF, NT-3 and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons, Neuron 8 (1992) 983–993. [7] Q. Yan, C. Matheson, J. Sun, M.J. Adeke, S.C. Weinstein, J.A. Miller, Distribution of intracerebral ventricularly administered neurotrophins in rat brain and its correlation with the Trk receptor expression, Exp. Neurol. 127 (1994) 23–36. [8] W.A. Banks, A.J. Kastin, Opposite direction of transport across the blood-brain barrier for Tyr-MIF-1 and MIF-1: comparison with morphine, Peptides 15 (1994) 23–29. [9] W.M. Pardidge, Y.-S. Kang, J.L. Buciak, Transport of human recombinant brain- derived neurotrophic factor (BDNF) through the rat blood-brain barrier in vivo using vectormediated peptide drug delivery, Pharm. Res. 11 (1994) 738–746. [10] J.F. Poduslo, G.L. Curran, Permeability at the blood-brain
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
297
and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF, Mol. Brain Res. 36 (1996) 280–286. W.A. Banks, J.B. Jaspan, A.J. Kastin, Selective, physiological transport of insulin across the blood-brain barrier: novel demonstration by species-specific radioimmunoassays, Peptides 18 (1997) 1257–1262. W.A. Banks, J.B. Jaspan, W. Huang, A.J. Kastin, Transport of insulin across the blood-brain barrier: saturability at euglycemic doses of insulin, Peptides 18 (1997) 1423–1429. W.A. Banks, J.B. Jaspan, A.J. Kastin, Effect of diabetes mellitus on the permeability of the blood-brain barrier to insulin, Peptides 18 (1997) 1577–1584. W. Pan, W.A. Banks, M.B. Fasold, J. Bluth, A.J. Kastin, Transport of BDNF across the blood–brain barrier, Neuropharmacol. (1998) in press. E.G. Gutierrez, W.A. Banks, A.J. Kastin, Murine tumor necrosis factor alpha is transported from blood to brain in the mouse, J. Neuroimmunol. 47 (1993) 169–176. W. Pan, J.E. Zadina, R.E. Harlan, J.T. Weber, W.A. Banks, A.J. Kastin, Tumor necrosis factor-a: a neuromodulator in the CNS, Neurosci. Biobehav. Rev. 21 (1997) 603–613. W. Pan, W.A. Banks, M.K. Kennedy, E.G. Gutierrez, A.J. Kastin, Differential permeability of the BBB in acute EAE: enhanced transport of TNF-a, Am. J. Physiol. 34 (1996) E636–E642. W. Pan, W.A. Banks, A.J. Kastin, Blood-brain barrier permeability to ebiratide and TNF in acute spinal cord injury, Exp. Neurol. 146 (1997) 367–373. W. Pan, W.A. Banks, A.J. Kastin, Permeability of the bloodbrain and blood-spinal cord barriers to interferons, J. Neuroimmunol. 76 (1997) 105–111. W.A. Banks, L. Ortiz, S.R. Plotkin, A.J. Kastin, Human interleukin (IL) 1a, murine IL-1a and murine IL-b are transported from blood to brain in the mouse by a shared saturable mechanism, J. Pharmacol. Exp. Ther. 259 (1991) 988–996. W.A. Banks, A.J. Kastin, D.A. Durham, Bidirectional transport of interleukin-1 alpha across the blood-brain barrier, Brain Res. Bull. 23 (1989) 433–437. W.A. Banks, A.J. Kastin, The interleukins-1a, -1b, and -2 do not acutely disrupt the murine blood-brain barrier, Int. J. Immunopharmacol. 14 (1992) 629–636. W.A. Banks, S.R. Plotkin, A.J. Kastin, Permeability of the blood-brain barrier to soluble cytokine receptors, Neuroimmunomodulation 2 (1995) 161–165. E.G. Gutierrez, W.A. Banks, A.J. Kastin, Blood-borne interleukin-1 receptor antagonist crosses the blood-brain barrier, J. Neuroimmunol. 55 (1994) 153–160. S.R. Plotkin, W.A. Banks, A.J. Kastin, Comparison of saturable transport and extracellular pathways in the passage of interleukin-1a across the blood-brain barrier, J. Neuroimmunol. 67 (1996) 41–47. L.M. Maness, W.A. Banks, J.E. Zadina, A.J. Kastin, Selective transport of blood-borne interleukin-1a into the posterior division of the septum of the mouse brain, Brain Res. 700 (1995) 83–88. W.A. Banks, A.J. Kastin, C.A. Ehrensing, Blood-borne
298
[28]
[29]
[30]
[31]
[32]
[33]
[34]
W. Pan, A. J. Kastin / Advanced Drug Delivery Reviews 36 (1999) 291 – 298 interleukin-1a is transported across the endothelial bloodspinal cord barrier of mice, J. Physiol. 479(2) (1994) 257– 264. W.A. Banks, A.J. Kastin, C.A. Ehrensing, Diurnal uptake of circulating interleukin-1a by brain, spinal cord, testis, and muscle, Neuroimmunomodulations 5 (1998) 36–41. P.J. Waguespack, W.A. Banks, A.J. Kastin, Interleukin-2 does not cross the blood-brain barrier by a saturable transport system, Brain Res. Bull. 34 (1994) 103–109. W.A. Banks, A.J. Kastin, E.G. Gutierrez, Penetration of interleukin-6 across the murine blood-brain barrier, Neurosci. Lett. 179 (1994) 53–56. W.A. Banks, A.J. Kastin, Reversible association of the cytokines MIP-1a and MIP-1b with the endothelia of the blood-brain barrier, Neurosci. Lett. 205 (1996) 202–206. G. Guillemin, F.D. Boussin, R. Le Grand, J. Croitoru, H. Coffigny, D. Dormont, Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains, Glia 16 (1996) 71–80. R.N. McLay, M. Kimura, W.A. Banks, A.J. Kastin, Granulocyte-macrophage colony-stimulating factor crosses the blood-brain and blood-spinal cord barriers, Brain 120 (1997) 2083–2091. A. Arimura, A. Somogyvari-Vigh, C. Weill, R.C. Fiore, I. Tatsuno, V. Bay, D.E. Brenneman, PACAP functions as a
[35]
[36]
[37]
[38]
[39] [40]
[41]
neurotrophic factor, Ann. NY Acad. Sci. 739 (1994) 228– 243. D. Uchida, A. Arimura, A. Somogyvari-Vigh, S. Shioda, W.A. Banks, Prevention of ischemia-induced death of hippocampal neurons by pituitary adenylate cyclase activating polypeptide, Brain Res. 736 (1996) 280–286. W.A. Banks, A.J. Kastin, A. Arimura, Effect of spinal cord injury on the permeability of the blood–brain and blood– spinal cord barriers to the neurotrophin PACAP, Exp. Neurol. 151 (1998) 116–123. F.L. Strand, S.J. Lee, T.S. Lee, L.A. Zuccarelli, F.J. Antonavitch, J. Kune, K.A. Williams, ACTH peptides modulate nerve development and regeneration, Rev. Neurosci. 4 (1993) 321–363. T. Shimura, S. Tabata, S. Hayashi, Brain transfer of a new neuromodulating ACTH analog, ebiratide, in rats, Peptides 12 (1991) 509–512. W.A. Banks, A.J. Kastin, Permeability of the blood-brain barrier to melanocortins, Peptides 16 (1995) 1157–1161. A.J. Kastin, R.D. Olson, E. Fritschka, D.H. Coy, Neuropeptides and the blood–brain barrier, in: J. Cervos-Navarro, E. Fritschka (Eds.), Cerebral Microcirculation and Metabolism, Raven Press, New York, 1981, pp. 139–145. W.A. Banks, A.J. Kastin, Passage of peptides across the blood-brain barrier: pathophysiological perspectives, Life Sci. 59 (1996) 1923–1943.
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Penetration of neurotrophins and cytokines across the blood–brain / blood–spinal cord barrier Weihong Pan*, Abba J. Kastin VA Medical Center and Tulane University School of Medicine, 1601 Perdido Street, New Orleans, LA 70146, USA
Abstract Now that peptides are no longer considered too large to cross the blood–brain barrier, attention has turned to the possibility that larger substances like polypeptides might also enter the central nervous system (CNS). This review summarizes evidence showing that many cytokines and neurotrophins not only enter the brain but also enter the spinal cord, sometimes faster than into the brain. 1999 Elsevier Science B.V. All rights reserved. Keywords: Neurotrophins; Cytokines; Blood–brain barrier; Spinal cord
Contents 1. Introduction ............................................................................................................................................................................ 2. Neurotrophins ......................................................................................................................................................................... 2.1. Nerve growth factor (NGF) and bNGF.............................................................................................................................. 2.2. Neurotrophin (NT)-5 ........................................................................................................................................................ 2.3. NT-3 ............................................................................................................................................................................... 2.4. Brain-derived neurotrophic factor (BDNF)......................................................................................................................... 3. Cytokines ............................................................................................................................................................................... 3.1. Tumor necrosis factor-a (TNF)......................................................................................................................................... 3.1.1. TNF entry into the brain ......................................................................................................................................... 3.1.2. TNF entry into the spinal cord in EAE..................................................................................................................... 3.1.3. TNF and ebiratide entry into the transected spinal cord ............................................................................................. 3.1.4. Rationale of upregulation of the saturable transport system for TNF .......................................................................... 3.2. Interferon (IFN)a and IFNg.............................................................................................................................................. 3.3. Interleukin (IL)-1a and IL-1b ........................................................................................................................................... 3.3.1. IL-1a entry into the spinal cord............................................................................................................................... 3.3.2. Circadian variation of IL-1a entry ........................................................................................................................... 3.4. IL-2 ................................................................................................................................................................................ 3.5. IL-6 ................................................................................................................................................................................ 3.6. Macrophage inflammatory protein (MIP)-1a and MIP-1b ................................................................................................... 3.7. Granulocyte-macrophage colony-stimulating factor (GM-CSF) entry into the spinal cord ...................................................... 3.8. Fibroblast growth factors (FGF)........................................................................................................................................ 4. Comparison with neurotrophic peptides .................................................................................................................................... 4.1. Pituitary adenylate cyclase-activating polypeptide (PACAP) ............................................................................................... *Corresponding author. Tel.: 1 1-504-5895928; fax: 1 1-504-5228559; e-mail: [email protected] 0169-409X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 98 )00086-6
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4.2. Ebiratide ......................................................................................................................................................................... 5. Conclusions ............................................................................................................................................................................ Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................
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1. Introduction
2. Neurotrophins
The blood–brain / blood–spinal cord barrier (BBB) serves as a dynamic interface between the periphery and CNS for the neurotrophins and cytokines, substances generally fitting the definition of polypeptides [1]. BBB research with these substances is relatively new. Just as our initial work showing that peptides can cross the BBB was met with skepticism, even among a few prominent researchers in the field, so there are researchers in the newer areas of neurotrophins and cytokines who are skeptical about these larger substances entering the CNS from the periphery. This review will summarize the overwhelming evidence now existing for the penetration of neurotrophins and cytokines into the brain and spinal cord. It will be restricted to studies of the endogenous forms of the neurotrophins and cytokines, not discussing studies involving synthetic chemically modified carriers for which neither mechanisms of transport nor toxicity has been determined. The review will be organized according to the individual endogenous substance. Most of the studies of entry into the CNS involve determination of the unidirectional influx rate by multiple-time regression analysis after i.v. bolus injection of femtomole quantities of the labeled neurotrophin (or cytokine). The details of this method, with emphasis on cytokines, as well as for exit from the brain are provided elsewhere [2–4]. The stability and distribution in brain parenchyma are tested for each compound with standard methods. By use of cerebral cortex, the possibility of leakage through circumventricular organs (CVOs) to bypass the BBB is excluded. The permeability of the BBB shows regional variation, as measured by radiolabeled albumin and sucrose. In general, the penetration of most cytokines and neurotrophic proteins across the BBB is much higher than what would be expected for simple diffusion.
Most members of the mammalian neurotrophin family cross the BBB through saturable transport systems to arrive intact in the CNS [5]. The radioactively labeled neurotrophins are relatively stable in blood and CNS and have variable permeability across the BBB. The rates of transport differ among the neurotrophins tested and among the regions examined. The initial volume of distribution in brain, which usually reflects the association with cerebral endothelial cells, also varies among neurotrophins and among regions. The studies rely on the use of 125 I for detection. It has been demonstrated by more than one group that neurotrophins labeled with 125 I remain biologically active [6,7]. In a study involving transport across the BBB of a peptide, which is much smaller than a neurotrophin and hence might be more susceptible to change by addition of an iodine, we found no difference in rate of entry or saturability between the tritiated and iodinated forms [8].
2.1. Nerve growth factor ( NGF) and b NGF The entry of the 7s NGF pentomer into the brain is rapid [5]. High-performance liquid chromatography (HPLC) shows that most of the injected [ 125 I]NGF remains intact in brain and serum 20 min after i.v. injection. Capillary depletion studies shows that iv injected NGF reaches the parenchyma of the brain. The rate of influx of NGF into the brain and the entire spinal cord is about twice as fast as for bNGF [5]. That the rate of entry into the spinal cord of the considerably larger NGF is much faster than that of its smaller bNGF subunit represents an unexpected finding with therapeutic implications. There are no significant differences in lipophilicity, so that neither molecular weight nor lipid solubility, key factors in simple diffusion, could explain this finding. Entry into spinal cord for both NGF and bNGF is more
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than twice as fast as into the brain. Entry of NGF is not inhibited by excess NGF when given i.v., but is saturable when perfused in buffer free of blood elements, including possible binding proteins.
2.2. Neurotrophin ( NT)-5 For NT5 the rate of entry into the brain and spinal cord is much slower than for NGF and slightly slower than for bNGF [5]. As with NGF and bNGF, the influx rate into the spinal cord is more than twice as fast as into the brain. The entry rate into the cervical region of the spinal cord is higher than for the lumbar or thoracic regions, in that order.
2.3. NT-3 For NT3 the entry rate is slower than that for NGF, bNGF, or NT5 [5]. Again, the entry is more than twice as fast as into brain and faster in the cervical than in the lumbar or thoracic areas. Entry is saturable in the brain but not in the spinal cord at the dose injected i.v.
2.4. Brain-derived neurotrophic factor ( BDNF) In contrast to a report finding no transport of unconjugated BDNF into brain [9], Poduslo and Curran [10] showed that BDNF enters brain at a rate comparable to the rate they found earlier for insulin and somewhat faster than for NGF, NT-3, and CNTF. Insulin enters the CNS relatively fast, saturably, and in a physiologically meaningful way [11–13]. Saturable transport of BDNF into the brain and spinal cord was found recently [14]. Influx is much greater in the cervical than in the thoracic or lumbar regions of the spinal cord, raising the possibility of differential entry not only by different neurotrophins but also by region of the cord. SDS–polyacrylamide gel electrophoresis (PAGE) shows the entering protein is intact, as had been found by Poduslo and Curran 60 min after uptake [10]. Capillary depletion studies confirm that most of the BDNF reaches the brain parenchyma. Efflux of BDNF from brain to blood occurs at the rate of the usual reabsorption of cerebrospinal fluid (CSF) and is not saturable. We found evidence in blood not only for protein
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binding but also for self-aggregation [14]. This might explain another unusual finding with BDNF, the paradoxical increase in the entry of [ 125 I]BDNF into the brain with co-injection of a small amount of unlabeled BDNF. It is possible that the excess unlabeled BDNF displaces the iodinated material from binding proteins or dissociated aggregates, among other explanations.
3. Cytokines
3.1. Tumor necrosis factor-a ( TNF) 3.1.1. TNF entry into the brain TNF enters the brain and CSF by a saturable system not shared with other cytokines [15]. Entry in intact form into the CSF, as shown by HPLC, confirms the results of capillary depletion studies in which it has been found not to be sequestered by capillaries. There is an age-related difference in permeability of the BBB to TNF. In neonatal rats, the entry rate of TNF is more than three times as fast as in adult mice. This is probably not explained by species differences since mice would be expected to transport the murine TNF used in the studies faster than rats [15]. In normal animals, there are reports that TNF may increase, decrease or cause no change in the BBB permeability [16]. This implies that TNF could affect its own transport system. 3.1.2. TNF entry into the spinal cord in EAE In a system with known disruption of the BBB— acute experimental autoimmune encephalomyelitis (EAE)—we showed that the transport of TNF into the spinal cord of these mice is greater than can be accounted for by disruption of the BBB measured with albumin and sucrose [17]. Although TNF circulates as a trimer similar in size to albumin, it penetrates much faster than albumin at a rate comparable to that of sucrose, a molecule more than 50 times smaller in size. The complete self-inhibition of the increased entry suggests an enhanced saturable transport system for TNF in EAE. The general disruption of the BBB in the acutely ill EAE mice, as measured by the ratio of TNF in
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CNS over blood at 10 min after i.v. injection, is greater in the thoracic and lumbar regions of the spinal cord than in the cervical region or the brain [17]. TNF does not further damage the BBB since the entry of albumin is not changed additionally. The saturability of TNF entry in all three regions of the spinal cord suggests an upregulation of the transport system.
3.1.3. TNF and ebiratide entry into the transected spinal cord During the acute phase of spinal cord injury by transection in the lumbar region, permeability to TNF is increased in different regions proximal to the lesion [18]. The increase of TNF penetration is greater than can be explained by simple disruption of the BBB and extends beyond the area injured. Specifically, increased entry of TNF occurs in regions (cervical and thoracic) and at times (24 h in the proximal lumbar region) unaccompanied by disruption of the BBB as measured by albumin and sucrose. In addition, the increased transport of TNF from blood to spinal cord proximal to the lumbar lesion is saturable. As would be expected by the selectivity of the saturation process, the excess TNF did not affect the entry of albumin. There is regional distribution of the enhanced permeability (expressed as the ratio of TNF / albumin) 2 h after spinal cord injury, and the correlating inhibition by excess unlabeled TNF. 3.1.4. Rationale of upregulation of the saturable transport system for TNF TNF is one of the few cytokines for which attention has been directed to the dual effects that may occur in many disease states [16]. Increased production of TNF and / or increased demand for it from the periphery may be essential in the early response of the CNS to insults, although excess TNF has been related to pathogenesis. It also is possible that TNF available in the early stages of disease can serve as a negative feedback to abolish the later overproduction that causes neuronal death. We found dual effects of TNF after exogenous administration in EAE. Low doses seem to have a positive modulatory effect, whereas higher doses aggravate the severity of EAE. Therefore, upregulation of the
transport system on the BBB may have a role in confining CNS disorders.
3.2. Interferon ( IFN)a and IFNg Intact IFNs are found in the brain up to 1 h after i.v. injection. The spinal cord has greater permeability to the IFNs than does the brain [19]. For each region of the spinal cord, the permeability to IFNa is greater than that for IFNg. In contrast to the saturable penetration of TNF into all regions of the brain and spinal cord, passage of IFNg is saturable only in the brain and cervical region of the spinal cord but not in the thoracic or lumbosacral area, and that of IFNa is not saturable in any region at the dose tested. This emphasizes the necessity of examining all regions of the spinal cord when studying the penetration of the BBB by different compounds and testing saturability, even when the entry rate is reasonably high.
3.3. Interleukin ( IL)-1a and IL-1b IL-1a and IL-1b enter the brain by a shared, saturable transport system [20]. There is no saturable egress of these cytokines from the brain. Intact human IL-1a has a faster rate of entry into brain tissue of the mouse than of the rat. In the mouse, the entry rate is faster for murine IL-1a than for human IL-1a, and is faster for murine IL-1a than for murine IL-1b [20,21]. As with all such studies, the inclusion of albumin eliminates non-specific leakage during the period of study [20]. Specific study of larger doses (2 mg / mouse) of IL-1a, IL-1b, and also IL-2 for up to 2 h after i.v. injection shows no increased permeability of the BBB [22]. An antibody directed toward the portion of the interleukin molecule that binds to the murine T lymphocyte receptor severely blocks entry of IL-1a into the brain and inhibits its clearance from blood [20]. In contrast, the antibody directed towards a nonbinding area of IL-1a does not inhibit the transport. Two different antibodies, directed at the sites on the murine T lymphocyte receptor that either bind or do not bind IL-1 show partial inhibition of the entry of IL-1a into the brain. The soluble murine receptor to IL-1 only partially
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blocks the entry of IL-1a into brain [23] and is not transported across the BBB. This suggests that the BBB transporter for IL-1 can strip IL-1 from its soluble receptor. In contrast, the soluble p75 human receptor to TNF totally blocks the entry of both human and murine TNF into brain. The endogenous IL-1 receptor antagonist is itself saturably transported across the BBB to enter the brain and CSF from the blood in intact form [24]. It shares the transport system for IL-1a and IL-1b, each of the three cytokines inhibiting each other’s transport. For the capillary depletion studies, showing penetration into brain parenchyma, portions of cortex devoid of CVOs were used. Although CVOs can concentrate IL-1a from the blood to a greater extent than cortex, this route cannot account for more than 5% of the total brain uptake of the cytokine [25]. IL-1a injected i.v. is saturably transported into the posterior division of the septum of the brain, on the pathway between the hippocampal formation and the midbrain [26]. IL-1 receptor antagonist, IL-1b, and TNFa are not concentrated in the septum and none of these three cytokines inhibit transport of IL-1a there.
3.3.1. IL-1a entry into the spinal cord IL-1a is a proinflammatory cytokine that may be involved in the early phase of spinal cord injury. In a previous study we found that intact IL-1a enters all three regions of the spinal cord, without significant differences [27]. In addition, it was determined that the material entering the brain and diffusing caudally could only account for about 1% of the total radioactivity found in the spinal cord after i.v. administration. 3.3.2. Circadian variation of IL-1a entry Recently, we found a circadian variation in the entry of IL-1a in several tissues [28]. The variation in entry into the spinal cord is greater than that into brain, testis, or muscle. Entry of IL-1a into spinal cord is fastest at 08:00 h and slowest at 24:00 h, the difference between these times being 10-fold. By contrast, there is no circadian rhythm for the disappearance curves in blood or for the spinal entry of human TNF, which in contrast to murine TNF does not cross the BBB. Thus, IL-1a shows a circadian
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pattern of entry into the spinal cord that is dependent on its specific uptake rather than on non-specific factors that would also have affected hTNF.
3.4. IL-2 Unlike the other cytokines, IL-2 is not saturably transported into the brain under the conditions studied [29]. Nevertheless, it does enter the CNS in intact form 10 times faster than albumin. Its entry is not affected by antibodies to IL-2, compatible with a nonspecific method of entry. At the doses used, it does not damage the BBB, and damage to the BBB secondary to hypertension elicited by epinephrine is not enhanced or prolonged by IL-2 [22].
3.5. IL-6 Although IL-6 is saturable transported into the brain, most of this cytokine is degraded before reaching the parenchyma of the brain [30]. In contrast, most of the IL-6 entering the CSF remains intact even 30 min after peripheral injection.
3.6. Macrophage inflammatory protein ( MIP)-1a and MIP-1b MIPs behave differently from the other cytokines studied. They bind reversibly with the luminal surface of the endothelial cells comprising the BBB [31]. This binding is greatly reduced by perfusion of the vascular space of the brain. The association with the vascular space does not increase over time nor is it self-inhibitable.
3.7. Granulocyte-macrophage colony-stimulating factor ( GM-CSF) entry into the spinal cord GM-CSF is a peripherally produced compound that can exert CNS effects in addition to hematopoietic effects. These include neuroregenerative actions [32]. We found that GM-CSF enters the spinal cord from the blood significantly faster than does the albumin control [33]. There is no apparent gender difference and species variation (mice and rats). The intact nature of the GM-CSF arriving in the CNS was shown by acid precipitation and HPLC, an effect that capillary depletion with perfusion showed could
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not be accounted for by any GM-CSF residual in the vasculature.
3.8. Fibroblast growth factors ( FGF) We measured entry into brain and cervical, thoracic, and lumbar regions of the spinal cord for acidic (a)FGF and basic (b)FGF (McLay and Kastin, unpublished results). The pattern of BBB permeability is different from the other compounds discussed in this review. Although both aFGF and bFGF enter faster than the albumin control, this entry is not self-inhibited by a large dose of unlabeled cytokine which is usually sufficient to suppress a transport system, indicating that entry occurs by non-saturable diffusion. Moreover, unlike most of the other cytokines, there seems to be considerable amount of reversible binding to the capillary endothelial cells that comprise the BBB; this was shown by the inclusion of a perfusion step in the capillary depletion procedure to wash out the vasculature.
4. Comparison with neurotrophic peptides
4.1. Pituitary adenylate cyclase-activating polypeptide ( PACAP) PACAP has neurotrophic properties such as preventing gp120-induced death of hippocampal neurons and CA1 neurons after global ischemia [34,35]. We found that, unlike FGF, the influx of iodinated PACAP is saturable for the cervical and thoracic regions of the spinal cord [36]. The use of radioactively labeled albumin again showed that the spinal cord provides a significant barrier to serum proteins. This means that the tissue / serum ratios of albumin reflect the vascular space of the tissues, which vary with region. The cervical cord shows the highest degree of vascularity and the thoracic the lowest. These ratios for the vascular marker albumin are not affected by PACAP. In the same study [36], complete transection of the spinal cord results in long-lasting effects on the uptake of PACAP that cannot be explained by disruption of the vascular barrier [5]. For example, unlike BDNF no change in the entry of PACAP into the lumbar cord, the site of transection, is found until
a week after injury when increased influx occurs in the cervical, thoracic, and distal, but not proximal, lumbar regions. If the effects are only due to traumatic disruption of the vascular barrier, they would have been expected to occur much earlier. Thirty minutes after transection there is a marked decrease in the transport of PACAP into the cervical cord that lasts for at least 3 days, changes not seen with albumin. A slower entry of PACAP, but not albumin, is found in the thoracic cord by 24 h but not in the lumbar cord. One speculative explanation for the transient decrease in entry of PACAP might involve a circulating factor that would be released after spinal cord injury. If this is related to the inflammatory phase of the trauma, it might be expected to decrease with time while PACAP transport is allowed to increase. This circulatory factor, such as a cytokine similar to those tested in some of the other studies (e.g. IFNg or IL-1a), could act on the BBB generally, only in specific areas, or only on selective transport systems. The difficulty with such an explanation, however, involves our observations that spinal cord injury does not decrease entry of TNFa or affect the entry of ebiratide in either direction. The transport of PACAP into the CNS after transection of the spinal cord is different from that found with TNF and ebiratide, a melanocortin analog with potent neurotrophic properties [17,18,37].
4.2. Ebiratide Ebiratide is a six-amino acid analog of an ACTH / a-MSH fragment that has neurotrophic properties [37–39]. Ebiratide has a faster transport rate than cytokines and neurotrophins in normal mice, and this rapid entry is unaffected by transection of the spinal cord in the lumbar region.
5. Conclusions Like peptides [40,41], most neurotrophins and cytokines are transported across the BBB from blood to brain. This transport is usually saturable and not confined to endothelial cells of the cerebral vasculature. It reflects intact material entering the CNS. Many neurotrophins and cytokines enter the spinal
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cord, where they could exert therapeutic effects, at a faster rate than into brain. Such transport represents further support for the dynamic regulatory functions of the BBB.
[11]
[12]
Acknowledgements [13]
The authors thank William A. Banks, M.D. for long-term collaboration and kind help, and Melita B. Fasold for editorial assistance.
[14]
[15]
References [16] [1] A.J. Kastin, J.E. Zadina, R.D. Olson, W.A. Banks, The history of neuropeptide research: version 5.a, in: J.N. Crawley, S. McLean (Eds.), Neuropeptides: Basic and Clinical Advances, Ann. NY Acad. Sci., New York, 1996, pp. 1–18. [2] W.A. Banks, A.J. Kastin, Measurement of transport of cytokines across the blood–brain barrier, in: P.M. Conn, E.B. De Souza (Eds.), Neurobiology of Cytokines, Part A, Academic Press, San Diego, 1993, pp. 67–77. [3] W.A. Banks, A.J. Kastin, 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, 1989, pp. 652–660. [4] W.A. Banks, M.B. Fasold, A.J. Kastin, Measurement of efflux rates from brain to blood, in: G.B. Irvine, C.H. Williams (Eds.), Methods in Molecular Biology, Neuropeptide Protocols, vol. 73, Humana Press, Totowa, NJ, 1997, pp. 353–360. [5] W. Pan, W.A. Banks, A.J. Kastin, Permeability of the blood– brain / spinal cord barrier to neurotrophins, Brain Res. 788 (1998) 87–94. [6] P.S. DiStefano, B. Friedman, C. Radziejewski, C. Alexander, P. Boland, C.M. Schick, R.M. Linsay, S.J. Wjegand, The neurotrophins BDNF, NT-3 and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons, Neuron 8 (1992) 983–993. [7] Q. Yan, C. Matheson, J. Sun, M.J. Adeke, S.C. Weinstein, J.A. Miller, Distribution of intracerebral ventricularly administered neurotrophins in rat brain and its correlation with the Trk receptor expression, Exp. Neurol. 127 (1994) 23–36. [8] W.A. Banks, A.J. Kastin, Opposite direction of transport across the blood-brain barrier for Tyr-MIF-1 and MIF-1: comparison with morphine, Peptides 15 (1994) 23–29. [9] W.M. Pardidge, Y.-S. Kang, J.L. Buciak, Transport of human recombinant brain- derived neurotrophic factor (BDNF) through the rat blood-brain barrier in vivo using vectormediated peptide drug delivery, Pharm. Res. 11 (1994) 738–746. [10] J.F. Poduslo, G.L. Curran, Permeability at the blood-brain
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
297
and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF, Mol. Brain Res. 36 (1996) 280–286. W.A. Banks, J.B. Jaspan, A.J. Kastin, Selective, physiological transport of insulin across the blood-brain barrier: novel demonstration by species-specific radioimmunoassays, Peptides 18 (1997) 1257–1262. W.A. Banks, J.B. Jaspan, W. Huang, A.J. Kastin, Transport of insulin across the blood-brain barrier: saturability at euglycemic doses of insulin, Peptides 18 (1997) 1423–1429. W.A. Banks, J.B. Jaspan, A.J. Kastin, Effect of diabetes mellitus on the permeability of the blood-brain barrier to insulin, Peptides 18 (1997) 1577–1584. W. Pan, W.A. Banks, M.B. Fasold, J. Bluth, A.J. Kastin, Transport of BDNF across the blood–brain barrier, Neuropharmacol. (1998) in press. E.G. Gutierrez, W.A. Banks, A.J. Kastin, Murine tumor necrosis factor alpha is transported from blood to brain in the mouse, J. Neuroimmunol. 47 (1993) 169–176. W. Pan, J.E. Zadina, R.E. Harlan, J.T. Weber, W.A. Banks, A.J. Kastin, Tumor necrosis factor-a: a neuromodulator in the CNS, Neurosci. Biobehav. Rev. 21 (1997) 603–613. W. Pan, W.A. Banks, M.K. Kennedy, E.G. Gutierrez, A.J. Kastin, Differential permeability of the BBB in acute EAE: enhanced transport of TNF-a, Am. J. Physiol. 34 (1996) E636–E642. W. Pan, W.A. Banks, A.J. Kastin, Blood-brain barrier permeability to ebiratide and TNF in acute spinal cord injury, Exp. Neurol. 146 (1997) 367–373. W. Pan, W.A. Banks, A.J. Kastin, Permeability of the bloodbrain and blood-spinal cord barriers to interferons, J. Neuroimmunol. 76 (1997) 105–111. W.A. Banks, L. Ortiz, S.R. Plotkin, A.J. Kastin, Human interleukin (IL) 1a, murine IL-1a and murine IL-b are transported from blood to brain in the mouse by a shared saturable mechanism, J. Pharmacol. Exp. Ther. 259 (1991) 988–996. W.A. Banks, A.J. Kastin, D.A. Durham, Bidirectional transport of interleukin-1 alpha across the blood-brain barrier, Brain Res. Bull. 23 (1989) 433–437. W.A. Banks, A.J. Kastin, The interleukins-1a, -1b, and -2 do not acutely disrupt the murine blood-brain barrier, Int. J. Immunopharmacol. 14 (1992) 629–636. W.A. Banks, S.R. Plotkin, A.J. Kastin, Permeability of the blood-brain barrier to soluble cytokine receptors, Neuroimmunomodulation 2 (1995) 161–165. E.G. Gutierrez, W.A. Banks, A.J. Kastin, Blood-borne interleukin-1 receptor antagonist crosses the blood-brain barrier, J. Neuroimmunol. 55 (1994) 153–160. S.R. Plotkin, W.A. Banks, A.J. Kastin, Comparison of saturable transport and extracellular pathways in the passage of interleukin-1a across the blood-brain barrier, J. Neuroimmunol. 67 (1996) 41–47. L.M. Maness, W.A. Banks, J.E. Zadina, A.J. Kastin, Selective transport of blood-borne interleukin-1a into the posterior division of the septum of the mouse brain, Brain Res. 700 (1995) 83–88. W.A. Banks, A.J. Kastin, C.A. Ehrensing, Blood-borne
298
[28]
[29]
[30]
[31]
[32]
[33]
[34]
W. Pan, A. J. Kastin / Advanced Drug Delivery Reviews 36 (1999) 291 – 298 interleukin-1a is transported across the endothelial bloodspinal cord barrier of mice, J. Physiol. 479(2) (1994) 257– 264. W.A. Banks, A.J. Kastin, C.A. Ehrensing, Diurnal uptake of circulating interleukin-1a by brain, spinal cord, testis, and muscle, Neuroimmunomodulations 5 (1998) 36–41. P.J. Waguespack, W.A. Banks, A.J. Kastin, Interleukin-2 does not cross the blood-brain barrier by a saturable transport system, Brain Res. Bull. 34 (1994) 103–109. W.A. Banks, A.J. Kastin, E.G. Gutierrez, Penetration of interleukin-6 across the murine blood-brain barrier, Neurosci. Lett. 179 (1994) 53–56. W.A. Banks, A.J. Kastin, Reversible association of the cytokines MIP-1a and MIP-1b with the endothelia of the blood-brain barrier, Neurosci. Lett. 205 (1996) 202–206. G. Guillemin, F.D. Boussin, R. Le Grand, J. Croitoru, H. Coffigny, D. Dormont, Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains, Glia 16 (1996) 71–80. R.N. McLay, M. Kimura, W.A. Banks, A.J. Kastin, Granulocyte-macrophage colony-stimulating factor crosses the blood-brain and blood-spinal cord barriers, Brain 120 (1997) 2083–2091. A. Arimura, A. Somogyvari-Vigh, C. Weill, R.C. Fiore, I. Tatsuno, V. Bay, D.E. Brenneman, PACAP functions as a
[35]
[36]
[37]
[38]
[39] [40]
[41]
neurotrophic factor, Ann. NY Acad. Sci. 739 (1994) 228– 243. D. Uchida, A. Arimura, A. Somogyvari-Vigh, S. Shioda, W.A. Banks, Prevention of ischemia-induced death of hippocampal neurons by pituitary adenylate cyclase activating polypeptide, Brain Res. 736 (1996) 280–286. W.A. Banks, A.J. Kastin, A. Arimura, Effect of spinal cord injury on the permeability of the blood–brain and blood– spinal cord barriers to the neurotrophin PACAP, Exp. Neurol. 151 (1998) 116–123. F.L. Strand, S.J. Lee, T.S. Lee, L.A. Zuccarelli, F.J. Antonavitch, J. Kune, K.A. Williams, ACTH peptides modulate nerve development and regeneration, Rev. Neurosci. 4 (1993) 321–363. T. Shimura, S. Tabata, S. Hayashi, Brain transfer of a new neuromodulating ACTH analog, ebiratide, in rats, Peptides 12 (1991) 509–512. W.A. Banks, A.J. Kastin, Permeability of the blood-brain barrier to melanocortins, Peptides 16 (1995) 1157–1161. A.J. Kastin, R.D. Olson, E. Fritschka, D.H. Coy, Neuropeptides and the blood–brain barrier, in: J. Cervos-Navarro, E. Fritschka (Eds.), Cerebral Microcirculation and Metabolism, Raven Press, New York, 1981, pp. 139–145. W.A. Banks, A.J. Kastin, Passage of peptides across the blood-brain barrier: pathophysiological perspectives, Life Sci. 59 (1996) 1923–1943.