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Inhibition of Experimental Autoimmune Encephalomyelitis by a Tyrosine Kinase Inhibitor¹

Gabriela Constantin^2,*,, Carlo Laudanna^*,, Stefan Brocke and Eugene C. Butcher^*

^* Laboratory of Immunology and Vascular Biology, Department of Pathology, and Digestive Disease Center, Stanford University School of Medicine, Stanford, CA 95305, and Center of Molecular Biology in Medicine, Foothill Research Center, Veterans Administration Medical Center, Palo Alto, CA 94304; Istituto di Patologia Generale, Universitá degli Studi di Verona, Strada le Grazie, Verona, Italy; and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892

	Abstract

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Migration of lymphocytes from the blood into the brain is a critical event in the pathogenesis of experimental autoimmune encephalomyelitis. Lymphocyte adhesion to brain endothelium is the first step in lymphocyte entry into the central nervous system, leading subsequently to myelin damage and paralysis. In this paper we show that the tyrosine kinase inhibitor, tyrphostin AG490, prevents binding of freshly isolated mouse lymph node cells and of in vivo activated lymphocytes to endothelium of inflamed brain in Stamper-Woodruff adhesion assays. Moreover, AG490 inhibits adhesion of encephalitogenic T cell lines to purified ICAM-1 and VCAM-1, molecules implicated in T cell recruitment into the central nervous system. In contrast, 2-h treatment of T cell lines with high doses of tyrphostin AG490 have no effect on the viability, intracellular calcium elevation induced by Con A or TCR cross-linking, proliferation, or TNF production by Ag-stimulated T cell lines. Systemic administration of AG490 prevents the accumulation of leukocytes in the brain and the development of experimental autoimmune encephalomyelitis induced by proteolipid protein, peptide 139–151-specific T cell lines in SJL/J mice. Blood leukocytes isolated from mice treated with tyrphostin AG490 are less adhesive on purified very late Ag-4 ligands compared with adhesion of leukocytes from control animals. Our results suggest that inhibition of signaling pathways involved in lymphocyte adhesion may represent a novel therapeutic approach for demyelinating diseases.

	Introduction

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Experimental autoimmune encephalomyelitis (EAE)³ is an inflammatory autoimmune disease mediated by CD4⁺ T lymphocytes reactive to brain Ags. The development of clinical and pathological signs of central nervous system (CNS) dysfunction in EAE is known to correlate with leukocyte infiltration of the brain (1). The ability of resting lymphocytes and monocytes to breach the blood-brain barrier is relatively rare. Activated T lymphocytes, however, can cross the blood-brain barrier regardless of their Ag specificity, but only T cells that recognize CNS Ag persist and can recruit other inflammatory cells (2). To migrate across the endothelium into the central nervous system, lymphocytes must first become attached firmly to the brain endothelium. This appears to occur after leukocyte activation by factors such as cytokines, and the subsequent binding of activation-dependent adhesion receptors on leukocytes with those on endothelial cells (1). These receptors and their counter-receptors are generally members of the integrin and Ig superfamilies. Several adhesion receptor-ligand pairs have been shown to be involved in T cell interaction with endothelium and extravasation into the CNS; these include ₄ß₁-VCAM-1 and _Lß₂-ICAM-1 (3, 4, 5).

Although the extracellular molecular interactions involving adhesion receptors have been well characterized, the intracellular signaling pathways controlling integrin-mediated adhesive events are less well elucidated (6). Protein tyrosine kinase (PTK) activity has been associated with integrin function during cellular adhesion (6, 7, 8, 9). Enhanced activity of tyrosine kinases has been associated with diseases such as cancer, psoriasis, and arteriosclerosis and with inflammatory responses such as septic shock (10). Tyrosine kinases and the signaling pathways they regulate have therefore been identified as targets for drug design, and recent progress in the development of PTK inhibitors demonstrates their therapeutic potential (10, 11).

Nothing is known about the signal transduction pathways involved in lymphocyte migration into the brain. In this paper we present data supporting the hypothesis that PTKs are involved in lymphocyte adhesion to brain endothelium. Moreover, when used in a murine model of EAE, PTK inhibitors are able to effectively prevent the accumulation of lymphocytes in the CNS and the development of EAE.

	Materials and Methods

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In vitro binding assays on inflamed brain sections

Normal lymphocytes were freshly isolated from peripheral lymph nodes, while in vivo activated lymphocytes were obtained from draining lymph nodes from SJL mice immunized in the footpads with CFA 10 days earlier (5). Lymphocytes were treated with 100 µM genistein (LC Services, Woburn, MA) for 1 h or with 150 µM tyrphostin (LC Services) for 2 h in RPMI supplemented with 1 mM sodium pyruvate, 1 mM glutamine, 5 x 10^-5 M 2-ME, and 10% BCS (HyClone, UT) containing 1% iron. Cells were pelleted and resuspended in binding assay medium represented by DMEM without sodium bicarbonate, containing 10 mM HEPES and 5% BCS (HyClone, UT), pH 7.2, at a concentration of 5 x 10⁶/100 µl. Binding assays were performed for 30 min at 25°C on freshly cut, unfixed, serial frozen sections from SJL mice with clinical EAE (5). The slides, containing two serial sections each (one for nontreated cells and one for cells treated with PTK inhibitor), were gyrated on a platform at 60 rpm. Binding of control and treated cells was quantitated on the same vessels in serial brain sections. Fifteen to twenty inflamed vessels were counted per slide, and 10–15 slides were used for each experimental condition. We evaluated only the vessels that had at least five adherent cells on control sections, as previously described (5). Analyses were performed in a single blind fashion.

In vitro binding assay on purified proteins

Slides were coated overnight at 4°C with purified mouse ICAM-1, VCAM-1, and fibronectin and were blocked for 10 min at 25°C with FBS. T cell lines were Ag stimulated for 3 days, and then a Ficoll gradient was performed. Viable T cell lines were treated or not treated with 100 µM tyrphostin AG490 or tyrphostin AG1478 for 2 h and then were added at 60 x 10³/25 µl/well and incubated for 20 min at 37°C. Binding assay medium was represented by DMEM without sodium bicarbonate, containing 10 mM HEPES and 5% BCS (HyClone, UT), pH 7.2. Then the slides were washed in PBS and fixed. Computer-assisted enumeration was performed (12). In other experiments, 0.3-ml blood samples were obtained from animals treated with AG490 or DMSO/DMEM 4–6 h after the last drug administration. Blood was collected in an Eppendorf tube containing PBS/heparin and 1% dextran to precipitate RBC; after 25 min, supernatants containing leukocytes were collected and centrifuged for 5 min at 400 x g, and plasma and leukocytes were separated (13). Cells were added at 100 x 10³/25 µl/well and incubated for 20 min at 37°C. Slides were washed and fixed, and then computer-assisted enumeration was performed. Background binding was minimal in all experiments and was subtracted.

Measurement of intracellular Ca²⁺ release

For TCR cross-linking 5 x 10⁶ T cell lines were Ag stimulated for 3 days, treated for 2 h with 200 µM tyrphostin AG490 at 37°C, pelleted, and then treated for 1 h in ice in PBS with a hamster anti-mouse TCR (PharMingen, San Diego, CA) at 20 µg/ml. After two washings, cells were resuspended in HBSS. After 30–60 s of stirring at 37°C in the cuvette of a Perkin-Elmer LS-50 luminescence spectrometer (Perkin-Elmer, Norwalk, CT), goat anti-hamster F(ab')₂ was added at a final concentration of 25 µg/ml.

Proliferation assay

PLP_139–151-specific T cell lines (G1 and G2) were Ag stimulated for 3 days and then treated for 2 h with various concentrations of tyrphostin AG490. Cells were pelleted and seeded at 5 x 10⁵/well in flat-bottom tissue culture 96-well plates for 16 h in fresh medium containing Ag. [³H]thymidine (1 µCi) was added in each well 8 h before the cultures were terminated. Cells were then collected, and samples were counted in a liquid scintillation counter.

TNF- measurement

PLP_139–151-specific T cell lines (G1 and G2) were Ag stimulated for 3 days and then treated for 2 h with various concentrations of tyrphostin AG490. Cells were pelleted and resuspended in fresh medium containing Ag at a concentration of 5 x 10⁶ cells/ml for 24 h. Supernatants were harvested, and TNF- was measured using an immunoassay kit for mouse TNF- (BioSource International, Camarillo, CA).

Transfer and evaluation of EAE

SJL/J females, 6–8 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). The production, characterization, and maintenance of G1 and G2 PLP_139–151-specific T cell lines were previously described (14, 15). In brief, SJL/J mice were immunized with 250 µg of peptide PLP_139–151 in CFA. Ten days later, draining lymph nodes were removed and stimulated with 30 µg/ml peptide for 4 days. T cell lines were obtained by stimulation of these cultures every 14 days with irradiated syngenic spleen cells at a ratio of 1/10 T cell vs irradiated spleen cells plus 30 µg/ml peptide for 3 days. Ag-stimulated T cell lines (5 x 10⁶) were injected into SJL mice. Mice were checked daily and scored for EAE according to the following scale (14): 0, no disease; 1, tail weakness; 2, paraparesis; 3, paraplegia; 4, paraplegia with forelimb weakness or paralysis; and 5, moribund or dead animals. Tyrphostin AG490 used for in vivo experiments was purchased in bulk (lot DU-101) from LC Services.

Statistical evaluation

Comparisons between groups were made using Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The association of tyrosine phosphorylation events with integrin-mediated adhesive interactions suggested that PTK inhibitors might modulate lymphocyte adhesion to brain endothelium. We performed in vitro binding assays on serial unfixed frozen sections of EAE brains, using a modification (5, 16) of the original technique described by Stamper and Woodruff (17). Freshly isolated resting lymph node cells and in vivo activated lymphocytes bound selectively to vessels from inflamed brain (3), but not to any vessels in sections of control brains. Various PTK inhibitors were examined for inhibitory activity in the in vitro assay (Table I). We first tested genistein, a widely used competitive inhibitor of the ATP binding site (10); genistein inhibited 43 and 50% of the attachment of freshly isolated or in vivo activated lymphocytes to inflamed brain endothelium. We then analyzed a panel of more selective low m.w. substrate-mimicking compounds, the tyrphostins. Many of these inhibitors have distinctive inhibitory activities with selectivity among tyrosine kinases, and they have hydrophobic characteristics that allow them to easily diffuse through the cell membrane (10, 18). Among the tyrphostins tested (Table I), AG490, previously described as a selective inhibitor of JAK-2 kinase (19), was the most effective inhibitor of lymphocyte binding to inflamed vessels, blocking almost 90% of lymphocyte adhesion (Fig. 1, A and B). Other tyrphostins, such as AG879, a selective inhibitor of pp140^c-trk (20); AG1478, a highly specific inhibitor of epidermal growth factor receptor (21); AG126, an inhibitor of tyrosine phosphorylation of p42 MAP-kinase (22); and the broad range tyrphostins, AG82 and AG18, had much less or no effect (Table I and Fig. 1, C and D). None of the PTK inhibitors altered cell viability. The inhibitory effect of AG490 was dose dependent (Table I), suggesting that AG490 has a specific effect on lymphocyte adhesion.

View larger version (131K): [in this window] [in a new window]   FIGURE 1. In vitro binding of lymphocytes to inflamed vessels in EAE mouse brain. Nontreated lymph node lymphocytes bound to inflamed vessels, to the vascular lumen (A), as previously described (3). In the same experiment, lymphocytes pretreated with 150 µM tyrphostin AG490 were incubated on serial sections of inflamed brain. AG490 inhibited lymphocyte binding to the inflamed venule (B). In serial inflamed brain sections, no significant difference was observed between binding of nontreated cells (C) and that of tyrphostin 1478-treated lymphocytes (D). See also Table I.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Effects of tyrphostin AG490 on calcium release, proliferation, and TNF- production. A, Increase in cytosolic free calcium in PLP139–151-specific T cell lines after treatment with Con A or TCR cross-linking. Assays were conducted as described previously (26). Fura-2/AM-loaded T cell lines stimulated with 100 µM Con A (arrow) showed no difference in the increase in intracellular calcium after treatment with 200 µM tyrphostin AG490 for 2 h. AG490 also had no effect on Ca2+ mobilization by TCR cross-linking (arrow). B, The effect of AG 490 on Ag-induced proliferation was determined as described in Materials and Methods. C, TNF- release was measured in supernatants from Ag-stimulated T cell lines treated with AG490 as described in Materials and Methods.

The ability of T cell lines specific for myelin Ags to transfer EAE was positively correlated with the amount of proinflammatory cytokines such as TNF- and lymphotoxin- (24). We found that TNF- production by our Ag-stimulated G1 and G2 PLP_139–151-specific T cell lines was unaffected by AG490 treatment (Fig. 2C).

Finally, the viability of G1 and G2 T cell lines specific for PLP_139–151 was assessed using the nuclear fluorescent dye YOPRO-1 (25). After 3 days of Ag stimulation, T cells were washed and incubated further with or without 200 µM AG490 for 2 h. After washing and culture for an additional 3 or 6 days, AG490-treated cultures contained 45 and 50%, respectively, more viable T cells, as assessed by flow cytometry of CD3-positive cells (data not shown). This observation is consistent with the reported ability of AG490 to increase the viability of germinal center B lymphocytes in vitro (26). Together, these results excluded nonspecific toxicity.

As tyrphostin AG490 blocked lymphocyte adhesive interactions implicated in EAE pathogenesis, we next determined its effects on disease (Table II). Encephalitogenic T cell lines specific for PLP_139–151 were treated with tyrphostin AG490 and injected i.v. into SJL mice. Because tyrphostins are reversible inhibitors, mice also received 50 µg of AG490 i.v. at the time of T cell transfer and 1.5 mg/day i.d. (Table II, Expt. 1). AG490 completely prevented the development of paralysis in all treated animals. Immunohistochemically, brains from untreated animals revealed extensive leukocyte infiltration (Fig. 3A), whereas infiltration was not detected in brains from animals treated with AG490 (Fig. 3B). In other experiments, untreated T cell lines were injected into mice treated daily with AG490 i.d. or/and i.p. Under these experimental conditions, the protective effect of tyrphostin was dose dependent. When 1.5 mg of tyrphostin was administered i.d. daily beginning on day 0, all mice developed disease but the day of onset was delayed (p < 0.001), and disease severity was reduced (p < 0.005; Table II, Expt. 2). When mice received 1.5 mg drug i.d. and 2 mg i.p. (3.5 mg daily in total), paralysis was averted in 50% of treated animals; in those that developed the disease, paralysis was delayed, and severity was dramatically reduced (p < 0.001; Table II, Expt. 3). When mice received AG490-treated T cell lines with no systemic tyrphostin administration, the onset of symptoms was delayed for up to 2 days, but no significant difference in clinical score was observed (not shown). This suggests that encephalitogenic T cell lines recuperated and that AG490 has no irreversible effect (not shown). As reported previously (19), administration of AG490 did not cause any visible toxicity itself, as assessed by the appearance of the treated animals, the hematological findings, or the macroscopic pathological analysis (not shown).

View larger version (75K): [in this window] [in a new window]   FIGURE 3. In vivo administration of tyrphostin AG490 prevents accumulation of leukocytes in the CNS. Brains were removed from several diseased animals and from AG490-treated animals with no clinical signs 20 days after transfer. The micrographs show hematoxylin-eosin-stained sections of comparable regions of the brain stem from animals from Expt. 1 (Table II). There was extensive inflammatory infiltration in brains from diseased animals (A; arrows), whereas no infiltrates could be detected in parenchyma of CNS from mice treated with AG490 (B).

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Adhesion of blood leukocytes can be inhibited by AG490 treatment. Leukocytes were obtained from blood samples collected from animals treated for 9–12 days with tyrphostin AG490 or DMEM/DMSO (Control) 4–6 h after the last drug administration. Treatment was performed as described in Table II, Expt. 3. Binding assays were assessed on 18-well glass slides coated with purified mouse VCAM-1 and fibronectin. Values are the mean counts of bound cells per 0.2 mm2 in four experiments (three wells per condition) ± SD.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Effect of AG490 treatment started at the beginning of clinical symptoms. Mice were injected with 10 x 106 G2 line cells to induce transfer EAE and were randomly distributed into two groups of six mice each. Nine days later, when clinical disease was apparent, one group was treated daily with 1.5 mg AG490 i.d. and 2 mg i.p. (3.5 mg daily in total/mouse). Mice from the control group received DMSO/DMEM. Animals received the treatment for 20 days post-transfer. Mice were evaluated for clinical signs of EAE daily as described in Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We then asked whether this inhibition might have clinical relevance in an EAE model. AG490 was able to prevent the transfer of EAE by encephalitogenic T cell lines, and this inhibitory effect of AG490 on the development of EAE was associated with a decreased adhesion of blood leukocytes to VCAM-1 and fibronectin, both ligands of the integrin VLA-4. In contrast, in vitro the same AG490 treatment applied to T cells before injection into the mice had no effect on the viability, intracellular calcium elevation induced by Con A or TCR cross-linking, proliferation, or TNF production by Ag-stimulated T cell lines. Thus, the mechanism of the prevention of EAE in AG490-treated animals may be explained by a reduction in leukocyte adhesiveness and therefore by a diminished entry of inflammatory cells into the CNS. We also observed a beneficial and significant effect on clinical scores even when AG490 was administered after the onset of disease. These findings support the idea that specific PTK inhibitors capable of blocking lymphocyte adhesion may represent effective agents for modulating EAE in the mouse model. Recent studies have confirmed that AG490 is also effective in an active EAE model, suggesting that inhibitors of PTKs may represent effective drugs in treating CNS inflammation (27).

Tyrphostins have been previously used in vivo. For example, tyrphostin AG126 blocks LPS-induced septic shock, and tyrphostin AG490 recently has been shown to inhibit the growth of human pre-B acute lymphoblastic leukemia in an SCID mouse model (19, 22). AG490 has not been studied previously in the context of leukocyte adhesion and autoimmune diseases. The aim of our work was a conceptual and clinically oriented study; further pharmakokinetic studies need to be performed in the future. Interestingly, AG490 is a potent and reportedly selective inhibitor of JAK-2 kinase, having no effect on the kinase activity of other PTKs, such as Src, Lck, Lyn, Btk, and Syk (19). Moreover, JAK-2 is a PTK involved in signaling by cytokines such as IL-3, granulocyte-macrophage CSF, and TNF, that are themselves able to activate integrin function and promote cell adhesion (28, 29, 30). Thus, JAK-2-dependent pathways may be involved in the adhesion of T cells to integrin ligands expressed on brain endothelium and may represent the AG490 target whose inhibition blocks EAE.

In conclusion, although we can never formally exclude the possibility that the in vivo effects of AG490 on disease can be mediated through inhibition of adhesion and additional mechanisms implicated in other aspects of the pathobiology of EAE, our results suggest that therapies designed to interfere with signal transduction mechanisms involved in integrin-dependent lymphocyte adhesion may be useful in treating autoimmune demyelinating diseases. Antibodies directed against ₄ and ß₂ integrins have been reported to modulate other autoimmune and inflammatory diseases, as well, suggesting that this novel approach may have broader application to immune system pathology.

	Footnotes

 
¹ This work was supported in part by grants from the National Institutes of Health, an award from the Department of Veteran Affairs, funds from Istituto Superiore di Sanitá, Proggetto Sclerosi Multipla (Rome, Italy), and fellowships from the National Multiple Sclerosis Society (New York, NY; to G.C.) and Fondazione Italiana Sclerosi Multipla (Genova, Italy; to G.C).

² Address correspondence and reprint requests to Dr. G. Constantin, Istituto di Patologia Generale, Universitá degli Studi di Verona, Strada le Grazie, Verona 37134, Italy. E-mail address:

³ Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; PTK, protein tyrosine kinase; BCS, bovine calf serum; PLP, proteolipid protein; VLA, very late antigen; i.d., intradermally.

Received for publication July 21, 1998. Accepted for publication September 22, 1998.

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