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(The FASEB Journal. 2001;15:187-194.)
© 2001 FASEB

An altered peptide ligand inhibits the activities of matrix metalloproteinase-9 and phospholipase C, and inhibits T cell interactions with VCAM-1 induced in vivo by a myasthenogenic T cell epitope

ANAT FABER-ELMANN, VALENTIN GRABOVSKY, MOLLY DAYAN, MICHAEL SELA, RONEN ALON and EDNA MOZES1

Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

1Correspondence: E-mail: edna.mozes{at}weizmann.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195–212 and p259–271, which are sequences of the human acetylcholine receptor, resulted in MG-associated immune responses. A dual altered peptide ligand (APL) composed of the two APLs of the myasthenogenic peptides inhibited, in vitro and in vivo, those responses. This study was aimed at understanding the mechanism(s) underlying the in vivo inhibitory properties of the dual APL. To this end, we analyzed T cells of mice that were immunized with p259–271 for their adhesiveness toward vascular cell adhesion molecule 1, for the activity of their secreted matrix metalloproteinases (MMPs), and for their intracellular phospholipase C (PLC) activity. Immunization with p259–271 triggered the above three activities and in vivo administration of the dual APL inhibited the latter. Thus, treatment of mice with the dual APL interferes with functions required for T cells to migrate and interact with the self-AChR. This is the first indication that very late antigen 4, MMP-9, and PLC are targets for immunomodulation of autoreactive T cells by altered peptide ligands.—Faber-Elmann, A., Grabovsky, V., Dayan, M., Sela, M., Alon, R., Mozes, E. An altered peptide ligand inhibits the activities of matrix metalloproteinase-9 and phospholipase C, and inhibits T cell interactions with VCAM-1 induced in vivo by a myasthenogenic T cell epitope.


Key Words: autoimmune myasthenia gravis/T cell receptor/immunomodulation/T cell adhesiveness


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MYASTHENIA GRAVIS (MG) is a T cell-regulated, antibody-mediated autoimmune disease. The abnormality in MG stems from a deficiency of acetylcholine receptors (AChR) at neuromuscular junctions due to the antibody-mediated autoimmune attack (1 2 3 4) .

Our previous work has shown that two peptides representing sequences of the human AChR {alpha}-subunit—p195-p212 and p259–271—were able to stimulate peripheral blood lymphocytes of patients with MG and serve as immunodominant T cell epitopes of SJL and BALB/c mice, respectively (5 , 6) . Altered myasthenogenic peptides, which are single amino acid substituted analogs of p195–212 (207Ala) and p259–271 (262Lys), as well as a dual APL composed of the tandemly arranged two single analogs (262Lys-207Ala), were synthesized and shown to inhibit the proliferative responses of both p195–212- and p259–271-specific T cell lines in vitro (5) . The single and dual APLs were also shown to be capable of inhibiting the proliferative responses of peripheral blood lymphocytes of MG patients to myasthenogenic peptides p195–212 and p259–271 (7) . Furthermore, the analogs inhibited in vivo priming to the myasthenogenic peptides (8 9 10) . The dual APL could reverse myasthenogenic manifestations in mice with experimental autoimmune MG induced by either pathogenic T cell lines (8) or the Torpedo AChR (11) . A possible mechanism for immunomodulation of autoreactive responses might be alteration of signal transduction by the peptide analogs that act either as partial agonists or as T cell antigen receptor (TCR) antagonists. In a previous report, we indeed showed that the dual APL serves as a partial agonist, since it could induce tyrosine phosphorylation of phospholipase C (PLC) in p259–271-specific T cell lines but not the generation of inositol phosphate second messengers (12) . Nevertheless, the mechanisms by which the dual APL exerts its effect in vivo have not been elucidated yet. Hence, the aim of this study was to attempt a better insight into these in vivo mechanisms.

For an autoimmune response to take place, the autoreactive T cells, once exposed to self-antigen in lymph nodes (LN), should home to target tissue in the periphery. These circulating cells first adhere to the endothelium, then extravasate through the vessel wall and penetrate the extracellular matrix (ECM) of the tissue toward the target organ (13 14 15 16) and, finally, interact with the relevant epitope. We therefore followed the effect of the myasthenogenic peptide p259–271 and the dual APL on representative key molecules participating in each of these steps. We tested interactions of LN-derived T cells with a major endothelial ligand, vascular cell adhesion molecule 1 (VCAM-1), the activity of matrix metalloproteinases implicated in the penetration of T cells toward the target organ, and the activity of PLC known to participate in signal transduction by antigen through the TCR. The study of these molecules is important whether the disease under investigation is T cell mediated or T cell regulated. Thus, in the case of MG, the T cells may use the above mechanisms either for homing to the lymphatic tissue or to the neuromuscular junction.

As a representative molecule participating in T cell adhesion to the endothelium, we chose VCAM-1. Very late antigen 4 (VLA-4), which is the receptor for VCAM-1, is present in constitutive high-affinity states on peripheral blood lymphocytes and can support both rolling and shear-resistant attachment of T cells to VCAM-1-expressing endothelium (17) . The importance of VCAM-1 and its receptor, the VLA-4 integrin, in autoimmune diseases has been demonstrated by anti-adhesion molecule-antibody therapy in several autoimmune diseases such as diabetes mellitus, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and Sjögren’s syndrome (18 19 20) .

The expression of several matrix metalloproteinases (MMPs) was shown to be regulated by members of the integrin family (21) . After arrest on VCAM-1-expressing endothelium, T cells use MMPs as key enzymes that participate in T cell transmigration across basement membranes and through the tissue ECM without apparent effect on the integrity of critical structures (22 23 24 25) . T cells are known to secrete both MMP-2 and MMP-9 (26) . The potential importance of the versatile activities of MMPs in autoimmune and inflammatory responses has been suggested by the inhibitory effects of specific MMP inhibitors, which have been shown to suppress damage to specialized tissue structures in several inflammatory and autoimmune diseases (26) .

As an intracellular marker of T cell activation by its specific antigen, we chose to assess one of the signal transduction enzymes activated by the antigen-specific TCR. The activation of PLC{gamma}1 is a key event that leads to a spectrum of effector functions of the stimulated T cells (27) . PLC{gamma}1 was also shown to participate in VLA-4 activation (28) . PLC activation results in hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP2) to the second messengers inositol-1,4,5-triphosphate (IP3) and diacyl glycerol, which are crucial for T cell differentiation and activation (27) . Upon TCR engagement, APLs were shown to activate signal transduction events, which are distinct from those induced by the immunogenic ligand and result in different phenotypes (27 , 29 30 31) . In a previous study we (12) showed that the myasthenogenic peptide p259–271 induces PLC activity in a peptide-specific T cell line and that the dual APL selectively inhibits the induced activity. We therefore chose PLC activity as a marker of the T cell activation by the myasthenogenic peptide.

In the present study, we show that immunization with the myasthenogenic peptide p259–271 increases the adhesiveness of LN-derived T cells to VCAM-1, enhances the secreted levels of MMP-9, and causes elevation in the TCR-associated PLC activity. In vivo administration of the dual APL inhibits all three peptide-induced processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic myasthenogenic peptides and their peptide analogs
The synthetic peptide p259–271 (VIVELIPSTSSAV) of the human AChR was synthesized and characterized as described (8) . The dual APL 262Lys-207Ala (VIVKLIPSTSSAVDTPYLDITYHFVAQRLPL) was designed as described (8) and synthesized (97% purity) by UCB Bioproducts (Brussels, Belgium).

Immunization and treatment of mice
Mice were injected intradermally into the hind foot pads with either 10 µg of p259–271 or ovalbumin (150 µg) used as control antigen (in CFA, Difco, Detroit, Mich.; 100 µl total volume), and popliteal LN cells were harvested 3–11 days later. For in vivo inhibition of priming, the dual APL or its reversed form (used as a specificity control) was administered either subcutaneously [(s.c.), 150 µg/0.1 ml phosphate-buffered saline (PBS)] or orally (500 µg/0.3 ml PBS), concomitant with or 2 days before immunization and concomitant with the immunization (pretreatment).

Proliferative response of LN cells
Popliteal LN cells (0.5x106/well) obtained from immunized mice were cultured in enriched RPMI 1640 medium supplemented with 1% normal mouse serum (NMS) (32) in the presence of various concentrations of p259–271 for 96 h. Then 0.5 µCi of [3H]thymidine was added; 16 h later plates were harvested onto filter paper and radioactivity was counted.

Preparation of LN-derived T cells for FACS analysis and for adhesion and PLC assays
Petri dishes were coated with 5 ml goat-anti-mouse-Ig (15 µg/ml in PBS) overnight at 4°C and washed three times. Popliteal LN cells obtained from immunized mice were incubated (in enriched RPMI containing 5% fetal calf serum) on coated plates for 70 min at 4°C. Macrophages were adhered to the plastic Petri dishes. The nonadherent cells, which were mainly T cells (95% as assessed by FACS analysis), were collected and washed in RPMI.

Shear flow assays
Laminar flow adhesion assays were performed as described previously (33 , 34) . Protein A (20 µg/ml in coating medium; PBS buffered with 20 mM bicarbonate pH 8.5) was spotted on a polystyrene plate for 2 h at 37°C, blocked with 2% HSA type V in PBS, and overlaid with 5 µg/ml of VCAM-1-Fc (VCAM-1 fused to the Fc portion of human IgG (35) , a kind gift of Dr. Sanchez-Madrid) suspended in PBS containing 2% HSA. The spotted plate was assembled as the lower stage of a parallel plate laminar flow chamber. All flow experiments were performed at 37°C. Freshly isolated LN-derived T cells were suspended in binding medium (HBSS, containing 10 mM HEPES (pH 7.4), 2 mg/ml bovine serum albumin, Ca2+, and Mg2+, each at 1 mM) and perfused into the chamber at a low flow rate yielding a shear stress of 0.25 dyne/cm2. As soon as cells reached the field of view, the shear stress was elevated to a physiological level of 1.5 dyne/cm2 and cells were allowed to accumulate on the VCAM-1-coated field for 1 min. Adherent cells that developed weak adherence to the ligand were immediately removed from the substrate by subjecting all adherent cells to a high shear stress of 7.5 dyne/cm2 for 10 s. The entire periods of perfusion were recorded on a videotape with a long integration LIS-700 CCD video camera (Applitech, Holon, Israel) and a Time Lapse SVHS Video recorder (AG-6730, Panasonic, Japan). Cells were tracked manually by analysis of replayed images. Cells failed to adhere to substrates coated with protein A alone. All adhesive interactions between perfused cells and the VCAM-1-coated substrates were blocked in the presence of EDTA, suggesting they were integrin specific.

FACS analysis
Cell surface expression of VLA-4 was detected by flow cytometry. Purified T cells (pooled from three mice per group) were incubated with 1 µg of purified rat anti-mouse CD49d (integrin {alpha}4 chain) monoclonal antibody (PharMingen, San Diego, Calif.), followed by incubation with FITC-conjugated rabbit-anti rat. Single-color fluorescence was analyzed by flow cytometry using the FACSort cytometer and CELL QUEST software (Becton Dickinson, Mountain View, Calif.).

Collection of conditioned media for the measurements of MMP-9 and MMP-2 activities
Three to 10 days after immunization with p259–271, popliteal LN cells obtained from immunized mice were cultured in 24-well plates (5x106 cells/ml) in enriched RPMI 1640 medium supplemented with 1% NMS. Twenty-four, 48, or 72 h later the supernatants were collected and tested for MMP-9 and MMP-2 activities.

Measurement of MMP-9 and MMP-2 activities
Activities of MMP-9 and MMP-2 was tested by gelatin zymography. Supernatants (18 µl) of cell cultures of different groups were applied to an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was polymerized with 1 mg/ml gelatin type A (Sigma, St. Louis, Mo.). Gels were washed once for 30 min in 2.5% Triton X-100 to remove the SDS and once for 30 min in the reaction buffer containing 50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, and 0.02% (w/v) Brij 35 (pH 7.5). The reaction buffer was changed to a fresh one and the gels were incubated at 37°C for 24 h. Gelatinolytic activity was visualized by staining the gels with 0.5% Coomassie brilliant blue.

Measurement of inositol phosphate accumulation
LN-derived T cells were suspended (20x106/5 ml) in RPMI depleted of inositol, containing 0.2% NMS and 6% fetal calf serum, which was dialyzed against PBS, and incubated with myo-2-[3H]inositol (6 µCi/ml; New England Nuclear, Boston, Mass.) overnight at 37°C. After labeling, cells were washed and incubated (20x106 per well in a 24-well plate) in a medium containing lithium chloride (15 mM) for 24 h with 5 x 106 antigen-presenting cells (APC) and the indicated peptides. Adherent cells from spleens of normal BALB/c mice served as APCs. Reactions were terminated by the addition of chloroform and methanol (1/2, v/v), and inositol metabolites were extracted. After the addition of chloroform and water, aqueous phases were removed and applied to an anion exchange column (AG 1-X8 resin, 100–200 mesh formate form; Bio-Rad, Hercules, Calif.). After the removal of free inositol and glycerophosphoinositide by elution with 5 mM inositol and 60 mM sodium formate-5 mM sodium tetraborate, inositol monophosphates (IP1) were eluted with 0.2 M ammonium formate-0.1 M formic acid; inositol (1 , 4) -biphosphates (IP2) and IP3 were eluted with 0.8 M ammonium formate-0.1µ formic acid. Radioactivity was assessed by liquid scintillation counting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vivo inhibition of priming of LN cells by the dual APL
Previous studies of our laboratory have shown that the dual APL administered intravenously, intraperitoneally, or orally to BALB/c mice immunized with p259–271 could inhibit the proliferative response of their LN cells in response to the addition of the myasthenogenic peptide in vitro (8) . In Fig. 1 we show that s.c. administration of the dual APL has an inhibitory effect on the proliferative responses to p259–271 comparable to that observed after oral administration of the APL. Similar results were obtained by pretreatment of the mice with the dual APL before and concomitant with the immunization.



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  		Figure 1. Inhibition of in vivo priming of LN cells to p259–271 by oral or s.c. administration of the dual APL. BALB/c mice were immunized with 10 µg of p259–271 in CFA with or without concomitant oral (500 µg/300 µl of PBS) or s.c. (150 µg/100 µl of PBS) administration of the dual APL. LN cells taken from the mice 10 days later were incubated in the presence of various concentrations (10–20 µM) of the myasthenogenic peptide and the proliferative response of the cells was measured (Materials and Methods). Results are expressed as the extent of T cell proliferation relative to p259–271 immunized mice, which was defined as 100% and 10915 ± 3800 above background levels. The results are the mean ± SE of three independent experiments.

Adhesion of in vivo-stimulated T cells to VCAM-1
The {alpha}4 integrins {alpha}4ß1 (VLA-4) and {alpha}4ß7 are the exclusive receptors for VCAM-1 on T cells. As the adhesiveness of T cells toward VCAM-1 does not always correlate with expression levels of its integrin receptors (34 , 36 , 37) , we applied a functional flow chamber assay to assess the potential of LN-derived T cells to interact with VCAM-1 under physiological shear flow. T cells from nonimmunized (naive), CFA-immunized, and p259–271- or the control antigen ovalbumin- (both antigens introduced in CFA) immunized mice were compared for their ability to interact with a VCAM-1-coated substrate. The results are demonstrated in Fig. 2A . The fraction of T cells that acquired high adhesiveness to VCAM-1 in LN of CFA-immunized mice was higher than that within T cells of naive mice (Fig. 2A ). Immunization with either p259–271 or ovalbumin caused a further (above the CFA-induced adhesiveness) 2.5-fold increase in the fraction of VCAM-1 adhesive T cells (Fig. 2A ). Up-regulation of the T cell adhesiveness to VCAM-1 by the p259–271 was dependent on the duration of the peptidic stimulation and was elevated toward day 11 (Fig. 2B ).



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  		Figure 2. Adherence of LN-derived T cells to VCAM-1 under physiological shear flow. A) T cells were derived from LN of nonimmunized (naive) mice or mice immunized for 11 days with the myasthenogenic peptide p259–271 or a control antigen-ovalbumin (both in CFA) and with CFA only. An equal number of T cells were perfused for 1 min over VCAM-1 substrate assembled in a flow chamber under a shear stress of 0.75 dyne/cm2 and the number of cells accumulated in multiple fields was determined. The number of attached cells in LN T cells derived from CFA-immunized mice was taken as 100%. Results are the mean ± SE of 2–5 experiments for each group. B) Mice were immunized with the myasthenogenic peptide p259–271 (in CFA) or with PBS/CFA for different time intervals and then killed the same day. The abilities of LN-derived T cells to form firm adherence to VCAM-1-coated substrate under shear flow was measured. This experiment is representative of four independent experiments. ({circ}) PBS/CFA; (•) p259–271/CFA. C) T cells obtained from LN of nonimmunized (naive) mice or from mice 11 days after immunization with the myasthenogenic peptide p259–271 in CFA or with CFA only were stained with anti {alpha}4 monoclonal antibodies (solid line) or with control antibodies (dashed line); the expression levels of {alpha}4 integrin were measured using flow cytometry. This experiment is representative of three independent experiments.

Ag-stimulated enhancement of T cell adhesiveness to VCAM-1 can be the result of either elevated expression levels of the VCAM-1 binding integrins, VLA-4 or {alpha}4ß7, or {alpha}4 integrin activation. To examine the first possibility, expression of the {alpha}4 integrin subunit on T cells derived from mice immunized with different antigens was analyzed by FACS. The majority of T cells from both naive and immunized mice expressed low to moderate levels of the {alpha}4 integrin (log fluorescence intensity<20). A small subset of cells expressed significantly higher {alpha}4 levels (log fluorescence intensity 20–90), and this subset was enriched by ~threefold after CFA injection as shown in Fig. 2C . Figure 2 also shows that immunization with the myasthenogenic peptide p259–271 further enriched {alpha}4 expression by only 30–40% beyond the expression levels by T cells derived from CFA immunized mice. It is unlikely that this elevation by itself accounts for the twofold elevation in T cell adhesiveness. We therefore assume that the elevation in the T cell adhesiveness results from an additional activation of the {alpha}4 integrin receptor.

It is noteworthy that the elevation of {alpha}4 expression by the CFA or by p259–271 could have reflected alterations in VLA-4 or {alpha}4ß7 expression levels. However, it was shown that {alpha}4ß7 is a much weaker receptor for VCAM-1 than VLA-4 (18) . We therefore assume that the augmented adhesiveness of T cells from CFA- and peptide-immunized mice was the result of elevation of VLA-4 expression and activity rather than the outcome of elevation in the expression or function of {alpha}4ß7.

Effect of immunization with p259–271 and treatment with the dual APL on the adhesion of LN T cells to VCAM-1
We have shown that the myasthenogenic peptide p259–271 (in CFA) increases the adhesion of the T cells to VCAM-1 (relative to CFA immunized mice) by expanding the population of LN-derived T cells with high adhesiveness toward VCAM-1. We next tested whether the dual APL can interfere with and inhibit the adhesion to VCAM-1 stimulated by the myasthenogenic antigen. As shown in Fig. 3A , adhesion to VCAM-1 of T cells from mice that were immunized with p259–271 and treated with the dual APL, either orally or s.c., was significantly reduced by the dual APL. The reduction was specific to the dual APL because the reversed sequence of the dual APL used as control did not affect significantly activated T cell adhesiveness to VCAM-1. Figure 3B demonstrates that the inhibitory effect of the dual APL was specific for the stimulation induced by the myasthenogenic peptide, since the dual APL did not inhibit ovalbumin-induced adhesion to VCAM-1 of T cells tested under identical experimental conditions. Our results show an up-regulated VLA-4 adhesiveness in LN-derived T cells after antigen stimulation that can be specifically reversed by the dual APL.



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  		Figure 3. Effect of the dual APL on the adherence to VCAM-1 of LN-derived T cells originated from p259–271 immunized mice. A) Mice were immunized with the myasthenogenic peptide p259–271 (in CFA), and the dual APL or its reversed form was given orally or s.c. B) Mice were immunized with ovalbumin and the dual APL was given orally or s.c. LN-derived T cells were isolated from mice 10 days later, and their ability to adhere to VCAM-1 under shear flow was measured as described in Fig. 2 . Results are the mean ± SE of four independent experiments in which the dual APL was given orally and of two independent experiments in which the dual APL was given s.c.

Effect of immunization with p259–271 and treatment with the dual APL on MMP activities in LN-derived T cells
To study the effect of antigen-specific immunization on MMP activities in LN-derived T cells, we immunized mice with the myasthenogenic peptide (in CFA) or with CFA alone. The animals were killed at the indicated time points, their LN cells were incubated in growth media containing 1% NMS, and the media conditioned by the cells was collected. As MMP-9 and MMP-2 degrade denatured collagen (gelatin) in addition to collagen, MMP-9 and MMP-2 activities were measured using gelatin zymography. As shown in Fig. 4 , the MMP activity in mice injected with CFA only [designated (-)] is very low and most is due to the NMS included in the medium. Immunization of mice with p259–271 [designated (+)] stimulated MMP-9 (but not MMP-2) activity in LN cells compared to mice injected with CFA only. Differences between the two groups were observed at all the time points tested postimmunization. To test whether the elevated MMP-9 activity originated from T cells, LN cells were depleted from macrophages and B cells, and their T cells were incubated without p259–271 for 24 h. Figure 5 shows that the elevation of MMP-9 activity after in vivo activation by either the myasthenogenic peptide or ovalbumin originates from T cells. Figure 5A shows that the peptide induced MMP-9 activity was markedly reduced in LN-derived T cells of immunized mice treated s.c. or orally with the dual APL. Figure 5B demonstrates that the inhibition by the dual APL is specific because the latter did not inhibit MMP-9 activity induced by the control antigen-ovalbumin.



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  		Figure 4. Kinetics of MMP-9 and MMP-2 activities in LN cells of mice that were immunized with the myasthenogenic peptide p259–271. Mice were immunized with the myasthenogenic peptide p259–271 [designated as (+)] or with PBS [designated as (-)] (both in CFA). LN cells were removed from mice at the indicated time points postimmunization and incubated for 24 h. Supernatants were collected and MMP-9 and MMP-2 activities were tested by gelatin zymography.



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  		Figure 5. Reduction in MMP-9 activity in LN-derived T cells of mice that were immunized with the myasthenogenic peptide p259–271 and treated with the dual APL. Mice were immunized with the myasthenogenic peptide p259–271 (A) or ovalbumin (B). The dual APL was given s.c. or orally. LN-derived T cells (see Materials and Methods) were incubated for 24 h and supernatants were tested for MMP-9 activity by gelatin zymography. The figure represents the results of two independent experiments.

Effect of immunization with p259–271 and treatment with the dual APL on PLC activity in LN-derived T cells
The intracellular mechanisms by which the dual APL exerts its effects on the T cells in vivo have not been studied yet. Therefore, we tested the response (in terms of PLC activity) of LN-derived T cells originated from p259–271-immunized mice that were treated in vivo or not treated with the dual APL, to in vitro stimulation by p259–271. As can be seen in Fig. 6 , in vitro triggering of T cells from p259–271 immunized mice (not treated with the dual APL) elevated the PLC activity by around 48%. However, in vitro presentation of p259–271 to T cells that were isolated from animals treated with the dual APL caused a reduction of ~31% in the PLC activity.



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  		Figure 6. Treatment of p259–271 immunized mice with the dual APL down-regulates PLC activity. LN-derived T cells isolated from the peptide immunized mice that were treated in vivo (s.c.) or not treated with the dual APL were prelabeled with myo-[2-3H]inositol, and incubated for 24 h with the myasthenogenic peptide p259–271 (10 µM) in the presence of APCs. The levels of the generated inositol phosphates were measured (Materials and Methods). Results are the mean ± SE of four independent experiments. The levels of inositol phosphates generated by T cells that were not activated in vitro by p259–271 were considered as 100% activation. 100% for IP1 was 3191 ± 1568 cpm in the different experiments, and 785 ± 239 cpm for IP2 + IP3.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of the present study are that immunization with the myasthenogenic peptide p259–271 results in elevation of all three parameters we tested: adhesion to VCAM-1, MMP-9 activity, and PLC activity. In vivo treatment of the p259–271-immunized mice with the dual APL (either orally or s.c.) down-regulated the peptide-specific acquisition of the above activities.

Our results show that immunization of mice with the myasthenogenic peptide elevates the adhesive potential to VCAM-1, a major vascular adhesion molecule that dominates the recruitment of effector T cells to inflammatory sites of nonlymphoid tissues (18) . The flow assay used in the present study simulates blood flow in venules and enables assessment of changes in the adhesiveness of the VLA-4 receptor to its endothelial ligand under physiological conditions. Using this functional assay along with FACS analysis of {alpha}4 integrin expression, we could show that elevated adhesion toward VCAM-1 induced by the myasthenogenic peptide is the result of elevated expression of {alpha}4 integrin receptors, combined with enhanced activation of these receptors toward VCAM-1. Treatment of the p259–271 immunized mice with the dual APL reversed specifically the stimulatory effects of the myasthenogenic peptide on both expression and activation of the {alpha}4 integrin. These results may suggest that T cells derived from LN of mice immunized with the myasthenogenic peptide and treated with the dual APL have a reduced capability to bind the endothelium, enter the relevant tissue, and be involved in the autoimmune response against the AChR. It is noteworthy that VLA-4 functions also as a key receptor for fibronectin, which is a major component of the ECM (38) . Thus, the dual APL might also suppress the adhesion of the T cells to fibronectin and their migration through the ECM.

Results of experiments performed in our laboratory showed that LN or spleen cells of mice immunized with either of the myasthenogenic peptides secreted Th1-type cytokines after in vitro triggering with the myasthenogenic peptides. Treatment with the dual APL inhibited the production of the Th1-type cytokines and caused the secretion of high levels of the immunosuppressive cytokine TGFß (39) . This observation suggests that after treatment with the dual APL, there are still cells with TCR that can bind the peptide. Thus, we assume that the reduced VLA-4 and MMP-9 activities observed in the present study are not the result of a decrease in the number of cells capable of responding to the p259–271 peptide, but rather may be due to the different characteristics of the T cell clones affected by the dual APL.

MMP-9 activity was also elevated in LN-derived T cells of mice immunized with the myasthenogenic peptide and was specifically inhibited in LN-derived T cells of mice that were treated with the dual APL. The physiological meaning of the induced activity might be that the autoreactive T cells that originated from p259–271-immunized mice have a greater ability to degrade collagen and to pave their way through the endothelium and the tissue toward the target organ. The reduced MMP-9 activity observed after treatment with the dual APL might predict a reduced capability of the AChR-specific T cells to penetrate the endothelium and the target tissue and to elicit an autoimmune response.

The integrins were shown to be physiological regulators of the expression of several MMPs (21) . Several studies have shown that Th1-type cytokines elevate MMP activity/expression, whereas Th2-type cytokines reduce the MMP levels. It has also been demonstrated (16 , 40) that MMP-2 is induced in a Th1 clone that is autoreactive to myelin basic protein upon adhesion to VCAM-1 on endothelial cells. Our results demonstrating MMP-9 activity are in line with these findings, since our model, immunization with p259–271, which is characterized by a Th1 phenotype (39) , increases the number of cells that adhere to VCAM-1 and secrete MMP-9, and these two responses are inhibited by the dual APL.

Treatment of the immunized mice either orally or s.c. yielded the same results regarding the inhibition of VLA-4 ligation to VCAM-1 and of MMP-9 activity potentiated by the myasthenogenic peptide. This suggests that modulation of these two processes plays an important role in the mechanism by which the dual APL exerts its effect regardless of its route of administration. In an identical experimental system, we could show that in vivo treatment with the dual APL reduces the rolling of LN-derived T cells on both E- and P-selectin (unpublished results). This strengthens our claim that the dual APL down-regulates the capability of the T cells to interact with the endothelium, which is a necessary step for their transmigration.

The interaction of the autoreactive T cell with its specific Ag should lead to clonal expansion, cytokine secretion, and other cellular functions resulting from the signal transmitted by the TCR. Our results show that LN-derived T cells from p259–271 immunized mice respond to in vitro presentation of p259–271 by the elevation of PLC activity. However, in vivo treatment of p259–271-immunized mice with the dual APL resulted rather in a reduction in PLC activity below the spontaneous activity levels observed in T cells not stimulated in vitro with p259–271. The latter reduction observed in PLC activity might be due to cytokines secreted by the p259–271-specific cells, which might also suppress the spontaneous generation of inositol phosphates by nonspecific T cells during the 24 h of culture. In this case, the p259–271-specific cells that interact with the antigen might suppress also other AChR reactive clones (bystander suppression). The physiological meaning of the latter effect is that p259–271-specific clones whose treatment did not prevent their penetration into the tissue will be down-regulated upon recognizing the p259–271 region of the AChR (at least in their PLC-dependent functions). The down-regulation of PLC activity by the dual APL may account for its capacity to inhibit in vivo MG-associated T cell responses.

Studies of T cell responses to altered peptide ligands have provided functional evidence that small changes in the original antigen cause the TCR to transduce a signal that is different from that transduced by the original antigen. Such small changes in the ligand can convert fully activating ligands into partially activating (partial agonists) or even inhibitory ones (antagonists). The ability of the TCR to transduce a partial signal results in partial T cell activation, and thus in different biochemical and cellular phenotypes (29 , 41 42 43) . In this work, we present evidence that the dual APL, which is an altered peptide ligand, down-regulates migratory properties acquired by subsets of T cells after antigenic stimulation, and thus interferes with the immune response of p259–271-specific clones. The experimental efficacy of the dual APL suggests that intervention with signaling and migration-associated events might be of therapeutic potential by reducing the capability of autoreactive T cells to elicit autoimmune responses.

The results presented in this paper show for the first time that exposure of antigen-stimulated T cells to an APL during the earliest phases of Ag challenge affects functions implicated in T cell transmigration and down-regulates signaling events induced by an autoantigen in the T cells. The cumulative result of the above might be a down-regulated response of autoreactive T cells in the dual APL treated animals.


   ACKNOWLEDGMENTS
 
This research was partly supported by Teva Pharmaceutical Industries Ltd., Israel (M.S. and E.M.). Part of this research was supported by the Minerva Foundation and the Israel Science Foundation (R.A.).

Received for publication November 18, 1999. Revision received April 11, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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