Permeability of the mouse blood–brain barrier to murine interleukin-2: predominance of a saturable efflux system

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Abstract

Interleukin (IL)-2, a T helper (TH)1 cell-derived glycoprotein with potent neuromodulatory effects, is implicated in the etiology and pathogenesis of various psychiatric and neurological disorders. Paralleling these findings, chronic IL-2 intravenous immunotherapy may induce similar psychopathological outcomes. The findings that acute or repeated injections of IL-2 induce motor and cognitive abnormalities in rodents are consistent with these clinical findings, and raise the possibility that IL-2 crosses the blood–brain barrier (BBB) to alter brain function. However, little is known about the ability of IL-2 to enter the brain or whether its effects vary with the chronicity of IL-2 treatment. Here, we found that radioactively labeled mouse IL-2 (I-IL-2) given intravenously entered the brain at a low rate (Ki=0.142 ± 0.044 μl/g-min) by a non-saturable process. Repeated injections of either IL-2 or vehicle altered the kinetics of entry without producing a net effect on IL-2 entry. When I-IL-2 was given by brain perfusion, the entry rate greatly increased over 10-fold to 2.2 ± 0.805 μl/g-min. This suggests a circulating factor is retarding the entry of IL-2 into the brain. A paradoxic increase in the rate of I-IL-2 entry into brain occurred when an excess of unlabeled IL-2 was included in the brain perfusate, suggesting a saturable CNS-to-blood efflux system. Intracerebroventricular injection of I-IL-2 with and without unlabeled IL-2 confirmed the presence of a saturable efflux system. We conclude that IL-2 entry into the brain is low because of the absence of a blood-to-brain transporter and further retarded by circulating factors and a CNS-to-blood efflux system. This is the first description of a saturable CNS-to-blood efflux system for a cytokine. We postulate that this efflux system may protect the brain from circulating IL-2.

Introduction

Interleukin-2 (IL-2) is a glycoprotein that is produced by activated TH1 cells. Among other things, it plays fundamental roles in regulating T and B cell proliferation and differentiation, and influences natural killer cell activity. IL-2 is also present in the central nervous system (CNS) and is a potent modulator of brain function. Centrally, IL-2 modulates dopamine release from mesencephalic and striatal slices (Alonso et al., 1993; Lapchak, 1992; Petitto et al., 1997), hippocampal acetylcholine and serotonin release (Hanisch et al., 1997; Pauli et al., 1998), NMDA-activated currents in the ventral tegmental area (Ye et al., 2001), as well as amygdalar and hypothalamic CRF activity (Raber et al., 1995). Effects of IL-2 on neurotransmitter release occur in a biphasic manner in that relatively low doses enhance and high doses suppress release. IL-2 also promotes the survival of cortical, striatal, and hippocampal neurons during neural development (Awatsuji et al., 1993; Sarder et al., 1993).

Peripheral administration of IL-2 results in marked alterations of central monoamine activity. For example, a single injection of IL-2 increases hypothalamic norepinephrine activity (Zalcman et al., 1994a), unit activity in the hypothalamus (Bartholomew and Hoffman, 1993), and dopamine turnover in the medial prefrontal cortex (Zalcman et al., 1994a). Moreover, unit activity in the hypothalamus (Bartholomew and Hoffman, 1993) and accumbal dopamine release are reduced following systemic IL-2 administration (Anisman et al., 1996; Song et al., 1999).

Peripherally administered IL-2 also induces marked neurobehavioral effects. A single injection of IL-2 potentiates novelty-induced exploratory activity and locomotion (Petitto et al., 1997; Zalcman et al., 1998; Zalcman, 2001). Behavioral and cognitive effects of IL-2 are influenced by a variety of factors, including the chronicity of IL-2 administration. For example, repeated peripheral injections of IL-2 induce marked alterations of exploratory activity, deficits in performance in the Morris Water Maze, and reduced rates of responding for intracranial self-stimulation in the medial forebrain bundle. These effects are not evident in animals receiving a single injection (Anisman et al., 1996; Hanisch et al., 1997; Lacosta et al., 1999; Lacosta et al., 2000; Zalcman, 2001). Zalcman (2002) recently demonstrated that whereas repeated injections of IL-2 induce a pronounced increase in stereotypical climbing behavior, single injections have no effect.

Paralleling the experimental evidence that IL-2 induces motor and cognitive changes in rodents, IL-2 is implicated in psychiatric outcomes, notably schizophrenic-like behavior and cognitive abnormalities. Increased levels of IL-2 and soluble IL-2 receptors have been detected in the cerebrospinal fluid (CSF) and serum of schizophrenic patients (Licinio et al., 1993; McCallister et al., 1995; Rapaport and Lohr, 1994), particularly those displaying motor abnormalities (Rapaport et al., 1997). The finding of increased levels of IL-2 in neuroleptic-free patients suggests that IL-2 plays a role in the disease process (Licinio et al., 1993). Furthermore, there is a positive correlation between increased CSF levels of IL-2 and the incidence of psychotic symptoms (McCallister et al., 1995). Paralleling these findings, schizophrenic-like behavior and cognitive abnormalities, among other psychiatric outcomes, have been observed in cancer and AIDS patients receiving chronic injections of IL-2 (Caraceni et al., 1992; Denicoff et al., 1987; Fent and Zbinden, 1987; West et al., 1987), see review in Meyers (1999).

Hence, IL-2 induces neurochemical and behavioral changes, and is implicated in the etiology and pathogenesis of various psychiatric disorders. Recent evidence indicates that peripheral administration of IL-2 induces marked increases in the incidence of stereotyped behavior, particularly in mice receiving repeated injections (Zalcman, 2002). Interestingly, profiles of IL-2-induced neurochemical and behavioral changes differ from those associated with proinflammatory cytokines (Zalcman et al., 1998). Hence, it is possible that the ability of IL-2 to enter the brain differs from other cytokines. Indeed, how IL-2 produces its central and behavioral effects is currently not understood. In general, peripherally administered cytokines can affect the brain by several mechanisms. These mechanisms include activation of afferent nerves, disruption of the blood–brain barrier (BBB), action at the circumventricular organs, and direct passage across the BBB (Alonso et al., 1993). For example, the ability of IL-1α to evoke deficits in memory after peripheral administration largely depend on its ability to cross the BBB (Banks et al., 2001). Whereas IL-1α, IL-1β, IL-6, and tumor necrosis factor-α are transported across the BBB by saturable systems (Banks et al., 1989, Banks et al., 1991, Banks et al., 1994; Gutierrez et al., 1993), preliminary evidence has found no saturable transport of human IL-2 across the mouse BBB (Waguespack et al., 1994).

Recently, a higher degree of species specificity for cytokines and their passage across the BBB has been noted than was originally thought. For example, human IL-1α crosses the mouse BBB but not the rat BBB (Plotkin et al., 2000). This raises the possibility that previous work with human IL-2 in the mouse may not reflect the fate of endogenous IL-2. Here, we determined whether murine IL-2 would cross the BBB of the mouse and whether effects vary with the chronicity of IL-2 administration.

Section snippets

Radioactive labeling of IL-2

Five micrograms of carrier-free, recombinant murine IL-2 (PeproTech, Rocky Hill, NJ) were labeled by the lactoperoxidase method. This was purified on a column of G-10 Sephadex by eluting 0.1 ml fractions with protein-free and chloride-free phosphate buffer solution. Human albumin was labeled with technetium with the use of stannous tartrate and purified on a column of G-10 Sephadex.

Measurement of blood-to-brain influx of intravenous IL-2

Male CD-1 mice (Charles River Labs, Wilmington, MA) weighing 20–30 g were anesthetized with an ip injection of 40%

Statistics

Means are reported with their n values and standard error of the mean (SEM), and compared by analysis of variance (ANOVA). Groups of more than two means were followed by Newman–Keuls post test and the p values were reported for relevant statistically significant differences. Regression lines were calculated by the least squares method with the Prism 3.0 program and are reported with their slopes, intercepts, the error terms (standard deviation of the mean) for the slopes, and intercepts, the

Measurement of blood-to-brain passage of intravenous IL-2

I-IL-2 entered the brain at a low rate with a Ki of 0.142 ± 0.044 μl/g-min and a Vi of 12.4 ± 0.7 μl/g (n=10, r=0.752, p<.05); Fig. 1. Coinjection of 1 μg/mouse unlabeled IL-2 did not have a significant effect on Ki (0.128 ± 0.37 μl/g-min) or Vi (11.7 ± 0.7 μl/g) and there was still a significant relation between brain/serum ratios and exposure time n=10, r=0.778, p<.01). The unlabeled material also did not alter clearance of I-IL-2 from the blood, which had a half-time disappearance of 11.4 min and a volume

Discussion

These studies examined the possibility that IL-2 can cross the BBB to induce its effects on the CNS. We found a low rate of passage that was not dependent on a saturable system. Chronic injection with IL-2 altered the kinetics of influx of IL-2, but did not result in any net increase in influx. Besides the absence of a saturable blood-to-brain transporter, we found two additional mechanisms that retard the accumulation by brain of blood-borne IL-2. First, brain perfusion studies found a much

Acknowledgements

Supported by VA Merit Review, R01 NS41863, R01 AA12743, and the National Alliance for Autism Research (NAAR).

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