Cytokine profile and T cell adhesiveness to endothelial selectins: in vivo induction by a myasthenogenic T cell epitope and immunomodulation by a dual altered peptide ligand
Anat Faber-Elmann,
Valentin Grabovsky,
Molly Dayan,
Michael Sela,
Ronen Alon and
Edna Mozes
Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
Correspondence to:
E. Mozes
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Abstract
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Myasthenia gravis (MG) is a T cell-regulated antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195212 and p259271, that are sequences of the human acetylcholine receptor
subunit was shown to induce experimental autoimmune MG (EAMG)-associated immune responses. A peptide composed of the two altered peptide ligands (APL) of the myasthenogenic peptides (designated as dual APL) inhibited, in vitro and in vivo, those responses. The objectives of this study were to examine (i) whether in vivo T cell activation by p259271 affects the cytokine profile and the T cell migration ability, and (ii) whether the latter are immunomodulated by in vivo administration of the dual APL. Our results showed that immunization of mice with p259271 enriched the population of lymph node and spleen cells with subsets of T cells with strong adhesiveness towards E- and P-selectins. This enrichment was associated with an acquisition of a Th1-type cytokine profile. Treatment of the immunized mice with the dual APL interfered with both the migratory potential of the autoreactive T cells, and the production of the Th1-type cytokines IL-2 and IFN-
(known to play a pathogenic role in MG and EAMG). T cells derived from APL-treated mice acquired a Th3-type cytokine profile, characterized by the secretion of the immunosuppresive cytokine transforming growth factor-ß. Thus, our results suggest that T cell selectin ligands and T cell-derived cytokines are involved in the induction and immunomodulation of EAMG- and MG-associated T cell responses.
Keywords: autoimmunity, cytokine, myasthenia gravis, selectin ligands, Th1, Th2, Th3, TCR
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Introduction
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Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Impaired neuromuscular transmission characteristic of MG results from antibodies against the nicotinic acetylcholine receptor (AChR) of skeletal muscle (1,2). Nevertheless, a role for T cells in MG and experimental autoimmune MG (EAMG) was shown in several studies (310). Our previous work demonstrated that two peptides representing sequences of the human acetylcholine receptor
subunit, p195212 and p259271, were able to stimulate peripheral blood lymphocytes of patients with MG, and to serve as immunodominant T cell epitopes of SJL and BALB/c mice respectively (11,12). Altered myasthenogenic peptides, which are single amino acid-substituted analogs of p195212 (207Ala) and p259271 (262Lys), as well as a dual altered peptide ligand (APL) composed of the tandemly arranged two single peptide analogs (262Lys-207Ala), were synthesized and were shown to inhibit the proliferative responses of both p195212- and p259271-specific T cell lines in vitro (11) as well as the in vivo priming to the myasthenogenic peptides (1315). The single and dual APL were also demonstrated to be capable of inhibiting the proliferative responses of peripheral blood lymphocytes of MG patients to both myasthenogenic peptides p195212 and p259271 (16). In addition, the dual APL could reverse myasthenogenic manifestations in mice with EAMG induced either by pathogenic T cell lines or by the Torpedo AChR (13,17,18). Nevertheless, the mechanisms by which the dual APL exerts its effect in vivo have not been elucidated yet. Hence, the aim of this study has been to attempt a better insight into these in vivo mechanisms.
A possible mechanism for the immunomodulation of autoreactive T cell responses by the dual APL might involve changes in cytokine secretion and in the T cell ability to roll on endothelial selectins. The migration of T cells from the blood vessel into target tissues requires the binding of T cells to the endothelium. Selectin ligands are enriched on subsets of T cells, and mediate their initial capture and rolling on vascular endothelium (19,20). Rolling interactions along the vessel wall, mediated by endothelial members of the selectin family, P- and E-selectins, allow the captured cells to survey the endothelium for additional activation signals, in particular stimulatory cytokines and chemokines. The rolling T cells use integrin family adhesion molecules to develop firm adhesion to the endothelium, a key step in their successful extravasation to the surrounding target tissue. In combination, selectins and integrins provide most of the information required for site-specific T cell recruitment.
For simplification, upon activation by a ligand, naive populations of T cells differentiate into three distinct functional subgroups of cells which differ with regard to their cytokine secretion profile. The population of Th1-type cells secrete mainly IL-2 and IFN-
, the population of Th2-type cells produce among others IL-4, IL-5 and IL-10, and Th3-type cells produce mainly transforming growth factor (TGF)-ß. There is evidence for a role of Th1-type cytokines in the pathogenesis of autoimmune diseases (21) including EAMG (7,22), and there are also studies showing that Th2- and Th3-type cytokines have a protective effect in EAMG (8,10), experimental autoimmune encephalomyelitis and insulin-dependent diabetes mellitus (21).
In addition to the different profiles of cytokine expression, Th1- and Th2-type cells may exhibit distinct trafficking patterns in vivo (23). It has been shown that in the same experimental system, Th1-type cells are recruited to their target sites, whereas Th2-type cells are not recruited to these sites. This differential recruitment is attributed to the expression of functional selectin ligands on Th1- but not Th2-type cells (2428). The role of E- and P-selectins in autoimmune diseases has not been elucidated yet. To date, information has been limited to the involvement of the latter in models of rheumatoid arthritis only (29,30). The in vivo importance of selectins in T cell migration to target sites in autoimmune diseases has been also demonstrated by the successful attenuation of the severity of autoimmune diseases following administration of anti-L-selectin antibodies (31). Nevertheless, the acquisition of endothelial selectin binding by specific T cells during the response to self antigen was never studied in any model of autoimmune disease, including EAMG.
In the present study, we examined the effect of in vivo administration of the dual APL on both cytokine secretion and selectin ligand function in T cells derived from lymph nodes (LN) and spleens of mice that were immunized with the myasthenogenic peptide p259271. Our results show that the dual APL shifts the cytokine profile of the LN and spleen cells obtained from p259271 mice from a Th1- to a Th3 type. We could also show that immunization of mice with the myasthenogenic peptide expands the population of LN- or spleen-derived T cells expressing functional receptors for P- and E-selectin. Treatment of animals with the dual APL (either orally or s.c.) inhibited the myasthenogenic peptide dependent acquisition of rolling on P- and E-selectin ligands under physiological shear flow.
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Methods
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Synthetic myasthenogenic peptides and their peptide analogs
The synthetic peptide p259271 (VIVELIPSTSSAV) that corresponds to a sequence of the human AChR
subunit was synthesized and characterized as described (13). The dual APL 262Lys207Ala (VIVKLIPSTSSAVDTPYLDITYHFVAQRLPL) was designed as described (13) and synthesized (97% purity) by UCB Bioproducts AS (Brussels, Belgium).
Immunization and treatment
Mice were injected i.d. into the hind foot pads with p259271 (10 µg) or ovalbumin (OVA; 100 µg) in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI; 100 µl total volume). The dual APL was administered either s.c. to the neck (150 µg/0.1 ml PBS) or orally (500 µg/0.3 ml PBS), 2 days before immunization and/or concomitant with the immunization.
Preparation of splenic- or LN-derived T cells for the adhesion assay
Petri dishes were coated with 5 ml goat anti-mouse Ig (15 µg/ml in PBS) overnight at 4°C and washed 3 times. Spleens or popliteal lymph nodes obtained from immunized mice were squeezed to single-cell suspensions, washed twice in RPMI and incubated (50x106/5 ml in enriched RPMI containing 5% FCS) on the coated plates for 70 min at 4°C. Macrophages were adhered to the plastic Petri dishes. The non-adherent cells, which were mainly T cells (95% as assessed by FACS analysis), were collected, washed in RPMI containing 5 mM EDTA, resuspended in divalent cation free-HBSS (10 mM HEPES pH 7.4, supplemented with 0.2% BSA; Sigma, St Louis, MO) and used in the adhesion assays.
Shear flow assays
Laminar flow adhesion assays were performed as previously described (32,33). To prepare selectin chimera substrates, a polystyrene dish was spotted with Protein A as described (34). Unbound Protein A was washed away and the surface was blocked with human serum albumin (34). The Protein A-coated spot was overlaid overnight at 4°C with E-selectinFc or P-selectinFc chimeras [produced as previously described (33), 510 µg/ml in 50 µl of culture supernatant]. The spotted plate was assembled as the lower stage of a parallel plate laminar flow chamber (32). All flow experiments were performed at 37°C. Protein A-coated plates overlaid with control culture supernatants lacked any adhesive reactivity in the flow assays described below (data not shown). Freshly isolated LN-derived T cells were suspended in binding medium (HBSS, containing 10 mM HEPES, pH 7.4, 2 mg/ml BSA, and Ca2+ and Mg2+ each at 1 mM) and introduced into the chamber at a low flow rate of 0.25 dyne/cm2 for 10 s. As soon as cells reached the field of view, the shear stress was elevated to a physiological level of 1.75 dyne/cm2 and cells were allowed to accumulate on the selectin-coated field under constant flow for 1 min. For detachment assays, T cells were allowed to accumulate for 1 min at a shear of 1.75 dyne/cm2 and the shear force was then increased every 5 s to a maximum of 12.2 dyne/cm2. The cells were visualized with a x10 objective of an inverted microscope. 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, Tokyo, Japan). T cells remaining bound and persistently rolling in multiple fields of view (0.17 mm2 area) were manually tracked by analysis of the videotaped images. A rolling cell can be distinguished from a non-interacting (or loosely interacting) cell by two definitions routinely used in our assays: (i) rolling cells were moving at velocities at least 100-fold slower than the velocities of non-interacting cells flowing over the tested fields and (ii) rolling should persist for at least 5 s, i.e. a cell which rolled for a shorter period was not included. The specific selectin dependency of rolling of the different experimental groups was verified by the complete inhibition of selectin-dependent rolling in the presence of the Ca2+ chelator, EDTA. The experiments were performed double blinded, i.e. the person that performed the adhesion experiments was not the one who evaluated the results.
Collection of conditioned media and measurements of cytokine levels
Ten days after immunization, popliteal LN and spleens were removed from mice and squeezed to single-cell suspensions. LN or spleen cells obtained from immunized mice were cultured in 24-well plates (5x106 cells/ml) in enriched RPMI 1640 medium supplemented with 1% normal mouse serum (for LN) or 5% FCS (for splenocytes), in the presence of p259271. After 24 or 48 h the supernatants were collected and tested for cytokine levels by ELISA using the appropriate capture antibodies to the various cytokines and horseradish peroxidase-conjugated anti-cytokines as detection antibodies (OptEIATM Set; PharMingen, San Diego, CA) according to the manufacturer's instructions. For TGF-ß1 measurements, in most experiments cells were grown for 72 h in RPMI 1640 medium supplemented with Nutridoma SP (Boehringer Mannheim, Indianapolis, IN) instead of serum. All samples were acidified in order to activate latent TGF-ß1 to immunoreactive TGF-ß1. Following neutralization TGF-ß1 levels were measured by using a combination of rhTGF-ß sRII/Fc chimera for capture of TGF-ß and biotinylated anti-human TGF-ß1 antibody as a detection antibody according to the manufacturer's instructions (R & D Systems, Minneapolis, MN).
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Results
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Induction of functional P- and E-selectin ligands in antigen-stimulated LN- and splenic-derived T cells
In order to follow the induction of functional selectin ligands on LN-derived T cells during early stages of exposure to the myasthenogenic peptide and its dual APL, we examined the adhesiveness of antigen-stimulated spleen- or LN-derived T cells towards E- and P-selectins under physiological shear flow. This technique simulates blood flow in venules. Freshly isolated spleens- or LN-derived T cells of non-immunized (naive), PBS/CFA-immunized, as well as p259271 peptide- or OVA- (both antigens in CFA) immunized mice were compared for their ability to interact under flow with the purified endothelial selectins, coated on substrates assembled on a wall of a flow chamber. The adhesiveness acquired by the compared groups of T cells towards endothelial selectins was determined by comparing the fraction of T cells (within each group) capable of establishing steady rolling on either P- or E-selectins. As can be seen in Fig. 1
, only a small fraction of LN-derived T cells of naive (non-immunized) mice could interact with E-selectin under flow (Fig. 1A
). However, immunization of mice with CFA enriched 4-fold (relative to naive animals) the LN population with T cells capable of rolling on E-selectin. Immunization with either the myasthenogenic peptide p259271 or a control antigen, OVA, led to a further 2- to 2.5-fold enrichment in the fraction of LN-derived T cells with high adhesiveness to E-selectin (Fig. 1A
). Figure 1
(A) also demonstrates the results obtained when adhesiveness towards P-selectin was measured in the same experimental system. It is noteworthy that the fraction of T cells enriched with high adhesiveness towards P-selectin was higher (3- to 3.5-fold, relative to immunization with CFA) than that observed for E-selectin. The p259271-specific up-regulation of the adhesiveness of LN-derived T cells to P- and E-selectins was found to depend on the time interval from the antigenic stimulation, and peaked on day 7 post-immunization (data not shown). Thus, during early stages of immunization with the myasthenogenic peptide, LN-derived T cells are enriched with ligands to both E- and P- selectins, above the levels obtained by CFA immunization alone.

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Fig. 1. Rolling of LN-derived T cells from immunized mice on E- and P-selectin. (A) T cells were derived from LN of non-immunized (naive) mice or mice that were immunized for 711 days with the myasthenogenic peptide p259271 or with a control antigen (OVA), both in CFA or with PBS/CFA only. Equal numbers of T cells were perfused for 1 min over E- or P-selectin substrate assembled in a flow chamber under a shear stress of 1.75 dyne/cm2 and the number of cells accumulated in multiple fields was determined. The number of rolling T cells derived from PBS/CFA-immunized mice was taken as 100% (9 cells/field, for both E- and P- selectin). The results are the mean ± SE of three to five independent experiments for each group, consisting of three to 12 animals per group (*P < 0.005, **P < 0.001 as compared to PBS/CFA). (B) Cells accumulated at a shear stress of 1.75 dyne/cm2 were subjected to detachment assay as described in Methods. The shear stress in which 50% of the cells detached from the substrate was defined as DC50. The results are the mean ± SE of four independent experiments on P-selectin and five independent experiments on E-selectin. Black bars, PBS/CFA; gray bars, p259271/CFA. (C) Mice were immunized with the myasthenogenic peptide p259271 or with PBS (both in CFA). LN cells were removed from mice 710 days later. The rolling velocities of the different groups were determined at a shear stress of 1.75 dyne/cm2. The results are the mean ± SE of three independent experiments. Black bars, PBS/CFA; gray bars, p259271/CFA.
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Up-regulation of T cell adhesiveness to each endothelial selectin under flow can be the result of: (i) enrichment of the LN or spleen population with T cell subsets that are capable of adhering to each endothelial selectin, and (ii) elevation of the functional selectin ligand density and/or avidity on individual T cells. Higher expression and avidity of functional ligand results in higher resistance to detachment by increased shear forces, as well as slower rolling velocities in a given shear stress (35). To examine the above possibilities, resistance of individual adherent T cells to elevated shear stresses which cause detachment from the selectin-coated substrate of LN-derived T cells from p259271- or CFA-immunized mice was compared. The results shown in Fig. 1
(B) indicate that while 50% of the peptide-stimulated T cells detached (ED50) from E-selectin at 7.5 dyne/cm2, control CFA-stimulated T cells detached from the E-selectin slightly more readily, at a lower, but non-significant (P = 0.13), shear stress, ED50 = 5 dyne/cm2 (Fig. 1B
). Similar small differences in the ED50 values for the peptide- versus CFA-stimulated T cells were also observed in detachment measurements performed on P-selectin (P = 0.28) (Fig. 1B
). We also compared the rolling velocities of the two groups on both selectins. As seen in Fig. 1
(C), no significant differences were observed between the rolling velocities of T cells from the CFA or the peptide immunized mice on either E- or P-selectins. Thus the contribution of the peptide-specific immunization is mainly to the enrichment of adhesive T cell subsets within the LN population rather than to elevation in receptor density on individual adhesive T cells.
Figure 2
demonstrates that immunization with the myasthenogenic peptide enriched the splenocytes in T cells with high adhesiveness towards both E- and P- selectins in a similar manner to that observed for LN cells.

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Fig. 2. Rolling of splenic-derived T cells from immunized mice on E- and P-selectin. T cells were derived from spleens of mice that were immunized for 711 days with the myasthenogenic peptide p259271 in CFA, or with PBS/CFA only. Equal numbers of T cells were perfused for 1 min over E- or P-selectin substrate assembled in a flow chamber under a shear stress of 1.75 dyne/cm2 and the number of cells accumulated in multiple fields was determined. The number of rolling T cells derived from CFA-immunized mice was taken as 100% (40 cells/field, for both E- and P- selectin). The results are the mean ± SE of three independent experiments for each group. *P = 0.05, **P = 0.03 as compared to PBS/CFA.
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Since the dual APL has been shown to inhibit p259271-induced T cell functions, we next examined the effect of in vivo treatment of p259271-immunized mice with the dual APL on the peptide-induced acquisition of selectin adhesiveness. The dual APL was administered to the immunized mice either orally or s.c. As can be seen in Fig. 3
(A), the dual APL could inhibit the p259271-induced acquisition of E-selectin ligands on LN-derived T cells as well as on splenic-derived T cells. The dual APL inhibited specifically the rolling induced by the myasthenogenic peptide since it did not inhibit the rolling induced by immunization with the control antigenOVA (Fig. 3A
). The values in the figures represent the results following the subtraction of the CFA background levels. Figure 3
(B) demonstrates the capacity of the dual APL to inhibit the rolling induced by the peptide on P-selectin. Similar to results with E-selectin, the dual APL did not inhibit the rolling on P-selectin induced by immunization with the control antigenOVA (Fig. 3B
)confirming the specificity of its inhibitory effect. The inhibition by the dual APL appeared to be more efficient on the p259271-dependent induction of adhesiveness to P-selectin (Fig. 3B
) than on adhesiveness to E-selectin (Fig. 3A
).
Effect of the dual APL on cytokine secretion by lymphocytes obtained from p259271-immunized mice
Th1-type cytokines were shown to induce selectin transcription and to regulate T cell adherence to endothelial selectins. We therefore tested whether the dual APL which inhibits the T cell rolling on selectins affects also the cytokine profile of LN- and splenic-derived T cells. For that purpose, we treated p259271-immunized mice orally or s.c. with the dual APL. Ten days post-immunization splenic and LN-derived lymphocytes were harvested and incubated in the presence of p259271. Culture supernatants were tested by ELISA for IL-2, IFN-
, IL-4, IL-5, IL-10 and TGF-ß levels. As can be seen in Fig. 4
, splenocytes of mice that were immunized with the myasthenogenic peptide p259271 secreted IFN-
(101 pg/ml), IL-2 (143 pg/ml) and TGF-ß (933 pg/ml) in response to in vitro stimulation with the myasthenogenic peptide. Oral treatment with the dual APL, but not with its reversed form, resulted in a diminished secretion of IL-2 and IFN-
from splenocytes of the treated mice while the secretion of TGF-ß was augmented. The dual APL affected specifically the cytokine secretion induced by the myasthenogenic peptide since it did not change the secretion induced by immunization with the control antigenOVA (Fig. 4
). Splenocytes from the p259271-immunized mice secreted, in addition to the Th1-type cytokines, IL-10 (~100 pg/ml). The latter was only slightly (~10%) elevated following treatment with the dual APL. In some experiments we could detect very low levels of IL-4 and IL-5 secretion from splenocytes, and the dual APL up-regulated the latter secretion (data not shown). Similar results were obtained for splenocytes derived from mice that were pretreated with the dual APL.
Since the myasthenogenic peptide induced also the rolling of LN-derived T cells on both E- and P-selectins, we examined the cytokine profile secreted by these cells and the effect of treatment with the dual APL on the cytokine secretion. As for the splenocytes, LN-derived lymphocytes secreted IL-2, IFN-
and TGF-ß in response to in vitro incubation with the myasthenogenic peptide (Table 1
). As can be seen from Table 1
, pretreatment with the dual APL either orally or s.c. markedly inhibited IL-2 and IFN-
secretion, and stimulated the production of TGF-ß. Levels of IL-10 secreted by the LN-derived lymphocytes were similar (~100 pg/ml) to those secreted by splenocytes. The latter were elevated, although not significantly, following treatment with the dual APL (Table 1
). Levels of IL-4 and IL-5 in most cases were below the detection sensitivity of the assay; however, in some experiments low levels of IL-4 and IL-5 could be detected in the supernatants of dual APL-treated mice (Table 1
). Similar results were obtained for LN cells derived from mice that were treated with the dual APL concomitant with the immunization (data not shown).
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Table 1. Cytokine levels in LN cells of mice that were immunized with p259271 and were treated with the dual APL
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Thus it appears that immunization with p259271 leads to secretion of Th1-type cytokines (IFN-
and IL-2) from both LN cells and splenocytes, while administration of the dual APL either orally or s.c. inhibits both IFN-
and IL-2 secretion, triggers mainly Th3-type (TGF-ß) response, and slightly elevates the Th2-type cytokines, IL-10 and IL-4.
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Discussion
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The results presented in this study show that immunization with the myasthenogenic peptide p259271 that corresponds to a sequence of the AChR
subunit leads to a Th1-type (IFN-
and IL-2) cytokine secretion by LN and spleen cells. The profile of Th1-type cytokines of both LN and spleen cells correlates with their acquisition of adhesiveness towards the endothelial P- and E-selectins. In vivo treatment of the p259271-immunized mice with the dual APL (either orally or s.c.) down-regulated the peptide-specific acquisition of selectin adhesiveness. The down-regulation was accompanied by a reduction in the levels of secreted Th1-type cytokines (IFN-
and IL-2) and by an elevation in the secretion of the Th3-type immunosuppresive cytokine TGF-ß. The results presented in this work suggest that exposure of T cells from mice immunized with the myasthenogenic peptide to the dual APL reduces their capability to bind the endothelium and thus to be involved in the AChR-specific autoimmune response. Previous studies of our laboratory demonstrated that the dual APL could reverse myasthenogenic manifestations in mice with EAMG induced either by pathogenic T cell lines specific to the myasthenogenic peptide or by the native Torpedo AChR (13,17,18). Therefore although in the present report we did not induce EAMG in mice, it is likely that the dual APL down-regulates disease manifestation by the mechanisms suggested in this report.
To our best knowledge, this is the first research which demonstrates (i) an increase in LN- or splenic-derived T cell adhesiveness towards endothelial selectins (measured under physiological flow ex vivo), as a result of an in vivo immunization with an autoantigen, and (ii) specific in vivo modulation of the induced adhesion by an APL.
According to our findings, LN and spleen cells from p259271-immunized mice secrete IL-2 and IFN-
. Although we performed short-term experiments and did not reach the stage of disease, our results are in agreement with studies showing that in a number of models of autoimmune diseases, Th1-type cells are involved in autoimmunity (21). Both IL-2 and IFN-
were shown to be involved in the pathogenesis of MG (8,36) and EAMG (4,8,10,18,22), and disease suppression by mucosal tolerance was associated with their down-regulation (7,18). These findings support the importance of the reduced levels of Th1-type cytokines that we observed following treatment of mice with the dual APL.
Mucosal tolerance (either oral or nasal), which silences antigen-specific T cells and shuts down autoaggressive immune responses, has been used successfully to prevent experimental autoimmune encephalomyelitis, experimental autoimmune neuritis, uveitis, arthritis, diabetes (37) and EAMG (7,38,39). Oral tolerance induction involves regulatory Th2/Th3-type cells (21). In our system, IL-10 secretion from both spleens and LN cells was augmented only slightly by the dual APL. The secretion of another Th2-type cytokine, IL-4, from both splenocytes and LN cells taken from p259271-immunized mice was undetectable in most experiments. However, secretion of a very low concentration of IL-4 could be detected following treatment of the animals with the dual APL. Similar results were obtained when SJL mice were immunized with the second myasthenogenic peptide, p195212, and treated with the dual APL administered orally (17). Reports concerning the role of IL-4 in EAMG are controversial; however, it has been shown (40) that IL-4 is not required either for the generation of a pathogenic anti-AChR humoral immune response or for progression of clinical EAMG in mice. Thus, quantitatively, the main cytokine elevated following treatment of mice with the dual APL is TGF-ß. TGF-ß in blood cells of MG patients was correlated with the remission of MG (8). EAMG suppression by oral or nasal administration of AChR was associated with up-regulation of TGF-ß (8,41). These findings are in line with our results, that show that both oral and s.c. administration of the dual APL enhance the levels of TGF-ß. TGF-ß was also shown to inhibit expression of adhesion molecules on inflamed endothelium as well as on lymphocytes, and to diminish their transmigration in vitro and in vivo (4244). These reported effects of TGF-ß may account at least in part for the inhibitory effect of the dual APL on the myasthenogenic peptide-induced rolling of T cells on P- and E-selectin.
APL were shown to activate signal transduction events which are distinct from those induced by the original antigen. These different signal transduction pathways result in different phenotypes including anergy, shift in cytokine profile, etc. (45-47). In a previous study (48) we showed that the myasthenogenic peptide p259271 induced phospholipase C (PLC) activity in a peptide-specific T cell line and that the dual APL selectively inhibited the induced activity. Moreover, we could show that the dual APL serves as a partial agonist, since it could induce tyrosine phosphorylation of PLC in p259271-specific T cell lines but not the generation of inositol phosphate second messengers. The fact that treatment of p259271-immunized mice with the dual APL resulted in elevated secretion of TGF-ß, suggests that the dual APL does not eliminate T cells with a p259271/dual APL-specific TCR. Thus we assume that the reduced selectin adhesiveness observed in the present study is not the result of a decrease in the number of cells capable of responding to p259271, but rather may be due to the different characteristics of the T cell clones affected by the dual APL.
According to our results, any immunization, either with a general antigen like OVA or with p259271, causes up-regulation of T cell adhesiveness to E- and P-selectins. However, the dual APL inhibited specifically the p259271-induced rolling. As was previously shown by us (12), this peptide is an immunodominant T cell epitope which elicits efficient T and B cell responses in BALB/c mice. Thus it is not surprising that the up-regulation achieved by the myasthenogenic peptide is similar to that achieved by OVA.
Our results show that the dual APL was more effective in inhibiting the T cell adhesion to P-selectin than to E- selectin. E-selectin recognizes many ligands while P-selectin interacts with a single glycoprotein ligand, PSGL-1 (49). Thus, it may be less complicated to inhibit the interactions of P-selectin with its only ligand than the interactions of E-selectin with its multiple ligands. In addition, the formation of fully functional ligands for both E- and P-selectins depends, among others, on a particular
1,3-fucosyltransferase (FucT-VII) (26,50,51), and elevated expression of this enzyme in Th1 but not in Th2 cells correlates with their ability to bind P- and E-selectin (51). Our findings are compatible with the above, since the dual APL which reduces the Th1-type cytokine levels also reduces the T cell adhesiveness towards both E- and P- selectins, and may also interfere with the post-translational modifications required for a selectin ligand to be functional. It is possible that p259271 up-regulates FucT-VII on p259271-stimulated T cell subsets, and as a result these T cells can generate high levels of functional ligands to both P- and E-selectin. TGF-ß, the cytokine induced by the dual APL, is likely to inhibit the p259271-specific induction of FucT-VII in the p259271-specific T cells. Similarly, our results showing that naive T cells do not bind E- and P-selectins are in line with results reported by Wagers et al. (26) and Lichtman et al. (52) that human naive CD4+ T cells do not bind to vascular selectins and express detectable FucT-VII mRNA.
Treatment of the immunized mice with the dual APL, either orally or s.c., inhibited the p259271-induced T cell rolling on selectins and shifted the cytokine profile. This suggests that modulation of these two processes is part of the mechanism by which the dual APL exerts its beneficial effects in vivo regardless of the route of its administration. The treatment with the dual APL that leads to production of TGF-ß by peptide-specific T cells and intervention with migratory capabilities of these cells might reduce the ability of the latter to elicit autoimmune responses. The overall outcome of in vivo treatment with the dual APL might be a down-regulated immune response of autoreactive T cells that prevents further activation of B cells secreting the pathogenic anti-AChR antibodies. The latter may be part of the mechanism by which the dual APL reverses myasthenogenic manifestations in mice with EAMG, induced either by pathogenic T cell lines specific to the myasthenogenic peptide or by Torpedo AChR (13,17,18).
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Acknowledgments
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This research was supported in part by Teva Pharmaceutical Industries Ltd, Israel (M. S. and E. M.), and in part by the Minerva Foundation and the Israel Science Foundation (R. A.).
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Abbreviations
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AChR acetylcholine receptor |
APL altered peptide ligand |
CFA complete Freund's adjuvant |
EAMG experimental autoimmune myasthenia gravis |
FucT-VII 1,3-fucosyltransferase |
LN lymph node |
MG myasthenia gravis |
OVA ovalbumin |
PLC phospholipase C |
TGF transforming growth factor |
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Notes
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Transmitting editor: J.-F. Bach
Received 27 March 2000,
accepted 14 August 2000.
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References
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