Mediated efflux of IgG molecules from brain to blood across the blood–brain barrier

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Abstract

Dextrans and albumin exit brain from blood following intra-cerebral injection by a slow process of convection with a halftime of 10–12 h in the rat. The present studies show that immunoglobulin G (IgG) molecules rapidly efflux from brain to blood across the blood–brain barrier (BBB) following intracerebral injection. The IgG efflux is rapid with a halftime of 48 min in the rat. The efflux of [3H]mouse IgG2a from brain to blood is competitively inhibited by intracerebral injection of unlabeled mouse IgG molecules, but is not inhibited by intracerebral injection of comparable doses of unlabeled rat albumin. The IgG efflux system has characteristics of an Fc receptor, as the efflux from brain is competitively inhibited by Fc fragments but is not blocked by F(ab′)2 fragments. Precipitation of brain homogenate by trichloroacetic acid indicates there is no significant metabolism of the IgG molecules during the experimental time period. In conclusion, these studies provide evidence for a BBB Fc receptor that mediates the reverse transcytosis of IgG molecules in the direction of brain to blood.

Introduction

Plasma proteins such as immunoglobulin G (IgG) molecules generally do not cross the brain capillary endothelial wall, which forms the blood–brain barrier (BBB) in vivo, under normal conditions (Brightman et al., 1970). However, certain monoclonal antibodies (MAbs) in the circulation do cross the BBB by a process of receptor-mediated transcytosis (Pardridge et al., 1991). In this case, the MAb is directed against a specific receptor located on the luminal membrane of the brain capillary endothelium, and the MAb binds to exofacial epitopes on the BBB receptor. Anti-transferrin receptor (TfR) or anti-insulin receptor (IR) MAbs undergo receptor-mediated transcytosis through the BBB by this process (Bickel et al., 1994). In contrast to the anti-TfR or anti-IR MAbs, the mouse (m) IgG2a isotype control antibody is not measurably taken up by brain in vivo because the general IgG molecule does not have access to specialized transport systems within the BBB (Pardridge et al., 1991).

The OX26 MAb to the rat TfR undergoes receptor-mediated transcytosis through the BBB, and this antibody has been used as a vector for drug targeting to the brain. In a recent study, an epidermal growth factor (EGF) peptide radiopharmaceutical was conjugated to the OX26 MAb and the EGF–OX26 conjugate bound selectively to tumor cells in brain that over-express the receptor for EGF. These studies revealed a clear demarcation in the zone of uptake of the OX26–EGF conjugate in the brain tumor relative to normal brain (Kurihara and Pardridge, 1999). In order for such a high signal/noise ratio to be obtained in the imaging of brain tumors in vivo, it would be necessary for there to be significant exodus of the OX26 MAb from normal brain. While the selective uptake of the OX26 MAb in the blood to brain direction has been well characterized in past studies, the extent to which the OX26 MAb effluxes from brain to blood is at present unknown. The efflux of the OX26 MAb following direct intracerebral injection was examined in the present studies with the brain efflux index (BEI) method (Terasaki, 1998). The efflux from brain to blood of either [125I]OX26 MAb or [3H]mIgG2a, which is the isotype control, was examined and compared to the rate of efflux of Mr 70 000 [3H]dextran. These studies described the unexpected finding of selective transport of IgG molecules across the BBB from brain to blood by a process that is saturated by IgG molecules, but is not inhibited by rat albumin.

Section snippets

Reagents

[125I]NaI (17.4 Ci/mg), [3H]dextran (182 mCi/g) and N-succinimidyl[2,3-3H]propionate (101 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL, USA). The dextran had a molecular mass of 50 000 to 90 000 (mean Mr 70 000). mIgG2a (κ) UPC10 myeloma ascites was purchased from Organon Teknika (Durham, NC, USA). Human Fc fragments and mIgG F(ab′)2 fragments were purchased from the Cappel Division of ICN Pharmaceuticals (Aurora, OH, USA). Chloramine T, mouse IgG3,κ (FLOPC-21)

Results

Following the intracerebral injection of [3H]70 000 dextran, >99% of the injected radioactivity was retained in brain for the first 60 min and by 90 min, the level of retention of labeled dextran in rat brain was 93±3% (Fig. 1). In contrast, either [125I]OX26 MAb or [3H]mIgG2a isotype control antibody rapidly effluxed from brain with a monoexponential rate of decay (Fig. 1). The time at which 50% of the injected radioactivity had effluxed from brain was 48 min for either the [3H]mIgG2a or the [3

Discussion

These studies describe an unexpected rapid rate of efflux from brain to blood of IgG molecules (Fig. 1). The data are consistent with the hypothesis of Fc-receptor mediated antibody efflux. The efflux is completely saturated by either intact IgG molecules or by Fc fragments (Fig. 2, Fig. 3), but is not inhibited by high concentrations of either rat albumin (Fig. 2) or mouse F(ab′)2 fragments (Fig. 3). The efflux of radioactivity from brain is not due to the efflux of labeled metabolites, as

Acknowledgements

This work was supported by a grant from the US Department of Energy. Daniel Jeong skillfully prepared the manuscript.

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