Permeability of the blood–brain and blood–spinal cord barriers to interferons

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Abstract

Interferons (IFNs) are cytokines that produce effects in the CNS even though their production occurs mainly in the periphery. Direct passage of IFNs from blood to CNS could be an important route by which circulating IFNs exert their central effects. In this report, we characterize the pharmacokinetics of the passage of IFNs through the blood–brain and blood–spinal cord barriers in four separate regions: whole brain and the cervical, thoracic and lumbosacral segments of the spinal cord. We found that the spinal cord had greater permeability to IFNs than did the brain. For each corresponding region, the permeability to IFNα was higher than that to IFNγ. Capillary depletion after cardiac perfusion showed that most of the injected IFN was not entrapped by the vasculature but entered the parenchyma of the brain. HPLC showed that most of the IFNγ entered in intact form. The passage of radioactively labeled IFNγ into the brain and cervical spinal cord was saturated by a low dose of unlabeled IFNγ, while passage into the thoracic and lumbosacral spinal cord was not saturated. In contrast, for another cytokine, tumor necrosis factor α (TNFα), a saturable transport system was present in distal spinal cord as well as the brain. The results show that IFNs and TNFα can enter the CNS from the periphery but with regional differences.

Introduction

Cytokines can exert a variety of effects on the central nervous system (CNS). Peripheral administration of interferon γ (IFNγ) induces major histocompatibility complex (MHC) I antigen expression in both microglia and endothelial cells (Xu and Ling, 1994) and MHC II antigen expression in microglia, especially in the subcortical white matter and circumventricular region of newborn rats (Xu and Ling, 1995). It has not been determined whether IFNγ causes these central changes directly by passage across the blood–brain and blood–spinal cord barriers (BBB).

Tumor necrosis factor α (TNFα) is another proinflammatory cytokine with similar effects as IFNγ in increasing autoimmunity and enhancing inflammation (Pan et al., 1997b). In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), TNFα can modulate the course of the disorder and inhibitors of TNFα decrease autoimmune demyelination (Monastra et al., 1993; Selmaj et al., 1991). TNFα receptors are present in the brain (Kinouchi et al., 1991) where there is a saturable transport system for TNFα from blood (Gutierrez et al., 1993).

In contrast to the proinflammatory effects of IFNγ and TNFα, IFNα has immunosuppressive effects (Panitch, 1992). Peripherally injected IFNα can be found in cerebrospinal fluid (Habif et al., 1975) and the receptor for IFNα has been localized to microglial cells in the brain (Yamada and Yamanaka, 1995). The greatest number of binding sites (Bmax) have been found in the hypothalamus and the least in the spinal cord (Janicki, 1992). It is possible that the CNS side-effects of IFNα as an anti-tumor agent (Quesada et al., 1986) could be related to its passage into the CNS.

The availability of cytokines to the spinal cord, although not well studied, may be significant in several aspects. First, the permeability of the spinal cord could be greater than that of the brain for cytokines so that their entry into the distal spinal cord could be involved in the development of immune and inflammatory processes there (Daniel et al., 1985). Second, in disorders such as EAE, disruption of the barrier is more prominent and occurs earlier in the distal spinal cord (Daniel et al., 1981; Juhler et al., 1984). Third, neurotrophic effects of cytokines, such as stimulation of synthesis of nerve growth factor by TNFα (Chao et al., 1995), may contribute to spinal cord regeneration.

Using sensitive techniques, we compared the permeability of the BBB to these three cytokines in the brain and the three main regions of the spinal cord.

Section snippets

Radioactive labeling and purification

Recombinant murine IFNα and IFNγ (CHO produced and tested for endotoxin, GIBCO BRL, Gaithersburg) and murine TNFα (R&D Systems, Minneapolis) were labeled with 125I by the enzymobead method (Biorad, Richmond). The labeled products were separated on a column of Sephadex G-10 and eluted with chloride-free phosphate buffer solution (PBS). The specific activities of the I-IFNs and I-TNFα were about 80 Ci/g.

Human serum albumin (Alb) was labeled with 99mTc (Tc-Alb) with the kit from Medi+Physics

Tissue/serum ratios of IFNα, IFNγ, and TNFα

The correlations between the tissue/serum ratios and exposure time for each iodinated cytokine were statistically significant in all regions, except for I-IFNγ in the lumbosacral spinal cord (p<0.07), as shown in Table 1.

In each region examined, there was a variation in the permeation of the three cytokines across the BBB. The most permeable was IFNα and the least permeable was IFNγ.

For each cytokine, the cervical spinal cord was the most permeable whereas the brain was the least. The

Discussion

In these experiments we examined the permeability of the blood–brain barrier and blood–spinal cord barrier to IFNγ, IFNα and TNFα, the amount of penetration into brain parenchyma relative to an association with the vascular space, the stability of the transported cytokine and the saturability of the transport system.

There were regional differences in the permeabilities of the blood–spinal cord barriers to the three cytokines. The cervical and lumbosacral spinal cord had higher tissue/serum

Acknowledgements

We are grateful to Melita B. Fasold for help with graphics and editing and to Dr. Jeannine A. Majde for intellectual encouragement. Supported by ONR (N00014-92-J-1384), VA Rehab and VA Merit Review.

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